monarch rna cleanup kit  (New England Biolabs)


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    Structured Review

    New England Biolabs monarch rna cleanup kit
    VSW-3 RNAP and its promoter. (A) Distance tree analysis of the representative ssRNAPs by Blast program. Distance from the root ‘○’: SP6 RNAP (3.374) > T7 RNAP (3.145) > KP34 RNAP (2.572) > VSW-3 RNAP (2.292) > Syn5 RNAP (1.118) suggests that VSW-3 RNAP is the second primitive after Syn5 RNAP, and evolved into a new branch of the evolutionary tree together with a predicted pollyC RNAP (3.055) from phage pollyC (YP_009622558.1). (B) SDS-PAGE gel analysis of purified VSW-3 RNAP (92.4 kDa including an N-terminal His-tag, 1 μM) and commercial T7 RNAP (New England Biolabs, 100 kDa, 1.5 μM), gel was stained with Coomassie blue. (C) Organization of phage VSW-3 genome and distribution of the predicted VSW-3 promoters (indicated by rightward arrows). (D) IVT of VSW-3 RNAP on the linearized <t>pUC19</t> plasmid with an insertion of predicted VSW-3 promoter (top gel). 5’-RACE revealed that the initial nucleotides of VSW-3 RNAP transcription in the predicted promoter is “GTA” (bottom sequencing result). (E) IVT on 5’-truncated DNA templates (left box) to determine the accurate promoter of VSW-3 RNAP. The <t>RNA</t> yield with each template (right gel) suggests that the 15 bp (5’-ATTGGGCCACCTATA-3’) sequence is the minimal promoter and the 18 bp (5’-TTAATTGGGCCACCTATA-3’) sequence is the full VSW-3 promoter.
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    Images

    1) Product Images from "In vitro transcription using psychrophilic phage VSW-3 RNA polymerase"

    Article Title: In vitro transcription using psychrophilic phage VSW-3 RNA polymerase

    Journal: bioRxiv

    doi: 10.1101/2020.09.14.297226

    VSW-3 RNAP and its promoter. (A) Distance tree analysis of the representative ssRNAPs by Blast program. Distance from the root ‘○’: SP6 RNAP (3.374) > T7 RNAP (3.145) > KP34 RNAP (2.572) > VSW-3 RNAP (2.292) > Syn5 RNAP (1.118) suggests that VSW-3 RNAP is the second primitive after Syn5 RNAP, and evolved into a new branch of the evolutionary tree together with a predicted pollyC RNAP (3.055) from phage pollyC (YP_009622558.1). (B) SDS-PAGE gel analysis of purified VSW-3 RNAP (92.4 kDa including an N-terminal His-tag, 1 μM) and commercial T7 RNAP (New England Biolabs, 100 kDa, 1.5 μM), gel was stained with Coomassie blue. (C) Organization of phage VSW-3 genome and distribution of the predicted VSW-3 promoters (indicated by rightward arrows). (D) IVT of VSW-3 RNAP on the linearized pUC19 plasmid with an insertion of predicted VSW-3 promoter (top gel). 5’-RACE revealed that the initial nucleotides of VSW-3 RNAP transcription in the predicted promoter is “GTA” (bottom sequencing result). (E) IVT on 5’-truncated DNA templates (left box) to determine the accurate promoter of VSW-3 RNAP. The RNA yield with each template (right gel) suggests that the 15 bp (5’-ATTGGGCCACCTATA-3’) sequence is the minimal promoter and the 18 bp (5’-TTAATTGGGCCACCTATA-3’) sequence is the full VSW-3 promoter.
    Figure Legend Snippet: VSW-3 RNAP and its promoter. (A) Distance tree analysis of the representative ssRNAPs by Blast program. Distance from the root ‘○’: SP6 RNAP (3.374) > T7 RNAP (3.145) > KP34 RNAP (2.572) > VSW-3 RNAP (2.292) > Syn5 RNAP (1.118) suggests that VSW-3 RNAP is the second primitive after Syn5 RNAP, and evolved into a new branch of the evolutionary tree together with a predicted pollyC RNAP (3.055) from phage pollyC (YP_009622558.1). (B) SDS-PAGE gel analysis of purified VSW-3 RNAP (92.4 kDa including an N-terminal His-tag, 1 μM) and commercial T7 RNAP (New England Biolabs, 100 kDa, 1.5 μM), gel was stained with Coomassie blue. (C) Organization of phage VSW-3 genome and distribution of the predicted VSW-3 promoters (indicated by rightward arrows). (D) IVT of VSW-3 RNAP on the linearized pUC19 plasmid with an insertion of predicted VSW-3 promoter (top gel). 5’-RACE revealed that the initial nucleotides of VSW-3 RNAP transcription in the predicted promoter is “GTA” (bottom sequencing result). (E) IVT on 5’-truncated DNA templates (left box) to determine the accurate promoter of VSW-3 RNAP. The RNA yield with each template (right gel) suggests that the 15 bp (5’-ATTGGGCCACCTATA-3’) sequence is the minimal promoter and the 18 bp (5’-TTAATTGGGCCACCTATA-3’) sequence is the full VSW-3 promoter.

    Techniques Used: SDS Page, Purification, Staining, Plasmid Preparation, Sequencing

    2) Product Images from "Synthesis of low immunogenicity RNA with high-temperature in vitro transcription"

    Article Title: Synthesis of low immunogenicity RNA with high-temperature in vitro transcription

    Journal: bioRxiv

    doi: 10.1101/815092

    Template-encoded Poly(A) tailing reduces antisense by-product formation. A) dsRNA immunoblot with J2 antibody and gel electrophoresis analysis of CLuc RNA synthesized from CLuc templates with varying length (30 bp, 60 bp, 120 bp) of poly-T sequence at 3’-end under standard conditions (5 mM rNTPs, 37°C for 1 hour). B) Immunoblot and native gel electrophoresis analysis of IVT reactions on 512B::CLuc chimeric template with poly-T (60 bp and 120 bp) sequence at the 3’-end. IVT reactions were performed at 37°C or 50°C.
    Figure Legend Snippet: Template-encoded Poly(A) tailing reduces antisense by-product formation. A) dsRNA immunoblot with J2 antibody and gel electrophoresis analysis of CLuc RNA synthesized from CLuc templates with varying length (30 bp, 60 bp, 120 bp) of poly-T sequence at 3’-end under standard conditions (5 mM rNTPs, 37°C for 1 hour). B) Immunoblot and native gel electrophoresis analysis of IVT reactions on 512B::CLuc chimeric template with poly-T (60 bp and 120 bp) sequence at the 3’-end. IVT reactions were performed at 37°C or 50°C.

    Techniques Used: Nucleic Acid Electrophoresis, Synthesized, Sequencing

    Truncation of the 3’-end of the 512B DNA templates results in reduction of the antisense RNA by-product formation. Immunoblot (with J2 antibody; 1:5000; Scicons) and native gel electrophoresis analyses of in vitro transcription reactions performed on 512B template with 3’-end truncations (50 and 200 base pairs). In vitro transcription reactions were performed with TsT7-1 at 37°C or 50°C.
    Figure Legend Snippet: Truncation of the 3’-end of the 512B DNA templates results in reduction of the antisense RNA by-product formation. Immunoblot (with J2 antibody; 1:5000; Scicons) and native gel electrophoresis analyses of in vitro transcription reactions performed on 512B template with 3’-end truncations (50 and 200 base pairs). In vitro transcription reactions were performed with TsT7-1 at 37°C or 50°C.

    Techniques Used: Nucleic Acid Electrophoresis, In Vitro

    3) Product Images from "A pro-metastatic tRNA fragment drives Nucleolin oligomerization and stabilization of bound metabolic mRNAs"

    Article Title: A pro-metastatic tRNA fragment drives Nucleolin oligomerization and stabilization of bound metabolic mRNAs

    Journal: bioRxiv

    doi: 10.1101/2021.04.26.441477

    5’-tRF Cys promotes Nucleolin binding to its target transcripts to enhance their stability. A. Quantification of rRNA levels upon inhibition of 5’-tRF Cys by RT-qPCR. B. Representative polysome profiles showing global translation status in 4T1 cells upon inhibition of 5’-tRF Cys . Mono, monosomes. Di, disomes. C. Percentage of Nucleolin peaks in different types of RNAs. RMSK, repeat masked RNAs. D. The number of Nucleolin-bound CLIP peaks in 5’, 3’ untranslated region (UTR) or coding sequencing (CDS) per 10 kb in the mouse genome. E. Cumulative distribution function (CDF) plots of log 2 FC in transcript abundance for all transcripts stratified by whether they were bound by Nucleolin (red) or not (grey). Statistical significance was determined by Kolmogorov–Smirnov (KS) test (P = 4.8e-13). F. Scatter plot comparing log 2 FC in transcript abundance upon inhibition of 5’-tRF Cys with two distinct 5’-tRF Cys antisense LNAs. Statistically significantly changed genes are marked in red. The blue dashed line represents the linear regression line for all data points. ρ, Spearman’s correlation coefficient. G. Scatter plot comparing log 2 FC in protein abundance and log 2 FC in transcript abundance between 5’-tRF Cys suppressed and control cells for all transcripts stratified by whether their Nucleolin binding is enhanced by 5’-tRF Cys (red) or not (grey). The blue dashed line represents the linear regression line for all data points. ρ, Spearman’s correlation coefficient. H, I. Representative western blot images of 5’-tRF Cys targets upon suppression of 5’-tRF Cys (H) or depletion of Nucleolin (I). J. Genome browser view of the aligned Nucleolin (Ncl)-CLIP tags (orange), RNA-Seq reads (red) and Ribo-Seq reads (green) within the 5’ UTR of Pafah1b1. The Y axis represents reads per million (RPM). TSS, transcription start site. K. Quantification by dual luciferase assays of the luminescence signals of reporters containing 5’ UTRs from 5’-tRF Cys targets relative to that from the control GAPDH. Statistical significance in A and K was determined by one-tail t-tests with Welch’s correction. ns, not significant. ***, p
    Figure Legend Snippet: 5’-tRF Cys promotes Nucleolin binding to its target transcripts to enhance their stability. A. Quantification of rRNA levels upon inhibition of 5’-tRF Cys by RT-qPCR. B. Representative polysome profiles showing global translation status in 4T1 cells upon inhibition of 5’-tRF Cys . Mono, monosomes. Di, disomes. C. Percentage of Nucleolin peaks in different types of RNAs. RMSK, repeat masked RNAs. D. The number of Nucleolin-bound CLIP peaks in 5’, 3’ untranslated region (UTR) or coding sequencing (CDS) per 10 kb in the mouse genome. E. Cumulative distribution function (CDF) plots of log 2 FC in transcript abundance for all transcripts stratified by whether they were bound by Nucleolin (red) or not (grey). Statistical significance was determined by Kolmogorov–Smirnov (KS) test (P = 4.8e-13). F. Scatter plot comparing log 2 FC in transcript abundance upon inhibition of 5’-tRF Cys with two distinct 5’-tRF Cys antisense LNAs. Statistically significantly changed genes are marked in red. The blue dashed line represents the linear regression line for all data points. ρ, Spearman’s correlation coefficient. G. Scatter plot comparing log 2 FC in protein abundance and log 2 FC in transcript abundance between 5’-tRF Cys suppressed and control cells for all transcripts stratified by whether their Nucleolin binding is enhanced by 5’-tRF Cys (red) or not (grey). The blue dashed line represents the linear regression line for all data points. ρ, Spearman’s correlation coefficient. H, I. Representative western blot images of 5’-tRF Cys targets upon suppression of 5’-tRF Cys (H) or depletion of Nucleolin (I). J. Genome browser view of the aligned Nucleolin (Ncl)-CLIP tags (orange), RNA-Seq reads (red) and Ribo-Seq reads (green) within the 5’ UTR of Pafah1b1. The Y axis represents reads per million (RPM). TSS, transcription start site. K. Quantification by dual luciferase assays of the luminescence signals of reporters containing 5’ UTRs from 5’-tRF Cys targets relative to that from the control GAPDH. Statistical significance in A and K was determined by one-tail t-tests with Welch’s correction. ns, not significant. ***, p

    Techniques Used: Binding Assay, Inhibition, Quantitative RT-PCR, Cross-linking Immunoprecipitation, Sequencing, Western Blot, RNA Sequencing Assay, Luciferase

    5’-tRF Cys promotes complex D assembly and Nucleolin oligomerization. A, B. Native gel analysis of Nucleolin complexes assembled from Pafah1b1 (A) or 5’-tRF Cys (B) using increasing amounts of Nucleolin protein. C. Quantification of complex D assembly as a function of Nucleolin concentration using purified Nucleolin protein. Bmax, specific maximum binding. h, Hill coefficient. Kd, equilibrium dissociation constant. D. Representative images of western blots of Nucleolin from Nucleolin IP that was pre-treated with different dilutions of micrococcal nuclease to remove endogenous RNAs before complexes were assembled at 30 °C and crosslinked with ethylene glycol bis (succinimidyl succinate). The number of blue dots represent the inferred number of Nucleolin monomers based on the molecular weight. E, F. Kinetics of Nucleolin complexes assembled from Pafah1b1 (E) or 5’-tRF Cys (F) using Nucleolin IP. See also Figure 5F . Asterisk denotes an RNA-protein complex that was detected only with Nucleolin IP but not Nucleolin protein. G, H. Native gel analysis of Nucleolin complexes assembled from Pafah1b1 (G) or 5’-tRF Cys (H) using increasing amount of Nucleolin IP. See also Figure 5G . I. Native gel analysis of Nucleolin complexes assembled using Nucleolin IP from Mthfd1l alone, or together with a wild-type (WT) or Nucleolin binding deficient (MUT) 5’-tRF Cys . Asterisk denotes an RNA-protein complex that was detected only with Nucleolin IP but not Nucleolin protein. J. Representative western blot of Nucleolin using Nucleolin IP incubated with or without Pafah1b1, or with both Pafah1b1 and 5’-tRF Cy at 30 °C before crosslinking with EGS. See also Figure 5I . K. Top, quantification of the protection provided by different forms of Nucleolin from degradation by a prototypical 5’- > 3’ exonuclease Terminator after conducting the assembly assay at 4 °C or 30 °C to form monomeric Nucleolin (complex A) or oligomeric Nucleolin (complex D) respectively. Bottom, representative image of denaturing PAGE analysis of the exonuclease degradation products.
    Figure Legend Snippet: 5’-tRF Cys promotes complex D assembly and Nucleolin oligomerization. A, B. Native gel analysis of Nucleolin complexes assembled from Pafah1b1 (A) or 5’-tRF Cys (B) using increasing amounts of Nucleolin protein. C. Quantification of complex D assembly as a function of Nucleolin concentration using purified Nucleolin protein. Bmax, specific maximum binding. h, Hill coefficient. Kd, equilibrium dissociation constant. D. Representative images of western blots of Nucleolin from Nucleolin IP that was pre-treated with different dilutions of micrococcal nuclease to remove endogenous RNAs before complexes were assembled at 30 °C and crosslinked with ethylene glycol bis (succinimidyl succinate). The number of blue dots represent the inferred number of Nucleolin monomers based on the molecular weight. E, F. Kinetics of Nucleolin complexes assembled from Pafah1b1 (E) or 5’-tRF Cys (F) using Nucleolin IP. See also Figure 5F . Asterisk denotes an RNA-protein complex that was detected only with Nucleolin IP but not Nucleolin protein. G, H. Native gel analysis of Nucleolin complexes assembled from Pafah1b1 (G) or 5’-tRF Cys (H) using increasing amount of Nucleolin IP. See also Figure 5G . I. Native gel analysis of Nucleolin complexes assembled using Nucleolin IP from Mthfd1l alone, or together with a wild-type (WT) or Nucleolin binding deficient (MUT) 5’-tRF Cys . Asterisk denotes an RNA-protein complex that was detected only with Nucleolin IP but not Nucleolin protein. J. Representative western blot of Nucleolin using Nucleolin IP incubated with or without Pafah1b1, or with both Pafah1b1 and 5’-tRF Cy at 30 °C before crosslinking with EGS. See also Figure 5I . K. Top, quantification of the protection provided by different forms of Nucleolin from degradation by a prototypical 5’- > 3’ exonuclease Terminator after conducting the assembly assay at 4 °C or 30 °C to form monomeric Nucleolin (complex A) or oligomeric Nucleolin (complex D) respectively. Bottom, representative image of denaturing PAGE analysis of the exonuclease degradation products.

    Techniques Used: Concentration Assay, Purification, Binding Assay, Western Blot, Molecular Weight, Incubation, Polyacrylamide Gel Electrophoresis

    4) Product Images from "A Digital CRISPR-based Method for the Rapid Detection and Absolute Quantification of Viral Nucleic Acids"

    Article Title: A Digital CRISPR-based Method for the Rapid Detection and Absolute Quantification of Viral Nucleic Acids

    Journal: medRxiv

    doi: 10.1101/2020.11.03.20223602

    Schematic illustration of RADICA. a , The workflow of RADICA sample partitioning on a chip for absolute quantification of nucleic acid targets. Generally, after the DNA/RNA extraction step, different kind of clinical samples can be used for detection and quantification of various targets. The sample mixture containing DNA/cDNA, RPA reagents, and Cas12a-crRNA-FQ probes is distributed randomly into thousands of partitions. In each partition, the DNA is amplified by RPA and detected by Cas12a-crRNA, resulting in a fluorescent signal in the partition. Based on the proportion of positive partitions and on Poisson distribution, the absolute copy number of the nucleic acid target is quantified. b , Illustration of RPA-Cas12a reaction in each positive partition. In each partition containing the target nucleic acid, the primers bind to the target nucleic acid and initiate amplification with the aid of recombinase and DNA polymerase. Because of the strand displacement of DNA polymerase, the exposed crRNA-targeted ssDNA sites are bound by Cas12a-crRNA complexes. Cas12a is then activated and cleaves the nearby FQ reporters to produce a fluorescence readout.
    Figure Legend Snippet: Schematic illustration of RADICA. a , The workflow of RADICA sample partitioning on a chip for absolute quantification of nucleic acid targets. Generally, after the DNA/RNA extraction step, different kind of clinical samples can be used for detection and quantification of various targets. The sample mixture containing DNA/cDNA, RPA reagents, and Cas12a-crRNA-FQ probes is distributed randomly into thousands of partitions. In each partition, the DNA is amplified by RPA and detected by Cas12a-crRNA, resulting in a fluorescent signal in the partition. Based on the proportion of positive partitions and on Poisson distribution, the absolute copy number of the nucleic acid target is quantified. b , Illustration of RPA-Cas12a reaction in each positive partition. In each partition containing the target nucleic acid, the primers bind to the target nucleic acid and initiate amplification with the aid of recombinase and DNA polymerase. Because of the strand displacement of DNA polymerase, the exposed crRNA-targeted ssDNA sites are bound by Cas12a-crRNA complexes. Cas12a is then activated and cleaves the nearby FQ reporters to produce a fluorescence readout.

    Techniques Used: Chromatin Immunoprecipitation, RNA Extraction, Recombinase Polymerase Amplification, Amplification, Fluorescence

    5) Product Images from "In vitro transcription using psychrophilic phage VSW-3 RNA polymerase"

    Article Title: In vitro transcription using psychrophilic phage VSW-3 RNA polymerase

    Journal: bioRxiv

    doi: 10.1101/2020.09.14.297226

    Optimal VSW-3 RNAP IVT conditions. (A) Screening for the optimal Mg 2+ /NTP concentration in the presence of 1 mM DTT. RNA yield with various optimal Mg 2+ /NTP concentration combination was further compared (gel in the dotted box). The stability of the optimal VSW-3 RNAP IVT buffer with 1 mM DTT was examined (gel in the solid box). (B) Screening for the optimal DTT/ Mg 2+ concentration for the stable and high-yield VSW-3 RNAP IVT buffer. The stability of the high-yield VSW-3 RNAP IVT buffer containing 16 mM Mg 2+ , 4 mM NTP and 5 mM DTT was examined (gel in the solid box). (C) The optimal reaction temperature of VSW-3 RNAP (25°C) for maximum run-off RNA yield. (D) The optimal enzyme concentration of VSW-3 RNAP (0.15 μM) for maximum run-off RNA yield. (E) Optimal IVT yield of VSW-3 RNAP with various reaction temperature/incubation time combinations. The maximum run-off RNA yield was obtained at 25°C for 12 hours. (F) Gray-scale quantitation of the run-off RNA transcripts in gel (E) . Diagram was made using GraphPad Prism. In all gels the bands corresponding to DNA templates were indicated by empty stars and the bands corresponding to run-off RNA transcripts were indicated by filled stars.
    Figure Legend Snippet: Optimal VSW-3 RNAP IVT conditions. (A) Screening for the optimal Mg 2+ /NTP concentration in the presence of 1 mM DTT. RNA yield with various optimal Mg 2+ /NTP concentration combination was further compared (gel in the dotted box). The stability of the optimal VSW-3 RNAP IVT buffer with 1 mM DTT was examined (gel in the solid box). (B) Screening for the optimal DTT/ Mg 2+ concentration for the stable and high-yield VSW-3 RNAP IVT buffer. The stability of the high-yield VSW-3 RNAP IVT buffer containing 16 mM Mg 2+ , 4 mM NTP and 5 mM DTT was examined (gel in the solid box). (C) The optimal reaction temperature of VSW-3 RNAP (25°C) for maximum run-off RNA yield. (D) The optimal enzyme concentration of VSW-3 RNAP (0.15 μM) for maximum run-off RNA yield. (E) Optimal IVT yield of VSW-3 RNAP with various reaction temperature/incubation time combinations. The maximum run-off RNA yield was obtained at 25°C for 12 hours. (F) Gray-scale quantitation of the run-off RNA transcripts in gel (E) . Diagram was made using GraphPad Prism. In all gels the bands corresponding to DNA templates were indicated by empty stars and the bands corresponding to run-off RNA transcripts were indicated by filled stars.

    Techniques Used: Concentration Assay, Incubation, Quantitation Assay

    Response of ssRNAPs to Class II terminator. (A) Using PCR-amplified templates for cas9-RNA IVT, obvious abortive RNA transcripts were observed for T7 RNAP and Syn5 RNAP but not VSW-3 RNAP (top gel). 3’-RACE revealed that the T7 RNAP transcription was terminated 9 nt downstream of a Class II terminator “ATCTGTT” (bottom sequencing result). (B) VSW-3 RNAP IVT was not terminated (no additional bands comparing lane 2 with lane 1) when a Class II terminator “ATCTGTT” was inserted into the middle of the copGFP RNA coding sequence.
    Figure Legend Snippet: Response of ssRNAPs to Class II terminator. (A) Using PCR-amplified templates for cas9-RNA IVT, obvious abortive RNA transcripts were observed for T7 RNAP and Syn5 RNAP but not VSW-3 RNAP (top gel). 3’-RACE revealed that the T7 RNAP transcription was terminated 9 nt downstream of a Class II terminator “ATCTGTT” (bottom sequencing result). (B) VSW-3 RNAP IVT was not terminated (no additional bands comparing lane 2 with lane 1) when a Class II terminator “ATCTGTT” was inserted into the middle of the copGFP RNA coding sequence.

    Techniques Used: Polymerase Chain Reaction, Amplification, Sequencing

    RNA 3’ extension and RdRp activity of T7 and VSW-3 RNAP. (A) The secondary structure of a sgRNA predicted with RNAfold software. (B) IVT synthesis of a sgRNA (targeting eGFP) by VSW-3 and T7 RNAP. (C) 3’-RACE of the sgRNAs transcripts from T7 and VSW-3 RNAP IVT. Only the 3’ region (red sequence on the top) of the full sgRNA in sequencing results was shown. The length of each sequence was noted. The sequences matching the exact run-off sgRNA (103 nt) was indicated by red stars. (D) Schematic showing the mechanism and origin (3’ self-templated extension by the RdRp activity of T7 RNAP) of the 16 nt 3’-extension in T7 RNAP products as in (C) . (E) T7 but not VSW-3 RNAP retains the RdRp activity to extend purified sgRNA (with terminal primer/template structure).
    Figure Legend Snippet: RNA 3’ extension and RdRp activity of T7 and VSW-3 RNAP. (A) The secondary structure of a sgRNA predicted with RNAfold software. (B) IVT synthesis of a sgRNA (targeting eGFP) by VSW-3 and T7 RNAP. (C) 3’-RACE of the sgRNAs transcripts from T7 and VSW-3 RNAP IVT. Only the 3’ region (red sequence on the top) of the full sgRNA in sequencing results was shown. The length of each sequence was noted. The sequences matching the exact run-off sgRNA (103 nt) was indicated by red stars. (D) Schematic showing the mechanism and origin (3’ self-templated extension by the RdRp activity of T7 RNAP) of the 16 nt 3’-extension in T7 RNAP products as in (C) . (E) T7 but not VSW-3 RNAP retains the RdRp activity to extend purified sgRNA (with terminal primer/template structure).

    Techniques Used: Activity Assay, Software, Sequencing, Purification

    VSW-3 RNAP and its promoter. (A) Distance tree analysis of the representative ssRNAPs by Blast program. Distance from the root ‘○’: SP6 RNAP (3.374) > T7 RNAP (3.145) > KP34 RNAP (2.572) > VSW-3 RNAP (2.292) > Syn5 RNAP (1.118) suggests that VSW-3 RNAP is the second primitive after Syn5 RNAP, and evolved into a new branch of the evolutionary tree together with a predicted pollyC RNAP (3.055) from phage pollyC (YP_009622558.1). (B) SDS-PAGE gel analysis of purified VSW-3 RNAP (92.4 kDa including an N-terminal His-tag, 1 μM) and commercial T7 RNAP (New England Biolabs, 100 kDa, 1.5 μM), gel was stained with Coomassie blue. (C) Organization of phage VSW-3 genome and distribution of the predicted VSW-3 promoters (indicated by rightward arrows). (D) IVT of VSW-3 RNAP on the linearized pUC19 plasmid with an insertion of predicted VSW-3 promoter (top gel). 5’-RACE revealed that the initial nucleotides of VSW-3 RNAP transcription in the predicted promoter is “GTA” (bottom sequencing result). (E) IVT on 5’-truncated DNA templates (left box) to determine the accurate promoter of VSW-3 RNAP. The RNA yield with each template (right gel) suggests that the 15 bp (5’-ATTGGGCCACCTATA-3’) sequence is the minimal promoter and the 18 bp (5’-TTAATTGGGCCACCTATA-3’) sequence is the full VSW-3 promoter.
    Figure Legend Snippet: VSW-3 RNAP and its promoter. (A) Distance tree analysis of the representative ssRNAPs by Blast program. Distance from the root ‘○’: SP6 RNAP (3.374) > T7 RNAP (3.145) > KP34 RNAP (2.572) > VSW-3 RNAP (2.292) > Syn5 RNAP (1.118) suggests that VSW-3 RNAP is the second primitive after Syn5 RNAP, and evolved into a new branch of the evolutionary tree together with a predicted pollyC RNAP (3.055) from phage pollyC (YP_009622558.1). (B) SDS-PAGE gel analysis of purified VSW-3 RNAP (92.4 kDa including an N-terminal His-tag, 1 μM) and commercial T7 RNAP (New England Biolabs, 100 kDa, 1.5 μM), gel was stained with Coomassie blue. (C) Organization of phage VSW-3 genome and distribution of the predicted VSW-3 promoters (indicated by rightward arrows). (D) IVT of VSW-3 RNAP on the linearized pUC19 plasmid with an insertion of predicted VSW-3 promoter (top gel). 5’-RACE revealed that the initial nucleotides of VSW-3 RNAP transcription in the predicted promoter is “GTA” (bottom sequencing result). (E) IVT on 5’-truncated DNA templates (left box) to determine the accurate promoter of VSW-3 RNAP. The RNA yield with each template (right gel) suggests that the 15 bp (5’-ATTGGGCCACCTATA-3’) sequence is the minimal promoter and the 18 bp (5’-TTAATTGGGCCACCTATA-3’) sequence is the full VSW-3 promoter.

    Techniques Used: SDS Page, Purification, Staining, Plasmid Preparation, Sequencing

    dsRNA by-products from T7 and VSW-3 RNAP IVT. (A) DNA templates for the IVT synthesis of various RNA as indicated on top of the gel. For each RNA, there are two DNA templates differ only in the promoter region to serve for VSW-3 and T7 RNAP IVT, respectively. DNA concentration and purity were compared in 1.5% agarose gel stained with ethidium bromide. (B) After template DNA was removed by DNase I treatment and purified with Monarch RNA Cleanup kit, 1μg of sox7, tdTomato, copGFP and Cas9 RNA transcribed by VSW-3 RNAP and T7 RNAP were analyzed in 1.5% agarose gel stained with ethidium bromide. The white and black colors for bands and background were converted in this gel picture to make the weak double-stranded and abortive RNA bands clearer. (C) Dot blot analysis of the RNA products (each 200 ng) as in (B) by VSW-3 RNAP and T7 RNAP with J2 monoclonal antibody. A prepared dsRNA (351 bp) was applied as quantitative standard (0.1 ng, 0.25 ng, 0.5 ng, 1.0 ng). (D) The gray value measurement and calculation of the X film image (top image in (C) ) by Image J software demonstrating the level of dsRNA contamination in T7 and VSW-3 RNAP transcripts.
    Figure Legend Snippet: dsRNA by-products from T7 and VSW-3 RNAP IVT. (A) DNA templates for the IVT synthesis of various RNA as indicated on top of the gel. For each RNA, there are two DNA templates differ only in the promoter region to serve for VSW-3 and T7 RNAP IVT, respectively. DNA concentration and purity were compared in 1.5% agarose gel stained with ethidium bromide. (B) After template DNA was removed by DNase I treatment and purified with Monarch RNA Cleanup kit, 1μg of sox7, tdTomato, copGFP and Cas9 RNA transcribed by VSW-3 RNAP and T7 RNAP were analyzed in 1.5% agarose gel stained with ethidium bromide. The white and black colors for bands and background were converted in this gel picture to make the weak double-stranded and abortive RNA bands clearer. (C) Dot blot analysis of the RNA products (each 200 ng) as in (B) by VSW-3 RNAP and T7 RNAP with J2 monoclonal antibody. A prepared dsRNA (351 bp) was applied as quantitative standard (0.1 ng, 0.25 ng, 0.5 ng, 1.0 ng). (D) The gray value measurement and calculation of the X film image (top image in (C) ) by Image J software demonstrating the level of dsRNA contamination in T7 and VSW-3 RNAP transcripts.

    Techniques Used: Concentration Assay, Agarose Gel Electrophoresis, Staining, Purification, Dot Blot, Software

    6) Product Images from "The cohesin regulator Stag1 promotes cell plasticity through heterochromatin regulation"

    Article Title: The cohesin regulator Stag1 promotes cell plasticity through heterochromatin regulation

    Journal: bioRxiv

    doi: 10.1101/2021.02.14.429938

    Diverse Transcription-Regulatory Control of Stag1 in ES cells. (A, B) 5’ Rapid Amplification of cDNA ends (RACE) for SA1 in ES and EpiLC cells. Arrows indicate bands which were cloned and sequenced. In A, red star indicates SATS TSS and red arrow indicates canonical TSS. In B: red indicates full length Stag1 with both SATS and can TSSs; dark blue indicates alternatively spliced variants, skipping of various exons in 5’ region; light blue indicates the TSSs at e6, e7. (C) 3’ RACE for SA1 in ES cells. Arrows indicate bands which were cloned and sequenced. Red indicates canonical full-length end; green indicates end in i25. (D) Top, schematic of the Stag1 gene annotation in mm10 and the identified TSS and TTSs from RACE indicated. Bottom, aligned sequence clones from the PCR mini-screen and their predicted impact on the SA1 protein, right. Green arrows and red bars within the transcripts indicate start of the coding sequence and the TTS respectively. Shown also are the regions which code for the AT hook and SCD domains. (E) Percent Spliced In (PSI) calculations based on VAST-Tools analysis of RNA-seq from multiple 2i (blue) and FCS (red) datasets (see Methods for details of libraries). Data are shown relative to Neural Stem cell (NSC) frequencies to highlight the events that are ES-specific. (F) Genome topology at the Stag1 locus. Hi-C contact maps in ES (2i) and NS cells of the 900kb region on chromosome 9 containing the Stag1 topologically associated domain (TAD). TADs are denoted with a vertical line and as repressed (orange) or active (blue). Shown also are tracks for Genes, Nanog and CTCF ChIP-seq as well as a track indicating the directionality of CTCF binding sites (red, forward; blue, reverse). Aligned to the Gene track are also the SA1 transcripts discovered above where red represents the untranslated regions and blue the coding body. UMI-4C-seq viewpoints (asterisks on the ChIP tracks) are positioned to the leftmost CTCF site (‘CTCF bait’) and to the Nanog site 40 kb upstream of the Stag1 canonical TSS (‘Nanog bait’). For each bait, UMI information for each cell type is shown as well as the comparative plots where red represents an enrichment of contacts in ES compared to NS. (G) Top, cartoon depicting functional domains within Stag1 protein, including the AT-hook (aa 3-58); Stromalin conserved domain (SCD, aa 296-381) and the C-terminus. Bottom, the predicted Stag1 protein isoforms based on transcript analysis with estimated sizes for each isoform. Purple boxes in the 105kDa and 90kDa isoforms represent retained introns. (H) PONDR tracks as before shown for the N-terminal truncated (top) and C-terminal truncated (bottom) transcripts. (I) Chromatin immunoprecipitation of SA1 from ES cells. (J) WB analysis of SA1 isoforms in chromatin fractions from ES cells treated with siscr and siSA1. H3 serves as a fraction and loading control. (K) Chromatin immunoprecipitation for the v5 tag in SA1 NG-FKBP ES cells treated with DMSO-only or dTAG. Note, SA1 bands now run 42kDa higher due to the addition of the tag. See also Figure S3, Table S1 and Table S2.
    Figure Legend Snippet: Diverse Transcription-Regulatory Control of Stag1 in ES cells. (A, B) 5’ Rapid Amplification of cDNA ends (RACE) for SA1 in ES and EpiLC cells. Arrows indicate bands which were cloned and sequenced. In A, red star indicates SATS TSS and red arrow indicates canonical TSS. In B: red indicates full length Stag1 with both SATS and can TSSs; dark blue indicates alternatively spliced variants, skipping of various exons in 5’ region; light blue indicates the TSSs at e6, e7. (C) 3’ RACE for SA1 in ES cells. Arrows indicate bands which were cloned and sequenced. Red indicates canonical full-length end; green indicates end in i25. (D) Top, schematic of the Stag1 gene annotation in mm10 and the identified TSS and TTSs from RACE indicated. Bottom, aligned sequence clones from the PCR mini-screen and their predicted impact on the SA1 protein, right. Green arrows and red bars within the transcripts indicate start of the coding sequence and the TTS respectively. Shown also are the regions which code for the AT hook and SCD domains. (E) Percent Spliced In (PSI) calculations based on VAST-Tools analysis of RNA-seq from multiple 2i (blue) and FCS (red) datasets (see Methods for details of libraries). Data are shown relative to Neural Stem cell (NSC) frequencies to highlight the events that are ES-specific. (F) Genome topology at the Stag1 locus. Hi-C contact maps in ES (2i) and NS cells of the 900kb region on chromosome 9 containing the Stag1 topologically associated domain (TAD). TADs are denoted with a vertical line and as repressed (orange) or active (blue). Shown also are tracks for Genes, Nanog and CTCF ChIP-seq as well as a track indicating the directionality of CTCF binding sites (red, forward; blue, reverse). Aligned to the Gene track are also the SA1 transcripts discovered above where red represents the untranslated regions and blue the coding body. UMI-4C-seq viewpoints (asterisks on the ChIP tracks) are positioned to the leftmost CTCF site (‘CTCF bait’) and to the Nanog site 40 kb upstream of the Stag1 canonical TSS (‘Nanog bait’). For each bait, UMI information for each cell type is shown as well as the comparative plots where red represents an enrichment of contacts in ES compared to NS. (G) Top, cartoon depicting functional domains within Stag1 protein, including the AT-hook (aa 3-58); Stromalin conserved domain (SCD, aa 296-381) and the C-terminus. Bottom, the predicted Stag1 protein isoforms based on transcript analysis with estimated sizes for each isoform. Purple boxes in the 105kDa and 90kDa isoforms represent retained introns. (H) PONDR tracks as before shown for the N-terminal truncated (top) and C-terminal truncated (bottom) transcripts. (I) Chromatin immunoprecipitation of SA1 from ES cells. (J) WB analysis of SA1 isoforms in chromatin fractions from ES cells treated with siscr and siSA1. H3 serves as a fraction and loading control. (K) Chromatin immunoprecipitation for the v5 tag in SA1 NG-FKBP ES cells treated with DMSO-only or dTAG. Note, SA1 bands now run 42kDa higher due to the addition of the tag. See also Figure S3, Table S1 and Table S2.

    Techniques Used: Rapid Amplification of cDNA Ends, Clone Assay, Sequencing, Polymerase Chain Reaction, RNA Sequencing Assay, Hi-C, Chromatin Immunoprecipitation, Binding Assay, Functional Assay, Western Blot

    7) Product Images from "An RNA polymerase ribozyme that synthesizes its own ancestor"

    Article Title: An RNA polymerase ribozyme that synthesizes its own ancestor

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

    doi: 10.1073/pnas.1914282117

    In vitro evolution of the 38-6 RNA polymerase ribozyme. ( A ) Scheme for selective amplification of polymerase ribozymes that synthesize a functional hammerhead ribozyme. (1) Attachment to the polymerase of an RNA primer (magenta), biotin (green), and the RNA substrate (orange) to be cleaved by the hammerhead. (2) Hybridization of the primer to an RNA template (brown) that encodes the hammerhead. (3) Extension of the primer by polymerization of NTPs (cyan), followed by biotin capture on streptavidin magnetic beads (gray). (4) Cleavage of the attached RNA substrate by the hammerhead, releasing the polymerase from the beads. (5) Recovery of functional polymerases. (6) Reverse transcription and PCR amplification. (7) Transcription to generate progeny polymerases. ( B ) Sequence and secondary structure of the hammerhead ribozyme (cyan), together with the primer used to initiate its synthesis and the RNA substrate. The arrow indicates the site of cleavage. ( C ). Stem elements P3–P7 within the core domain are labeled.
    Figure Legend Snippet: In vitro evolution of the 38-6 RNA polymerase ribozyme. ( A ) Scheme for selective amplification of polymerase ribozymes that synthesize a functional hammerhead ribozyme. (1) Attachment to the polymerase of an RNA primer (magenta), biotin (green), and the RNA substrate (orange) to be cleaved by the hammerhead. (2) Hybridization of the primer to an RNA template (brown) that encodes the hammerhead. (3) Extension of the primer by polymerization of NTPs (cyan), followed by biotin capture on streptavidin magnetic beads (gray). (4) Cleavage of the attached RNA substrate by the hammerhead, releasing the polymerase from the beads. (5) Recovery of functional polymerases. (6) Reverse transcription and PCR amplification. (7) Transcription to generate progeny polymerases. ( B ) Sequence and secondary structure of the hammerhead ribozyme (cyan), together with the primer used to initiate its synthesis and the RNA substrate. The arrow indicates the site of cleavage. ( C ). Stem elements P3–P7 within the core domain are labeled.

    Techniques Used: In Vitro, Amplification, Functional Assay, Hybridization, Magnetic Beads, Polymerase Chain Reaction, Sequencing, Labeling

    8) Product Images from "Enhanced Anticoagulation Activity of Functional RNA Origami"

    Article Title: Enhanced Anticoagulation Activity of Functional RNA Origami

    Journal: bioRxiv

    doi: 10.1101/2020.09.29.319590

    Reversibility of RNA origami anticoagulation via ssDNA and ssPNA antidotes. (A) 2D illustration of active and inactive 2HF-2NN1 anticoagulant. Exosite-1 and 2 aptamers are highlighted in purple and blue, respectively. (B) Recovery of thrombin activity by addition of specific DNA and PNA antidotes. Percent activity remaining +/− standard deviation.
    Figure Legend Snippet: Reversibility of RNA origami anticoagulation via ssDNA and ssPNA antidotes. (A) 2D illustration of active and inactive 2HF-2NN1 anticoagulant. Exosite-1 and 2 aptamers are highlighted in purple and blue, respectively. (B) Recovery of thrombin activity by addition of specific DNA and PNA antidotes. Percent activity remaining +/− standard deviation.

    Techniques Used: Activity Assay, Standard Deviation

    9) Product Images from "Nanoparticle-Mediated In Situ Molecular Reprogramming of Immune Checkpoint Interactions for Cancer Immunotherapy"

    Article Title: Nanoparticle-Mediated In Situ Molecular Reprogramming of Immune Checkpoint Interactions for Cancer Immunotherapy

    Journal: ACS Nano

    doi: 10.1021/acsnano.1c04456

    SNALPs have an irregular structure with evidence of internal concentric rings and both mRNA and siRNA are loaded into SNALPs with minimal interference between molecules. (A) SNALPs were formulated with siRNA, mRNA, or a combination of both RNA molecules as previously described. SNALPs were drop-cast on to a graphene grid and imaging was carried out using a Tecnai Osiris transmission electron microscopy. Images represent a single event representative of the wider field. (B) To confirm that both types of nucleic acid can be loaded into SNALPs and that there is minimal hindrance between either molecule, SNALPs coformulating mRNA, and siRNA were treated with RNase H (1 mg/mL) to degrade non-encapsulated/external RNA. The enzyme was inactivated with heat and EDTA (1.25 mM), the SNALP was dissociated by incubation with 10% ( v / v ) heparin. The RNA was purified with Monarch RNA Cleanup Kit and run on a 2% agarose gel at 225 V for 25 min. (C) Resulting gel image. Free siRNA and mRNA were run as size markers. A mix of the two free nucleic acids (Mix) corresponding to the starting ratio of nucleic acid (50:50) at a quantity equal to the amount obtained from the SNALP was run alongside the RNA extracted from the SNALP (dissociated SNALP). (D) The intensities of the bands for both Mix and dissociated SNALP were measured using imageJ software.
    Figure Legend Snippet: SNALPs have an irregular structure with evidence of internal concentric rings and both mRNA and siRNA are loaded into SNALPs with minimal interference between molecules. (A) SNALPs were formulated with siRNA, mRNA, or a combination of both RNA molecules as previously described. SNALPs were drop-cast on to a graphene grid and imaging was carried out using a Tecnai Osiris transmission electron microscopy. Images represent a single event representative of the wider field. (B) To confirm that both types of nucleic acid can be loaded into SNALPs and that there is minimal hindrance between either molecule, SNALPs coformulating mRNA, and siRNA were treated with RNase H (1 mg/mL) to degrade non-encapsulated/external RNA. The enzyme was inactivated with heat and EDTA (1.25 mM), the SNALP was dissociated by incubation with 10% ( v / v ) heparin. The RNA was purified with Monarch RNA Cleanup Kit and run on a 2% agarose gel at 225 V for 25 min. (C) Resulting gel image. Free siRNA and mRNA were run as size markers. A mix of the two free nucleic acids (Mix) corresponding to the starting ratio of nucleic acid (50:50) at a quantity equal to the amount obtained from the SNALP was run alongside the RNA extracted from the SNALP (dissociated SNALP). (D) The intensities of the bands for both Mix and dissociated SNALP were measured using imageJ software.

    Techniques Used: Imaging, Transmission Assay, Electron Microscopy, Incubation, Purification, Agarose Gel Electrophoresis, Software

    10) Product Images from "Synthesis of low immunogenicity RNA with high-temperature in vitro transcription"

    Article Title: Synthesis of low immunogenicity RNA with high-temperature in vitro transcription

    Journal: RNA

    doi: 10.1261/rna.073858.119

    Template-encoded poly(A) tailing reduces antisense by-product formation. ( A ) dsRNA immunoblot with J2 antibody and gel electrophoresis analysis of CLuc RNA synthesized from CLuc templates with varying length (30, 60, 120 bp) of poly(T) sequence at 3′ end under standard conditions. ( B ) Immunoblot and native gel electrophoresis analysis of IVT reactions on 512B::CLuc chimeric template with poly(T) (60 and 120 bp) sequence at the 3′ end. IVT reactions were performed at 37°C or 50°C.
    Figure Legend Snippet: Template-encoded poly(A) tailing reduces antisense by-product formation. ( A ) dsRNA immunoblot with J2 antibody and gel electrophoresis analysis of CLuc RNA synthesized from CLuc templates with varying length (30, 60, 120 bp) of poly(T) sequence at 3′ end under standard conditions. ( B ) Immunoblot and native gel electrophoresis analysis of IVT reactions on 512B::CLuc chimeric template with poly(T) (60 and 120 bp) sequence at the 3′ end. IVT reactions were performed at 37°C or 50°C.

    Techniques Used: Nucleic Acid Electrophoresis, Synthesized, Sequencing

    11) Product Images from "Klebsiella Phage KP34 RNA Polymerase and Its Use in RNA Synthesis"

    Article Title: Klebsiella Phage KP34 RNA Polymerase and Its Use in RNA Synthesis

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2019.02487

    Identification of the KP34 RNAP promoter. (A) The DNA fragment (Template 1) containing previously predicated promoters failed to serve as transcription template for KP34 RNAP to produce RNA in vitro , while the DNA fragment covering the RNAP gene and downstream gap region (Template 2) was active as transcription template. RNA products are shown as bright bands on the 2% TAE agarose gel. (B) 5′-RACE analysis of the position of transcription initiation. 5′ sequence of KP34 transcripts were matched to KP34 genome. Major sequences were in solid box and minor sequences in dotted box. Their upstream region containing putative promoters is shown in bold. (C) Comparison of run-off RNA synthesis by T7 and KP34 RNAP under the control of various promoters. A DNA template containing a T7 promoter (5′-TAATACGACTCACTATA-3′) was incubated with 100 nM T7 RNAP, and three DNA templates containing either a KP34 strong promoter S1 (5′-TAATGTTACAGGAGTA-3′), a KP34 strong promoter S2 (5′-TGATGTTACAGGAGTA-3′), or a KP34 weak promoter W (5′-ACTTTGGACATCCG TCAAGT-3′) were incubated with 100 nM KP34 RNAP to direct to the transcription of their downstream sequence that encodes the same 37 nt RNA. [α-32P]ATP was added into reactions for imaging and visualization. Reaction products were separated by a 25% TBE-Urea denaturing gel. (D) Identification of the full KP34 strong promoter. A KP34 strong promoter S1 (5′-TAATGTTACAGGAGTA-3′) or the 3′ 14 nt common sequence of the two KP34 strong promoters (5′-ATGTTA CAGGAGTA-3′) was inserted into plasmid pUC19 to direct the transcription of their downstream sequences, respectively. A run-off transcript of ∼2700 nt and a terminated transcript of ∼1000 nt (terminated by a predictable T7 class I hairpin terminator structure) were expected from the linearized form of these plasmids if the inserted promoter is sufficient to direct transcription by KP34 RNAP. M: ssRNA Ladder. (E) KP34 promoters in the genome (location of strong promoter (S) pointed by solid arrow and weak promoter (W) by dotted arrow) and comparison of typical ssRNAP promoters. Conserved sequence among ssRNAP promoters are in bold and those homologous between Syn5 promoter and KP34 weak promoter are underlined.
    Figure Legend Snippet: Identification of the KP34 RNAP promoter. (A) The DNA fragment (Template 1) containing previously predicated promoters failed to serve as transcription template for KP34 RNAP to produce RNA in vitro , while the DNA fragment covering the RNAP gene and downstream gap region (Template 2) was active as transcription template. RNA products are shown as bright bands on the 2% TAE agarose gel. (B) 5′-RACE analysis of the position of transcription initiation. 5′ sequence of KP34 transcripts were matched to KP34 genome. Major sequences were in solid box and minor sequences in dotted box. Their upstream region containing putative promoters is shown in bold. (C) Comparison of run-off RNA synthesis by T7 and KP34 RNAP under the control of various promoters. A DNA template containing a T7 promoter (5′-TAATACGACTCACTATA-3′) was incubated with 100 nM T7 RNAP, and three DNA templates containing either a KP34 strong promoter S1 (5′-TAATGTTACAGGAGTA-3′), a KP34 strong promoter S2 (5′-TGATGTTACAGGAGTA-3′), or a KP34 weak promoter W (5′-ACTTTGGACATCCG TCAAGT-3′) were incubated with 100 nM KP34 RNAP to direct to the transcription of their downstream sequence that encodes the same 37 nt RNA. [α-32P]ATP was added into reactions for imaging and visualization. Reaction products were separated by a 25% TBE-Urea denaturing gel. (D) Identification of the full KP34 strong promoter. A KP34 strong promoter S1 (5′-TAATGTTACAGGAGTA-3′) or the 3′ 14 nt common sequence of the two KP34 strong promoters (5′-ATGTTA CAGGAGTA-3′) was inserted into plasmid pUC19 to direct the transcription of their downstream sequences, respectively. A run-off transcript of ∼2700 nt and a terminated transcript of ∼1000 nt (terminated by a predictable T7 class I hairpin terminator structure) were expected from the linearized form of these plasmids if the inserted promoter is sufficient to direct transcription by KP34 RNAP. M: ssRNA Ladder. (E) KP34 promoters in the genome (location of strong promoter (S) pointed by solid arrow and weak promoter (W) by dotted arrow) and comparison of typical ssRNAP promoters. Conserved sequence among ssRNAP promoters are in bold and those homologous between Syn5 promoter and KP34 weak promoter are underlined.

    Techniques Used: In Vitro, Agarose Gel Electrophoresis, Sequencing, Incubation, Imaging, Plasmid Preparation

    Synthesis of a 50 nt RNA containing 3′ hairpin structure by various RNAPs. (A) The 50 nt RNA sequence is shown at the top of the gel. The three DNA templates containing the same coding sequences for the 50 nt run-off RNA transcripts under the control of either a T7 promoter, a KP34 strong promoter, or a Syn5 promoter were incubated with 0.2 μM T7 RNAP, 1 μM KP34 RNAP, or 1 μM Syn5 RNAP, respectively. Incubation with KP34 and T7 RNAP was at 37°C for 1 h and incubation with Syn5 RNAP was at 24°C for 1 h. Reaction products were separated by a 12% TBE native gel and then stained with ethidium bromide. M: ssRNA Ladder. (B) RNA-Seq analysis of the 3′ termini of T7 RNAP transcripts. 3′ termini of sequences with reads more than 1% of total reads were aligned and shown. Percentage of major sequences in total sequencing results are noted and percentage of the correct product is in bold. A dotted line cut indicates the precise terminus encoded by DNA template, and the number of extended nt is shown as n + x. Bold sequences indicate complementary sequences in each RNA specie resulted from extension of a possible 3′ self-primed structure. (C) Similar as B, RNA-Seq analysis of the 3′ termini of KP34 RNAP transcripts. Number of missing nt at the 3′ terminus of major sequences is shown as n–x.
    Figure Legend Snippet: Synthesis of a 50 nt RNA containing 3′ hairpin structure by various RNAPs. (A) The 50 nt RNA sequence is shown at the top of the gel. The three DNA templates containing the same coding sequences for the 50 nt run-off RNA transcripts under the control of either a T7 promoter, a KP34 strong promoter, or a Syn5 promoter were incubated with 0.2 μM T7 RNAP, 1 μM KP34 RNAP, or 1 μM Syn5 RNAP, respectively. Incubation with KP34 and T7 RNAP was at 37°C for 1 h and incubation with Syn5 RNAP was at 24°C for 1 h. Reaction products were separated by a 12% TBE native gel and then stained with ethidium bromide. M: ssRNA Ladder. (B) RNA-Seq analysis of the 3′ termini of T7 RNAP transcripts. 3′ termini of sequences with reads more than 1% of total reads were aligned and shown. Percentage of major sequences in total sequencing results are noted and percentage of the correct product is in bold. A dotted line cut indicates the precise terminus encoded by DNA template, and the number of extended nt is shown as n + x. Bold sequences indicate complementary sequences in each RNA specie resulted from extension of a possible 3′ self-primed structure. (C) Similar as B, RNA-Seq analysis of the 3′ termini of KP34 RNAP transcripts. Number of missing nt at the 3′ terminus of major sequences is shown as n–x.

    Techniques Used: Sequencing, Incubation, Staining, RNA Sequencing Assay

    Synthesis of an sgRNA by T7 and KP34 RNAP. (A) The sgRNA sequence is shown at the top of the gel. The three DNA templates containing the same coding sequences for the sgRNA under the control of either a T7 promoter, a KP34 strong promoter, or a Syn5 promoter were incubated with 0.2 μM T7 RNAP, 1 μM KP34 RNAP, or 1 μM Syn5 RNAP, respectively. Incubation with KP34 and T7 RNAP was at 37°C for 1 h and incubation with Syn5 RNAP was at 24°C for 1 h. Reaction products were separated by a 12% TBE native gel and then stained with ethidium bromide. M: ssRNA Ladder. (B) 3′-RACE analysis of the 3′ termini of T7 RNAP transcripts. 3′ termini of obtained sequences were aligned and shown. A dotted line cut indicates the precise terminus encoded by DNA template and number of extended or missing nt is shown as n + x or n–x. Bold sequences indicate complementary sequences in each RNA resulted from extension of possible 3′ self-primed structures. (C) Similar as B, 3′-RACE analysis of the 3′ termini of KP34 RNAP transcripts.
    Figure Legend Snippet: Synthesis of an sgRNA by T7 and KP34 RNAP. (A) The sgRNA sequence is shown at the top of the gel. The three DNA templates containing the same coding sequences for the sgRNA under the control of either a T7 promoter, a KP34 strong promoter, or a Syn5 promoter were incubated with 0.2 μM T7 RNAP, 1 μM KP34 RNAP, or 1 μM Syn5 RNAP, respectively. Incubation with KP34 and T7 RNAP was at 37°C for 1 h and incubation with Syn5 RNAP was at 24°C for 1 h. Reaction products were separated by a 12% TBE native gel and then stained with ethidium bromide. M: ssRNA Ladder. (B) 3′-RACE analysis of the 3′ termini of T7 RNAP transcripts. 3′ termini of obtained sequences were aligned and shown. A dotted line cut indicates the precise terminus encoded by DNA template and number of extended or missing nt is shown as n + x or n–x. Bold sequences indicate complementary sequences in each RNA resulted from extension of possible 3′ self-primed structures. (C) Similar as B, 3′-RACE analysis of the 3′ termini of KP34 RNAP transcripts.

    Techniques Used: Sequencing, Incubation, Staining

    12) Product Images from "In vitro transcription using psychrophilic phage VSW-3 RNA polymerase"

    Article Title: In vitro transcription using psychrophilic phage VSW-3 RNA polymerase

    Journal: bioRxiv

    doi: 10.1101/2020.09.14.297226

    Response of ssRNAPs to Class II terminator. (A) Using PCR-amplified templates for cas9-RNA IVT, obvious abortive RNA transcripts were observed for T7 RNAP and Syn5 RNAP but not VSW-3 RNAP (top gel). 3’-RACE revealed that the T7 RNAP transcription was terminated 9 nt downstream of a Class II terminator “ATCTGTT” (bottom sequencing result). (B) VSW-3 RNAP IVT was not terminated (no additional bands comparing lane 2 with lane 1) when a Class II terminator “ATCTGTT” was inserted into the middle of the copGFP RNA coding sequence.
    Figure Legend Snippet: Response of ssRNAPs to Class II terminator. (A) Using PCR-amplified templates for cas9-RNA IVT, obvious abortive RNA transcripts were observed for T7 RNAP and Syn5 RNAP but not VSW-3 RNAP (top gel). 3’-RACE revealed that the T7 RNAP transcription was terminated 9 nt downstream of a Class II terminator “ATCTGTT” (bottom sequencing result). (B) VSW-3 RNAP IVT was not terminated (no additional bands comparing lane 2 with lane 1) when a Class II terminator “ATCTGTT” was inserted into the middle of the copGFP RNA coding sequence.

    Techniques Used: Polymerase Chain Reaction, Amplification, Sequencing

    RNA 3’ extension and RdRp activity of T7 and VSW-3 RNAP. (A) The secondary structure of a sgRNA predicted with RNAfold software. (B) IVT synthesis of a sgRNA (targeting eGFP) by VSW-3 and T7 RNAP. (C) 3’-RACE of the sgRNAs transcripts from T7 and VSW-3 RNAP IVT. Only the 3’ region (red sequence on the top) of the full sgRNA in sequencing results was shown. The length of each sequence was noted. The sequences matching the exact run-off sgRNA (103 nt) was indicated by red stars. (D) Schematic showing the mechanism and origin (3’ self-templated extension by the RdRp activity of T7 RNAP) of the 16 nt 3’-extension in T7 RNAP products as in (C) . (E) T7 but not VSW-3 RNAP retains the RdRp activity to extend purified sgRNA (with terminal primer/template structure).
    Figure Legend Snippet: RNA 3’ extension and RdRp activity of T7 and VSW-3 RNAP. (A) The secondary structure of a sgRNA predicted with RNAfold software. (B) IVT synthesis of a sgRNA (targeting eGFP) by VSW-3 and T7 RNAP. (C) 3’-RACE of the sgRNAs transcripts from T7 and VSW-3 RNAP IVT. Only the 3’ region (red sequence on the top) of the full sgRNA in sequencing results was shown. The length of each sequence was noted. The sequences matching the exact run-off sgRNA (103 nt) was indicated by red stars. (D) Schematic showing the mechanism and origin (3’ self-templated extension by the RdRp activity of T7 RNAP) of the 16 nt 3’-extension in T7 RNAP products as in (C) . (E) T7 but not VSW-3 RNAP retains the RdRp activity to extend purified sgRNA (with terminal primer/template structure).

    Techniques Used: Activity Assay, Software, Sequencing, Purification

    VSW-3 RNAP and its promoter. (A) Distance tree analysis of the representative ssRNAPs by Blast program. Distance from the root ‘○’: SP6 RNAP (3.374) > T7 RNAP (3.145) > KP34 RNAP (2.572) > VSW-3 RNAP (2.292) > Syn5 RNAP (1.118) suggests that VSW-3 RNAP is the second primitive after Syn5 RNAP, and evolved into a new branch of the evolutionary tree together with a predicted pollyC RNAP (3.055) from phage pollyC (YP_009622558.1). (B) SDS-PAGE gel analysis of purified VSW-3 RNAP (92.4 kDa including an N-terminal His-tag, 1 μM) and commercial T7 RNAP (New England Biolabs, 100 kDa, 1.5 μM), gel was stained with Coomassie blue. (C) Organization of phage VSW-3 genome and distribution of the predicted VSW-3 promoters (indicated by rightward arrows). (D) IVT of VSW-3 RNAP on the linearized pUC19 plasmid with an insertion of predicted VSW-3 promoter (top gel). 5’-RACE revealed that the initial nucleotides of VSW-3 RNAP transcription in the predicted promoter is “GTA” (bottom sequencing result). (E) IVT on 5’-truncated DNA templates (left box) to determine the accurate promoter of VSW-3 RNAP. The RNA yield with each template (right gel) suggests that the 15 bp (5’-ATTGGGCCACCTATA-3’) sequence is the minimal promoter and the 18 bp (5’-TTAATTGGGCCACCTATA-3’) sequence is the full VSW-3 promoter.
    Figure Legend Snippet: VSW-3 RNAP and its promoter. (A) Distance tree analysis of the representative ssRNAPs by Blast program. Distance from the root ‘○’: SP6 RNAP (3.374) > T7 RNAP (3.145) > KP34 RNAP (2.572) > VSW-3 RNAP (2.292) > Syn5 RNAP (1.118) suggests that VSW-3 RNAP is the second primitive after Syn5 RNAP, and evolved into a new branch of the evolutionary tree together with a predicted pollyC RNAP (3.055) from phage pollyC (YP_009622558.1). (B) SDS-PAGE gel analysis of purified VSW-3 RNAP (92.4 kDa including an N-terminal His-tag, 1 μM) and commercial T7 RNAP (New England Biolabs, 100 kDa, 1.5 μM), gel was stained with Coomassie blue. (C) Organization of phage VSW-3 genome and distribution of the predicted VSW-3 promoters (indicated by rightward arrows). (D) IVT of VSW-3 RNAP on the linearized pUC19 plasmid with an insertion of predicted VSW-3 promoter (top gel). 5’-RACE revealed that the initial nucleotides of VSW-3 RNAP transcription in the predicted promoter is “GTA” (bottom sequencing result). (E) IVT on 5’-truncated DNA templates (left box) to determine the accurate promoter of VSW-3 RNAP. The RNA yield with each template (right gel) suggests that the 15 bp (5’-ATTGGGCCACCTATA-3’) sequence is the minimal promoter and the 18 bp (5’-TTAATTGGGCCACCTATA-3’) sequence is the full VSW-3 promoter.

    Techniques Used: SDS Page, Purification, Staining, Plasmid Preparation, Sequencing

    dsRNA by-products from T7 and VSW-3 RNAP IVT. (A) DNA templates for the IVT synthesis of various RNA as indicated on top of the gel. For each RNA, there are two DNA templates differ only in the promoter region to serve for VSW-3 and T7 RNAP IVT, respectively. DNA concentration and purity were compared in 1.5% agarose gel stained with ethidium bromide. (B) After template DNA was removed by DNase I treatment and purified with Monarch RNA Cleanup kit, 1μg of sox7, tdTomato, copGFP and Cas9 RNA transcribed by VSW-3 RNAP and T7 RNAP were analyzed in 1.5% agarose gel stained with ethidium bromide. The white and black colors for bands and background were converted in this gel picture to make the weak double-stranded and abortive RNA bands clearer. (C) Dot blot analysis of the RNA products (each 200 ng) as in (B) by VSW-3 RNAP and T7 RNAP with J2 monoclonal antibody. A prepared dsRNA (351 bp) was applied as quantitative standard (0.1 ng, 0.25 ng, 0.5 ng, 1.0 ng). (D) The gray value measurement and calculation of the X film image (top image in (C) ) by Image J software demonstrating the level of dsRNA contamination in T7 and VSW-3 RNAP transcripts.
    Figure Legend Snippet: dsRNA by-products from T7 and VSW-3 RNAP IVT. (A) DNA templates for the IVT synthesis of various RNA as indicated on top of the gel. For each RNA, there are two DNA templates differ only in the promoter region to serve for VSW-3 and T7 RNAP IVT, respectively. DNA concentration and purity were compared in 1.5% agarose gel stained with ethidium bromide. (B) After template DNA was removed by DNase I treatment and purified with Monarch RNA Cleanup kit, 1μg of sox7, tdTomato, copGFP and Cas9 RNA transcribed by VSW-3 RNAP and T7 RNAP were analyzed in 1.5% agarose gel stained with ethidium bromide. The white and black colors for bands and background were converted in this gel picture to make the weak double-stranded and abortive RNA bands clearer. (C) Dot blot analysis of the RNA products (each 200 ng) as in (B) by VSW-3 RNAP and T7 RNAP with J2 monoclonal antibody. A prepared dsRNA (351 bp) was applied as quantitative standard (0.1 ng, 0.25 ng, 0.5 ng, 1.0 ng). (D) The gray value measurement and calculation of the X film image (top image in (C) ) by Image J software demonstrating the level of dsRNA contamination in T7 and VSW-3 RNAP transcripts.

    Techniques Used: Concentration Assay, Agarose Gel Electrophoresis, Staining, Purification, Dot Blot, Software

    13) Product Images from "Active coacervate droplets as a model for membraneless organelles and protocells"

    Article Title: Active coacervate droplets as a model for membraneless organelles and protocells

    Journal: Nature Communications

    doi: 10.1038/s41467-020-18815-9

    Functional RNA inside fuel-driven droplets. a Schematic representation of the experimental procedure for dynamic droplets with functional RNA. b Confocal micrographs of 23 mM precursor, 4.1 mM poly-U, 25 mM EDC with 0.2 µM Cy5-RNA (SunY ribozyme, Hammerhead ribozyme or Broccoli aptamer), 5 min after addition of EDC. c Confocal micrographs of the SunY containing solution described in d (with 27 mM EDC) at different time points before or after EDC addition. d Fluorescence intensity of solutions containing the Broccoli aptamer with or without DFHB1T (ligand), in the presence and absence of droplets. Maximum fluorescence intensity at 504 nm. Standard conditions with 2 mM MgCl 2 , 30 mM KCl, and 1.5 µM Broccoli aptamer. Addition of 10 mM EDC (fuel) to induce droplet formation. e Confocal and bright field micrographs under the conditions described in d , 5 min after the addition of 15 mM EDC. Experiments were perfomed for n = 2. Source data are provided as a Source Data file.
    Figure Legend Snippet: Functional RNA inside fuel-driven droplets. a Schematic representation of the experimental procedure for dynamic droplets with functional RNA. b Confocal micrographs of 23 mM precursor, 4.1 mM poly-U, 25 mM EDC with 0.2 µM Cy5-RNA (SunY ribozyme, Hammerhead ribozyme or Broccoli aptamer), 5 min after addition of EDC. c Confocal micrographs of the SunY containing solution described in d (with 27 mM EDC) at different time points before or after EDC addition. d Fluorescence intensity of solutions containing the Broccoli aptamer with or without DFHB1T (ligand), in the presence and absence of droplets. Maximum fluorescence intensity at 504 nm. Standard conditions with 2 mM MgCl 2 , 30 mM KCl, and 1.5 µM Broccoli aptamer. Addition of 10 mM EDC (fuel) to induce droplet formation. e Confocal and bright field micrographs under the conditions described in d , 5 min after the addition of 15 mM EDC. Experiments were perfomed for n = 2. Source data are provided as a Source Data file.

    Techniques Used: Functional Assay, Fluorescence

    14) Product Images from "In vitro transcription using psychrophilic phage VSW-3 RNA polymerase"

    Article Title: In vitro transcription using psychrophilic phage VSW-3 RNA polymerase

    Journal: bioRxiv

    doi: 10.1101/2020.09.14.297226

    Optimal VSW-3 RNAP IVT conditions. (A) Screening for the optimal Mg 2+ /NTP concentration in the presence of 1 mM DTT. RNA yield with various optimal Mg 2+ /NTP concentration combination was further compared (gel in the dotted box). The stability of the optimal VSW-3 RNAP IVT buffer with 1 mM DTT was examined (gel in the solid box). (B) Screening for the optimal DTT/ Mg 2+ concentration for the stable and high-yield VSW-3 RNAP IVT buffer. The stability of the high-yield VSW-3 RNAP IVT buffer containing 16 mM Mg 2+ , 4 mM NTP and 5 mM DTT was examined (gel in the solid box). (C) The optimal reaction temperature of VSW-3 RNAP (25°C) for maximum run-off RNA yield. (D) The optimal enzyme concentration of VSW-3 RNAP (0.15 μM) for maximum run-off RNA yield. (E) Optimal IVT yield of VSW-3 RNAP with various reaction temperature/incubation time combinations. The maximum run-off RNA yield was obtained at 25°C for 12 hours. (F) Gray-scale quantitation of the run-off RNA transcripts in gel (E) . Diagram was made using GraphPad Prism. In all gels the bands corresponding to DNA templates were indicated by empty stars and the bands corresponding to run-off RNA transcripts were indicated by filled stars.
    Figure Legend Snippet: Optimal VSW-3 RNAP IVT conditions. (A) Screening for the optimal Mg 2+ /NTP concentration in the presence of 1 mM DTT. RNA yield with various optimal Mg 2+ /NTP concentration combination was further compared (gel in the dotted box). The stability of the optimal VSW-3 RNAP IVT buffer with 1 mM DTT was examined (gel in the solid box). (B) Screening for the optimal DTT/ Mg 2+ concentration for the stable and high-yield VSW-3 RNAP IVT buffer. The stability of the high-yield VSW-3 RNAP IVT buffer containing 16 mM Mg 2+ , 4 mM NTP and 5 mM DTT was examined (gel in the solid box). (C) The optimal reaction temperature of VSW-3 RNAP (25°C) for maximum run-off RNA yield. (D) The optimal enzyme concentration of VSW-3 RNAP (0.15 μM) for maximum run-off RNA yield. (E) Optimal IVT yield of VSW-3 RNAP with various reaction temperature/incubation time combinations. The maximum run-off RNA yield was obtained at 25°C for 12 hours. (F) Gray-scale quantitation of the run-off RNA transcripts in gel (E) . Diagram was made using GraphPad Prism. In all gels the bands corresponding to DNA templates were indicated by empty stars and the bands corresponding to run-off RNA transcripts were indicated by filled stars.

    Techniques Used: Concentration Assay, Incubation, Quantitation Assay

    VSW-3 RNAP and its promoter. (A) Distance tree analysis of the representative ssRNAPs by Blast program. Distance from the root ‘○’: SP6 RNAP (3.374) > T7 RNAP (3.145) > KP34 RNAP (2.572) > VSW-3 RNAP (2.292) > Syn5 RNAP (1.118) suggests that VSW-3 RNAP is the second primitive after Syn5 RNAP, and evolved into a new branch of the evolutionary tree together with a predicted pollyC RNAP (3.055) from phage pollyC (YP_009622558.1). (B) SDS-PAGE gel analysis of purified VSW-3 RNAP (92.4 kDa including an N-terminal His-tag, 1 μM) and commercial T7 RNAP (New England Biolabs, 100 kDa, 1.5 μM), gel was stained with Coomassie blue. (C) Organization of phage VSW-3 genome and distribution of the predicted VSW-3 promoters (indicated by rightward arrows). (D) IVT of VSW-3 RNAP on the linearized pUC19 plasmid with an insertion of predicted VSW-3 promoter (top gel). 5’-RACE revealed that the initial nucleotides of VSW-3 RNAP transcription in the predicted promoter is “GTA” (bottom sequencing result). (E) IVT on 5’-truncated DNA templates (left box) to determine the accurate promoter of VSW-3 RNAP. The RNA yield with each template (right gel) suggests that the 15 bp (5’-ATTGGGCCACCTATA-3’) sequence is the minimal promoter and the 18 bp (5’-TTAATTGGGCCACCTATA-3’) sequence is the full VSW-3 promoter.
    Figure Legend Snippet: VSW-3 RNAP and its promoter. (A) Distance tree analysis of the representative ssRNAPs by Blast program. Distance from the root ‘○’: SP6 RNAP (3.374) > T7 RNAP (3.145) > KP34 RNAP (2.572) > VSW-3 RNAP (2.292) > Syn5 RNAP (1.118) suggests that VSW-3 RNAP is the second primitive after Syn5 RNAP, and evolved into a new branch of the evolutionary tree together with a predicted pollyC RNAP (3.055) from phage pollyC (YP_009622558.1). (B) SDS-PAGE gel analysis of purified VSW-3 RNAP (92.4 kDa including an N-terminal His-tag, 1 μM) and commercial T7 RNAP (New England Biolabs, 100 kDa, 1.5 μM), gel was stained with Coomassie blue. (C) Organization of phage VSW-3 genome and distribution of the predicted VSW-3 promoters (indicated by rightward arrows). (D) IVT of VSW-3 RNAP on the linearized pUC19 plasmid with an insertion of predicted VSW-3 promoter (top gel). 5’-RACE revealed that the initial nucleotides of VSW-3 RNAP transcription in the predicted promoter is “GTA” (bottom sequencing result). (E) IVT on 5’-truncated DNA templates (left box) to determine the accurate promoter of VSW-3 RNAP. The RNA yield with each template (right gel) suggests that the 15 bp (5’-ATTGGGCCACCTATA-3’) sequence is the minimal promoter and the 18 bp (5’-TTAATTGGGCCACCTATA-3’) sequence is the full VSW-3 promoter.

    Techniques Used: SDS Page, Purification, Staining, Plasmid Preparation, Sequencing

    dsRNA by-products from T7 and VSW-3 RNAP IVT. (A) DNA templates for the IVT synthesis of various RNA as indicated on top of the gel. For each RNA, there are two DNA templates differ only in the promoter region to serve for VSW-3 and T7 RNAP IVT, respectively. DNA concentration and purity were compared in 1.5% agarose gel stained with ethidium bromide. (B) After template DNA was removed by DNase I treatment and purified with Monarch RNA Cleanup kit, 1μg of sox7, tdTomato, copGFP and Cas9 RNA transcribed by VSW-3 RNAP and T7 RNAP were analyzed in 1.5% agarose gel stained with ethidium bromide. The white and black colors for bands and background were converted in this gel picture to make the weak double-stranded and abortive RNA bands clearer. (C) Dot blot analysis of the RNA products (each 200 ng) as in (B) by VSW-3 RNAP and T7 RNAP with J2 monoclonal antibody. A prepared dsRNA (351 bp) was applied as quantitative standard (0.1 ng, 0.25 ng, 0.5 ng, 1.0 ng). (D) The gray value measurement and calculation of the X film image (top image in (C) ) by Image J software demonstrating the level of dsRNA contamination in T7 and VSW-3 RNAP transcripts.
    Figure Legend Snippet: dsRNA by-products from T7 and VSW-3 RNAP IVT. (A) DNA templates for the IVT synthesis of various RNA as indicated on top of the gel. For each RNA, there are two DNA templates differ only in the promoter region to serve for VSW-3 and T7 RNAP IVT, respectively. DNA concentration and purity were compared in 1.5% agarose gel stained with ethidium bromide. (B) After template DNA was removed by DNase I treatment and purified with Monarch RNA Cleanup kit, 1μg of sox7, tdTomato, copGFP and Cas9 RNA transcribed by VSW-3 RNAP and T7 RNAP were analyzed in 1.5% agarose gel stained with ethidium bromide. The white and black colors for bands and background were converted in this gel picture to make the weak double-stranded and abortive RNA bands clearer. (C) Dot blot analysis of the RNA products (each 200 ng) as in (B) by VSW-3 RNAP and T7 RNAP with J2 monoclonal antibody. A prepared dsRNA (351 bp) was applied as quantitative standard (0.1 ng, 0.25 ng, 0.5 ng, 1.0 ng). (D) The gray value measurement and calculation of the X film image (top image in (C) ) by Image J software demonstrating the level of dsRNA contamination in T7 and VSW-3 RNAP transcripts.

    Techniques Used: Concentration Assay, Agarose Gel Electrophoresis, Staining, Purification, Dot Blot, Software

    15) Product Images from "hNOP2/NSUN1 Regulates Ribosome Biogenesis through Stabilization of snoRNP Complexes and Cytosine-5 Methylation of 28S rRNA"

    Article Title: hNOP2/NSUN1 Regulates Ribosome Biogenesis through Stabilization of snoRNP Complexes and Cytosine-5 Methylation of 28S rRNA

    Journal: bioRxiv

    doi: 10.1101/2021.11.12.468419

    Human NOP2/NSUN1 miCLIP design. (A) Alignment of the protein sequences of human NSUN family members showing conserved cysteine residues (highlighted) required for catalytic activity. The mutated cysteine in motif IV used for the miCLIP is indicated as C459 (B) Cartoon showing that only NOP2/NUSN1 C459A mutant forms irreversible covalent crosslinks with its RNA substrate. (C) FLAG-tagged NOP2/NSUN1 Wildtype and C459A mutant were immunoprecipitated with a FLAG antibody and immunoblotted with FLAG antibody. (D) FLAG-tagged NOP2/NSUN1 Wildtype and C459A mutant were immunoprecipitated by FLAG antibody and 3’-end labeled with pCp-biotin for detection of co-precipitated RNA. After membrane transfer, the pCp-biotin labeled RNA was detected with Streptavidin-IR800. Immunoprecipitated NOP2/NSUN1 Wildtype and C459A mutant were detected by immunoblotting with a FLAG antibody. Right panel show a high exposure of the pCp-biotin labeled RNA with relative densitometry analysis. (E) Schematic overview of NOP2/NSUN1 miCLIP-sequencing. Cells were lysed, digested with RNase, immunoprecipitated with FLAG antibody, separated on SDS-PAGE gel and transferred to membrane. NOP2/NSUN1 Wildtype and C459A mutant associated RNAs as well as their respective size matched inputs (SMInputs) were extracted from the membrane and processed for sequencing libraries.
    Figure Legend Snippet: Human NOP2/NSUN1 miCLIP design. (A) Alignment of the protein sequences of human NSUN family members showing conserved cysteine residues (highlighted) required for catalytic activity. The mutated cysteine in motif IV used for the miCLIP is indicated as C459 (B) Cartoon showing that only NOP2/NUSN1 C459A mutant forms irreversible covalent crosslinks with its RNA substrate. (C) FLAG-tagged NOP2/NSUN1 Wildtype and C459A mutant were immunoprecipitated with a FLAG antibody and immunoblotted with FLAG antibody. (D) FLAG-tagged NOP2/NSUN1 Wildtype and C459A mutant were immunoprecipitated by FLAG antibody and 3’-end labeled with pCp-biotin for detection of co-precipitated RNA. After membrane transfer, the pCp-biotin labeled RNA was detected with Streptavidin-IR800. Immunoprecipitated NOP2/NSUN1 Wildtype and C459A mutant were detected by immunoblotting with a FLAG antibody. Right panel show a high exposure of the pCp-biotin labeled RNA with relative densitometry analysis. (E) Schematic overview of NOP2/NSUN1 miCLIP-sequencing. Cells were lysed, digested with RNase, immunoprecipitated with FLAG antibody, separated on SDS-PAGE gel and transferred to membrane. NOP2/NSUN1 Wildtype and C459A mutant associated RNAs as well as their respective size matched inputs (SMInputs) were extracted from the membrane and processed for sequencing libraries.

    Techniques Used: Activity Assay, Mutagenesis, Immunoprecipitation, Labeling, Sequencing, SDS Page

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    New England Biolabs monarch rna cleanup kit
    VSW-3 RNAP and its promoter. (A) Distance tree analysis of the representative ssRNAPs by Blast program. Distance from the root ‘○’: SP6 RNAP (3.374) > T7 RNAP (3.145) > KP34 RNAP (2.572) > VSW-3 RNAP (2.292) > Syn5 RNAP (1.118) suggests that VSW-3 RNAP is the second primitive after Syn5 RNAP, and evolved into a new branch of the evolutionary tree together with a predicted pollyC RNAP (3.055) from phage pollyC (YP_009622558.1). (B) SDS-PAGE gel analysis of purified VSW-3 RNAP (92.4 kDa including an N-terminal His-tag, 1 μM) and commercial T7 RNAP (New England Biolabs, 100 kDa, 1.5 μM), gel was stained with Coomassie blue. (C) Organization of phage VSW-3 genome and distribution of the predicted VSW-3 promoters (indicated by rightward arrows). (D) IVT of VSW-3 RNAP on the linearized <t>pUC19</t> plasmid with an insertion of predicted VSW-3 promoter (top gel). 5’-RACE revealed that the initial nucleotides of VSW-3 RNAP transcription in the predicted promoter is “GTA” (bottom sequencing result). (E) IVT on 5’-truncated DNA templates (left box) to determine the accurate promoter of VSW-3 RNAP. The <t>RNA</t> yield with each template (right gel) suggests that the 15 bp (5’-ATTGGGCCACCTATA-3’) sequence is the minimal promoter and the 18 bp (5’-TTAATTGGGCCACCTATA-3’) sequence is the full VSW-3 promoter.
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    VSW-3 RNAP and its promoter. (A) Distance tree analysis of the representative ssRNAPs by Blast program. Distance from the root ‘○’: SP6 RNAP (3.374) > T7 RNAP (3.145) > KP34 RNAP (2.572) > VSW-3 RNAP (2.292) > Syn5 RNAP (1.118) suggests that VSW-3 RNAP is the second primitive after Syn5 RNAP, and evolved into a new branch of the evolutionary tree together with a predicted pollyC RNAP (3.055) from phage pollyC (YP_009622558.1). (B) SDS-PAGE gel analysis of purified VSW-3 RNAP (92.4 kDa including an N-terminal His-tag, 1 μM) and commercial T7 RNAP (New England Biolabs, 100 kDa, 1.5 μM), gel was stained with Coomassie blue. (C) Organization of phage VSW-3 genome and distribution of the predicted VSW-3 promoters (indicated by rightward arrows). (D) IVT of VSW-3 RNAP on the linearized pUC19 plasmid with an insertion of predicted VSW-3 promoter (top gel). 5’-RACE revealed that the initial nucleotides of VSW-3 RNAP transcription in the predicted promoter is “GTA” (bottom sequencing result). (E) IVT on 5’-truncated DNA templates (left box) to determine the accurate promoter of VSW-3 RNAP. The RNA yield with each template (right gel) suggests that the 15 bp (5’-ATTGGGCCACCTATA-3’) sequence is the minimal promoter and the 18 bp (5’-TTAATTGGGCCACCTATA-3’) sequence is the full VSW-3 promoter.

    Journal: bioRxiv

    Article Title: In vitro transcription using psychrophilic phage VSW-3 RNA polymerase

    doi: 10.1101/2020.09.14.297226

    Figure Lengend Snippet: VSW-3 RNAP and its promoter. (A) Distance tree analysis of the representative ssRNAPs by Blast program. Distance from the root ‘○’: SP6 RNAP (3.374) > T7 RNAP (3.145) > KP34 RNAP (2.572) > VSW-3 RNAP (2.292) > Syn5 RNAP (1.118) suggests that VSW-3 RNAP is the second primitive after Syn5 RNAP, and evolved into a new branch of the evolutionary tree together with a predicted pollyC RNAP (3.055) from phage pollyC (YP_009622558.1). (B) SDS-PAGE gel analysis of purified VSW-3 RNAP (92.4 kDa including an N-terminal His-tag, 1 μM) and commercial T7 RNAP (New England Biolabs, 100 kDa, 1.5 μM), gel was stained with Coomassie blue. (C) Organization of phage VSW-3 genome and distribution of the predicted VSW-3 promoters (indicated by rightward arrows). (D) IVT of VSW-3 RNAP on the linearized pUC19 plasmid with an insertion of predicted VSW-3 promoter (top gel). 5’-RACE revealed that the initial nucleotides of VSW-3 RNAP transcription in the predicted promoter is “GTA” (bottom sequencing result). (E) IVT on 5’-truncated DNA templates (left box) to determine the accurate promoter of VSW-3 RNAP. The RNA yield with each template (right gel) suggests that the 15 bp (5’-ATTGGGCCACCTATA-3’) sequence is the minimal promoter and the 18 bp (5’-TTAATTGGGCCACCTATA-3’) sequence is the full VSW-3 promoter.

    Article Snippet: The transcripts (pUC19-RNA) were then purified with Monarch RNA Cleanup kit.

    Techniques: SDS Page, Purification, Staining, Plasmid Preparation, Sequencing

    Template-encoded Poly(A) tailing reduces antisense by-product formation. A) dsRNA immunoblot with J2 antibody and gel electrophoresis analysis of CLuc RNA synthesized from CLuc templates with varying length (30 bp, 60 bp, 120 bp) of poly-T sequence at 3’-end under standard conditions (5 mM rNTPs, 37°C for 1 hour). B) Immunoblot and native gel electrophoresis analysis of IVT reactions on 512B::CLuc chimeric template with poly-T (60 bp and 120 bp) sequence at the 3’-end. IVT reactions were performed at 37°C or 50°C.

    Journal: bioRxiv

    Article Title: Synthesis of low immunogenicity RNA with high-temperature in vitro transcription

    doi: 10.1101/815092

    Figure Lengend Snippet: Template-encoded Poly(A) tailing reduces antisense by-product formation. A) dsRNA immunoblot with J2 antibody and gel electrophoresis analysis of CLuc RNA synthesized from CLuc templates with varying length (30 bp, 60 bp, 120 bp) of poly-T sequence at 3’-end under standard conditions (5 mM rNTPs, 37°C for 1 hour). B) Immunoblot and native gel electrophoresis analysis of IVT reactions on 512B::CLuc chimeric template with poly-T (60 bp and 120 bp) sequence at the 3’-end. IVT reactions were performed at 37°C or 50°C.

    Article Snippet: For the 512B transcripts, reactions were cleaned up with the Monarch® RNA Cleanup Kit (New England Biolabs).

    Techniques: Nucleic Acid Electrophoresis, Synthesized, Sequencing

    Truncation of the 3’-end of the 512B DNA templates results in reduction of the antisense RNA by-product formation. Immunoblot (with J2 antibody; 1:5000; Scicons) and native gel electrophoresis analyses of in vitro transcription reactions performed on 512B template with 3’-end truncations (50 and 200 base pairs). In vitro transcription reactions were performed with TsT7-1 at 37°C or 50°C.

    Journal: bioRxiv

    Article Title: Synthesis of low immunogenicity RNA with high-temperature in vitro transcription

    doi: 10.1101/815092

    Figure Lengend Snippet: Truncation of the 3’-end of the 512B DNA templates results in reduction of the antisense RNA by-product formation. Immunoblot (with J2 antibody; 1:5000; Scicons) and native gel electrophoresis analyses of in vitro transcription reactions performed on 512B template with 3’-end truncations (50 and 200 base pairs). In vitro transcription reactions were performed with TsT7-1 at 37°C or 50°C.

    Article Snippet: For the 512B transcripts, reactions were cleaned up with the Monarch® RNA Cleanup Kit (New England Biolabs).

    Techniques: Nucleic Acid Electrophoresis, In Vitro

    5’-tRF Cys promotes Nucleolin binding to its target transcripts to enhance their stability. A. Quantification of rRNA levels upon inhibition of 5’-tRF Cys by RT-qPCR. B. Representative polysome profiles showing global translation status in 4T1 cells upon inhibition of 5’-tRF Cys . Mono, monosomes. Di, disomes. C. Percentage of Nucleolin peaks in different types of RNAs. RMSK, repeat masked RNAs. D. The number of Nucleolin-bound CLIP peaks in 5’, 3’ untranslated region (UTR) or coding sequencing (CDS) per 10 kb in the mouse genome. E. Cumulative distribution function (CDF) plots of log 2 FC in transcript abundance for all transcripts stratified by whether they were bound by Nucleolin (red) or not (grey). Statistical significance was determined by Kolmogorov–Smirnov (KS) test (P = 4.8e-13). F. Scatter plot comparing log 2 FC in transcript abundance upon inhibition of 5’-tRF Cys with two distinct 5’-tRF Cys antisense LNAs. Statistically significantly changed genes are marked in red. The blue dashed line represents the linear regression line for all data points. ρ, Spearman’s correlation coefficient. G. Scatter plot comparing log 2 FC in protein abundance and log 2 FC in transcript abundance between 5’-tRF Cys suppressed and control cells for all transcripts stratified by whether their Nucleolin binding is enhanced by 5’-tRF Cys (red) or not (grey). The blue dashed line represents the linear regression line for all data points. ρ, Spearman’s correlation coefficient. H, I. Representative western blot images of 5’-tRF Cys targets upon suppression of 5’-tRF Cys (H) or depletion of Nucleolin (I). J. Genome browser view of the aligned Nucleolin (Ncl)-CLIP tags (orange), RNA-Seq reads (red) and Ribo-Seq reads (green) within the 5’ UTR of Pafah1b1. The Y axis represents reads per million (RPM). TSS, transcription start site. K. Quantification by dual luciferase assays of the luminescence signals of reporters containing 5’ UTRs from 5’-tRF Cys targets relative to that from the control GAPDH. Statistical significance in A and K was determined by one-tail t-tests with Welch’s correction. ns, not significant. ***, p

    Journal: bioRxiv

    Article Title: A pro-metastatic tRNA fragment drives Nucleolin oligomerization and stabilization of bound metabolic mRNAs

    doi: 10.1101/2021.04.26.441477

    Figure Lengend Snippet: 5’-tRF Cys promotes Nucleolin binding to its target transcripts to enhance their stability. A. Quantification of rRNA levels upon inhibition of 5’-tRF Cys by RT-qPCR. B. Representative polysome profiles showing global translation status in 4T1 cells upon inhibition of 5’-tRF Cys . Mono, monosomes. Di, disomes. C. Percentage of Nucleolin peaks in different types of RNAs. RMSK, repeat masked RNAs. D. The number of Nucleolin-bound CLIP peaks in 5’, 3’ untranslated region (UTR) or coding sequencing (CDS) per 10 kb in the mouse genome. E. Cumulative distribution function (CDF) plots of log 2 FC in transcript abundance for all transcripts stratified by whether they were bound by Nucleolin (red) or not (grey). Statistical significance was determined by Kolmogorov–Smirnov (KS) test (P = 4.8e-13). F. Scatter plot comparing log 2 FC in transcript abundance upon inhibition of 5’-tRF Cys with two distinct 5’-tRF Cys antisense LNAs. Statistically significantly changed genes are marked in red. The blue dashed line represents the linear regression line for all data points. ρ, Spearman’s correlation coefficient. G. Scatter plot comparing log 2 FC in protein abundance and log 2 FC in transcript abundance between 5’-tRF Cys suppressed and control cells for all transcripts stratified by whether their Nucleolin binding is enhanced by 5’-tRF Cys (red) or not (grey). The blue dashed line represents the linear regression line for all data points. ρ, Spearman’s correlation coefficient. H, I. Representative western blot images of 5’-tRF Cys targets upon suppression of 5’-tRF Cys (H) or depletion of Nucleolin (I). J. Genome browser view of the aligned Nucleolin (Ncl)-CLIP tags (orange), RNA-Seq reads (red) and Ribo-Seq reads (green) within the 5’ UTR of Pafah1b1. The Y axis represents reads per million (RPM). TSS, transcription start site. K. Quantification by dual luciferase assays of the luminescence signals of reporters containing 5’ UTRs from 5’-tRF Cys targets relative to that from the control GAPDH. Statistical significance in A and K was determined by one-tail t-tests with Welch’s correction. ns, not significant. ***, p

    Article Snippet: After purified with Monarch RNA Cleanup Kit (NEB Biolabs), RNAs were polyadenylated with E. coli poly(A) polymerase (NEB Biolabs), and capped and 2’-O-methylated with Vaccinia Capping System (NEB Biolabs).

    Techniques: Binding Assay, Inhibition, Quantitative RT-PCR, Cross-linking Immunoprecipitation, Sequencing, Western Blot, RNA Sequencing Assay, Luciferase

    5’-tRF Cys promotes complex D assembly and Nucleolin oligomerization. A, B. Native gel analysis of Nucleolin complexes assembled from Pafah1b1 (A) or 5’-tRF Cys (B) using increasing amounts of Nucleolin protein. C. Quantification of complex D assembly as a function of Nucleolin concentration using purified Nucleolin protein. Bmax, specific maximum binding. h, Hill coefficient. Kd, equilibrium dissociation constant. D. Representative images of western blots of Nucleolin from Nucleolin IP that was pre-treated with different dilutions of micrococcal nuclease to remove endogenous RNAs before complexes were assembled at 30 °C and crosslinked with ethylene glycol bis (succinimidyl succinate). The number of blue dots represent the inferred number of Nucleolin monomers based on the molecular weight. E, F. Kinetics of Nucleolin complexes assembled from Pafah1b1 (E) or 5’-tRF Cys (F) using Nucleolin IP. See also Figure 5F . Asterisk denotes an RNA-protein complex that was detected only with Nucleolin IP but not Nucleolin protein. G, H. Native gel analysis of Nucleolin complexes assembled from Pafah1b1 (G) or 5’-tRF Cys (H) using increasing amount of Nucleolin IP. See also Figure 5G . I. Native gel analysis of Nucleolin complexes assembled using Nucleolin IP from Mthfd1l alone, or together with a wild-type (WT) or Nucleolin binding deficient (MUT) 5’-tRF Cys . Asterisk denotes an RNA-protein complex that was detected only with Nucleolin IP but not Nucleolin protein. J. Representative western blot of Nucleolin using Nucleolin IP incubated with or without Pafah1b1, or with both Pafah1b1 and 5’-tRF Cy at 30 °C before crosslinking with EGS. See also Figure 5I . K. Top, quantification of the protection provided by different forms of Nucleolin from degradation by a prototypical 5’- > 3’ exonuclease Terminator after conducting the assembly assay at 4 °C or 30 °C to form monomeric Nucleolin (complex A) or oligomeric Nucleolin (complex D) respectively. Bottom, representative image of denaturing PAGE analysis of the exonuclease degradation products.

    Journal: bioRxiv

    Article Title: A pro-metastatic tRNA fragment drives Nucleolin oligomerization and stabilization of bound metabolic mRNAs

    doi: 10.1101/2021.04.26.441477

    Figure Lengend Snippet: 5’-tRF Cys promotes complex D assembly and Nucleolin oligomerization. A, B. Native gel analysis of Nucleolin complexes assembled from Pafah1b1 (A) or 5’-tRF Cys (B) using increasing amounts of Nucleolin protein. C. Quantification of complex D assembly as a function of Nucleolin concentration using purified Nucleolin protein. Bmax, specific maximum binding. h, Hill coefficient. Kd, equilibrium dissociation constant. D. Representative images of western blots of Nucleolin from Nucleolin IP that was pre-treated with different dilutions of micrococcal nuclease to remove endogenous RNAs before complexes were assembled at 30 °C and crosslinked with ethylene glycol bis (succinimidyl succinate). The number of blue dots represent the inferred number of Nucleolin monomers based on the molecular weight. E, F. Kinetics of Nucleolin complexes assembled from Pafah1b1 (E) or 5’-tRF Cys (F) using Nucleolin IP. See also Figure 5F . Asterisk denotes an RNA-protein complex that was detected only with Nucleolin IP but not Nucleolin protein. G, H. Native gel analysis of Nucleolin complexes assembled from Pafah1b1 (G) or 5’-tRF Cys (H) using increasing amount of Nucleolin IP. See also Figure 5G . I. Native gel analysis of Nucleolin complexes assembled using Nucleolin IP from Mthfd1l alone, or together with a wild-type (WT) or Nucleolin binding deficient (MUT) 5’-tRF Cys . Asterisk denotes an RNA-protein complex that was detected only with Nucleolin IP but not Nucleolin protein. J. Representative western blot of Nucleolin using Nucleolin IP incubated with or without Pafah1b1, or with both Pafah1b1 and 5’-tRF Cy at 30 °C before crosslinking with EGS. See also Figure 5I . K. Top, quantification of the protection provided by different forms of Nucleolin from degradation by a prototypical 5’- > 3’ exonuclease Terminator after conducting the assembly assay at 4 °C or 30 °C to form monomeric Nucleolin (complex A) or oligomeric Nucleolin (complex D) respectively. Bottom, representative image of denaturing PAGE analysis of the exonuclease degradation products.

    Article Snippet: After purified with Monarch RNA Cleanup Kit (NEB Biolabs), RNAs were polyadenylated with E. coli poly(A) polymerase (NEB Biolabs), and capped and 2’-O-methylated with Vaccinia Capping System (NEB Biolabs).

    Techniques: Concentration Assay, Purification, Binding Assay, Western Blot, Molecular Weight, Incubation, Polyacrylamide Gel Electrophoresis

    Schematic illustration of RADICA. a , The workflow of RADICA sample partitioning on a chip for absolute quantification of nucleic acid targets. Generally, after the DNA/RNA extraction step, different kind of clinical samples can be used for detection and quantification of various targets. The sample mixture containing DNA/cDNA, RPA reagents, and Cas12a-crRNA-FQ probes is distributed randomly into thousands of partitions. In each partition, the DNA is amplified by RPA and detected by Cas12a-crRNA, resulting in a fluorescent signal in the partition. Based on the proportion of positive partitions and on Poisson distribution, the absolute copy number of the nucleic acid target is quantified. b , Illustration of RPA-Cas12a reaction in each positive partition. In each partition containing the target nucleic acid, the primers bind to the target nucleic acid and initiate amplification with the aid of recombinase and DNA polymerase. Because of the strand displacement of DNA polymerase, the exposed crRNA-targeted ssDNA sites are bound by Cas12a-crRNA complexes. Cas12a is then activated and cleaves the nearby FQ reporters to produce a fluorescence readout.

    Journal: medRxiv

    Article Title: A Digital CRISPR-based Method for the Rapid Detection and Absolute Quantification of Viral Nucleic Acids

    doi: 10.1101/2020.11.03.20223602

    Figure Lengend Snippet: Schematic illustration of RADICA. a , The workflow of RADICA sample partitioning on a chip for absolute quantification of nucleic acid targets. Generally, after the DNA/RNA extraction step, different kind of clinical samples can be used for detection and quantification of various targets. The sample mixture containing DNA/cDNA, RPA reagents, and Cas12a-crRNA-FQ probes is distributed randomly into thousands of partitions. In each partition, the DNA is amplified by RPA and detected by Cas12a-crRNA, resulting in a fluorescent signal in the partition. Based on the proportion of positive partitions and on Poisson distribution, the absolute copy number of the nucleic acid target is quantified. b , Illustration of RPA-Cas12a reaction in each positive partition. In each partition containing the target nucleic acid, the primers bind to the target nucleic acid and initiate amplification with the aid of recombinase and DNA polymerase. Because of the strand displacement of DNA polymerase, the exposed crRNA-targeted ssDNA sites are bound by Cas12a-crRNA complexes. Cas12a is then activated and cleaves the nearby FQ reporters to produce a fluorescence readout.

    Article Snippet: The synthesized crRNA was purified using Monarch® RNA Cleanup Kit (New England Biolabs) after treatment with DNase I (RNase-free, New England Biolabs), Thermolabile Exonuclease I (New England Biolabs), and T5 Exonuclease (New England Biolabs).

    Techniques: Chromatin Immunoprecipitation, RNA Extraction, Recombinase Polymerase Amplification, Amplification, Fluorescence