rnase h  (Thermo Fisher)


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    Name:
    Ribonuclease H
    Description:
    Ribonuclease H RNase H is an endoribonuclease that specifically degrades the RNA strand of an RNA DNA hybrid to produce 5 phosphate terminated oligoribonucleotides and single stranded DNA Applications Removal of mRNA during second strand cDNA synthesis Removal of poly A sequences from mRNA in the presence of oligo dT Oligodeoxyribonucleotide directed cleavage of RNA Source Purified from E coli expressing the E coli RNase H gene on a plasmid Performance and quality testing Ribonuclease nonspecific endodeoxyribonuclease 3 and 5 exodeoxyribonuclease Unit definition One unit hydrolyzes 1 nmol of RNA in 3H labeled poly A poly dT to acid soluble material in 20 min at 37°C Unit reaction conditions 20 mM Tris HCl pH 7 5 0 1 M KCl 10 mM MgCl2 0 1 mM DTT 5 w v sucrose 0 5 nmol 3H labeled poly A poly dT and enzyme in 50 µL for 20 min at 37°C
    Catalog Number:
    18021014
    Price:
    None
    Applications:
    PCR & Real-Time PCR|Reverse Transcription
    Category:
    Proteins Enzymes Peptides
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    Structured Review

    Thermo Fisher rnase h
    Polyadenylation state of Fmr1 mRNA variants in the CGG KI mouse brain. ( A ) Left panel: schematic view of the polyadenylation assay (PAT). According to di Penta et al . ( 42 ), the poly(A) tails are tagged by incubating the RNA with a (T) 12 -tag oligonucleotide, blocked at the 3′-end, in the presence of dNTPs and Klenow enzyme to fill in the complementary tag sequence. The RNA is then denatured and annealed to a DNA primer, identical to the tag, to start a reverse transcription (RT). The cDNA is then amplified using a gene-specific forward primer and the reverse tag oligo. Right panel: cartoon of a polyadenylation profile obtained with a PAT assay. The PCR of a polyadenylated mRNA gives rise to a smear while the same mRNA deadenylated with oligodT and <t>RNase</t> H prior to poly(A) tagging is used as a negative control and gives a sharp band. ( B ) Upper panel: β-actin mRNA in WT (lane 1) and CGG KI (lane 3). Deadenylated RNA is shown as negative control (lane 2) and the deadenylated form is indicated by black arrows. Lower panel: dispersion graph representing the distribution of the β-actin polyadenylated transcripts in WT (black line) and CGG KI (grey line). The signal intensity along the lane has been plotted against the poly(A) tail length, estimated from the molecular markers loaded on the same gel. ( C ) PAT for all three poly(A) Fmr1 mRNA variants. Because of close proximity, the transcripts containing sites V and VI cannot be discriminated and therefore they are not taken into exam (upper panel). Black arrows points to the deadenylated form. The polyadenylation of transcripts using site IV from WT (lane 1) and CGG KI (lane 3) has been independently acquired and highlighted in the box below. Deadenylated RNA treated as mentioned above, is shown as negative control (lane 2). Right panel: dispersion graph for Fmr1 variants using site IV in WT (black line) and CGG KI (grey line). ( D ) Left panel: PAT for Fmr1 variants using site VI in WT (lane 1) and CGG KI (lane 3) brain. Deadenylated RNA as above is used as negative control (lane 2). Right panel: Dispersion graph representing the distribution of the polyadenylated transcripts using site VI in WT (black line) and CGG KI (grey line).
    Ribonuclease H RNase H is an endoribonuclease that specifically degrades the RNA strand of an RNA DNA hybrid to produce 5 phosphate terminated oligoribonucleotides and single stranded DNA Applications Removal of mRNA during second strand cDNA synthesis Removal of poly A sequences from mRNA in the presence of oligo dT Oligodeoxyribonucleotide directed cleavage of RNA Source Purified from E coli expressing the E coli RNase H gene on a plasmid Performance and quality testing Ribonuclease nonspecific endodeoxyribonuclease 3 and 5 exodeoxyribonuclease Unit definition One unit hydrolyzes 1 nmol of RNA in 3H labeled poly A poly dT to acid soluble material in 20 min at 37°C Unit reaction conditions 20 mM Tris HCl pH 7 5 0 1 M KCl 10 mM MgCl2 0 1 mM DTT 5 w v sucrose 0 5 nmol 3H labeled poly A poly dT and enzyme in 50 µL for 20 min at 37°C
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    Images

    1) Product Images from "Differential usage of transcriptional start sites and polyadenylation sites in FMR1 premutation alleles †"

    Article Title: Differential usage of transcriptional start sites and polyadenylation sites in FMR1 premutation alleles †

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr100

    Polyadenylation state of Fmr1 mRNA variants in the CGG KI mouse brain. ( A ) Left panel: schematic view of the polyadenylation assay (PAT). According to di Penta et al . ( 42 ), the poly(A) tails are tagged by incubating the RNA with a (T) 12 -tag oligonucleotide, blocked at the 3′-end, in the presence of dNTPs and Klenow enzyme to fill in the complementary tag sequence. The RNA is then denatured and annealed to a DNA primer, identical to the tag, to start a reverse transcription (RT). The cDNA is then amplified using a gene-specific forward primer and the reverse tag oligo. Right panel: cartoon of a polyadenylation profile obtained with a PAT assay. The PCR of a polyadenylated mRNA gives rise to a smear while the same mRNA deadenylated with oligodT and RNase H prior to poly(A) tagging is used as a negative control and gives a sharp band. ( B ) Upper panel: β-actin mRNA in WT (lane 1) and CGG KI (lane 3). Deadenylated RNA is shown as negative control (lane 2) and the deadenylated form is indicated by black arrows. Lower panel: dispersion graph representing the distribution of the β-actin polyadenylated transcripts in WT (black line) and CGG KI (grey line). The signal intensity along the lane has been plotted against the poly(A) tail length, estimated from the molecular markers loaded on the same gel. ( C ) PAT for all three poly(A) Fmr1 mRNA variants. Because of close proximity, the transcripts containing sites V and VI cannot be discriminated and therefore they are not taken into exam (upper panel). Black arrows points to the deadenylated form. The polyadenylation of transcripts using site IV from WT (lane 1) and CGG KI (lane 3) has been independently acquired and highlighted in the box below. Deadenylated RNA treated as mentioned above, is shown as negative control (lane 2). Right panel: dispersion graph for Fmr1 variants using site IV in WT (black line) and CGG KI (grey line). ( D ) Left panel: PAT for Fmr1 variants using site VI in WT (lane 1) and CGG KI (lane 3) brain. Deadenylated RNA as above is used as negative control (lane 2). Right panel: Dispersion graph representing the distribution of the polyadenylated transcripts using site VI in WT (black line) and CGG KI (grey line).
    Figure Legend Snippet: Polyadenylation state of Fmr1 mRNA variants in the CGG KI mouse brain. ( A ) Left panel: schematic view of the polyadenylation assay (PAT). According to di Penta et al . ( 42 ), the poly(A) tails are tagged by incubating the RNA with a (T) 12 -tag oligonucleotide, blocked at the 3′-end, in the presence of dNTPs and Klenow enzyme to fill in the complementary tag sequence. The RNA is then denatured and annealed to a DNA primer, identical to the tag, to start a reverse transcription (RT). The cDNA is then amplified using a gene-specific forward primer and the reverse tag oligo. Right panel: cartoon of a polyadenylation profile obtained with a PAT assay. The PCR of a polyadenylated mRNA gives rise to a smear while the same mRNA deadenylated with oligodT and RNase H prior to poly(A) tagging is used as a negative control and gives a sharp band. ( B ) Upper panel: β-actin mRNA in WT (lane 1) and CGG KI (lane 3). Deadenylated RNA is shown as negative control (lane 2) and the deadenylated form is indicated by black arrows. Lower panel: dispersion graph representing the distribution of the β-actin polyadenylated transcripts in WT (black line) and CGG KI (grey line). The signal intensity along the lane has been plotted against the poly(A) tail length, estimated from the molecular markers loaded on the same gel. ( C ) PAT for all three poly(A) Fmr1 mRNA variants. Because of close proximity, the transcripts containing sites V and VI cannot be discriminated and therefore they are not taken into exam (upper panel). Black arrows points to the deadenylated form. The polyadenylation of transcripts using site IV from WT (lane 1) and CGG KI (lane 3) has been independently acquired and highlighted in the box below. Deadenylated RNA treated as mentioned above, is shown as negative control (lane 2). Right panel: dispersion graph for Fmr1 variants using site IV in WT (black line) and CGG KI (grey line). ( D ) Left panel: PAT for Fmr1 variants using site VI in WT (lane 1) and CGG KI (lane 3) brain. Deadenylated RNA as above is used as negative control (lane 2). Right panel: Dispersion graph representing the distribution of the polyadenylated transcripts using site VI in WT (black line) and CGG KI (grey line).

    Techniques Used: Sequencing, Amplification, Polymerase Chain Reaction, Negative Control

    2) Product Images from "Unstructured 5′-tails act through ribosome standby to override inhibitory structure at ribosome binding sites"

    Article Title: Unstructured 5′-tails act through ribosome standby to override inhibitory structure at ribosome binding sites

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky073

    The 5′ standby tails need to be single-stranded to promote translation and initiation complex formation. ( A ) The effect of blocking of the (CA)6 and (CA)8 standby tail by an antisense oligo (at 6.7 times molar excess), or a control oligo, was assayed by in vitro translation. The mRNAs used are indicated, and GFP was detected by Western blot. ( B ) Formation of a heteroduplex between the antisense oligo and the standby tail of (CA)8 mRNA was tested by RNase H cleavage (Materials and Methods). Cleavage was observed after reverse transcription using a 5′-labeled oligo annealed downstream. Cleavage near the base of the stem is observed only in the presence of the antisense oligo and RNase H (far right lane). ( C ) The toeprint experiment was conducted on several mRNA variants, with 30S and tRNA fMet , with or without antisense or control oligo (Materials and Methods), as indicated. The position of the characteristic toeprint at +15 is shown. UAGC shows a sequencing ladder for reference. The gel is representative of three technical replicates.
    Figure Legend Snippet: The 5′ standby tails need to be single-stranded to promote translation and initiation complex formation. ( A ) The effect of blocking of the (CA)6 and (CA)8 standby tail by an antisense oligo (at 6.7 times molar excess), or a control oligo, was assayed by in vitro translation. The mRNAs used are indicated, and GFP was detected by Western blot. ( B ) Formation of a heteroduplex between the antisense oligo and the standby tail of (CA)8 mRNA was tested by RNase H cleavage (Materials and Methods). Cleavage was observed after reverse transcription using a 5′-labeled oligo annealed downstream. Cleavage near the base of the stem is observed only in the presence of the antisense oligo and RNase H (far right lane). ( C ) The toeprint experiment was conducted on several mRNA variants, with 30S and tRNA fMet , with or without antisense or control oligo (Materials and Methods), as indicated. The position of the characteristic toeprint at +15 is shown. UAGC shows a sequencing ladder for reference. The gel is representative of three technical replicates.

    Techniques Used: Blocking Assay, In Vitro, Western Blot, Labeling, Sequencing

    3) Product Images from "An optimized protocol for microarray validation by quantitative PCR using amplified amino allyl labeled RNA"

    Article Title: An optimized protocol for microarray validation by quantitative PCR using amplified amino allyl labeled RNA

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-11-542

    RNaseH improves qPCR efficiency . Different quantities of AA-aRNA from universal reference RNA (100-1000 ng) were used as inputs into either the original RT protocol or the optimized RT protocol, with or without RNase H treatment. Resulting cDNAs were diluted 10-fold and subjected to qPCR using primer pairs specific for VEGFB, MMP9, TFRC, HAMP and GAPDH. Cq values were plotted against the Log of the concentration of the AA-aRNA used for RT and linear regression was applied. qPCR efficiency (E) was calculated by the slope of the regression line. A slope of -3.2 indicates optimal efficiency. qPCR linearity (R 2 ) corresponds to the correlation coefficient of the regression line. A coefficient R 2 of 1 indicates optimal linearity. (A) Representative experiment using VEGFB primers. (B) Plots representing qPCR efficiency as a function of linearity for the 5 genes tested. Optimum conditions are indicated at the intersection of dotted lines corresponding to E = -3.2 and R 2 = 1. E was improved by RNase H treatment.
    Figure Legend Snippet: RNaseH improves qPCR efficiency . Different quantities of AA-aRNA from universal reference RNA (100-1000 ng) were used as inputs into either the original RT protocol or the optimized RT protocol, with or without RNase H treatment. Resulting cDNAs were diluted 10-fold and subjected to qPCR using primer pairs specific for VEGFB, MMP9, TFRC, HAMP and GAPDH. Cq values were plotted against the Log of the concentration of the AA-aRNA used for RT and linear regression was applied. qPCR efficiency (E) was calculated by the slope of the regression line. A slope of -3.2 indicates optimal efficiency. qPCR linearity (R 2 ) corresponds to the correlation coefficient of the regression line. A coefficient R 2 of 1 indicates optimal linearity. (A) Representative experiment using VEGFB primers. (B) Plots representing qPCR efficiency as a function of linearity for the 5 genes tested. Optimum conditions are indicated at the intersection of dotted lines corresponding to E = -3.2 and R 2 = 1. E was improved by RNase H treatment.

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

    Protocol optimization improves RT yield . One μg RNA (A) and 100 ng AA-aRNA (B) obtained from universal reference RNA were used as inputs to either the original RT protocol (black bars), the optimized RT protocol without RNase H treatment (white bars), or the modified RT protocol with RNase H treatment (hatched bars). Resulting cDNAs were subjected to qPCR using primer pairs specific for VEGFB, MMP9, TFRC, HAMP and GAPDH. Threshold Cq values for each gene and the mean ± SD of the 5 genes are indicated. RT protocol optimization decreased Cq values and RNase H did not induce a further decrease. * P
    Figure Legend Snippet: Protocol optimization improves RT yield . One μg RNA (A) and 100 ng AA-aRNA (B) obtained from universal reference RNA were used as inputs to either the original RT protocol (black bars), the optimized RT protocol without RNase H treatment (white bars), or the modified RT protocol with RNase H treatment (hatched bars). Resulting cDNAs were subjected to qPCR using primer pairs specific for VEGFB, MMP9, TFRC, HAMP and GAPDH. Threshold Cq values for each gene and the mean ± SD of the 5 genes are indicated. RT protocol optimization decreased Cq values and RNase H did not induce a further decrease. * P

    Techniques Used: Modification, Real-time Polymerase Chain Reaction

    4) Product Images from "Genetic and Biochemical Assays Reveal a Key Role for Replication Restart Proteins in Group II Intron Retrohoming"

    Article Title: Genetic and Biochemical Assays Reveal a Key Role for Replication Restart Proteins in Group II Intron Retrohoming

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1003469

    Model for function of host factors in group II intron retrohoming in E. coli . In initial steps, the group II intron lariat RNA reverse splices into the top strand of the DNA target site, while the intron-encoded RT cuts the bottom DNA strand and uses the 3′ end of the cleaved strand as a primer for target DNA-primed reverse transcription of the intron RNA. During or after cDNA synthesis, a host RNase H (RNase H1) degrades the intron RNA template strand. Extension of the intron cDNA into the 5′ exon displaces the bottom-DNA strand resulting in a branched intermediate that is recognized by the replication restart proteins PriA or PriC, with PriA preferentially recognizing intermediates with short gaps in the bottom strand and PriC preferentially recognizing intermediates with long gaps in the bottom strand. PriA and PriC then initiate a replisome loading cascade involving the sequential recruitment of the replicative helicase DnaB, the primase DnaG, and the replicative polymerase Pol III for second-strand DNA synthesis. Ssb stabilizes single-stranded DNA in gapped regions and interacts with PriA to stimulate the loading of DnaB. The 5′→3′ exonuclease activity of Pol I contributes to the removal of residual RNA primers and its DNA polymerase activity may contribute to filling in gaps, and a host DNA ligase (LigA) seals nicks in the top and bottom strands. Although bottom-strand synthesis is completely dependent on group II RT activity ( Figure 3D ), biochemical assays show that it is strongly inhibited in a DNA primase (DnaG) mutant and moderately inhibited in repair DNA polymerase DinB and PolB mutants, suggesting a previously unsuspected role for host factors in initiating bottom-strand (cDNA) synthesis. Deletion of RecJ moderately inhibits synthesis of full-length bottom strands in extracts, consistent with a role in resection of the 5′-overhang resulting from the staggered cleavage of the DNA substrate by group II intron RNPs [12] .
    Figure Legend Snippet: Model for function of host factors in group II intron retrohoming in E. coli . In initial steps, the group II intron lariat RNA reverse splices into the top strand of the DNA target site, while the intron-encoded RT cuts the bottom DNA strand and uses the 3′ end of the cleaved strand as a primer for target DNA-primed reverse transcription of the intron RNA. During or after cDNA synthesis, a host RNase H (RNase H1) degrades the intron RNA template strand. Extension of the intron cDNA into the 5′ exon displaces the bottom-DNA strand resulting in a branched intermediate that is recognized by the replication restart proteins PriA or PriC, with PriA preferentially recognizing intermediates with short gaps in the bottom strand and PriC preferentially recognizing intermediates with long gaps in the bottom strand. PriA and PriC then initiate a replisome loading cascade involving the sequential recruitment of the replicative helicase DnaB, the primase DnaG, and the replicative polymerase Pol III for second-strand DNA synthesis. Ssb stabilizes single-stranded DNA in gapped regions and interacts with PriA to stimulate the loading of DnaB. The 5′→3′ exonuclease activity of Pol I contributes to the removal of residual RNA primers and its DNA polymerase activity may contribute to filling in gaps, and a host DNA ligase (LigA) seals nicks in the top and bottom strands. Although bottom-strand synthesis is completely dependent on group II RT activity ( Figure 3D ), biochemical assays show that it is strongly inhibited in a DNA primase (DnaG) mutant and moderately inhibited in repair DNA polymerase DinB and PolB mutants, suggesting a previously unsuspected role for host factors in initiating bottom-strand (cDNA) synthesis. Deletion of RecJ moderately inhibits synthesis of full-length bottom strands in extracts, consistent with a role in resection of the 5′-overhang resulting from the staggered cleavage of the DNA substrate by group II intron RNPs [12] .

    Techniques Used: DNA Synthesis, Activity Assay, Mutagenesis

    5) Product Images from "Two-dimensional intact mitochondrial DNA agarose electrophoresis reveals the structural complexity of the mammalian mitochondrial genome"

    Article Title: Two-dimensional intact mitochondrial DNA agarose electrophoresis reveals the structural complexity of the mammalian mitochondrial genome

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gks1324

    Mitochondrial DNA topoisomers are associated with RNA in 2D-IMAGE profiles. ( A ) 2D-IMAGE profile of wild-type MEF DNA without RNAse treatment. New topoisomers relative to those in Figure 3 are assigned letters. ( B ) RNase H treatment, which digests RNA:DNA hybrids, reveals the vertical spikes of DNA that are sensitive to S1 nuclease ( 23–25 ). Molecules that decrease on digestion are indicated with dashed arrows, whereas those that increase are indicated with solid arrows. Numbers correspond to topoisomers in Figure 3 . ( C ) RNase A treatment, which digests heterogeneous RNA after U and C bases, reveals the typical 2D-IMAGE pattern shown in Figure 2 . Dashed arrows indicate a reduction in signal, and solid arrows indicate increased signal. ( D ) Quantitation of change in abundance of topoisomers shown in panels B and C relative to untreated.
    Figure Legend Snippet: Mitochondrial DNA topoisomers are associated with RNA in 2D-IMAGE profiles. ( A ) 2D-IMAGE profile of wild-type MEF DNA without RNAse treatment. New topoisomers relative to those in Figure 3 are assigned letters. ( B ) RNase H treatment, which digests RNA:DNA hybrids, reveals the vertical spikes of DNA that are sensitive to S1 nuclease ( 23–25 ). Molecules that decrease on digestion are indicated with dashed arrows, whereas those that increase are indicated with solid arrows. Numbers correspond to topoisomers in Figure 3 . ( C ) RNase A treatment, which digests heterogeneous RNA after U and C bases, reveals the typical 2D-IMAGE pattern shown in Figure 2 . Dashed arrows indicate a reduction in signal, and solid arrows indicate increased signal. ( D ) Quantitation of change in abundance of topoisomers shown in panels B and C relative to untreated.

    Techniques Used: Quantitation Assay

    6) Product Images from "A Role for DEAD Box 1 at DNA Double-Strand Breaks ▿A Role for DEAD Box 1 at DNA Double-Strand Breaks ▿ †"

    Article Title: A Role for DEAD Box 1 at DNA Double-Strand Breaks ▿A Role for DEAD Box 1 at DNA Double-Strand Breaks ▿ †

    Journal:

    doi: 10.1128/MCB.01053-08

    RNase H treatment dissociates DDX1 from IRIF. HeLa cells were treated with IR (5 Gy) and incubated at 37°C for 1 h to allow the IRIF to form. Cells were then permeabilized (using 2% Tween 20) (A) and treated with DNase I (B), RNase A (C),
    Figure Legend Snippet: RNase H treatment dissociates DDX1 from IRIF. HeLa cells were treated with IR (5 Gy) and incubated at 37°C for 1 h to allow the IRIF to form. Cells were then permeabilized (using 2% Tween 20) (A) and treated with DNase I (B), RNase A (C),

    Techniques Used: Incubation

    7) Product Images from "RNA Interference-Guided Targeting of Hepatitis C Virus Replication with Antisense Locked Nucleic Acid-Based Oligonucleotides Containing 8-oxo-dG Modifications"

    Article Title: RNA Interference-Guided Targeting of Hepatitis C Virus Replication with Antisense Locked Nucleic Acid-Based Oligonucleotides Containing 8-oxo-dG Modifications

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0128686

    RNase H-mediated degradation of pre-formed ASO:RNA duplexes and in vitro -synthesized RNAs targeted by ASOs. (A) Schematic of the experimental setup for panels B and C. Applicable for some ASOs: Y, 8-oxo-dG residue; +, LNA sugar base. (B, C) Cleavage of pre-formed ASO:target RNA duplexes by RNase H. (B) Five femtomoles of 33 P-labeled substrate was treated with RNase H for the indicated times. The reaction products were collected, denatured by heating at 95°C for 2 min and analyzed by PAGE in native 15% gels. Arrows at right point to the substrate (S) and major cleavage product(s) (P). Results from one of three independent reproducible experiments are shown. (C) Kinetics of RNase H cleavage of different ASO:RNA duplexes. The amounts of radioactivity remaining in the uncleaved substrate were quantified using a Typhoon Trio instrument. Quantifications were performed for each gel. The obtained values were normalized to the radioactivity present in the substrate before adding RNase H (set to 100%). Each point corresponds to the average of three independent experiments. Error bars indicate the standard deviation. (D) Cleavage of FR3131 RNA by RNase H in the presence of different ASOs. The RNA and ASOs were mixed and incubated at 37°C for 10 min; then, RNase H was added to the reaction mixture. RNA samples were collected at the indicated time points and analyzed by electrophoresis in native 0.8% TAE agarose gels. The results from one of three independent reproducible experiments are shown. S: substrate; P1 and P2: cleavage products.
    Figure Legend Snippet: RNase H-mediated degradation of pre-formed ASO:RNA duplexes and in vitro -synthesized RNAs targeted by ASOs. (A) Schematic of the experimental setup for panels B and C. Applicable for some ASOs: Y, 8-oxo-dG residue; +, LNA sugar base. (B, C) Cleavage of pre-formed ASO:target RNA duplexes by RNase H. (B) Five femtomoles of 33 P-labeled substrate was treated with RNase H for the indicated times. The reaction products were collected, denatured by heating at 95°C for 2 min and analyzed by PAGE in native 15% gels. Arrows at right point to the substrate (S) and major cleavage product(s) (P). Results from one of three independent reproducible experiments are shown. (C) Kinetics of RNase H cleavage of different ASO:RNA duplexes. The amounts of radioactivity remaining in the uncleaved substrate were quantified using a Typhoon Trio instrument. Quantifications were performed for each gel. The obtained values were normalized to the radioactivity present in the substrate before adding RNase H (set to 100%). Each point corresponds to the average of three independent experiments. Error bars indicate the standard deviation. (D) Cleavage of FR3131 RNA by RNase H in the presence of different ASOs. The RNA and ASOs were mixed and incubated at 37°C for 10 min; then, RNase H was added to the reaction mixture. RNA samples were collected at the indicated time points and analyzed by electrophoresis in native 0.8% TAE agarose gels. The results from one of three independent reproducible experiments are shown. S: substrate; P1 and P2: cleavage products.

    Techniques Used: Allele-specific Oligonucleotide, In Vitro, Synthesized, Labeling, Polyacrylamide Gel Electrophoresis, Radioactivity, Standard Deviation, Incubation, Electrophoresis

    8) Product Images from "Mechanisms of Resistance to an Amino Acid Antibiotic That Targets Translation"

    Article Title: Mechanisms of Resistance to an Amino Acid Antibiotic That Targets Translation

    Journal: ACS chemical biology

    doi: 10.1021/cb7002253

    RNase H cleavage of the E. coli lysC leader RNA. Labeled RNAs generated in the presence of increasing concentrations of lysine (a) or AEC (b) were incubated with a DNA oligonucleotide complimentary to the 3′ side of helix 1 to allow hybridization,
    Figure Legend Snippet: RNase H cleavage of the E. coli lysC leader RNA. Labeled RNAs generated in the presence of increasing concentrations of lysine (a) or AEC (b) were incubated with a DNA oligonucleotide complimentary to the 3′ side of helix 1 to allow hybridization,

    Techniques Used: Labeling, Generated, Incubation, Hybridization

    9) Product Images from "A competitive formation of DNA:RNA hybrid G-quadruplex is responsible to the mitochondrial transcription termination at the DNA replication priming site"

    Article Title: A competitive formation of DNA:RNA hybrid G-quadruplex is responsible to the mitochondrial transcription termination at the DNA replication priming site

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gku764

    HQ formation in transcribed plasmid containing a human mtDNA fragment starting from the light strand promoter (LSP) and containing the CSB II and CSB I. ( A ) Scheme of the plasmid and detection of G-quadruplex formation by ligand-induced photocleavage. Plasmid transcribed in 50 mM of K + or Li + solution in the presence or absence of 40% (w/v) PEG 200 with GTP or dzGTP was subjected to Zn-TTAPc-mediated photocleavage, then cut at the Mun I restriction site, and filled in at the recessive 3′ end with a dATP followed by a fluorescein-dUTP, before being resolved on a denaturing gel. The marker (M) was a single-stranded synthetic DNA equivalent to the fragment between the Mun I site and the 3′ end of the G 5 AG 7 motif. Filled and open bars indicate G-quadruplex-specific cleavage signals. ( B ) Detection of RNA in HQ by photo-crosslinking. Transcription was conducted using normal GTP or dzGTP and 4S-UTP in solution containing 50-mM K + or Li + . With or without a prior RNase H digestion, transcribed plasmid was crosslinked and precipitated. Then a 5′-FAM-labeled primer (5′-CCAGCCTGCGG­CGAGTG-3′) was annealed to the non-template DNA strand downstream of CSB II, followed by extension with DNA sequenase. Extension products were resolved on a denaturing gel. G and T ladders were obtained by primer extension on the non-template DNA strand with ddCTP and ddATP, respectively. Filled and open bars indicate crosslinking sites. ( C ) Detection of G-quadruplex formation by RNA polymerase arrest assay. A plasmid containing convergent T7 and SP6 promoters and the correspondent terminators (top scheme) was transcribed by SP6 RNA polymerase in 50-mM K + or Li + solution without (lanes 1 and 2) or with (lanes 4 and 5) a prior transcription with T7 RNA polymerase in the same solution. The T7 transcription was stopped by competitive DNA specific to the T7 polymerase before the SP6 transcription was initiated. Fluorescein-UTP was supplied with SP6 RNA polymerase. RNA transcripts were resolved on a denaturing gel and visualized by the incorporated fluorescein-UTP. The marker represents SP6 transcript terminated right before the CSB II obtained by transcription of a linear DNA amplified from the plasmid.
    Figure Legend Snippet: HQ formation in transcribed plasmid containing a human mtDNA fragment starting from the light strand promoter (LSP) and containing the CSB II and CSB I. ( A ) Scheme of the plasmid and detection of G-quadruplex formation by ligand-induced photocleavage. Plasmid transcribed in 50 mM of K + or Li + solution in the presence or absence of 40% (w/v) PEG 200 with GTP or dzGTP was subjected to Zn-TTAPc-mediated photocleavage, then cut at the Mun I restriction site, and filled in at the recessive 3′ end with a dATP followed by a fluorescein-dUTP, before being resolved on a denaturing gel. The marker (M) was a single-stranded synthetic DNA equivalent to the fragment between the Mun I site and the 3′ end of the G 5 AG 7 motif. Filled and open bars indicate G-quadruplex-specific cleavage signals. ( B ) Detection of RNA in HQ by photo-crosslinking. Transcription was conducted using normal GTP or dzGTP and 4S-UTP in solution containing 50-mM K + or Li + . With or without a prior RNase H digestion, transcribed plasmid was crosslinked and precipitated. Then a 5′-FAM-labeled primer (5′-CCAGCCTGCGG­CGAGTG-3′) was annealed to the non-template DNA strand downstream of CSB II, followed by extension with DNA sequenase. Extension products were resolved on a denaturing gel. G and T ladders were obtained by primer extension on the non-template DNA strand with ddCTP and ddATP, respectively. Filled and open bars indicate crosslinking sites. ( C ) Detection of G-quadruplex formation by RNA polymerase arrest assay. A plasmid containing convergent T7 and SP6 promoters and the correspondent terminators (top scheme) was transcribed by SP6 RNA polymerase in 50-mM K + or Li + solution without (lanes 1 and 2) or with (lanes 4 and 5) a prior transcription with T7 RNA polymerase in the same solution. The T7 transcription was stopped by competitive DNA specific to the T7 polymerase before the SP6 transcription was initiated. Fluorescein-UTP was supplied with SP6 RNA polymerase. RNA transcripts were resolved on a denaturing gel and visualized by the incorporated fluorescein-UTP. The marker represents SP6 transcript terminated right before the CSB II obtained by transcription of a linear DNA amplified from the plasmid.

    Techniques Used: Plasmid Preparation, Marker, Labeling, Amplification

    Identification of G-quadruplexes in plasmid at the wild and mutated CSB II by ( A ) ligand-induced photocleavage and ( B ) photo-crosslinking. (A) Plasmids transcribed in 50-mM K +  solution were treated with RNase A (A) or A and H (AH), incubated with Zn-TTAPc, and irradiated with UV light. The plasmids were then cut with Mun I, labeled at the 3′ recessive end with an FAM dye by a fill-in reaction using fluorescein-dUTP. Marker was prepared in the same way using a synthetic dsDNA that has the same sequence as the plasmid at the correspondent region (scheme at bottom). The labeling may add one or two Ts, resulting in two bands. Cleavage fragments were resolved on a denaturing gel. (B) Plasmids were transcribed in 50-mM K +  with 4-S-UTP and the other three NTPs, treated with RNase H, followed by UV irradiation. A 5′-FAM-labeled primer was extended on the non-template DNA strand that stalled at the crosslinking sites. Extension products were resolved on a denaturing gel.
    Figure Legend Snippet: Identification of G-quadruplexes in plasmid at the wild and mutated CSB II by ( A ) ligand-induced photocleavage and ( B ) photo-crosslinking. (A) Plasmids transcribed in 50-mM K + solution were treated with RNase A (A) or A and H (AH), incubated with Zn-TTAPc, and irradiated with UV light. The plasmids were then cut with Mun I, labeled at the 3′ recessive end with an FAM dye by a fill-in reaction using fluorescein-dUTP. Marker was prepared in the same way using a synthetic dsDNA that has the same sequence as the plasmid at the correspondent region (scheme at bottom). The labeling may add one or two Ts, resulting in two bands. Cleavage fragments were resolved on a denaturing gel. (B) Plasmids were transcribed in 50-mM K + with 4-S-UTP and the other three NTPs, treated with RNase H, followed by UV irradiation. A 5′-FAM-labeled primer was extended on the non-template DNA strand that stalled at the crosslinking sites. Extension products were resolved on a denaturing gel.

    Techniques Used: Plasmid Preparation, Incubation, Irradiation, Labeling, Marker, Sequencing

    Stability of HQ and DQ formed in synthetic CSB II oligonucleotides in 50-mM K + . ( A ) Melting profile of intramolecular DNA DQ and chimeric DNA:RNA HQ. Sequences used are shown on the left. Each of them carried a fluorescent donor FAM at the 5′ end and an accepter TAMRA at the 3′ end. The curves in the graph show the first derivative of FAM fluorescence over temperature as a function of temperature. ( B ) Protection of DNA by the formation of DQ or HQ. DQ or HQ formed in the single-stranded DNA or dimeric DNA:RNA partial duplex protected the DNA from being hydrolyzed from the 3′ end by Exo I exonuclease (scheme at left). Three substrates (Wild, M3G and HQ) were treated with Exo I in a single tube and those survived the hydrolysis were resolved on a denaturing gel. The RNA in the duplex region of the HQ substrate was hydrolyzed by RNase H prior to the exonuclease digestion. The DNAs were visualized by the FAM dye covalently labeled at their 5′ end, digitized, and the results are given on the right. The numbers above the bars indicate the average of the two time points. The DNA oligomer or moiety is shown in uppercase and that of RNA in lowercase in (A,B).
    Figure Legend Snippet: Stability of HQ and DQ formed in synthetic CSB II oligonucleotides in 50-mM K + . ( A ) Melting profile of intramolecular DNA DQ and chimeric DNA:RNA HQ. Sequences used are shown on the left. Each of them carried a fluorescent donor FAM at the 5′ end and an accepter TAMRA at the 3′ end. The curves in the graph show the first derivative of FAM fluorescence over temperature as a function of temperature. ( B ) Protection of DNA by the formation of DQ or HQ. DQ or HQ formed in the single-stranded DNA or dimeric DNA:RNA partial duplex protected the DNA from being hydrolyzed from the 3′ end by Exo I exonuclease (scheme at left). Three substrates (Wild, M3G and HQ) were treated with Exo I in a single tube and those survived the hydrolysis were resolved on a denaturing gel. The RNA in the duplex region of the HQ substrate was hydrolyzed by RNase H prior to the exonuclease digestion. The DNAs were visualized by the FAM dye covalently labeled at their 5′ end, digitized, and the results are given on the right. The numbers above the bars indicate the average of the two time points. The DNA oligomer or moiety is shown in uppercase and that of RNA in lowercase in (A,B).

    Techniques Used: Fluorescence, Labeling

    10) Product Images from "Poly(A) Tail Length Control in Saccharomyces cerevisiae Occurs by Message-Specific Deadenylation"

    Article Title: Poly(A) Tail Length Control in Saccharomyces cerevisiae Occurs by Message-Specific Deadenylation

    Journal: Molecular and Cellular Biology

    doi:

    Transcriptional pulse of the PGK1 and MFA2 mRNAs in pan mutant and wild-type yeast strains. (A) Wild-type yeast strain YAS2286 and pan mutant yeast strain YAS2287 were pregrown in raffinose and sucrose-containing medium and then shifted to galactose (Gal)-containing medium to induce PGK1pG mRNA. Time points were taken at 0, 4, 8, and 12 min following the galactose (Gal) shift. Lanes 1 and 12, DNA markers (M) with sizes (in nucleotides) indicated to the left; lanes 2 and 7, PGK1pG mRNA treated with RNase H and oligo(dT) to remove the poly(A) tail (A 0 ); lanes 13 and 14, the wild-type (WT) and pan mutant PGK1pG mRNA produced after a 12-min galactose induction were electrophoresed side by side to more easily visualize poly(A) tail length differences. (B) Transcriptional pulse was carried out as described above for panel A, except MFA2pG mRNA was induced in the wild-type yeast strain YAS2284 and the pan mutant yeast strain YAS2285.
    Figure Legend Snippet: Transcriptional pulse of the PGK1 and MFA2 mRNAs in pan mutant and wild-type yeast strains. (A) Wild-type yeast strain YAS2286 and pan mutant yeast strain YAS2287 were pregrown in raffinose and sucrose-containing medium and then shifted to galactose (Gal)-containing medium to induce PGK1pG mRNA. Time points were taken at 0, 4, 8, and 12 min following the galactose (Gal) shift. Lanes 1 and 12, DNA markers (M) with sizes (in nucleotides) indicated to the left; lanes 2 and 7, PGK1pG mRNA treated with RNase H and oligo(dT) to remove the poly(A) tail (A 0 ); lanes 13 and 14, the wild-type (WT) and pan mutant PGK1pG mRNA produced after a 12-min galactose induction were electrophoresed side by side to more easily visualize poly(A) tail length differences. (B) Transcriptional pulse was carried out as described above for panel A, except MFA2pG mRNA was induced in the wild-type yeast strain YAS2284 and the pan mutant yeast strain YAS2285.

    Techniques Used: Mutagenesis, Produced

    11) Product Images from "Modulation of p53 Expression Using Antisense Oligonucleotides Complementary to the 5?-Terminal Region of p53 mRNA In Vitro and in the Living Cells"

    Article Title: Modulation of p53 Expression Using Antisense Oligonucleotides Complementary to the 5?-Terminal Region of p53 mRNA In Vitro and in the Living Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0078863

    RNase H assay in rabbit reticulocyte lysate in the presence of antisense oligomers no. 1 and 7b targeted to ΔNp53utr RNA. The 5′-end-[ 32 P]-labelled ΔNp53utr-Luc RNA was incubated in RRL and subsequently antisense oligomers no. 1 and no. 7b in their unmodified (UM) and modified (2′- O Me or GAP) form were added to the mixture. After 10 and 30 min incubation at 30°C, RNA was isolated and resolved on 8% polyacrylamide gel in denaturing conditions. The ΔNp53utr-Luc RNA was also subjected to limited hydrolysis by RNase T1 in denaturing conditions to determine the positions of RNase H cleavages. Lanes (−) and H 2 O indicate control reactions in the absence of antisense nucleotide in RRL and water, respectively. Lane (+) denotes the reaction in the presence of a control antisense oligonucleotide which is complementary to the Firefly luciferase sequence.
    Figure Legend Snippet: RNase H assay in rabbit reticulocyte lysate in the presence of antisense oligomers no. 1 and 7b targeted to ΔNp53utr RNA. The 5′-end-[ 32 P]-labelled ΔNp53utr-Luc RNA was incubated in RRL and subsequently antisense oligomers no. 1 and no. 7b in their unmodified (UM) and modified (2′- O Me or GAP) form were added to the mixture. After 10 and 30 min incubation at 30°C, RNA was isolated and resolved on 8% polyacrylamide gel in denaturing conditions. The ΔNp53utr-Luc RNA was also subjected to limited hydrolysis by RNase T1 in denaturing conditions to determine the positions of RNase H cleavages. Lanes (−) and H 2 O indicate control reactions in the absence of antisense nucleotide in RRL and water, respectively. Lane (+) denotes the reaction in the presence of a control antisense oligonucleotide which is complementary to the Firefly luciferase sequence.

    Techniques Used: Rnase H Assay, Incubation, Modification, Isolation, Luciferase, Sequencing

    Mapping of the accessibility of the 5′-terminal region of ΔNp53utr-Luc transcript to oligonucleotide hybridization. Semi-random libraries of DNA 6-mers and RNase H hydrolysis of RNA-DNA hybrids were used to search for sites accessible to hybridization in the 5′-terminal region of ΔNp53utr-Luc transcript. ( A ) The cleavage sites were identified by reverse transcription with the 5′-end-[ 32 P]-labelled DNA primer that was bound to nucleotides 232–257 of the ΔNp53utr-Luc sequence. The cDNA products were analyzed on 8% polyacrylamide gels. Selected cytosine and guanosine residues are marked on the left and the short and long run of the gel is shown. Lane (−) denotes the control reaction in the absence of semi-random oligonucleotide libraries. A, T, C, G – sequencing reaction with adenosine, thymidine, cytosine and guanosine dideoxy terminating nucleotides, respectively. ( B ) The cleavage sites occurring in the presence of libraries a , c , g , t displayed on the RNA secondary structure model. Continuous grey lines along the RNA sequence show the most probable positions of oligonucleotide hybridization on the ΔNp53utr-Luc transcript. The designed specific antisense oligonucleotides are marked with black lines and numbered respectively.
    Figure Legend Snippet: Mapping of the accessibility of the 5′-terminal region of ΔNp53utr-Luc transcript to oligonucleotide hybridization. Semi-random libraries of DNA 6-mers and RNase H hydrolysis of RNA-DNA hybrids were used to search for sites accessible to hybridization in the 5′-terminal region of ΔNp53utr-Luc transcript. ( A ) The cleavage sites were identified by reverse transcription with the 5′-end-[ 32 P]-labelled DNA primer that was bound to nucleotides 232–257 of the ΔNp53utr-Luc sequence. The cDNA products were analyzed on 8% polyacrylamide gels. Selected cytosine and guanosine residues are marked on the left and the short and long run of the gel is shown. Lane (−) denotes the control reaction in the absence of semi-random oligonucleotide libraries. A, T, C, G – sequencing reaction with adenosine, thymidine, cytosine and guanosine dideoxy terminating nucleotides, respectively. ( B ) The cleavage sites occurring in the presence of libraries a , c , g , t displayed on the RNA secondary structure model. Continuous grey lines along the RNA sequence show the most probable positions of oligonucleotide hybridization on the ΔNp53utr-Luc transcript. The designed specific antisense oligonucleotides are marked with black lines and numbered respectively.

    Techniques Used: Hybridization, Sequencing

    12) Product Images from "An allosteric-feedback mechanism for protein-assisted group I intron splicing"

    Article Title: An allosteric-feedback mechanism for protein-assisted group I intron splicing

    Journal:

    doi: 10.1261/rna.307907

    Time-resolved mapping of I- Ani I binding of  A.n.  COB pre-RNA. ( A ) Oligonucleotide accessibility. Internally labeled RNA was incubated with I- Ani I for 10–300 sec and then probed by the addition of a DNA oligonucleotide and RNase H. Digestion occurred
    Figure Legend Snippet: Time-resolved mapping of I- Ani I binding of A.n. COB pre-RNA. ( A ) Oligonucleotide accessibility. Internally labeled RNA was incubated with I- Ani I for 10–300 sec and then probed by the addition of a DNA oligonucleotide and RNase H. Digestion occurred

    Techniques Used: Binding Assay, Labeling, Incubation, Size-exclusion Chromatography

    13) Product Images from "APOBEC3G and APOBEC3F Act in Concert To Extinguish HIV-1 Replication"

    Article Title: APOBEC3G and APOBEC3F Act in Concert To Extinguish HIV-1 Replication

    Journal: Journal of Virology

    doi: 10.1128/JVI.03275-15

    Mutations in HIV RNase H are present only in virus from mouse G4. HIV-1 RNase H sequence amplified from the plasma of viremic humanized mice infected with either JRCSFvifH42/43D or JRCSFvifW79S showed that only virus from mouse G4 had mutations in RNase H. One G-to-A mutation was present in RNase H from mouse G4 (highlighted in orange) that resulted in a valine-to-isoleucine amino acid substitution.
    Figure Legend Snippet: Mutations in HIV RNase H are present only in virus from mouse G4. HIV-1 RNase H sequence amplified from the plasma of viremic humanized mice infected with either JRCSFvifH42/43D or JRCSFvifW79S showed that only virus from mouse G4 had mutations in RNase H. One G-to-A mutation was present in RNase H from mouse G4 (highlighted in orange) that resulted in a valine-to-isoleucine amino acid substitution.

    Techniques Used: Sequencing, Amplification, Mouse Assay, Infection, Mutagenesis

    14) Product Images from "Defining the Factors That Contribute to On-Target Specificity of Antisense Oligonucleotides"

    Article Title: Defining the Factors That Contribute to On-Target Specificity of Antisense Oligonucleotides

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0101752

    Effect of E. coli RNase H overexpression on on-target antisense activity. HEK 293 cells harboring the SOD/TO minigene or the SOD/TO minigene and pcDNA3.1-RHA were treated with ASOs at concentration between 0.5 and 150 nM. Following transfection and TET induction of the minigene, target RNA reduction was measured by qRT/PCR. Data are presented as percent mock-transfected control for SOD/TO (solid line) and SOD/TO-RHA cells (dashed line).
    Figure Legend Snippet: Effect of E. coli RNase H overexpression on on-target antisense activity. HEK 293 cells harboring the SOD/TO minigene or the SOD/TO minigene and pcDNA3.1-RHA were treated with ASOs at concentration between 0.5 and 150 nM. Following transfection and TET induction of the minigene, target RNA reduction was measured by qRT/PCR. Data are presented as percent mock-transfected control for SOD/TO (solid line) and SOD/TO-RHA cells (dashed line).

    Techniques Used: Over Expression, Activity Assay, Concentration Assay, Transfection, Quantitative RT-PCR

    Effect of E. coli RNase H overexpression on off-target antisense activity. HEK 293 cells harboring the minigene or the SOD 282_DL minigene and pcDNA3.1-RHA were treated with ASOs at concentrations between 0.5 and 150 nM. Following transfection and TET induction of the minigene, target RNA reduction was determined by qRT/PCR. Data are presented as percent mock-transfected control for SOD 282_DL (solid line) and SOD 282_DLH cells (dashed line).
    Figure Legend Snippet: Effect of E. coli RNase H overexpression on off-target antisense activity. HEK 293 cells harboring the minigene or the SOD 282_DL minigene and pcDNA3.1-RHA were treated with ASOs at concentrations between 0.5 and 150 nM. Following transfection and TET induction of the minigene, target RNA reduction was determined by qRT/PCR. Data are presented as percent mock-transfected control for SOD 282_DL (solid line) and SOD 282_DLH cells (dashed line).

    Techniques Used: Over Expression, Activity Assay, Transfection, Quantitative RT-PCR

    Cellular off-target cleavage is observed only with RNase H1 overexpression. A modified RLM-RACE protocol was used to determine sites of target-specific and off-target cleavage. A) Exon 5 target-specific RACE cleavage products. (B) Quantitative RACE of exon 5 target-specific cleavage products for SOD/TO cells (black bars) and SOD/TO-RHA cells (gray bars). Results are given as threshold cycle (cT) for the amplification reaction with or without overexpression of RNase H. C) Exon 4 off-target RACE cleavage products. D) Quantitative RACE of exon 4 off-target cleavage products (cT). E) Human RNase H1 was overexpressed in HeLa SOD/TO cells by infecting with adenoviral human RNase H1 for 48 hours   [4] . Cells were then treated with 50 nM ASO 38, and RLM_RACE was performed as described above. 293 cells with or without  E. coli  RNase H; HeLa cells with or without human RNase H1. F) RACE products were gel purified and sequenced using gene specific RACE primers. Arrows indicate the predominant cleavage site.
    Figure Legend Snippet: Cellular off-target cleavage is observed only with RNase H1 overexpression. A modified RLM-RACE protocol was used to determine sites of target-specific and off-target cleavage. A) Exon 5 target-specific RACE cleavage products. (B) Quantitative RACE of exon 5 target-specific cleavage products for SOD/TO cells (black bars) and SOD/TO-RHA cells (gray bars). Results are given as threshold cycle (cT) for the amplification reaction with or without overexpression of RNase H. C) Exon 4 off-target RACE cleavage products. D) Quantitative RACE of exon 4 off-target cleavage products (cT). E) Human RNase H1 was overexpressed in HeLa SOD/TO cells by infecting with adenoviral human RNase H1 for 48 hours [4] . Cells were then treated with 50 nM ASO 38, and RLM_RACE was performed as described above. 293 cells with or without E. coli RNase H; HeLa cells with or without human RNase H1. F) RACE products were gel purified and sequenced using gene specific RACE primers. Arrows indicate the predominant cleavage site.

    Techniques Used: Over Expression, Modification, Amplification, Allele-specific Oligonucleotide, Purification

    15) Product Images from "Dynamics and consequences of spliceosome E complex formation"

    Article Title: Dynamics and consequences of spliceosome E complex formation

    Journal: eLife

    doi: 10.7554/eLife.27592

    Single molecule analysis of U1 binding after ablation of the 5' end of the snRNA. ( A ) Cartoon of a two-color CoSMoS experiment for observing U1 binding dynamics. U1 was labeled with two green-excited fluorophores while the RNA was immobilized to the slide surface and contained a single, red-excited Cy5 fluorophore. ( B ) Confirmation of U1 ablation by primer extension. Primer extension of the U2 snRNA is included as a loading control. Quantification of the band intensities indicate that ≥96% of the U1 snRNA was cleaved by RNase H. ( C ) Bar graph comparing the relative number of U1 binding events observed on RNAs 3 or 7, in the presence or absence of CA and/or the RNase H ablation oligo. ( D–F ) Bar graph comparison of the fit parameters (τ 1 , panel D; τ 2 , panel E; the τ 2 amplitude A 2 , panel F) obtained from analysis of the dwell time distributions of U1 binding events. Details of the fit parameters for data shown in ( D–F ) can be found in Supplementary file 2 . Error bars in ( C ) represent the error in counting statistics as given by the variance of a binomial distribution. Bars in ( D–F ) represent the fit parameters ± S.D.
    Figure Legend Snippet: Single molecule analysis of U1 binding after ablation of the 5' end of the snRNA. ( A ) Cartoon of a two-color CoSMoS experiment for observing U1 binding dynamics. U1 was labeled with two green-excited fluorophores while the RNA was immobilized to the slide surface and contained a single, red-excited Cy5 fluorophore. ( B ) Confirmation of U1 ablation by primer extension. Primer extension of the U2 snRNA is included as a loading control. Quantification of the band intensities indicate that ≥96% of the U1 snRNA was cleaved by RNase H. ( C ) Bar graph comparing the relative number of U1 binding events observed on RNAs 3 or 7, in the presence or absence of CA and/or the RNase H ablation oligo. ( D–F ) Bar graph comparison of the fit parameters (τ 1 , panel D; τ 2 , panel E; the τ 2 amplitude A 2 , panel F) obtained from analysis of the dwell time distributions of U1 binding events. Details of the fit parameters for data shown in ( D–F ) can be found in Supplementary file 2 . Error bars in ( C ) represent the error in counting statistics as given by the variance of a binomial distribution. Bars in ( D–F ) represent the fit parameters ± S.D.

    Techniques Used: Binding Assay, Labeling

    16) Product Images from "Detecting RNA-RNA interactions in E. coli using a modified CLASH method"

    Article Title: Detecting RNA-RNA interactions in E. coli using a modified CLASH method

    Journal: BMC Genomics

    doi: 10.1186/s12864-017-3725-3

    Schematic overview of the modified protocol. a , wet experiment. Irradiated with 365 nm UV, RNAs were cross-linked by AMT at the paired region, and survive DNase I, RNase T1 and RNase H treatments which digest DNA and single strand RNA. Cross-linked RNAs were ligated by T4 RNA ligase 1. After photoreversal of cross-linkages by 254 nm UV, the ligated RNAs could be sequenced and identified. b , bioinformatics analysis
    Figure Legend Snippet: Schematic overview of the modified protocol. a , wet experiment. Irradiated with 365 nm UV, RNAs were cross-linked by AMT at the paired region, and survive DNase I, RNase T1 and RNase H treatments which digest DNA and single strand RNA. Cross-linked RNAs were ligated by T4 RNA ligase 1. After photoreversal of cross-linkages by 254 nm UV, the ligated RNAs could be sequenced and identified. b , bioinformatics analysis

    Techniques Used: Modification, Irradiation

    17) Product Images from "De Novo Initiation of RNA Synthesis by the RNA-Dependent RNA Polymerase (NS5B) of Hepatitis C Virus"

    Article Title: De Novo Initiation of RNA Synthesis by the RNA-Dependent RNA Polymerase (NS5B) of Hepatitis C Virus

    Journal: Journal of Virology

    doi:

    Analyses of RNA products by RNase H digestion. (A) Schematic presentation of the RNase H assay. After the RdRp reaction, RNA samples were mixed with a fivefold excess [to input D(+) RNA template] of unlabeled D(−) RNA, denatured, and reannealed. Hybridization between the input D(+) RNA template and the added in D(−) RNA frees [α- 32 P]UTP-labeled RNA product, which then hybridizes to the synthetic oligonucleotide and is cleaved by RNase H. Two cleaved RNA products, estimated at 277 and 80 nt, are expected if the monomer RNA product is digested by RNase H. (B) Analyses of RNase H-digested RNA products. RdRp and RNase H reactions were performed as described in Materials and Methods. Half of the sample was directly used for RNase H digestion in the presence of 0 (lane 2), 0.5 (lane 3), and 2.0 μg (lane 4) of oligo/DCoH. The other half was mixed with D(−) RNA, denatured, reannealed, and then digested with RNase H in the presence of 0 (lane 5), 0.5 (lane 6), and 2.0 μg (lane 7) of oligo/DCoH. Lanes 10 to 14 are longer exposures of lanes 5 to 9 to show the smaller digested band. The RNA size markers (left; GIBCO BRL) are shown in lanes 1, 8, and 13. The purified monomer RNA product was run in lane 9 (and is also shown in lane 14) (M). D, self-primed RNA dimer product. Two RNase H-cleaved RNA products with estimated lengths of 277 and 80 nt are indicated on the right. (C) Analyses of purified monomer RNA product by RNase H digestion. After the RdRp reaction, the [α- 32 P]UTP-labeled monomer RNA product was purified from a 6% polyacrylamide–7M urea gel, eluted out by electrophoresis, and collected by ethanol precipitation. The monomer RNA was then analyzed by RNase H digestion by the same strategy as that for panel A. The RNase H-digested monomer RNA was resolved in a 6% denaturing PAGE–7 M urea gel. The RNA size marker is shown in lane 1. M, monomer RNA product; 277, RNase H-cleaved RNA with 277 nt.
    Figure Legend Snippet: Analyses of RNA products by RNase H digestion. (A) Schematic presentation of the RNase H assay. After the RdRp reaction, RNA samples were mixed with a fivefold excess [to input D(+) RNA template] of unlabeled D(−) RNA, denatured, and reannealed. Hybridization between the input D(+) RNA template and the added in D(−) RNA frees [α- 32 P]UTP-labeled RNA product, which then hybridizes to the synthetic oligonucleotide and is cleaved by RNase H. Two cleaved RNA products, estimated at 277 and 80 nt, are expected if the monomer RNA product is digested by RNase H. (B) Analyses of RNase H-digested RNA products. RdRp and RNase H reactions were performed as described in Materials and Methods. Half of the sample was directly used for RNase H digestion in the presence of 0 (lane 2), 0.5 (lane 3), and 2.0 μg (lane 4) of oligo/DCoH. The other half was mixed with D(−) RNA, denatured, reannealed, and then digested with RNase H in the presence of 0 (lane 5), 0.5 (lane 6), and 2.0 μg (lane 7) of oligo/DCoH. Lanes 10 to 14 are longer exposures of lanes 5 to 9 to show the smaller digested band. The RNA size markers (left; GIBCO BRL) are shown in lanes 1, 8, and 13. The purified monomer RNA product was run in lane 9 (and is also shown in lane 14) (M). D, self-primed RNA dimer product. Two RNase H-cleaved RNA products with estimated lengths of 277 and 80 nt are indicated on the right. (C) Analyses of purified monomer RNA product by RNase H digestion. After the RdRp reaction, the [α- 32 P]UTP-labeled monomer RNA product was purified from a 6% polyacrylamide–7M urea gel, eluted out by electrophoresis, and collected by ethanol precipitation. The monomer RNA was then analyzed by RNase H digestion by the same strategy as that for panel A. The RNase H-digested monomer RNA was resolved in a 6% denaturing PAGE–7 M urea gel. The RNA size marker is shown in lane 1. M, monomer RNA product; 277, RNase H-cleaved RNA with 277 nt.

    Techniques Used: Rnase H Assay, Hybridization, Labeling, Purification, Electrophoresis, Ethanol Precipitation, Polyacrylamide Gel Electrophoresis, Marker

    18) Product Images from "Multiple physical forms of excised group II intron RNAs in wheat mitochondria"

    Article Title: Multiple physical forms of excised group II intron RNAs in wheat mitochondria

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl328

    Determination of in vivo abundance of lariat (and/or circularized) versus linear excised introns by RNase H analysis. Wheat mitochondrial RNA (24 h) was annealed with oligomers as shown in schematics (arrows a–h) for ( A ) nad7 intron 4, ( B ) nad2 intron 4, ( C ) cox2 intron and ( D ) nad1 intron 2, prior to RNase H treatment. Positions of oligomer probes used in subsequent northern blot analysis are shown by overlined asterisks. Size markers are shown, black dots represent the positions of native excised introns, black arrowheads indicate positions expected for hybridizing products from lariat (or circularized) intron forms and white arrowheads for linear intron forms.
    Figure Legend Snippet: Determination of in vivo abundance of lariat (and/or circularized) versus linear excised introns by RNase H analysis. Wheat mitochondrial RNA (24 h) was annealed with oligomers as shown in schematics (arrows a–h) for ( A ) nad7 intron 4, ( B ) nad2 intron 4, ( C ) cox2 intron and ( D ) nad1 intron 2, prior to RNase H treatment. Positions of oligomer probes used in subsequent northern blot analysis are shown by overlined asterisks. Size markers are shown, black dots represent the positions of native excised introns, black arrowheads indicate positions expected for hybridizing products from lariat (or circularized) intron forms and white arrowheads for linear intron forms.

    Techniques Used: In Vivo, Northern Blot

    19) Product Images from "Secondary Structure and Hybridization Accessibility of Hepatitis C Virus 3?-Terminal Sequences"

    Article Title: Secondary Structure and Hybridization Accessibility of Hepatitis C Virus 3?-Terminal Sequences

    Journal: Journal of Virology

    doi: 10.1128/JVI.76.19.9563-9574.2002

    Hybridization accessibility mapping of the HCV negative-strand 3′ terminus. SP6 in vitro transcripts from Xba I-digested template pG5 were renatured in TMK buffer and digested with 0.2 or 2 U of RNase H at 37°C in the presence of a 300-fold molar excess of fully randomized (N) or nucleotide-specific (C, T, A, and G) chimeric 11-mer oligonucleotide libraries. (A) Overview of RNase H cleavage pattern for the entire 3′-terminal region. cDNAs were derived from MMLV-mediated extension of primer P2. Lanes: 1, negative control, HCV RNA omitted; 2, RNase H (2 U) mock digest, library omitted; 3 to 14, nucleotide-specific library digests and dideoxy sequencing products; 15 to 17, N library digests. (B) Detail of the cleavage pattern for RNA digested with 2 U of RNase H. cDNAs were derived from extension of primers P2 (lower left), P118 (upper left), and P237 (right). Lanes: 1, mock digest, library omitted; 2, mock digest, RNase H omitted; 3, N library digest; 4, negative control, HCV RNA omitted; 5 to 12, nucleotide-specific library digests and dideoxy sequencing products.
    Figure Legend Snippet: Hybridization accessibility mapping of the HCV negative-strand 3′ terminus. SP6 in vitro transcripts from Xba I-digested template pG5 were renatured in TMK buffer and digested with 0.2 or 2 U of RNase H at 37°C in the presence of a 300-fold molar excess of fully randomized (N) or nucleotide-specific (C, T, A, and G) chimeric 11-mer oligonucleotide libraries. (A) Overview of RNase H cleavage pattern for the entire 3′-terminal region. cDNAs were derived from MMLV-mediated extension of primer P2. Lanes: 1, negative control, HCV RNA omitted; 2, RNase H (2 U) mock digest, library omitted; 3 to 14, nucleotide-specific library digests and dideoxy sequencing products; 15 to 17, N library digests. (B) Detail of the cleavage pattern for RNA digested with 2 U of RNase H. cDNAs were derived from extension of primers P2 (lower left), P118 (upper left), and P237 (right). Lanes: 1, mock digest, library omitted; 2, mock digest, RNase H omitted; 3, N library digest; 4, negative control, HCV RNA omitted; 5 to 12, nucleotide-specific library digests and dideoxy sequencing products.

    Techniques Used: Hybridization, In Vitro, Derivative Assay, Negative Control, Sequencing

    20) Product Images from "Transcription and RNA editing in a soluble in vitro system from Physarum mitochondria"

    Article Title: Transcription and RNA editing in a soluble in vitro system from Physarum mitochondria

    Journal: Nucleic Acids Research

    doi:

    Characterization of TEC. ( A ) Dot blot hybridization analysis of RNAs synthesized by TEC. DNAs from the nuclear genes actin and tubulin, the mitochondrial genes coI, coII and α-ATPase, and the cloning vector were immobilized and hybridized to labeled transcripts synthesized by TEC as described in Materials and Methods. ( B ) RNase H digestion of S1 nuclease protected α-ATPase mRNA synthesized in the absence of a cold nucleotide chase. Lane 1, no oligonucleotide, no RNase H; lane 2, oligonucleotide A in the presence of RNase H; lane 3, oligonucleotide B in the presence of RNase H. Sizes of the expected cleavage products are shown in the diagram to the right. ( C ) Nucleotide requirements for transcription by TEC. Run-on RNA synthesis in the presence of all four ribonucleotides (lane 1); 20 µM [α- 32 P]UTP only (lane 2); 20 µM [α- 32 P]UTP, 500 µM CTP and GTP (lane 3); 20 µM [α- 32 P]UTP and 500 µM ATP and GTP (lane 4); or 20 µM [α- 32 P]UTP and 500 µM CTP and ATP (lane 5).
    Figure Legend Snippet: Characterization of TEC. ( A ) Dot blot hybridization analysis of RNAs synthesized by TEC. DNAs from the nuclear genes actin and tubulin, the mitochondrial genes coI, coII and α-ATPase, and the cloning vector were immobilized and hybridized to labeled transcripts synthesized by TEC as described in Materials and Methods. ( B ) RNase H digestion of S1 nuclease protected α-ATPase mRNA synthesized in the absence of a cold nucleotide chase. Lane 1, no oligonucleotide, no RNase H; lane 2, oligonucleotide A in the presence of RNase H; lane 3, oligonucleotide B in the presence of RNase H. Sizes of the expected cleavage products are shown in the diagram to the right. ( C ) Nucleotide requirements for transcription by TEC. Run-on RNA synthesis in the presence of all four ribonucleotides (lane 1); 20 µM [α- 32 P]UTP only (lane 2); 20 µM [α- 32 P]UTP, 500 µM CTP and GTP (lane 3); 20 µM [α- 32 P]UTP and 500 µM ATP and GTP (lane 4); or 20 µM [α- 32 P]UTP and 500 µM CTP and ATP (lane 5).

    Techniques Used: Dot Blot, Hybridization, Synthesized, Clone Assay, Plasmid Preparation, Labeling

    21) Product Images from "Polypyrimidine Tract Binding Protein Functions as a Negative Regulator of Feline Calicivirus Translation"

    Article Title: Polypyrimidine Tract Binding Protein Functions as a Negative Regulator of Feline Calicivirus Translation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0009562

    RNAi mediated knockdown of PTB inhibit the translation from the FCV subgenomic RNA in cells. (A) Genome schematic of the FCV genome highlighting the position of the three FCV open reading frames and the NS6-7 specific primer which bound to nucleotides between 3215–3244 used for RNase H-directed inactivation of the FCV genomic RNA. (B) In vitro translation of FCV RNA after RNase H digestion using an FCV genomic RNA specific DNA oligonucleotide (FP) and a control DNA oligonucleotide (CP). Immunoprecipitation (VP1-IP) with α-major capsid (VP1) from the in vitro translation of FCV RNA after RNase H digestion using an FCV genomic RNA specific DNA oligonucleotide and a control DNA oligonucleotide. The proteins were labeled with 35 S methionine and the SDS-PAGE analysis was exposed to a phosphorimager screen. (C) Western blot for the major capsid protein (VP1) in infected CRFK cells, purified virus, non-transfected CRFK cells and CRFK cells treated with PTB or GFP siRNAs that have been transfected with FCV RNA digested with RNase H using an FCV specific DNA oligonucleotide. Control blots for PTB and GAPDH are shown to demonstrate PTB knockdown and equal loading of samples. Results displayed were obtained from cells incubated at 37°C but identical results were also observed when the experiments were performed at 32°C (data not shown) An asterisk is used to highlight a non-specific protein with reactivity to anti-VP1 antisera.
    Figure Legend Snippet: RNAi mediated knockdown of PTB inhibit the translation from the FCV subgenomic RNA in cells. (A) Genome schematic of the FCV genome highlighting the position of the three FCV open reading frames and the NS6-7 specific primer which bound to nucleotides between 3215–3244 used for RNase H-directed inactivation of the FCV genomic RNA. (B) In vitro translation of FCV RNA after RNase H digestion using an FCV genomic RNA specific DNA oligonucleotide (FP) and a control DNA oligonucleotide (CP). Immunoprecipitation (VP1-IP) with α-major capsid (VP1) from the in vitro translation of FCV RNA after RNase H digestion using an FCV genomic RNA specific DNA oligonucleotide and a control DNA oligonucleotide. The proteins were labeled with 35 S methionine and the SDS-PAGE analysis was exposed to a phosphorimager screen. (C) Western blot for the major capsid protein (VP1) in infected CRFK cells, purified virus, non-transfected CRFK cells and CRFK cells treated with PTB or GFP siRNAs that have been transfected with FCV RNA digested with RNase H using an FCV specific DNA oligonucleotide. Control blots for PTB and GAPDH are shown to demonstrate PTB knockdown and equal loading of samples. Results displayed were obtained from cells incubated at 37°C but identical results were also observed when the experiments were performed at 32°C (data not shown) An asterisk is used to highlight a non-specific protein with reactivity to anti-VP1 antisera.

    Techniques Used: In Vitro, Immunoprecipitation, Labeling, SDS Page, Western Blot, Infection, Purification, Transfection, Incubation

    22) Product Images from "Nucleophosmin deposition during mRNA 3? end processing influences poly(A) tail length"

    Article Title: Nucleophosmin deposition during mRNA 3? end processing influences poly(A) tail length

    Journal: The EMBO Journal

    doi: 10.1038/emboj.2011.272

    Knockdown of NPM1 results in an increase in mRNA poly(A) tail length. Total RNA was isolated from untreated HeLa cells (HeLa lanes), HeLa cells containing a vector only control (LKO.1 lanes) or HeLa cells knocked down for NPM1 (NPM1 KD lanes) and analysed by linker ligation-mediated PCR-based poly(A) tail length assay. A set of samples was treated with oligo dT and RNAse H to remove the poly(A) tail before analysis (RNase H/d(T) lanes). The PAT lanes represent samples where mRNAs with intact poly(A) tails were analysed. Primers for β-actin mRNA were used in ( A ), hnRNP H mRNA in ( B ) and rps5 mRNA in ( C ). PCR products were analysed on a 5% acrylamide gel. The positions of the unadenylated RNA (A 0 ), normally polyadenylated (pA) and hyperadenylated (pA++) are indicated on the left.
    Figure Legend Snippet: Knockdown of NPM1 results in an increase in mRNA poly(A) tail length. Total RNA was isolated from untreated HeLa cells (HeLa lanes), HeLa cells containing a vector only control (LKO.1 lanes) or HeLa cells knocked down for NPM1 (NPM1 KD lanes) and analysed by linker ligation-mediated PCR-based poly(A) tail length assay. A set of samples was treated with oligo dT and RNAse H to remove the poly(A) tail before analysis (RNase H/d(T) lanes). The PAT lanes represent samples where mRNAs with intact poly(A) tails were analysed. Primers for β-actin mRNA were used in ( A ), hnRNP H mRNA in ( B ) and rps5 mRNA in ( C ). PCR products were analysed on a 5% acrylamide gel. The positions of the unadenylated RNA (A 0 ), normally polyadenylated (pA) and hyperadenylated (pA++) are indicated on the left.

    Techniques Used: Isolation, Plasmid Preparation, Ligation, Polymerase Chain Reaction, Acrylamide Gel Assay

    23) Product Images from "Investigating a New Generation of Ribozymes in Order to Target HCV"

    Article Title: Investigating a New Generation of Ribozymes in Order to Target HCV

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0009627

    Schematic representation of the 3-step procedure used for the identification of the sites possessing the greatest targeting potential in the HCV 5′-UTR. Step 1 involved a bioinformatic analysis that included both the prediction of the secondary structure and the identification of the 7 nt streches most likely to be bound by the ribozyme's P1 region using both the RNA structure 3.7 and Oligowalk softwares. Step 2 involved the selection of the sequences that fulfill the HDV ribozyme requirements. Step 3 involved the RNase H hydrolysis assay. The autoradiogram shown corresponds to a typical 5% polyacrylamide gel performed for the analysis of 6 potential sites. The positions of proposed cleavage sites are identified at the top of the gel. The negative control performed in the absence of any oligonucleotide is indicated by the letter C.
    Figure Legend Snippet: Schematic representation of the 3-step procedure used for the identification of the sites possessing the greatest targeting potential in the HCV 5′-UTR. Step 1 involved a bioinformatic analysis that included both the prediction of the secondary structure and the identification of the 7 nt streches most likely to be bound by the ribozyme's P1 region using both the RNA structure 3.7 and Oligowalk softwares. Step 2 involved the selection of the sequences that fulfill the HDV ribozyme requirements. Step 3 involved the RNase H hydrolysis assay. The autoradiogram shown corresponds to a typical 5% polyacrylamide gel performed for the analysis of 6 potential sites. The positions of proposed cleavage sites are identified at the top of the gel. The negative control performed in the absence of any oligonucleotide is indicated by the letter C.

    Techniques Used: Selection, Hydrolysis Assay, Negative Control

    24) Product Images from "PARTICLE triplexes cluster in the tumor suppressor WWOX and may extend throughout the human genome"

    Article Title: PARTICLE triplexes cluster in the tumor suppressor WWOX and may extend throughout the human genome

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-07295-5

    PARTICLE  forms a triplex with  WWOX . ( A ) Triplex-forming oligonucleotide motifs within  PARTICLE  (green) identified using Triplexator software to target triplex sites in  WWOX  (blue and red) to form a triple helix. Subscript base indicates a mismatch in the sequence. ( B ) Representative surface plasmon resonance (SPR) sensorgrams illustrating spectral shift following  PARTICLE  triplex formation with the duplex  WWOX  region ( PT -ds W , red line, schematic B1) in comparison to controls (scrambled  PARTICLE  on duplex  WWOX  region; s PT -ds W , blue line, left, schematic B2);  PARTICLE  on single stranded  WWOX  target ( PT -ss W , purple, middle, schematic B3);  PARTICLE  on duplex shuffled  WWOX  region ( PT -sf W , green, right, schematic B4)), n  >  3. ( C ) Electrophoretic mobility shift assays (EMSA) with gels (cropped) showing triplex formation (T) with incremental concentrations (0, 0.2, 2, 20 nM horizontal black triangle) of TFO:  PARTICLE  627-646 RNA with duplex  WWOX  target region and absence with single  WWOX  (TTS_hit_1) oligo or following treatment with RNase H.
    Figure Legend Snippet: PARTICLE forms a triplex with WWOX . ( A ) Triplex-forming oligonucleotide motifs within PARTICLE (green) identified using Triplexator software to target triplex sites in WWOX (blue and red) to form a triple helix. Subscript base indicates a mismatch in the sequence. ( B ) Representative surface plasmon resonance (SPR) sensorgrams illustrating spectral shift following PARTICLE triplex formation with the duplex WWOX region ( PT -ds W , red line, schematic B1) in comparison to controls (scrambled PARTICLE on duplex WWOX region; s PT -ds W , blue line, left, schematic B2); PARTICLE on single stranded WWOX target ( PT -ss W , purple, middle, schematic B3); PARTICLE on duplex shuffled WWOX region ( PT -sf W , green, right, schematic B4)), n  >  3. ( C ) Electrophoretic mobility shift assays (EMSA) with gels (cropped) showing triplex formation (T) with incremental concentrations (0, 0.2, 2, 20 nM horizontal black triangle) of TFO: PARTICLE 627-646 RNA with duplex WWOX target region and absence with single WWOX (TTS_hit_1) oligo or following treatment with RNase H.

    Techniques Used: Software, Sequencing, SPR Assay, Electrophoretic Mobility Shift Assay

    25) Product Images from "Characterization of a Novel Association between Two Trypanosome-Specific Proteins and 5S rRNA"

    Article Title: Characterization of a Novel Association between Two Trypanosome-Specific Proteins and 5S rRNA

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0030029

    Analysis of the solution structure of T. brucei 5S rRNA by RNase H digestion. Panel A : Oligonucleotides used and their coverage of the T. brucei 5S rRNA structure. Panel B : Radiolabeled 5S rRNA was incubated with each of these oligonucleotides and then treated with RNase H. The reactions were then resolved by electrophoresis on a denaturing urea gel.
    Figure Legend Snippet: Analysis of the solution structure of T. brucei 5S rRNA by RNase H digestion. Panel A : Oligonucleotides used and their coverage of the T. brucei 5S rRNA structure. Panel B : Radiolabeled 5S rRNA was incubated with each of these oligonucleotides and then treated with RNase H. The reactions were then resolved by electrophoresis on a denaturing urea gel.

    Techniques Used: Incubation, Electrophoresis

    P34 and P37 protect the Loop C of 5S rRNA. RNase H protection assays were performed with full-length 5S rRNA in the absence (lanes 3–6) or presence (lanes 7–9) of protein. The arrow indicates full-length RNA and the asterisks indicate cleavage products.
    Figure Legend Snippet: P34 and P37 protect the Loop C of 5S rRNA. RNase H protection assays were performed with full-length 5S rRNA in the absence (lanes 3–6) or presence (lanes 7–9) of protein. The arrow indicates full-length RNA and the asterisks indicate cleavage products.

    Techniques Used:

    26) Product Images from "Peptide-oligonucleotide conjugates exhibiting pyrimidine-X cleavage specificity efficiently silence miRNA target acting synergistically with RNase H"

    Article Title: Peptide-oligonucleotide conjugates exhibiting pyrimidine-X cleavage specificity efficiently silence miRNA target acting synergistically with RNase H

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-33331-z

    Cleavage of miR-17 by miR-17-specific conjugate 4 and RNase H. ( A) Patterns of 5′-[ 32 P]-miR-17 cleavage in complex with conjugate 4 or in complex with oligonucleotide 4 (ON 4) by RNase H. Autoradiograph of 18% polyacrylamide/8 M urea gel. Duplexes of 5′-[ 32 P]-miR-17 (10 μM) and oligonucleotide or conjugate (5 μM) were incubated at 37 °C for 24 h with RNase H (100 U/ml). Lanes Im and T1 — imidazole ladder and partial RNA digestion with RNase T1, respectively; control — RNA was incubated in the absence of oligonucleotide/conjugate and in the presence of RNase H. ( B) Kinetics of miR-17 cleavage by conjugates 4 , miR-17/ON 4 cleavage by RNase H, and miR-17 cleavage by conjugate 4 together with RNase H. ( C) Positions of miR-17 cleavage by RNase H and conjugate 4 . Cleavage at specific sites is indicated by arrows; pep — catalytic construction. ( D) Diagram showing contribution of conjugate 4 and RNase H in the total cleavage of miR-17 for 8 h. Dark grey bars and white bars show contribution of RNase H and conjugate 4 to the miR-17 cleavage, respectively.
    Figure Legend Snippet: Cleavage of miR-17 by miR-17-specific conjugate 4 and RNase H. ( A) Patterns of 5′-[ 32 P]-miR-17 cleavage in complex with conjugate 4 or in complex with oligonucleotide 4 (ON 4) by RNase H. Autoradiograph of 18% polyacrylamide/8 M urea gel. Duplexes of 5′-[ 32 P]-miR-17 (10 μM) and oligonucleotide or conjugate (5 μM) were incubated at 37 °C for 24 h with RNase H (100 U/ml). Lanes Im and T1 — imidazole ladder and partial RNA digestion with RNase T1, respectively; control — RNA was incubated in the absence of oligonucleotide/conjugate and in the presence of RNase H. ( B) Kinetics of miR-17 cleavage by conjugates 4 , miR-17/ON 4 cleavage by RNase H, and miR-17 cleavage by conjugate 4 together with RNase H. ( C) Positions of miR-17 cleavage by RNase H and conjugate 4 . Cleavage at specific sites is indicated by arrows; pep — catalytic construction. ( D) Diagram showing contribution of conjugate 4 and RNase H in the total cleavage of miR-17 for 8 h. Dark grey bars and white bars show contribution of RNase H and conjugate 4 to the miR-17 cleavage, respectively.

    Techniques Used: Autoradiography, Incubation

    27) Product Images from "RNA Interference-Guided Targeting of Hepatitis C Virus Replication with Antisense Locked Nucleic Acid-Based Oligonucleotides Containing 8-oxo-dG Modifications"

    Article Title: RNA Interference-Guided Targeting of Hepatitis C Virus Replication with Antisense Locked Nucleic Acid-Based Oligonucleotides Containing 8-oxo-dG Modifications

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0128686

    RNase H-mediated degradation of pre-formed ASO:RNA duplexes and in vitro -synthesized RNAs targeted by ASOs. (A) Schematic of the experimental setup for panels B and C. Applicable for some ASOs: Y, 8-oxo-dG residue; +, LNA sugar base. (B, C) Cleavage of pre-formed ASO:target RNA duplexes by RNase H. (B) Five femtomoles of 33 P-labeled substrate was treated with RNase H for the indicated times. The reaction products were collected, denatured by heating at 95°C for 2 min and analyzed by PAGE in native 15% gels. Arrows at right point to the substrate (S) and major cleavage product(s) (P). Results from one of three independent reproducible experiments are shown. (C) Kinetics of RNase H cleavage of different ASO:RNA duplexes. The amounts of radioactivity remaining in the uncleaved substrate were quantified using a Typhoon Trio instrument. Quantifications were performed for each gel. The obtained values were normalized to the radioactivity present in the substrate before adding RNase H (set to 100%). Each point corresponds to the average of three independent experiments. Error bars indicate the standard deviation. (D) Cleavage of FR3131 RNA by RNase H in the presence of different ASOs. The RNA and ASOs were mixed and incubated at 37°C for 10 min; then, RNase H was added to the reaction mixture. RNA samples were collected at the indicated time points and analyzed by electrophoresis in native 0.8% TAE agarose gels. The results from one of three independent reproducible experiments are shown. S: substrate; P1 and P2: cleavage products.
    Figure Legend Snippet: RNase H-mediated degradation of pre-formed ASO:RNA duplexes and in vitro -synthesized RNAs targeted by ASOs. (A) Schematic of the experimental setup for panels B and C. Applicable for some ASOs: Y, 8-oxo-dG residue; +, LNA sugar base. (B, C) Cleavage of pre-formed ASO:target RNA duplexes by RNase H. (B) Five femtomoles of 33 P-labeled substrate was treated with RNase H for the indicated times. The reaction products were collected, denatured by heating at 95°C for 2 min and analyzed by PAGE in native 15% gels. Arrows at right point to the substrate (S) and major cleavage product(s) (P). Results from one of three independent reproducible experiments are shown. (C) Kinetics of RNase H cleavage of different ASO:RNA duplexes. The amounts of radioactivity remaining in the uncleaved substrate were quantified using a Typhoon Trio instrument. Quantifications were performed for each gel. The obtained values were normalized to the radioactivity present in the substrate before adding RNase H (set to 100%). Each point corresponds to the average of three independent experiments. Error bars indicate the standard deviation. (D) Cleavage of FR3131 RNA by RNase H in the presence of different ASOs. The RNA and ASOs were mixed and incubated at 37°C for 10 min; then, RNase H was added to the reaction mixture. RNA samples were collected at the indicated time points and analyzed by electrophoresis in native 0.8% TAE agarose gels. The results from one of three independent reproducible experiments are shown. S: substrate; P1 and P2: cleavage products.

    Techniques Used: Allele-specific Oligonucleotide, In Vitro, Synthesized, Labeling, Polyacrylamide Gel Electrophoresis, Radioactivity, Standard Deviation, Incubation, Electrophoresis

    28) Product Images from "Simultaneous Visualization of Both Signaling Cascade Activity and End-Point Gene Expression in Single Cells"

    Article Title: Simultaneous Visualization of Both Signaling Cascade Activity and End-Point Gene Expression in Single Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0020148

    Method overview. (A) Upon stimulation with PDGF-BB the PDGFRβ becomes autophosphorylated at various sites. This promotes downstream signaling to ERK activation. Phosphorylated ERK translocates into the nucleus and enhances DUSP6 expression. Upon fixation of the cells DUSP6 mRNA is reverse transcribed utilizing an LNA-modified primer. Subsequently the cDNA is made accessible through RNase H digestion. The single standed cDNA stays attached to the mRNA-primer by the 5′-end of the primer that contains the LNA bases, which is not recognized by RNase H – to allow hybridization of padlock probes. (B) After ligation of the padlock probe to a circular DNA molecule, primary antibodies directed against the PDGFRβ and phosphorylated tyrosines, followed by addition of secondary PLA probes, are applied for detection of the phosphorylated PDGFRβ. If the antibodies and probes are bound in close proximity two circularization oligonucleotides can hybridize to the PLA probes. (C) This molecule can subsequently also be joined by ligation, giving rise to another circular DNA molecule. (D) Both DNA circles are then simultaneously amplified by RCA, primed from either the cDNA or the oligonucleotide attached to the PLA probe. The resulting bundles of DNA (amplified ∼1000 times) are still attached to the place where the RCA was primed and can be detected by hybridization of fluorescence labeled oligonucleotides (different fluorophores (green and pink stars) for the two types of circles), resulting in bright spots easily distinguishable from background.
    Figure Legend Snippet: Method overview. (A) Upon stimulation with PDGF-BB the PDGFRβ becomes autophosphorylated at various sites. This promotes downstream signaling to ERK activation. Phosphorylated ERK translocates into the nucleus and enhances DUSP6 expression. Upon fixation of the cells DUSP6 mRNA is reverse transcribed utilizing an LNA-modified primer. Subsequently the cDNA is made accessible through RNase H digestion. The single standed cDNA stays attached to the mRNA-primer by the 5′-end of the primer that contains the LNA bases, which is not recognized by RNase H – to allow hybridization of padlock probes. (B) After ligation of the padlock probe to a circular DNA molecule, primary antibodies directed against the PDGFRβ and phosphorylated tyrosines, followed by addition of secondary PLA probes, are applied for detection of the phosphorylated PDGFRβ. If the antibodies and probes are bound in close proximity two circularization oligonucleotides can hybridize to the PLA probes. (C) This molecule can subsequently also be joined by ligation, giving rise to another circular DNA molecule. (D) Both DNA circles are then simultaneously amplified by RCA, primed from either the cDNA or the oligonucleotide attached to the PLA probe. The resulting bundles of DNA (amplified ∼1000 times) are still attached to the place where the RCA was primed and can be detected by hybridization of fluorescence labeled oligonucleotides (different fluorophores (green and pink stars) for the two types of circles), resulting in bright spots easily distinguishable from background.

    Techniques Used: Activation Assay, Expressing, Modification, Hybridization, Ligation, Proximity Ligation Assay, Amplification, Fluorescence, Labeling

    29) Product Images from "DROSHA targets its own transcript to modulate alternative splicing"

    Article Title: DROSHA targets its own transcript to modulate alternative splicing

    Journal: RNA

    doi: 10.1261/rna.059808.116

    The human DROSHA hairpin is responsible for alternative splicing of DROSHA exon 7. ( A ) Schematic representation of the DROSHA gene region from exons 6–8. Boxes, horizontal lines, and the red bar across the exon 7–intron 7 junction depict exons, introns, and the DROSHA hairpin, respectively. PCR primers to detect exon 7 splicing or the 3′ processing product are represented by blue and gray arrows, respectively. It is noted that a vector-specific forward primer was used instead of Ex6F for the analysis of minigene splicing. The 5′ splice site of intron 7 and its base-pairing to U1 snRNA are also shown. ( B ) Splicing of DROSHA exon 7 in human and murine cells and its recapitulation in minigene systems. Percentages of the exon 7-skipped isoform and standard error of the mean (SEM) from three independent experiments are presented below the gel. ( C ) Hairpin swapping assay. The DROSHA hairpins were interchanged between the human and mouse minigenes and exon 7 splicing from these hybrid constructs was analyzed. H and M stand for human and mouse, respectively. 5′ RLM-RACE was also performed to indicate hairpin cleavage in cells. Percentages of the exon 7-skipped isoform and SEM from three biologically independent experiments are presented below the gel. GAPDH and Neo r serve as loading controls. ( D ) Psoralen crosslinking assay. Radiolabeled hairpin RNAs were incubated with HeLa nuclear extract (NE) under splicing conditions and irradiated with 365-nm UV light in the presence of psoralen. The U1 snRNA: DROSHA hairpin adducts are indicated by arrowheads. The shortened adducts resulting from RNase H digestion with an oligonucleotide complementary to U1 snRNA are marked by an arrow. Relative intensities of the adducts from five independent experiments are plotted on the graph. (**) Statistical significance of P ≤ 0.005 as determined by Student's t -test. Error bars represent SEM.
    Figure Legend Snippet: The human DROSHA hairpin is responsible for alternative splicing of DROSHA exon 7. ( A ) Schematic representation of the DROSHA gene region from exons 6–8. Boxes, horizontal lines, and the red bar across the exon 7–intron 7 junction depict exons, introns, and the DROSHA hairpin, respectively. PCR primers to detect exon 7 splicing or the 3′ processing product are represented by blue and gray arrows, respectively. It is noted that a vector-specific forward primer was used instead of Ex6F for the analysis of minigene splicing. The 5′ splice site of intron 7 and its base-pairing to U1 snRNA are also shown. ( B ) Splicing of DROSHA exon 7 in human and murine cells and its recapitulation in minigene systems. Percentages of the exon 7-skipped isoform and standard error of the mean (SEM) from three independent experiments are presented below the gel. ( C ) Hairpin swapping assay. The DROSHA hairpins were interchanged between the human and mouse minigenes and exon 7 splicing from these hybrid constructs was analyzed. H and M stand for human and mouse, respectively. 5′ RLM-RACE was also performed to indicate hairpin cleavage in cells. Percentages of the exon 7-skipped isoform and SEM from three biologically independent experiments are presented below the gel. GAPDH and Neo r serve as loading controls. ( D ) Psoralen crosslinking assay. Radiolabeled hairpin RNAs were incubated with HeLa nuclear extract (NE) under splicing conditions and irradiated with 365-nm UV light in the presence of psoralen. The U1 snRNA: DROSHA hairpin adducts are indicated by arrowheads. The shortened adducts resulting from RNase H digestion with an oligonucleotide complementary to U1 snRNA are marked by an arrow. Relative intensities of the adducts from five independent experiments are plotted on the graph. (**) Statistical significance of P ≤ 0.005 as determined by Student's t -test. Error bars represent SEM.

    Techniques Used: Polymerase Chain Reaction, Plasmid Preparation, Construct, Incubation, Irradiation

    30) Product Images from "RNase-sensitive DNA modification(s) initiates S. pombe mating-type switching"

    Article Title: RNase-sensitive DNA modification(s) initiates S. pombe mating-type switching

    Journal: Genes & Development

    doi: 10.1101/gad.289404

    Analysis of replication intermediates at mat1 . ( A ) Primer extension on Okazaki fragments from the wild-type (JZ105), smt -0 (JZ108), swi1 (SV5), swi3 (SV1), and swi7 (SP469) strains. The left panel shows the analysis of the wild-type and smt-0 strains. The position of the homology domain H1 and the site of the imprint are given to the right of the autoradiograph. The drawing below shows the annealing position of the primer used for the assay on the wild-type DNA and lack of specific annealing to smt-0 DNA. The right panel shows the analysis of swi1, swi3 , and swi7 mutants. Black lines indicate the position of the analyzed region relative to the left panel. The drawing below indicates that in this experiment the parental DNA, shown by dotted lines, was digested away by λ-exonuclease before primer extension. ( B ) Mapping of the pause site on the lagging strand on gel-purified paused intermediates. Strains are given in A . The sequence of the region (upper strand) is shown to the right , and the arrows indicate the position of the bands observed. The drawing below shows that RNase H removes RNA primers at 5′-ends of pausing intermediates of the lagging strand. The drawing also indicates that in this experiment primer can anneal both to nascent and to parental DNA. ( C ) Mapping of the pause site on the leading strand. The nonswitching strain smt-0 (JZ108) was used for clarity of the analysis, to avoid detecting imprinting and switching intermediates. The sequence of the analyzed region is shown to the right . The position of the BstNI restriction site on the upper strand is labeled, and the region of pausing is designated by the brackets. The line drawing below displays the position of the BstNI and SfaNI sites and the size of the full-length fragment of the lower strand. The strands, visualized on the gel, are shown as thick lines, and the mapped 3′-end of the nascent leading strand is shown as an empty arrowhead.
    Figure Legend Snippet: Analysis of replication intermediates at mat1 . ( A ) Primer extension on Okazaki fragments from the wild-type (JZ105), smt -0 (JZ108), swi1 (SV5), swi3 (SV1), and swi7 (SP469) strains. The left panel shows the analysis of the wild-type and smt-0 strains. The position of the homology domain H1 and the site of the imprint are given to the right of the autoradiograph. The drawing below shows the annealing position of the primer used for the assay on the wild-type DNA and lack of specific annealing to smt-0 DNA. The right panel shows the analysis of swi1, swi3 , and swi7 mutants. Black lines indicate the position of the analyzed region relative to the left panel. The drawing below indicates that in this experiment the parental DNA, shown by dotted lines, was digested away by λ-exonuclease before primer extension. ( B ) Mapping of the pause site on the lagging strand on gel-purified paused intermediates. Strains are given in A . The sequence of the region (upper strand) is shown to the right , and the arrows indicate the position of the bands observed. The drawing below shows that RNase H removes RNA primers at 5′-ends of pausing intermediates of the lagging strand. The drawing also indicates that in this experiment primer can anneal both to nascent and to parental DNA. ( C ) Mapping of the pause site on the leading strand. The nonswitching strain smt-0 (JZ108) was used for clarity of the analysis, to avoid detecting imprinting and switching intermediates. The sequence of the analyzed region is shown to the right . The position of the BstNI restriction site on the upper strand is labeled, and the region of pausing is designated by the brackets. The line drawing below displays the position of the BstNI and SfaNI sites and the size of the full-length fragment of the lower strand. The strands, visualized on the gel, are shown as thick lines, and the mapped 3′-end of the nascent leading strand is shown as an empty arrowhead.

    Techniques Used: Autoradiography, Purification, Sequencing, Labeling

    31) Product Images from "BRCA2 controls DNA:RNA hybrid level at DSBs by mediating RNase H2 recruitment"

    Article Title: BRCA2 controls DNA:RNA hybrid level at DSBs by mediating RNase H2 recruitment

    Journal: Nature Communications

    doi: 10.1038/s41467-018-07799-2

    DNA:RNA hybrids form at DSBs independently of the genomic context. a Schematic representation of DNA:RNA hybrids (in red) that can be generated upon the hybridization of mRNA (top) or dilncRNAs (bottom) with resected DNA ends at the I-PpoI cut site within DAB1 gene. b DRIP-qPCR analysis at the I-PpoI cut site within a genic ( DAB1 gene) or c nongenic locus in HeLa cells transfected with the I-PpoI nuclease. d DRIP-qPCR analysis at a nongenic AsiSI cut site in DIvA cells. Bar graphs in b , c and d show fold induction of DNA:RNA hybrid levels in cut samples relative to uncut. RNase H treatment was performed on cut samples to demonstrate specificity of the signal. Error bars represent s.e.m. ( n ≥ 3 independent experiments). * P
    Figure Legend Snippet: DNA:RNA hybrids form at DSBs independently of the genomic context. a Schematic representation of DNA:RNA hybrids (in red) that can be generated upon the hybridization of mRNA (top) or dilncRNAs (bottom) with resected DNA ends at the I-PpoI cut site within DAB1 gene. b DRIP-qPCR analysis at the I-PpoI cut site within a genic ( DAB1 gene) or c nongenic locus in HeLa cells transfected with the I-PpoI nuclease. d DRIP-qPCR analysis at a nongenic AsiSI cut site in DIvA cells. Bar graphs in b , c and d show fold induction of DNA:RNA hybrid levels in cut samples relative to uncut. RNase H treatment was performed on cut samples to demonstrate specificity of the signal. Error bars represent s.e.m. ( n ≥ 3 independent experiments). * P

    Techniques Used: Generated, Hybridization, Real-time Polymerase Chain Reaction, Transfection

    DNA:RNA hybrids are directly recognized by BRCA1 in vitro and in vivo. a Representative pictures of super-resolution imaging analysis of BRCA1 (cyan) and DNA:RNA hybrids (yellow) colocalization in S-phase synchronized NCS-treated U2OS cells. Scale bar: 5 μm. b Dot plot shows the normalized number of overlaps relative to random of BRCA1 and DNA:RNA hybrids signals in S-phase U2OS cells treated with DSMO or NCS. At least n = 40 events were counted from three independent experiments. Lines represent mean ± s.e.m. c Electrophoretic mobility shift assay (EMSA) of purified recombinant human BRCA1 or BRCA1-BARD1 with end-labeled (*) double-stranded DNA or DNA:RNA substrates. d Graph showing the percentage of protein-bound substrate at respective protein concentrations. Error bars represent s.e.m. ( n = 2 independent experiments). e Representative images of BRCA1 foci co-stained with cyclin A, as S/G2-phase marker, in irradiated (2 Gy) U2OS cells over-expressing GFP or GFP-RNase H1 (GFP-RH1). Scale bar: 5 μm. f Dot plot shows the number of foci in e . At least n = 80 cells were counted from at least three independent experiments. Lines represent mean ± s.e.m. g Representative images of BRCA1 foci co-stained with cyclin A, as S/G2-phase marker, in irradiated (2 Gy) U2OS cells treated with RNase H prior to fixation. Scale bar: 10 μm. h Dot plot shows the number of foci in g . At least n = 80 cells were counted from three independent experiments. Lines represent mean ± s.e.m. * P
    Figure Legend Snippet: DNA:RNA hybrids are directly recognized by BRCA1 in vitro and in vivo. a Representative pictures of super-resolution imaging analysis of BRCA1 (cyan) and DNA:RNA hybrids (yellow) colocalization in S-phase synchronized NCS-treated U2OS cells. Scale bar: 5 μm. b Dot plot shows the normalized number of overlaps relative to random of BRCA1 and DNA:RNA hybrids signals in S-phase U2OS cells treated with DSMO or NCS. At least n = 40 events were counted from three independent experiments. Lines represent mean ± s.e.m. c Electrophoretic mobility shift assay (EMSA) of purified recombinant human BRCA1 or BRCA1-BARD1 with end-labeled (*) double-stranded DNA or DNA:RNA substrates. d Graph showing the percentage of protein-bound substrate at respective protein concentrations. Error bars represent s.e.m. ( n = 2 independent experiments). e Representative images of BRCA1 foci co-stained with cyclin A, as S/G2-phase marker, in irradiated (2 Gy) U2OS cells over-expressing GFP or GFP-RNase H1 (GFP-RH1). Scale bar: 5 μm. f Dot plot shows the number of foci in e . At least n = 80 cells were counted from at least three independent experiments. Lines represent mean ± s.e.m. g Representative images of BRCA1 foci co-stained with cyclin A, as S/G2-phase marker, in irradiated (2 Gy) U2OS cells treated with RNase H prior to fixation. Scale bar: 10 μm. h Dot plot shows the number of foci in g . At least n = 80 cells were counted from three independent experiments. Lines represent mean ± s.e.m. * P

    Techniques Used: In Vitro, In Vivo, Imaging, Electrophoretic Mobility Shift Assay, Purification, Recombinant, Labeling, Staining, Marker, Irradiation, Expressing

    32) Product Images from "Enhanced RNA cleavage within bulge-loops by an artificial ribonuclease"

    Article Title: Enhanced RNA cleavage within bulge-loops by an artificial ribonuclease

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gki264

    Probing of M2-96 RNA:oligodeoxynucleotide complexes with RNase H ( A ) and with RNase A ( B ). Radioautographs of denaturing 18% PAAG. Numbers of oligonucleotides are shown on the top. Lane L, imidazole ladder; lane T1, RNA cleavage with RNase T1 under denaturing conditions; and C, control, incubation of the RNA with RNase H or RNase A in the absence of oligodeoxynucleotide. Lines on the right-hand side of each lane indicate the position of the designed bulge (red) and double-stranded regions (blue). Cleavage conditions are described in Materials and Methods.
    Figure Legend Snippet: Probing of M2-96 RNA:oligodeoxynucleotide complexes with RNase H ( A ) and with RNase A ( B ). Radioautographs of denaturing 18% PAAG. Numbers of oligonucleotides are shown on the top. Lane L, imidazole ladder; lane T1, RNA cleavage with RNase T1 under denaturing conditions; and C, control, incubation of the RNA with RNase H or RNase A in the absence of oligodeoxynucleotide. Lines on the right-hand side of each lane indicate the position of the designed bulge (red) and double-stranded regions (blue). Cleavage conditions are described in Materials and Methods.

    Techniques Used: Incubation

    33) Product Images from "Translational Silencing of Ceruloplasmin Requires the Essential Elements of mRNA Circularization: Poly(A) Tail, Poly(A)-Binding Protein, and Eukaryotic Translation Initiation Factor 4G"

    Article Title: Translational Silencing of Ceruloplasmin Requires the Essential Elements of mRNA Circularization: Poly(A) Tail, Poly(A)-Binding Protein, and Eukaryotic Translation Initiation Factor 4G

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.21.19.6440-6449.2001

    Role of poly(A) tail in translational silencing of endogenous Cp mRNA. U937 cells (5 × 10 8 ) were treated with IFN-γ (500 U/ml) for 8 or 24 h. Poly(A)-containing mRNA was isolated from total RNA (100 μg) extracted from cells treated for 8 h. The poly(A) tail was removed by incubation with oligo(dT) (18-mer), and then double-stranded regions of DNA-RNA hybrids were digested by incubation with RNase H. The reaction was terminated by addition of 10 mM EDTA followed by ethanol precipitation. (A) The Cp transcript length was determined by Northern blot hybridization using radiolabeled Cp cDNA as probe. The two major transcripts are indicated by arrow. (B) To verify the absence of a poly(A) tail, aliquots of RNase H-treated and untreated cellular mRNA were subjected to reverse transcription using Superscript and oligo(dT) followed by PCR amplification using primers encompassing the full-length Cp 3′-UTR. (C) Intact and deadenylated cellular mRNA were subjected to in vitro translation in a rabbit reticulocyte lysate with [ 35 S]methionine in the presence of cytosolic extracts (4 μg of protein) from U937 cells treated with IFN-γ for 8 or 24 h (the rightmost pair of lanes show the effect of replicate 24-h extracts on translation of deadenylated RNA). Newly synthesized, [ 35 S]Cp was immunoprecipitated (IP) with rabbit anti-human Cp IgG, resolved by SDS-PAGE, and detected by fluorography (arrow). (D) To show specificity of the translational inhibition by U937 cell extracts, aliquots of the rabbit reticulocyte lysates that were not subjected to immunoprecipitation were resolved by SDS-PAGE and fluorography.
    Figure Legend Snippet: Role of poly(A) tail in translational silencing of endogenous Cp mRNA. U937 cells (5 × 10 8 ) were treated with IFN-γ (500 U/ml) for 8 or 24 h. Poly(A)-containing mRNA was isolated from total RNA (100 μg) extracted from cells treated for 8 h. The poly(A) tail was removed by incubation with oligo(dT) (18-mer), and then double-stranded regions of DNA-RNA hybrids were digested by incubation with RNase H. The reaction was terminated by addition of 10 mM EDTA followed by ethanol precipitation. (A) The Cp transcript length was determined by Northern blot hybridization using radiolabeled Cp cDNA as probe. The two major transcripts are indicated by arrow. (B) To verify the absence of a poly(A) tail, aliquots of RNase H-treated and untreated cellular mRNA were subjected to reverse transcription using Superscript and oligo(dT) followed by PCR amplification using primers encompassing the full-length Cp 3′-UTR. (C) Intact and deadenylated cellular mRNA were subjected to in vitro translation in a rabbit reticulocyte lysate with [ 35 S]methionine in the presence of cytosolic extracts (4 μg of protein) from U937 cells treated with IFN-γ for 8 or 24 h (the rightmost pair of lanes show the effect of replicate 24-h extracts on translation of deadenylated RNA). Newly synthesized, [ 35 S]Cp was immunoprecipitated (IP) with rabbit anti-human Cp IgG, resolved by SDS-PAGE, and detected by fluorography (arrow). (D) To show specificity of the translational inhibition by U937 cell extracts, aliquots of the rabbit reticulocyte lysates that were not subjected to immunoprecipitation were resolved by SDS-PAGE and fluorography.

    Techniques Used: Isolation, Incubation, Ethanol Precipitation, Northern Blot, Hybridization, Polymerase Chain Reaction, Amplification, In Vitro, Synthesized, Immunoprecipitation, SDS Page, Inhibition

    34) Product Images from "Increasing gene discovery and coverage using RNA-seq of globin RNA reduced porcine blood samples"

    Article Title: Increasing gene discovery and coverage using RNA-seq of globin RNA reduced porcine blood samples

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-15-954

    The average proportions of HBA and HBB reads to total mapped reads in pre- and post-GR samples. Solid and pattern bars show the average (± s.d.) proportions of globin reads in pre- and post-GR samples, respectively. RNase H mediated GR protocol decreased both proportions of HBA and HBB reads to mapped reads significantly (Paired Wilcoxon signed rank test, p
    Figure Legend Snippet: The average proportions of HBA and HBB reads to total mapped reads in pre- and post-GR samples. Solid and pattern bars show the average (± s.d.) proportions of globin reads in pre- and post-GR samples, respectively. RNase H mediated GR protocol decreased both proportions of HBA and HBB reads to mapped reads significantly (Paired Wilcoxon signed rank test, p

    Techniques Used:

    35) Product Images from "Peptide-oligonucleotide conjugates exhibiting pyrimidine-X cleavage specificity efficiently silence miRNA target acting synergistically with RNase H"

    Article Title: Peptide-oligonucleotide conjugates exhibiting pyrimidine-X cleavage specificity efficiently silence miRNA target acting synergistically with RNase H

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-33331-z

    Cleavage of miR-17 by miR-17-specific conjugate 4 and RNase H. ( A) Patterns of 5′-[ 32 P]-miR-17 cleavage in complex with conjugate 4 or in complex with oligonucleotide 4 (ON 4) by RNase H. Autoradiograph of 18% polyacrylamide/8 M urea gel. Duplexes of 5′-[ 32 P]-miR-17 (10 μM) and oligonucleotide or conjugate (5 μM) were incubated at 37 °C for 24 h with RNase H (100 U/ml). Lanes Im and T1 — imidazole ladder and partial RNA digestion with RNase T1, respectively; control — RNA was incubated in the absence of oligonucleotide/conjugate and in the presence of RNase H. ( B) Kinetics of miR-17 cleavage by conjugates 4 , miR-17/ON 4 cleavage by RNase H, and miR-17 cleavage by conjugate 4 together with RNase H. ( C) Positions of miR-17 cleavage by RNase H and conjugate 4 . Cleavage at specific sites is indicated by arrows; pep — catalytic construction. ( D) Diagram showing contribution of conjugate 4 and RNase H in the total cleavage of miR-17 for 8 h. Dark grey bars and white bars show contribution of RNase H and conjugate 4 to the miR-17 cleavage, respectively.
    Figure Legend Snippet: Cleavage of miR-17 by miR-17-specific conjugate 4 and RNase H. ( A) Patterns of 5′-[ 32 P]-miR-17 cleavage in complex with conjugate 4 or in complex with oligonucleotide 4 (ON 4) by RNase H. Autoradiograph of 18% polyacrylamide/8 M urea gel. Duplexes of 5′-[ 32 P]-miR-17 (10 μM) and oligonucleotide or conjugate (5 μM) were incubated at 37 °C for 24 h with RNase H (100 U/ml). Lanes Im and T1 — imidazole ladder and partial RNA digestion with RNase T1, respectively; control — RNA was incubated in the absence of oligonucleotide/conjugate and in the presence of RNase H. ( B) Kinetics of miR-17 cleavage by conjugates 4 , miR-17/ON 4 cleavage by RNase H, and miR-17 cleavage by conjugate 4 together with RNase H. ( C) Positions of miR-17 cleavage by RNase H and conjugate 4 . Cleavage at specific sites is indicated by arrows; pep — catalytic construction. ( D) Diagram showing contribution of conjugate 4 and RNase H in the total cleavage of miR-17 for 8 h. Dark grey bars and white bars show contribution of RNase H and conjugate 4 to the miR-17 cleavage, respectively.

    Techniques Used: Autoradiography, Incubation

    36) Product Images from "RNA–DNA hybrids promote the expansion of Friedreich's ataxia (GAA)n repeats via break-induced replication"

    Article Title: RNA–DNA hybrids promote the expansion of Friedreich's ataxia (GAA)n repeats via break-induced replication

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky099

    RNase H knockout increases the expansion rate of transcribed (GAA) n repeats. ( A ) Schematic of the GAA;CD system to select for repeat expansion events in yeast. A total of 100 (GAA) n repeats were cloned into an artificially split URA3 gene such that expansion events abrogate splicing and result in resistance to 5-FOA. ARS306 : autonomously replicating sequence on Chr III. P GAL1 : Galactose inducible promoter. TRP1 : auxotrophic marker for selection of strains bearing the construct. ‘Up’ indicates the region amplified by primers used for RT-qPCR. ( B ) (GAA) 100 expansion rates in strains bearing the construct in A. Error bars represent 95% confidence intervals. Cells were grown non-selectively on glucose (low) or galactose (high) to modulate transcription. ‘**’ Indicates non-overlapping 95% confidence intervals compared to WT under the same conditions. Numbers indicate fold change in expansion rate in rnh1Δ, rnh201Δ strains compared to WT.
    Figure Legend Snippet: RNase H knockout increases the expansion rate of transcribed (GAA) n repeats. ( A ) Schematic of the GAA;CD system to select for repeat expansion events in yeast. A total of 100 (GAA) n repeats were cloned into an artificially split URA3 gene such that expansion events abrogate splicing and result in resistance to 5-FOA. ARS306 : autonomously replicating sequence on Chr III. P GAL1 : Galactose inducible promoter. TRP1 : auxotrophic marker for selection of strains bearing the construct. ‘Up’ indicates the region amplified by primers used for RT-qPCR. ( B ) (GAA) 100 expansion rates in strains bearing the construct in A. Error bars represent 95% confidence intervals. Cells were grown non-selectively on glucose (low) or galactose (high) to modulate transcription. ‘**’ Indicates non-overlapping 95% confidence intervals compared to WT under the same conditions. Numbers indicate fold change in expansion rate in rnh1Δ, rnh201Δ strains compared to WT.

    Techniques Used: Knock-Out, Clone Assay, Sequencing, Marker, Selection, Construct, Amplification, Quantitative RT-PCR

    Altering the direction of transcription-replication collisions or inverting the repetitive run alone does not change the effect of RNase H deletion on repeat expansion rate. ( A ) Schematic of our GAA;HO construct where transcription and replication are oriented head-on. (TTC) 100 serves as the lagging strand template and (GAA) 100 remains on the transcriptional coding strand. ( B ) Repeat expansion rates in strains bearing different constructs for selection of repeat expansion events. Error bars represent 95% confidence intervals. Cells were grown non-selectively on glucose (low) or galactose (high) to modulate transcription. Numbers indicate fold increase in expansion rate due to galactose induction. ( C ) Schematic of our TTC;CD construct, in which only the repeats have been flipped such that (TTC) 100 now serves as both the lagging strand template and the transcriptional coding strand. ( D ) Repeat expansion rates in WT and RNase H deficient strains bearing our different constructs for selecting for repeat expansion events. Error bars represent 95% confidence intervals. Cells were grown non-selectively on glucose (low) or galactose (high) to modulate transcription. ‘**’ Indicates non-overlapping 95% confidence intervals compared to corresponding WT strain and conditions ‘*’ Indicates non-overlapping 84% confidence intervals compared to corresponding WT strain and conditions. Numbers indicate fold change in expansion rate in rnh1Δ, rnh201Δ strains compared to WT.
    Figure Legend Snippet: Altering the direction of transcription-replication collisions or inverting the repetitive run alone does not change the effect of RNase H deletion on repeat expansion rate. ( A ) Schematic of our GAA;HO construct where transcription and replication are oriented head-on. (TTC) 100 serves as the lagging strand template and (GAA) 100 remains on the transcriptional coding strand. ( B ) Repeat expansion rates in strains bearing different constructs for selection of repeat expansion events. Error bars represent 95% confidence intervals. Cells were grown non-selectively on glucose (low) or galactose (high) to modulate transcription. Numbers indicate fold increase in expansion rate due to galactose induction. ( C ) Schematic of our TTC;CD construct, in which only the repeats have been flipped such that (TTC) 100 now serves as both the lagging strand template and the transcriptional coding strand. ( D ) Repeat expansion rates in WT and RNase H deficient strains bearing our different constructs for selecting for repeat expansion events. Error bars represent 95% confidence intervals. Cells were grown non-selectively on glucose (low) or galactose (high) to modulate transcription. ‘**’ Indicates non-overlapping 95% confidence intervals compared to corresponding WT strain and conditions ‘*’ Indicates non-overlapping 84% confidence intervals compared to corresponding WT strain and conditions. Numbers indicate fold change in expansion rate in rnh1Δ, rnh201Δ strains compared to WT.

    Techniques Used: Construct, Selection

    RNase H knockout does not affect the expansion rate of non-transcribed (GAA) n repeats. ( A ) Schematic of our system to select for expansion of non-transcribed repeats in yeast. A total of 100 (GAA) n repeats were cloned into the region between the UAS and TSS of a GAL1 promoter driving expression of the CAN1 gene. Repeat expansion events diminish promoter activity allowing for selection on media containing canvanine. ARS306 : autonomously replicating sequence on Chr III. TRP1 : auxotrophic marker for selection of strains bearing the construct. ( B ) (GAA) 100 expansion rates in strains bearing the construct in A. Error bars represent 95% confidence intervals. Cells were grown non-selectively on glucose (low) or galactose (high) to modulate transcription. Differences between WT and rnh1Δ, rnh201Δ strains were not significant.
    Figure Legend Snippet: RNase H knockout does not affect the expansion rate of non-transcribed (GAA) n repeats. ( A ) Schematic of our system to select for expansion of non-transcribed repeats in yeast. A total of 100 (GAA) n repeats were cloned into the region between the UAS and TSS of a GAL1 promoter driving expression of the CAN1 gene. Repeat expansion events diminish promoter activity allowing for selection on media containing canvanine. ARS306 : autonomously replicating sequence on Chr III. TRP1 : auxotrophic marker for selection of strains bearing the construct. ( B ) (GAA) 100 expansion rates in strains bearing the construct in A. Error bars represent 95% confidence intervals. Cells were grown non-selectively on glucose (low) or galactose (high) to modulate transcription. Differences between WT and rnh1Δ, rnh201Δ strains were not significant.

    Techniques Used: Knock-Out, Clone Assay, Expressing, Activity Assay, Selection, Sequencing, Marker, Construct

    RNase H knockout increases (GAA) n contraction rate via mechanisms that differ from expansion. ( A ) Schematic of our construct for selecting for contraction events of transcribed (GAA) 128 repeats. Our URA3 construct from Figure 1A was altered such that strains start out Ura − and become Ura + after repeat contraction. ( B ) Schematic of our construct for selecting for contraction events on the non-transcribed (GAA) 128 repeat. The negative selective CAN1 gene from the cassette in Figure 2A was replaced by the positive selection HIS3 marker. Strains with the (GAA) 128 repeat are His − . Contraction of the repeats between the UAS and TSS leads to promoter activation and a His + phenotype. ( C ) (GAA) 128 contraction rates in strains bearing the construct in A. Error bars represent 95% confidence intervals. ‘**’ Indicates non-overlapping 95% confidence intervals compared to WT. ‘∧∧’ Indicates non-overlapping 95% confidence intervals compared to WT grown on glucose. ( D ) (GAA) 128 contraction rates in strains bearing the construct in B. Error bars represent 95% confidence intervals. ‘∧∧’ Indicates non-overlapping 95% confidence intervals compared to WT grown on glucose.
    Figure Legend Snippet: RNase H knockout increases (GAA) n contraction rate via mechanisms that differ from expansion. ( A ) Schematic of our construct for selecting for contraction events of transcribed (GAA) 128 repeats. Our URA3 construct from Figure 1A was altered such that strains start out Ura − and become Ura + after repeat contraction. ( B ) Schematic of our construct for selecting for contraction events on the non-transcribed (GAA) 128 repeat. The negative selective CAN1 gene from the cassette in Figure 2A was replaced by the positive selection HIS3 marker. Strains with the (GAA) 128 repeat are His − . Contraction of the repeats between the UAS and TSS leads to promoter activation and a His + phenotype. ( C ) (GAA) 128 contraction rates in strains bearing the construct in A. Error bars represent 95% confidence intervals. ‘**’ Indicates non-overlapping 95% confidence intervals compared to WT. ‘∧∧’ Indicates non-overlapping 95% confidence intervals compared to WT grown on glucose. ( D ) (GAA) 128 contraction rates in strains bearing the construct in B. Error bars represent 95% confidence intervals. ‘∧∧’ Indicates non-overlapping 95% confidence intervals compared to WT grown on glucose.

    Techniques Used: Knock-Out, Construct, Selection, Marker, Activation Assay

    37) Product Images from "Cytosolic RNA:DNA hybrids activate the cGAS–STING axis"

    Article Title: Cytosolic RNA:DNA hybrids activate the cGAS–STING axis

    Journal: The EMBO Journal

    doi: 10.15252/embj.201488726

    Synthetic annealed poly(rA):poly(dT) hybrid induces type I interferon response Synthetic poly(rA) (lane 1) and poly(dT) (lane 2) oligos were annealed, and the samples were run on a 1% agarose gel. Arrows indicate the annealed poly(rA):poly(dT) (lane 3) RNA:DNA hybrid. Different amounts of synthetic annealed poly(rA):poly(dT) hybrid was spotted on a nitrocellulose membrane and probed with anti-hybrid S9.6 antibody. Poly(rA):poly(dT) was either undigested (1) or digested with DNase I (2), RNase A (3) or RNase H (4), and the digested samples were analyzed on a 1% agarose gel. Human peripheral blood mononuclear cells (PBMCs) were transfected with or without poly(rA), poly(dT) or poly(rA):poly(dT) hybrid or poly(dA):poly(dT) dsDNA for 6 h. RT–PCR was performed to check for the expression of IFNB . Figure shows values from two biological replicates. Data plot shows mean with SEM. Human PBMCs from two different donors were transfected with or without poly(rA), poly(dT) or poly(rA):poly(dT) for 16 h, and the amount of secreted IP-10 was measured by ELISA. Each experiment was performed in duplicate. Data plot shows mean with SEM. Differentiated human monocyte THP-1 cells were transfected with or without poly(rA):poly(dT) hybrid, poly(dA):poly(dT) dsDNA and RT–PCR was performed to check for the expression of IFNB . Each experiment was performed in duplicate. Data are representative of one of two independent experiments. Data plot shows mean with SEM. Differentiated human monocyte THP-1 cells with Gaussia luciferase knockin under IFIT1 promoter were transfected with or without poly(rA):poly(dT) hybrid, poly(dA):poly(dT) dsDNA and luciferase assay was performed. Data shown are from two biological replicates. Data plot shows mean with SEM. Differentiated human monocyte THP-1 cells with Gaussia luciferase knockin under IFIT1 promoter were transfected with different amounts of poly(rA):poly(dT) hybrid or poly(dA):poly(dT) dsDNA and luciferase assay was performed. Data shown are from two biological replicates. Data plot shows mean with SEM.
    Figure Legend Snippet: Synthetic annealed poly(rA):poly(dT) hybrid induces type I interferon response Synthetic poly(rA) (lane 1) and poly(dT) (lane 2) oligos were annealed, and the samples were run on a 1% agarose gel. Arrows indicate the annealed poly(rA):poly(dT) (lane 3) RNA:DNA hybrid. Different amounts of synthetic annealed poly(rA):poly(dT) hybrid was spotted on a nitrocellulose membrane and probed with anti-hybrid S9.6 antibody. Poly(rA):poly(dT) was either undigested (1) or digested with DNase I (2), RNase A (3) or RNase H (4), and the digested samples were analyzed on a 1% agarose gel. Human peripheral blood mononuclear cells (PBMCs) were transfected with or without poly(rA), poly(dT) or poly(rA):poly(dT) hybrid or poly(dA):poly(dT) dsDNA for 6 h. RT–PCR was performed to check for the expression of IFNB . Figure shows values from two biological replicates. Data plot shows mean with SEM. Human PBMCs from two different donors were transfected with or without poly(rA), poly(dT) or poly(rA):poly(dT) for 16 h, and the amount of secreted IP-10 was measured by ELISA. Each experiment was performed in duplicate. Data plot shows mean with SEM. Differentiated human monocyte THP-1 cells were transfected with or without poly(rA):poly(dT) hybrid, poly(dA):poly(dT) dsDNA and RT–PCR was performed to check for the expression of IFNB . Each experiment was performed in duplicate. Data are representative of one of two independent experiments. Data plot shows mean with SEM. Differentiated human monocyte THP-1 cells with Gaussia luciferase knockin under IFIT1 promoter were transfected with or without poly(rA):poly(dT) hybrid, poly(dA):poly(dT) dsDNA and luciferase assay was performed. Data shown are from two biological replicates. Data plot shows mean with SEM. Differentiated human monocyte THP-1 cells with Gaussia luciferase knockin under IFIT1 promoter were transfected with different amounts of poly(rA):poly(dT) hybrid or poly(dA):poly(dT) dsDNA and luciferase assay was performed. Data shown are from two biological replicates. Data plot shows mean with SEM.

    Techniques Used: Agarose Gel Electrophoresis, Transfection, Reverse Transcription Polymerase Chain Reaction, Expressing, Enzyme-linked Immunosorbent Assay, Luciferase, Knock-In

    38) Product Images from "Rapid decay of unstable Leishmania mRNAs bearing a conserved retroposon signature 3?-UTR motif is initiated by a site-specific endonucleolytic cleavage without prior deadenylation"

    Article Title: Rapid decay of unstable Leishmania mRNAs bearing a conserved retroposon signature 3?-UTR motif is initiated by a site-specific endonucleolytic cleavage without prior deadenylation

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkq349

    Rapid degradation of unstable SIDER2-bearing transcripts is not initiated by a shortening of the poly(A) tail. ( A ) Schematic representation of the deadenylation assay. LUC transcripts are specifically cleaved at 300 nt from the poly(A) tail using oligonucleotide-directed RNase H cleavage. The resulting 3′-products containing the poly(A) tail are visualized by northern blot using a probe complementary to the last 300 nt of transcripts 3810 and 1270 , respectively. Poly(A) tail lengths of chimeric LUC transcripts were analyzed at different time points after transcriptional arrest using ActD. In each experiment, one sample was treated with oligo(dT) and served as a control for a completely deadenylated mRNA species. Another RNA sample that was not treated with RNase H was used as negative control. Deadenylation profiles of the unstable SIDER2-containing transcripts ( B ) LUC -3′-UTR3810 and ( C ) LUC -3′-UTR1270 and of stable LUC chimeric mRNAs lacking either the complete ( D ) SIDER3810 or ( E ) signature II. Histone 4A mRNA was used as a loading control together with an ethidium bromide staining to visualize rRNA. Decay kinetics of the corresponding uncut LUC mRNAs (from identical samples) and their half-lives ( t 1/2 ) are shown below the blots to demonstrate the fate of the full-length LUC transcripts (uncut) in comparison to the cleaved 3′-ends including the poly(A) tails. LUC mRNA levels were normalized to those of the α-tubulin mRNA. The numbers indicated below the blots represent the relative LUC transcript abundance with respect to time point 0 (before addition of the transcription inhibitor Act D). Deadenylation assays shown here are representative of three independent experiments that yielded similar results.
    Figure Legend Snippet: Rapid degradation of unstable SIDER2-bearing transcripts is not initiated by a shortening of the poly(A) tail. ( A ) Schematic representation of the deadenylation assay. LUC transcripts are specifically cleaved at 300 nt from the poly(A) tail using oligonucleotide-directed RNase H cleavage. The resulting 3′-products containing the poly(A) tail are visualized by northern blot using a probe complementary to the last 300 nt of transcripts 3810 and 1270 , respectively. Poly(A) tail lengths of chimeric LUC transcripts were analyzed at different time points after transcriptional arrest using ActD. In each experiment, one sample was treated with oligo(dT) and served as a control for a completely deadenylated mRNA species. Another RNA sample that was not treated with RNase H was used as negative control. Deadenylation profiles of the unstable SIDER2-containing transcripts ( B ) LUC -3′-UTR3810 and ( C ) LUC -3′-UTR1270 and of stable LUC chimeric mRNAs lacking either the complete ( D ) SIDER3810 or ( E ) signature II. Histone 4A mRNA was used as a loading control together with an ethidium bromide staining to visualize rRNA. Decay kinetics of the corresponding uncut LUC mRNAs (from identical samples) and their half-lives ( t 1/2 ) are shown below the blots to demonstrate the fate of the full-length LUC transcripts (uncut) in comparison to the cleaved 3′-ends including the poly(A) tails. LUC mRNA levels were normalized to those of the α-tubulin mRNA. The numbers indicated below the blots represent the relative LUC transcript abundance with respect to time point 0 (before addition of the transcription inhibitor Act D). Deadenylation assays shown here are representative of three independent experiments that yielded similar results.

    Techniques Used: Northern Blot, Negative Control, Staining, Activated Clotting Time Assay

    39) Product Images from "Lariat capping as a tool to manipulate the 5′ end of individual yeast mRNA species in vivo"

    Article Title: Lariat capping as a tool to manipulate the 5′ end of individual yeast mRNA species in vivo

    Journal: RNA

    doi: 10.1261/rna.059337.116

    Transcripts are lariat-capped cotranscriptionally and processed to have oligo(A) tails. ( A ) Outline of the RNase H assay applied to measure poly(A) tail lengths of GFP mRNAs. Relative positions of the oligo used to cleave the GFP mRNA, the d(T)NN oligo used to trim the poly(A) tail, and the Northern hybridization probe oligo are indicated. ( B ) RNase H/Northern assay of whole cell RNA extracted from wt cells expressing m 7 G GFP-, LC GFP-, and LCmutGFP-mRNA as indicated. The mobility shift between samples treated without (−) and with (+) oligo d(T)NN indicates the length of the poly(A) tail. ( C ) RNase H/Northern assay of whole cell RNA from cells expressing m 7 G GFP- or LC GFP-mRNA in the ccr4 Δ and ccr4 Δ pan2 Δ mutant cell backgrounds as indicated. The gel was run at identical conditions and to the same length as in B as judged by the xylene cyanol and bromophenol blue dye markers. ( D ) Box-plot showing lengths of poly(A) tails from individual clones of PCR products derived from 3′ RACE experiments of RNA from B and C . ( E ) Map of amplicons used for nascent RNA analysis by qRT-PCR. Three primer sets were used to generate amplicon a located upstream of the IPS (the LCrz processing site), amplicon b spanning the IPS, and amplicon c targeting the GFP part of the transcript downstream from the IPS. ( F ) qRT-PCR of nascent RNA. All amplicon signals were normalized to that of amplicon c and plotted as the ratio between LC- and LCmut-RNA (uncleaved). The error bars indicate the standard error of the mean (SEM), n = 3.
    Figure Legend Snippet: Transcripts are lariat-capped cotranscriptionally and processed to have oligo(A) tails. ( A ) Outline of the RNase H assay applied to measure poly(A) tail lengths of GFP mRNAs. Relative positions of the oligo used to cleave the GFP mRNA, the d(T)NN oligo used to trim the poly(A) tail, and the Northern hybridization probe oligo are indicated. ( B ) RNase H/Northern assay of whole cell RNA extracted from wt cells expressing m 7 G GFP-, LC GFP-, and LCmutGFP-mRNA as indicated. The mobility shift between samples treated without (−) and with (+) oligo d(T)NN indicates the length of the poly(A) tail. ( C ) RNase H/Northern assay of whole cell RNA from cells expressing m 7 G GFP- or LC GFP-mRNA in the ccr4 Δ and ccr4 Δ pan2 Δ mutant cell backgrounds as indicated. The gel was run at identical conditions and to the same length as in B as judged by the xylene cyanol and bromophenol blue dye markers. ( D ) Box-plot showing lengths of poly(A) tails from individual clones of PCR products derived from 3′ RACE experiments of RNA from B and C . ( E ) Map of amplicons used for nascent RNA analysis by qRT-PCR. Three primer sets were used to generate amplicon a located upstream of the IPS (the LCrz processing site), amplicon b spanning the IPS, and amplicon c targeting the GFP part of the transcript downstream from the IPS. ( F ) qRT-PCR of nascent RNA. All amplicon signals were normalized to that of amplicon c and plotted as the ratio between LC- and LCmut-RNA (uncleaved). The error bars indicate the standard error of the mean (SEM), n = 3.

    Techniques Used: Rnase H Assay, Northern Blot, Hybridization, Expressing, Mobility Shift, Mutagenesis, Clone Assay, Polymerase Chain Reaction, Derivative Assay, Quantitative RT-PCR, Amplification

    40) Product Images from "Yeast poly(A)-binding protein, Pab1, and PAN, a poly(A) nuclease complex recruited by Pab1, connect mRNA biogenesis to export"

    Article Title: Yeast poly(A)-binding protein, Pab1, and PAN, a poly(A) nuclease complex recruited by Pab1, connect mRNA biogenesis to export

    Journal: Genes & Development

    doi: 10.1101/gad.1267005

    Hyperpolyadenylation of SSA4 mRNA in PAB1 null bypass strains. ( A ) Comparison of SSA4 mRNA poly(A) tail lengths in spb8 Δ mutant strains. SSA4 mRNA RNase H assays were performed on 5 μg of total RNA from each strain either without (–) (lanes 1,3,5,7 ), or with (+) (lanes 2,4,6,8 ) 400 ng oligo (dT 50 ). (Lanes 1,2 ) FY86 (WT). (Lanes 3,4 ) yAS2324 ( spb8 Δ). (Lanes 5,6 ) yAS2315 ( spb8 Δ pab1 Δ). (Lanes 7,8 ) yEDy201 ( spb8 Δ pab1 Δ rrp6 Δ). Lanes 1–4 represent a shorter exposure time as compared with lanes 5–8 . ( B ) Comparison of SSA4 mRNA poly(A) tail lengths in pab1 Δ bypass suppressor strains. SSA4 RNase H assays were performed on 5 μg of total RNA extracted from each strain after shifting to 42°C for 15 min. Strains used were yAS2315 ( spb8 Δ pab1 Δ, lane 3 ), EDy200 ( rpl39 Δ pab1 Δ, lane 5 ), EDy196 ( pat1 Δ pab1 Δ, lane 6 ), CMHy251 ( rrp6 Δ pab1 Δ, lane 7 ), FY86 (WT, lanes 3,8 ), and FY86 (WT) incubated with 400 ng oligo (dT 50 ) prior to RNase digestion (lanes 2,9 ).
    Figure Legend Snippet: Hyperpolyadenylation of SSA4 mRNA in PAB1 null bypass strains. ( A ) Comparison of SSA4 mRNA poly(A) tail lengths in spb8 Δ mutant strains. SSA4 mRNA RNase H assays were performed on 5 μg of total RNA from each strain either without (–) (lanes 1,3,5,7 ), or with (+) (lanes 2,4,6,8 ) 400 ng oligo (dT 50 ). (Lanes 1,2 ) FY86 (WT). (Lanes 3,4 ) yAS2324 ( spb8 Δ). (Lanes 5,6 ) yAS2315 ( spb8 Δ pab1 Δ). (Lanes 7,8 ) yEDy201 ( spb8 Δ pab1 Δ rrp6 Δ). Lanes 1–4 represent a shorter exposure time as compared with lanes 5–8 . ( B ) Comparison of SSA4 mRNA poly(A) tail lengths in pab1 Δ bypass suppressor strains. SSA4 RNase H assays were performed on 5 μg of total RNA extracted from each strain after shifting to 42°C for 15 min. Strains used were yAS2315 ( spb8 Δ pab1 Δ, lane 3 ), EDy200 ( rpl39 Δ pab1 Δ, lane 5 ), EDy196 ( pat1 Δ pab1 Δ, lane 6 ), CMHy251 ( rrp6 Δ pab1 Δ, lane 7 ), FY86 (WT, lanes 3,8 ), and FY86 (WT) incubated with 400 ng oligo (dT 50 ) prior to RNase digestion (lanes 2,9 ).

    Techniques Used: Mutagenesis, Incubation

    Related Articles

    Negative Control:

    Article Title: Differential usage of transcriptional start sites and polyadenylation sites in FMR1 premutation alleles †
    Article Snippet: .. As a negative control, RNAs were deadenylated by incubating with Oligo(dT)12–18 (Invitrogen) and RNase H (Fermentas) at 37°C for 90 min, purified by phenol-chloroform and subjected to the same tagging reaction. .. Graphs shown in B–D were elaborated following a gel scanning (ImageQuant300, GE Healthcare) using the ImageQuant software (GE Healthcare).

    Ethanol Precipitation:

    Article Title: Genetic and Biochemical Assays Reveal a Key Role for Replication Restart Proteins in Group II Intron Retrohoming
    Article Snippet: .. For RNase treatment, 0.4 units RNase H (Invitrogen) and 0.1 µg RNase A (Roche) were added, and the sample was incubated for 30 min at 37°C before the ethanol-precipitation step. ..

    Purification:

    Article Title: Differential usage of transcriptional start sites and polyadenylation sites in FMR1 premutation alleles †
    Article Snippet: .. As a negative control, RNAs were deadenylated by incubating with Oligo(dT)12–18 (Invitrogen) and RNase H (Fermentas) at 37°C for 90 min, purified by phenol-chloroform and subjected to the same tagging reaction. .. Graphs shown in B–D were elaborated following a gel scanning (ImageQuant300, GE Healthcare) using the ImageQuant software (GE Healthcare).

    Real-time Polymerase Chain Reaction:

    Article Title: Bone Marrow-Derived Mesenchymal Stem Cells Repaired but Did Not Prevent Gentamicin-Induced Acute Kidney Injury through Paracrine Effects in Rats
    Article Snippet: .. RNA isolation, Reverse transcription and Quantitative Real Time PCR Total RNA from the BMSC culture medium treated with RNase (40 µg/ml) was extracted using a Trizol technique (Invitrogen Life Technologies) according to the manufacturer's instructions and published protocol . .. Reverse transcription was performed using a High Capacity cDNA Reverse Transcription kit for real-time PCR (Applied Biosystems).

    Incubation:

    Article Title: Genetic and Biochemical Assays Reveal a Key Role for Replication Restart Proteins in Group II Intron Retrohoming
    Article Snippet: .. For RNase treatment, 0.4 units RNase H (Invitrogen) and 0.1 µg RNase A (Roche) were added, and the sample was incubated for 30 min at 37°C before the ethanol-precipitation step. ..

    Article Title: Unstructured 5′-tails act through ribosome standby to override inhibitory structure at ribosome binding sites
    Article Snippet: .. RNase H (5 U; Thermo Scientific) and RNase H buffer (200 mM Tris–HCl [pH 7.8], 400 mM KCl, 80 mM MgCl2 , 10 mM DTT) were added to a total volume of 20 μl, and incubated for 15 min at 37°C. ..

    Article Title: A Role for DEAD Box 1 at DNA Double-Strand Breaks ▿A Role for DEAD Box 1 at DNA Double-Strand Breaks ▿ †
    Article Snippet: .. Cells were exposed to 5 Gy of IR and incubated at 37°C for 1 h. The cells were then permeabilized (in a solution of 2% Tween 20 in PBS for 10 min at room temperature) and treated with DNase I (20 U; Roche), RNase A (0.1 mg; USB Corporation), or RNase H (5 U; USB Corporation) in 100 μl of PBS containing 5 mM MgCl2 per coverslip for 15 min at room temperature. ..

    Article Title: An optimized protocol for microarray validation by quantitative PCR using amplified amino allyl labeled RNA
    Article Snippet: .. Finally, 1 μL (2 U) of RNase H (Ambion) was added, and incubation was continued for 20 min at 37°C. .. Quantitative real-time PCR (qPCR) cDNAs obtained from RT of RNA or AA-aRNA were diluted 10-fold and 4 μL were mixed with 16 μL of SYBR® Green Master Mix (Biorad, Nazareth, Belgium) containing 300 nM of each primer (final volume 20 μL).

    other:

    Article Title: Two-dimensional intact mitochondrial DNA agarose electrophoresis reveals the structural complexity of the mammalian mitochondrial genome
    Article Snippet: DNA treatment For enzymatic treatment of DNA, samples were diluted into the appropriate buffer provided by each manufacturer and treated with the indicated units of enzyme at the following temperatures and times: topoisomerase I (Invitrogen; 20 U at 37°C for 1 h); topoisomerase II (USB Affymetrix; 40 U at 37°C for 1 h); topoisomerase IV (Inspiralis; 20 U at 37°C for 1 h); DNA gyrase (New England Biolabs; 10 U at 37°C for 1 h); BglII (Fermentas; 4 U at 37°C for 30 min); S1 nuclease (Promega; 0.9 U at 37°C for 30 min); E scherichia coli exonuclease I (New England Biolabs; 20 U at 37°C for 30 min); RNase A (Ambion; 2 µl at 24°C for 1 h, used to generate data in and Supplementary Figures S1 and S2 ); RNase H (Fermentas; 15 U at 37°C for 30 min).

    Isolation:

    Article Title: Microvesicles Derived from Mesenchymal Stem Cells Enhance Survival in a Lethal Model of Acute Kidney Injury
    Article Snippet: .. Total RNA was isolated from MVs, treated or not with RNase, using the mirVana RNA isolation kit (Ambion) according to the manufacturer’s protocol. .. RNA integrity and structure and the efficacy of RNase treatment were evaluated by Agilent 2100 bioanalyzer (Agilent Technologies Inc., Santa Clara, CA), using the eukaryotic total RNA 6000 Pico Kit (Agilent Tech.).

    Article Title: Bone Marrow-Derived Mesenchymal Stem Cells Repaired but Did Not Prevent Gentamicin-Induced Acute Kidney Injury through Paracrine Effects in Rats
    Article Snippet: .. RNA isolation, Reverse transcription and Quantitative Real Time PCR Total RNA from the BMSC culture medium treated with RNase (40 µg/ml) was extracted using a Trizol technique (Invitrogen Life Technologies) according to the manufacturer's instructions and published protocol . .. Reverse transcription was performed using a High Capacity cDNA Reverse Transcription kit for real-time PCR (Applied Biosystems).

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    Thermo Fisher rnase h
    The 5′ standby tails need to be single-stranded to promote translation and initiation complex formation. ( A ) The effect of blocking of the (CA)6 and (CA)8 standby tail by an antisense oligo (at 6.7 times molar excess), or a control oligo, was assayed by in vitro translation. The mRNAs used are indicated, and GFP was detected by Western blot. ( B ) Formation of a heteroduplex between the antisense oligo and the standby tail of (CA)8 mRNA was tested by <t>RNase</t> H cleavage (Materials and Methods). Cleavage was observed after reverse transcription using a 5′-labeled oligo annealed downstream. Cleavage near the base of the stem is observed only in the presence of the antisense oligo and RNase H (far right lane). ( C ) The toeprint experiment was conducted on several mRNA variants, with 30S and tRNA fMet , with or without antisense or control oligo (Materials and Methods), as indicated. The position of the characteristic toeprint at +15 is shown. UAGC shows a sequencing ladder for reference. The gel is representative of three technical replicates.
    Rnase H, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 554 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    84
    Thermo Fisher rnase h 5 u µl
    m 5 C mRNA methylation is enriched at transcriptionally active sites with DNA damage. a U2OS-TRE cells transfected with TA-KR/TA-Cherry/tetR-KR/tetR-Cherry plasmids were exposed to light for 30 min for KR activation and allowed to recover for 1 h before harvest (scale bar: 10 μm). Quantification of frequency of cells in 500 cells with m 5 C foci from three independent experiments, mean ± SD (upper right). Fold increase of m 5 C mean intensity = mean intensity of m 5 C at TA-KR/mean intensity of background ( n = 20, mean ± SD) (lower right). b U2OS-TRE cells were transfected with TA-KR/TA-Cherry to induce local oxidative damage or for the control condition. Cells were then stained for m 5 C with four different anti-m 5 C antibodies. Frequency of m 5 C-positive cells in 500 cells was quantified ( n = 3, mean ± SD). c U2OS-TRE cells transfected with TA-KR were digested with <t>RNaseH1,</t> RNaseA, or DNase I and stained for m 5 C quantification (scale bar: 10 μm). d The mRNA from Flp-in 293 cells treated with or without 2 mM H 2 O 2 for 40 min was used for m 5 C measurement via dot blot. Quantification of m 5 C levels (mean ± SD) from three independent experiments normalized with Ctrl and methylene blue is shown. e 32 P-labeled mRNA monophosphate nucleosides were run on 2D gels for 2D-TLC analysis. In vitro-transcribed 4B mRNA with or without m 5 C was run in parallel. Representative images from three sets of independent experiments are shown with arrows showing the directions of each solvent run. Position of each nucleotide and m 5 C are labeled (Left). f 32 P-labeled mRNA monophosphate nucleosides from U2OS cells with or without 2 mM H 2 O 2 for 40 min were run on 2D gels for 2D-TLC analysis. Representative images from three sets of independent experiments. Associated quantification of relative increase in m 5 C in peroxide-treated cells compared to control, normalized to nucleotide C (right). Statistical analysis was performed with the unpaired two tailed Student’s t -test. * p
    Rnase H 5 U µl, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    The 5′ standby tails need to be single-stranded to promote translation and initiation complex formation. ( A ) The effect of blocking of the (CA)6 and (CA)8 standby tail by an antisense oligo (at 6.7 times molar excess), or a control oligo, was assayed by in vitro translation. The mRNAs used are indicated, and GFP was detected by Western blot. ( B ) Formation of a heteroduplex between the antisense oligo and the standby tail of (CA)8 mRNA was tested by RNase H cleavage (Materials and Methods). Cleavage was observed after reverse transcription using a 5′-labeled oligo annealed downstream. Cleavage near the base of the stem is observed only in the presence of the antisense oligo and RNase H (far right lane). ( C ) The toeprint experiment was conducted on several mRNA variants, with 30S and tRNA fMet , with or without antisense or control oligo (Materials and Methods), as indicated. The position of the characteristic toeprint at +15 is shown. UAGC shows a sequencing ladder for reference. The gel is representative of three technical replicates.

    Journal: Nucleic Acids Research

    Article Title: Unstructured 5′-tails act through ribosome standby to override inhibitory structure at ribosome binding sites

    doi: 10.1093/nar/gky073

    Figure Lengend Snippet: The 5′ standby tails need to be single-stranded to promote translation and initiation complex formation. ( A ) The effect of blocking of the (CA)6 and (CA)8 standby tail by an antisense oligo (at 6.7 times molar excess), or a control oligo, was assayed by in vitro translation. The mRNAs used are indicated, and GFP was detected by Western blot. ( B ) Formation of a heteroduplex between the antisense oligo and the standby tail of (CA)8 mRNA was tested by RNase H cleavage (Materials and Methods). Cleavage was observed after reverse transcription using a 5′-labeled oligo annealed downstream. Cleavage near the base of the stem is observed only in the presence of the antisense oligo and RNase H (far right lane). ( C ) The toeprint experiment was conducted on several mRNA variants, with 30S and tRNA fMet , with or without antisense or control oligo (Materials and Methods), as indicated. The position of the characteristic toeprint at +15 is shown. UAGC shows a sequencing ladder for reference. The gel is representative of three technical replicates.

    Article Snippet: RNase H (5 U; Thermo Scientific) and RNase H buffer (200 mM Tris–HCl [pH 7.8], 400 mM KCl, 80 mM MgCl2 , 10 mM DTT) were added to a total volume of 20 μl, and incubated for 15 min at 37°C.

    Techniques: Blocking Assay, In Vitro, Western Blot, Labeling, Sequencing

    RNase H-mediated degradation of pre-formed ASO:RNA duplexes and in vitro -synthesized RNAs targeted by ASOs. (A) Schematic of the experimental setup for panels B and C. Applicable for some ASOs: Y, 8-oxo-dG residue; +, LNA sugar base. (B, C) Cleavage of pre-formed ASO:target RNA duplexes by RNase H. (B) Five femtomoles of 33 P-labeled substrate was treated with RNase H for the indicated times. The reaction products were collected, denatured by heating at 95°C for 2 min and analyzed by PAGE in native 15% gels. Arrows at right point to the substrate (S) and major cleavage product(s) (P). Results from one of three independent reproducible experiments are shown. (C) Kinetics of RNase H cleavage of different ASO:RNA duplexes. The amounts of radioactivity remaining in the uncleaved substrate were quantified using a Typhoon Trio instrument. Quantifications were performed for each gel. The obtained values were normalized to the radioactivity present in the substrate before adding RNase H (set to 100%). Each point corresponds to the average of three independent experiments. Error bars indicate the standard deviation. (D) Cleavage of FR3131 RNA by RNase H in the presence of different ASOs. The RNA and ASOs were mixed and incubated at 37°C for 10 min; then, RNase H was added to the reaction mixture. RNA samples were collected at the indicated time points and analyzed by electrophoresis in native 0.8% TAE agarose gels. The results from one of three independent reproducible experiments are shown. S: substrate; P1 and P2: cleavage products.

    Journal: PLoS ONE

    Article Title: RNA Interference-Guided Targeting of Hepatitis C Virus Replication with Antisense Locked Nucleic Acid-Based Oligonucleotides Containing 8-oxo-dG Modifications

    doi: 10.1371/journal.pone.0128686

    Figure Lengend Snippet: RNase H-mediated degradation of pre-formed ASO:RNA duplexes and in vitro -synthesized RNAs targeted by ASOs. (A) Schematic of the experimental setup for panels B and C. Applicable for some ASOs: Y, 8-oxo-dG residue; +, LNA sugar base. (B, C) Cleavage of pre-formed ASO:target RNA duplexes by RNase H. (B) Five femtomoles of 33 P-labeled substrate was treated with RNase H for the indicated times. The reaction products were collected, denatured by heating at 95°C for 2 min and analyzed by PAGE in native 15% gels. Arrows at right point to the substrate (S) and major cleavage product(s) (P). Results from one of three independent reproducible experiments are shown. (C) Kinetics of RNase H cleavage of different ASO:RNA duplexes. The amounts of radioactivity remaining in the uncleaved substrate were quantified using a Typhoon Trio instrument. Quantifications were performed for each gel. The obtained values were normalized to the radioactivity present in the substrate before adding RNase H (set to 100%). Each point corresponds to the average of three independent experiments. Error bars indicate the standard deviation. (D) Cleavage of FR3131 RNA by RNase H in the presence of different ASOs. The RNA and ASOs were mixed and incubated at 37°C for 10 min; then, RNase H was added to the reaction mixture. RNA samples were collected at the indicated time points and analyzed by electrophoresis in native 0.8% TAE agarose gels. The results from one of three independent reproducible experiments are shown. S: substrate; P1 and P2: cleavage products.

    Article Snippet: This target RNA was pre-incubated with D4676, DM4676, LD4676 or LDM4676 for 10 min at 37°C; next, RNase H was added to the reaction mixture.

    Techniques: Allele-specific Oligonucleotide, In Vitro, Synthesized, Labeling, Polyacrylamide Gel Electrophoresis, Radioactivity, Standard Deviation, Incubation, Electrophoresis

    HQ formation in transcribed plasmid containing a human mtDNA fragment starting from the light strand promoter (LSP) and containing the CSB II and CSB I. ( A ) Scheme of the plasmid and detection of G-quadruplex formation by ligand-induced photocleavage. Plasmid transcribed in 50 mM of K + or Li + solution in the presence or absence of 40% (w/v) PEG 200 with GTP or dzGTP was subjected to Zn-TTAPc-mediated photocleavage, then cut at the Mun I restriction site, and filled in at the recessive 3′ end with a dATP followed by a fluorescein-dUTP, before being resolved on a denaturing gel. The marker (M) was a single-stranded synthetic DNA equivalent to the fragment between the Mun I site and the 3′ end of the G 5 AG 7 motif. Filled and open bars indicate G-quadruplex-specific cleavage signals. ( B ) Detection of RNA in HQ by photo-crosslinking. Transcription was conducted using normal GTP or dzGTP and 4S-UTP in solution containing 50-mM K + or Li + . With or without a prior RNase H digestion, transcribed plasmid was crosslinked and precipitated. Then a 5′-FAM-labeled primer (5′-CCAGCCTGCGG­CGAGTG-3′) was annealed to the non-template DNA strand downstream of CSB II, followed by extension with DNA sequenase. Extension products were resolved on a denaturing gel. G and T ladders were obtained by primer extension on the non-template DNA strand with ddCTP and ddATP, respectively. Filled and open bars indicate crosslinking sites. ( C ) Detection of G-quadruplex formation by RNA polymerase arrest assay. A plasmid containing convergent T7 and SP6 promoters and the correspondent terminators (top scheme) was transcribed by SP6 RNA polymerase in 50-mM K + or Li + solution without (lanes 1 and 2) or with (lanes 4 and 5) a prior transcription with T7 RNA polymerase in the same solution. The T7 transcription was stopped by competitive DNA specific to the T7 polymerase before the SP6 transcription was initiated. Fluorescein-UTP was supplied with SP6 RNA polymerase. RNA transcripts were resolved on a denaturing gel and visualized by the incorporated fluorescein-UTP. The marker represents SP6 transcript terminated right before the CSB II obtained by transcription of a linear DNA amplified from the plasmid.

    Journal: Nucleic Acids Research

    Article Title: A competitive formation of DNA:RNA hybrid G-quadruplex is responsible to the mitochondrial transcription termination at the DNA replication priming site

    doi: 10.1093/nar/gku764

    Figure Lengend Snippet: HQ formation in transcribed plasmid containing a human mtDNA fragment starting from the light strand promoter (LSP) and containing the CSB II and CSB I. ( A ) Scheme of the plasmid and detection of G-quadruplex formation by ligand-induced photocleavage. Plasmid transcribed in 50 mM of K + or Li + solution in the presence or absence of 40% (w/v) PEG 200 with GTP or dzGTP was subjected to Zn-TTAPc-mediated photocleavage, then cut at the Mun I restriction site, and filled in at the recessive 3′ end with a dATP followed by a fluorescein-dUTP, before being resolved on a denaturing gel. The marker (M) was a single-stranded synthetic DNA equivalent to the fragment between the Mun I site and the 3′ end of the G 5 AG 7 motif. Filled and open bars indicate G-quadruplex-specific cleavage signals. ( B ) Detection of RNA in HQ by photo-crosslinking. Transcription was conducted using normal GTP or dzGTP and 4S-UTP in solution containing 50-mM K + or Li + . With or without a prior RNase H digestion, transcribed plasmid was crosslinked and precipitated. Then a 5′-FAM-labeled primer (5′-CCAGCCTGCGG­CGAGTG-3′) was annealed to the non-template DNA strand downstream of CSB II, followed by extension with DNA sequenase. Extension products were resolved on a denaturing gel. G and T ladders were obtained by primer extension on the non-template DNA strand with ddCTP and ddATP, respectively. Filled and open bars indicate crosslinking sites. ( C ) Detection of G-quadruplex formation by RNA polymerase arrest assay. A plasmid containing convergent T7 and SP6 promoters and the correspondent terminators (top scheme) was transcribed by SP6 RNA polymerase in 50-mM K + or Li + solution without (lanes 1 and 2) or with (lanes 4 and 5) a prior transcription with T7 RNA polymerase in the same solution. The T7 transcription was stopped by competitive DNA specific to the T7 polymerase before the SP6 transcription was initiated. Fluorescein-UTP was supplied with SP6 RNA polymerase. RNA transcripts were resolved on a denaturing gel and visualized by the incorporated fluorescein-UTP. The marker represents SP6 transcript terminated right before the CSB II obtained by transcription of a linear DNA amplified from the plasmid.

    Article Snippet: To differentiate the possible contribution of R-loop, crosslinking was performed before and after a post-transcription digestion with RNase H to cleave the R-loop ( ).

    Techniques: Plasmid Preparation, Marker, Labeling, Amplification

    Identification of G-quadruplexes in plasmid at the wild and mutated CSB II by ( A ) ligand-induced photocleavage and ( B ) photo-crosslinking. (A) Plasmids transcribed in 50-mM K +  solution were treated with RNase A (A) or A and H (AH), incubated with Zn-TTAPc, and irradiated with UV light. The plasmids were then cut with Mun I, labeled at the 3′ recessive end with an FAM dye by a fill-in reaction using fluorescein-dUTP. Marker was prepared in the same way using a synthetic dsDNA that has the same sequence as the plasmid at the correspondent region (scheme at bottom). The labeling may add one or two Ts, resulting in two bands. Cleavage fragments were resolved on a denaturing gel. (B) Plasmids were transcribed in 50-mM K +  with 4-S-UTP and the other three NTPs, treated with RNase H, followed by UV irradiation. A 5′-FAM-labeled primer was extended on the non-template DNA strand that stalled at the crosslinking sites. Extension products were resolved on a denaturing gel.

    Journal: Nucleic Acids Research

    Article Title: A competitive formation of DNA:RNA hybrid G-quadruplex is responsible to the mitochondrial transcription termination at the DNA replication priming site

    doi: 10.1093/nar/gku764

    Figure Lengend Snippet: Identification of G-quadruplexes in plasmid at the wild and mutated CSB II by ( A ) ligand-induced photocleavage and ( B ) photo-crosslinking. (A) Plasmids transcribed in 50-mM K + solution were treated with RNase A (A) or A and H (AH), incubated with Zn-TTAPc, and irradiated with UV light. The plasmids were then cut with Mun I, labeled at the 3′ recessive end with an FAM dye by a fill-in reaction using fluorescein-dUTP. Marker was prepared in the same way using a synthetic dsDNA that has the same sequence as the plasmid at the correspondent region (scheme at bottom). The labeling may add one or two Ts, resulting in two bands. Cleavage fragments were resolved on a denaturing gel. (B) Plasmids were transcribed in 50-mM K + with 4-S-UTP and the other three NTPs, treated with RNase H, followed by UV irradiation. A 5′-FAM-labeled primer was extended on the non-template DNA strand that stalled at the crosslinking sites. Extension products were resolved on a denaturing gel.

    Article Snippet: To differentiate the possible contribution of R-loop, crosslinking was performed before and after a post-transcription digestion with RNase H to cleave the R-loop ( ).

    Techniques: Plasmid Preparation, Incubation, Irradiation, Labeling, Marker, Sequencing

    Stability of HQ and DQ formed in synthetic CSB II oligonucleotides in 50-mM K + . ( A ) Melting profile of intramolecular DNA DQ and chimeric DNA:RNA HQ. Sequences used are shown on the left. Each of them carried a fluorescent donor FAM at the 5′ end and an accepter TAMRA at the 3′ end. The curves in the graph show the first derivative of FAM fluorescence over temperature as a function of temperature. ( B ) Protection of DNA by the formation of DQ or HQ. DQ or HQ formed in the single-stranded DNA or dimeric DNA:RNA partial duplex protected the DNA from being hydrolyzed from the 3′ end by Exo I exonuclease (scheme at left). Three substrates (Wild, M3G and HQ) were treated with Exo I in a single tube and those survived the hydrolysis were resolved on a denaturing gel. The RNA in the duplex region of the HQ substrate was hydrolyzed by RNase H prior to the exonuclease digestion. The DNAs were visualized by the FAM dye covalently labeled at their 5′ end, digitized, and the results are given on the right. The numbers above the bars indicate the average of the two time points. The DNA oligomer or moiety is shown in uppercase and that of RNA in lowercase in (A,B).

    Journal: Nucleic Acids Research

    Article Title: A competitive formation of DNA:RNA hybrid G-quadruplex is responsible to the mitochondrial transcription termination at the DNA replication priming site

    doi: 10.1093/nar/gku764

    Figure Lengend Snippet: Stability of HQ and DQ formed in synthetic CSB II oligonucleotides in 50-mM K + . ( A ) Melting profile of intramolecular DNA DQ and chimeric DNA:RNA HQ. Sequences used are shown on the left. Each of them carried a fluorescent donor FAM at the 5′ end and an accepter TAMRA at the 3′ end. The curves in the graph show the first derivative of FAM fluorescence over temperature as a function of temperature. ( B ) Protection of DNA by the formation of DQ or HQ. DQ or HQ formed in the single-stranded DNA or dimeric DNA:RNA partial duplex protected the DNA from being hydrolyzed from the 3′ end by Exo I exonuclease (scheme at left). Three substrates (Wild, M3G and HQ) were treated with Exo I in a single tube and those survived the hydrolysis were resolved on a denaturing gel. The RNA in the duplex region of the HQ substrate was hydrolyzed by RNase H prior to the exonuclease digestion. The DNAs were visualized by the FAM dye covalently labeled at their 5′ end, digitized, and the results are given on the right. The numbers above the bars indicate the average of the two time points. The DNA oligomer or moiety is shown in uppercase and that of RNA in lowercase in (A,B).

    Article Snippet: To differentiate the possible contribution of R-loop, crosslinking was performed before and after a post-transcription digestion with RNase H to cleave the R-loop ( ).

    Techniques: Fluorescence, Labeling

    m 5 C mRNA methylation is enriched at transcriptionally active sites with DNA damage. a U2OS-TRE cells transfected with TA-KR/TA-Cherry/tetR-KR/tetR-Cherry plasmids were exposed to light for 30 min for KR activation and allowed to recover for 1 h before harvest (scale bar: 10 μm). Quantification of frequency of cells in 500 cells with m 5 C foci from three independent experiments, mean ± SD (upper right). Fold increase of m 5 C mean intensity = mean intensity of m 5 C at TA-KR/mean intensity of background ( n = 20, mean ± SD) (lower right). b U2OS-TRE cells were transfected with TA-KR/TA-Cherry to induce local oxidative damage or for the control condition. Cells were then stained for m 5 C with four different anti-m 5 C antibodies. Frequency of m 5 C-positive cells in 500 cells was quantified ( n = 3, mean ± SD). c U2OS-TRE cells transfected with TA-KR were digested with RNaseH1, RNaseA, or DNase I and stained for m 5 C quantification (scale bar: 10 μm). d The mRNA from Flp-in 293 cells treated with or without 2 mM H 2 O 2 for 40 min was used for m 5 C measurement via dot blot. Quantification of m 5 C levels (mean ± SD) from three independent experiments normalized with Ctrl and methylene blue is shown. e 32 P-labeled mRNA monophosphate nucleosides were run on 2D gels for 2D-TLC analysis. In vitro-transcribed 4B mRNA with or without m 5 C was run in parallel. Representative images from three sets of independent experiments are shown with arrows showing the directions of each solvent run. Position of each nucleotide and m 5 C are labeled (Left). f 32 P-labeled mRNA monophosphate nucleosides from U2OS cells with or without 2 mM H 2 O 2 for 40 min were run on 2D gels for 2D-TLC analysis. Representative images from three sets of independent experiments. Associated quantification of relative increase in m 5 C in peroxide-treated cells compared to control, normalized to nucleotide C (right). Statistical analysis was performed with the unpaired two tailed Student’s t -test. * p

    Journal: Nature Communications

    Article Title: m5C modification of mRNA serves a DNA damage code to promote homologous recombination

    doi: 10.1038/s41467-020-16722-7

    Figure Lengend Snippet: m 5 C mRNA methylation is enriched at transcriptionally active sites with DNA damage. a U2OS-TRE cells transfected with TA-KR/TA-Cherry/tetR-KR/tetR-Cherry plasmids were exposed to light for 30 min for KR activation and allowed to recover for 1 h before harvest (scale bar: 10 μm). Quantification of frequency of cells in 500 cells with m 5 C foci from three independent experiments, mean ± SD (upper right). Fold increase of m 5 C mean intensity = mean intensity of m 5 C at TA-KR/mean intensity of background ( n = 20, mean ± SD) (lower right). b U2OS-TRE cells were transfected with TA-KR/TA-Cherry to induce local oxidative damage or for the control condition. Cells were then stained for m 5 C with four different anti-m 5 C antibodies. Frequency of m 5 C-positive cells in 500 cells was quantified ( n = 3, mean ± SD). c U2OS-TRE cells transfected with TA-KR were digested with RNaseH1, RNaseA, or DNase I and stained for m 5 C quantification (scale bar: 10 μm). d The mRNA from Flp-in 293 cells treated with or without 2 mM H 2 O 2 for 40 min was used for m 5 C measurement via dot blot. Quantification of m 5 C levels (mean ± SD) from three independent experiments normalized with Ctrl and methylene blue is shown. e 32 P-labeled mRNA monophosphate nucleosides were run on 2D gels for 2D-TLC analysis. In vitro-transcribed 4B mRNA with or without m 5 C was run in parallel. Representative images from three sets of independent experiments are shown with arrows showing the directions of each solvent run. Position of each nucleotide and m 5 C are labeled (Left). f 32 P-labeled mRNA monophosphate nucleosides from U2OS cells with or without 2 mM H 2 O 2 for 40 min were run on 2D gels for 2D-TLC analysis. Representative images from three sets of independent experiments. Associated quantification of relative increase in m 5 C in peroxide-treated cells compared to control, normalized to nucleotide C (right). Statistical analysis was performed with the unpaired two tailed Student’s t -test. * p

    Article Snippet: For RNaseA treatment: after heat treatment, cells were incubated with 100 μg/mL RNaseA in 100 μL RNase digestion buffer (5 mM EDTA, 300 mM NaCl, 10 mM Tris-HCl, pH 7.5) at room temperature for 25 min. For RNaseH1 treatment, the cells were incubated with 15 U RNaseH1 (Cat#: EN0201, ThermoFisher Scientific) in 100 μL reaction buffer (200 mM Tris-HCl, pH 7.8, 400 mM KCl, 80 mM MgCl2 , 10 mM DTT) at room temperature for 25 min. For DNase I treatment, cells were incubated with 20 U (1 μL) DNase I in 100 μL buffer (10 mM Tris-HCl, 2.5 mM MgCl2 , 0.5 mM CaCl2 , pH 7.5) at 37 °C for 30 min followed by heat treatment.

    Techniques: Methylation, Transfection, Activation Assay, Staining, Dot Blot, Labeling, Thin Layer Chromatography, In Vitro, Two Tailed Test