t4 pnk  (New England Biolabs)


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    Structured Review

    New England Biolabs t4 pnk
    Overview of metabarcoding and library preparation steps and formation of tag-jumps in a typical ‘shotgun’ Illumina library protocol and our presented Tagsteady library protocol. 1) Metabarcoding PCR with 5’ nucleotide tagged primers. To allow detection of tag-jumps, only unique twin-tag combinations is visualised. Following pooling of PCR reactions, differently tagged single-stranded amplicons can form heteroduplexes with non-complementary tag overhangs. 2) In a typical ‘shotgun’ Illumina library protocol (left), T4 DNA polymerase is used for blunt-ending, <t>T4</t> PNK for 5’ phosphorylation and Taq polymerase for 3’ adenylation. In this type of end-repair, 3’ overhangs (in heteroduplexes) will become substrate for the 3’→5’ exonuclease activity of T4 DNA Polymerase. The opposite strand, the 5’ overhangs (i.e. the inherent tag), will then act as a template for extension, causing the tag to ‘jump’ from one strand to the other (asterisk) (see van Orsouw et al. 2007 ; Schnell, Bohmann, and Gilbert 2015 ). The Tagsteady end-repair (right) only contains T4 PNK and Klenow exo- (thus no exonuclease activity) and therefore tag-jumps cannot arise. 3) After end repair, T4 DNA Ligase is used to ligate Illumina sequencing adapters (here depicted as Illumina Y-shaped adapters). 4) Often post-ligation PCR is carried out, causing further tag-jumps as a result of incomplete primer extension. Post-ligation PCR is not necessary with the Tagsteady protocol as it uses PCR-free full length adapters. 5) Sequencing of libraries on an Illumina sequencing platform. 6) Following initial sequence read processing, sequences within each amplicon library are sorted according to primer and tag sequences to assess levels of sequences carrying new combinations of used tags (tag-jumps).
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    Images

    1) Product Images from "Tagsteady: a metabarcoding library preparation protocol to avoid false assignment of sequences to samples"

    Article Title: Tagsteady: a metabarcoding library preparation protocol to avoid false assignment of sequences to samples

    Journal: bioRxiv

    doi: 10.1101/2020.01.22.915009

    Overview of metabarcoding and library preparation steps and formation of tag-jumps in a typical ‘shotgun’ Illumina library protocol and our presented Tagsteady library protocol. 1) Metabarcoding PCR with 5’ nucleotide tagged primers. To allow detection of tag-jumps, only unique twin-tag combinations is visualised. Following pooling of PCR reactions, differently tagged single-stranded amplicons can form heteroduplexes with non-complementary tag overhangs. 2) In a typical ‘shotgun’ Illumina library protocol (left), T4 DNA polymerase is used for blunt-ending, T4 PNK for 5’ phosphorylation and Taq polymerase for 3’ adenylation. In this type of end-repair, 3’ overhangs (in heteroduplexes) will become substrate for the 3’→5’ exonuclease activity of T4 DNA Polymerase. The opposite strand, the 5’ overhangs (i.e. the inherent tag), will then act as a template for extension, causing the tag to ‘jump’ from one strand to the other (asterisk) (see van Orsouw et al. 2007 ; Schnell, Bohmann, and Gilbert 2015 ). The Tagsteady end-repair (right) only contains T4 PNK and Klenow exo- (thus no exonuclease activity) and therefore tag-jumps cannot arise. 3) After end repair, T4 DNA Ligase is used to ligate Illumina sequencing adapters (here depicted as Illumina Y-shaped adapters). 4) Often post-ligation PCR is carried out, causing further tag-jumps as a result of incomplete primer extension. Post-ligation PCR is not necessary with the Tagsteady protocol as it uses PCR-free full length adapters. 5) Sequencing of libraries on an Illumina sequencing platform. 6) Following initial sequence read processing, sequences within each amplicon library are sorted according to primer and tag sequences to assess levels of sequences carrying new combinations of used tags (tag-jumps).
    Figure Legend Snippet: Overview of metabarcoding and library preparation steps and formation of tag-jumps in a typical ‘shotgun’ Illumina library protocol and our presented Tagsteady library protocol. 1) Metabarcoding PCR with 5’ nucleotide tagged primers. To allow detection of tag-jumps, only unique twin-tag combinations is visualised. Following pooling of PCR reactions, differently tagged single-stranded amplicons can form heteroduplexes with non-complementary tag overhangs. 2) In a typical ‘shotgun’ Illumina library protocol (left), T4 DNA polymerase is used for blunt-ending, T4 PNK for 5’ phosphorylation and Taq polymerase for 3’ adenylation. In this type of end-repair, 3’ overhangs (in heteroduplexes) will become substrate for the 3’→5’ exonuclease activity of T4 DNA Polymerase. The opposite strand, the 5’ overhangs (i.e. the inherent tag), will then act as a template for extension, causing the tag to ‘jump’ from one strand to the other (asterisk) (see van Orsouw et al. 2007 ; Schnell, Bohmann, and Gilbert 2015 ). The Tagsteady end-repair (right) only contains T4 PNK and Klenow exo- (thus no exonuclease activity) and therefore tag-jumps cannot arise. 3) After end repair, T4 DNA Ligase is used to ligate Illumina sequencing adapters (here depicted as Illumina Y-shaped adapters). 4) Often post-ligation PCR is carried out, causing further tag-jumps as a result of incomplete primer extension. Post-ligation PCR is not necessary with the Tagsteady protocol as it uses PCR-free full length adapters. 5) Sequencing of libraries on an Illumina sequencing platform. 6) Following initial sequence read processing, sequences within each amplicon library are sorted according to primer and tag sequences to assess levels of sequences carrying new combinations of used tags (tag-jumps).

    Techniques Used: Polymerase Chain Reaction, Activity Assay, Sequencing, Ligation, Amplification

    2) Product Images from "Perturbation of base excision repair sensitizes breast cancer cells to APOBEC3 deaminase-mediated mutations"

    Article Title: Perturbation of base excision repair sensitizes breast cancer cells to APOBEC3 deaminase-mediated mutations

    Journal: eLife

    doi: 10.7554/eLife.51605

    Purification and activity assays of PNKP and Polβ. ( A ) Purified Polβ-His 6 (17 ng) and PNKP-His 6 (127 ng) from E. coli were subjected to PAGE and stained with Coomassie blue. ( B ) Incorporation of [α- 32 P]-dCTP by Polβ using APE1-generated product. ddC, di-deoxynucleotide; P, product. ( C ) Schematic of the preparation of S (substrate) and subsequent enzymatic reactions for testing PNKP activity. ( D ) Efficiency of oligonucleotide labeling, annealing, and ligation leading to S indicated in ( C ). ( E ) Fpg (NEB, 1 U) completely digested S and the 3’ phosphate was completely removed by PNKP (12.7 ng and 127 ng, lanes 2 and 3), or by T4 PNK (NEB, 0.1 U and 1 U, lanes 7 and 8). NEIL2 (272 ng) only partially digested S and its 3’P was resistant to the PNKP phosphatase (lanes 4 and 5).
    Figure Legend Snippet: Purification and activity assays of PNKP and Polβ. ( A ) Purified Polβ-His 6 (17 ng) and PNKP-His 6 (127 ng) from E. coli were subjected to PAGE and stained with Coomassie blue. ( B ) Incorporation of [α- 32 P]-dCTP by Polβ using APE1-generated product. ddC, di-deoxynucleotide; P, product. ( C ) Schematic of the preparation of S (substrate) and subsequent enzymatic reactions for testing PNKP activity. ( D ) Efficiency of oligonucleotide labeling, annealing, and ligation leading to S indicated in ( C ). ( E ) Fpg (NEB, 1 U) completely digested S and the 3’ phosphate was completely removed by PNKP (12.7 ng and 127 ng, lanes 2 and 3), or by T4 PNK (NEB, 0.1 U and 1 U, lanes 7 and 8). NEIL2 (272 ng) only partially digested S and its 3’P was resistant to the PNKP phosphatase (lanes 4 and 5).

    Techniques Used: Purification, Activity Assay, Polyacrylamide Gel Electrophoresis, Staining, Generated, Oligonucleotide Labeling, Ligation

    3) Product Images from "Single Nucleotide Resolution RNA–Protein Cross-Linking Mass Spectrometry: A Simple Extension of the CLIR-MS Workflow"

    Article Title: Single Nucleotide Resolution RNA–Protein Cross-Linking Mass Spectrometry: A Simple Extension of the CLIR-MS Workflow

    Journal: Analytical Chemistry

    doi: 10.1021/acs.analchem.1c02384

    Post-digest Labeling Reaction Scheme (A) The γ-phosphate of 18 O 4 -γ-ATP is transferred to the 5′-hydroxy group of a cross-linked RNA oligonucleotide. T4-PNK also catalyzes a 3′-dephosphorylation reaction. (B) Simulation of the isotopic distribution 33 of a cross-linked peptide (PEPTIDER cross-linked to a GU dinucleotide, doubly charged) for a 1:1 mixture of light and heavy ATP using single-phosphate labeling.
    Figure Legend Snippet: Post-digest Labeling Reaction Scheme (A) The γ-phosphate of 18 O 4 -γ-ATP is transferred to the 5′-hydroxy group of a cross-linked RNA oligonucleotide. T4-PNK also catalyzes a 3′-dephosphorylation reaction. (B) Simulation of the isotopic distribution 33 of a cross-linked peptide (PEPTIDER cross-linked to a GU dinucleotide, doubly charged) for a 1:1 mixture of light and heavy ATP using single-phosphate labeling.

    Techniques Used: Labeling, De-Phosphorylation Assay

    4) Product Images from "Transcriptional arrest: Escherichia coli RNA polymerase translocates backward, leaving the 3? end of the RNA intact and extruded"

    Article Title: Transcriptional arrest: Escherichia coli RNA polymerase translocates backward, leaving the 3? end of the RNA intact and extruded

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

    doi:

    Effect of arrest on the transcript arrangement in RNAP. ( A ) RNase A footprinting of the transcript in active EC 26 and in arrested EC 27 . The RNA in the complexes was internally labeled at positions +26A or +12C. Each sample was treated with RNase A and fractionated by centrifugation into soluble (s) and matrix-associated (p) fractions before gel analysis. Sequences of the transcripts are shown alongside the autoradiograms, asterisks mark positions of labeling, and arrows show major cuts introduced into the RNA (bold shaded line) by RNase A. The cylinders represent the transcript segments protected by RNAP in the active and arrested complexes. Nonfractionated samples (t) are included as controls. ( B ) 5′-Terminal phosphorylation of the transcripts. RNA-labeled EC 20 and EC 27 (lanes 6 and 4) and arrested fraction of EC 27 purified by chase (lane 2) were treated with T4 polynucleotide kinase in the presence of ATP (lanes 5, 3, and 1). The symbol (P) indicates the phosphorylated transcripts. Arrows indicate the mobility of phosphorylated and nonphosphorylated transcripts.
    Figure Legend Snippet: Effect of arrest on the transcript arrangement in RNAP. ( A ) RNase A footprinting of the transcript in active EC 26 and in arrested EC 27 . The RNA in the complexes was internally labeled at positions +26A or +12C. Each sample was treated with RNase A and fractionated by centrifugation into soluble (s) and matrix-associated (p) fractions before gel analysis. Sequences of the transcripts are shown alongside the autoradiograms, asterisks mark positions of labeling, and arrows show major cuts introduced into the RNA (bold shaded line) by RNase A. The cylinders represent the transcript segments protected by RNAP in the active and arrested complexes. Nonfractionated samples (t) are included as controls. ( B ) 5′-Terminal phosphorylation of the transcripts. RNA-labeled EC 20 and EC 27 (lanes 6 and 4) and arrested fraction of EC 27 purified by chase (lane 2) were treated with T4 polynucleotide kinase in the presence of ATP (lanes 5, 3, and 1). The symbol (P) indicates the phosphorylated transcripts. Arrows indicate the mobility of phosphorylated and nonphosphorylated transcripts.

    Techniques Used: Footprinting, Labeling, Centrifugation, Purification

    5) Product Images from "Axl-Gas6 Interaction Counteracts E1A-Mediated Cell Growth Suppression and Proapoptotic Activity"

    Article Title: Axl-Gas6 Interaction Counteracts E1A-Mediated Cell Growth Suppression and Proapoptotic Activity

    Journal: Molecular and Cellular Biology

    doi:

    (A) The 150-bp RT-PCR products representing the activation loop of the catalytic domains of various tyrosine kinases. RNA was isolated by using the TRIzol reagent. Single-stranded cDNA was synthesized with a GIBCO-BRL kit. Primer 1 was labeled with [γ- 32 P]ATP and T4 polynucleotide kinase before PCR. The RT-PCR products were analyzed by 8% PAGE. DNA was stained with ethidium bromide at 1 μg/ml. SKOV3.ip1 is a subline of the SKOV3 ovarian cancer cell line; ip1-efs is ip1 cells transfected with an E1A frameshift mutant construct. (B) Tyrosine kinase display assay showing downregulation of a tyrosine kinase, designated TK-Alu I, in E1A-transfected cells. The RT-PCR products shown in panel A were excised and eluted from the gel. The eluted DNAs of equal radioactivity were digested with the restriction enzyme Alu I and then fractionated with a 6% DNA sequencing gel. 2774 is the ovarian cancer cell line 2774-C10. (C) Tyrosine kinase display assay of bacterial clones obtained from the tyrosine kinase cDNA library screening. The TK-Alu I probe was PCR labeled and used to screen a cDNA library of tyrosine kinases. The positive clones were picked out, and the plasmid DNA was purified and subjected to PCR as described for panel A. The products of both RT-PCR from ip1-efs cells and plasmid PCR from bacterial clones were digested with Alu I and then resolved with a 6% DNA sequencing gel. The TK-Alu I band was seen in all of the plasmid PCR clones. (D) Immunoblotting analysis showing decreased level of the Axl receptor protein in E1A-expressing ip1 and 2774 cells. Western blot analysis was performed as described in Materials and Methods, and the Axl receptor was detected by treating the transblot with an anti-Axl antibody. As a gel loading control, the same blot was reprobed with an anti-α-actin antibody.
    Figure Legend Snippet: (A) The 150-bp RT-PCR products representing the activation loop of the catalytic domains of various tyrosine kinases. RNA was isolated by using the TRIzol reagent. Single-stranded cDNA was synthesized with a GIBCO-BRL kit. Primer 1 was labeled with [γ- 32 P]ATP and T4 polynucleotide kinase before PCR. The RT-PCR products were analyzed by 8% PAGE. DNA was stained with ethidium bromide at 1 μg/ml. SKOV3.ip1 is a subline of the SKOV3 ovarian cancer cell line; ip1-efs is ip1 cells transfected with an E1A frameshift mutant construct. (B) Tyrosine kinase display assay showing downregulation of a tyrosine kinase, designated TK-Alu I, in E1A-transfected cells. The RT-PCR products shown in panel A were excised and eluted from the gel. The eluted DNAs of equal radioactivity were digested with the restriction enzyme Alu I and then fractionated with a 6% DNA sequencing gel. 2774 is the ovarian cancer cell line 2774-C10. (C) Tyrosine kinase display assay of bacterial clones obtained from the tyrosine kinase cDNA library screening. The TK-Alu I probe was PCR labeled and used to screen a cDNA library of tyrosine kinases. The positive clones were picked out, and the plasmid DNA was purified and subjected to PCR as described for panel A. The products of both RT-PCR from ip1-efs cells and plasmid PCR from bacterial clones were digested with Alu I and then resolved with a 6% DNA sequencing gel. The TK-Alu I band was seen in all of the plasmid PCR clones. (D) Immunoblotting analysis showing decreased level of the Axl receptor protein in E1A-expressing ip1 and 2774 cells. Western blot analysis was performed as described in Materials and Methods, and the Axl receptor was detected by treating the transblot with an anti-Axl antibody. As a gel loading control, the same blot was reprobed with an anti-α-actin antibody.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Activation Assay, Isolation, Synthesized, Labeling, Polymerase Chain Reaction, Polyacrylamide Gel Electrophoresis, Staining, Transfection, Mutagenesis, Construct, Radioactivity, DNA Sequencing, Clone Assay, cDNA Library Assay, Plasmid Preparation, Purification, Expressing, Western Blot

    6) Product Images from "Direct and Base Excision Repair-Mediated Regulation of a GC-Rich cis-Element in Response to 5-Formylcytosine and 5-Carboxycytosine"

    Article Title: Direct and Base Excision Repair-Mediated Regulation of a GC-Rich cis-Element in Response to 5-Formylcytosine and 5-Carboxycytosine

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms222011025

    Effects of 5-methylcytosine (5-mC), 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-fC) and 5-carboxycytosine (5-caC) in the purine-rich strand on the GC box activity. ( a ) Scheme of the reporter enhanced green fluorescence protein ( EGFP ) gene under the control of a GC box as the only upstream activating element. Synthetic oligonucleotide containing cytosine modifications at the unique CpG site (position indicated with an asterisk) were incorporated into the gap generated with the Nb.BsrDI nicking endonuclease (cleavage sites indicated with arrowheads). ( b ) Procedure for the incorporation of synthetic oligonucleotides containing C/5-mC/5-hmC/5-fC/5-caC (*) into the gap generated by depletion of the purine-rich strand of the targeted GC box. Aliquots of the same annealing reactions were incubated with or without T4 polynucleotide kinase (PNK) to validate full replacement of the native DNA strand. DNA strand labeling denotes transcribed strand (TS) of the EGFP gene and the non-transcribed strand (NTS); broken arrow indicates the transcription start site and direction. ( c ) Agarose gel electrophoresis of the reporter constructs generated by targeted incorporation of C/5-mC/5-hmC/5-fC/5-caC into plasmid DNA. Arrows indicate the open circular (oc) and covalently closed (cc) forms. ( d ) Expression time course of constructs containing 5-mC/5-hmC/5-fC/5-caC in transfected HeLa cells. All values (mean ± SD) are calculated relative to the expression of the control construct harboring synthetic oligonucleotide containing cytosine for n = 6 independent experiments (the 12-h point was skipped in three of the experiments). Representative flow cytometry data is shown in Supplementary Materials Figure S1 .
    Figure Legend Snippet: Effects of 5-methylcytosine (5-mC), 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-fC) and 5-carboxycytosine (5-caC) in the purine-rich strand on the GC box activity. ( a ) Scheme of the reporter enhanced green fluorescence protein ( EGFP ) gene under the control of a GC box as the only upstream activating element. Synthetic oligonucleotide containing cytosine modifications at the unique CpG site (position indicated with an asterisk) were incorporated into the gap generated with the Nb.BsrDI nicking endonuclease (cleavage sites indicated with arrowheads). ( b ) Procedure for the incorporation of synthetic oligonucleotides containing C/5-mC/5-hmC/5-fC/5-caC (*) into the gap generated by depletion of the purine-rich strand of the targeted GC box. Aliquots of the same annealing reactions were incubated with or without T4 polynucleotide kinase (PNK) to validate full replacement of the native DNA strand. DNA strand labeling denotes transcribed strand (TS) of the EGFP gene and the non-transcribed strand (NTS); broken arrow indicates the transcription start site and direction. ( c ) Agarose gel electrophoresis of the reporter constructs generated by targeted incorporation of C/5-mC/5-hmC/5-fC/5-caC into plasmid DNA. Arrows indicate the open circular (oc) and covalently closed (cc) forms. ( d ) Expression time course of constructs containing 5-mC/5-hmC/5-fC/5-caC in transfected HeLa cells. All values (mean ± SD) are calculated relative to the expression of the control construct harboring synthetic oligonucleotide containing cytosine for n = 6 independent experiments (the 12-h point was skipped in three of the experiments). Representative flow cytometry data is shown in Supplementary Materials Figure S1 .

    Techniques Used: Activity Assay, Fluorescence, Generated, Incubation, Labeling, Agarose Gel Electrophoresis, Construct, Plasmid Preparation, Expressing, Transfection, Flow Cytometry

    7) Product Images from "The interplay of RNA:DNA hybrid structure and G-quadruplexes determines the outcome of R-loop-replisome collisions"

    Article Title: The interplay of RNA:DNA hybrid structure and G-quadruplexes determines the outcome of R-loop-replisome collisions

    Journal: bioRxiv

    doi: 10.1101/2021.07.16.452753

    R-loop transcripts can prime leading strand restart after CD R-loop-replisome collisions. ( A ) Replication products obtained on mock- or T4 PNK-treated CD R-loop-containing templates in absence or presence RNase H1. ( B ) Products obtained on CD T4 PNK-treated R-loop templates in absence or presence of DDK. Relative signal intensity for restart product is quantified on the right. ( C ) Replication products obtained on mock- or T4 PNK-treated CD R-loop-containing templates in absence or presence RNase H1. ( D ) Schematic illustrating replication products observed in A. ( E ) RNase H1 titration into reactions with CD R-loop template. ( F ) Model for leading strand restart at R-loop transcript after replisome encounter with CD R-loop harboring 5’ RNA flap and RNA nick.
    Figure Legend Snippet: R-loop transcripts can prime leading strand restart after CD R-loop-replisome collisions. ( A ) Replication products obtained on mock- or T4 PNK-treated CD R-loop-containing templates in absence or presence RNase H1. ( B ) Products obtained on CD T4 PNK-treated R-loop templates in absence or presence of DDK. Relative signal intensity for restart product is quantified on the right. ( C ) Replication products obtained on mock- or T4 PNK-treated CD R-loop-containing templates in absence or presence RNase H1. ( D ) Schematic illustrating replication products observed in A. ( E ) RNase H1 titration into reactions with CD R-loop template. ( F ) Model for leading strand restart at R-loop transcript after replisome encounter with CD R-loop harboring 5’ RNA flap and RNA nick.

    Techniques Used: Titration

    R-loop transcripts can prime leading strand restart after CD R-loop-replisome collisions. ( A ) Polymerase assay with T4 PNK-treated CD R-loop template in presence of Pol α, Pol ε, or Pol δ, as indicated. All reactions include RFC, PCNA, RPA, dNTPs, NTPs and α-[ 32 P]-dATP. The concentrations of reaction components are equivalent to those used in the replication assay. ( B ) Replication products obtained on RNase H1-treated CD R-loop templates with or without Pol δ. Reactions were performed either in the presence of 1 nM (lanes 1+2) or 0.06 nM (lanes 3+4) RNase H1 to induce resolution or nicking of the RNA:DNA hybrid, respectively. ( C ) Left: Purified RNase H2. Right: Replication products obtained on CD and HO templates in the presence of sub-saturating levels of RNase H2.
    Figure Legend Snippet: R-loop transcripts can prime leading strand restart after CD R-loop-replisome collisions. ( A ) Polymerase assay with T4 PNK-treated CD R-loop template in presence of Pol α, Pol ε, or Pol δ, as indicated. All reactions include RFC, PCNA, RPA, dNTPs, NTPs and α-[ 32 P]-dATP. The concentrations of reaction components are equivalent to those used in the replication assay. ( B ) Replication products obtained on RNase H1-treated CD R-loop templates with or without Pol δ. Reactions were performed either in the presence of 1 nM (lanes 1+2) or 0.06 nM (lanes 3+4) RNase H1 to induce resolution or nicking of the RNA:DNA hybrid, respectively. ( C ) Left: Purified RNase H2. Right: Replication products obtained on CD and HO templates in the presence of sub-saturating levels of RNase H2.

    Techniques Used: Recombinase Polymerase Amplification, Purification

    8) Product Images from "Detection of circulating extracellular mRNAs by modified small-RNA-sequencing analysis"

    Article Title: Detection of circulating extracellular mRNAs by modified small-RNA-sequencing analysis

    Journal: JCI Insight

    doi: 10.1172/jci.insight.127317

    Treatment of total extracellular RNA with T4 polynucleotide kinase followed by small-RNA-sequencing. ( A ) Total RNA was isolated from 450 μl serum or platelet-depleted EDTA, acid citrate dextrose (ACD), and heparin plasma from 6 healthy individuals and purified using silica-based spin columns. Half of the RNA was treated with T4 polynucleotide kinase (T4 PNK) and repurified (PNK treated), and multiplexed small-RNA-sequencing (sRNA-seq) libraries were prepared separately for the untreated (libraries 1 and 3) and PNK-treated RNA (libraries 2 and 4). ( B ) Differences in read annotation in the 4 sample types for untreated RNA and PNK-treated RNA using initial annotation settings (reads 12–42 nt, up to 2 mismatches, multimapping). ( C ) Differences in ex‑mRNA capture between untreated and PNK-treated RNA using final annotation criteria (reads > 15 nt, no mismatch and up to 2 mapping locations). Box plots show the median and first and third quartiles (bottom and top hinges). Whiskers extend at most ×1.5 interquartile range from the hinges; any data outside this are shown as individual outlier points. Shown are results from n = 6 individual samples per condition.
    Figure Legend Snippet: Treatment of total extracellular RNA with T4 polynucleotide kinase followed by small-RNA-sequencing. ( A ) Total RNA was isolated from 450 μl serum or platelet-depleted EDTA, acid citrate dextrose (ACD), and heparin plasma from 6 healthy individuals and purified using silica-based spin columns. Half of the RNA was treated with T4 polynucleotide kinase (T4 PNK) and repurified (PNK treated), and multiplexed small-RNA-sequencing (sRNA-seq) libraries were prepared separately for the untreated (libraries 1 and 3) and PNK-treated RNA (libraries 2 and 4). ( B ) Differences in read annotation in the 4 sample types for untreated RNA and PNK-treated RNA using initial annotation settings (reads 12–42 nt, up to 2 mismatches, multimapping). ( C ) Differences in ex‑mRNA capture between untreated and PNK-treated RNA using final annotation criteria (reads > 15 nt, no mismatch and up to 2 mapping locations). Box plots show the median and first and third quartiles (bottom and top hinges). Whiskers extend at most ×1.5 interquartile range from the hinges; any data outside this are shown as individual outlier points. Shown are results from n = 6 individual samples per condition.

    Techniques Used: RNA Sequencing Assay, Isolation, Purification

    9) Product Images from "Rhythmic binding of Topoisomerase I impacts on the transcription of Bmal1 and circadian period"

    Article Title: Rhythmic binding of Topoisomerase I impacts on the transcription of Bmal1 and circadian period

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gks779

    Top1 binds to intermediate region between ROREs in Bmal1 promoter region. Top1-mediated cleavage assay ( A ). DNA fragment around the ROREs (nucleotides −88 to −22: 5′-GATTGGTCGGAAAGTAGGTTAGTGGTGCGACATTTAGGGAAGGCAGAAAGTAGGTCAGGGACGGAGG-3′) was end-labeled with [γ- 32 P]ATP using T4 polynucleotide kinase. DNA fragment was reacted with either 50 units of Top1 alone or with 2–50 units of Top1 plus 0.5 mM camptothecin. Purified DNA was resolved on 8% polyacrylamide–urea gels. CPT, camptothecin. EMSA using probe, nucleotides −88 to −22, 15 units of Top1 protein and a 100-fold molar excess of the following competitors (Comp): AT, control oligonucleotides, (dA) 30 and (dT) 30 ; unlabeled probe, nucleotides −88 to −22; −67 to −43, nucleotides −67 to −4 ( B ). Arrowhead, shifted band.
    Figure Legend Snippet: Top1 binds to intermediate region between ROREs in Bmal1 promoter region. Top1-mediated cleavage assay ( A ). DNA fragment around the ROREs (nucleotides −88 to −22: 5′-GATTGGTCGGAAAGTAGGTTAGTGGTGCGACATTTAGGGAAGGCAGAAAGTAGGTCAGGGACGGAGG-3′) was end-labeled with [γ- 32 P]ATP using T4 polynucleotide kinase. DNA fragment was reacted with either 50 units of Top1 alone or with 2–50 units of Top1 plus 0.5 mM camptothecin. Purified DNA was resolved on 8% polyacrylamide–urea gels. CPT, camptothecin. EMSA using probe, nucleotides −88 to −22, 15 units of Top1 protein and a 100-fold molar excess of the following competitors (Comp): AT, control oligonucleotides, (dA) 30 and (dT) 30 ; unlabeled probe, nucleotides −88 to −22; −67 to −43, nucleotides −67 to −4 ( B ). Arrowhead, shifted band.

    Techniques Used: Cleavage Assay, Labeling, Purification, Cycling Probe Technology

    10) Product Images from "Crystal structure of a PIWI protein suggests mechanisms for siRNA recognition and slicer activity"

    Article Title: Crystal structure of a PIWI protein suggests mechanisms for siRNA recognition and slicer activity

    Journal: The EMBO Journal

    doi: 10.1038/sj.emboj.7600488

    AfPiwi forms a distinct complex with an siRNA-like RNA duplex. ( A ) Sequence and structure of the self-complementary RNA oligonucleotide used in this study. The RNA was labelled with  32 P at the 5′ end ( * ) using T4 PNK. ( B ) EMSA assessing complex formation between the end-labelled RNA (
    Figure Legend Snippet: AfPiwi forms a distinct complex with an siRNA-like RNA duplex. ( A ) Sequence and structure of the self-complementary RNA oligonucleotide used in this study. The RNA was labelled with 32 P at the 5′ end ( * ) using T4 PNK. ( B ) EMSA assessing complex formation between the end-labelled RNA (

    Techniques Used: Sequencing

    11) Product Images from "A library-based method to rapidly analyse chromatin accessibility at multiple genomic regions"

    Article Title: A library-based method to rapidly analyse chromatin accessibility at multiple genomic regions

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp037

    Overview of the experimental steps required to create and analyse a chromatin accessibility library. ( A ) Step 1: fungal mycelia pre-grown under specific conditions or isolated DNA ( in vitro controls) are processed as described in Materials and methods section and digested with MNase or restriction enzymes of choice. Step 2: digested DNA is blunt-ended and phosphorylated by subsequent treatment of the chromatin with Klenow fragment polymerase, T4 polynucleotide kinase. This step produces blunt-ended DNA fragments for ligation with adaptors. Step 3: DNA fragments are ligated with double-stranded adaptors A and B, originating from oligonucleotides Adaptor-A short and Adaptor-A long or Adaptor-B short and Adaptor-B long , where adaptor oligonucleotide B long is biotinylated for later retention on the streptavidin beads. In this step, fragments containing all adaptor combinations (A-A, A-B and B-B) are generated. Step 4: the ligation step leaves nicks at the 3′-terminus that are repaired by Bst polymerase treatment. Step 5: all fragments containing biotinylated adaptor B are captured on streptavidin-coated magnetic beads. At this step, adaptor A-A fragments are lost. Step 6: after a washing step, the retained fragments (adaptors A-B and B-B fragments) are denatured at 95°C. The denaturation step results in the release of single strands which exclusively carry A-B adaptor fragments. Step 7: the single-stranded A-B adaptor fragment library is amplified by a nested PCR approach to give the final A-B fragment library. The input and output fragment libraries are quality controlled by amplification with single A and B, as well as mixed A-B primers. Only the A-B primer mix should result in the amplification of fragments in the range of 200–1000 bp (see Panel B). Step 8: the resulting A-B adaptor fragment library is diluted and aliquots are used for analytical PCR amplifications for fragment size analysis of specific loci of interest. In the final analytical PCR step, either gene-specific or adaptor-specific primers can be labelled for subsequent capillary sequencer analysis. The chromatograms are finally analysed by image analysis software. ( B ) Example of quality control of A-B adaptor fragment libraries. Two input chromatin fragment libraries without adaptor ligation (lanes 1 and 2) are compared to two output libraries with adaptor ligation as described in Materials and methods section (lanes 3 and 4). Libraries originating from nitrate-grown cells (lanes 1 and 3) as well as from ammonium-grown cells (lanes 2 and 4) are shown as an example. M, DNA size marker.
    Figure Legend Snippet: Overview of the experimental steps required to create and analyse a chromatin accessibility library. ( A ) Step 1: fungal mycelia pre-grown under specific conditions or isolated DNA ( in vitro controls) are processed as described in Materials and methods section and digested with MNase or restriction enzymes of choice. Step 2: digested DNA is blunt-ended and phosphorylated by subsequent treatment of the chromatin with Klenow fragment polymerase, T4 polynucleotide kinase. This step produces blunt-ended DNA fragments for ligation with adaptors. Step 3: DNA fragments are ligated with double-stranded adaptors A and B, originating from oligonucleotides Adaptor-A short and Adaptor-A long or Adaptor-B short and Adaptor-B long , where adaptor oligonucleotide B long is biotinylated for later retention on the streptavidin beads. In this step, fragments containing all adaptor combinations (A-A, A-B and B-B) are generated. Step 4: the ligation step leaves nicks at the 3′-terminus that are repaired by Bst polymerase treatment. Step 5: all fragments containing biotinylated adaptor B are captured on streptavidin-coated magnetic beads. At this step, adaptor A-A fragments are lost. Step 6: after a washing step, the retained fragments (adaptors A-B and B-B fragments) are denatured at 95°C. The denaturation step results in the release of single strands which exclusively carry A-B adaptor fragments. Step 7: the single-stranded A-B adaptor fragment library is amplified by a nested PCR approach to give the final A-B fragment library. The input and output fragment libraries are quality controlled by amplification with single A and B, as well as mixed A-B primers. Only the A-B primer mix should result in the amplification of fragments in the range of 200–1000 bp (see Panel B). Step 8: the resulting A-B adaptor fragment library is diluted and aliquots are used for analytical PCR amplifications for fragment size analysis of specific loci of interest. In the final analytical PCR step, either gene-specific or adaptor-specific primers can be labelled for subsequent capillary sequencer analysis. The chromatograms are finally analysed by image analysis software. ( B ) Example of quality control of A-B adaptor fragment libraries. Two input chromatin fragment libraries without adaptor ligation (lanes 1 and 2) are compared to two output libraries with adaptor ligation as described in Materials and methods section (lanes 3 and 4). Libraries originating from nitrate-grown cells (lanes 1 and 3) as well as from ammonium-grown cells (lanes 2 and 4) are shown as an example. M, DNA size marker.

    Techniques Used: Isolation, In Vitro, Ligation, Generated, Magnetic Beads, Amplification, Nested PCR, Polymerase Chain Reaction, Software, Marker

    12) Product Images from "RISC is a 5? phosphomonoester-producing RNA endonuclease"

    Article Title: RISC is a 5? phosphomonoester-producing RNA endonuclease

    Journal: Genes & Development

    doi: 10.1101/gad.1187904

    Target RNA is cleaved endonucleolytically producing 5′-phosphate and 3′-hydroxyl termini. ( A ) Preparation of site-specifically labeled substrates and cleavage assay. 5′- 32 P-labeled and 3′ aminolinker (L) protected 12-nt oligoribonucleotide was ligated to nonphosphorylated 9-nt oligoribonucleotide using T4 RNA ligase. An aliquot of the ligation product was further 5′- 32 P-labeled using T4 polynucleotide kinase. The purified substrates were incubated with affinity-purified RISC programmed with single-stranded guide siRNA. ( B ) PhosphorImaging of cleavage reactions incubated for 2 h at 30°C, and resolved on a 20% denaturing polyacrylamide gel. 5′- 32 P-labeled 9- and 12-nt oligoribonucleotides were loaded as marker in lanes 1 and 2 , respectively. The cleavage reactions with single- and double-labeled 21-nt substrate are loaded in lanes 4 and 5 , respectively. Lane 3 contains the 12-nt cleavage product isolated from a prior cleavage reaction. ( C ) Two-dimensional thin layer chromatography analysis of the ribonuclease T2-digested RISC-cleavage product. The oval depicts the unlabeled pAp as detected by UV shadowing. The radioactive signal comigrates with the 5′ 32 pAp released by ribonuclease T2 digestion from the gel-purified 12-nt cleavage product.
    Figure Legend Snippet: Target RNA is cleaved endonucleolytically producing 5′-phosphate and 3′-hydroxyl termini. ( A ) Preparation of site-specifically labeled substrates and cleavage assay. 5′- 32 P-labeled and 3′ aminolinker (L) protected 12-nt oligoribonucleotide was ligated to nonphosphorylated 9-nt oligoribonucleotide using T4 RNA ligase. An aliquot of the ligation product was further 5′- 32 P-labeled using T4 polynucleotide kinase. The purified substrates were incubated with affinity-purified RISC programmed with single-stranded guide siRNA. ( B ) PhosphorImaging of cleavage reactions incubated for 2 h at 30°C, and resolved on a 20% denaturing polyacrylamide gel. 5′- 32 P-labeled 9- and 12-nt oligoribonucleotides were loaded as marker in lanes 1 and 2 , respectively. The cleavage reactions with single- and double-labeled 21-nt substrate are loaded in lanes 4 and 5 , respectively. Lane 3 contains the 12-nt cleavage product isolated from a prior cleavage reaction. ( C ) Two-dimensional thin layer chromatography analysis of the ribonuclease T2-digested RISC-cleavage product. The oval depicts the unlabeled pAp as detected by UV shadowing. The radioactive signal comigrates with the 5′ 32 pAp released by ribonuclease T2 digestion from the gel-purified 12-nt cleavage product.

    Techniques Used: Labeling, Cleavage Assay, Ligation, Purification, Incubation, Affinity Purification, Marker, Isolation, Thin Layer Chromatography

    13) Product Images from "A Type III-B Cmr effector complex catalyzes the synthesis of cyclic oligoadenylate second messengers by cooperative substrate binding"

    Article Title: A Type III-B Cmr effector complex catalyzes the synthesis of cyclic oligoadenylate second messengers by cooperative substrate binding

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky844

    Michaelis–Menten modeling of the ATP reaction by Cmr-α–RNP. ( A ) Four nanomolar Cmr–α was incubated with increasing concentrations of ATP and the ATP incorporation rate ( V ) was plotted to the substrate concentration [ S ] (blue line). Error bar represents S.D. of three independent experiments. The V versus [S] relationship was further fitted into the Michaelis–Menten model (red line). F -test shows the P value
    Figure Legend Snippet: Michaelis–Menten modeling of the ATP reaction by Cmr-α–RNP. ( A ) Four nanomolar Cmr–α was incubated with increasing concentrations of ATP and the ATP incorporation rate ( V ) was plotted to the substrate concentration [ S ] (blue line). Error bar represents S.D. of three independent experiments. The V versus [S] relationship was further fitted into the Michaelis–Menten model (red line). F -test shows the P value

    Techniques Used: Incubation, Concentration Assay

    Both Palm1 and Palm2 domains of Cmr2α function in c-A4 generation. ( A ) Schematic of Cmr–2α domain structure. The conserved amino acids in HD, Palm1 and Palm2 domains (subjected to mutagenesis) are indicated. ( B ) Relative cOA synthesis activity of the wild-type Cmr-α–RNP (WT) and its mutant derivatives. ( C ) ATP binding affinity of the wild-type Cmr-α–RNP (WT) and its mutant derivatives. Cmr-α HD , Cmr-α P1 and Cmr-α P2 : effector complexes carrying substitution mutations in the HD, Palm 1 or Palm 2 domain of Cmr2α. The amount of synthesized cOA/bound ATP by the wild-type Cmr-α complex was arbitrarily set to 1.
    Figure Legend Snippet: Both Palm1 and Palm2 domains of Cmr2α function in c-A4 generation. ( A ) Schematic of Cmr–2α domain structure. The conserved amino acids in HD, Palm1 and Palm2 domains (subjected to mutagenesis) are indicated. ( B ) Relative cOA synthesis activity of the wild-type Cmr-α–RNP (WT) and its mutant derivatives. ( C ) ATP binding affinity of the wild-type Cmr-α–RNP (WT) and its mutant derivatives. Cmr-α HD , Cmr-α P1 and Cmr-α P2 : effector complexes carrying substitution mutations in the HD, Palm 1 or Palm 2 domain of Cmr2α. The amount of synthesized cOA/bound ATP by the wild-type Cmr-α complex was arbitrarily set to 1.

    Techniques Used: Mutagenesis, Activity Assay, Binding Assay, Synthesized

    A model for Cmr-α-mediated c-A4 synthesis. Cmr-α is represented with Palm1 (P1), Palm2 (P2) and D2 (D2) domains of the Cmr–2α, three of the five conserved domains identified in the P. furiosus Cmr2 ( 57 ). Binding of the cognate target RNA to Cmr-α yields a ternary Cmr-α complex ( A ). Upon the binding of the first ATP molecule to the ternary Cmr-α, the substrate-enzyme intermediate adopts a conformational change and becomes more accessible to a second ATP molecule ( B and C ). Nucleophilic attack from the 3′-hydroxyl group (3′-OH) of the first ATP molecule to the 5′-triphosphate group (5′-P) of the second ATP molecule yields a phosphodiester bond between the two nucleotides ( D ). Cmr-α–RNP translocates on the 2-nt intermediate, freeing one of the ATP-binding sites ( E ). The process is repeated, leading to the formation of the third and the fourth phosphodiester bond (E and F). Finally, the substrate-free active site in Cmr-α recaptures the first nucleotide of the poly-A4 RNA ( F and G ) and circularizes the tetraadenylate in a condensation reaction.
    Figure Legend Snippet: A model for Cmr-α-mediated c-A4 synthesis. Cmr-α is represented with Palm1 (P1), Palm2 (P2) and D2 (D2) domains of the Cmr–2α, three of the five conserved domains identified in the P. furiosus Cmr2 ( 57 ). Binding of the cognate target RNA to Cmr-α yields a ternary Cmr-α complex ( A ). Upon the binding of the first ATP molecule to the ternary Cmr-α, the substrate-enzyme intermediate adopts a conformational change and becomes more accessible to a second ATP molecule ( B and C ). Nucleophilic attack from the 3′-hydroxyl group (3′-OH) of the first ATP molecule to the 5′-triphosphate group (5′-P) of the second ATP molecule yields a phosphodiester bond between the two nucleotides ( D ). Cmr-α–RNP translocates on the 2-nt intermediate, freeing one of the ATP-binding sites ( E ). The process is repeated, leading to the formation of the third and the fourth phosphodiester bond (E and F). Finally, the substrate-free active site in Cmr-α recaptures the first nucleotide of the poly-A4 RNA ( F and G ) and circularizes the tetraadenylate in a condensation reaction.

    Techniques Used: Binding Assay

    14) Product Images from "The wobble nucleotide-excising anticodon nuclease RloC is governed by the zinc-hook and DNA-dependent ATPase of its Rad50-like region"

    Article Title: The wobble nucleotide-excising anticodon nuclease RloC is governed by the zinc-hook and DNA-dependent ATPase of its Rad50-like region

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gks593

    Gka RloC’s ATPase activates its ACNase. Gka RloC’s ACNase of the IMAC fraction was assayed in vitro in panels ( A )–( C ) and ( E ) essentially as described in Materials and Methods but in the absence of added DNA. ( A ) Dependence of Gka RloC’s ACNase activity on ATP’s level. ( B ) Gka RloC’s ACNase activity was assayed in the presence of 500 µM of the indicated nucleotides. ( C ) Time courses of Gka RloC’s ACNase activity in the presence of 0.5 mM ATP and indicated amounts of AMPPNP. ( D ) In vivo ACNase activity of the indicated Gka RloC alleles. Left panel—RNA extracted from cells expressing these alleles was 5′-end labelled using T4 Pnk and separated by denaturing PAGE. Right panel—the expression of the indicated Gka RloC alleles were monitored by Western using an anti-His tag monoclonal antibody ( 4 ). ( E ) Nucleotide specificity of Gka RloC’s ACNase activation. The activation reaction was performed in the presence of the indicated nucleotides (GTP and ATP at 0.5 mM each, dTTP at 5 µM).
    Figure Legend Snippet: Gka RloC’s ATPase activates its ACNase. Gka RloC’s ACNase of the IMAC fraction was assayed in vitro in panels ( A )–( C ) and ( E ) essentially as described in Materials and Methods but in the absence of added DNA. ( A ) Dependence of Gka RloC’s ACNase activity on ATP’s level. ( B ) Gka RloC’s ACNase activity was assayed in the presence of 500 µM of the indicated nucleotides. ( C ) Time courses of Gka RloC’s ACNase activity in the presence of 0.5 mM ATP and indicated amounts of AMPPNP. ( D ) In vivo ACNase activity of the indicated Gka RloC alleles. Left panel—RNA extracted from cells expressing these alleles was 5′-end labelled using T4 Pnk and separated by denaturing PAGE. Right panel—the expression of the indicated Gka RloC alleles were monitored by Western using an anti-His tag monoclonal antibody ( 4 ). ( E ) Nucleotide specificity of Gka RloC’s ACNase activation. The activation reaction was performed in the presence of the indicated nucleotides (GTP and ATP at 0.5 mM each, dTTP at 5 µM).

    Techniques Used: In Vitro, Activity Assay, In Vivo, Expressing, Polyacrylamide Gel Electrophoresis, Western Blot, Activation Assay

    15) Product Images from "Transcriptional arrest: Escherichia coli RNA polymerase translocates backward, leaving the 3? end of the RNA intact and extruded"

    Article Title: Transcriptional arrest: Escherichia coli RNA polymerase translocates backward, leaving the 3? end of the RNA intact and extruded

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

    doi:

    Effect of arrest on the transcript arrangement in RNAP. ( A ) RNase A footprinting of the transcript in active EC 26 and in arrested EC 27 . The RNA in the complexes was internally labeled at positions +26A or +12C. Each sample was treated with RNase A and fractionated by centrifugation into soluble (s) and matrix-associated (p) fractions before gel analysis. Sequences of the transcripts are shown alongside the autoradiograms, asterisks mark positions of labeling, and arrows show major cuts introduced into the RNA (bold shaded line) by RNase A. The cylinders represent the transcript segments protected by RNAP in the active and arrested complexes. Nonfractionated samples (t) are included as controls. ( B ) 5′-Terminal phosphorylation of the transcripts. RNA-labeled EC 20 and EC 27 (lanes 6 and 4) and arrested fraction of EC 27 purified by chase (lane 2) were treated with T4 polynucleotide kinase in the presence of ATP (lanes 5, 3, and 1). The symbol (P) indicates the phosphorylated transcripts. Arrows indicate the mobility of phosphorylated and nonphosphorylated transcripts.
    Figure Legend Snippet: Effect of arrest on the transcript arrangement in RNAP. ( A ) RNase A footprinting of the transcript in active EC 26 and in arrested EC 27 . The RNA in the complexes was internally labeled at positions +26A or +12C. Each sample was treated with RNase A and fractionated by centrifugation into soluble (s) and matrix-associated (p) fractions before gel analysis. Sequences of the transcripts are shown alongside the autoradiograms, asterisks mark positions of labeling, and arrows show major cuts introduced into the RNA (bold shaded line) by RNase A. The cylinders represent the transcript segments protected by RNAP in the active and arrested complexes. Nonfractionated samples (t) are included as controls. ( B ) 5′-Terminal phosphorylation of the transcripts. RNA-labeled EC 20 and EC 27 (lanes 6 and 4) and arrested fraction of EC 27 purified by chase (lane 2) were treated with T4 polynucleotide kinase in the presence of ATP (lanes 5, 3, and 1). The symbol (P) indicates the phosphorylated transcripts. Arrows indicate the mobility of phosphorylated and nonphosphorylated transcripts.

    Techniques Used: Footprinting, Labeling, Centrifugation, Purification

    16) Product Images from "A universal method to produce in vitro transcripts with homogeneous 3? ends"

    Article Title: A universal method to produce in vitro transcripts with homogeneous 3? ends

    Journal: Nucleic Acids Research

    doi:

    Removal of the terminal 2′,3′ cyclic phosphate group of the released tRNA resulting from the HDV ribozyme cleavage reaction. Treatment with T4 polynucleotide kinase (T4 PNK) leads to the removal of the phosphate group and therefore to a reduced net charge of the RNA. This can be observed by a reduced electrophoretic mobility on a denaturing 10% polyacrylamide gel in comparison with the untreated control (mock incubation without T4 PNK).
    Figure Legend Snippet: Removal of the terminal 2′,3′ cyclic phosphate group of the released tRNA resulting from the HDV ribozyme cleavage reaction. Treatment with T4 polynucleotide kinase (T4 PNK) leads to the removal of the phosphate group and therefore to a reduced net charge of the RNA. This can be observed by a reduced electrophoretic mobility on a denaturing 10% polyacrylamide gel in comparison with the untreated control (mock incubation without T4 PNK).

    Techniques Used: Incubation

    17) Product Images from "Usb1 controls U6 snRNP assembly through evolutionarily divergent cyclic phosphodiesterase activities"

    Article Title: Usb1 controls U6 snRNP assembly through evolutionarily divergent cyclic phosphodiesterase activities

    Journal: Nature Communications

    doi: 10.1038/s41467-017-00484-w

    yUsb1 acts as a 3′–5′exonuclease and CPDase in vitro. a U6 snRNA is synthesized by RNA Polymerase III. Transcription termination produces a heterogeneous U6 with a 4–8 nucleotide U-tail. Processing by yUsb1 shortens the U-tail and leaves a phosphoryl group. b Usb1 removes nucleotides from the 3′ end of RNAs. The 5′-labeled U6 95–112+3U oligonucleotide cis -diol substrate (lane 2) is insensitive to CIP (lane 3) or T4 PNK (lane 4) treatment. Incubation with yUsb1 for 1 h results in a shorter product (lane 5). Similar reactivity of the product to both CIP (lane 6) and T4 PNK (lane 7) indicates that the product is a noncyclic phosphate. An alkaline hydrolysis ladder (lane 1) shows the mobility of oligonucleotide products of different lengths. ( c , top ) One-dimensional 31 P NMR spectra of 2′,3′-cUMP shows a single peak at 20 ppm. A 3′ UMP standard has a single peak at 3.4 ppm. When 2′,3′-cUMP is incubated with AtRNL, which leaves a 2′ phosphate 8 , there is a single peak at 3.2 ppm. Incubation of 2′,3′-cUMP with yUsb1 produces a new signal at 3.4 ppm ( c , bottom ) Zoom of dashed region in top panel. d Time course of Usb1 processing on RNAs with different 3′ end modifications. yUsb1 is most active on RNA substrates with a cis -diol (lanes 1–4), less active on those with a 2′,3′-cyclic phosphate ( > p; lanes 5–8) or 2′ phosphates (2′P; lanes 9–12), and is inactive on 3′ phosphate ends (3′P; lanes 13–16). e Model describing the dual activities of yUsb1
    Figure Legend Snippet: yUsb1 acts as a 3′–5′exonuclease and CPDase in vitro. a U6 snRNA is synthesized by RNA Polymerase III. Transcription termination produces a heterogeneous U6 with a 4–8 nucleotide U-tail. Processing by yUsb1 shortens the U-tail and leaves a phosphoryl group. b Usb1 removes nucleotides from the 3′ end of RNAs. The 5′-labeled U6 95–112+3U oligonucleotide cis -diol substrate (lane 2) is insensitive to CIP (lane 3) or T4 PNK (lane 4) treatment. Incubation with yUsb1 for 1 h results in a shorter product (lane 5). Similar reactivity of the product to both CIP (lane 6) and T4 PNK (lane 7) indicates that the product is a noncyclic phosphate. An alkaline hydrolysis ladder (lane 1) shows the mobility of oligonucleotide products of different lengths. ( c , top ) One-dimensional 31 P NMR spectra of 2′,3′-cUMP shows a single peak at 20 ppm. A 3′ UMP standard has a single peak at 3.4 ppm. When 2′,3′-cUMP is incubated with AtRNL, which leaves a 2′ phosphate 8 , there is a single peak at 3.2 ppm. Incubation of 2′,3′-cUMP with yUsb1 produces a new signal at 3.4 ppm ( c , bottom ) Zoom of dashed region in top panel. d Time course of Usb1 processing on RNAs with different 3′ end modifications. yUsb1 is most active on RNA substrates with a cis -diol (lanes 1–4), less active on those with a 2′,3′-cyclic phosphate ( > p; lanes 5–8) or 2′ phosphates (2′P; lanes 9–12), and is inactive on 3′ phosphate ends (3′P; lanes 13–16). e Model describing the dual activities of yUsb1

    Techniques Used: In Vitro, Synthesized, Labeling, Incubation, Nuclear Magnetic Resonance

    18) Product Images from "A Simple and Cost-Effective Approach for In Vitro Production of Sliced siRNAs as Potent Triggers for RNAi"

    Article Title: A Simple and Cost-Effective Approach for In Vitro Production of Sliced siRNAs as Potent Triggers for RNAi

    Journal: Molecular Therapy. Nucleic Acids

    doi: 10.1016/j.omtn.2017.07.008

    Manipulation of 5′ppp-Triggered Interferon Response HEK293 cells were transfected with poly(I:C) or several tsli-siRNAs. The final concentration of 10 nM for each RNAi reagent was used in transfection for qPCR assay. Gene expression level changes in OAS1, IRF9, CDKL, and IFNB relative to GAPDH were measured by qPCR. (A) Mild interferon response was observed from all four tsli-siRNAs, with tsli-RRM2 having the strongest response among them. G-tsli-Stat3 exhibited a much stronger response than tsli-Stat3, and GG-tsli-Stat3 reversed this effect to some extent. (B) CIP treatment minimized the strong interferon response by G-tsli-Stat3. (C) CIP treatment minimized and T4 PNK treatment elevated the interferon response by tsli-RRM2. Fold changes in gene expression were normalized to untreated HEK293 cells. Details of qPCR procedure and results calculation were provided in the Materials and Methods . Error bars indicate SD.
    Figure Legend Snippet: Manipulation of 5′ppp-Triggered Interferon Response HEK293 cells were transfected with poly(I:C) or several tsli-siRNAs. The final concentration of 10 nM for each RNAi reagent was used in transfection for qPCR assay. Gene expression level changes in OAS1, IRF9, CDKL, and IFNB relative to GAPDH were measured by qPCR. (A) Mild interferon response was observed from all four tsli-siRNAs, with tsli-RRM2 having the strongest response among them. G-tsli-Stat3 exhibited a much stronger response than tsli-Stat3, and GG-tsli-Stat3 reversed this effect to some extent. (B) CIP treatment minimized the strong interferon response by G-tsli-Stat3. (C) CIP treatment minimized and T4 PNK treatment elevated the interferon response by tsli-RRM2. Fold changes in gene expression were normalized to untreated HEK293 cells. Details of qPCR procedure and results calculation were provided in the Materials and Methods . Error bars indicate SD.

    Techniques Used: Transfection, Concentration Assay, Real-time Polymerase Chain Reaction, Expressing

    19) Product Images from "A universal method to produce in vitro transcripts with homogeneous 3? ends"

    Article Title: A universal method to produce in vitro transcripts with homogeneous 3? ends

    Journal: Nucleic Acids Research

    doi:

    Removal of the terminal 2′,3′ cyclic phosphate group of the released tRNA resulting from the HDV ribozyme cleavage reaction. Treatment with T4 polynucleotide kinase (T4 PNK) leads to the removal of the phosphate group and therefore to a reduced net charge of the RNA. This can be observed by a reduced electrophoretic mobility on a denaturing 10% polyacrylamide gel in comparison with the untreated control (mock incubation without T4 PNK).
    Figure Legend Snippet: Removal of the terminal 2′,3′ cyclic phosphate group of the released tRNA resulting from the HDV ribozyme cleavage reaction. Treatment with T4 polynucleotide kinase (T4 PNK) leads to the removal of the phosphate group and therefore to a reduced net charge of the RNA. This can be observed by a reduced electrophoretic mobility on a denaturing 10% polyacrylamide gel in comparison with the untreated control (mock incubation without T4 PNK).

    Techniques Used: Incubation

    20) Product Images from "PARP3 is a sensor of nicked nucleosomes and monoribosylates histone H2BGlu2"

    Article Title: PARP3 is a sensor of nicked nucleosomes and monoribosylates histone H2BGlu2

    Journal: Nature Communications

    doi: 10.1038/ncomms12404

    PARP3 monoribosylates H2B in damaged chromatin. ( a , left) 10μg of the chicken chromatin employed in these experiments was fractionated by SDS–PAGE and stained with Coomassie blue. (right) One microgram of soluble MNase-treated chicken chromatin or 50-mer oligonucleotide duplex (200 nM) harbouring a nick with 3′-P/5′-OH termini was mock-treated (0) or treated with 1, 0.5 or 0.25 U T4 PNK to restore 3′-OH/5′-P termini. These DNA substrates were then incubated with 100 nM hPARP3 and 12.5 μM biotin-NAD + for 30 min and biotinylated products separated by 15% SDS–PAGE and detected with streptavidin-HRP. ( b ) 1 μg chicken chromatin or the indicated recombinant histone was incubated with 100 nM hPARP3 in the presence of 300 nM 32 P-NAD + or 12.5 μM biotin-NAD and oligonucleotide harbouring either a DSB (middle) or SSB (right) and the reaction products fractionated by 15% SDS–PAGE and detected by autoradiography or streptavidin-HRP. (left) An aliquot of the chicken chromatin and recombinant histones was fractionated by SDS–PAGE and stained with Coomassie blue. ( c , left) Aliquots of recombinant histone standards were fractionated separately or together as an octamer on triton-acid urea gels and analysed by staining with Coomassie blue. (right) The products of the PARP3 ribosylation reactions conducted in b were fractionated on triton-acid urea gels and analysed by autoradiography. HRP, horseradish peroxidase.
    Figure Legend Snippet: PARP3 monoribosylates H2B in damaged chromatin. ( a , left) 10μg of the chicken chromatin employed in these experiments was fractionated by SDS–PAGE and stained with Coomassie blue. (right) One microgram of soluble MNase-treated chicken chromatin or 50-mer oligonucleotide duplex (200 nM) harbouring a nick with 3′-P/5′-OH termini was mock-treated (0) or treated with 1, 0.5 or 0.25 U T4 PNK to restore 3′-OH/5′-P termini. These DNA substrates were then incubated with 100 nM hPARP3 and 12.5 μM biotin-NAD + for 30 min and biotinylated products separated by 15% SDS–PAGE and detected with streptavidin-HRP. ( b ) 1 μg chicken chromatin or the indicated recombinant histone was incubated with 100 nM hPARP3 in the presence of 300 nM 32 P-NAD + or 12.5 μM biotin-NAD and oligonucleotide harbouring either a DSB (middle) or SSB (right) and the reaction products fractionated by 15% SDS–PAGE and detected by autoradiography or streptavidin-HRP. (left) An aliquot of the chicken chromatin and recombinant histones was fractionated by SDS–PAGE and stained with Coomassie blue. ( c , left) Aliquots of recombinant histone standards were fractionated separately or together as an octamer on triton-acid urea gels and analysed by staining with Coomassie blue. (right) The products of the PARP3 ribosylation reactions conducted in b were fractionated on triton-acid urea gels and analysed by autoradiography. HRP, horseradish peroxidase.

    Techniques Used: SDS Page, Staining, Incubation, TNKS1 Histone Ribosylation Assay, Recombinant, Autoradiography

    21) Product Images from "Swi6/HP1 binding to RNA-DNA hybrids initiates heterochromatin assembly at the centromeric dg-dh repeats in Fission Yeast"

    Article Title: Swi6/HP1 binding to RNA-DNA hybrids initiates heterochromatin assembly at the centromeric dg-dh repeats in Fission Yeast

    Journal: bioRxiv

    doi: 10.1101/2020.10.21.349050

    Specificity of binding of Swi6 to the dg.dh repeat sequences in vivo. ( A ) Expression of GFP-Swi6 and GFP-Swi6 3K→3A in swi6Δ cells. Extracts prepared from cells of swi6Δ strain expressing the control vector, GFP-Swi6 and GFP-Swi6 3K→3A were subjected to western blotting with anti-GFP (upper panel) and α-tubulin (lower panel) antibody. ( B ) Slot blot hybridization.RNAs extracted from cells expressing TAP-tagged Tas3 immunprecipitated with anti-CBP antibody and those expressing GFP-tagged Swi6 or GFP-tagged Swi6 3K→3A , immunoprecipitated with anti-GFP antibody, were radiolabeled at the 5’-end with [γ− 32 P]ATP and T4 polynucleotide kinase and used as a probe for slot blots on which equal amounts of the DNAs (100ng) corresponding to actl, dg, dh and dhk (cenH ) were blotted. The bound signal was visualized by autoradiography. ( C ) The size fractionated radiolabelled siRNA samples from indicated strains were subjected to electrophoresis in 8% acrylamide/7M urea gels and visualized by autoradiography. In parallel, unlabeled samples were similarly processed blotted and probed with radiolabelled snoRNAs (lower panel), followed by autoradiography. ( D ) No bidirectional transcription is observed in cells expressing swi6 3K→3A mutant. RNAs extracted from wt, dcr1Δ, and swi6Δ strain harboring the empty vector, or vector expressing GFP-Swi6 or GFP-Swi6 3K→3A were subjected to RTPCR for the Forward strand and Reverse strand for the dh and act1. The PCR products were visualized by agarose gel electrophoresis and staining with ethidium bromide.
    Figure Legend Snippet: Specificity of binding of Swi6 to the dg.dh repeat sequences in vivo. ( A ) Expression of GFP-Swi6 and GFP-Swi6 3K→3A in swi6Δ cells. Extracts prepared from cells of swi6Δ strain expressing the control vector, GFP-Swi6 and GFP-Swi6 3K→3A were subjected to western blotting with anti-GFP (upper panel) and α-tubulin (lower panel) antibody. ( B ) Slot blot hybridization.RNAs extracted from cells expressing TAP-tagged Tas3 immunprecipitated with anti-CBP antibody and those expressing GFP-tagged Swi6 or GFP-tagged Swi6 3K→3A , immunoprecipitated with anti-GFP antibody, were radiolabeled at the 5’-end with [γ− 32 P]ATP and T4 polynucleotide kinase and used as a probe for slot blots on which equal amounts of the DNAs (100ng) corresponding to actl, dg, dh and dhk (cenH ) were blotted. The bound signal was visualized by autoradiography. ( C ) The size fractionated radiolabelled siRNA samples from indicated strains were subjected to electrophoresis in 8% acrylamide/7M urea gels and visualized by autoradiography. In parallel, unlabeled samples were similarly processed blotted and probed with radiolabelled snoRNAs (lower panel), followed by autoradiography. ( D ) No bidirectional transcription is observed in cells expressing swi6 3K→3A mutant. RNAs extracted from wt, dcr1Δ, and swi6Δ strain harboring the empty vector, or vector expressing GFP-Swi6 or GFP-Swi6 3K→3A were subjected to RTPCR for the Forward strand and Reverse strand for the dh and act1. The PCR products were visualized by agarose gel electrophoresis and staining with ethidium bromide.

    Techniques Used: Binding Assay, In Vivo, Expressing, Plasmid Preparation, Western Blot, Dot Blot, Hybridization, Immunoprecipitation, Autoradiography, Electrophoresis, Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining

    22) Product Images from "Deep small RNA sequencing from the nematode Ascaris reveals conservation, functional diversification, and novel developmental profiles"

    Article Title: Deep small RNA sequencing from the nematode Ascaris reveals conservation, functional diversification, and novel developmental profiles

    Journal: Genome Research

    doi: 10.1101/gr.121426.111

    Characteristics of Ascaris small RNAs. ( A ) 5′ end-labeled Ascaris small RNAs. Low-molecular-weight (LMW) enriched RNAs were treated with calf alkaline phosphatase and then 5′ end labeled with 32 P using T4 polynucleotide kinase. RNAs in
    Figure Legend Snippet: Characteristics of Ascaris small RNAs. ( A ) 5′ end-labeled Ascaris small RNAs. Low-molecular-weight (LMW) enriched RNAs were treated with calf alkaline phosphatase and then 5′ end labeled with 32 P using T4 polynucleotide kinase. RNAs in

    Techniques Used: Labeling, Molecular Weight

    23) Product Images from "System-wide analyses of the fission yeast poly(A)+ RNA interactome reveal insights into organization and function of RNA–protein complexes"

    Article Title: System-wide analyses of the fission yeast poly(A)+ RNA interactome reveal insights into organization and function of RNA–protein complexes

    Journal: Genome Research

    doi: 10.1101/gr.257006.119

    Only a subpopulation of the substoichiometric RBP Cyp4 interacts with RNA. ( A ) Crosslinking and immunoprecipitation (CLIP) analysis of FLAG-tagged proteins captured from WCEs of 4sU-labeled UV-crosslinked cells (3J/cm 2 ). After RNase digest, 5′ ends of crosslinked RNAs were radioactively labeled using T4 polynucleotide kinase and [γ- 32 P]ATP, and complexes were separated by gel electrophoresis followed by membrane transfer. A nontagged strain was included as control. All shown WCE samples are from the same membrane at the same exposure; irrelevant lanes were removed. ( B ) Volcano plot of a comparative RIC experiment for mtl1-1 as in 4E. Poly(A) + RNA association of Cyp4 is increased compared to WT. ( C ) CLIP analysis of FLAG-tagged Cyp4 in wild-type or mtl1-1 as in A . Non-irradiated cells and a nontagged strain were included as controls. (*) Cyp4 species that migrates at a higher apparent molecular weight. Although the band is apparently stabilized by crosslinking, it is present in noCL samples where no associated RNA can be detected (lanes 9 and 14 ). All shown WCE samples are from the same membrane at the same exposure; irrelevant lanes were removed.
    Figure Legend Snippet: Only a subpopulation of the substoichiometric RBP Cyp4 interacts with RNA. ( A ) Crosslinking and immunoprecipitation (CLIP) analysis of FLAG-tagged proteins captured from WCEs of 4sU-labeled UV-crosslinked cells (3J/cm 2 ). After RNase digest, 5′ ends of crosslinked RNAs were radioactively labeled using T4 polynucleotide kinase and [γ- 32 P]ATP, and complexes were separated by gel electrophoresis followed by membrane transfer. A nontagged strain was included as control. All shown WCE samples are from the same membrane at the same exposure; irrelevant lanes were removed. ( B ) Volcano plot of a comparative RIC experiment for mtl1-1 as in 4E. Poly(A) + RNA association of Cyp4 is increased compared to WT. ( C ) CLIP analysis of FLAG-tagged Cyp4 in wild-type or mtl1-1 as in A . Non-irradiated cells and a nontagged strain were included as controls. (*) Cyp4 species that migrates at a higher apparent molecular weight. Although the band is apparently stabilized by crosslinking, it is present in noCL samples where no associated RNA can be detected (lanes 9 and 14 ). All shown WCE samples are from the same membrane at the same exposure; irrelevant lanes were removed.

    Techniques Used: Immunoprecipitation, Cross-linking Immunoprecipitation, Labeling, Nucleic Acid Electrophoresis, Irradiation, Molecular Weight

    24) Product Images from "A novel method for the efficient and selective identification of 5-hydroxymethylcytosine in genomic DNA"

    Article Title: A novel method for the efficient and selective identification of 5-hydroxymethylcytosine in genomic DNA

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr051

    The β-gt can specifically modify 5hmC residues at a high efficiency. ( a ) Oligonucleotides that were either incubated in the presence or absence of the β-gt were digested with Taq I, treated with alkaline phosphatase, 5′-end labeled using T4 polynucleotide kinase and digested to 5′-mononucleotides using DNase I and Snake Venom Phosphodiesterase. Radiolabeled mononucleotides were analyzed by two-dimensional TLC. C, 3′-deoxyribocytosine-5′-monophosphate; T, 3′-deoxyribothymidine-5′-monophosphate; 5meC, 3′-deoxyribo-N5-methylcytosine-5′-monophosphate; 5hmC, 3′-deoxyribo-N5-hydroxymethylcytosine-5′-monophosphate. ( b ) HPLC coupled to tandem mass spectrometry was used to measure the efficiency of the β-gt reaction. Substrates analyzed were 2.7 kb linear PCR products of pUC18: the dC substrate contained only cytosine residues; the 5meC substrate was created by methylating the CpG dinucleotide of the cytosine substrate; the 5hmC substrate was created by using d5hmC in place of dCTP in the PCR reactions; the β-glu-5hmC substrate was created by incubating the 5hmC substrate with the β-gt in the presence of UDP-glucose. Control DNA was prepared from salmon sperm. LC/MS/MS chromatograms of the cytosine residues from each of the substrates are presented. Abbreviations: dC, 3′-deoxyribocytosine; 5me(dC), 3′-deoxyribo-N5-methylcytosine; 5hm(dC), 3′-deoxyribo-N5-hydroxymethylcytosine; 5-glu-hm(dC), 3′-deoxyribo-N5-(β- d -glucosyl(hydroxymethyl))cytosine. Asterisks indictes that cytosines are only 5meC modified at CpG sequences.
    Figure Legend Snippet: The β-gt can specifically modify 5hmC residues at a high efficiency. ( a ) Oligonucleotides that were either incubated in the presence or absence of the β-gt were digested with Taq I, treated with alkaline phosphatase, 5′-end labeled using T4 polynucleotide kinase and digested to 5′-mononucleotides using DNase I and Snake Venom Phosphodiesterase. Radiolabeled mononucleotides were analyzed by two-dimensional TLC. C, 3′-deoxyribocytosine-5′-monophosphate; T, 3′-deoxyribothymidine-5′-monophosphate; 5meC, 3′-deoxyribo-N5-methylcytosine-5′-monophosphate; 5hmC, 3′-deoxyribo-N5-hydroxymethylcytosine-5′-monophosphate. ( b ) HPLC coupled to tandem mass spectrometry was used to measure the efficiency of the β-gt reaction. Substrates analyzed were 2.7 kb linear PCR products of pUC18: the dC substrate contained only cytosine residues; the 5meC substrate was created by methylating the CpG dinucleotide of the cytosine substrate; the 5hmC substrate was created by using d5hmC in place of dCTP in the PCR reactions; the β-glu-5hmC substrate was created by incubating the 5hmC substrate with the β-gt in the presence of UDP-glucose. Control DNA was prepared from salmon sperm. LC/MS/MS chromatograms of the cytosine residues from each of the substrates are presented. Abbreviations: dC, 3′-deoxyribocytosine; 5me(dC), 3′-deoxyribo-N5-methylcytosine; 5hm(dC), 3′-deoxyribo-N5-hydroxymethylcytosine; 5-glu-hm(dC), 3′-deoxyribo-N5-(β- d -glucosyl(hydroxymethyl))cytosine. Asterisks indictes that cytosines are only 5meC modified at CpG sequences.

    Techniques Used: Incubation, Labeling, Thin Layer Chromatography, High Performance Liquid Chromatography, Mass Spectrometry, Polymerase Chain Reaction, Liquid Chromatography with Mass Spectroscopy, Modification

    25) Product Images from "Programmable RNA cleavage and recognition by a natural CRISPR-Cas9 system from Neisseria meningitidis"

    Article Title: Programmable RNA cleavage and recognition by a natural CRISPR-Cas9 system from Neisseria meningitidis

    Journal: Molecular cell

    doi: 10.1016/j.molcel.2018.01.025

    NmeCas9-catalyzed RNA cleavage is programmable (A) A schematic depicting the ssRNA target 25 and three matching (crRNAs, crRNA-sp25-wt, walk-2, and walk+3). Yellow, crRNA spacer; grey, crRNA repeat; bold, rPAM; green, FAM label; red arrows, predominant RNA cleavage sites mapped out in (B). (B) RNA cleavage site mapping experiment. SsRNA target 25 and the three crRNAs used are shown in (A). The NmeCas9 cleavage products and RNase T1 and hydrolysis ladders were subjected to 3′ de-phosphorylation by T4 Polynucleotide kinase, and separated by 15% denaturing PAGE. T1, RNase T1 ladder; OH − , hydrolysis ladder. −2, walk-2; +3, walk+3; wt, crRNA-sp25. Sites of G cleavage by RNase T1 are indicated; sites of NmeCas9 cleavage (G30, A27, G25 for the three crRNAs, respectively) are marked by red arrows. (C) Serial single nt mutants of crRNA-sp25 were analyzed for NmeCas9-catalyzed RNA cleavage. M1 through M19, single nt mutation introduced into every other position of the crRNA spacer. The location and sequence of each mutation (in red) are shown at the top. Yellow, crRNA spacer; grey, crRNA repeat; red arrow, RNA cleavage site. Non-cog, crRNA-sp23. (D) A schematic depicting the ssRNA target 9 and the two matching crRNAs (crRNA-sp9-wt and walk-1). Yellow, crRNA spacer; grey, crRNA repeat; bold, rPAM; red arrows, predicted RNA cleavage sites; green, FAM label; magenta, Cy5 label. (E) NmeCas9’s ribonuclease activity is re-programmable on a different RNA substrate. The two crRNAs shown in (D) were assayed for in vitro cleavage on ssRNA target 9. The same denaturing gel is subjected to FAM (left) and Cy5 (right) detection.
    Figure Legend Snippet: NmeCas9-catalyzed RNA cleavage is programmable (A) A schematic depicting the ssRNA target 25 and three matching (crRNAs, crRNA-sp25-wt, walk-2, and walk+3). Yellow, crRNA spacer; grey, crRNA repeat; bold, rPAM; green, FAM label; red arrows, predominant RNA cleavage sites mapped out in (B). (B) RNA cleavage site mapping experiment. SsRNA target 25 and the three crRNAs used are shown in (A). The NmeCas9 cleavage products and RNase T1 and hydrolysis ladders were subjected to 3′ de-phosphorylation by T4 Polynucleotide kinase, and separated by 15% denaturing PAGE. T1, RNase T1 ladder; OH − , hydrolysis ladder. −2, walk-2; +3, walk+3; wt, crRNA-sp25. Sites of G cleavage by RNase T1 are indicated; sites of NmeCas9 cleavage (G30, A27, G25 for the three crRNAs, respectively) are marked by red arrows. (C) Serial single nt mutants of crRNA-sp25 were analyzed for NmeCas9-catalyzed RNA cleavage. M1 through M19, single nt mutation introduced into every other position of the crRNA spacer. The location and sequence of each mutation (in red) are shown at the top. Yellow, crRNA spacer; grey, crRNA repeat; red arrow, RNA cleavage site. Non-cog, crRNA-sp23. (D) A schematic depicting the ssRNA target 9 and the two matching crRNAs (crRNA-sp9-wt and walk-1). Yellow, crRNA spacer; grey, crRNA repeat; bold, rPAM; red arrows, predicted RNA cleavage sites; green, FAM label; magenta, Cy5 label. (E) NmeCas9’s ribonuclease activity is re-programmable on a different RNA substrate. The two crRNAs shown in (D) were assayed for in vitro cleavage on ssRNA target 9. The same denaturing gel is subjected to FAM (left) and Cy5 (right) detection.

    Techniques Used: De-Phosphorylation Assay, Polyacrylamide Gel Electrophoresis, Mutagenesis, Sequencing, Activity Assay, In Vitro

    26) Product Images from "A universal method to produce in vitro transcripts with homogeneous 3? ends"

    Article Title: A universal method to produce in vitro transcripts with homogeneous 3? ends

    Journal: Nucleic Acids Research

    doi:

    Removal of the terminal 2′,3′ cyclic phosphate group of the released tRNA resulting from the HDV ribozyme cleavage reaction. Treatment with T4 polynucleotide kinase (T4 PNK) leads to the removal of the phosphate group and therefore to a reduced net charge of the RNA. This can be observed by a reduced electrophoretic mobility on a denaturing 10% polyacrylamide gel in comparison with the untreated control (mock incubation without T4 PNK).
    Figure Legend Snippet: Removal of the terminal 2′,3′ cyclic phosphate group of the released tRNA resulting from the HDV ribozyme cleavage reaction. Treatment with T4 polynucleotide kinase (T4 PNK) leads to the removal of the phosphate group and therefore to a reduced net charge of the RNA. This can be observed by a reduced electrophoretic mobility on a denaturing 10% polyacrylamide gel in comparison with the untreated control (mock incubation without T4 PNK).

    Techniques Used: Incubation

    27) Product Images from "The interplay of RNA:DNA hybrid structure and G-quadruplexes determines the outcome of R-loop-replisome collisions"

    Article Title: The interplay of RNA:DNA hybrid structure and G-quadruplexes determines the outcome of R-loop-replisome collisions

    Journal: bioRxiv

    doi: 10.1101/2021.07.16.452753

    R-loop transcripts can prime leading strand restart after CD R-loop-replisome collisions. ( A ) Replication products obtained on mock- or T4 PNK-treated CD R-loop-containing templates in absence or presence RNase H1. ( B ) Products obtained on CD T4 PNK-treated R-loop templates in absence or presence of DDK. Relative signal intensity for restart product is quantified on the right. ( C ) Replication products obtained on mock- or T4 PNK-treated CD R-loop-containing templates in absence or presence RNase H1. ( D ) Schematic illustrating replication products observed in A. ( E ) RNase H1 titration into reactions with CD R-loop template. ( F ) Model for leading strand restart at R-loop transcript after replisome encounter with CD R-loop harboring 5’ RNA flap and RNA nick.
    Figure Legend Snippet: R-loop transcripts can prime leading strand restart after CD R-loop-replisome collisions. ( A ) Replication products obtained on mock- or T4 PNK-treated CD R-loop-containing templates in absence or presence RNase H1. ( B ) Products obtained on CD T4 PNK-treated R-loop templates in absence or presence of DDK. Relative signal intensity for restart product is quantified on the right. ( C ) Replication products obtained on mock- or T4 PNK-treated CD R-loop-containing templates in absence or presence RNase H1. ( D ) Schematic illustrating replication products observed in A. ( E ) RNase H1 titration into reactions with CD R-loop template. ( F ) Model for leading strand restart at R-loop transcript after replisome encounter with CD R-loop harboring 5’ RNA flap and RNA nick.

    Techniques Used: Titration

    R-loop transcripts can prime leading strand restart after CD R-loop-replisome collisions. ( A ) Polymerase assay with T4 PNK-treated CD R-loop template in presence of Pol α, Pol ε, or Pol δ, as indicated. All reactions include RFC, PCNA, RPA, dNTPs, NTPs and α-[ 32 P]-dATP. The concentrations of reaction components are equivalent to those used in the replication assay. ( B ) Replication products obtained on RNase H1-treated CD R-loop templates with or without Pol δ. Reactions were performed either in the presence of 1 nM (lanes 1+2) or 0.06 nM (lanes 3+4) RNase H1 to induce resolution or nicking of the RNA:DNA hybrid, respectively. ( C ) Left: Purified RNase H2. Right: Replication products obtained on CD and HO templates in the presence of sub-saturating levels of RNase H2.
    Figure Legend Snippet: R-loop transcripts can prime leading strand restart after CD R-loop-replisome collisions. ( A ) Polymerase assay with T4 PNK-treated CD R-loop template in presence of Pol α, Pol ε, or Pol δ, as indicated. All reactions include RFC, PCNA, RPA, dNTPs, NTPs and α-[ 32 P]-dATP. The concentrations of reaction components are equivalent to those used in the replication assay. ( B ) Replication products obtained on RNase H1-treated CD R-loop templates with or without Pol δ. Reactions were performed either in the presence of 1 nM (lanes 1+2) or 0.06 nM (lanes 3+4) RNase H1 to induce resolution or nicking of the RNA:DNA hybrid, respectively. ( C ) Left: Purified RNase H2. Right: Replication products obtained on CD and HO templates in the presence of sub-saturating levels of RNase H2.

    Techniques Used: Recombinase Polymerase Amplification, Purification

    28) Product Images from "Phosphorylation of Large T Antigen Regulates Merkel Cell Polyomavirus Replication"

    Article Title: Phosphorylation of Large T Antigen Regulates Merkel Cell Polyomavirus Replication

    Journal: Cancers

    doi: 10.3390/cancers6031464

    MCPyV LT phospho-mutants bind the viral Ori with different affinities. ( A ) Schematic of the MCPyV Ori and the EMSA Probe. Only one strand of DNA is shown for clarity. The MCPyV Ori sequence was cloned from the R17a isolate of MCPyV into a pcDNA4c vector [ 14 ]. This origin was used for replication assays ( Figure 3 and Figure 4 ). Consensus GAGGC pentanucleotide repeats which are recognized by the OBD of LT are marked with arrows and numbered as was reported by Kwun et al. [ 31 ]. Arrows with dashed lines indicate imperfect pentanucleotides. The EMSA Probe was generated by PCR amplification of the indicated region of the MCPyV Ori. This PCR product was 5' end-labeled with [ 32 P-γ] ATP using T4 polynucleotide kinase (indicated by red asterisk); ( B ) Western blot of purified MCPyV proteins (0.25 µg) used in EMSA. The buffer control contained residual TEV protease (also in LT samples); ( C ) Electromobility shift assays were performed with the EMSA probe in ( A ) and increasing amounts of MCPyV wild type or phospho-mutant LT affinity purified from HEK 293 cells. Reactions with buffer and residual TEV protease served as a negative control (first lane). Positions of free probe and LT bound probe are indicated. Data in ( B , C ) are representative of at least three experiments.
    Figure Legend Snippet: MCPyV LT phospho-mutants bind the viral Ori with different affinities. ( A ) Schematic of the MCPyV Ori and the EMSA Probe. Only one strand of DNA is shown for clarity. The MCPyV Ori sequence was cloned from the R17a isolate of MCPyV into a pcDNA4c vector [ 14 ]. This origin was used for replication assays ( Figure 3 and Figure 4 ). Consensus GAGGC pentanucleotide repeats which are recognized by the OBD of LT are marked with arrows and numbered as was reported by Kwun et al. [ 31 ]. Arrows with dashed lines indicate imperfect pentanucleotides. The EMSA Probe was generated by PCR amplification of the indicated region of the MCPyV Ori. This PCR product was 5' end-labeled with [ 32 P-γ] ATP using T4 polynucleotide kinase (indicated by red asterisk); ( B ) Western blot of purified MCPyV proteins (0.25 µg) used in EMSA. The buffer control contained residual TEV protease (also in LT samples); ( C ) Electromobility shift assays were performed with the EMSA probe in ( A ) and increasing amounts of MCPyV wild type or phospho-mutant LT affinity purified from HEK 293 cells. Reactions with buffer and residual TEV protease served as a negative control (first lane). Positions of free probe and LT bound probe are indicated. Data in ( B , C ) are representative of at least three experiments.

    Techniques Used: Sequencing, Clone Assay, Plasmid Preparation, Generated, Polymerase Chain Reaction, Amplification, Labeling, Western Blot, Purification, Mutagenesis, Affinity Purification, Negative Control

    29) Product Images from "tRNAs Promote Nuclear Import of HIV-1 Intracellular Reverse Transcription Complexes Nuclear Import of HIV-1 Complex Relies on a Surprise Player: tRNA"

    Article Title: tRNAs Promote Nuclear Import of HIV-1 Intracellular Reverse Transcription Complexes Nuclear Import of HIV-1 Complex Relies on a Surprise Player: tRNA

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.0040332

    The 60S NA Fraction Contains Small RNA Molecules (A) Equal amounts of nucleic acids purified from the active Phenyl-Sepharose fraction (60S NA) were subjected to nuclease S7 (NS7) or DNAse-free RNAse (RNAse) treatment and analyzed by 15% denaturing PAGE followed by silver staining. Total HeLa RNA (totRNA) was used as control for nuclease and RNAse treatments. ST1, oligonucleotide size markers (range, 8–32 nucleotides); ST2, size markers pBP322DNA-MspI. (B) 60S NA loses its ability stimulate RTC nuclear import after nuclease or RNAse treatment. Nuclear import of YOYO-1 labelled RTCs in permeabilized primary human macrophages in the presence of 1× energy-regenerating system and 60S (0.5 mg/ml), 60S NA (1 μg), 60S NA digested with NS7, 60S NA digested with RNAse (1-μg starting material), 21mer siRNA (1 μg), total HeLa RNA (totRNA, 1μg), or buffer (ctr –). (C) 60S NA fraction can be specifically 3′-end radiolabelled by T4 RNA ligase. Following 3′-end labelling with 5′-[ 32 P]pCp, samples were analyzed by 15% denaturing PAGE and visualized by Storm 860 PhosphoImager. Total HeLa RNA (totRNA) was used as a control for T4 RNA ligase reaction. ST1 and ST2 are 5′-end radiolabelled oligonucleotide size markers as in (A).
    Figure Legend Snippet: The 60S NA Fraction Contains Small RNA Molecules (A) Equal amounts of nucleic acids purified from the active Phenyl-Sepharose fraction (60S NA) were subjected to nuclease S7 (NS7) or DNAse-free RNAse (RNAse) treatment and analyzed by 15% denaturing PAGE followed by silver staining. Total HeLa RNA (totRNA) was used as control for nuclease and RNAse treatments. ST1, oligonucleotide size markers (range, 8–32 nucleotides); ST2, size markers pBP322DNA-MspI. (B) 60S NA loses its ability stimulate RTC nuclear import after nuclease or RNAse treatment. Nuclear import of YOYO-1 labelled RTCs in permeabilized primary human macrophages in the presence of 1× energy-regenerating system and 60S (0.5 mg/ml), 60S NA (1 μg), 60S NA digested with NS7, 60S NA digested with RNAse (1-μg starting material), 21mer siRNA (1 μg), total HeLa RNA (totRNA, 1μg), or buffer (ctr –). (C) 60S NA fraction can be specifically 3′-end radiolabelled by T4 RNA ligase. Following 3′-end labelling with 5′-[ 32 P]pCp, samples were analyzed by 15% denaturing PAGE and visualized by Storm 860 PhosphoImager. Total HeLa RNA (totRNA) was used as a control for T4 RNA ligase reaction. ST1 and ST2 are 5′-end radiolabelled oligonucleotide size markers as in (A).

    Techniques Used: Purification, Polyacrylamide Gel Electrophoresis, Silver Staining

    30) Product Images from "Single Nucleotide Resolution RNA–Protein Cross-Linking Mass Spectrometry: A Simple Extension of the CLIR-MS Workflow"

    Article Title: Single Nucleotide Resolution RNA–Protein Cross-Linking Mass Spectrometry: A Simple Extension of the CLIR-MS Workflow

    Journal: Analytical Chemistry

    doi: 10.1021/acs.analchem.1c02384

    Post-digest Labeling Reaction Scheme (A) The γ-phosphate of 18 O 4 -γ-ATP is transferred to the 5′-hydroxy group of a cross-linked RNA oligonucleotide. T4-PNK also catalyzes a 3′-dephosphorylation reaction. (B) Simulation of the isotopic distribution 33 of a cross-linked peptide (PEPTIDER cross-linked to a GU dinucleotide, doubly charged) for a 1:1 mixture of light and heavy ATP using single-phosphate labeling.
    Figure Legend Snippet: Post-digest Labeling Reaction Scheme (A) The γ-phosphate of 18 O 4 -γ-ATP is transferred to the 5′-hydroxy group of a cross-linked RNA oligonucleotide. T4-PNK also catalyzes a 3′-dephosphorylation reaction. (B) Simulation of the isotopic distribution 33 of a cross-linked peptide (PEPTIDER cross-linked to a GU dinucleotide, doubly charged) for a 1:1 mixture of light and heavy ATP using single-phosphate labeling.

    Techniques Used: Labeling, De-Phosphorylation Assay

    31) Product Images from "Ribozyme-enhanced single-stranded Ago2-processed interfering RNA triggers efficient gene silencing with fewer off-target effects"

    Article Title: Ribozyme-enhanced single-stranded Ago2-processed interfering RNA triggers efficient gene silencing with fewer off-target effects

    Journal: Nature Communications

    doi: 10.1038/ncomms9430

    Cleavage at the 3′ end of the siRNA precursor by the HDV ribozyme enhances its expression and knockdown efficiency. ( a ) Secondary structure of saiRNA with HDV ribozyme at the 3′ end. The left part represents the saiRNA, with the guide sequence in red. The right part represents the HDV ribozyme. The blue arrow indicates the HDV ribozyme cleavage site. ( b ) Cleavage of the HDV ribozyme in vitro . The saiRNAs fused with a wild-type (saiRNA-RZ) or mutant (saiRNA-mRZ) HDV ribozyme at the 3′ end were transcribed in vitro by the T7 RNA polymerase. The transcripts were treated with T4 PNK without ATP and then analysed on a 20% denaturing polyacrylamide gel by ethidium bromide (EB) staining. ( c ) Schematic diagram of the shRNA and saiRNA with or without the HDV ribozyme (HDV-RZ) downstream of the 3′ end of the siRNA precursor. Expression of the siRNA precursors in mammalian cells was driven by an H1 promoter. The blue arrow indicates the cleavage site of HDV-RZ, and nucleotides marked in red represent the guide strand. ( d ) Knockdown efficiency and processing of shGP and saiGP transcribed by the H1 promoter as described in c . ( e , f ) Knockdown efficiency and processing of shRNA and saiRNA targeting the laminC (LC) and P53 genes in HEK293 cells. Luciferase and Northern blotting assays were performed as in d . Changes in protein levels on siRNA expression were determined by western blotting assays with antibodies recognizing laminC or p53. β-actin served as the loading control. ( g ) Effect of transfection dosages on the repression activity of shGP and saiGP-RZ. ( h ) Knockdown efficiency of the endogenous P53 gene by shRNA or saiRNA stably expressed in HEK293 cells transduced with lentiviral vectors. HEK293 cells were transduced with lentivirus encoding shp53, saip53 or saip53-RZ at different MOIs and selected by puromycin for 6 days. Expression of the P53 gene was measured by western blotting as in f . All the error bars represent the s.d. of three independent measurements.
    Figure Legend Snippet: Cleavage at the 3′ end of the siRNA precursor by the HDV ribozyme enhances its expression and knockdown efficiency. ( a ) Secondary structure of saiRNA with HDV ribozyme at the 3′ end. The left part represents the saiRNA, with the guide sequence in red. The right part represents the HDV ribozyme. The blue arrow indicates the HDV ribozyme cleavage site. ( b ) Cleavage of the HDV ribozyme in vitro . The saiRNAs fused with a wild-type (saiRNA-RZ) or mutant (saiRNA-mRZ) HDV ribozyme at the 3′ end were transcribed in vitro by the T7 RNA polymerase. The transcripts were treated with T4 PNK without ATP and then analysed on a 20% denaturing polyacrylamide gel by ethidium bromide (EB) staining. ( c ) Schematic diagram of the shRNA and saiRNA with or without the HDV ribozyme (HDV-RZ) downstream of the 3′ end of the siRNA precursor. Expression of the siRNA precursors in mammalian cells was driven by an H1 promoter. The blue arrow indicates the cleavage site of HDV-RZ, and nucleotides marked in red represent the guide strand. ( d ) Knockdown efficiency and processing of shGP and saiGP transcribed by the H1 promoter as described in c . ( e , f ) Knockdown efficiency and processing of shRNA and saiRNA targeting the laminC (LC) and P53 genes in HEK293 cells. Luciferase and Northern blotting assays were performed as in d . Changes in protein levels on siRNA expression were determined by western blotting assays with antibodies recognizing laminC or p53. β-actin served as the loading control. ( g ) Effect of transfection dosages on the repression activity of shGP and saiGP-RZ. ( h ) Knockdown efficiency of the endogenous P53 gene by shRNA or saiRNA stably expressed in HEK293 cells transduced with lentiviral vectors. HEK293 cells were transduced with lentivirus encoding shp53, saip53 or saip53-RZ at different MOIs and selected by puromycin for 6 days. Expression of the P53 gene was measured by western blotting as in f . All the error bars represent the s.d. of three independent measurements.

    Techniques Used: Expressing, Sequencing, In Vitro, Mutagenesis, Staining, shRNA, Luciferase, Northern Blot, Western Blot, Transfection, Activity Assay, Stable Transfection, Transduction

    32) Product Images from "High-throughput determination of RNA structure by proximity ligation"

    Article Title: High-throughput determination of RNA structure by proximity ligation

    Journal: Nature biotechnology

    doi: 10.1038/nbt.3289

    RNA Proximity Ligation identifies structurally proximate regions within the complex secondary structures of S. cerevisiae ribosomal RNAs. a.) A schematic representation of the RPL method. Whole cells are spheroplasted with zymolyase and RNA is allowed to react with endogenous RNases. RNA ends are repaired in situ via T4 PNK to yield 5′-phosphate termini. Complexes are ligated overnight in the presence of T4 RNA Ligase I. Ligation products are cleaned up via acid guanidinium-phenol and subsequent DNase treatment, and subjected to Illumina TruSeq RNA-seq library preparation. These libraries are sequenced to map and count ligation junctions; b.-c.) We examined the distribution of ligation junctions as a function of distance from known base-pair partners in the 25S/5.8S rRNA and 18S rRNAs. Ligation products capture the structural proximity implied by base-pairing relationships, as evidenced by the enrichment for ligation junctions immediately near paired bases. Y-axes are shown as ligation counts per million reads analyzed. d.) Contact probability map for the eukaryotic 5.8S/25S rRNA based on RPL scores, which are calculated from the frequencies of ligation events between pairs of 21 nt windows ( Methods ). Lower inset : Ligation events, shown for bases 1300 to 1475 of the LSU rRNA in orange, primarily occur across digested single-stranded loops. RPL scores effectively smooth this noisy signal and are enriched for pairs of interacting regions. Plotted here are the 8,463 ligation events where both nucleotides fall within the displayed domain (compared to 17,029 ligation events where one nucleotide falls within the displayed domain and one does not, not shown). Right inset: RPL scores localize known pseudo-knots in the LSU rRNA structure, such as the interaction between bases 1727-1812 (shown in red) and bases 1941 – 2038 (shown in blue).
    Figure Legend Snippet: RNA Proximity Ligation identifies structurally proximate regions within the complex secondary structures of S. cerevisiae ribosomal RNAs. a.) A schematic representation of the RPL method. Whole cells are spheroplasted with zymolyase and RNA is allowed to react with endogenous RNases. RNA ends are repaired in situ via T4 PNK to yield 5′-phosphate termini. Complexes are ligated overnight in the presence of T4 RNA Ligase I. Ligation products are cleaned up via acid guanidinium-phenol and subsequent DNase treatment, and subjected to Illumina TruSeq RNA-seq library preparation. These libraries are sequenced to map and count ligation junctions; b.-c.) We examined the distribution of ligation junctions as a function of distance from known base-pair partners in the 25S/5.8S rRNA and 18S rRNAs. Ligation products capture the structural proximity implied by base-pairing relationships, as evidenced by the enrichment for ligation junctions immediately near paired bases. Y-axes are shown as ligation counts per million reads analyzed. d.) Contact probability map for the eukaryotic 5.8S/25S rRNA based on RPL scores, which are calculated from the frequencies of ligation events between pairs of 21 nt windows ( Methods ). Lower inset : Ligation events, shown for bases 1300 to 1475 of the LSU rRNA in orange, primarily occur across digested single-stranded loops. RPL scores effectively smooth this noisy signal and are enriched for pairs of interacting regions. Plotted here are the 8,463 ligation events where both nucleotides fall within the displayed domain (compared to 17,029 ligation events where one nucleotide falls within the displayed domain and one does not, not shown). Right inset: RPL scores localize known pseudo-knots in the LSU rRNA structure, such as the interaction between bases 1727-1812 (shown in red) and bases 1941 – 2038 (shown in blue).

    Techniques Used: Ligation, In Situ, RNA Sequencing Assay

    33) Product Images from "A Simple and Cost-Effective Approach for In Vitro Production of Sliced siRNAs as Potent Triggers for RNAi"

    Article Title: A Simple and Cost-Effective Approach for In Vitro Production of Sliced siRNAs as Potent Triggers for RNAi

    Journal: Molecular Therapy. Nucleic Acids

    doi: 10.1016/j.omtn.2017.07.008

    Manipulation of 5′ppp-Triggered Interferon Response HEK293 cells were transfected with poly(I:C) or several tsli-siRNAs. The final concentration of 10 nM for each RNAi reagent was used in transfection for qPCR assay. Gene expression level changes in OAS1, IRF9, CDKL, and IFNB relative to GAPDH were measured by qPCR. (A) Mild interferon response was observed from all four tsli-siRNAs, with tsli-RRM2 having the strongest response among them. G-tsli-Stat3 exhibited a much stronger response than tsli-Stat3, and GG-tsli-Stat3 reversed this effect to some extent. (B) CIP treatment minimized the strong interferon response by G-tsli-Stat3. (C) CIP treatment minimized and T4 PNK treatment elevated the interferon response by tsli-RRM2. Fold changes in gene expression were normalized to untreated HEK293 cells. Details of qPCR procedure and results calculation were provided in the Materials and Methods . Error bars indicate SD.
    Figure Legend Snippet: Manipulation of 5′ppp-Triggered Interferon Response HEK293 cells were transfected with poly(I:C) or several tsli-siRNAs. The final concentration of 10 nM for each RNAi reagent was used in transfection for qPCR assay. Gene expression level changes in OAS1, IRF9, CDKL, and IFNB relative to GAPDH were measured by qPCR. (A) Mild interferon response was observed from all four tsli-siRNAs, with tsli-RRM2 having the strongest response among them. G-tsli-Stat3 exhibited a much stronger response than tsli-Stat3, and GG-tsli-Stat3 reversed this effect to some extent. (B) CIP treatment minimized the strong interferon response by G-tsli-Stat3. (C) CIP treatment minimized and T4 PNK treatment elevated the interferon response by tsli-RRM2. Fold changes in gene expression were normalized to untreated HEK293 cells. Details of qPCR procedure and results calculation were provided in the Materials and Methods . Error bars indicate SD.

    Techniques Used: Transfection, Concentration Assay, Real-time Polymerase Chain Reaction, Expressing

    34) Product Images from "A new G-triplex-based strategy for sensitivity enhancement of the detection of endonuclease activity and inhibition †"

    Article Title: A new G-triplex-based strategy for sensitivity enhancement of the detection of endonuclease activity and inhibition †

    Journal: RSC Advances

    doi: 10.1039/d1ra04203c

    Responses of G3 (2.5 μM) to the presence of various enzymes (100 U mL −1 ) including EcoRI endonuclease, T4 polynucleotide kinase, BamHI endonuclease, endonuclease IV, and Nt.BstNBI.
    Figure Legend Snippet: Responses of G3 (2.5 μM) to the presence of various enzymes (100 U mL −1 ) including EcoRI endonuclease, T4 polynucleotide kinase, BamHI endonuclease, endonuclease IV, and Nt.BstNBI.

    Techniques Used:

    35) Product Images from "Single Nucleotide Resolution RNA–Protein Cross-Linking Mass Spectrometry: A Simple Extension of the CLIR-MS Workflow"

    Article Title: Single Nucleotide Resolution RNA–Protein Cross-Linking Mass Spectrometry: A Simple Extension of the CLIR-MS Workflow

    Journal: Analytical Chemistry

    doi: 10.1021/acs.analchem.1c02384

    Post-digest Labeling Reaction Scheme (A) The γ-phosphate of 18 O 4 -γ-ATP is transferred to the 5′-hydroxy group of a cross-linked RNA oligonucleotide. T4-PNK also catalyzes a 3′-dephosphorylation reaction. (B) Simulation of the isotopic distribution 33 of a cross-linked peptide (PEPTIDER cross-linked to a GU dinucleotide, doubly charged) for a 1:1 mixture of light and heavy ATP using single-phosphate labeling.
    Figure Legend Snippet: Post-digest Labeling Reaction Scheme (A) The γ-phosphate of 18 O 4 -γ-ATP is transferred to the 5′-hydroxy group of a cross-linked RNA oligonucleotide. T4-PNK also catalyzes a 3′-dephosphorylation reaction. (B) Simulation of the isotopic distribution 33 of a cross-linked peptide (PEPTIDER cross-linked to a GU dinucleotide, doubly charged) for a 1:1 mixture of light and heavy ATP using single-phosphate labeling.

    Techniques Used: Labeling, De-Phosphorylation Assay

    36) Product Images from "Single Nucleotide Resolution RNA–Protein Cross-Linking Mass Spectrometry: A Simple Extension of the CLIR-MS Workflow"

    Article Title: Single Nucleotide Resolution RNA–Protein Cross-Linking Mass Spectrometry: A Simple Extension of the CLIR-MS Workflow

    Journal: Analytical Chemistry

    doi: 10.1021/acs.analchem.1c02384

    Post-digest Labeling Reaction Scheme (A) The γ-phosphate of 18 O 4 -γ-ATP is transferred to the 5′-hydroxy group of a cross-linked RNA oligonucleotide. T4-PNK also catalyzes a 3′-dephosphorylation reaction. (B) Simulation of the isotopic distribution 33 of a cross-linked peptide (PEPTIDER cross-linked to a GU dinucleotide, doubly charged) for a 1:1 mixture of light and heavy ATP using single-phosphate labeling.
    Figure Legend Snippet: Post-digest Labeling Reaction Scheme (A) The γ-phosphate of 18 O 4 -γ-ATP is transferred to the 5′-hydroxy group of a cross-linked RNA oligonucleotide. T4-PNK also catalyzes a 3′-dephosphorylation reaction. (B) Simulation of the isotopic distribution 33 of a cross-linked peptide (PEPTIDER cross-linked to a GU dinucleotide, doubly charged) for a 1:1 mixture of light and heavy ATP using single-phosphate labeling.

    Techniques Used: Labeling, De-Phosphorylation Assay

    37) Product Images from "Cleavage of DNA and RNA by PLD3 and PLD4 limits autoinflammatory triggering by multiple sensors"

    Article Title: Cleavage of DNA and RNA by PLD3 and PLD4 limits autoinflammatory triggering by multiple sensors

    Journal: Nature Communications

    doi: 10.1038/s41467-021-26150-w

    Accumulation of small nucleic acids in Pld3 −/− Pld4 −/− and Unc93b1 3d/3d Pld3 −/− Pld4 −/− cells. Sequence length distributions of RNA isolated from the livers of a 2-week-old C57BL/6 (B6), Pld3 −/− , Pld4 −/− or Pld3 −/− Pld4 −/− mice, b Two-month-old B6, Tlr9 CpG11/CpG11 , Tlr9 CpG11/CpG11 Pld3 −/− Pld4 −/− or Unc93b1 3d/3d Pld3 −/− Pld4 −/− mice. Shown are means and SEM of three mice/groups. In a, p values show a significant difference in Pld3 −/− Pld4 −/− over C57BL/6 by unpaired two-tailed T -test. In ( b ), p values are shown for Tlr9 CpG11/CpG11 Pld3 −/− Pld4 −/− or Unc93b1 3d/3d Pld3 −/− Pld4 −/− sequences, compared to C57BL/6 controls. Raw data for a , b are provided in Supplementary Tables 3 and 4 . c Biochemical analysis of lysosome-associated nucleic acids. Lysosomal fractions were enriched from spleen or liver tissue of Unc93b1 3d/3d Pld3 −/− Pld4 −/− or Unc93b1 3d/3d mice, or from HEK Blue-hTLR9 cells that were PLD3-sufficient or -deficient, nucleic acids were 5′ labeled with [ 32 P] phosphate using T4 polynucleotide kinase and electrophoresed on histidine-buffered 20% polyacrylamide gel. Radioactivity was revealed by phosphorimaging. Marker lanes show oligo dT lengths in nt.
    Figure Legend Snippet: Accumulation of small nucleic acids in Pld3 −/− Pld4 −/− and Unc93b1 3d/3d Pld3 −/− Pld4 −/− cells. Sequence length distributions of RNA isolated from the livers of a 2-week-old C57BL/6 (B6), Pld3 −/− , Pld4 −/− or Pld3 −/− Pld4 −/− mice, b Two-month-old B6, Tlr9 CpG11/CpG11 , Tlr9 CpG11/CpG11 Pld3 −/− Pld4 −/− or Unc93b1 3d/3d Pld3 −/− Pld4 −/− mice. Shown are means and SEM of three mice/groups. In a, p values show a significant difference in Pld3 −/− Pld4 −/− over C57BL/6 by unpaired two-tailed T -test. In ( b ), p values are shown for Tlr9 CpG11/CpG11 Pld3 −/− Pld4 −/− or Unc93b1 3d/3d Pld3 −/− Pld4 −/− sequences, compared to C57BL/6 controls. Raw data for a , b are provided in Supplementary Tables 3 and 4 . c Biochemical analysis of lysosome-associated nucleic acids. Lysosomal fractions were enriched from spleen or liver tissue of Unc93b1 3d/3d Pld3 −/− Pld4 −/− or Unc93b1 3d/3d mice, or from HEK Blue-hTLR9 cells that were PLD3-sufficient or -deficient, nucleic acids were 5′ labeled with [ 32 P] phosphate using T4 polynucleotide kinase and electrophoresed on histidine-buffered 20% polyacrylamide gel. Radioactivity was revealed by phosphorimaging. Marker lanes show oligo dT lengths in nt.

    Techniques Used: Sequencing, Isolation, Mouse Assay, Two Tailed Test, Labeling, Radioactivity, Marker

    38) Product Images from "Kaposi's Sarcoma-Associated Herpesvirus Rta Tetramers Make High-Affinity Interactions with Repetitive DNA Elements in the Mta Promoter To Stimulate DNA Binding of RBP-Jk/CSL ▿Kaposi's Sarcoma-Associated Herpesvirus Rta Tetramers Make High-Affinity Interactions with Repetitive DNA Elements in the Mta Promoter To Stimulate DNA Binding of RBP-Jk/CSL ▿ †"

    Article Title: Kaposi's Sarcoma-Associated Herpesvirus Rta Tetramers Make High-Affinity Interactions with Repetitive DNA Elements in the Mta Promoter To Stimulate DNA Binding of RBP-Jk/CSL ▿Kaposi's Sarcoma-Associated Herpesvirus Rta Tetramers Make High-Affinity Interactions with Repetitive DNA Elements in the Mta Promoter To Stimulate DNA Binding of RBP-Jk/CSL ▿ †

    Journal: Journal of Virology

    doi: 10.1128/JVI.05479-11

    Purified Rta makes extended contacts to the Mta promoter that flank the RBP-Jk binding site. (A) Footprinting of the indicated proteins to the top strand of the Mta promoter was performed using 3 × 10 3 cpm DNA labeled with T4 polynucleotide kinase.
    Figure Legend Snippet: Purified Rta makes extended contacts to the Mta promoter that flank the RBP-Jk binding site. (A) Footprinting of the indicated proteins to the top strand of the Mta promoter was performed using 3 × 10 3 cpm DNA labeled with T4 polynucleotide kinase.

    Techniques Used: Purification, Binding Assay, Footprinting, Labeling

    39) Product Images from "High-throughput screening of soluble recombinant proteins"

    Article Title: High-throughput screening of soluble recombinant proteins

    Journal: Protein Science : A Publication of the Protein Society

    doi:

    Moleclular cloning strategy. Four PCR primers and reactions were used in two separate tubes. An equal amount of the two PCR products were mixed, and then the 5` ends were phosphorylated with T4 polynucleotide kinase. After denaturing (95°C for 5 min) and renaturing (65°C for 10 min), ∼25% of the final products carry EcoRI (5`) and XhoI (3`) cohesive ends and are ready for ligation with the vectors.
    Figure Legend Snippet: Moleclular cloning strategy. Four PCR primers and reactions were used in two separate tubes. An equal amount of the two PCR products were mixed, and then the 5` ends were phosphorylated with T4 polynucleotide kinase. After denaturing (95°C for 5 min) and renaturing (65°C for 10 min), ∼25% of the final products carry EcoRI (5`) and XhoI (3`) cohesive ends and are ready for ligation with the vectors.

    Techniques Used: Clone Assay, Polymerase Chain Reaction, Ligation

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    New England Biolabs t4 pnk
    Overview of metabarcoding and library preparation steps and formation of tag-jumps in a typical ‘shotgun’ Illumina library protocol and our presented Tagsteady library protocol. 1) Metabarcoding PCR with 5’ nucleotide tagged primers. To allow detection of tag-jumps, only unique twin-tag combinations is visualised. Following pooling of PCR reactions, differently tagged single-stranded amplicons can form heteroduplexes with non-complementary tag overhangs. 2) In a typical ‘shotgun’ Illumina library protocol (left), T4 DNA polymerase is used for blunt-ending, <t>T4</t> PNK for 5’ phosphorylation and Taq polymerase for 3’ adenylation. In this type of end-repair, 3’ overhangs (in heteroduplexes) will become substrate for the 3’→5’ exonuclease activity of T4 DNA Polymerase. The opposite strand, the 5’ overhangs (i.e. the inherent tag), will then act as a template for extension, causing the tag to ‘jump’ from one strand to the other (asterisk) (see van Orsouw et al. 2007 ; Schnell, Bohmann, and Gilbert 2015 ). The Tagsteady end-repair (right) only contains T4 PNK and Klenow exo- (thus no exonuclease activity) and therefore tag-jumps cannot arise. 3) After end repair, T4 DNA Ligase is used to ligate Illumina sequencing adapters (here depicted as Illumina Y-shaped adapters). 4) Often post-ligation PCR is carried out, causing further tag-jumps as a result of incomplete primer extension. Post-ligation PCR is not necessary with the Tagsteady protocol as it uses PCR-free full length adapters. 5) Sequencing of libraries on an Illumina sequencing platform. 6) Following initial sequence read processing, sequences within each amplicon library are sorted according to primer and tag sequences to assess levels of sequences carrying new combinations of used tags (tag-jumps).
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    Overview of metabarcoding and library preparation steps and formation of tag-jumps in a typical ‘shotgun’ Illumina library protocol and our presented Tagsteady library protocol. 1) Metabarcoding PCR with 5’ nucleotide tagged primers. To allow detection of tag-jumps, only unique twin-tag combinations is visualised. Following pooling of PCR reactions, differently tagged single-stranded amplicons can form heteroduplexes with non-complementary tag overhangs. 2) In a typical ‘shotgun’ Illumina library protocol (left), T4 DNA polymerase is used for blunt-ending, T4 PNK for 5’ phosphorylation and Taq polymerase for 3’ adenylation. In this type of end-repair, 3’ overhangs (in heteroduplexes) will become substrate for the 3’→5’ exonuclease activity of T4 DNA Polymerase. The opposite strand, the 5’ overhangs (i.e. the inherent tag), will then act as a template for extension, causing the tag to ‘jump’ from one strand to the other (asterisk) (see van Orsouw et al. 2007 ; Schnell, Bohmann, and Gilbert 2015 ). The Tagsteady end-repair (right) only contains T4 PNK and Klenow exo- (thus no exonuclease activity) and therefore tag-jumps cannot arise. 3) After end repair, T4 DNA Ligase is used to ligate Illumina sequencing adapters (here depicted as Illumina Y-shaped adapters). 4) Often post-ligation PCR is carried out, causing further tag-jumps as a result of incomplete primer extension. Post-ligation PCR is not necessary with the Tagsteady protocol as it uses PCR-free full length adapters. 5) Sequencing of libraries on an Illumina sequencing platform. 6) Following initial sequence read processing, sequences within each amplicon library are sorted according to primer and tag sequences to assess levels of sequences carrying new combinations of used tags (tag-jumps).

    Journal: bioRxiv

    Article Title: Tagsteady: a metabarcoding library preparation protocol to avoid false assignment of sequences to samples

    doi: 10.1101/2020.01.22.915009

    Figure Lengend Snippet: Overview of metabarcoding and library preparation steps and formation of tag-jumps in a typical ‘shotgun’ Illumina library protocol and our presented Tagsteady library protocol. 1) Metabarcoding PCR with 5’ nucleotide tagged primers. To allow detection of tag-jumps, only unique twin-tag combinations is visualised. Following pooling of PCR reactions, differently tagged single-stranded amplicons can form heteroduplexes with non-complementary tag overhangs. 2) In a typical ‘shotgun’ Illumina library protocol (left), T4 DNA polymerase is used for blunt-ending, T4 PNK for 5’ phosphorylation and Taq polymerase for 3’ adenylation. In this type of end-repair, 3’ overhangs (in heteroduplexes) will become substrate for the 3’→5’ exonuclease activity of T4 DNA Polymerase. The opposite strand, the 5’ overhangs (i.e. the inherent tag), will then act as a template for extension, causing the tag to ‘jump’ from one strand to the other (asterisk) (see van Orsouw et al. 2007 ; Schnell, Bohmann, and Gilbert 2015 ). The Tagsteady end-repair (right) only contains T4 PNK and Klenow exo- (thus no exonuclease activity) and therefore tag-jumps cannot arise. 3) After end repair, T4 DNA Ligase is used to ligate Illumina sequencing adapters (here depicted as Illumina Y-shaped adapters). 4) Often post-ligation PCR is carried out, causing further tag-jumps as a result of incomplete primer extension. Post-ligation PCR is not necessary with the Tagsteady protocol as it uses PCR-free full length adapters. 5) Sequencing of libraries on an Illumina sequencing platform. 6) Following initial sequence read processing, sequences within each amplicon library are sorted according to primer and tag sequences to assess levels of sequences carrying new combinations of used tags (tag-jumps).

    Article Snippet: End-repair without T4 DNA Polymerase: for treatments −/+ and −/−, an end-repair mastermix was made by combining 4 μl T4 DNA ligase reaction buffer (New England Biolabs, NEB, Ipswich, Massachusetts, US), 0.5 μl dATP (10mM) (Thermo-Fisher), 2 μl reaction booster mix (consisting of 25 % PEG-4000 (Sigma Aldrich, 50%), 2 mg/ml BSA (Thermo-Fisher) and 400 mM NaCl) , 2 μl T4 PNK (NEB, cat#M0201S, 10 U/μl) and 1.5 μl Klenow Fragment (3’- > 5’ exo-) (NEB, cat#M0212S, 5 U/μl) per amplicon pool reaction.

    Techniques: Polymerase Chain Reaction, Activity Assay, Sequencing, Ligation, Amplification

    Purification and activity assays of PNKP and Polβ. ( A ) Purified Polβ-His 6 (17 ng) and PNKP-His 6 (127 ng) from E. coli were subjected to PAGE and stained with Coomassie blue. ( B ) Incorporation of [α- 32 P]-dCTP by Polβ using APE1-generated product. ddC, di-deoxynucleotide; P, product. ( C ) Schematic of the preparation of S (substrate) and subsequent enzymatic reactions for testing PNKP activity. ( D ) Efficiency of oligonucleotide labeling, annealing, and ligation leading to S indicated in ( C ). ( E ) Fpg (NEB, 1 U) completely digested S and the 3’ phosphate was completely removed by PNKP (12.7 ng and 127 ng, lanes 2 and 3), or by T4 PNK (NEB, 0.1 U and 1 U, lanes 7 and 8). NEIL2 (272 ng) only partially digested S and its 3’P was resistant to the PNKP phosphatase (lanes 4 and 5).

    Journal: eLife

    Article Title: Perturbation of base excision repair sensitizes breast cancer cells to APOBEC3 deaminase-mediated mutations

    doi: 10.7554/eLife.51605

    Figure Lengend Snippet: Purification and activity assays of PNKP and Polβ. ( A ) Purified Polβ-His 6 (17 ng) and PNKP-His 6 (127 ng) from E. coli were subjected to PAGE and stained with Coomassie blue. ( B ) Incorporation of [α- 32 P]-dCTP by Polβ using APE1-generated product. ddC, di-deoxynucleotide; P, product. ( C ) Schematic of the preparation of S (substrate) and subsequent enzymatic reactions for testing PNKP activity. ( D ) Efficiency of oligonucleotide labeling, annealing, and ligation leading to S indicated in ( C ). ( E ) Fpg (NEB, 1 U) completely digested S and the 3’ phosphate was completely removed by PNKP (12.7 ng and 127 ng, lanes 2 and 3), or by T4 PNK (NEB, 0.1 U and 1 U, lanes 7 and 8). NEIL2 (272 ng) only partially digested S and its 3’P was resistant to the PNKP phosphatase (lanes 4 and 5).

    Article Snippet: The products were purified by PCI extraction and ethanol precipitation and treated with PNKP (25 mM HEPES-KOH, pH 7.6, 50 mM NaCl, 0.5 mM EDTA, 0.5 mM DTT, 100 ng/ml BSA, 5% Glycerol) or T4 PNK (70 mM Tris-HCl, 10 mM MgCl2 , 5 mM DTT, pH 6.0) at 37°C for 30 min.

    Techniques: Purification, Activity Assay, Polyacrylamide Gel Electrophoresis, Staining, Generated, Oligonucleotide Labeling, Ligation

    Post-digest Labeling Reaction Scheme (A) The γ-phosphate of 18 O 4 -γ-ATP is transferred to the 5′-hydroxy group of a cross-linked RNA oligonucleotide. T4-PNK also catalyzes a 3′-dephosphorylation reaction. (B) Simulation of the isotopic distribution 33 of a cross-linked peptide (PEPTIDER cross-linked to a GU dinucleotide, doubly charged) for a 1:1 mixture of light and heavy ATP using single-phosphate labeling.

    Journal: Analytical Chemistry

    Article Title: Single Nucleotide Resolution RNA–Protein Cross-Linking Mass Spectrometry: A Simple Extension of the CLIR-MS Workflow

    doi: 10.1021/acs.analchem.1c02384

    Figure Lengend Snippet: Post-digest Labeling Reaction Scheme (A) The γ-phosphate of 18 O 4 -γ-ATP is transferred to the 5′-hydroxy group of a cross-linked RNA oligonucleotide. T4-PNK also catalyzes a 3′-dephosphorylation reaction. (B) Simulation of the isotopic distribution 33 of a cross-linked peptide (PEPTIDER cross-linked to a GU dinucleotide, doubly charged) for a 1:1 mixture of light and heavy ATP using single-phosphate labeling.

    Article Snippet: T4-PNK exhibits two reactions: (1) phosphorylation of the 5′-hydroxy group and (2) dephosphorylation of the 3′-phosphate group ( ).

    Techniques: Labeling, De-Phosphorylation Assay

    Effect of arrest on the transcript arrangement in RNAP. ( A ) RNase A footprinting of the transcript in active EC 26 and in arrested EC 27 . The RNA in the complexes was internally labeled at positions +26A or +12C. Each sample was treated with RNase A and fractionated by centrifugation into soluble (s) and matrix-associated (p) fractions before gel analysis. Sequences of the transcripts are shown alongside the autoradiograms, asterisks mark positions of labeling, and arrows show major cuts introduced into the RNA (bold shaded line) by RNase A. The cylinders represent the transcript segments protected by RNAP in the active and arrested complexes. Nonfractionated samples (t) are included as controls. ( B ) 5′-Terminal phosphorylation of the transcripts. RNA-labeled EC 20 and EC 27 (lanes 6 and 4) and arrested fraction of EC 27 purified by chase (lane 2) were treated with T4 polynucleotide kinase in the presence of ATP (lanes 5, 3, and 1). The symbol (P) indicates the phosphorylated transcripts. Arrows indicate the mobility of phosphorylated and nonphosphorylated transcripts.

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

    Article Title: Transcriptional arrest: Escherichia coli RNA polymerase translocates backward, leaving the 3? end of the RNA intact and extruded

    doi:

    Figure Lengend Snippet: Effect of arrest on the transcript arrangement in RNAP. ( A ) RNase A footprinting of the transcript in active EC 26 and in arrested EC 27 . The RNA in the complexes was internally labeled at positions +26A or +12C. Each sample was treated with RNase A and fractionated by centrifugation into soluble (s) and matrix-associated (p) fractions before gel analysis. Sequences of the transcripts are shown alongside the autoradiograms, asterisks mark positions of labeling, and arrows show major cuts introduced into the RNA (bold shaded line) by RNase A. The cylinders represent the transcript segments protected by RNAP in the active and arrested complexes. Nonfractionated samples (t) are included as controls. ( B ) 5′-Terminal phosphorylation of the transcripts. RNA-labeled EC 20 and EC 27 (lanes 6 and 4) and arrested fraction of EC 27 purified by chase (lane 2) were treated with T4 polynucleotide kinase in the presence of ATP (lanes 5, 3, and 1). The symbol (P) indicates the phosphorylated transcripts. Arrows indicate the mobility of phosphorylated and nonphosphorylated transcripts.

    Article Snippet: EC11 (the numerical index denotes the length of the transcript) was treated with 10 units of T4 polynucleotide kinase (New England Biolabs) and 50 μCi of [α-32 P]ATP (4500 Ci/mmol; ICN Biomedicals, Costa Mesa, CA) for 10 min to label the DNA, washed with TB, and walked to the desired position.

    Techniques: Footprinting, Labeling, Centrifugation, Purification