t7 rna polymerase  (New England Biolabs)


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    Name:
    T7 RNA Polymerase
    Description:
    T7 RNA Polymerase 25 000 units
    Catalog Number:
    m0251l
    Price:
    282
    Size:
    25 000 units
    Category:
    RNA Polymerases
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    Structured Review

    New England Biolabs t7 rna polymerase
    T7 RNA Polymerase
    T7 RNA Polymerase 25 000 units
    https://www.bioz.com/result/t7 rna polymerase/product/New England Biolabs
    Average 99 stars, based on 42 article reviews
    Price from $9.99 to $1999.99
    t7 rna polymerase - by Bioz Stars, 2020-10
    99/100 stars

    Images

    1) Product Images from "A Reverse Genetics System for Zika Virus Based on a Simple Molecular Cloning Strategy"

    Article Title: A Reverse Genetics System for Zika Virus Based on a Simple Molecular Cloning Strategy

    Journal: Viruses

    doi: 10.3390/v10070368

    Construction and stability of synthetic full length Zika virus (synZIKV) cDNA clones. ( A ) Schematic representation of the synZIKV MR766 construct and the four fragments used to assemble the genome. The 5′ and 3′UTRs are indicated with bold black lines, the promoter for the T7 RNA polymerase with a black arrow. Restriction sites used for the assembly of the fragments are indicated. An enlargement of fragment #1 is shown below with putative CEPs (score > 0.85) indicated by red arrow heads. CEP 1 was not mutated (indicated with the pink arrow head). ( B ) Same as in panel ( A ) but for synZIKV-H/PF/2013. ( C ) Restriction patterns of pFK-synZIKV constructs obtained after digest with EcoRI (MR766) or XmnI (H/PF/2013) and agarose gel electrophoresis. Plasmids were analysed directly after assembly (original prep) and after five passages (P5) in E. coli (five DNA clones of P5 are shown).
    Figure Legend Snippet: Construction and stability of synthetic full length Zika virus (synZIKV) cDNA clones. ( A ) Schematic representation of the synZIKV MR766 construct and the four fragments used to assemble the genome. The 5′ and 3′UTRs are indicated with bold black lines, the promoter for the T7 RNA polymerase with a black arrow. Restriction sites used for the assembly of the fragments are indicated. An enlargement of fragment #1 is shown below with putative CEPs (score > 0.85) indicated by red arrow heads. CEP 1 was not mutated (indicated with the pink arrow head). ( B ) Same as in panel ( A ) but for synZIKV-H/PF/2013. ( C ) Restriction patterns of pFK-synZIKV constructs obtained after digest with EcoRI (MR766) or XmnI (H/PF/2013) and agarose gel electrophoresis. Plasmids were analysed directly after assembly (original prep) and after five passages (P5) in E. coli (five DNA clones of P5 are shown).

    Techniques Used: Clone Assay, Construct, Agarose Gel Electrophoresis

    2) Product Images from "Preferential Amplification of Pathogenic Sequences"

    Article Title: Preferential Amplification of Pathogenic Sequences

    Journal: Scientific Reports

    doi: 10.1038/srep11047

    Schematic representation of the PATHseq (Preferential Amplification of Pathogenic Sequences) method. ( 1 ) Total mRNAs from clinical sample, including human mRNAs and relatively scarce pathogenic mRNAs; ( 2 ) Total mRNAs are transcribed into first strand cDNAs with P1 primer; ( 3 ) RNase H cleaves RNAs in RNA-DNA duplex; ( 4 ) Reverse transcriptase (RT) synthesizes secondary cDNA strands with P2 primers; ( 5 ) T7 RNA polymerase synthesizes RNAs in the presence of T7 promoter; ( 6 ) Synthesized anti-sense RNAs; ( 7 ) Synthesized RNAs are hybridized to human reference (non-pathogenic) cDNA library coated on a solid phase. RNase H cleaves bound RNAs (human RNAs) in RNA-DNA duplex; ( 8 ) Pathogenic RNAs are enriched; ( 9 ) Reverse transcription; ( 10 ) RNase H cleaves RNAs in RNA-DNA duplex; ( 11 ) T7 RNA polymerase synthesizes RNAs; ( 12 ) New RNAs synthesized from enriched pathogenic RNAs are amplified 100-1000 fold.
    Figure Legend Snippet: Schematic representation of the PATHseq (Preferential Amplification of Pathogenic Sequences) method. ( 1 ) Total mRNAs from clinical sample, including human mRNAs and relatively scarce pathogenic mRNAs; ( 2 ) Total mRNAs are transcribed into first strand cDNAs with P1 primer; ( 3 ) RNase H cleaves RNAs in RNA-DNA duplex; ( 4 ) Reverse transcriptase (RT) synthesizes secondary cDNA strands with P2 primers; ( 5 ) T7 RNA polymerase synthesizes RNAs in the presence of T7 promoter; ( 6 ) Synthesized anti-sense RNAs; ( 7 ) Synthesized RNAs are hybridized to human reference (non-pathogenic) cDNA library coated on a solid phase. RNase H cleaves bound RNAs (human RNAs) in RNA-DNA duplex; ( 8 ) Pathogenic RNAs are enriched; ( 9 ) Reverse transcription; ( 10 ) RNase H cleaves RNAs in RNA-DNA duplex; ( 11 ) T7 RNA polymerase synthesizes RNAs; ( 12 ) New RNAs synthesized from enriched pathogenic RNAs are amplified 100-1000 fold.

    Techniques Used: Amplification, Synthesized, cDNA Library Assay

    3) Product Images from "A Reverse Genetics System for Zika Virus Based on a Simple Molecular Cloning Strategy"

    Article Title: A Reverse Genetics System for Zika Virus Based on a Simple Molecular Cloning Strategy

    Journal: Viruses

    doi: 10.3390/v10070368

    Construction and stability of synthetic full length Zika virus (synZIKV) cDNA clones. ( A ) Schematic representation of the synZIKV MR766 construct and the four fragments used to assemble the genome. The 5′ and 3′UTRs are indicated with bold black lines, the promoter for the T7 RNA polymerase with a black arrow. Restriction sites used for the assembly of the fragments are indicated. An enlargement of fragment #1 is shown below with putative CEPs (score > 0.85) indicated by red arrow heads. CEP 1 was not mutated (indicated with the pink arrow head). ( B ) Same as in panel ( A ) but for synZIKV-H/PF/2013. ( C ) Restriction patterns of pFK-synZIKV constructs obtained after digest with EcoRI (MR766) or XmnI (H/PF/2013) and agarose gel electrophoresis. Plasmids were analysed directly after assembly (original prep) and after five passages (P5) in E. coli (five DNA clones of P5 are shown).
    Figure Legend Snippet: Construction and stability of synthetic full length Zika virus (synZIKV) cDNA clones. ( A ) Schematic representation of the synZIKV MR766 construct and the four fragments used to assemble the genome. The 5′ and 3′UTRs are indicated with bold black lines, the promoter for the T7 RNA polymerase with a black arrow. Restriction sites used for the assembly of the fragments are indicated. An enlargement of fragment #1 is shown below with putative CEPs (score > 0.85) indicated by red arrow heads. CEP 1 was not mutated (indicated with the pink arrow head). ( B ) Same as in panel ( A ) but for synZIKV-H/PF/2013. ( C ) Restriction patterns of pFK-synZIKV constructs obtained after digest with EcoRI (MR766) or XmnI (H/PF/2013) and agarose gel electrophoresis. Plasmids were analysed directly after assembly (original prep) and after five passages (P5) in E. coli (five DNA clones of P5 are shown).

    Techniques Used: Clone Assay, Construct, Agarose Gel Electrophoresis

    4) Product Images from "Filamentation and restoration of normal growth in Escherichia coli using a combined CRISPRi sgRNA/antisense RNA approach"

    Article Title: Filamentation and restoration of normal growth in Escherichia coli using a combined CRISPRi sgRNA/antisense RNA approach

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0198058

    CRISPRi-based growth control. (A) Schematic representation of E . coli switching into filamentous growth after induction of single guide RNA (sgRNA) via IPTG and dCas9 via aTc. The dCas9-sgRNA complex blocks the expression of FtsZ, stopping the formation of the septal ring that is essential for cell division in E . coli . Cell division can be rescued by inducing appropriate antisense sgRNAs (‘anti-sgRNA’) with AHL and by removing the inducers for the dCas9 and sgRNA. (B) Scheme of sgRNA forming a complex with dCas9. The anti-sgRNA can inhibit formation of the complex by binding to the sgRNA. (C) Details of the genetic constructs involved: the CRISPRi plasmid codes for dCas9 under aTc-inducible promoters and three different sgRNAs under T7 promoters which target three different promoters of the ftsZ gene on the genome of the E . coli . T7 RNA polymerase is inducible with IPTG. The ‘anti-sgRNA plasmid’ codes for anti-sgRNAs under the control of an AHL-inducible promoter. The sponge elements on the plasmids act as decoy binding sites for the corresponding dCas9-sgRNA complexes.
    Figure Legend Snippet: CRISPRi-based growth control. (A) Schematic representation of E . coli switching into filamentous growth after induction of single guide RNA (sgRNA) via IPTG and dCas9 via aTc. The dCas9-sgRNA complex blocks the expression of FtsZ, stopping the formation of the septal ring that is essential for cell division in E . coli . Cell division can be rescued by inducing appropriate antisense sgRNAs (‘anti-sgRNA’) with AHL and by removing the inducers for the dCas9 and sgRNA. (B) Scheme of sgRNA forming a complex with dCas9. The anti-sgRNA can inhibit formation of the complex by binding to the sgRNA. (C) Details of the genetic constructs involved: the CRISPRi plasmid codes for dCas9 under aTc-inducible promoters and three different sgRNAs under T7 promoters which target three different promoters of the ftsZ gene on the genome of the E . coli . T7 RNA polymerase is inducible with IPTG. The ‘anti-sgRNA plasmid’ codes for anti-sgRNAs under the control of an AHL-inducible promoter. The sponge elements on the plasmids act as decoy binding sites for the corresponding dCas9-sgRNA complexes.

    Techniques Used: Expressing, Binding Assay, Construct, Plasmid Preparation, Activated Clotting Time Assay

    5) Product Images from "The Hepatitis C Virus RNA 3?-Untranslated Region Strongly Enhances Translation Directed by the Internal Ribosome Entry Site ▿"

    Article Title: The Hepatitis C Virus RNA 3?-Untranslated Region Strongly Enhances Translation Directed by the Internal Ribosome Entry Site ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.00675-06

    The HCV 3′-UTR enhances translation of full-length genomes. (A) Structure of the transfected RNAs. Full-length HCV genome RNAs including the 3′-UTR (upper panel) or not (lower panel) were in vitro transcribed using T7 RNA polymerase. Precise
    Figure Legend Snippet: The HCV 3′-UTR enhances translation of full-length genomes. (A) Structure of the transfected RNAs. Full-length HCV genome RNAs including the 3′-UTR (upper panel) or not (lower panel) were in vitro transcribed using T7 RNA polymerase. Precise

    Techniques Used: Transfection, In Vitro

    Analysis of the effect of the HCV 3′-UTR on translation efficiency in vitro. (A) The HCV reporter constructs. RNAs (arrows) were obtained by in vitro transcription using T7 RNA polymerase either from linearized plasmids or from PCR fragments (lines).
    Figure Legend Snippet: Analysis of the effect of the HCV 3′-UTR on translation efficiency in vitro. (A) The HCV reporter constructs. RNAs (arrows) were obtained by in vitro transcription using T7 RNA polymerase either from linearized plasmids or from PCR fragments (lines).

    Techniques Used: In Vitro, Construct, Polymerase Chain Reaction

    6) Product Images from "RNA primer–primase complexes serve as the signal for polymerase recycling and Okazaki fragment initiation in T4 phage DNA replication"

    Article Title: RNA primer–primase complexes serve as the signal for polymerase recycling and Okazaki fragment initiation in T4 phage DNA replication

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1620459114

    Synthesis of a physiological RNA primer. ( A ) The physiological RNA primer (pppRNA) with the sequence 5′-ppp-rGrCrCrGrA-3′ with a 5′-triphosphate moiety was synthesized using a standard run-off transcription protocol with T7 RNA polymerase. The resulting RNA product was size fractionated on a 7.5 M urea–20% polyacrylamide gel. ( B ) Mass spectroscopy data acquired on a Waters Micromass LCT Premier time-of-flight mass spectrometer equipped with a Waters Alliance 2695 Separations Module and using electrospray ionization confirmed the pentaribonucleotide product by excellent agreement between the measured and calculated molecular masses.
    Figure Legend Snippet: Synthesis of a physiological RNA primer. ( A ) The physiological RNA primer (pppRNA) with the sequence 5′-ppp-rGrCrCrGrA-3′ with a 5′-triphosphate moiety was synthesized using a standard run-off transcription protocol with T7 RNA polymerase. The resulting RNA product was size fractionated on a 7.5 M urea–20% polyacrylamide gel. ( B ) Mass spectroscopy data acquired on a Waters Micromass LCT Premier time-of-flight mass spectrometer equipped with a Waters Alliance 2695 Separations Module and using electrospray ionization confirmed the pentaribonucleotide product by excellent agreement between the measured and calculated molecular masses.

    Techniques Used: Sequencing, Synthesized, Mass Spectrometry

    7) Product Images from "Protein Expression Redirects Vesicular Stomatitis Virus RNA Synthesis to Cytoplasmic Inclusions"

    Article Title: Protein Expression Redirects Vesicular Stomatitis Virus RNA Synthesis to Cytoplasmic Inclusions

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1000958

    VSV RNA polymerase incorporates BrUTP during transcription in vitro and in vivo . ( A ) Incorporation of BrUTP into RNA synthesized in vitro . An autoradiograph of an acid agarose-urea gel is shown, depicting RNA transcribed by T7 RNA polymerase from a plasmid encoding VSV N (lanes 1–5) or synthesized by detergent activated virus in vitro (lanes 6–10) in the presence of increasing concentrations (0, 0.1, 0.5 or 1 mM) of BrUTP. The products of the reactions are indicated alongside the gel. ( B ) The samples of panel A were immune precipitated using an antibody raised against bromodeoxyuridine prior to acid-agarose gel electrophoresis. ( C ) The samples of panel A were isolated by oligo-dT chromatography prior to acid-agarose gel electrophoresis. ( D ) BSR-T7 cells were infected with wild-type VSV and, where indicated (+), transfected 4 hpi with BrUTP (5mM final concentration). Cells were exposed to [ 3 H]-uridine for 5 hours and RNA was isolated prior to acid-agarose gel electrophoresis. Where indicated (IP) the RNA was immunoprecipitated as in panel (B).
    Figure Legend Snippet: VSV RNA polymerase incorporates BrUTP during transcription in vitro and in vivo . ( A ) Incorporation of BrUTP into RNA synthesized in vitro . An autoradiograph of an acid agarose-urea gel is shown, depicting RNA transcribed by T7 RNA polymerase from a plasmid encoding VSV N (lanes 1–5) or synthesized by detergent activated virus in vitro (lanes 6–10) in the presence of increasing concentrations (0, 0.1, 0.5 or 1 mM) of BrUTP. The products of the reactions are indicated alongside the gel. ( B ) The samples of panel A were immune precipitated using an antibody raised against bromodeoxyuridine prior to acid-agarose gel electrophoresis. ( C ) The samples of panel A were isolated by oligo-dT chromatography prior to acid-agarose gel electrophoresis. ( D ) BSR-T7 cells were infected with wild-type VSV and, where indicated (+), transfected 4 hpi with BrUTP (5mM final concentration). Cells were exposed to [ 3 H]-uridine for 5 hours and RNA was isolated prior to acid-agarose gel electrophoresis. Where indicated (IP) the RNA was immunoprecipitated as in panel (B).

    Techniques Used: In Vitro, In Vivo, Synthesized, Autoradiography, Plasmid Preparation, Agarose Gel Electrophoresis, Isolation, Chromatography, Infection, Transfection, Concentration Assay, Immunoprecipitation

    8) Product Images from "iSpinach: a fluorogenic RNA aptamer optimized for in vitro applications"

    Article Title: iSpinach: a fluorogenic RNA aptamer optimized for in vitro applications

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw083

    Real-time monitoring of RNA synthesis and ribozyme activity. ( A ) Constructs used. DNA fragments coding for iSpinach, Spinach2 or iXm1 ribozyme were combined in different ways and placed under the control of T7 RNA polymerase promoter sequence (Pro T7 ). ( B ) Real-time ribozyme activity monitoring. The different constructs were in vitro transcribed in the presence of ribozyme fluorogenic substrate (S21-Atto) and the fluorescence monitored at 37°C. Product generation rate \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$\left( {\frac{{{\rm dP}}}{{{\rm dt}}}} \right)$\end{document} and uncatalyzed reaction rate ( k uncat ) were determined as the slope of the linear phase of reaction respectively in the presence and absence of ribozyme. ( C ) Real-time transcription monitoring. The in vitro transcription mixture used in B. was supplemented with DFHBI (instead of S21-Atto) and the fluorescence monitored at 37°C. The synthesis rate (σ) was determined as the slope of the linear phase.
    Figure Legend Snippet: Real-time monitoring of RNA synthesis and ribozyme activity. ( A ) Constructs used. DNA fragments coding for iSpinach, Spinach2 or iXm1 ribozyme were combined in different ways and placed under the control of T7 RNA polymerase promoter sequence (Pro T7 ). ( B ) Real-time ribozyme activity monitoring. The different constructs were in vitro transcribed in the presence of ribozyme fluorogenic substrate (S21-Atto) and the fluorescence monitored at 37°C. Product generation rate \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$\left( {\frac{{{\rm dP}}}{{{\rm dt}}}} \right)$\end{document} and uncatalyzed reaction rate ( k uncat ) were determined as the slope of the linear phase of reaction respectively in the presence and absence of ribozyme. ( C ) Real-time transcription monitoring. The in vitro transcription mixture used in B. was supplemented with DFHBI (instead of S21-Atto) and the fluorescence monitored at 37°C. The synthesis rate (σ) was determined as the slope of the linear phase.

    Techniques Used: Activity Assay, Construct, Sequencing, In Vitro, Fluorescence

    9) Product Images from "Quantitative Assessment of RNA-Protein Interactions with High Throughput Sequencing - RNA Affinity Profiling (HiTS-RAP)"

    Article Title: Quantitative Assessment of RNA-Protein Interactions with High Throughput Sequencing - RNA Affinity Profiling (HiTS-RAP)

    Journal: Nature protocols

    doi: 10.1038/nprot.2015.074

    DNA template and RNA transcript of HiTS-RAP. (A) Schematics of the DNA template used for HiTS-RAP and the resulting halted RNA transcript. DNA template encoding the RNA of interest (green) is flanked by the Illumina flowcell adaptor 1 (gray) and T7 RNA polymerase promoter (orange) upstream, and by the Illumina sequencing primer annealing site (purple), Tus-binding Ter site (red), and Illumina flowcell adaptor 2 downstream. Illumina flowcell adaptors 1 and 2 are required for cluster generation on Illumina GA flowcell. T7 RNA polymerase promoter is required for transcription of the RNA of interest. The Illumina sequencing primer is used for sequencing the DNA template of the RNA of interest and serves as a docking site for the T7 RNA polymerase when it is halted. Tus protein binds to Ter site and halts the transcribing RNA polymerase. Direction of transcription and sequencing are indicated by orange and purple arrows, respectively. Tus-bound Ter site that is non-permissive (halting) and permissive (read-through) to RNA polymerase are indicated by solid and open red triangles, respectively. The halted RNA transcript includes a triplet G derived from the T7 promoter, followed by the RNA of interest and some of the Illumina sequencing primer. The 3’-end of RNA transcript, indicated by a dashed line, is inaccessible. (B) Construction of DNA templates for HiTS-RAP. DNA template is constructed by PCR in two steps using 2 sets of nested oligos. Forward oligos introduce T7 promoter (step 1) and Illumina flowcell adaptor (step 2), whereas reverse oligos introduce Illumina sequencing primer (step 1), and Ter site and Illumina flowcell adaptor 2 (step 2). (C) Sequence of the HiTS-RAP DNA template for GFP aptamer. GFP aptamer encoding sequence is in green, and the rest of the sequences are colored as in (A). The transcription start site is indicated by +1 and a broken arrow. (D) Sequence of the halted RNA transcript from GFP aptamer template. Sequences are colored as in (C). Uppercase indicates the region of the halted RNA transcript that is accessible, and the lowercase indicates a region that is likely to be buried in T7 RNA polymerase and thus inaccessible by other proteins.
    Figure Legend Snippet: DNA template and RNA transcript of HiTS-RAP. (A) Schematics of the DNA template used for HiTS-RAP and the resulting halted RNA transcript. DNA template encoding the RNA of interest (green) is flanked by the Illumina flowcell adaptor 1 (gray) and T7 RNA polymerase promoter (orange) upstream, and by the Illumina sequencing primer annealing site (purple), Tus-binding Ter site (red), and Illumina flowcell adaptor 2 downstream. Illumina flowcell adaptors 1 and 2 are required for cluster generation on Illumina GA flowcell. T7 RNA polymerase promoter is required for transcription of the RNA of interest. The Illumina sequencing primer is used for sequencing the DNA template of the RNA of interest and serves as a docking site for the T7 RNA polymerase when it is halted. Tus protein binds to Ter site and halts the transcribing RNA polymerase. Direction of transcription and sequencing are indicated by orange and purple arrows, respectively. Tus-bound Ter site that is non-permissive (halting) and permissive (read-through) to RNA polymerase are indicated by solid and open red triangles, respectively. The halted RNA transcript includes a triplet G derived from the T7 promoter, followed by the RNA of interest and some of the Illumina sequencing primer. The 3’-end of RNA transcript, indicated by a dashed line, is inaccessible. (B) Construction of DNA templates for HiTS-RAP. DNA template is constructed by PCR in two steps using 2 sets of nested oligos. Forward oligos introduce T7 promoter (step 1) and Illumina flowcell adaptor (step 2), whereas reverse oligos introduce Illumina sequencing primer (step 1), and Ter site and Illumina flowcell adaptor 2 (step 2). (C) Sequence of the HiTS-RAP DNA template for GFP aptamer. GFP aptamer encoding sequence is in green, and the rest of the sequences are colored as in (A). The transcription start site is indicated by +1 and a broken arrow. (D) Sequence of the halted RNA transcript from GFP aptamer template. Sequences are colored as in (C). Uppercase indicates the region of the halted RNA transcript that is accessible, and the lowercase indicates a region that is likely to be buried in T7 RNA polymerase and thus inaccessible by other proteins.

    Techniques Used: Sequencing, Binding Assay, Derivative Assay, Construct, Polymerase Chain Reaction, Introduce

    10) Product Images from "Synthesis of 2′-Fluoro RNA by Syn5 RNA polymerase"

    Article Title: Synthesis of 2′-Fluoro RNA by Syn5 RNA polymerase

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkv367

    Impact of initial sequence of transcript on the yield of transcripts synthesized by Syn5 RNA polymerase. (A) Influence of different sequences at various positions in the first 12 nucleotides at the 5′ end of transcripts on the yield of products synthesized by Syn5 RNA polymerase. The DNA template used in each reaction is indicated at the top of each lane. All of the templates were derived from template T4, from which the first 12 nucleotides at the 5′ end of the RNA product are shown at the top of the gel. The variations in the sequence of RNA products from template T4 synthesized on each template are color coded; variations in the first three nucleotides are in orange, nucleotides 4–6 in blue, nucleotides 7-9 in purple and nucleotides 10–12 in green. The position of the migration of the DNA templates and the RNA products are marked on the left of the gel. (B) Influence of different sequences at various positions in the first 10 nucleotides at the 5′ end of transcripts on the yield of products synthesized by Syn5 and T7 RNA polymerases. Transcription reactions were carried out using Syn5 RNA polymerase (lanes 1–7, 13 and 14) and T7 RNA polymerase (lanes 8–12). The templates used for each reaction and the sequence of the first 10 nucleotides at the 5′ end of each RNA are shown at the bottom of the gel. Variations in RNA products encoded by each template are in blue background. (C) Influence of NTP concentration on the yield of products synthesized by Syn5 RNA polymerase. Transcription reactions were carried out on templates T32 (lanes 1–3), T2 (lanes 4–6) and T33 (lanes 7–9). The first three nucleotides of the transcript synthesized on each of these templates are UGA (T32), GGG (T2) and GGG (T33), as indicated at the bottom of the gel. The concentration of the NTP being varied in each reaction mixture is shown at the top; the concentrations of the other three NTPs were fixed at 4 mM. The position of the migration of the DNA templates and the RNA products are marked on the left of the gel. (D) and (E) Incorporation of 2′-F-dCMP and/or 2′-F-dUMP into transcripts synthesized by Syn5 and T7 RNA polymerases. The templates used for each reaction (T18, T17, T28 and T4) and the sequence of the first 12 nucleotides at the 5′ end of each RNA are shown at the bottom of the gel. The NTP analog present in each reaction is indicated at the top of the gel where ‘C/U’ corresponds to reactions carried out in the presence of both 2′-F-dCTP and 2′-F-dUTP.
    Figure Legend Snippet: Impact of initial sequence of transcript on the yield of transcripts synthesized by Syn5 RNA polymerase. (A) Influence of different sequences at various positions in the first 12 nucleotides at the 5′ end of transcripts on the yield of products synthesized by Syn5 RNA polymerase. The DNA template used in each reaction is indicated at the top of each lane. All of the templates were derived from template T4, from which the first 12 nucleotides at the 5′ end of the RNA product are shown at the top of the gel. The variations in the sequence of RNA products from template T4 synthesized on each template are color coded; variations in the first three nucleotides are in orange, nucleotides 4–6 in blue, nucleotides 7-9 in purple and nucleotides 10–12 in green. The position of the migration of the DNA templates and the RNA products are marked on the left of the gel. (B) Influence of different sequences at various positions in the first 10 nucleotides at the 5′ end of transcripts on the yield of products synthesized by Syn5 and T7 RNA polymerases. Transcription reactions were carried out using Syn5 RNA polymerase (lanes 1–7, 13 and 14) and T7 RNA polymerase (lanes 8–12). The templates used for each reaction and the sequence of the first 10 nucleotides at the 5′ end of each RNA are shown at the bottom of the gel. Variations in RNA products encoded by each template are in blue background. (C) Influence of NTP concentration on the yield of products synthesized by Syn5 RNA polymerase. Transcription reactions were carried out on templates T32 (lanes 1–3), T2 (lanes 4–6) and T33 (lanes 7–9). The first three nucleotides of the transcript synthesized on each of these templates are UGA (T32), GGG (T2) and GGG (T33), as indicated at the bottom of the gel. The concentration of the NTP being varied in each reaction mixture is shown at the top; the concentrations of the other three NTPs were fixed at 4 mM. The position of the migration of the DNA templates and the RNA products are marked on the left of the gel. (D) and (E) Incorporation of 2′-F-dCMP and/or 2′-F-dUMP into transcripts synthesized by Syn5 and T7 RNA polymerases. The templates used for each reaction (T18, T17, T28 and T4) and the sequence of the first 12 nucleotides at the 5′ end of each RNA are shown at the bottom of the gel. The NTP analog present in each reaction is indicated at the top of the gel where ‘C/U’ corresponds to reactions carried out in the presence of both 2′-F-dCTP and 2′-F-dUTP.

    Techniques Used: Sequencing, Synthesized, Derivative Assay, Migration, Concentration Assay

    11) Product Images from "Sensitive detection of a bacterial pathogen using allosteric probe-initiated catalysis and CRISPR-Cas13a amplification reaction"

    Article Title: Sensitive detection of a bacterial pathogen using allosteric probe-initiated catalysis and CRISPR-Cas13a amplification reaction

    Journal: Nature Communications

    doi: 10.1038/s41467-019-14135-9

    Analysis of APC-Cas. a Illustration and representative laser scanning confocal microscope (LSCM) images of dual-labeled AP binding to the S . Enteritidis. b Electrophoretic analysis of the feasibility of APC-Cas for S . Enteritidis detection. M: DNA marker, L1: AP; L2: AP + primer; L3: AP + primer + KF DNA polymerase; L4: AP + primer + KF DNA polymerase + S . Enteritidis; L5: AP + primer + KF DNA polymerase + S . Enteritidis + T7 RNA polymerase. ‘ + ’ means presence, ‘-’ means absence. c Fluorescence measurement of LbuCas13a activity. RNase A was used as positive control for the degradation of RNA reporter probe. d Comparison of seven APs with varied stem-length. Data represent mean ± s.d., n = 3, three technical replicates.
    Figure Legend Snippet: Analysis of APC-Cas. a Illustration and representative laser scanning confocal microscope (LSCM) images of dual-labeled AP binding to the S . Enteritidis. b Electrophoretic analysis of the feasibility of APC-Cas for S . Enteritidis detection. M: DNA marker, L1: AP; L2: AP + primer; L3: AP + primer + KF DNA polymerase; L4: AP + primer + KF DNA polymerase + S . Enteritidis; L5: AP + primer + KF DNA polymerase + S . Enteritidis + T7 RNA polymerase. ‘ + ’ means presence, ‘-’ means absence. c Fluorescence measurement of LbuCas13a activity. RNase A was used as positive control for the degradation of RNA reporter probe. d Comparison of seven APs with varied stem-length. Data represent mean ± s.d., n = 3, three technical replicates.

    Techniques Used: Microscopy, Labeling, Binding Assay, Marker, Fluorescence, Activity Assay, Positive Control

    12) Product Images from "Broad-Spectrum Protection against Tombusviruses Elicited by Defective Interfering RNAs in Transgenic Plants"

    Article Title: Broad-Spectrum Protection against Tombusviruses Elicited by Defective Interfering RNAs in Transgenic Plants

    Journal: Journal of Virology

    doi:

    Processing of TBSV B10 DI RNA flanked by ribozymes. (A) A map of the TBSV genome is illustrated at the top, with the six coding regions (p33, p92, p41, p22, p19, and pX) identified. The four conserved regions of the 595-nt B10 DI RNA are represented below the map by blocks (I, II, III, and IV) to indicate the sequence motifs derived from different regions of the TBSV genome. (B) Schematic illustration of ribozyme processing. The B10 DI RNA was flanked at its 5′ end by the ASBVd ribozyme (5′Rz) and at its 3′ end by the TRSV ribozyme (3′Rz). Sites of ribozyme cleavage are indicated by small arrows flanking the DI RNA. Intermediate and final products (B10 DI, 5′Rz, and 3′Rz) of the in vitro processing reaction are illustrated. (C) Evaluation of ribozyme processing in vitro. Time course experiments showing the in vitro processing of the B10 DI RNA by flanking ribozymes. The positions of the processed and unprocessed DI RNAs, as well as the locations of the liberated ribozymes (5′Rz and 3′Rz), are indicated. The in vitro transcription reactions were performed at 37°C with T7 RNA polymerase in the presence of [ 32 P]UTP, and the samples were separated on 5% acrylamide gels. Short (4-h) and long (20-h) exposures were used to permit visualization of the individual bands.
    Figure Legend Snippet: Processing of TBSV B10 DI RNA flanked by ribozymes. (A) A map of the TBSV genome is illustrated at the top, with the six coding regions (p33, p92, p41, p22, p19, and pX) identified. The four conserved regions of the 595-nt B10 DI RNA are represented below the map by blocks (I, II, III, and IV) to indicate the sequence motifs derived from different regions of the TBSV genome. (B) Schematic illustration of ribozyme processing. The B10 DI RNA was flanked at its 5′ end by the ASBVd ribozyme (5′Rz) and at its 3′ end by the TRSV ribozyme (3′Rz). Sites of ribozyme cleavage are indicated by small arrows flanking the DI RNA. Intermediate and final products (B10 DI, 5′Rz, and 3′Rz) of the in vitro processing reaction are illustrated. (C) Evaluation of ribozyme processing in vitro. Time course experiments showing the in vitro processing of the B10 DI RNA by flanking ribozymes. The positions of the processed and unprocessed DI RNAs, as well as the locations of the liberated ribozymes (5′Rz and 3′Rz), are indicated. The in vitro transcription reactions were performed at 37°C with T7 RNA polymerase in the presence of [ 32 P]UTP, and the samples were separated on 5% acrylamide gels. Short (4-h) and long (20-h) exposures were used to permit visualization of the individual bands.

    Techniques Used: Sequencing, Derivative Assay, In Vitro

    13) Product Images from "Filamentation and restoration of normal growth in Escherichia coli using a combined CRISPRi sgRNA/antisense RNA approach"

    Article Title: Filamentation and restoration of normal growth in Escherichia coli using a combined CRISPRi sgRNA/antisense RNA approach

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0198058

    CRISPRi-based growth control. (A) Schematic representation of E . coli switching into filamentous growth after induction of single guide RNA (sgRNA) via IPTG and dCas9 via aTc. The dCas9-sgRNA complex blocks the expression of FtsZ, stopping the formation of the septal ring that is essential for cell division in E . coli . Cell division can be rescued by inducing appropriate antisense sgRNAs (‘anti-sgRNA’) with AHL and by removing the inducers for the dCas9 and sgRNA. (B) Scheme of sgRNA forming a complex with dCas9. The anti-sgRNA can inhibit formation of the complex by binding to the sgRNA. (C) Details of the genetic constructs involved: the CRISPRi plasmid codes for dCas9 under aTc-inducible promoters and three different sgRNAs under T7 promoters which target three different promoters of the ftsZ gene on the genome of the E . coli . T7 RNA polymerase is inducible with IPTG. The ‘anti-sgRNA plasmid’ codes for anti-sgRNAs under the control of an AHL-inducible promoter. The sponge elements on the plasmids act as decoy binding sites for the corresponding dCas9-sgRNA complexes.
    Figure Legend Snippet: CRISPRi-based growth control. (A) Schematic representation of E . coli switching into filamentous growth after induction of single guide RNA (sgRNA) via IPTG and dCas9 via aTc. The dCas9-sgRNA complex blocks the expression of FtsZ, stopping the formation of the septal ring that is essential for cell division in E . coli . Cell division can be rescued by inducing appropriate antisense sgRNAs (‘anti-sgRNA’) with AHL and by removing the inducers for the dCas9 and sgRNA. (B) Scheme of sgRNA forming a complex with dCas9. The anti-sgRNA can inhibit formation of the complex by binding to the sgRNA. (C) Details of the genetic constructs involved: the CRISPRi plasmid codes for dCas9 under aTc-inducible promoters and three different sgRNAs under T7 promoters which target three different promoters of the ftsZ gene on the genome of the E . coli . T7 RNA polymerase is inducible with IPTG. The ‘anti-sgRNA plasmid’ codes for anti-sgRNAs under the control of an AHL-inducible promoter. The sponge elements on the plasmids act as decoy binding sites for the corresponding dCas9-sgRNA complexes.

    Techniques Used: Expressing, Binding Assay, Construct, Plasmid Preparation, Activated Clotting Time Assay

    14) Product Images from "A transcription and translation-coupled DNA replication system using rolling-circle replication"

    Article Title: A transcription and translation-coupled DNA replication system using rolling-circle replication

    Journal: Scientific Reports

    doi: 10.1038/srep10404

    Transcription- and translation-coupled DNA (TTcDR) replication. To perform the TTcDR reaction, circular plasmid DNA encoding phi29 DNA polymerase was incubated with the translation system optimized in a previous study 11 , including dNTPs, yeast ppiase, T7 RNA polymerase, and [ 32 P]-dCTP, for 12 h at 30 °C. An aliquot of the mixture after incubation was used in 1% agarose gel electrophoresis and autoradiography. The arrowhead indicates the product of the TTcDR reaction. Lane 1: lambda-BstPI marker. Lane 2: TTcDR reaction without plasmid DNA. Lane 3: TTcDR reaction with plasmid DNA. Lane 4: DNA polymerization with a purified phi29 in phi29 standard buffer.
    Figure Legend Snippet: Transcription- and translation-coupled DNA (TTcDR) replication. To perform the TTcDR reaction, circular plasmid DNA encoding phi29 DNA polymerase was incubated with the translation system optimized in a previous study 11 , including dNTPs, yeast ppiase, T7 RNA polymerase, and [ 32 P]-dCTP, for 12 h at 30 °C. An aliquot of the mixture after incubation was used in 1% agarose gel electrophoresis and autoradiography. The arrowhead indicates the product of the TTcDR reaction. Lane 1: lambda-BstPI marker. Lane 2: TTcDR reaction without plasmid DNA. Lane 3: TTcDR reaction with plasmid DNA. Lane 4: DNA polymerization with a purified phi29 in phi29 standard buffer.

    Techniques Used: Plasmid Preparation, Incubation, Agarose Gel Electrophoresis, Autoradiography, Marker, Purification

    Schematic representation of the transcription- and translation-coupled DNA replication system. Circular DNA encoding phi29 DNA polymerase under control of the T7 promoter is incubated with the reconstituted translation system including T7 RNA polymerase. mRNA is transcribed from the DNA, and phi29 DNA polymerase is translated. The polymerase attaches to the circular DNA and initiates the polymerization of a long single-stranded RNA in a rolling-circle manner. The polymerase further synthesizes the complementary strand to produce double-stranded DNA, which is a long repeat of the circular DNA sequence. The next round of transcription and translation occurs from the double-stranded DNA.
    Figure Legend Snippet: Schematic representation of the transcription- and translation-coupled DNA replication system. Circular DNA encoding phi29 DNA polymerase under control of the T7 promoter is incubated with the reconstituted translation system including T7 RNA polymerase. mRNA is transcribed from the DNA, and phi29 DNA polymerase is translated. The polymerase attaches to the circular DNA and initiates the polymerization of a long single-stranded RNA in a rolling-circle manner. The polymerase further synthesizes the complementary strand to produce double-stranded DNA, which is a long repeat of the circular DNA sequence. The next round of transcription and translation occurs from the double-stranded DNA.

    Techniques Used: Incubation, Sequencing

    15) Product Images from "The Stem-Loop Binding Protein Is Required for Efficient Translation of Histone mRNA In Vivo and In Vitro"

    Article Title: The Stem-Loop Binding Protein Is Required for Efficient Translation of Histone mRNA In Vivo and In Vitro

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.22.20.7093-7104.2002

    SLBP stimulates histone mRNA translation in vitro. (A) Structure of the 3′ end of the luciferase reporter mRNAs. The Luc-SL mRNA ends in the histone stem-loop that binds SLBP, the Luc-TL mRNA ends in a stem-loop that does not bind SLBP, and the Luc-polyA mRNA ends in a poly(A) tail 50 nt long. ORF, open reading frame. (B) Schematic of the in vitro translation assay. An aliquot of RRL was incubated either with buffer or with SLBP DNA in the presence of T7 RNA polymerase and [ 35 S]methionine. After incubation for 90 min, an aliquot of the lysate was mixed with a fresh aliquot of reticulocyte lysate together with a luciferase uncapped reporter mRNA and a polyadenylated uncapped CAT mRNA as an internal standard. After incubation for 90 min at 30°C, the reaction products were analyzed by SDS-PAGE and the in vitro-synthesized proteins were detected by autoradiography. An aliquot of the assay was analyzed for luciferase activity by luminometry. (C) Lysates containing no SLBP (lanes 1 to 3), xSLBP2 (lanes 4 to 6), or xSLBP1 (lanes 7 to 9) were incubated with the standard polyadenylated CAT mRNA and the Luc-TL (lanes 1, 4, and 7), Luc-SL (lanes 2, 5, and 8), or Luc-polyA (lanes 3, 6, and 9) mRNA. The reaction products were analyzed by gel electrophoresis, and the proteins were detected by autoradiography. (D) The autoradiogram (panel C) was quantified, and relative luciferase activity was calculated. In addition, an aliquot of each reaction mixture was analyzed for luciferase activity with a luminometer. Quantification of the results by PhosphorImager and by luciferase assay yielded identical results. Panel D shows the results of an average of five experiments with five different batches of RRL, and the error bars represent the standard deviation. The fold activation is the result of luciferase activity from the Luc-SL and Luc-polyA mRNAs divided by the luciferase activity from the Luc-TL mRNA.
    Figure Legend Snippet: SLBP stimulates histone mRNA translation in vitro. (A) Structure of the 3′ end of the luciferase reporter mRNAs. The Luc-SL mRNA ends in the histone stem-loop that binds SLBP, the Luc-TL mRNA ends in a stem-loop that does not bind SLBP, and the Luc-polyA mRNA ends in a poly(A) tail 50 nt long. ORF, open reading frame. (B) Schematic of the in vitro translation assay. An aliquot of RRL was incubated either with buffer or with SLBP DNA in the presence of T7 RNA polymerase and [ 35 S]methionine. After incubation for 90 min, an aliquot of the lysate was mixed with a fresh aliquot of reticulocyte lysate together with a luciferase uncapped reporter mRNA and a polyadenylated uncapped CAT mRNA as an internal standard. After incubation for 90 min at 30°C, the reaction products were analyzed by SDS-PAGE and the in vitro-synthesized proteins were detected by autoradiography. An aliquot of the assay was analyzed for luciferase activity by luminometry. (C) Lysates containing no SLBP (lanes 1 to 3), xSLBP2 (lanes 4 to 6), or xSLBP1 (lanes 7 to 9) were incubated with the standard polyadenylated CAT mRNA and the Luc-TL (lanes 1, 4, and 7), Luc-SL (lanes 2, 5, and 8), or Luc-polyA (lanes 3, 6, and 9) mRNA. The reaction products were analyzed by gel electrophoresis, and the proteins were detected by autoradiography. (D) The autoradiogram (panel C) was quantified, and relative luciferase activity was calculated. In addition, an aliquot of each reaction mixture was analyzed for luciferase activity with a luminometer. Quantification of the results by PhosphorImager and by luciferase assay yielded identical results. Panel D shows the results of an average of five experiments with five different batches of RRL, and the error bars represent the standard deviation. The fold activation is the result of luciferase activity from the Luc-SL and Luc-polyA mRNAs divided by the luciferase activity from the Luc-TL mRNA.

    Techniques Used: In Vitro, Luciferase, Incubation, SDS Page, Synthesized, Autoradiography, Activity Assay, Nucleic Acid Electrophoresis, Standard Deviation, Activation Assay

    16) Product Images from "Reverse Sanger sequencing of RNA by MALDI-TOF mass spectrometry after solid phase purification"

    Article Title: Reverse Sanger sequencing of RNA by MALDI-TOF mass spectrometry after solid phase purification

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gnh089

    MALDI mass fragmentation spectrum of RNA in vitro transcript (23mer) obtained by SVP-cleavage after solid-phase purification. Transcription was performed with T7 RNA polymerase utilizing 100% α-S-ATP. ?: Sequence-independent signals in the lower mass range.
    Figure Legend Snippet: MALDI mass fragmentation spectrum of RNA in vitro transcript (23mer) obtained by SVP-cleavage after solid-phase purification. Transcription was performed with T7 RNA polymerase utilizing 100% α-S-ATP. ?: Sequence-independent signals in the lower mass range.

    Techniques Used: In Vitro, Purification, Sequencing

    MALDI mass spectra of unmodified and modified full-length transcripts (23mer) isolated by solid phase purification. ( A ) RNA in vitro transcriptions performed with T7 RNA polymerase and with 100% ATP (black) and 100% α-S-ATP (green); ( B ) containing 25% α-S-ATP, which results in a heterogeneity of the products.
    Figure Legend Snippet: MALDI mass spectra of unmodified and modified full-length transcripts (23mer) isolated by solid phase purification. ( A ) RNA in vitro transcriptions performed with T7 RNA polymerase and with 100% ATP (black) and 100% α-S-ATP (green); ( B ) containing 25% α-S-ATP, which results in a heterogeneity of the products.

    Techniques Used: Modification, Isolation, Purification, In Vitro

    MALDI mass spectra of the RNA in vitro transcription performed with pGEM 3Zf linearized with ( A ) HindIII (sequence of 60mer: 5′-PPP-GGGCGAAUUCGAGCUCGGUACCCGGGGAUCCUCUAGAGUCGACCUGCAGGCAUGCAAGCU-3′) or with ( B ) SmaI (sequence of 23mer: 5′-PPP-GGGCGAAUUCGAGCUCGGUACCC-3′) and transcribed with T7 RNA polymerase utilizing unmodified NTP after EtOH-precipitation and ZipTip purification. The byproducts, abortive products (2–12 nt) and slippage products (G 4 –G 7 ), are labeled according to their length.
    Figure Legend Snippet: MALDI mass spectra of the RNA in vitro transcription performed with pGEM 3Zf linearized with ( A ) HindIII (sequence of 60mer: 5′-PPP-GGGCGAAUUCGAGCUCGGUACCCGGGGAUCCUCUAGAGUCGACCUGCAGGCAUGCAAGCU-3′) or with ( B ) SmaI (sequence of 23mer: 5′-PPP-GGGCGAAUUCGAGCUCGGUACCC-3′) and transcribed with T7 RNA polymerase utilizing unmodified NTP after EtOH-precipitation and ZipTip purification. The byproducts, abortive products (2–12 nt) and slippage products (G 4 –G 7 ), are labeled according to their length.

    Techniques Used: In Vitro, Sequencing, Purification, Labeling

    17) Product Images from "Juglone, an inhibitor of the peptidyl-prolyl isomerase Pin1, also directly blocks transcription"

    Article Title: Juglone, an inhibitor of the peptidyl-prolyl isomerase Pin1, also directly blocks transcription

    Journal: Nucleic Acids Research

    doi:

    The substrate specificity of juglone. ( A ) The effect of juglone on restriction enzyme digestions. Plasmid pCDNA3-HA was digested by Bam HI (B), Eco RI (R), Kpn I (K), Xba I (X), Hin dIII (H), Sca I (Sca) and Sal I (Sal) with (+) or without (–) the addition of juglone (50 µM). The uncut plasmid (un) served as a control. ( B ) The effect of juglone on the transcription of T7 RNA polymerase. Reactions were as described in Materials and Methods.
    Figure Legend Snippet: The substrate specificity of juglone. ( A ) The effect of juglone on restriction enzyme digestions. Plasmid pCDNA3-HA was digested by Bam HI (B), Eco RI (R), Kpn I (K), Xba I (X), Hin dIII (H), Sca I (Sca) and Sal I (Sal) with (+) or without (–) the addition of juglone (50 µM). The uncut plasmid (un) served as a control. ( B ) The effect of juglone on the transcription of T7 RNA polymerase. Reactions were as described in Materials and Methods.

    Techniques Used: Plasmid Preparation

    18) Product Images from "Altering the spectrum of immunoglobulin V gene somatic hypermutation by modifying the active site of AID"

    Article Title: Altering the spectrum of immunoglobulin V gene somatic hypermutation by modifying the active site of AID

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20092238

    Mutation spectra from in vitro transcription-coupled mutation assays. (A) The T7- lacZ transcription-coupled substrate in which a T7 promoter is inserted upstream of the lac promoter in M13mp19 ( Fig. S5 B ). Nucleotide position 1 is defined as the start of the lac promoter. The adjacent table shows the 5′-flanking nucleotide preferences of the C mutations produced by GST-AID 1 or GST-AID 1 */3G in lac target DNA during in vitro transcription by phage T7 RNA polymerase. (B) Mutation distribution along the T7 transcription-coupled lacZ target DNA with mutation at each nucleotide position expressed as the percentage of total mutations. Two heavily mutated positions are off-scale: their percentage mutations are indicated. Because mutation analysis was restricted to Lac − plaques, this selection results in a skewing in favor of lac -inactivating mutations, although most mutated templates carried 1–3 mutations in the target region. Positions at which C deamination yields a stop codon are indicated by an asterisk. (C) The location and local context of the five most frequently mutated C residues along the transcribed lac target. All the mutated residues shown are located on the top (nontranscribed) strand. (D) Transcription-linked mutation of a T7-linked GFP-Vλ target. The substrate DNA is a derivative of plasmid pCR-Blunt II-TOPO in which a region of IgVλ (residues 115–164 or 228–263) has been inserted between the T7 promoter and a GFP reporter with T7-catalyzed transcription of the IgVλ fragment being in the same sense as IgVλ transcription in B cells ( Fig. S5 C ). For each construct (pCR-GFP-Vλ 115−164 -T7 and pCR-GFP-Vλ 228−263 -T7), the tables compare the percentage of total mutations within the target Vλ region that occur at selected individual positions with the equivalent percentage mutation values for the same positions over the same target regions in the M13 or B cell mutation assays. (E) Comparison of the mutation distributions obtained in the T7-coupled mutation assay over IgVλ residues 115–164 (for AID 1 ) or 228–263 (for AID 1 */3G) to the distributions obtained in DT40 B cells (left) or in the gapped duplex assay (right). In these comparisons, analysis is restricted to nontranscribed strand mutations. Positions of individual hotspots are indicated in italics. Red, 5′-purine flank; blue, 5′-pyrimidine flank.
    Figure Legend Snippet: Mutation spectra from in vitro transcription-coupled mutation assays. (A) The T7- lacZ transcription-coupled substrate in which a T7 promoter is inserted upstream of the lac promoter in M13mp19 ( Fig. S5 B ). Nucleotide position 1 is defined as the start of the lac promoter. The adjacent table shows the 5′-flanking nucleotide preferences of the C mutations produced by GST-AID 1 or GST-AID 1 */3G in lac target DNA during in vitro transcription by phage T7 RNA polymerase. (B) Mutation distribution along the T7 transcription-coupled lacZ target DNA with mutation at each nucleotide position expressed as the percentage of total mutations. Two heavily mutated positions are off-scale: their percentage mutations are indicated. Because mutation analysis was restricted to Lac − plaques, this selection results in a skewing in favor of lac -inactivating mutations, although most mutated templates carried 1–3 mutations in the target region. Positions at which C deamination yields a stop codon are indicated by an asterisk. (C) The location and local context of the five most frequently mutated C residues along the transcribed lac target. All the mutated residues shown are located on the top (nontranscribed) strand. (D) Transcription-linked mutation of a T7-linked GFP-Vλ target. The substrate DNA is a derivative of plasmid pCR-Blunt II-TOPO in which a region of IgVλ (residues 115–164 or 228–263) has been inserted between the T7 promoter and a GFP reporter with T7-catalyzed transcription of the IgVλ fragment being in the same sense as IgVλ transcription in B cells ( Fig. S5 C ). For each construct (pCR-GFP-Vλ 115−164 -T7 and pCR-GFP-Vλ 228−263 -T7), the tables compare the percentage of total mutations within the target Vλ region that occur at selected individual positions with the equivalent percentage mutation values for the same positions over the same target regions in the M13 or B cell mutation assays. (E) Comparison of the mutation distributions obtained in the T7-coupled mutation assay over IgVλ residues 115–164 (for AID 1 ) or 228–263 (for AID 1 */3G) to the distributions obtained in DT40 B cells (left) or in the gapped duplex assay (right). In these comparisons, analysis is restricted to nontranscribed strand mutations. Positions of individual hotspots are indicated in italics. Red, 5′-purine flank; blue, 5′-pyrimidine flank.

    Techniques Used: Mutagenesis, In Vitro, Produced, Selection, Plasmid Preparation, Polymerase Chain Reaction, Construct

    19) Product Images from "Design and application of cotranscriptional non-enzymatic RNA circuits and signal transducers"

    Article Title: Design and application of cotranscriptional non-enzymatic RNA circuits and signal transducers

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gku074

    Operation of cotranscriptionally generated RNA CHA circuits without any downstream purification and design optimization for detection of DNA target. ( a ) Fifty nanograms each of the indicated pairs of hairpin 1 and 2 transcription templates was cotranscribed with or without 10 ng of C1 transcription template for 1 h at 42°C using T7 RNA polymerase. Following transcription, 2 µl of the reaction mix was directly incubated in 1× TNaK buffer containing 20 U of RNaseOUT and 0.5 µM ROX reference dye along with 400 nM RepF (annealed with 5× excess RepQ) fluorescent DNA reporter duplex for quantitating RNA CHA in real-time at 52°C. ( b ) Schematic depicting SDA of DNA. The single-stranded template DNA (black arrow) consists of a sequence (C*) complementary to the RNA CHA catalyst followed by the nicking enzyme recognition sequence (NE) that is present on the non-cleaved DNA strand and a primer binding site. Following primer binding (step 1), the DNA polymerase synthesizes the complementary strand that now completes the duplex NE site and contains the RNA CHA catalyst sequence (C). Nicking enzyme then binds the duplex NE site (step 2) and cleaves the newly synthesized strand at the NE site. The new 3′-OH group generated at the nick site is then extended by the DNA polymerase (step 3) while displacing the previously synthesized strand. The displaced ssDNA amplicon can then catalyze RNA CHA. ( c ) Schematic of DNA target sequence design for catalysis of RNA CHA. Single toehold (domain 1*) DNA target C1 (generated by SDA from the template TLTRSDA) with the same domain architecture as the RNA C1 is an inefficient catalyst of RNA CHA. Extended DNA target C1234 (generated by SDA from the template 1234LTRSDA) presenting two toeholds for RNA H1 successfully catalyzes RNA CHA.
    Figure Legend Snippet: Operation of cotranscriptionally generated RNA CHA circuits without any downstream purification and design optimization for detection of DNA target. ( a ) Fifty nanograms each of the indicated pairs of hairpin 1 and 2 transcription templates was cotranscribed with or without 10 ng of C1 transcription template for 1 h at 42°C using T7 RNA polymerase. Following transcription, 2 µl of the reaction mix was directly incubated in 1× TNaK buffer containing 20 U of RNaseOUT and 0.5 µM ROX reference dye along with 400 nM RepF (annealed with 5× excess RepQ) fluorescent DNA reporter duplex for quantitating RNA CHA in real-time at 52°C. ( b ) Schematic depicting SDA of DNA. The single-stranded template DNA (black arrow) consists of a sequence (C*) complementary to the RNA CHA catalyst followed by the nicking enzyme recognition sequence (NE) that is present on the non-cleaved DNA strand and a primer binding site. Following primer binding (step 1), the DNA polymerase synthesizes the complementary strand that now completes the duplex NE site and contains the RNA CHA catalyst sequence (C). Nicking enzyme then binds the duplex NE site (step 2) and cleaves the newly synthesized strand at the NE site. The new 3′-OH group generated at the nick site is then extended by the DNA polymerase (step 3) while displacing the previously synthesized strand. The displaced ssDNA amplicon can then catalyze RNA CHA. ( c ) Schematic of DNA target sequence design for catalysis of RNA CHA. Single toehold (domain 1*) DNA target C1 (generated by SDA from the template TLTRSDA) with the same domain architecture as the RNA C1 is an inefficient catalyst of RNA CHA. Extended DNA target C1234 (generated by SDA from the template 1234LTRSDA) presenting two toeholds for RNA H1 successfully catalyzes RNA CHA.

    Techniques Used: Generated, Purification, Incubation, Sequencing, Binding Assay, Synthesized, Amplification

    Synthesis and execution of RNA CHA circuit. ( a ) LHRz and RHRz-mediated cotranscriptional RNA cleavage releases the internal circuit components H1, H2 and C1. Fifity nanograms of PCR-generated transcription templates for H1, H2 and C1 was transcribed in 50 µl of reactions by T7 RNA polymerase for 2 h at 42°C. Two microliters of the resulting transcripts was analyzed by electrophoresis on a 10% denaturing polyacrylamide gel. Single-stranded DNA oligonucleotides were used as size markers. ( b ) RNA hairpins undergo catalyzed assembly into RNA duplexes. Gel-purified RNA catalyst C1 and the hairpins H1 and H2 were combined as indicated and incubated in 1× TNaK buffer containing 20 U of RNaseOUT for 150 min at 42°C (lanes 1–4), 52°C (lanes 5–8) or 62°C (lanes 9–12). The reactions were then analyzed on a 10% native polyacrylamide gel. Fifteen nanograms of C1 RNA was included in lane 13 as a control. Single-stranded DNA oligonucleotides were used as size markers.
    Figure Legend Snippet: Synthesis and execution of RNA CHA circuit. ( a ) LHRz and RHRz-mediated cotranscriptional RNA cleavage releases the internal circuit components H1, H2 and C1. Fifity nanograms of PCR-generated transcription templates for H1, H2 and C1 was transcribed in 50 µl of reactions by T7 RNA polymerase for 2 h at 42°C. Two microliters of the resulting transcripts was analyzed by electrophoresis on a 10% denaturing polyacrylamide gel. Single-stranded DNA oligonucleotides were used as size markers. ( b ) RNA hairpins undergo catalyzed assembly into RNA duplexes. Gel-purified RNA catalyst C1 and the hairpins H1 and H2 were combined as indicated and incubated in 1× TNaK buffer containing 20 U of RNaseOUT for 150 min at 42°C (lanes 1–4), 52°C (lanes 5–8) or 62°C (lanes 9–12). The reactions were then analyzed on a 10% native polyacrylamide gel. Fifteen nanograms of C1 RNA was included in lane 13 as a control. Single-stranded DNA oligonucleotides were used as size markers.

    Techniques Used: Polymerase Chain Reaction, Generated, Electrophoresis, Purification, Incubation

    Cotranscriptionally generated RNA CHA as signal transducer for nucleic acid diagnostics. ( a ) End-point sequence-specific detection of SDA-generated ssDNA targets by RNA CHA. Samples with or without 10 nM template 1234LTRSDA were amplified by SDA for 90 min at 37°C in 25 µl of reaction volumes. Reactions were then incubated at 95°C for 5 min and stored at room temperature before assay by RNA CHA. Five microliters of these SDA products was then probed with 2 µl of Sephadex G25 column-purified cotranscribed mH1:H2 RNA CHA circuit. RNA CHA cotranscriptions were performed with T7 RNA polymerase using 50 ng each of the mH1 and H2 transcription templates for 1 h at 42°C. End-point RNA CHA detection reactions were assembled in 1× TNaK buffer containing 20 U of RNaseOUT, 0.5 µM ROX reference dye and 100 nM RepF (annealed with 5× excess RepQ) fluorescent DNA reporter duplex for quantitating RNA CHA in real-time at 52°C. Negative control reactions lacking RNA CHA components or containing 2 µl of either only mH1 or H2 were also tested. ( b ) Real-time signal transduction of ssDNA-generating SDA by cotranscribed mH1:H2 RNA CHA. High temperature (55°C) SDA reactions were set up with or without 10 nM 1234HTRSDA template in 20 µl of volume containing 0.5 µM ROX reference dye and 75 nM RepF (annealed with 5× excess RepQ) fluorescent DNA reporter duplex for quantitating RNA CHA in real-time. Real-time sequence-specific signal transduction was achieved by adding 2 µl of unpurified mH1:H2 RNA CHA circuits cotranscribed from 50 ng of each transcription template to the SDA reactions. Control SDA reactions containing no RNA CHA components or 2 µl of either only mH1 or H2 were also tested.
    Figure Legend Snippet: Cotranscriptionally generated RNA CHA as signal transducer for nucleic acid diagnostics. ( a ) End-point sequence-specific detection of SDA-generated ssDNA targets by RNA CHA. Samples with or without 10 nM template 1234LTRSDA were amplified by SDA for 90 min at 37°C in 25 µl of reaction volumes. Reactions were then incubated at 95°C for 5 min and stored at room temperature before assay by RNA CHA. Five microliters of these SDA products was then probed with 2 µl of Sephadex G25 column-purified cotranscribed mH1:H2 RNA CHA circuit. RNA CHA cotranscriptions were performed with T7 RNA polymerase using 50 ng each of the mH1 and H2 transcription templates for 1 h at 42°C. End-point RNA CHA detection reactions were assembled in 1× TNaK buffer containing 20 U of RNaseOUT, 0.5 µM ROX reference dye and 100 nM RepF (annealed with 5× excess RepQ) fluorescent DNA reporter duplex for quantitating RNA CHA in real-time at 52°C. Negative control reactions lacking RNA CHA components or containing 2 µl of either only mH1 or H2 were also tested. ( b ) Real-time signal transduction of ssDNA-generating SDA by cotranscribed mH1:H2 RNA CHA. High temperature (55°C) SDA reactions were set up with or without 10 nM 1234HTRSDA template in 20 µl of volume containing 0.5 µM ROX reference dye and 75 nM RepF (annealed with 5× excess RepQ) fluorescent DNA reporter duplex for quantitating RNA CHA in real-time. Real-time sequence-specific signal transduction was achieved by adding 2 µl of unpurified mH1:H2 RNA CHA circuits cotranscribed from 50 ng of each transcription template to the SDA reactions. Control SDA reactions containing no RNA CHA components or 2 µl of either only mH1 or H2 were also tested.

    Techniques Used: Generated, Sequencing, Amplification, Incubation, Purification, Negative Control, Transduction

    Design of non-enzymatic catalyzed RNA hairpin assembly circuit. ( a ) Schematic of catalyzed nucleic acid hairpin assembly circuit adapted from ( 2 ). The circuit composed of hairpins H1 and H2 is turned on in the presence of the input sequence (C1). C1 catalyzes the assembly of H1 and H2 into an H1:H2 duplex and is itself recycled. Circuit output (H1:H2 duplex) is quantitated as increasing fluorescence intensity of a labeled oligonucleotide probe (RepF) on displacement of its complementary quencher oligonucleotide (RepQ) by the H1:H2 duplex. ( b ) Design of T7 RNA polymerase-driven transcription templates for enzymatic synthesis of RNA CHA circuit components with precise 5′- and 3′-ends. Transcription template for each component, H1, H2 and C1, is flanked on both the left (L) and the right (R) sides by hammerhead ribozymes (HRz). The size (in nucleotides) of each component and its ribozyme flanks is indicated under each schematic. Secondary structures of the resulting chimeric RNA at 42°C before ribozyme processing are depicted (green, A; blue, C; black, G; red, U). The RNA structures were generated using NUPACK ( 13–16 ).
    Figure Legend Snippet: Design of non-enzymatic catalyzed RNA hairpin assembly circuit. ( a ) Schematic of catalyzed nucleic acid hairpin assembly circuit adapted from ( 2 ). The circuit composed of hairpins H1 and H2 is turned on in the presence of the input sequence (C1). C1 catalyzes the assembly of H1 and H2 into an H1:H2 duplex and is itself recycled. Circuit output (H1:H2 duplex) is quantitated as increasing fluorescence intensity of a labeled oligonucleotide probe (RepF) on displacement of its complementary quencher oligonucleotide (RepQ) by the H1:H2 duplex. ( b ) Design of T7 RNA polymerase-driven transcription templates for enzymatic synthesis of RNA CHA circuit components with precise 5′- and 3′-ends. Transcription template for each component, H1, H2 and C1, is flanked on both the left (L) and the right (R) sides by hammerhead ribozymes (HRz). The size (in nucleotides) of each component and its ribozyme flanks is indicated under each schematic. Secondary structures of the resulting chimeric RNA at 42°C before ribozyme processing are depicted (green, A; blue, C; black, G; red, U). The RNA structures were generated using NUPACK ( 13–16 ).

    Techniques Used: Sequencing, Fluorescence, Labeling, Generated

    Application of RNA CHA circuit as an OR logic processor. ( a ) Schematic of RNA CHA circuit operation in response to either catalyst C1 OR C2. The RNA hairpin H1B serves as the OR gate, and circuit output is measured fluorimetrically using Spinach.ST1 RNA aptamer beacon. ( b ) Circuit components (H1B and H2 RNA hairpins), reporter RNA (Spinach.ST1) and the inputs C1 and C2 were transcribed from 500 ng of duplex DNA transcription templates using T7 RNA polymerase. Transcription templates were prepared using the same procedure as Figure 8 . Following filtration through Sephadex G25, 3 µl/transcript (or 1.5 µl each of C1 and C2 when added together in a reaction) was mixed in the indicated combinations in 1× TNaK buffer containing 70 µM DFHBI and 20 U of RNaseOUT. Circuits were operated at 37°C, and outputs were measured fluorimetrically.
    Figure Legend Snippet: Application of RNA CHA circuit as an OR logic processor. ( a ) Schematic of RNA CHA circuit operation in response to either catalyst C1 OR C2. The RNA hairpin H1B serves as the OR gate, and circuit output is measured fluorimetrically using Spinach.ST1 RNA aptamer beacon. ( b ) Circuit components (H1B and H2 RNA hairpins), reporter RNA (Spinach.ST1) and the inputs C1 and C2 were transcribed from 500 ng of duplex DNA transcription templates using T7 RNA polymerase. Transcription templates were prepared using the same procedure as Figure 8 . Following filtration through Sephadex G25, 3 µl/transcript (or 1.5 µl each of C1 and C2 when added together in a reaction) was mixed in the indicated combinations in 1× TNaK buffer containing 70 µM DFHBI and 20 U of RNaseOUT. Circuits were operated at 37°C, and outputs were measured fluorimetrically.

    Techniques Used: Filtration

    Cotranscriptional RNA CHA and circuit design optimization for cotranscription. ( a ) Cotranscribed RNA circuit components undergo catalyzed hairpin assembly without requiring gel purification of individual reactants. Fifty nanograms each of H1 and H2 transcription templates, along with titrating amounts of C1 transcription template, was cotranscribed for 1 h at 42°C using T7 RNA polymerase followed by passage through Illustra MicroSpin Sephadex G25 columns. Transcription templates were amplified from cloned inserts using primers pCR2.1.F and pCR2.1.R specific to plasmid sequences flanking the inserts. Two microliter aliquots of the cotranscribed RNA mixtures were then incubated in 15 µl of volume with 400 nM RepF annealed with 5× excess (2 µM) RepQ fluorescent DNA reporter duplex in 1× TNaK buffer containing 20 U of RNaseOUT and 0.5 µM ROX reference dye to quantitate formation of H1:H2 RNA duplexes at 52°C. Average data from triplicate experiments are represented. ( b and c ) Schematic depicting sequences of RNA hairpins H1 and H2 with one- or two-base engineered mismatches. Mismatched H1 (mH1) presents a two-base mismatch between its domain 4* and domain 4 of H2. The hairpins mAH1 and mGH1 each contain a single mismatched base between their domain 4* and the domain 4 of H2. The mutated H2 hairpin m2H2 presents two mismatched bases between its domain 2* and the H1 domain 2.
    Figure Legend Snippet: Cotranscriptional RNA CHA and circuit design optimization for cotranscription. ( a ) Cotranscribed RNA circuit components undergo catalyzed hairpin assembly without requiring gel purification of individual reactants. Fifty nanograms each of H1 and H2 transcription templates, along with titrating amounts of C1 transcription template, was cotranscribed for 1 h at 42°C using T7 RNA polymerase followed by passage through Illustra MicroSpin Sephadex G25 columns. Transcription templates were amplified from cloned inserts using primers pCR2.1.F and pCR2.1.R specific to plasmid sequences flanking the inserts. Two microliter aliquots of the cotranscribed RNA mixtures were then incubated in 15 µl of volume with 400 nM RepF annealed with 5× excess (2 µM) RepQ fluorescent DNA reporter duplex in 1× TNaK buffer containing 20 U of RNaseOUT and 0.5 µM ROX reference dye to quantitate formation of H1:H2 RNA duplexes at 52°C. Average data from triplicate experiments are represented. ( b and c ) Schematic depicting sequences of RNA hairpins H1 and H2 with one- or two-base engineered mismatches. Mismatched H1 (mH1) presents a two-base mismatch between its domain 4* and domain 4 of H2. The hairpins mAH1 and mGH1 each contain a single mismatched base between their domain 4* and the domain 4 of H2. The mutated H2 hairpin m2H2 presents two mismatched bases between its domain 2* and the H1 domain 2.

    Techniques Used: Gel Purification, Amplification, Clone Assay, Plasmid Preparation, Incubation

    An entirely RNA-based CHA circuit operation and fluorimetric detection. ( a ) CHA circuit components (hairpins H1B and H2 and catalyst C1) and the RNA reporter Spinach.ST1 were separately transcribed by T7 RNA polymerase from 500 ng of PCR-generated duplex DNA transcription templates. H1B, H2 and C1 transcription templates were amplified using primers complementary to the exact ends of the cloned inserts (H1B.amp.F:H1B.amp.R, H2.amp.F:H2.amp.R and C1.amp.F:C1.amp.R, respectively) rather than the flanking plasmid. Spinach.ST transcription templates were amplified using primers specific to the flanking plasmid sequence at the 5′-end (pCR2.1.F) and the primer sphT.U.R specific to the 3′-end sequence of Spinach.ST. Transcription reactions were filtered through Sephadex G25 columns before circuit assembly. Three microliters of H1B, H2, C1 and Spinach.ST1 transcripts was mixed in indicated combinations and incubated in 1× TNaK buffer containing 70 µM DFHBI and 20 U of RNaseOUT. Circuit output was measured as increasing fluorescence intensity over time at 37°C. ( b–d ) Performance of DNA reporter duplex H1BF:H1BQ (b) versus Spinach.ST1 (c) in measuring RNA CHA circuit output. Indicated concentrations of gel-purified RNA hairpins H1B and H2 were incubated with equal concentration of H1BF:H1BQ or gel-purified Spinach.ST1 (+ 70 µM DFHBI) in the presence of titrating concentrations of pure C1 RNA. All circuits were operated in 1× TNaK buffer containing 20 U of RNaseOUT at 37°C, and average data from triplicate experiments are represented. Signal-to-noise ratio of H1BF:H1BQ versus Spinach.ST1 over the time course of RNA CHA detection is plotted in (d).
    Figure Legend Snippet: An entirely RNA-based CHA circuit operation and fluorimetric detection. ( a ) CHA circuit components (hairpins H1B and H2 and catalyst C1) and the RNA reporter Spinach.ST1 were separately transcribed by T7 RNA polymerase from 500 ng of PCR-generated duplex DNA transcription templates. H1B, H2 and C1 transcription templates were amplified using primers complementary to the exact ends of the cloned inserts (H1B.amp.F:H1B.amp.R, H2.amp.F:H2.amp.R and C1.amp.F:C1.amp.R, respectively) rather than the flanking plasmid. Spinach.ST transcription templates were amplified using primers specific to the flanking plasmid sequence at the 5′-end (pCR2.1.F) and the primer sphT.U.R specific to the 3′-end sequence of Spinach.ST. Transcription reactions were filtered through Sephadex G25 columns before circuit assembly. Three microliters of H1B, H2, C1 and Spinach.ST1 transcripts was mixed in indicated combinations and incubated in 1× TNaK buffer containing 70 µM DFHBI and 20 U of RNaseOUT. Circuit output was measured as increasing fluorescence intensity over time at 37°C. ( b–d ) Performance of DNA reporter duplex H1BF:H1BQ (b) versus Spinach.ST1 (c) in measuring RNA CHA circuit output. Indicated concentrations of gel-purified RNA hairpins H1B and H2 were incubated with equal concentration of H1BF:H1BQ or gel-purified Spinach.ST1 (+ 70 µM DFHBI) in the presence of titrating concentrations of pure C1 RNA. All circuits were operated in 1× TNaK buffer containing 20 U of RNaseOUT at 37°C, and average data from triplicate experiments are represented. Signal-to-noise ratio of H1BF:H1BQ versus Spinach.ST1 over the time course of RNA CHA detection is plotted in (d).

    Techniques Used: Polymerase Chain Reaction, Generated, Amplification, Clone Assay, Plasmid Preparation, Sequencing, Incubation, Fluorescence, Purification, Concentration Assay

    20) Product Images from "A specific, promoter-independent activity of T7 RNA polymerase suggests a general model for DNA/RNA editing in single subunit RNA Polymerases"

    Article Title: A specific, promoter-independent activity of T7 RNA polymerase suggests a general model for DNA/RNA editing in single subunit RNA Polymerases

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-32231-6

    Specificity of oligonucleotide labeling by T7 RNA polymerase. ( A ) Autoradiograph of 15% polyacrylamide gel which shows 3′ end labeling by T7 RNAP on DNA oligonucleotides (36mer) in the presence of the α- 32 P GTP (right two lanes) or α- 32 P ATP (left two lanes). ( B ) The sequence and potential looped structure of the DNA oligonucleotides labeled in ( A ).
    Figure Legend Snippet: Specificity of oligonucleotide labeling by T7 RNA polymerase. ( A ) Autoradiograph of 15% polyacrylamide gel which shows 3′ end labeling by T7 RNAP on DNA oligonucleotides (36mer) in the presence of the α- 32 P GTP (right two lanes) or α- 32 P ATP (left two lanes). ( B ) The sequence and potential looped structure of the DNA oligonucleotides labeled in ( A ).

    Techniques Used: Oligonucleotide Labeling, Autoradiography, End Labeling, Sequencing, Labeling

    21) Product Images from "A hybrid G-quadruplex structure formed between RNA and DNA explains the extraordinary stability of the mitochondrial R-loop"

    Article Title: A hybrid G-quadruplex structure formed between RNA and DNA explains the extraordinary stability of the mitochondrial R-loop

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gks802

    A longer R-loop can form during transcription of human mtDNA with phage T7 RNA polymerase. ( A ) Transcription of a human mtDNA template containing wildtype or mutant CSB II with T7 RNA pol in the presence of either GTP (lanes 1–3 and 7–9) or 7-deaza-GTP (lanes 4–6 and 10–12). A hybrid species of ∼120 bp is revealed upon RNase A treatment (lane 2) and is sensitive to hRNaseH1 (lane 3). This longer hybrid is dependent on CSB II and not observed in the presence of 7-deaza-GTP (lanes 4–6). ( B ) Schematic presentation of the RNA–DNA hybrid G-quadruplex that forms between the RNA and the non-template DNA strand during transcription of mtDNA. Under some conditions, an extended R-loop may be formed, similar to that reported in ( 22 ).
    Figure Legend Snippet: A longer R-loop can form during transcription of human mtDNA with phage T7 RNA polymerase. ( A ) Transcription of a human mtDNA template containing wildtype or mutant CSB II with T7 RNA pol in the presence of either GTP (lanes 1–3 and 7–9) or 7-deaza-GTP (lanes 4–6 and 10–12). A hybrid species of ∼120 bp is revealed upon RNase A treatment (lane 2) and is sensitive to hRNaseH1 (lane 3). This longer hybrid is dependent on CSB II and not observed in the presence of 7-deaza-GTP (lanes 4–6). ( B ) Schematic presentation of the RNA–DNA hybrid G-quadruplex that forms between the RNA and the non-template DNA strand during transcription of mtDNA. Under some conditions, an extended R-loop may be formed, similar to that reported in ( 22 ).

    Techniques Used: Mutagenesis

    22) Product Images from "Using droplet-based microfluidics to improve the catalytic properties of RNA under multiple-turnover conditions"

    Article Title: Using droplet-based microfluidics to improve the catalytic properties of RNA under multiple-turnover conditions

    Journal: RNA

    doi: 10.1261/rna.048033.114

    Model selection. ( A ) Gene constructs. Genes coding for the extended X-motif ribozyme and an inactive control RNA are shown. Both genes are of the same length and under the control of a T7 RNA polymerase promoter (ProT7) and code for RNA bearing 5′
    Figure Legend Snippet: Model selection. ( A ) Gene constructs. Genes coding for the extended X-motif ribozyme and an inactive control RNA are shown. Both genes are of the same length and under the control of a T7 RNA polymerase promoter (ProT7) and code for RNA bearing 5′

    Techniques Used: Selection, Construct

    Structure and activity of the original X-motif and its extended form. ( A ) Organization of extended X-motif coding gene. The extended X-motif coding sequence was placed under the control of a T7 RNA polymerase promoter (ProT7). Annealing sites of amplification
    Figure Legend Snippet: Structure and activity of the original X-motif and its extended form. ( A ) Organization of extended X-motif coding gene. The extended X-motif coding sequence was placed under the control of a T7 RNA polymerase promoter (ProT7). Annealing sites of amplification

    Techniques Used: Activity Assay, Sequencing, Amplification

    23) Product Images from "The Hepatitis C Virus RNA 3?-Untranslated Region Strongly Enhances Translation Directed by the Internal Ribosome Entry Site ▿"

    Article Title: The Hepatitis C Virus RNA 3?-Untranslated Region Strongly Enhances Translation Directed by the Internal Ribosome Entry Site ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.00675-06

    The HCV 3′-UTR enhances translation of full-length genomes. (A) Structure of the transfected RNAs. Full-length HCV genome RNAs including the 3′-UTR (upper panel) or not (lower panel) were in vitro transcribed using T7 RNA polymerase. Precise
    Figure Legend Snippet: The HCV 3′-UTR enhances translation of full-length genomes. (A) Structure of the transfected RNAs. Full-length HCV genome RNAs including the 3′-UTR (upper panel) or not (lower panel) were in vitro transcribed using T7 RNA polymerase. Precise

    Techniques Used: Transfection, In Vitro

    Analysis of the effect of the HCV 3′-UTR on translation efficiency in vitro. (A) The HCV reporter constructs. RNAs (arrows) were obtained by in vitro transcription using T7 RNA polymerase either from linearized plasmids or from PCR fragments (lines).
    Figure Legend Snippet: Analysis of the effect of the HCV 3′-UTR on translation efficiency in vitro. (A) The HCV reporter constructs. RNAs (arrows) were obtained by in vitro transcription using T7 RNA polymerase either from linearized plasmids or from PCR fragments (lines).

    Techniques Used: In Vitro, Construct, Polymerase Chain Reaction

    24) Product Images from "Label-free single-cell protein quantification using a drop-based mix-and-read system"

    Article Title: Label-free single-cell protein quantification using a drop-based mix-and-read system

    Journal: Scientific Reports

    doi: 10.1038/srep12756

    Detection and quantification of antibody in single hybridoma cells using drop-based PAIGE. ( a ) Scheme of PAIGE for detecting an antigen (Ag)-specific antibody (Ab). Two fusion proteins, AD-Ag and A/G-DB, are constitutively expressed under T7 promoter (T7) from input DNA templates by T7 RNA polymerase (step 1). The antibody binds to both Ag and A/G, forming a ternary complex that binds to the upstream-activation sequence (UAS) on the reporter DNA and recruits AD near the promoter-bound RNA polymerase (RNAP) (step 2). AD activates RNAP (step 3) to express the reporter gene, which produces GFP (step 4). ( b ) The workflow of drop-based PAIGE. Single cells (megenta) are encapsulated with PAIGE and a lysis buffer in drops (blue). After off-chip incubation, drops are re-injected into a fluorescence-activated drop-sorting (FADS) device to measure their fluorescence and sort them based on a fluorescence threshold. ( c ) Titration curves of pure anti-myc in microwell (blue square) and drop-based (red circle) PAIGE. The dashed line shows the average number of anti-Myc molecules in a single cell. The fluorescence of anti-Myc in microwells or drops is normalized by that of microwells or drops without anti-Myc. The normalized activated fluorescence, F , is obtained by subtracting one. ( d ) Heat map showing the distribution of drops in terms of their normalized activated fluorescence, F , and width recorded as the PMT’s pulse duration (left). The corresponding fluorescence histogram is shown on the right. The inset fluorescence microscopy image shows that after incubation a small fraction of the drops is bright.
    Figure Legend Snippet: Detection and quantification of antibody in single hybridoma cells using drop-based PAIGE. ( a ) Scheme of PAIGE for detecting an antigen (Ag)-specific antibody (Ab). Two fusion proteins, AD-Ag and A/G-DB, are constitutively expressed under T7 promoter (T7) from input DNA templates by T7 RNA polymerase (step 1). The antibody binds to both Ag and A/G, forming a ternary complex that binds to the upstream-activation sequence (UAS) on the reporter DNA and recruits AD near the promoter-bound RNA polymerase (RNAP) (step 2). AD activates RNAP (step 3) to express the reporter gene, which produces GFP (step 4). ( b ) The workflow of drop-based PAIGE. Single cells (megenta) are encapsulated with PAIGE and a lysis buffer in drops (blue). After off-chip incubation, drops are re-injected into a fluorescence-activated drop-sorting (FADS) device to measure their fluorescence and sort them based on a fluorescence threshold. ( c ) Titration curves of pure anti-myc in microwell (blue square) and drop-based (red circle) PAIGE. The dashed line shows the average number of anti-Myc molecules in a single cell. The fluorescence of anti-Myc in microwells or drops is normalized by that of microwells or drops without anti-Myc. The normalized activated fluorescence, F , is obtained by subtracting one. ( d ) Heat map showing the distribution of drops in terms of their normalized activated fluorescence, F , and width recorded as the PMT’s pulse duration (left). The corresponding fluorescence histogram is shown on the right. The inset fluorescence microscopy image shows that after incubation a small fraction of the drops is bright.

    Techniques Used: Activation Assay, Sequencing, Lysis, Chromatin Immunoprecipitation, Incubation, Injection, Fluorescence, Titration, Microscopy

    25) Product Images from "Encoding folding paths of RNA switches"

    Article Title: Encoding folding paths of RNA switches

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl1036

    Opposite co-transcriptional folding paths of a pair of RNA switches with ‘direct’ and ‘reverse’ sequences (i.e. 5′-ABCD-3′ versus 5′-DCBA-3′). Structures 1D and 1R (respectively, 2D and 2R) of the direct and reverse switches are energetically equivalent because of helix symmetries; dashed lines indicate mirror symmetry of Pa, Pb, Pc and Pd which are therefore conserved under sequence reversal relating direct and reverse switches. Despite these strong similarities between D and R structures at equilibrium, direct and reverse switches display ‘opposite’ co-transcriptional folding paths (direct switch into structure 1D and reverse switch into structure 2R) guided through a helix encoded persistence (left) or exchange (right) during in vitro transcription using T7 RNA polymerase (see Materials and Methods).
    Figure Legend Snippet: Opposite co-transcriptional folding paths of a pair of RNA switches with ‘direct’ and ‘reverse’ sequences (i.e. 5′-ABCD-3′ versus 5′-DCBA-3′). Structures 1D and 1R (respectively, 2D and 2R) of the direct and reverse switches are energetically equivalent because of helix symmetries; dashed lines indicate mirror symmetry of Pa, Pb, Pc and Pd which are therefore conserved under sequence reversal relating direct and reverse switches. Despite these strong similarities between D and R structures at equilibrium, direct and reverse switches display ‘opposite’ co-transcriptional folding paths (direct switch into structure 1D and reverse switch into structure 2R) guided through a helix encoded persistence (left) or exchange (right) during in vitro transcription using T7 RNA polymerase (see Materials and Methods).

    Techniques Used: Sequencing, In Vitro

    26) Product Images from "Juglone, an inhibitor of the peptidyl-prolyl isomerase Pin1, also directly blocks transcription"

    Article Title: Juglone, an inhibitor of the peptidyl-prolyl isomerase Pin1, also directly blocks transcription

    Journal: Nucleic Acids Research

    doi:

    The substrate specificity of juglone. ( A ) The effect of juglone on restriction enzyme digestions. Plasmid pCDNA3-HA was digested by Bam HI (B), Eco RI (R), Kpn I (K), Xba I (X), Hin dIII (H), Sca I (Sca) and Sal I (Sal) with (+) or without (–) the addition of juglone (50 µM). The uncut plasmid (un) served as a control. ( B ) The effect of juglone on the transcription of T7 RNA polymerase. Reactions were as described in Materials and Methods.
    Figure Legend Snippet: The substrate specificity of juglone. ( A ) The effect of juglone on restriction enzyme digestions. Plasmid pCDNA3-HA was digested by Bam HI (B), Eco RI (R), Kpn I (K), Xba I (X), Hin dIII (H), Sca I (Sca) and Sal I (Sal) with (+) or without (–) the addition of juglone (50 µM). The uncut plasmid (un) served as a control. ( B ) The effect of juglone on the transcription of T7 RNA polymerase. Reactions were as described in Materials and Methods.

    Techniques Used: Plasmid Preparation

    27) Product Images from "A specific, promoter-independent activity of T7 RNA polymerase suggests a general model for DNA/RNA editing in single subunit RNA Polymerases"

    Article Title: A specific, promoter-independent activity of T7 RNA polymerase suggests a general model for DNA/RNA editing in single subunit RNA Polymerases

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-32231-6

    Specificity of oligonucleotide labeling by T7 RNA polymerase. ( A ) Autoradiograph of 15% polyacrylamide gel which shows 3′ end labeling by T7 RNAP on DNA oligonucleotides (36mer) in the presence of the α- 32 P GTP (right two lanes) or α- 32 P ATP (left two lanes). ( B ) The sequence and potential looped structure of the DNA oligonucleotides labeled in ( A ).
    Figure Legend Snippet: Specificity of oligonucleotide labeling by T7 RNA polymerase. ( A ) Autoradiograph of 15% polyacrylamide gel which shows 3′ end labeling by T7 RNAP on DNA oligonucleotides (36mer) in the presence of the α- 32 P GTP (right two lanes) or α- 32 P ATP (left two lanes). ( B ) The sequence and potential looped structure of the DNA oligonucleotides labeled in ( A ).

    Techniques Used: Oligonucleotide Labeling, Autoradiography, End Labeling, Sequencing, Labeling

    28) Product Images from "A specific, promoter-independent activity of T7 RNA polymerase suggests a general model for DNA/RNA editing in single subunit RNA Polymerases"

    Article Title: A specific, promoter-independent activity of T7 RNA polymerase suggests a general model for DNA/RNA editing in single subunit RNA Polymerases

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-32231-6

    Specificity of oligonucleotide labeling by T7 RNA polymerase. ( A ) Autoradiograph of 15% polyacrylamide gel which shows 3′ end labeling by T7 RNAP on DNA oligonucleotides (36mer) in the presence of the α- 32 P GTP (right two lanes) or α- 32 P ATP (left two lanes). ( B ) The sequence and potential looped structure of the DNA oligonucleotides labeled in ( A ).
    Figure Legend Snippet: Specificity of oligonucleotide labeling by T7 RNA polymerase. ( A ) Autoradiograph of 15% polyacrylamide gel which shows 3′ end labeling by T7 RNAP on DNA oligonucleotides (36mer) in the presence of the α- 32 P GTP (right two lanes) or α- 32 P ATP (left two lanes). ( B ) The sequence and potential looped structure of the DNA oligonucleotides labeled in ( A ).

    Techniques Used: Oligonucleotide Labeling, Autoradiography, End Labeling, Sequencing, Labeling

    29) Product Images from "Generation of siRNA Nanosheets for Efficient RNA Interference"

    Article Title: Generation of siRNA Nanosheets for Efficient RNA Interference

    Journal: Scientific Reports

    doi: 10.1038/srep25146

    Schematic illustration of the synthetic process of siRNA-NS. ( a ) Design for siRNA-NS bearing siRNA precursors. Complementary rolling circle transcription (cRCT) is carried out by T7 RNA polymerase with circular DNA 1 and circular DNA 2 , partially complementary to circular DNA 1 . The cRCT process is followed by evaporation-induced self-assembly, completing the synthetic process of RNA membrane. ( b ) Schematic illustration and digital images of GelRed-stained RNA membrane after ultrasonication. As sonication proceeds, RNA membrane lost its initial form and torn into large pieces (~20 s). After 60 s of sonication, RNA membrane was broken into smaller pieces with large sediments still remaining. At the time point of 180 s after sonication, no sediment was observed with naked eye.
    Figure Legend Snippet: Schematic illustration of the synthetic process of siRNA-NS. ( a ) Design for siRNA-NS bearing siRNA precursors. Complementary rolling circle transcription (cRCT) is carried out by T7 RNA polymerase with circular DNA 1 and circular DNA 2 , partially complementary to circular DNA 1 . The cRCT process is followed by evaporation-induced self-assembly, completing the synthetic process of RNA membrane. ( b ) Schematic illustration and digital images of GelRed-stained RNA membrane after ultrasonication. As sonication proceeds, RNA membrane lost its initial form and torn into large pieces (~20 s). After 60 s of sonication, RNA membrane was broken into smaller pieces with large sediments still remaining. At the time point of 180 s after sonication, no sediment was observed with naked eye.

    Techniques Used: Evaporation, Staining, Sonication

    30) Product Images from "Synthetic Circular RNA Functions as a miR-21 Sponge to Suppress Gastric Carcinoma Cell Proliferation"

    Article Title: Synthetic Circular RNA Functions as a miR-21 Sponge to Suppress Gastric Carcinoma Cell Proliferation

    Journal: Molecular Therapy. Nucleic Acids

    doi: 10.1016/j.omtn.2018.09.010

    Workflow for Producing Synthetic miR-21 circRNA Sponge Each binding site sequence is perfectly complementary to the miR-21 seed region, but contains a bulge at positions 9–12 to prevent RNAi-type cleavage and degradation. One PCR cycle facilitates synthesis to generate a double-stranded DNA PCR fragment, which is then cloned into the TOPO PCR cloning vector (Invitrogen). The T7 RNA polymerase binding site located just 5′ to the PCR insert is used to generate, via T7 RNA polymerase, large quantities (150 μg) of linear RNA containing the miR sponge sequence described above. Calf intestinal phosphatase dephosphorylates the 5′ end of the RNA transcript, and T4 polynucleotide kinase (in the presence of ATP) generates RNA molecules suitable for ligation. Incubation with T4 RNA ligase results in RNA circularization.
    Figure Legend Snippet: Workflow for Producing Synthetic miR-21 circRNA Sponge Each binding site sequence is perfectly complementary to the miR-21 seed region, but contains a bulge at positions 9–12 to prevent RNAi-type cleavage and degradation. One PCR cycle facilitates synthesis to generate a double-stranded DNA PCR fragment, which is then cloned into the TOPO PCR cloning vector (Invitrogen). The T7 RNA polymerase binding site located just 5′ to the PCR insert is used to generate, via T7 RNA polymerase, large quantities (150 μg) of linear RNA containing the miR sponge sequence described above. Calf intestinal phosphatase dephosphorylates the 5′ end of the RNA transcript, and T4 polynucleotide kinase (in the presence of ATP) generates RNA molecules suitable for ligation. Incubation with T4 RNA ligase results in RNA circularization.

    Techniques Used: Binding Assay, Sequencing, Polymerase Chain Reaction, Clone Assay, Plasmid Preparation, Ligation, Incubation

    31) Product Images from "Juglone, an inhibitor of the peptidyl-prolyl isomerase Pin1, also directly blocks transcription"

    Article Title: Juglone, an inhibitor of the peptidyl-prolyl isomerase Pin1, also directly blocks transcription

    Journal: Nucleic Acids Research

    doi:

    The substrate specificity of juglone. ( A ) The effect of juglone on restriction enzyme digestions. Plasmid pCDNA3-HA was digested by Bam HI (B), Eco RI (R), Kpn I (K), Xba I (X), Hin dIII (H), Sca I (Sca) and Sal I (Sal) with (+) or without (–) the addition of juglone (50 µM). The uncut plasmid (un) served as a control. ( B ) The effect of juglone on the transcription of T7 RNA polymerase. Reactions were as described in Materials and Methods.
    Figure Legend Snippet: The substrate specificity of juglone. ( A ) The effect of juglone on restriction enzyme digestions. Plasmid pCDNA3-HA was digested by Bam HI (B), Eco RI (R), Kpn I (K), Xba I (X), Hin dIII (H), Sca I (Sca) and Sal I (Sal) with (+) or without (–) the addition of juglone (50 µM). The uncut plasmid (un) served as a control. ( B ) The effect of juglone on the transcription of T7 RNA polymerase. Reactions were as described in Materials and Methods.

    Techniques Used: Plasmid Preparation

    32) Product Images from "Distinct Poly(rC) Binding Protein KH Domain Determinants for Poliovirus Translation Initiation and Viral RNA Replication"

    Article Title: Distinct Poly(rC) Binding Protein KH Domain Determinants for Poliovirus Translation Initiation and Viral RNA Replication

    Journal: Journal of Virology

    doi: 10.1128/JVI.76.23.12008-12022.2002

    Genetic organization of the dicistronic replicon RNA, RibPVE2A(MluI), transcribed from the T7 RNA polymerase-based transcription construct pT7RibPVE2A(MluI). pT7RibPVE2A(MluI) encodes an RNA that contains the entire 5′ NCR and P1 coding region of PV separated from the P2/P3 coding regions, the 3′ NCR, and a poly(A) ( > 30 nt in length) by the EMCV IRES element. A cis -acting hammerhead ribozyme enzymatically cleaves itself (and the two guanosine residues of the T7 promoter) from the RNA, leaving the transcript with an authentic 5′ end. The 3′ terminus of the transcript contains four nonviral nucleotides imparted by the Mlu I restriction endonuclease site utilized for template linearization. The inserted fragment (see inset) encodes a UGA stop codon (Stop) followed by a 7-nt spacer, the EMCV IRES element (EMCV nt 260 to 834) followed by the first 15 nt of the EMCV open reading frame, and an additional tyrosine codon (Y) to provide a cis cleavage site for the generation of an authentic P2 N terminus following cleavage by the adjacent 2A proteinase (2A pro ) activity. The initiation codon of the second cistron is indicated (M).
    Figure Legend Snippet: Genetic organization of the dicistronic replicon RNA, RibPVE2A(MluI), transcribed from the T7 RNA polymerase-based transcription construct pT7RibPVE2A(MluI). pT7RibPVE2A(MluI) encodes an RNA that contains the entire 5′ NCR and P1 coding region of PV separated from the P2/P3 coding regions, the 3′ NCR, and a poly(A) ( > 30 nt in length) by the EMCV IRES element. A cis -acting hammerhead ribozyme enzymatically cleaves itself (and the two guanosine residues of the T7 promoter) from the RNA, leaving the transcript with an authentic 5′ end. The 3′ terminus of the transcript contains four nonviral nucleotides imparted by the Mlu I restriction endonuclease site utilized for template linearization. The inserted fragment (see inset) encodes a UGA stop codon (Stop) followed by a 7-nt spacer, the EMCV IRES element (EMCV nt 260 to 834) followed by the first 15 nt of the EMCV open reading frame, and an additional tyrosine codon (Y) to provide a cis cleavage site for the generation of an authentic P2 N terminus following cleavage by the adjacent 2A proteinase (2A pro ) activity. The initiation codon of the second cistron is indicated (M).

    Techniques Used: Construct, Activity Assay

    33) Product Images from "Heterogeneous Nuclear Ribonucleoprotein R Cooperates with Mediator to Facilitate Transcription Reinitiation on the c-Fos Gene"

    Article Title: Heterogeneous Nuclear Ribonucleoprotein R Cooperates with Mediator to Facilitate Transcription Reinitiation on the c-Fos Gene

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0072496

    hnRNP R facilitates transcription reinitiation in the presence of Mediator. (A) RNase protection assays were performed using the 32 P-labeled RNA probes against the 5′ or 3′ region of the G-free cassette. After hybridization of each 32 P-labeled probe and the transcripts, the products were treated by RNase A and T1 to remove the unhybridized regions. The digested products were separated on an 8% denaturing polyacrylamide gel and detected by autoradiography. As depicted schematically, the first- and second-round transcripts are transcribed by the Pol II system from the supercoiled c-fos template. The two c-fos transcripts produce a single band (I) when the 5′ probe is used for the RNase protection assay while they produce two bands (II and III) when the 3′ probe is used. By contrast, a single control transcript is transcribed by T7 RNA polymerase from the linear control template. This T7 promoter-based transcript produces a single band (IV) even when the 3′ probe is used for the same RNase protection assay. The results of the RNase protection assays are shown in the right panels, and the positions of the expected products are indicated on the right (I, II, III and IV). The used probe (5′ or 3′ RNA probe) is indicated on the top and loaded in lane 1 or 4. No transcript RNA indicates that the 5′ or 3′ RNA probe was treated by RNaseA/T1 without hybridization with any transcript RNAs, and c-fos (-hnRNP R/Med) or c-fos (+hnRNP R/Med) indicates the transcripts from c-fos promoter-driven transcription in the absence or presence of hnRNP R and Med(0.85), respectively. T7 indicates the transcript produced from T7 promoter-driven transcription. (B) GTFs, Pol II, the four activators (SRF, Elk-1, CREB and ATF1), PC4 and the template pfMC2AT(390) were incubated at 30°C for 40 min to form preinitiation complex, and then ATP, CTP, UTP and 3′- O -methyl-GTP (NTPs) were added to initiate transcription. After additional 60-min incubation, 20 mM EDTA pH 8.0 and 0.2% SDS were added to stop the reaction. hnRNP R and 3xF:Mediator were added as indicated in the figure. 0.01% Sarkosyl was added before preinitiation complex formation (A: 0 min) or immediately after initiation of transcription (B: 42 min, C: 48 min). (C) The transcripts from the reactions were separated on a 5% denaturing polyacrylamide gel and detected by autoradiography. The positions of the 390-nt transcript (arrow) and the second-round transcript (arrowhead) are indicated on the right. (D) Time-course analyses of the reinitiation products were performed using the pfMC2AT (390) or pfMC2AT (200) template. t indicates the incubation time (min) after the addition of the nucleotides. The upper panel shows the outline of the experiment. The positions of the first- (arrow), second- (arrowhead) and third-round transcripts (white arrowhead) are indicated on the right.
    Figure Legend Snippet: hnRNP R facilitates transcription reinitiation in the presence of Mediator. (A) RNase protection assays were performed using the 32 P-labeled RNA probes against the 5′ or 3′ region of the G-free cassette. After hybridization of each 32 P-labeled probe and the transcripts, the products were treated by RNase A and T1 to remove the unhybridized regions. The digested products were separated on an 8% denaturing polyacrylamide gel and detected by autoradiography. As depicted schematically, the first- and second-round transcripts are transcribed by the Pol II system from the supercoiled c-fos template. The two c-fos transcripts produce a single band (I) when the 5′ probe is used for the RNase protection assay while they produce two bands (II and III) when the 3′ probe is used. By contrast, a single control transcript is transcribed by T7 RNA polymerase from the linear control template. This T7 promoter-based transcript produces a single band (IV) even when the 3′ probe is used for the same RNase protection assay. The results of the RNase protection assays are shown in the right panels, and the positions of the expected products are indicated on the right (I, II, III and IV). The used probe (5′ or 3′ RNA probe) is indicated on the top and loaded in lane 1 or 4. No transcript RNA indicates that the 5′ or 3′ RNA probe was treated by RNaseA/T1 without hybridization with any transcript RNAs, and c-fos (-hnRNP R/Med) or c-fos (+hnRNP R/Med) indicates the transcripts from c-fos promoter-driven transcription in the absence or presence of hnRNP R and Med(0.85), respectively. T7 indicates the transcript produced from T7 promoter-driven transcription. (B) GTFs, Pol II, the four activators (SRF, Elk-1, CREB and ATF1), PC4 and the template pfMC2AT(390) were incubated at 30°C for 40 min to form preinitiation complex, and then ATP, CTP, UTP and 3′- O -methyl-GTP (NTPs) were added to initiate transcription. After additional 60-min incubation, 20 mM EDTA pH 8.0 and 0.2% SDS were added to stop the reaction. hnRNP R and 3xF:Mediator were added as indicated in the figure. 0.01% Sarkosyl was added before preinitiation complex formation (A: 0 min) or immediately after initiation of transcription (B: 42 min, C: 48 min). (C) The transcripts from the reactions were separated on a 5% denaturing polyacrylamide gel and detected by autoradiography. The positions of the 390-nt transcript (arrow) and the second-round transcript (arrowhead) are indicated on the right. (D) Time-course analyses of the reinitiation products were performed using the pfMC2AT (390) or pfMC2AT (200) template. t indicates the incubation time (min) after the addition of the nucleotides. The upper panel shows the outline of the experiment. The positions of the first- (arrow), second- (arrowhead) and third-round transcripts (white arrowhead) are indicated on the right.

    Techniques Used: Labeling, Hybridization, Autoradiography, Rnase Protection Assay, Produced, Incubation

    34) Product Images from "Heterogeneous Nuclear Ribonucleoprotein R Cooperates with Mediator to Facilitate Transcription Reinitiation on the c-Fos Gene"

    Article Title: Heterogeneous Nuclear Ribonucleoprotein R Cooperates with Mediator to Facilitate Transcription Reinitiation on the c-Fos Gene

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0072496

    hnRNP R facilitates transcription reinitiation in the presence of Mediator. (A) RNase protection assays were performed using the 32 P-labeled RNA probes against the 5′ or 3′ region of the G-free cassette. After hybridization of each 32 P-labeled probe and the transcripts, the products were treated by RNase A and T1 to remove the unhybridized regions. The digested products were separated on an 8% denaturing polyacrylamide gel and detected by autoradiography. As depicted schematically, the first- and second-round transcripts are transcribed by the Pol II system from the supercoiled c-fos template. The two c-fos transcripts produce a single band (I) when the 5′ probe is used for the RNase protection assay while they produce two bands (II and III) when the 3′ probe is used. By contrast, a single control transcript is transcribed by T7 RNA polymerase from the linear control template. This T7 promoter-based transcript produces a single band (IV) even when the 3′ probe is used for the same RNase protection assay. The results of the RNase protection assays are shown in the right panels, and the positions of the expected products are indicated on the right (I, II, III and IV). The used probe (5′ or 3′ RNA probe) is indicated on the top and loaded in lane 1 or 4. No transcript RNA indicates that the 5′ or 3′ RNA probe was treated by RNaseA/T1 without hybridization with any transcript RNAs, and c-fos (-hnRNP R/Med) or c-fos (+hnRNP R/Med) indicates the transcripts from c-fos promoter-driven transcription in the absence or presence of hnRNP R and Med(0.85), respectively. T7 indicates the transcript produced from T7 promoter-driven transcription. (B) GTFs, Pol II, the four activators (SRF, Elk-1, CREB and ATF1), PC4 and the template pfMC2AT(390) were incubated at 30°C for 40 min to form preinitiation complex, and then ATP, CTP, UTP and 3′- O -methyl-GTP (NTPs) were added to initiate transcription. After additional 60-min incubation, 20 mM EDTA pH 8.0 and 0.2% SDS were added to stop the reaction. hnRNP R and 3xF:Mediator were added as indicated in the figure. 0.01% Sarkosyl was added before preinitiation complex formation (A: 0 min) or immediately after initiation of transcription (B: 42 min, C: 48 min). (C) The transcripts from the reactions were separated on a 5% denaturing polyacrylamide gel and detected by autoradiography. The positions of the 390-nt transcript (arrow) and the second-round transcript (arrowhead) are indicated on the right. (D) Time-course analyses of the reinitiation products were performed using the pfMC2AT (390) or pfMC2AT (200) template. t indicates the incubation time (min) after the addition of the nucleotides. The upper panel shows the outline of the experiment. The positions of the first- (arrow), second- (arrowhead) and third-round transcripts (white arrowhead) are indicated on the right.
    Figure Legend Snippet: hnRNP R facilitates transcription reinitiation in the presence of Mediator. (A) RNase protection assays were performed using the 32 P-labeled RNA probes against the 5′ or 3′ region of the G-free cassette. After hybridization of each 32 P-labeled probe and the transcripts, the products were treated by RNase A and T1 to remove the unhybridized regions. The digested products were separated on an 8% denaturing polyacrylamide gel and detected by autoradiography. As depicted schematically, the first- and second-round transcripts are transcribed by the Pol II system from the supercoiled c-fos template. The two c-fos transcripts produce a single band (I) when the 5′ probe is used for the RNase protection assay while they produce two bands (II and III) when the 3′ probe is used. By contrast, a single control transcript is transcribed by T7 RNA polymerase from the linear control template. This T7 promoter-based transcript produces a single band (IV) even when the 3′ probe is used for the same RNase protection assay. The results of the RNase protection assays are shown in the right panels, and the positions of the expected products are indicated on the right (I, II, III and IV). The used probe (5′ or 3′ RNA probe) is indicated on the top and loaded in lane 1 or 4. No transcript RNA indicates that the 5′ or 3′ RNA probe was treated by RNaseA/T1 without hybridization with any transcript RNAs, and c-fos (-hnRNP R/Med) or c-fos (+hnRNP R/Med) indicates the transcripts from c-fos promoter-driven transcription in the absence or presence of hnRNP R and Med(0.85), respectively. T7 indicates the transcript produced from T7 promoter-driven transcription. (B) GTFs, Pol II, the four activators (SRF, Elk-1, CREB and ATF1), PC4 and the template pfMC2AT(390) were incubated at 30°C for 40 min to form preinitiation complex, and then ATP, CTP, UTP and 3′- O -methyl-GTP (NTPs) were added to initiate transcription. After additional 60-min incubation, 20 mM EDTA pH 8.0 and 0.2% SDS were added to stop the reaction. hnRNP R and 3xF:Mediator were added as indicated in the figure. 0.01% Sarkosyl was added before preinitiation complex formation (A: 0 min) or immediately after initiation of transcription (B: 42 min, C: 48 min). (C) The transcripts from the reactions were separated on a 5% denaturing polyacrylamide gel and detected by autoradiography. The positions of the 390-nt transcript (arrow) and the second-round transcript (arrowhead) are indicated on the right. (D) Time-course analyses of the reinitiation products were performed using the pfMC2AT (390) or pfMC2AT (200) template. t indicates the incubation time (min) after the addition of the nucleotides. The upper panel shows the outline of the experiment. The positions of the first- (arrow), second- (arrowhead) and third-round transcripts (white arrowhead) are indicated on the right.

    Techniques Used: Labeling, Hybridization, Autoradiography, Rnase Protection Assay, Produced, Incubation

    35) Product Images from "iSpinach: a fluorogenic RNA aptamer optimized for in vitro applications"

    Article Title: iSpinach: a fluorogenic RNA aptamer optimized for in vitro applications

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw083

    Real-time monitoring of RNA synthesis and ribozyme activity. ( A ) Constructs used. DNA fragments coding for iSpinach, Spinach2 or iXm1 ribozyme were combined in different ways and placed under the control of T7 RNA polymerase promoter sequence (Pro T7 ). ( B ) Real-time ribozyme activity monitoring. The different constructs were in vitro transcribed in the presence of ribozyme fluorogenic substrate (S21-Atto) and the fluorescence monitored at 37°C. Product generation rate \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$\left( {\frac{{{\rm dP}}}{{{\rm dt}}}} \right)$\end{document} and uncatalyzed reaction rate ( k uncat ) were determined as the slope of the linear phase of reaction respectively in the presence and absence of ribozyme. ( C ) Real-time transcription monitoring. The in vitro transcription mixture used in B. was supplemented with DFHBI (instead of S21-Atto) and the fluorescence monitored at 37°C. The synthesis rate (σ) was determined as the slope of the linear phase.
    Figure Legend Snippet: Real-time monitoring of RNA synthesis and ribozyme activity. ( A ) Constructs used. DNA fragments coding for iSpinach, Spinach2 or iXm1 ribozyme were combined in different ways and placed under the control of T7 RNA polymerase promoter sequence (Pro T7 ). ( B ) Real-time ribozyme activity monitoring. The different constructs were in vitro transcribed in the presence of ribozyme fluorogenic substrate (S21-Atto) and the fluorescence monitored at 37°C. Product generation rate \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$\left( {\frac{{{\rm dP}}}{{{\rm dt}}}} \right)$\end{document} and uncatalyzed reaction rate ( k uncat ) were determined as the slope of the linear phase of reaction respectively in the presence and absence of ribozyme. ( C ) Real-time transcription monitoring. The in vitro transcription mixture used in B. was supplemented with DFHBI (instead of S21-Atto) and the fluorescence monitored at 37°C. The synthesis rate (σ) was determined as the slope of the linear phase.

    Techniques Used: Activity Assay, Construct, Sequencing, In Vitro, Fluorescence

    36) Product Images from "Transcription Impacts the Efficiency of mRNA Translation via Co-transcriptional N6-adenosine Methylation"

    Article Title: Transcription Impacts the Efficiency of mRNA Translation via Co-transcriptional N6-adenosine Methylation

    Journal: Cell

    doi: 10.1016/j.cell.2017.03.031

    Analysis of Rluc mRNAs, Related to Figures 2 , 3 , 5 , and 6 (A and B) Rluc mRNAs transcribed by ASNSD1 (A) or SZT2 (B) promoters either lacking or bearing an artificial TATA box were subjected to 5′RACE analysis in order to determine their TSSs. Reads were plotted in a quantitative manner using IGV software. Note the first ATG of the Rluc ORF and the precise focusing of the TSS by the TATA box. (C) Evaluation of the effect of the different 5′UTRs on TE. Rluc transcripts bearing the different 5′UTRs identified in A,B (see the numbered arrows) were transcribed in vitro using T7 RNA polymerase, polyadenylated and transfected into MCF7 cells in fold-wise amounts. Renilla luciferase activity was measured 24 hr later and plotted in a relative manner to draw the trend lines that represent the respective ratios of translation. The rightmost panel shows the absolute Rluc signals; n = 4. (D) Rluc mRNAs expressed from either induced or non-induced TRex-Rluc gene were subjected to 5′RACE analysis. Reads (numbers of sequenced colonies) were plotted in a quantitative manner on a scale ranging from TATA box (−243) to ATG (+1). (E) Rluc transcripts produced in vitro using HeLa extract in optimal conditions (1.5mM Mg++), upon high MgCl 2 (3mM Mg++) or from promoter with mutated TATA element (muTATA) were subjected to 5′- and 3′-RACE analyses. The reads from both assays were analyzed and plotted in a quantitative manner using IGV software. (F) Transcripts described in (E) were subjected to analysis of the length of polyA-tails. (G) The TRex-Rluc cassette was induced for 24 hr in cells transfected with the indicated siRNAs. After isolation of total RNA, the 5′- and 3′ ends of the Rluc mRNAs were determined as detailed in (E).
    Figure Legend Snippet: Analysis of Rluc mRNAs, Related to Figures 2 , 3 , 5 , and 6 (A and B) Rluc mRNAs transcribed by ASNSD1 (A) or SZT2 (B) promoters either lacking or bearing an artificial TATA box were subjected to 5′RACE analysis in order to determine their TSSs. Reads were plotted in a quantitative manner using IGV software. Note the first ATG of the Rluc ORF and the precise focusing of the TSS by the TATA box. (C) Evaluation of the effect of the different 5′UTRs on TE. Rluc transcripts bearing the different 5′UTRs identified in A,B (see the numbered arrows) were transcribed in vitro using T7 RNA polymerase, polyadenylated and transfected into MCF7 cells in fold-wise amounts. Renilla luciferase activity was measured 24 hr later and plotted in a relative manner to draw the trend lines that represent the respective ratios of translation. The rightmost panel shows the absolute Rluc signals; n = 4. (D) Rluc mRNAs expressed from either induced or non-induced TRex-Rluc gene were subjected to 5′RACE analysis. Reads (numbers of sequenced colonies) were plotted in a quantitative manner on a scale ranging from TATA box (−243) to ATG (+1). (E) Rluc transcripts produced in vitro using HeLa extract in optimal conditions (1.5mM Mg++), upon high MgCl 2 (3mM Mg++) or from promoter with mutated TATA element (muTATA) were subjected to 5′- and 3′-RACE analyses. The reads from both assays were analyzed and plotted in a quantitative manner using IGV software. (F) Transcripts described in (E) were subjected to analysis of the length of polyA-tails. (G) The TRex-Rluc cassette was induced for 24 hr in cells transfected with the indicated siRNAs. After isolation of total RNA, the 5′- and 3′ ends of the Rluc mRNAs were determined as detailed in (E).

    Techniques Used: Software, In Vitro, Transfection, Luciferase, Activity Assay, Produced, Isolation

    37) Product Images from "Design and application of cotranscriptional non-enzymatic RNA circuits and signal transducers"

    Article Title: Design and application of cotranscriptional non-enzymatic RNA circuits and signal transducers

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gku074

    Operation of cotranscriptionally generated RNA CHA circuits without any downstream purification and design optimization for detection of DNA target. ( a ) Fifty nanograms each of the indicated pairs of hairpin 1 and 2 transcription templates was cotranscribed with or without 10 ng of C1 transcription template for 1 h at 42°C using T7 RNA polymerase. Following transcription, 2 µl of the reaction mix was directly incubated in 1× TNaK buffer containing 20 U of RNaseOUT and 0.5 µM ROX reference dye along with 400 nM RepF (annealed with 5× excess RepQ) fluorescent DNA reporter duplex for quantitating RNA CHA in real-time at 52°C. ( b ) Schematic depicting SDA of DNA. The single-stranded template DNA (black arrow) consists of a sequence (C*) complementary to the RNA CHA catalyst followed by the nicking enzyme recognition sequence (NE) that is present on the non-cleaved DNA strand and a primer binding site. Following primer binding (step 1), the DNA polymerase synthesizes the complementary strand that now completes the duplex NE site and contains the RNA CHA catalyst sequence (C). Nicking enzyme then binds the duplex NE site (step 2) and cleaves the newly synthesized strand at the NE site. The new 3′-OH group generated at the nick site is then extended by the DNA polymerase (step 3) while displacing the previously synthesized strand. The displaced ssDNA amplicon can then catalyze RNA CHA. ( c ) Schematic of DNA target sequence design for catalysis of RNA CHA. Single toehold (domain 1*) DNA target C1 (generated by SDA from the template TLTRSDA) with the same domain architecture as the RNA C1 is an inefficient catalyst of RNA CHA. Extended DNA target C1234 (generated by SDA from the template 1234LTRSDA) presenting two toeholds for RNA H1 successfully catalyzes RNA CHA.
    Figure Legend Snippet: Operation of cotranscriptionally generated RNA CHA circuits without any downstream purification and design optimization for detection of DNA target. ( a ) Fifty nanograms each of the indicated pairs of hairpin 1 and 2 transcription templates was cotranscribed with or without 10 ng of C1 transcription template for 1 h at 42°C using T7 RNA polymerase. Following transcription, 2 µl of the reaction mix was directly incubated in 1× TNaK buffer containing 20 U of RNaseOUT and 0.5 µM ROX reference dye along with 400 nM RepF (annealed with 5× excess RepQ) fluorescent DNA reporter duplex for quantitating RNA CHA in real-time at 52°C. ( b ) Schematic depicting SDA of DNA. The single-stranded template DNA (black arrow) consists of a sequence (C*) complementary to the RNA CHA catalyst followed by the nicking enzyme recognition sequence (NE) that is present on the non-cleaved DNA strand and a primer binding site. Following primer binding (step 1), the DNA polymerase synthesizes the complementary strand that now completes the duplex NE site and contains the RNA CHA catalyst sequence (C). Nicking enzyme then binds the duplex NE site (step 2) and cleaves the newly synthesized strand at the NE site. The new 3′-OH group generated at the nick site is then extended by the DNA polymerase (step 3) while displacing the previously synthesized strand. The displaced ssDNA amplicon can then catalyze RNA CHA. ( c ) Schematic of DNA target sequence design for catalysis of RNA CHA. Single toehold (domain 1*) DNA target C1 (generated by SDA from the template TLTRSDA) with the same domain architecture as the RNA C1 is an inefficient catalyst of RNA CHA. Extended DNA target C1234 (generated by SDA from the template 1234LTRSDA) presenting two toeholds for RNA H1 successfully catalyzes RNA CHA.

    Techniques Used: Generated, Purification, Incubation, Sequencing, Binding Assay, Synthesized, Amplification

    Synthesis and execution of RNA CHA circuit. ( a ) LHRz and RHRz-mediated cotranscriptional RNA cleavage releases the internal circuit components H1, H2 and C1. Fifity nanograms of PCR-generated transcription templates for H1, H2 and C1 was transcribed in 50 µl of reactions by T7 RNA polymerase for 2 h at 42°C. Two microliters of the resulting transcripts was analyzed by electrophoresis on a 10% denaturing polyacrylamide gel. Single-stranded DNA oligonucleotides were used as size markers. ( b ) RNA hairpins undergo catalyzed assembly into RNA duplexes. Gel-purified RNA catalyst C1 and the hairpins H1 and H2 were combined as indicated and incubated in 1× TNaK buffer containing 20 U of RNaseOUT for 150 min at 42°C (lanes 1–4), 52°C (lanes 5–8) or 62°C (lanes 9–12). The reactions were then analyzed on a 10% native polyacrylamide gel. Fifteen nanograms of C1 RNA was included in lane 13 as a control. Single-stranded DNA oligonucleotides were used as size markers.
    Figure Legend Snippet: Synthesis and execution of RNA CHA circuit. ( a ) LHRz and RHRz-mediated cotranscriptional RNA cleavage releases the internal circuit components H1, H2 and C1. Fifity nanograms of PCR-generated transcription templates for H1, H2 and C1 was transcribed in 50 µl of reactions by T7 RNA polymerase for 2 h at 42°C. Two microliters of the resulting transcripts was analyzed by electrophoresis on a 10% denaturing polyacrylamide gel. Single-stranded DNA oligonucleotides were used as size markers. ( b ) RNA hairpins undergo catalyzed assembly into RNA duplexes. Gel-purified RNA catalyst C1 and the hairpins H1 and H2 were combined as indicated and incubated in 1× TNaK buffer containing 20 U of RNaseOUT for 150 min at 42°C (lanes 1–4), 52°C (lanes 5–8) or 62°C (lanes 9–12). The reactions were then analyzed on a 10% native polyacrylamide gel. Fifteen nanograms of C1 RNA was included in lane 13 as a control. Single-stranded DNA oligonucleotides were used as size markers.

    Techniques Used: Polymerase Chain Reaction, Generated, Electrophoresis, Purification, Incubation

    Cotranscriptionally generated RNA CHA as signal transducer for nucleic acid diagnostics. ( a ) End-point sequence-specific detection of SDA-generated ssDNA targets by RNA CHA. Samples with or without 10 nM template 1234LTRSDA were amplified by SDA for 90 min at 37°C in 25 µl of reaction volumes. Reactions were then incubated at 95°C for 5 min and stored at room temperature before assay by RNA CHA. Five microliters of these SDA products was then probed with 2 µl of Sephadex G25 column-purified cotranscribed mH1:H2 RNA CHA circuit. RNA CHA cotranscriptions were performed with T7 RNA polymerase using 50 ng each of the mH1 and H2 transcription templates for 1 h at 42°C. End-point RNA CHA detection reactions were assembled in 1× TNaK buffer containing 20 U of RNaseOUT, 0.5 µM ROX reference dye and 100 nM RepF (annealed with 5× excess RepQ) fluorescent DNA reporter duplex for quantitating RNA CHA in real-time at 52°C. Negative control reactions lacking RNA CHA components or containing 2 µl of either only mH1 or H2 were also tested. ( b ) Real-time signal transduction of ssDNA-generating SDA by cotranscribed mH1:H2 RNA CHA. High temperature (55°C) SDA reactions were set up with or without 10 nM 1234HTRSDA template in 20 µl of volume containing 0.5 µM ROX reference dye and 75 nM RepF (annealed with 5× excess RepQ) fluorescent DNA reporter duplex for quantitating RNA CHA in real-time. Real-time sequence-specific signal transduction was achieved by adding 2 µl of unpurified mH1:H2 RNA CHA circuits cotranscribed from 50 ng of each transcription template to the SDA reactions. Control SDA reactions containing no RNA CHA components or 2 µl of either only mH1 or H2 were also tested.
    Figure Legend Snippet: Cotranscriptionally generated RNA CHA as signal transducer for nucleic acid diagnostics. ( a ) End-point sequence-specific detection of SDA-generated ssDNA targets by RNA CHA. Samples with or without 10 nM template 1234LTRSDA were amplified by SDA for 90 min at 37°C in 25 µl of reaction volumes. Reactions were then incubated at 95°C for 5 min and stored at room temperature before assay by RNA CHA. Five microliters of these SDA products was then probed with 2 µl of Sephadex G25 column-purified cotranscribed mH1:H2 RNA CHA circuit. RNA CHA cotranscriptions were performed with T7 RNA polymerase using 50 ng each of the mH1 and H2 transcription templates for 1 h at 42°C. End-point RNA CHA detection reactions were assembled in 1× TNaK buffer containing 20 U of RNaseOUT, 0.5 µM ROX reference dye and 100 nM RepF (annealed with 5× excess RepQ) fluorescent DNA reporter duplex for quantitating RNA CHA in real-time at 52°C. Negative control reactions lacking RNA CHA components or containing 2 µl of either only mH1 or H2 were also tested. ( b ) Real-time signal transduction of ssDNA-generating SDA by cotranscribed mH1:H2 RNA CHA. High temperature (55°C) SDA reactions were set up with or without 10 nM 1234HTRSDA template in 20 µl of volume containing 0.5 µM ROX reference dye and 75 nM RepF (annealed with 5× excess RepQ) fluorescent DNA reporter duplex for quantitating RNA CHA in real-time. Real-time sequence-specific signal transduction was achieved by adding 2 µl of unpurified mH1:H2 RNA CHA circuits cotranscribed from 50 ng of each transcription template to the SDA reactions. Control SDA reactions containing no RNA CHA components or 2 µl of either only mH1 or H2 were also tested.

    Techniques Used: Generated, Sequencing, Amplification, Incubation, Purification, Negative Control, Transduction

    Design of non-enzymatic catalyzed RNA hairpin assembly circuit. ( a ) Schematic of catalyzed nucleic acid hairpin assembly circuit adapted from ( 2 ). The circuit composed of hairpins H1 and H2 is turned on in the presence of the input sequence (C1). C1 catalyzes the assembly of H1 and H2 into an H1:H2 duplex and is itself recycled. Circuit output (H1:H2 duplex) is quantitated as increasing fluorescence intensity of a labeled oligonucleotide probe (RepF) on displacement of its complementary quencher oligonucleotide (RepQ) by the H1:H2 duplex. ( b ) Design of T7 RNA polymerase-driven transcription templates for enzymatic synthesis of RNA CHA circuit components with precise 5′- and 3′-ends. Transcription template for each component, H1, H2 and C1, is flanked on both the left (L) and the right (R) sides by hammerhead ribozymes (HRz). The size (in nucleotides) of each component and its ribozyme flanks is indicated under each schematic. Secondary structures of the resulting chimeric RNA at 42°C before ribozyme processing are depicted (green, A; blue, C; black, G; red, U). The RNA structures were generated using NUPACK ( 13–16 ).
    Figure Legend Snippet: Design of non-enzymatic catalyzed RNA hairpin assembly circuit. ( a ) Schematic of catalyzed nucleic acid hairpin assembly circuit adapted from ( 2 ). The circuit composed of hairpins H1 and H2 is turned on in the presence of the input sequence (C1). C1 catalyzes the assembly of H1 and H2 into an H1:H2 duplex and is itself recycled. Circuit output (H1:H2 duplex) is quantitated as increasing fluorescence intensity of a labeled oligonucleotide probe (RepF) on displacement of its complementary quencher oligonucleotide (RepQ) by the H1:H2 duplex. ( b ) Design of T7 RNA polymerase-driven transcription templates for enzymatic synthesis of RNA CHA circuit components with precise 5′- and 3′-ends. Transcription template for each component, H1, H2 and C1, is flanked on both the left (L) and the right (R) sides by hammerhead ribozymes (HRz). The size (in nucleotides) of each component and its ribozyme flanks is indicated under each schematic. Secondary structures of the resulting chimeric RNA at 42°C before ribozyme processing are depicted (green, A; blue, C; black, G; red, U). The RNA structures were generated using NUPACK ( 13–16 ).

    Techniques Used: Sequencing, Fluorescence, Labeling, Generated

    Application of RNA CHA circuit as an OR logic processor. ( a ) Schematic of RNA CHA circuit operation in response to either catalyst C1 OR C2. The RNA hairpin H1B serves as the OR gate, and circuit output is measured fluorimetrically using Spinach.ST1 RNA aptamer beacon. ( b ) Circuit components (H1B and H2 RNA hairpins), reporter RNA (Spinach.ST1) and the inputs C1 and C2 were transcribed from 500 ng of duplex DNA transcription templates using T7 RNA polymerase. Transcription templates were prepared using the same procedure as Figure 8 . Following filtration through Sephadex G25, 3 µl/transcript (or 1.5 µl each of C1 and C2 when added together in a reaction) was mixed in the indicated combinations in 1× TNaK buffer containing 70 µM DFHBI and 20 U of RNaseOUT. Circuits were operated at 37°C, and outputs were measured fluorimetrically.
    Figure Legend Snippet: Application of RNA CHA circuit as an OR logic processor. ( a ) Schematic of RNA CHA circuit operation in response to either catalyst C1 OR C2. The RNA hairpin H1B serves as the OR gate, and circuit output is measured fluorimetrically using Spinach.ST1 RNA aptamer beacon. ( b ) Circuit components (H1B and H2 RNA hairpins), reporter RNA (Spinach.ST1) and the inputs C1 and C2 were transcribed from 500 ng of duplex DNA transcription templates using T7 RNA polymerase. Transcription templates were prepared using the same procedure as Figure 8 . Following filtration through Sephadex G25, 3 µl/transcript (or 1.5 µl each of C1 and C2 when added together in a reaction) was mixed in the indicated combinations in 1× TNaK buffer containing 70 µM DFHBI and 20 U of RNaseOUT. Circuits were operated at 37°C, and outputs were measured fluorimetrically.

    Techniques Used: Filtration

    Cotranscriptional RNA CHA and circuit design optimization for cotranscription. ( a ) Cotranscribed RNA circuit components undergo catalyzed hairpin assembly without requiring gel purification of individual reactants. Fifty nanograms each of H1 and H2 transcription templates, along with titrating amounts of C1 transcription template, was cotranscribed for 1 h at 42°C using T7 RNA polymerase followed by passage through Illustra MicroSpin Sephadex G25 columns. Transcription templates were amplified from cloned inserts using primers pCR2.1.F and pCR2.1.R specific to plasmid sequences flanking the inserts. Two microliter aliquots of the cotranscribed RNA mixtures were then incubated in 15 µl of volume with 400 nM RepF annealed with 5× excess (2 µM) RepQ fluorescent DNA reporter duplex in 1× TNaK buffer containing 20 U of RNaseOUT and 0.5 µM ROX reference dye to quantitate formation of H1:H2 RNA duplexes at 52°C. Average data from triplicate experiments are represented. ( b and c ) Schematic depicting sequences of RNA hairpins H1 and H2 with one- or two-base engineered mismatches. Mismatched H1 (mH1) presents a two-base mismatch between its domain 4* and domain 4 of H2. The hairpins mAH1 and mGH1 each contain a single mismatched base between their domain 4* and the domain 4 of H2. The mutated H2 hairpin m2H2 presents two mismatched bases between its domain 2* and the H1 domain 2.
    Figure Legend Snippet: Cotranscriptional RNA CHA and circuit design optimization for cotranscription. ( a ) Cotranscribed RNA circuit components undergo catalyzed hairpin assembly without requiring gel purification of individual reactants. Fifty nanograms each of H1 and H2 transcription templates, along with titrating amounts of C1 transcription template, was cotranscribed for 1 h at 42°C using T7 RNA polymerase followed by passage through Illustra MicroSpin Sephadex G25 columns. Transcription templates were amplified from cloned inserts using primers pCR2.1.F and pCR2.1.R specific to plasmid sequences flanking the inserts. Two microliter aliquots of the cotranscribed RNA mixtures were then incubated in 15 µl of volume with 400 nM RepF annealed with 5× excess (2 µM) RepQ fluorescent DNA reporter duplex in 1× TNaK buffer containing 20 U of RNaseOUT and 0.5 µM ROX reference dye to quantitate formation of H1:H2 RNA duplexes at 52°C. Average data from triplicate experiments are represented. ( b and c ) Schematic depicting sequences of RNA hairpins H1 and H2 with one- or two-base engineered mismatches. Mismatched H1 (mH1) presents a two-base mismatch between its domain 4* and domain 4 of H2. The hairpins mAH1 and mGH1 each contain a single mismatched base between their domain 4* and the domain 4 of H2. The mutated H2 hairpin m2H2 presents two mismatched bases between its domain 2* and the H1 domain 2.

    Techniques Used: Gel Purification, Amplification, Clone Assay, Plasmid Preparation, Incubation

    An entirely RNA-based CHA circuit operation and fluorimetric detection. ( a ) CHA circuit components (hairpins H1B and H2 and catalyst C1) and the RNA reporter Spinach.ST1 were separately transcribed by T7 RNA polymerase from 500 ng of PCR-generated duplex DNA transcription templates. H1B, H2 and C1 transcription templates were amplified using primers complementary to the exact ends of the cloned inserts (H1B.amp.F:H1B.amp.R, H2.amp.F:H2.amp.R and C1.amp.F:C1.amp.R, respectively) rather than the flanking plasmid. Spinach.ST transcription templates were amplified using primers specific to the flanking plasmid sequence at the 5′-end (pCR2.1.F) and the primer sphT.U.R specific to the 3′-end sequence of Spinach.ST. Transcription reactions were filtered through Sephadex G25 columns before circuit assembly. Three microliters of H1B, H2, C1 and Spinach.ST1 transcripts was mixed in indicated combinations and incubated in 1× TNaK buffer containing 70 µM DFHBI and 20 U of RNaseOUT. Circuit output was measured as increasing fluorescence intensity over time at 37°C. ( b–d ) Performance of DNA reporter duplex H1BF:H1BQ (b) versus Spinach.ST1 (c) in measuring RNA CHA circuit output. Indicated concentrations of gel-purified RNA hairpins H1B and H2 were incubated with equal concentration of H1BF:H1BQ or gel-purified Spinach.ST1 (+ 70 µM DFHBI) in the presence of titrating concentrations of pure C1 RNA. All circuits were operated in 1× TNaK buffer containing 20 U of RNaseOUT at 37°C, and average data from triplicate experiments are represented. Signal-to-noise ratio of H1BF:H1BQ versus Spinach.ST1 over the time course of RNA CHA detection is plotted in (d).
    Figure Legend Snippet: An entirely RNA-based CHA circuit operation and fluorimetric detection. ( a ) CHA circuit components (hairpins H1B and H2 and catalyst C1) and the RNA reporter Spinach.ST1 were separately transcribed by T7 RNA polymerase from 500 ng of PCR-generated duplex DNA transcription templates. H1B, H2 and C1 transcription templates were amplified using primers complementary to the exact ends of the cloned inserts (H1B.amp.F:H1B.amp.R, H2.amp.F:H2.amp.R and C1.amp.F:C1.amp.R, respectively) rather than the flanking plasmid. Spinach.ST transcription templates were amplified using primers specific to the flanking plasmid sequence at the 5′-end (pCR2.1.F) and the primer sphT.U.R specific to the 3′-end sequence of Spinach.ST. Transcription reactions were filtered through Sephadex G25 columns before circuit assembly. Three microliters of H1B, H2, C1 and Spinach.ST1 transcripts was mixed in indicated combinations and incubated in 1× TNaK buffer containing 70 µM DFHBI and 20 U of RNaseOUT. Circuit output was measured as increasing fluorescence intensity over time at 37°C. ( b–d ) Performance of DNA reporter duplex H1BF:H1BQ (b) versus Spinach.ST1 (c) in measuring RNA CHA circuit output. Indicated concentrations of gel-purified RNA hairpins H1B and H2 were incubated with equal concentration of H1BF:H1BQ or gel-purified Spinach.ST1 (+ 70 µM DFHBI) in the presence of titrating concentrations of pure C1 RNA. All circuits were operated in 1× TNaK buffer containing 20 U of RNaseOUT at 37°C, and average data from triplicate experiments are represented. Signal-to-noise ratio of H1BF:H1BQ versus Spinach.ST1 over the time course of RNA CHA detection is plotted in (d).

    Techniques Used: Polymerase Chain Reaction, Generated, Amplification, Clone Assay, Plasmid Preparation, Sequencing, Incubation, Fluorescence, Purification, Concentration Assay

    38) Product Images from "Design and application of cotranscriptional non-enzymatic RNA circuits and signal transducers"

    Article Title: Design and application of cotranscriptional non-enzymatic RNA circuits and signal transducers

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gku074

    Operation of cotranscriptionally generated RNA CHA circuits without any downstream purification and design optimization for detection of DNA target. ( a ) Fifty nanograms each of the indicated pairs of hairpin 1 and 2 transcription templates was cotranscribed with or without 10 ng of C1 transcription template for 1 h at 42°C using T7 RNA polymerase. Following transcription, 2 µl of the reaction mix was directly incubated in 1× TNaK buffer containing 20 U of RNaseOUT and 0.5 µM ROX reference dye along with 400 nM RepF (annealed with 5× excess RepQ) fluorescent DNA reporter duplex for quantitating RNA CHA in real-time at 52°C. ( b ) Schematic depicting SDA of DNA. The single-stranded template DNA (black arrow) consists of a sequence (C*) complementary to the RNA CHA catalyst followed by the nicking enzyme recognition sequence (NE) that is present on the non-cleaved DNA strand and a primer binding site. Following primer binding (step 1), the DNA polymerase synthesizes the complementary strand that now completes the duplex NE site and contains the RNA CHA catalyst sequence (C). Nicking enzyme then binds the duplex NE site (step 2) and cleaves the newly synthesized strand at the NE site. The new 3′-OH group generated at the nick site is then extended by the DNA polymerase (step 3) while displacing the previously synthesized strand. The displaced ssDNA amplicon can then catalyze RNA CHA. ( c ) Schematic of DNA target sequence design for catalysis of RNA CHA. Single toehold (domain 1*) DNA target C1 (generated by SDA from the template TLTRSDA) with the same domain architecture as the RNA C1 is an inefficient catalyst of RNA CHA. Extended DNA target C1234 (generated by SDA from the template 1234LTRSDA) presenting two toeholds for RNA H1 successfully catalyzes RNA CHA.
    Figure Legend Snippet: Operation of cotranscriptionally generated RNA CHA circuits without any downstream purification and design optimization for detection of DNA target. ( a ) Fifty nanograms each of the indicated pairs of hairpin 1 and 2 transcription templates was cotranscribed with or without 10 ng of C1 transcription template for 1 h at 42°C using T7 RNA polymerase. Following transcription, 2 µl of the reaction mix was directly incubated in 1× TNaK buffer containing 20 U of RNaseOUT and 0.5 µM ROX reference dye along with 400 nM RepF (annealed with 5× excess RepQ) fluorescent DNA reporter duplex for quantitating RNA CHA in real-time at 52°C. ( b ) Schematic depicting SDA of DNA. The single-stranded template DNA (black arrow) consists of a sequence (C*) complementary to the RNA CHA catalyst followed by the nicking enzyme recognition sequence (NE) that is present on the non-cleaved DNA strand and a primer binding site. Following primer binding (step 1), the DNA polymerase synthesizes the complementary strand that now completes the duplex NE site and contains the RNA CHA catalyst sequence (C). Nicking enzyme then binds the duplex NE site (step 2) and cleaves the newly synthesized strand at the NE site. The new 3′-OH group generated at the nick site is then extended by the DNA polymerase (step 3) while displacing the previously synthesized strand. The displaced ssDNA amplicon can then catalyze RNA CHA. ( c ) Schematic of DNA target sequence design for catalysis of RNA CHA. Single toehold (domain 1*) DNA target C1 (generated by SDA from the template TLTRSDA) with the same domain architecture as the RNA C1 is an inefficient catalyst of RNA CHA. Extended DNA target C1234 (generated by SDA from the template 1234LTRSDA) presenting two toeholds for RNA H1 successfully catalyzes RNA CHA.

    Techniques Used: Generated, Purification, Incubation, Sequencing, Binding Assay, Synthesized, Amplification

    Synthesis and execution of RNA CHA circuit. ( a ) LHRz and RHRz-mediated cotranscriptional RNA cleavage releases the internal circuit components H1, H2 and C1. Fifity nanograms of PCR-generated transcription templates for H1, H2 and C1 was transcribed in 50 µl of reactions by T7 RNA polymerase for 2 h at 42°C. Two microliters of the resulting transcripts was analyzed by electrophoresis on a 10% denaturing polyacrylamide gel. Single-stranded DNA oligonucleotides were used as size markers. ( b ) RNA hairpins undergo catalyzed assembly into RNA duplexes. Gel-purified RNA catalyst C1 and the hairpins H1 and H2 were combined as indicated and incubated in 1× TNaK buffer containing 20 U of RNaseOUT for 150 min at 42°C (lanes 1–4), 52°C (lanes 5–8) or 62°C (lanes 9–12). The reactions were then analyzed on a 10% native polyacrylamide gel. Fifteen nanograms of C1 RNA was included in lane 13 as a control. Single-stranded DNA oligonucleotides were used as size markers.
    Figure Legend Snippet: Synthesis and execution of RNA CHA circuit. ( a ) LHRz and RHRz-mediated cotranscriptional RNA cleavage releases the internal circuit components H1, H2 and C1. Fifity nanograms of PCR-generated transcription templates for H1, H2 and C1 was transcribed in 50 µl of reactions by T7 RNA polymerase for 2 h at 42°C. Two microliters of the resulting transcripts was analyzed by electrophoresis on a 10% denaturing polyacrylamide gel. Single-stranded DNA oligonucleotides were used as size markers. ( b ) RNA hairpins undergo catalyzed assembly into RNA duplexes. Gel-purified RNA catalyst C1 and the hairpins H1 and H2 were combined as indicated and incubated in 1× TNaK buffer containing 20 U of RNaseOUT for 150 min at 42°C (lanes 1–4), 52°C (lanes 5–8) or 62°C (lanes 9–12). The reactions were then analyzed on a 10% native polyacrylamide gel. Fifteen nanograms of C1 RNA was included in lane 13 as a control. Single-stranded DNA oligonucleotides were used as size markers.

    Techniques Used: Polymerase Chain Reaction, Generated, Electrophoresis, Purification, Incubation

    Cotranscriptionally generated RNA CHA as signal transducer for nucleic acid diagnostics. ( a ) End-point sequence-specific detection of SDA-generated ssDNA targets by RNA CHA. Samples with or without 10 nM template 1234LTRSDA were amplified by SDA for 90 min at 37°C in 25 µl of reaction volumes. Reactions were then incubated at 95°C for 5 min and stored at room temperature before assay by RNA CHA. Five microliters of these SDA products was then probed with 2 µl of Sephadex G25 column-purified cotranscribed mH1:H2 RNA CHA circuit. RNA CHA cotranscriptions were performed with T7 RNA polymerase using 50 ng each of the mH1 and H2 transcription templates for 1 h at 42°C. End-point RNA CHA detection reactions were assembled in 1× TNaK buffer containing 20 U of RNaseOUT, 0.5 µM ROX reference dye and 100 nM RepF (annealed with 5× excess RepQ) fluorescent DNA reporter duplex for quantitating RNA CHA in real-time at 52°C. Negative control reactions lacking RNA CHA components or containing 2 µl of either only mH1 or H2 were also tested. ( b ) Real-time signal transduction of ssDNA-generating SDA by cotranscribed mH1:H2 RNA CHA. High temperature (55°C) SDA reactions were set up with or without 10 nM 1234HTRSDA template in 20 µl of volume containing 0.5 µM ROX reference dye and 75 nM RepF (annealed with 5× excess RepQ) fluorescent DNA reporter duplex for quantitating RNA CHA in real-time. Real-time sequence-specific signal transduction was achieved by adding 2 µl of unpurified mH1:H2 RNA CHA circuits cotranscribed from 50 ng of each transcription template to the SDA reactions. Control SDA reactions containing no RNA CHA components or 2 µl of either only mH1 or H2 were also tested.
    Figure Legend Snippet: Cotranscriptionally generated RNA CHA as signal transducer for nucleic acid diagnostics. ( a ) End-point sequence-specific detection of SDA-generated ssDNA targets by RNA CHA. Samples with or without 10 nM template 1234LTRSDA were amplified by SDA for 90 min at 37°C in 25 µl of reaction volumes. Reactions were then incubated at 95°C for 5 min and stored at room temperature before assay by RNA CHA. Five microliters of these SDA products was then probed with 2 µl of Sephadex G25 column-purified cotranscribed mH1:H2 RNA CHA circuit. RNA CHA cotranscriptions were performed with T7 RNA polymerase using 50 ng each of the mH1 and H2 transcription templates for 1 h at 42°C. End-point RNA CHA detection reactions were assembled in 1× TNaK buffer containing 20 U of RNaseOUT, 0.5 µM ROX reference dye and 100 nM RepF (annealed with 5× excess RepQ) fluorescent DNA reporter duplex for quantitating RNA CHA in real-time at 52°C. Negative control reactions lacking RNA CHA components or containing 2 µl of either only mH1 or H2 were also tested. ( b ) Real-time signal transduction of ssDNA-generating SDA by cotranscribed mH1:H2 RNA CHA. High temperature (55°C) SDA reactions were set up with or without 10 nM 1234HTRSDA template in 20 µl of volume containing 0.5 µM ROX reference dye and 75 nM RepF (annealed with 5× excess RepQ) fluorescent DNA reporter duplex for quantitating RNA CHA in real-time. Real-time sequence-specific signal transduction was achieved by adding 2 µl of unpurified mH1:H2 RNA CHA circuits cotranscribed from 50 ng of each transcription template to the SDA reactions. Control SDA reactions containing no RNA CHA components or 2 µl of either only mH1 or H2 were also tested.

    Techniques Used: Generated, Sequencing, Amplification, Incubation, Purification, Negative Control, Transduction

    Design of non-enzymatic catalyzed RNA hairpin assembly circuit. ( a ) Schematic of catalyzed nucleic acid hairpin assembly circuit adapted from ( 2 ). The circuit composed of hairpins H1 and H2 is turned on in the presence of the input sequence (C1). C1 catalyzes the assembly of H1 and H2 into an H1:H2 duplex and is itself recycled. Circuit output (H1:H2 duplex) is quantitated as increasing fluorescence intensity of a labeled oligonucleotide probe (RepF) on displacement of its complementary quencher oligonucleotide (RepQ) by the H1:H2 duplex. ( b ) Design of T7 RNA polymerase-driven transcription templates for enzymatic synthesis of RNA CHA circuit components with precise 5′- and 3′-ends. Transcription template for each component, H1, H2 and C1, is flanked on both the left (L) and the right (R) sides by hammerhead ribozymes (HRz). The size (in nucleotides) of each component and its ribozyme flanks is indicated under each schematic. Secondary structures of the resulting chimeric RNA at 42°C before ribozyme processing are depicted (green, A; blue, C; black, G; red, U). The RNA structures were generated using NUPACK ( 13–16 ).
    Figure Legend Snippet: Design of non-enzymatic catalyzed RNA hairpin assembly circuit. ( a ) Schematic of catalyzed nucleic acid hairpin assembly circuit adapted from ( 2 ). The circuit composed of hairpins H1 and H2 is turned on in the presence of the input sequence (C1). C1 catalyzes the assembly of H1 and H2 into an H1:H2 duplex and is itself recycled. Circuit output (H1:H2 duplex) is quantitated as increasing fluorescence intensity of a labeled oligonucleotide probe (RepF) on displacement of its complementary quencher oligonucleotide (RepQ) by the H1:H2 duplex. ( b ) Design of T7 RNA polymerase-driven transcription templates for enzymatic synthesis of RNA CHA circuit components with precise 5′- and 3′-ends. Transcription template for each component, H1, H2 and C1, is flanked on both the left (L) and the right (R) sides by hammerhead ribozymes (HRz). The size (in nucleotides) of each component and its ribozyme flanks is indicated under each schematic. Secondary structures of the resulting chimeric RNA at 42°C before ribozyme processing are depicted (green, A; blue, C; black, G; red, U). The RNA structures were generated using NUPACK ( 13–16 ).

    Techniques Used: Sequencing, Fluorescence, Labeling, Generated

    Application of RNA CHA circuit as an OR logic processor. ( a ) Schematic of RNA CHA circuit operation in response to either catalyst C1 OR C2. The RNA hairpin H1B serves as the OR gate, and circuit output is measured fluorimetrically using Spinach.ST1 RNA aptamer beacon. ( b ) Circuit components (H1B and H2 RNA hairpins), reporter RNA (Spinach.ST1) and the inputs C1 and C2 were transcribed from 500 ng of duplex DNA transcription templates using T7 RNA polymerase. Transcription templates were prepared using the same procedure as Figure 8 . Following filtration through Sephadex G25, 3 µl/transcript (or 1.5 µl each of C1 and C2 when added together in a reaction) was mixed in the indicated combinations in 1× TNaK buffer containing 70 µM DFHBI and 20 U of RNaseOUT. Circuits were operated at 37°C, and outputs were measured fluorimetrically.
    Figure Legend Snippet: Application of RNA CHA circuit as an OR logic processor. ( a ) Schematic of RNA CHA circuit operation in response to either catalyst C1 OR C2. The RNA hairpin H1B serves as the OR gate, and circuit output is measured fluorimetrically using Spinach.ST1 RNA aptamer beacon. ( b ) Circuit components (H1B and H2 RNA hairpins), reporter RNA (Spinach.ST1) and the inputs C1 and C2 were transcribed from 500 ng of duplex DNA transcription templates using T7 RNA polymerase. Transcription templates were prepared using the same procedure as Figure 8 . Following filtration through Sephadex G25, 3 µl/transcript (or 1.5 µl each of C1 and C2 when added together in a reaction) was mixed in the indicated combinations in 1× TNaK buffer containing 70 µM DFHBI and 20 U of RNaseOUT. Circuits were operated at 37°C, and outputs were measured fluorimetrically.

    Techniques Used: Filtration

    Cotranscriptional RNA CHA and circuit design optimization for cotranscription. ( a ) Cotranscribed RNA circuit components undergo catalyzed hairpin assembly without requiring gel purification of individual reactants. Fifty nanograms each of H1 and H2 transcription templates, along with titrating amounts of C1 transcription template, was cotranscribed for 1 h at 42°C using T7 RNA polymerase followed by passage through Illustra MicroSpin Sephadex G25 columns. Transcription templates were amplified from cloned inserts using primers pCR2.1.F and pCR2.1.R specific to plasmid sequences flanking the inserts. Two microliter aliquots of the cotranscribed RNA mixtures were then incubated in 15 µl of volume with 400 nM RepF annealed with 5× excess (2 µM) RepQ fluorescent DNA reporter duplex in 1× TNaK buffer containing 20 U of RNaseOUT and 0.5 µM ROX reference dye to quantitate formation of H1:H2 RNA duplexes at 52°C. Average data from triplicate experiments are represented. ( b and c ) Schematic depicting sequences of RNA hairpins H1 and H2 with one- or two-base engineered mismatches. Mismatched H1 (mH1) presents a two-base mismatch between its domain 4* and domain 4 of H2. The hairpins mAH1 and mGH1 each contain a single mismatched base between their domain 4* and the domain 4 of H2. The mutated H2 hairpin m2H2 presents two mismatched bases between its domain 2* and the H1 domain 2.
    Figure Legend Snippet: Cotranscriptional RNA CHA and circuit design optimization for cotranscription. ( a ) Cotranscribed RNA circuit components undergo catalyzed hairpin assembly without requiring gel purification of individual reactants. Fifty nanograms each of H1 and H2 transcription templates, along with titrating amounts of C1 transcription template, was cotranscribed for 1 h at 42°C using T7 RNA polymerase followed by passage through Illustra MicroSpin Sephadex G25 columns. Transcription templates were amplified from cloned inserts using primers pCR2.1.F and pCR2.1.R specific to plasmid sequences flanking the inserts. Two microliter aliquots of the cotranscribed RNA mixtures were then incubated in 15 µl of volume with 400 nM RepF annealed with 5× excess (2 µM) RepQ fluorescent DNA reporter duplex in 1× TNaK buffer containing 20 U of RNaseOUT and 0.5 µM ROX reference dye to quantitate formation of H1:H2 RNA duplexes at 52°C. Average data from triplicate experiments are represented. ( b and c ) Schematic depicting sequences of RNA hairpins H1 and H2 with one- or two-base engineered mismatches. Mismatched H1 (mH1) presents a two-base mismatch between its domain 4* and domain 4 of H2. The hairpins mAH1 and mGH1 each contain a single mismatched base between their domain 4* and the domain 4 of H2. The mutated H2 hairpin m2H2 presents two mismatched bases between its domain 2* and the H1 domain 2.

    Techniques Used: Gel Purification, Amplification, Clone Assay, Plasmid Preparation, Incubation

    An entirely RNA-based CHA circuit operation and fluorimetric detection. ( a ) CHA circuit components (hairpins H1B and H2 and catalyst C1) and the RNA reporter Spinach.ST1 were separately transcribed by T7 RNA polymerase from 500 ng of PCR-generated duplex DNA transcription templates. H1B, H2 and C1 transcription templates were amplified using primers complementary to the exact ends of the cloned inserts (H1B.amp.F:H1B.amp.R, H2.amp.F:H2.amp.R and C1.amp.F:C1.amp.R, respectively) rather than the flanking plasmid. Spinach.ST transcription templates were amplified using primers specific to the flanking plasmid sequence at the 5′-end (pCR2.1.F) and the primer sphT.U.R specific to the 3′-end sequence of Spinach.ST. Transcription reactions were filtered through Sephadex G25 columns before circuit assembly. Three microliters of H1B, H2, C1 and Spinach.ST1 transcripts was mixed in indicated combinations and incubated in 1× TNaK buffer containing 70 µM DFHBI and 20 U of RNaseOUT. Circuit output was measured as increasing fluorescence intensity over time at 37°C. ( b–d ) Performance of DNA reporter duplex H1BF:H1BQ (b) versus Spinach.ST1 (c) in measuring RNA CHA circuit output. Indicated concentrations of gel-purified RNA hairpins H1B and H2 were incubated with equal concentration of H1BF:H1BQ or gel-purified Spinach.ST1 (+ 70 µM DFHBI) in the presence of titrating concentrations of pure C1 RNA. All circuits were operated in 1× TNaK buffer containing 20 U of RNaseOUT at 37°C, and average data from triplicate experiments are represented. Signal-to-noise ratio of H1BF:H1BQ versus Spinach.ST1 over the time course of RNA CHA detection is plotted in (d).
    Figure Legend Snippet: An entirely RNA-based CHA circuit operation and fluorimetric detection. ( a ) CHA circuit components (hairpins H1B and H2 and catalyst C1) and the RNA reporter Spinach.ST1 were separately transcribed by T7 RNA polymerase from 500 ng of PCR-generated duplex DNA transcription templates. H1B, H2 and C1 transcription templates were amplified using primers complementary to the exact ends of the cloned inserts (H1B.amp.F:H1B.amp.R, H2.amp.F:H2.amp.R and C1.amp.F:C1.amp.R, respectively) rather than the flanking plasmid. Spinach.ST transcription templates were amplified using primers specific to the flanking plasmid sequence at the 5′-end (pCR2.1.F) and the primer sphT.U.R specific to the 3′-end sequence of Spinach.ST. Transcription reactions were filtered through Sephadex G25 columns before circuit assembly. Three microliters of H1B, H2, C1 and Spinach.ST1 transcripts was mixed in indicated combinations and incubated in 1× TNaK buffer containing 70 µM DFHBI and 20 U of RNaseOUT. Circuit output was measured as increasing fluorescence intensity over time at 37°C. ( b–d ) Performance of DNA reporter duplex H1BF:H1BQ (b) versus Spinach.ST1 (c) in measuring RNA CHA circuit output. Indicated concentrations of gel-purified RNA hairpins H1B and H2 were incubated with equal concentration of H1BF:H1BQ or gel-purified Spinach.ST1 (+ 70 µM DFHBI) in the presence of titrating concentrations of pure C1 RNA. All circuits were operated in 1× TNaK buffer containing 20 U of RNaseOUT at 37°C, and average data from triplicate experiments are represented. Signal-to-noise ratio of H1BF:H1BQ versus Spinach.ST1 over the time course of RNA CHA detection is plotted in (d).

    Techniques Used: Polymerase Chain Reaction, Generated, Amplification, Clone Assay, Plasmid Preparation, Sequencing, Incubation, Fluorescence, Purification, Concentration Assay

    39) Product Images from "A specific, promoter-independent activity of T7 RNA polymerase suggests a general model for DNA/RNA editing in single subunit RNA Polymerases"

    Article Title: A specific, promoter-independent activity of T7 RNA polymerase suggests a general model for DNA/RNA editing in single subunit RNA Polymerases

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-32231-6

    Specificity of oligonucleotide labeling by T7 RNA polymerase. ( A ) Autoradiograph of 15% polyacrylamide gel which shows 3′ end labeling by T7 RNAP on DNA oligonucleotides (36mer) in the presence of the α- 32 P GTP (right two lanes) or α- 32 P ATP (left two lanes). ( B ) The sequence and potential looped structure of the DNA oligonucleotides labeled in ( A ).
    Figure Legend Snippet: Specificity of oligonucleotide labeling by T7 RNA polymerase. ( A ) Autoradiograph of 15% polyacrylamide gel which shows 3′ end labeling by T7 RNAP on DNA oligonucleotides (36mer) in the presence of the α- 32 P GTP (right two lanes) or α- 32 P ATP (left two lanes). ( B ) The sequence and potential looped structure of the DNA oligonucleotides labeled in ( A ).

    Techniques Used: Oligonucleotide Labeling, Autoradiography, End Labeling, Sequencing, Labeling

    40) Product Images from "A hybrid G-quadruplex structure formed between RNA and DNA explains the extraordinary stability of the mitochondrial R-loop"

    Article Title: A hybrid G-quadruplex structure formed between RNA and DNA explains the extraordinary stability of the mitochondrial R-loop

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gks802

    A longer R-loop can form during transcription of human mtDNA with phage T7 RNA polymerase. ( A ) Transcription of a human mtDNA template containing wildtype or mutant CSB II with T7 RNA pol in the presence of either GTP (lanes 1–3 and 7–9) or 7-deaza-GTP (lanes 4–6 and 10–12). A hybrid species of ∼120 bp is revealed upon RNase A treatment (lane 2) and is sensitive to hRNaseH1 (lane 3). This longer hybrid is dependent on CSB II and not observed in the presence of 7-deaza-GTP (lanes 4–6). ( B ) Schematic presentation of the RNA–DNA hybrid G-quadruplex that forms between the RNA and the non-template DNA strand during transcription of mtDNA. Under some conditions, an extended R-loop may be formed, similar to that reported in ( 22 ).
    Figure Legend Snippet: A longer R-loop can form during transcription of human mtDNA with phage T7 RNA polymerase. ( A ) Transcription of a human mtDNA template containing wildtype or mutant CSB II with T7 RNA pol in the presence of either GTP (lanes 1–3 and 7–9) or 7-deaza-GTP (lanes 4–6 and 10–12). A hybrid species of ∼120 bp is revealed upon RNase A treatment (lane 2) and is sensitive to hRNaseH1 (lane 3). This longer hybrid is dependent on CSB II and not observed in the presence of 7-deaza-GTP (lanes 4–6). ( B ) Schematic presentation of the RNA–DNA hybrid G-quadruplex that forms between the RNA and the non-template DNA strand during transcription of mtDNA. Under some conditions, an extended R-loop may be formed, similar to that reported in ( 22 ).

    Techniques Used: Mutagenesis

    Related Articles

    Amplification:

    Article Title: The Hepatitis C Virus RNA 3?-Untranslated Region Strongly Enhances Translation Directed by the Internal Ribosome Entry Site ▿
    Article Snippet: .. The amplified DNA fragments were gel purified, extracted with phenol-chloroform, ethanol precipitated, dissolved in water, and used for transcription using T7 RNA polymerase (New England Biolabs). pRL-FMDV-wt-FL was linearized with XbaI downstream of the RLuc sequence and transcribed with T7 RNA polymerase to obtain RLuc control RNA for cotransfections. .. Reactions were treated with RNase-free DNase I to digest DNA templates, and RNA was purified with RNeasy kits (QIAGEN).

    In Vitro:

    Article Title: A Reverse Genetics System for Zika Virus Based on a Simple Molecular Cloning Strategy
    Article Snippet: .. The in vitro transcription reaction was carried out with 10 μg of linearized plasmid DNA in a total volume of 100 μL containing 20 μL 5× RRL buffer (400 mM HEPES (pH 7.5), 60 mM MgCl2 , 10 mM spermidine and 200 mM DTT), NTP-Mix (3.125 mM ATP, CTP and UTP and 1.56 mM GTP), 1 U/μL RNasin (Promega, Madison, WI, USA), 2 U/μL T7 RNA polymerase (New England Biolabs) and 1 mM anti-reverse cap analogue (ARCA; 3′-O-Me- m7G(5′)ppp(5′)G; New England Biolabs). .. After incubation at 37 °C for 2.5 h, 1 U/μL T7 RNA polymerase was added followed by additional 2.5 h incubation at 37 °C.

    Article Title: Filamentation and restoration of normal growth in Escherichia coli using a combined CRISPRi sgRNA/antisense RNA approach
    Article Snippet: .. Anti-sgRNA and sgRNA where transcribed in vitro by T7 RNA Polymerase (NEB) from linear DNA (IDT) overnight and then extracted with Phenol-Chloroform. .. The concentration of the RNA was determined by comparing a SYBR Green II stained band in a denaturing PAGE (8M Urea at 45°C) to the RNA Ladder (NEB, N0364S).

    Synthesized:

    Article Title: Protein Expression Redirects Vesicular Stomatitis Virus RNA Synthesis to Cytoplasmic Inclusions
    Article Snippet: .. As a control, transcripts were also synthesized by T7 RNA polymerase (New England Biolabs, Beverly MA) using the previously described VSV expression plasmid pN . .. Incorporation of BrUTP into RNA in cells Approximately 30,000 BSR-T7 cells grown on cover slips in 24 well plates were infected with VSV at the specified MOI (3–500).

    Purification:

    Article Title: Native purification and labeling of RNA for single molecule fluorescence studies
    Article Snippet: .. 3 Plasmids encoding for the T7 RNA polymerase gene for protein overexpression and purification are available ( ) or the enzyme is available for purchase through New England Biolabs. ..

    Article Title: The Hepatitis C Virus RNA 3?-Untranslated Region Strongly Enhances Translation Directed by the Internal Ribosome Entry Site ▿
    Article Snippet: .. The amplified DNA fragments were gel purified, extracted with phenol-chloroform, ethanol precipitated, dissolved in water, and used for transcription using T7 RNA polymerase (New England Biolabs). pRL-FMDV-wt-FL was linearized with XbaI downstream of the RLuc sequence and transcribed with T7 RNA polymerase to obtain RLuc control RNA for cotransfections. .. Reactions were treated with RNase-free DNase I to digest DNA templates, and RNA was purified with RNeasy kits (QIAGEN).

    Incubation:

    Article Title: A Reverse Genetics System for Zika Virus Based on a Simple Molecular Cloning Strategy
    Article Snippet: .. After incubation at 37 °C for 2.5 h, 1 U/μL T7 RNA polymerase was added followed by additional 2.5 h incubation at 37 °C. .. DNA was digested with DNaseI for one hour and RNA was purified by acidic phenol-chloroform extraction and isopropanol precipitation.

    Expressing:

    Article Title: Protein Expression Redirects Vesicular Stomatitis Virus RNA Synthesis to Cytoplasmic Inclusions
    Article Snippet: .. As a control, transcripts were also synthesized by T7 RNA polymerase (New England Biolabs, Beverly MA) using the previously described VSV expression plasmid pN . .. Incorporation of BrUTP into RNA in cells Approximately 30,000 BSR-T7 cells grown on cover slips in 24 well plates were infected with VSV at the specified MOI (3–500).

    Sequencing:

    Article Title: The Hepatitis C Virus RNA 3?-Untranslated Region Strongly Enhances Translation Directed by the Internal Ribosome Entry Site ▿
    Article Snippet: .. The amplified DNA fragments were gel purified, extracted with phenol-chloroform, ethanol precipitated, dissolved in water, and used for transcription using T7 RNA polymerase (New England Biolabs). pRL-FMDV-wt-FL was linearized with XbaI downstream of the RLuc sequence and transcribed with T7 RNA polymerase to obtain RLuc control RNA for cotransfections. .. Reactions were treated with RNase-free DNase I to digest DNA templates, and RNA was purified with RNeasy kits (QIAGEN).

    Recombinant:

    Article Title: RNA primer–primase complexes serve as the signal for polymerase recycling and Okazaki fragment initiation in T4 phage DNA replication
    Article Snippet: .. The T7 RNA transcription was performed overnight at 37 °C in a reaction mixture containing 3.3 μM dsDNA template, 7.5 mM of each rNTP, 34 mM MgCl2 , 1 U/μL Recombinant RNasin Ribonuclease Inhibitor (Promega), 10 mM DTT, 0.1 U/μL inorganic pyrophosphatase, and 10 U/μL T7 RNA polymerase (New England Biolabs) in 1× RNAPol Reaction Buffer (40 mM Tris⋅HCl, pH 7.9, 6 mM MgCl2 , 2 mM spermidine, 10 mM DTT). ..

    Over Expression:

    Article Title: Native purification and labeling of RNA for single molecule fluorescence studies
    Article Snippet: .. 3 Plasmids encoding for the T7 RNA polymerase gene for protein overexpression and purification are available ( ) or the enzyme is available for purchase through New England Biolabs. ..

    Plasmid Preparation:

    Article Title: Protein Expression Redirects Vesicular Stomatitis Virus RNA Synthesis to Cytoplasmic Inclusions
    Article Snippet: .. As a control, transcripts were also synthesized by T7 RNA polymerase (New England Biolabs, Beverly MA) using the previously described VSV expression plasmid pN . .. Incorporation of BrUTP into RNA in cells Approximately 30,000 BSR-T7 cells grown on cover slips in 24 well plates were infected with VSV at the specified MOI (3–500).

    Article Title: A Reverse Genetics System for Zika Virus Based on a Simple Molecular Cloning Strategy
    Article Snippet: .. The in vitro transcription reaction was carried out with 10 μg of linearized plasmid DNA in a total volume of 100 μL containing 20 μL 5× RRL buffer (400 mM HEPES (pH 7.5), 60 mM MgCl2 , 10 mM spermidine and 200 mM DTT), NTP-Mix (3.125 mM ATP, CTP and UTP and 1.56 mM GTP), 1 U/μL RNasin (Promega, Madison, WI, USA), 2 U/μL T7 RNA polymerase (New England Biolabs) and 1 mM anti-reverse cap analogue (ARCA; 3′-O-Me- m7G(5′)ppp(5′)G; New England Biolabs). .. After incubation at 37 °C for 2.5 h, 1 U/μL T7 RNA polymerase was added followed by additional 2.5 h incubation at 37 °C.

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    New England Biolabs t7 rna polymerase
    Construction and stability of synthetic full length Zika virus (synZIKV) cDNA clones. ( A ) Schematic representation of the synZIKV MR766 construct and the four fragments used to assemble the genome. The 5′ and 3′UTRs are indicated with bold black lines, the promoter for the <t>T7</t> RNA polymerase with a black arrow. Restriction sites used for the assembly of the fragments are indicated. An enlargement of fragment #1 is shown below with putative CEPs (score > 0.85) indicated by red arrow heads. CEP 1 was not mutated (indicated with the pink arrow head). ( B ) Same as in panel ( A ) but for synZIKV-H/PF/2013. ( C ) Restriction patterns of pFK-synZIKV constructs obtained after digest with EcoRI (MR766) or XmnI (H/PF/2013) and agarose gel electrophoresis. Plasmids were analysed directly after assembly (original prep) and after five passages (P5) in E. coli (five DNA clones of P5 are shown).
    T7 Rna Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 42 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs dnase i
    TNA SELEX to generate OTA-binding aptamers. The initial ssDNA library is amplified using a forward primer modified with a PEG spacer and polyT tail to enable separation and recovery by denaturing PAGE. The PEGylated DNA template is then annealed to the FAM-labelled TNA primer and extended using KOD RI polymerase to generate the TNA library for each selection round. The TNA library is incubated with OTA-functionalized magnetic beads, and bound sequences recovered by either heat (rounds 1–4) or ligand elution (rounds 5–9). These sequences are then treated with <t>DNase</t> I to digest any remaining DNA template. The TNA is then reverse transcribed back into DNA using Bst DNA polymerase and PCR amplified for the next round of selection.
    Dnase I, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs t7 rnap
    Single-molecule fluorescence cotranscriptional folding assay. ( A ) Model for TPP-induced structural transition of the E. coli ThiM riboswitch. The fluorophore-labeling positions for single-molecule studies are indicated by green and red boxes (green for Cy3 or Dy547, red for Dy647). ( B ) Experimental scheme. Dy647-labeled seed RNA was incubated with a template DNA strand and phage <t>T7</t> RNAP in a tube for 50 min at 37 °C. To assemble a full EC, Cy3-labeled UTP and a nontemplate DNA strand were added to the tube and incubated for 20 min. ECs were immobilized on a polymer-passivated quartz surface using streptavidin–biotin interactions. Elongation was resumed by injecting NTP, while RNA folding was observed using a single-molecule FRET microscope. ( C ) Single-molecule fluorescence images of EC ( Top ) and control (ctrl) images of nonspecifically bound Cy3-UTP ( Bottom ). The colocalized Cy3 and Cy5 spots are enclosed by circles. The percentage of acceptor spots colocalized with donor spots was estimated as 57 ± 14% from 10 measurements.
    T7 Rnap, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Construction and stability of synthetic full length Zika virus (synZIKV) cDNA clones. ( A ) Schematic representation of the synZIKV MR766 construct and the four fragments used to assemble the genome. The 5′ and 3′UTRs are indicated with bold black lines, the promoter for the T7 RNA polymerase with a black arrow. Restriction sites used for the assembly of the fragments are indicated. An enlargement of fragment #1 is shown below with putative CEPs (score > 0.85) indicated by red arrow heads. CEP 1 was not mutated (indicated with the pink arrow head). ( B ) Same as in panel ( A ) but for synZIKV-H/PF/2013. ( C ) Restriction patterns of pFK-synZIKV constructs obtained after digest with EcoRI (MR766) or XmnI (H/PF/2013) and agarose gel electrophoresis. Plasmids were analysed directly after assembly (original prep) and after five passages (P5) in E. coli (five DNA clones of P5 are shown).

    Journal: Viruses

    Article Title: A Reverse Genetics System for Zika Virus Based on a Simple Molecular Cloning Strategy

    doi: 10.3390/v10070368

    Figure Lengend Snippet: Construction and stability of synthetic full length Zika virus (synZIKV) cDNA clones. ( A ) Schematic representation of the synZIKV MR766 construct and the four fragments used to assemble the genome. The 5′ and 3′UTRs are indicated with bold black lines, the promoter for the T7 RNA polymerase with a black arrow. Restriction sites used for the assembly of the fragments are indicated. An enlargement of fragment #1 is shown below with putative CEPs (score > 0.85) indicated by red arrow heads. CEP 1 was not mutated (indicated with the pink arrow head). ( B ) Same as in panel ( A ) but for synZIKV-H/PF/2013. ( C ) Restriction patterns of pFK-synZIKV constructs obtained after digest with EcoRI (MR766) or XmnI (H/PF/2013) and agarose gel electrophoresis. Plasmids were analysed directly after assembly (original prep) and after five passages (P5) in E. coli (five DNA clones of P5 are shown).

    Article Snippet: After incubation at 37 °C for 2.5 h, 1 U/μL T7 RNA polymerase was added followed by additional 2.5 h incubation at 37 °C.

    Techniques: Clone Assay, Construct, Agarose Gel Electrophoresis

    CRISPRi-based growth control. (A) Schematic representation of E . coli switching into filamentous growth after induction of single guide RNA (sgRNA) via IPTG and dCas9 via aTc. The dCas9-sgRNA complex blocks the expression of FtsZ, stopping the formation of the septal ring that is essential for cell division in E . coli . Cell division can be rescued by inducing appropriate antisense sgRNAs (‘anti-sgRNA’) with AHL and by removing the inducers for the dCas9 and sgRNA. (B) Scheme of sgRNA forming a complex with dCas9. The anti-sgRNA can inhibit formation of the complex by binding to the sgRNA. (C) Details of the genetic constructs involved: the CRISPRi plasmid codes for dCas9 under aTc-inducible promoters and three different sgRNAs under T7 promoters which target three different promoters of the ftsZ gene on the genome of the E . coli . T7 RNA polymerase is inducible with IPTG. The ‘anti-sgRNA plasmid’ codes for anti-sgRNAs under the control of an AHL-inducible promoter. The sponge elements on the plasmids act as decoy binding sites for the corresponding dCas9-sgRNA complexes.

    Journal: PLoS ONE

    Article Title: Filamentation and restoration of normal growth in Escherichia coli using a combined CRISPRi sgRNA/antisense RNA approach

    doi: 10.1371/journal.pone.0198058

    Figure Lengend Snippet: CRISPRi-based growth control. (A) Schematic representation of E . coli switching into filamentous growth after induction of single guide RNA (sgRNA) via IPTG and dCas9 via aTc. The dCas9-sgRNA complex blocks the expression of FtsZ, stopping the formation of the septal ring that is essential for cell division in E . coli . Cell division can be rescued by inducing appropriate antisense sgRNAs (‘anti-sgRNA’) with AHL and by removing the inducers for the dCas9 and sgRNA. (B) Scheme of sgRNA forming a complex with dCas9. The anti-sgRNA can inhibit formation of the complex by binding to the sgRNA. (C) Details of the genetic constructs involved: the CRISPRi plasmid codes for dCas9 under aTc-inducible promoters and three different sgRNAs under T7 promoters which target three different promoters of the ftsZ gene on the genome of the E . coli . T7 RNA polymerase is inducible with IPTG. The ‘anti-sgRNA plasmid’ codes for anti-sgRNAs under the control of an AHL-inducible promoter. The sponge elements on the plasmids act as decoy binding sites for the corresponding dCas9-sgRNA complexes.

    Article Snippet: Anti-sgRNA and sgRNA where transcribed in vitro by T7 RNA Polymerase (NEB) from linear DNA (IDT) overnight and then extracted with Phenol-Chloroform.

    Techniques: Expressing, Binding Assay, Construct, Plasmid Preparation, Activated Clotting Time Assay

    TNA SELEX to generate OTA-binding aptamers. The initial ssDNA library is amplified using a forward primer modified with a PEG spacer and polyT tail to enable separation and recovery by denaturing PAGE. The PEGylated DNA template is then annealed to the FAM-labelled TNA primer and extended using KOD RI polymerase to generate the TNA library for each selection round. The TNA library is incubated with OTA-functionalized magnetic beads, and bound sequences recovered by either heat (rounds 1–4) or ligand elution (rounds 5–9). These sequences are then treated with DNase I to digest any remaining DNA template. The TNA is then reverse transcribed back into DNA using Bst DNA polymerase and PCR amplified for the next round of selection.

    Journal: Nucleic Acids Research

    Article Title: In vitro selection of an XNA aptamer capable of small-molecule recognition

    doi: 10.1093/nar/gky667

    Figure Lengend Snippet: TNA SELEX to generate OTA-binding aptamers. The initial ssDNA library is amplified using a forward primer modified with a PEG spacer and polyT tail to enable separation and recovery by denaturing PAGE. The PEGylated DNA template is then annealed to the FAM-labelled TNA primer and extended using KOD RI polymerase to generate the TNA library for each selection round. The TNA library is incubated with OTA-functionalized magnetic beads, and bound sequences recovered by either heat (rounds 1–4) or ligand elution (rounds 5–9). These sequences are then treated with DNase I to digest any remaining DNA template. The TNA is then reverse transcribed back into DNA using Bst DNA polymerase and PCR amplified for the next round of selection.

    Article Snippet: TNA stability assay FAM-labeled DNA aptamer A08 and TNA aptamer A04T.2 were prepared in three nuclease challenge conditions: 1.5 U DNase I (RNase-free, New England Biolabs) in 1× DNase I reaction buffer (10 mM Tris–HCl, 2.5 mM MgCl2 , 0.5 mM CaCl2 , pH 7.6), 50% (v/v) human blood serum (normal pool, Fisher BioReagents) in 1× Dulbecco's PBS, 0.5 mg/ml pooled human liver microsomes (XenoTech) in 1× selection buffer.

    Techniques: Binding Assay, Amplification, Modification, Polyacrylamide Gel Electrophoresis, Selection, Incubation, Magnetic Beads, Polymerase Chain Reaction

    Comparison of the biostability of FAM-labeled TNA aptamer A04T.2 and DNA aptamer A08. ( A ) Denaturing PAGE analysis of the TNA (T) and DNA (D) aptamers after incubation in conditions of increasing nuclease stringency: selection buffer (control), 1.5 U DNase I, 50% human blood serum in PBS, and 0.5 mg/mL human liver microsomes. Samples were incubated under these conditions for 3 days at 37°C. ( B ) Bead-binding assay to determine retention of aptamer binding in the presence of nucleases. Each column and error bar represents the average and standard deviation of two trials.

    Journal: Nucleic Acids Research

    Article Title: In vitro selection of an XNA aptamer capable of small-molecule recognition

    doi: 10.1093/nar/gky667

    Figure Lengend Snippet: Comparison of the biostability of FAM-labeled TNA aptamer A04T.2 and DNA aptamer A08. ( A ) Denaturing PAGE analysis of the TNA (T) and DNA (D) aptamers after incubation in conditions of increasing nuclease stringency: selection buffer (control), 1.5 U DNase I, 50% human blood serum in PBS, and 0.5 mg/mL human liver microsomes. Samples were incubated under these conditions for 3 days at 37°C. ( B ) Bead-binding assay to determine retention of aptamer binding in the presence of nucleases. Each column and error bar represents the average and standard deviation of two trials.

    Article Snippet: TNA stability assay FAM-labeled DNA aptamer A08 and TNA aptamer A04T.2 were prepared in three nuclease challenge conditions: 1.5 U DNase I (RNase-free, New England Biolabs) in 1× DNase I reaction buffer (10 mM Tris–HCl, 2.5 mM MgCl2 , 0.5 mM CaCl2 , pH 7.6), 50% (v/v) human blood serum (normal pool, Fisher BioReagents) in 1× Dulbecco's PBS, 0.5 mg/ml pooled human liver microsomes (XenoTech) in 1× selection buffer.

    Techniques: Labeling, Polyacrylamide Gel Electrophoresis, Incubation, Selection, Binding Assay, Standard Deviation

    Single-molecule fluorescence cotranscriptional folding assay. ( A ) Model for TPP-induced structural transition of the E. coli ThiM riboswitch. The fluorophore-labeling positions for single-molecule studies are indicated by green and red boxes (green for Cy3 or Dy547, red for Dy647). ( B ) Experimental scheme. Dy647-labeled seed RNA was incubated with a template DNA strand and phage T7 RNAP in a tube for 50 min at 37 °C. To assemble a full EC, Cy3-labeled UTP and a nontemplate DNA strand were added to the tube and incubated for 20 min. ECs were immobilized on a polymer-passivated quartz surface using streptavidin–biotin interactions. Elongation was resumed by injecting NTP, while RNA folding was observed using a single-molecule FRET microscope. ( C ) Single-molecule fluorescence images of EC ( Top ) and control (ctrl) images of nonspecifically bound Cy3-UTP ( Bottom ). The colocalized Cy3 and Cy5 spots are enclosed by circles. The percentage of acceptor spots colocalized with donor spots was estimated as 57 ± 14% from 10 measurements.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Single-molecule FRET studies on the cotranscriptional folding of a thiamine pyrophosphate riboswitch

    doi: 10.1073/pnas.1712983115

    Figure Lengend Snippet: Single-molecule fluorescence cotranscriptional folding assay. ( A ) Model for TPP-induced structural transition of the E. coli ThiM riboswitch. The fluorophore-labeling positions for single-molecule studies are indicated by green and red boxes (green for Cy3 or Dy547, red for Dy647). ( B ) Experimental scheme. Dy647-labeled seed RNA was incubated with a template DNA strand and phage T7 RNAP in a tube for 50 min at 37 °C. To assemble a full EC, Cy3-labeled UTP and a nontemplate DNA strand were added to the tube and incubated for 20 min. ECs were immobilized on a polymer-passivated quartz surface using streptavidin–biotin interactions. Elongation was resumed by injecting NTP, while RNA folding was observed using a single-molecule FRET microscope. ( C ) Single-molecule fluorescence images of EC ( Top ) and control (ctrl) images of nonspecifically bound Cy3-UTP ( Bottom ). The colocalized Cy3 and Cy5 spots are enclosed by circles. The percentage of acceptor spots colocalized with donor spots was estimated as 57 ± 14% from 10 measurements.

    Article Snippet: The seed RNA (800 nM) was incubated in a tube with a template DNA strand (200 nM) and T7 RNAP (40 nM; New England Biolabs) for 50 min at 37 °C.

    Techniques: Fluorescence, Labeling, Incubation, Microscopy