thermostable rnase h  (New England Biolabs)


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    New England Biolabs thermostable rnase h
    DDR at ALT telomeres induced by FANCM deficiency is mediated by XPF. a Representative images of TRF2 and XPF co-immunostaining in U2OS cells transfected with FANCM siRNAs for 3 days. Arrows indicate the colocalization events. b Quantification of colocalization events in ( a ). The number of XPF-TRF2 colocalization per nucleus (left). The intensity of XPF foci that are colocalized with TRF2 (right). P -values by two-sided Mann-Whitney test. Bars, mean ± SEM. n , cell number (left) or number of XPF foci (right). Data of three independent experiments. c DRIP-qPCR to detect telomeric R-loops in U2OS cells after transfection with control, XPF, or FANCM siRNAs for 3 days. Relative R-loop levels were normalized to the same amount of genomic DNA treated with <t>RNase</t> <t>H</t> prior to DRIP. P -values by two tailed Student’s t -test. Bars, mean ± SD. Representative of three independent experiments. Other replicates show similar trends and are provided in the Source Data file. d Quantification of APBs in U2OS cells transfected with siRNAs. APB foci were determined by large TRF2 foci with PML staining shown in Supplementary Fig. 5d . n , cell number. Bars, mean ± SEM. P -values by two-sided Mann-Whitney test. Data of three independent experiments. e Representative images of immuno-DNA FISH to detect the co-localization of γH2AX and telomeres in U2OS cells after transfection with control, XPF, or FANCM siRNAs for 3 days. Arrows indicate the colocalization events. f Quantification of γH2AX foci at telomeres in ( e ). n , cell number. Bars, mean ± SEM. P -values by two-sided Mann-Whitney test. Data of two independent experiments. g Quantification of top 5% telomere intensity foci in ( e ). n , cell number. Bars, medians. P -values by two-sided Mann-Whitney test. Representative of three independent experiments. Other replicates show similar trends and are provided in the Source Data file. a – g Mean or median values of each group shown on the top of figures.
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    Images

    1) Product Images from "XPF activates break-induced telomere synthesis"

    Article Title: XPF activates break-induced telomere synthesis

    Journal: Nature Communications

    doi: 10.1038/s41467-022-33428-0

    DDR at ALT telomeres induced by FANCM deficiency is mediated by XPF. a Representative images of TRF2 and XPF co-immunostaining in U2OS cells transfected with FANCM siRNAs for 3 days. Arrows indicate the colocalization events. b Quantification of colocalization events in ( a ). The number of XPF-TRF2 colocalization per nucleus (left). The intensity of XPF foci that are colocalized with TRF2 (right). P -values by two-sided Mann-Whitney test. Bars, mean ± SEM. n , cell number (left) or number of XPF foci (right). Data of three independent experiments. c DRIP-qPCR to detect telomeric R-loops in U2OS cells after transfection with control, XPF, or FANCM siRNAs for 3 days. Relative R-loop levels were normalized to the same amount of genomic DNA treated with RNase H prior to DRIP. P -values by two tailed Student’s t -test. Bars, mean ± SD. Representative of three independent experiments. Other replicates show similar trends and are provided in the Source Data file. d Quantification of APBs in U2OS cells transfected with siRNAs. APB foci were determined by large TRF2 foci with PML staining shown in Supplementary Fig. 5d . n , cell number. Bars, mean ± SEM. P -values by two-sided Mann-Whitney test. Data of three independent experiments. e Representative images of immuno-DNA FISH to detect the co-localization of γH2AX and telomeres in U2OS cells after transfection with control, XPF, or FANCM siRNAs for 3 days. Arrows indicate the colocalization events. f Quantification of γH2AX foci at telomeres in ( e ). n , cell number. Bars, mean ± SEM. P -values by two-sided Mann-Whitney test. Data of two independent experiments. g Quantification of top 5% telomere intensity foci in ( e ). n , cell number. Bars, medians. P -values by two-sided Mann-Whitney test. Representative of three independent experiments. Other replicates show similar trends and are provided in the Source Data file. a – g Mean or median values of each group shown on the top of figures.
    Figure Legend Snippet: DDR at ALT telomeres induced by FANCM deficiency is mediated by XPF. a Representative images of TRF2 and XPF co-immunostaining in U2OS cells transfected with FANCM siRNAs for 3 days. Arrows indicate the colocalization events. b Quantification of colocalization events in ( a ). The number of XPF-TRF2 colocalization per nucleus (left). The intensity of XPF foci that are colocalized with TRF2 (right). P -values by two-sided Mann-Whitney test. Bars, mean ± SEM. n , cell number (left) or number of XPF foci (right). Data of three independent experiments. c DRIP-qPCR to detect telomeric R-loops in U2OS cells after transfection with control, XPF, or FANCM siRNAs for 3 days. Relative R-loop levels were normalized to the same amount of genomic DNA treated with RNase H prior to DRIP. P -values by two tailed Student’s t -test. Bars, mean ± SD. Representative of three independent experiments. Other replicates show similar trends and are provided in the Source Data file. d Quantification of APBs in U2OS cells transfected with siRNAs. APB foci were determined by large TRF2 foci with PML staining shown in Supplementary Fig. 5d . n , cell number. Bars, mean ± SEM. P -values by two-sided Mann-Whitney test. Data of three independent experiments. e Representative images of immuno-DNA FISH to detect the co-localization of γH2AX and telomeres in U2OS cells after transfection with control, XPF, or FANCM siRNAs for 3 days. Arrows indicate the colocalization events. f Quantification of γH2AX foci at telomeres in ( e ). n , cell number. Bars, mean ± SEM. P -values by two-sided Mann-Whitney test. Data of two independent experiments. g Quantification of top 5% telomere intensity foci in ( e ). n , cell number. Bars, medians. P -values by two-sided Mann-Whitney test. Representative of three independent experiments. Other replicates show similar trends and are provided in the Source Data file. a – g Mean or median values of each group shown on the top of figures.

    Techniques Used: Immunostaining, Transfection, MANN-WHITNEY, Real-time Polymerase Chain Reaction, Two Tailed Test, Staining, Fluorescence In Situ Hybridization

    2) Product Images from "RNase H-based analysis of synthetic mRNA 5′ cap incorporation"

    Article Title: RNase H-based analysis of synthetic mRNA 5′ cap incorporation

    Journal: RNA

    doi: 10.1261/rna.079173.122

    Directed RNA cleavage with RNase H. ( A ) Previously reported RNase H cleavage sites (17–19). ( B ) Synthetic RNA oligonucleotide containing the first 33 nt of an artificial FLuc transcript (syn FLuc -AC) and targeting oligos FLuc TO-25 designed based on A . Inverted triangles represent RNase H cleavage sites reported in the literature ( A ) and in this study ( B ). Deoxynucleotides in TOs are colored in blue; ribonucleotides are colored in green. The predicted loop region of the synthetic RNA oligonucleotides based on an RNA folding algorithm (RNAFold, University of Vienna) is indicated. ( C ) RNA cleavage using E. coli or Thermus thermophilus RNase H at 37°C. The cleavage efficiency of both enzymes was similar but Tth RNase H generates more uniform cuts than the E. coli enzyme.
    Figure Legend Snippet: Directed RNA cleavage with RNase H. ( A ) Previously reported RNase H cleavage sites (17–19). ( B ) Synthetic RNA oligonucleotide containing the first 33 nt of an artificial FLuc transcript (syn FLuc -AC) and targeting oligos FLuc TO-25 designed based on A . Inverted triangles represent RNase H cleavage sites reported in the literature ( A ) and in this study ( B ). Deoxynucleotides in TOs are colored in blue; ribonucleotides are colored in green. The predicted loop region of the synthetic RNA oligonucleotides based on an RNA folding algorithm (RNAFold, University of Vienna) is indicated. ( C ) RNA cleavage using E. coli or Thermus thermophilus RNase H at 37°C. The cleavage efficiency of both enzymes was similar but Tth RNase H generates more uniform cuts than the E. coli enzyme.

    Techniques Used:

    Fluorescent labeling of RNase H 5′ cleavage products and analyses. ( A ) Schematic representation of targeting, cleavage, and labeling. The targeting oligo was designed to guide RNase H to generate a 1-nt 3′ recessive end, which can be filled in by a fluorescently labeled deoxynucleotide using the Klenow fragment. In this example, a FAM-labeled dCTP was incorporated into the 3′ end of the FLuc RNase H cleavage fragment and directly analyzed by urea PAGE ( B ) or capillary electrophoresis ( C ) after enrichment. ( B ) Polyacrylamide gel analysis of cleaved RNA fragments. The 1.7 kb FLuc transcript capped using the vaccinia RNA capping enzyme (VCE) was subjected to the RNase H/Klenow fill-in treatments. Reactions were analyzed directly by urea PAGE followed by laser scanning of total RNA stain using SYBR Gold ( left panel) or fluorescent signal ( right panel). The targeting oligo TO-1 was invisible when the gel was scanned using the FAM channel and did not interfere with quantitation of the 5′ cleavage products. ( C ) Resolution and quantification of capping and capping intermediates using capillary electrophoresis. The FLuc transcript was capped using a low concentration of VCE (10 nM) and subjected to the RNase H/Klenow fill-in reactions. After enrichment, the RNA was analyzed using capillary electrophoresis. In addition to substrate 5′ triphosphate (ppp-) and the product m7Gppp-capped forms, enzymatic intermediate products 5′ diphosphate (pp-) and the unmethyl-cap (Gppp-) can be resolved and quantified. ( D ) Synthetic mRNA cap analysis using gel electrophoresis, capillary electrophoresis, and LC-MS intact mass analysis produce comparable results. After RNase H/Klenow fill-in reactions and enrichment, an uncapped or partially capped FLuc transcript was analyzed using all three available methods. Capillary electrophoresis and LC-MS yielded comparable results in quantification of substrate (ppp-), product (m7Gppp-), and intermediate products (pp- and Gppp-). Urea-PAGE does not resolve pp- from ppp- or Gppp- from m7Gppp-. Considering ppp- and pp- as uncapped and Gppp- and m7Gppp- as capped species, quantitation of fluorescently labeled RNase H cleavage products using urea-PAGE generate results comparable to CE or LC-MS, despite the lack of resolution for intermediate products.
    Figure Legend Snippet: Fluorescent labeling of RNase H 5′ cleavage products and analyses. ( A ) Schematic representation of targeting, cleavage, and labeling. The targeting oligo was designed to guide RNase H to generate a 1-nt 3′ recessive end, which can be filled in by a fluorescently labeled deoxynucleotide using the Klenow fragment. In this example, a FAM-labeled dCTP was incorporated into the 3′ end of the FLuc RNase H cleavage fragment and directly analyzed by urea PAGE ( B ) or capillary electrophoresis ( C ) after enrichment. ( B ) Polyacrylamide gel analysis of cleaved RNA fragments. The 1.7 kb FLuc transcript capped using the vaccinia RNA capping enzyme (VCE) was subjected to the RNase H/Klenow fill-in treatments. Reactions were analyzed directly by urea PAGE followed by laser scanning of total RNA stain using SYBR Gold ( left panel) or fluorescent signal ( right panel). The targeting oligo TO-1 was invisible when the gel was scanned using the FAM channel and did not interfere with quantitation of the 5′ cleavage products. ( C ) Resolution and quantification of capping and capping intermediates using capillary electrophoresis. The FLuc transcript was capped using a low concentration of VCE (10 nM) and subjected to the RNase H/Klenow fill-in reactions. After enrichment, the RNA was analyzed using capillary electrophoresis. In addition to substrate 5′ triphosphate (ppp-) and the product m7Gppp-capped forms, enzymatic intermediate products 5′ diphosphate (pp-) and the unmethyl-cap (Gppp-) can be resolved and quantified. ( D ) Synthetic mRNA cap analysis using gel electrophoresis, capillary electrophoresis, and LC-MS intact mass analysis produce comparable results. After RNase H/Klenow fill-in reactions and enrichment, an uncapped or partially capped FLuc transcript was analyzed using all three available methods. Capillary electrophoresis and LC-MS yielded comparable results in quantification of substrate (ppp-), product (m7Gppp-), and intermediate products (pp- and Gppp-). Urea-PAGE does not resolve pp- from ppp- or Gppp- from m7Gppp-. Considering ppp- and pp- as uncapped and Gppp- and m7Gppp- as capped species, quantitation of fluorescently labeled RNase H cleavage products using urea-PAGE generate results comparable to CE or LC-MS, despite the lack of resolution for intermediate products.

    Techniques Used: Labeling, Polyacrylamide Gel Electrophoresis, Electrophoresis, Staining, Quantitation Assay, Concentration Assay, Nucleic Acid Electrophoresis, Liquid Chromatography with Mass Spectroscopy

    Uniform RNase H cleavage with designed targeting oligos. ( A ) 5′ sequence of a 1.7 kb in vitro FL uc transcript containing an artificial 5′ UTR and the corresponding targeting oligo TO-1. The targeting oligo contains six deoxynucleotides (blue) at the 5′ end followed by 19 ribonucleotides (green) and a desthiobiotin (DTB) group at the 3′ end. The size of the RNase H cleavage products is shown. ( B ) Frequency of cleavage events expressed as percentage of detected cleavage product using LC-MS. Median of cleavage frequencies was 8%, 91%, and 1% at (A|A), (A|C), and (C|U), respectively.
    Figure Legend Snippet: Uniform RNase H cleavage with designed targeting oligos. ( A ) 5′ sequence of a 1.7 kb in vitro FL uc transcript containing an artificial 5′ UTR and the corresponding targeting oligo TO-1. The targeting oligo contains six deoxynucleotides (blue) at the 5′ end followed by 19 ribonucleotides (green) and a desthiobiotin (DTB) group at the 3′ end. The size of the RNase H cleavage products is shown. ( B ) Frequency of cleavage events expressed as percentage of detected cleavage product using LC-MS. Median of cleavage frequencies was 8%, 91%, and 1% at (A|A), (A|C), and (C|U), respectively.

    Techniques Used: Sequencing, In Vitro, Liquid Chromatography with Mass Spectroscopy

    A general scheme of RNase H-based RNA cap analysis. A DNA–RNA or DNA-2′- O -methyl-ribonucleotide chimera is designed to be complementary to part of the 5′ end of the target RNA molecule such that the chimera stays annealed to the cleavage fragment after RNase H cleavage. The chimera (called targeting oligo or TO in this paper) contains a 3′-desthiobiotin group. After denaturation and annealing, RNase H cleaves at a predefined site within the RNA-TO duplex and generates a one-base recessive end at the 3′ end of the cleaved RNA. Because RNase H cleavage results in a 3′ hydroxyl group (24), this recessive 3′ end can be filled in with a fluorescently labeled deoxynucleotide using the Klenow fragment of DNA polymerase I. The fluorescently labeled 5′ cleavage fragment can be analyzed by denaturing PAGE directly without enrichment. The 5′ duplex cleavage fragment can be enriched using streptavidin magnetic beads. The enriched RNase H cleavage products can be analyzed by LC-MS or capillary electrophoresis (if filled in with a fluorescent deoxynucleotide).
    Figure Legend Snippet: A general scheme of RNase H-based RNA cap analysis. A DNA–RNA or DNA-2′- O -methyl-ribonucleotide chimera is designed to be complementary to part of the 5′ end of the target RNA molecule such that the chimera stays annealed to the cleavage fragment after RNase H cleavage. The chimera (called targeting oligo or TO in this paper) contains a 3′-desthiobiotin group. After denaturation and annealing, RNase H cleaves at a predefined site within the RNA-TO duplex and generates a one-base recessive end at the 3′ end of the cleaved RNA. Because RNase H cleavage results in a 3′ hydroxyl group (24), this recessive 3′ end can be filled in with a fluorescently labeled deoxynucleotide using the Klenow fragment of DNA polymerase I. The fluorescently labeled 5′ cleavage fragment can be analyzed by denaturing PAGE directly without enrichment. The 5′ duplex cleavage fragment can be enriched using streptavidin magnetic beads. The enriched RNase H cleavage products can be analyzed by LC-MS or capillary electrophoresis (if filled in with a fluorescent deoxynucleotide).

    Techniques Used: Labeling, Polyacrylamide Gel Electrophoresis, Magnetic Beads, Liquid Chromatography with Mass Spectroscopy, Electrophoresis

    The effect of pseudouridine on RNase H cleavage. ( A ) A Cypridina luciferase transcript ( CLuc ; 1.8 kb) was cleaved using Tth RNase H in conjunction with CLuc TO-26, which directs RNase H, a cleavage site containing a uridine residue ( Supplemental Fig. 6 ). The size of the cleavage products and cleavage sites are indicated. ( B ) When unsubstituted, median cleavage frequency at the C|UC site was 73% (25 nt) and at the CU|C site was 27% (26 nt). When all uridines were substituted with pseudouridine, cleavage at the C|ΨC site decreased to a median frequency of 34%, with 66% cleavage at the CΨ|C site.
    Figure Legend Snippet: The effect of pseudouridine on RNase H cleavage. ( A ) A Cypridina luciferase transcript ( CLuc ; 1.8 kb) was cleaved using Tth RNase H in conjunction with CLuc TO-26, which directs RNase H, a cleavage site containing a uridine residue ( Supplemental Fig. 6 ). The size of the cleavage products and cleavage sites are indicated. ( B ) When unsubstituted, median cleavage frequency at the C|UC site was 73% (25 nt) and at the CU|C site was 27% (26 nt). When all uridines were substituted with pseudouridine, cleavage at the C|ΨC site decreased to a median frequency of 34%, with 66% cleavage at the CΨ|C site.

    Techniques Used: Luciferase

    Enrichment of RNase H cleavage products by size and affinity selection. The RNase H cleavage product (input) was first size-selected by two rounds of NEBNext magnetic beads. The clarified unbound fraction of the second round of size selection (ub2) was then added directly to streptavidin magnetic beads for affinity selection. After the first wash using a standard wash solution containing 1 M NaCl (W1), the resuspended bead slurry was divided into two fractions. One fraction was washed three more times using the standard wash buffer (W4_HS) and eluted using biotin (Eluate_biotin). The other fraction of the slurry was washed three more times using a low NaCl wash solution (W4_LS) followed by elution using the same volume of water (Eluate_water). Similar amounts of RNase H cleavage product and TO were eluted (also see Supplemental Fig. 4 ).
    Figure Legend Snippet: Enrichment of RNase H cleavage products by size and affinity selection. The RNase H cleavage product (input) was first size-selected by two rounds of NEBNext magnetic beads. The clarified unbound fraction of the second round of size selection (ub2) was then added directly to streptavidin magnetic beads for affinity selection. After the first wash using a standard wash solution containing 1 M NaCl (W1), the resuspended bead slurry was divided into two fractions. One fraction was washed three more times using the standard wash buffer (W4_HS) and eluted using biotin (Eluate_biotin). The other fraction of the slurry was washed three more times using a low NaCl wash solution (W4_LS) followed by elution using the same volume of water (Eluate_water). Similar amounts of RNase H cleavage product and TO were eluted (also see Supplemental Fig. 4 ).

    Techniques Used: Selection, Magnetic Beads

    Selection of targeting oligos for uniform RNase H cleavage. ( A ) In general, a surrogate RNA oligonucleotide containing the first 30–50 nt of the target transcript is chemically synthesized with a 5′ FAM group. A series of targeting oligos (TOs) are designed and chemically synthesized. The TOs used in the selection exercise did not require a desthiobiotin group, because unlike the long 3′ cleavage products of in vitro transcripts, the 3′ cleavage products of the surrogate RNA were short and did not interfere with LC-MS analysis. After RNase H cleavage, the fluorescently labeled 5′ cleavage fragments can be analyzed by urea PAGE and LC-MS intact mass analysis. ( B ) For consistency, the phosphodiester bonds are numbered around the nucleotide hybridized to the 5′ deoxynucleotide of the TO ( top panel; a cytidine in this case). Urea PAGE showed that RNase H cleavage is most efficient with FLuc TO-25, FLuc TO-26, and FLuc TO-27. Multiple cleavage products were observed with FLuc TO-24, FLuc TO-25, and FLuc TO-26 ( middle panel). LC-MS intact mass analysis of the cleavage products is shown in the lower panel. In the schematics, phosphodiester bonds are shown as “-”. Deoxynucleotides in the TOs are shown as gray bars; ribonucleotides are shown as yellow bars. Numbers represent frequency of cleavage detected at corresponding phosphodiester bonds obtained in triplicated experiments.
    Figure Legend Snippet: Selection of targeting oligos for uniform RNase H cleavage. ( A ) In general, a surrogate RNA oligonucleotide containing the first 30–50 nt of the target transcript is chemically synthesized with a 5′ FAM group. A series of targeting oligos (TOs) are designed and chemically synthesized. The TOs used in the selection exercise did not require a desthiobiotin group, because unlike the long 3′ cleavage products of in vitro transcripts, the 3′ cleavage products of the surrogate RNA were short and did not interfere with LC-MS analysis. After RNase H cleavage, the fluorescently labeled 5′ cleavage fragments can be analyzed by urea PAGE and LC-MS intact mass analysis. ( B ) For consistency, the phosphodiester bonds are numbered around the nucleotide hybridized to the 5′ deoxynucleotide of the TO ( top panel; a cytidine in this case). Urea PAGE showed that RNase H cleavage is most efficient with FLuc TO-25, FLuc TO-26, and FLuc TO-27. Multiple cleavage products were observed with FLuc TO-24, FLuc TO-25, and FLuc TO-26 ( middle panel). LC-MS intact mass analysis of the cleavage products is shown in the lower panel. In the schematics, phosphodiester bonds are shown as “-”. Deoxynucleotides in the TOs are shown as gray bars; ribonucleotides are shown as yellow bars. Numbers represent frequency of cleavage detected at corresponding phosphodiester bonds obtained in triplicated experiments.

    Techniques Used: Selection, Synthesized, In Vitro, Liquid Chromatography with Mass Spectroscopy, Labeling, Polyacrylamide Gel Electrophoresis

    Deconvoluted mass spectrums of capping analysis. An enzymatically capped Cap-1 CLuc ( A ) or FLuc transcript ( B ) was processed with RNase H and the single-step affinity enrichment and analyzed by LC-MS as described in the main text. The RNase H cleavage products with relevant 5′ groups were identified by their distinct deconvoluted mass values. The area under the identified mass peaks was used to calculate the relative percentage of each species in the sample. A mass corresponded to 24 nt + pG in the Cap-1 form was detected in the FLuc transcript. The addition of a pG was probably the result of T7 polymerase slippage at the 5′ end of the transcript, which is composed of three consecutive guanosine residues.
    Figure Legend Snippet: Deconvoluted mass spectrums of capping analysis. An enzymatically capped Cap-1 CLuc ( A ) or FLuc transcript ( B ) was processed with RNase H and the single-step affinity enrichment and analyzed by LC-MS as described in the main text. The RNase H cleavage products with relevant 5′ groups were identified by their distinct deconvoluted mass values. The area under the identified mass peaks was used to calculate the relative percentage of each species in the sample. A mass corresponded to 24 nt + pG in the Cap-1 form was detected in the FLuc transcript. The addition of a pG was probably the result of T7 polymerase slippage at the 5′ end of the transcript, which is composed of three consecutive guanosine residues.

    Techniques Used: Liquid Chromatography with Mass Spectroscopy

    3) Product Images from "RNase H-based analysis of synthetic mRNA 5′ cap incorporation"

    Article Title: RNase H-based analysis of synthetic mRNA 5′ cap incorporation

    Journal: RNA

    doi: 10.1261/rna.079173.122

    Directed RNA cleavage with RNase H. ( A ) Previously reported RNase H cleavage sites (17–19). ( B ) Synthetic RNA oligonucleotide containing the first 33 nt of an artificial FLuc transcript (syn FLuc -AC) and targeting oligos FLuc TO-25 designed based on A . Inverted triangles represent RNase H cleavage sites reported in the literature ( A ) and in this study ( B ). Deoxynucleotides in TOs are colored in blue; ribonucleotides are colored in green. The predicted loop region of the synthetic RNA oligonucleotides based on an RNA folding algorithm (RNAFold, University of Vienna) is indicated. ( C ) RNA cleavage using E. coli or Thermus thermophilus RNase H at 37°C. The cleavage efficiency of both enzymes was similar but Tth RNase H generates more uniform cuts than the E. coli enzyme.
    Figure Legend Snippet: Directed RNA cleavage with RNase H. ( A ) Previously reported RNase H cleavage sites (17–19). ( B ) Synthetic RNA oligonucleotide containing the first 33 nt of an artificial FLuc transcript (syn FLuc -AC) and targeting oligos FLuc TO-25 designed based on A . Inverted triangles represent RNase H cleavage sites reported in the literature ( A ) and in this study ( B ). Deoxynucleotides in TOs are colored in blue; ribonucleotides are colored in green. The predicted loop region of the synthetic RNA oligonucleotides based on an RNA folding algorithm (RNAFold, University of Vienna) is indicated. ( C ) RNA cleavage using E. coli or Thermus thermophilus RNase H at 37°C. The cleavage efficiency of both enzymes was similar but Tth RNase H generates more uniform cuts than the E. coli enzyme.

    Techniques Used:

    Fluorescent labeling of RNase H 5′ cleavage products and analyses. ( A ) Schematic representation of targeting, cleavage, and labeling. The targeting oligo was designed to guide RNase H to generate a 1-nt 3′ recessive end, which can be filled in by a fluorescently labeled deoxynucleotide using the Klenow fragment. In this example, a FAM-labeled dCTP was incorporated into the 3′ end of the FLuc RNase H cleavage fragment and directly analyzed by urea PAGE ( B ) or capillary electrophoresis ( C ) after enrichment. ( B ) Polyacrylamide gel analysis of cleaved RNA fragments. The 1.7 kb FLuc transcript capped using the vaccinia RNA capping enzyme (VCE) was subjected to the RNase H/Klenow fill-in treatments. Reactions were analyzed directly by urea PAGE followed by laser scanning of total RNA stain using SYBR Gold ( left panel) or fluorescent signal ( right panel). The targeting oligo TO-1 was invisible when the gel was scanned using the FAM channel and did not interfere with quantitation of the 5′ cleavage products. ( C ) Resolution and quantification of capping and capping intermediates using capillary electrophoresis. The FLuc transcript was capped using a low concentration of VCE (10 nM) and subjected to the RNase H/Klenow fill-in reactions. After enrichment, the RNA was analyzed using capillary electrophoresis. In addition to substrate 5′ triphosphate (ppp-) and the product m7Gppp-capped forms, enzymatic intermediate products 5′ diphosphate (pp-) and the unmethyl-cap (Gppp-) can be resolved and quantified. ( D ) Synthetic mRNA cap analysis using gel electrophoresis, capillary electrophoresis, and LC-MS intact mass analysis produce comparable results. After RNase H/Klenow fill-in reactions and enrichment, an uncapped or partially capped FLuc transcript was analyzed using all three available methods. Capillary electrophoresis and LC-MS yielded comparable results in quantification of substrate (ppp-), product (m7Gppp-), and intermediate products (pp- and Gppp-). Urea-PAGE does not resolve pp- from ppp- or Gppp- from m7Gppp-. Considering ppp- and pp- as uncapped and Gppp- and m7Gppp- as capped species, quantitation of fluorescently labeled RNase H cleavage products using urea-PAGE generate results comparable to CE or LC-MS, despite the lack of resolution for intermediate products.
    Figure Legend Snippet: Fluorescent labeling of RNase H 5′ cleavage products and analyses. ( A ) Schematic representation of targeting, cleavage, and labeling. The targeting oligo was designed to guide RNase H to generate a 1-nt 3′ recessive end, which can be filled in by a fluorescently labeled deoxynucleotide using the Klenow fragment. In this example, a FAM-labeled dCTP was incorporated into the 3′ end of the FLuc RNase H cleavage fragment and directly analyzed by urea PAGE ( B ) or capillary electrophoresis ( C ) after enrichment. ( B ) Polyacrylamide gel analysis of cleaved RNA fragments. The 1.7 kb FLuc transcript capped using the vaccinia RNA capping enzyme (VCE) was subjected to the RNase H/Klenow fill-in treatments. Reactions were analyzed directly by urea PAGE followed by laser scanning of total RNA stain using SYBR Gold ( left panel) or fluorescent signal ( right panel). The targeting oligo TO-1 was invisible when the gel was scanned using the FAM channel and did not interfere with quantitation of the 5′ cleavage products. ( C ) Resolution and quantification of capping and capping intermediates using capillary electrophoresis. The FLuc transcript was capped using a low concentration of VCE (10 nM) and subjected to the RNase H/Klenow fill-in reactions. After enrichment, the RNA was analyzed using capillary electrophoresis. In addition to substrate 5′ triphosphate (ppp-) and the product m7Gppp-capped forms, enzymatic intermediate products 5′ diphosphate (pp-) and the unmethyl-cap (Gppp-) can be resolved and quantified. ( D ) Synthetic mRNA cap analysis using gel electrophoresis, capillary electrophoresis, and LC-MS intact mass analysis produce comparable results. After RNase H/Klenow fill-in reactions and enrichment, an uncapped or partially capped FLuc transcript was analyzed using all three available methods. Capillary electrophoresis and LC-MS yielded comparable results in quantification of substrate (ppp-), product (m7Gppp-), and intermediate products (pp- and Gppp-). Urea-PAGE does not resolve pp- from ppp- or Gppp- from m7Gppp-. Considering ppp- and pp- as uncapped and Gppp- and m7Gppp- as capped species, quantitation of fluorescently labeled RNase H cleavage products using urea-PAGE generate results comparable to CE or LC-MS, despite the lack of resolution for intermediate products.

    Techniques Used: Labeling, Polyacrylamide Gel Electrophoresis, Electrophoresis, Staining, Quantitation Assay, Concentration Assay, Nucleic Acid Electrophoresis, Liquid Chromatography with Mass Spectroscopy

    Uniform RNase H cleavage with designed targeting oligos. ( A ) 5′ sequence of a 1.7 kb in vitro FL uc transcript containing an artificial 5′ UTR and the corresponding targeting oligo TO-1. The targeting oligo contains six deoxynucleotides (blue) at the 5′ end followed by 19 ribonucleotides (green) and a desthiobiotin (DTB) group at the 3′ end. The size of the RNase H cleavage products is shown. ( B ) Frequency of cleavage events expressed as percentage of detected cleavage product using LC-MS. Median of cleavage frequencies was 8%, 91%, and 1% at (A|A), (A|C), and (C|U), respectively.
    Figure Legend Snippet: Uniform RNase H cleavage with designed targeting oligos. ( A ) 5′ sequence of a 1.7 kb in vitro FL uc transcript containing an artificial 5′ UTR and the corresponding targeting oligo TO-1. The targeting oligo contains six deoxynucleotides (blue) at the 5′ end followed by 19 ribonucleotides (green) and a desthiobiotin (DTB) group at the 3′ end. The size of the RNase H cleavage products is shown. ( B ) Frequency of cleavage events expressed as percentage of detected cleavage product using LC-MS. Median of cleavage frequencies was 8%, 91%, and 1% at (A|A), (A|C), and (C|U), respectively.

    Techniques Used: Sequencing, In Vitro, Liquid Chromatography with Mass Spectroscopy

    A general scheme of RNase H-based RNA cap analysis. A DNA–RNA or DNA-2′- O -methyl-ribonucleotide chimera is designed to be complementary to part of the 5′ end of the target RNA molecule such that the chimera stays annealed to the cleavage fragment after RNase H cleavage. The chimera (called targeting oligo or TO in this paper) contains a 3′-desthiobiotin group. After denaturation and annealing, RNase H cleaves at a predefined site within the RNA-TO duplex and generates a one-base recessive end at the 3′ end of the cleaved RNA. Because RNase H cleavage results in a 3′ hydroxyl group (24), this recessive 3′ end can be filled in with a fluorescently labeled deoxynucleotide using the Klenow fragment of DNA polymerase I. The fluorescently labeled 5′ cleavage fragment can be analyzed by denaturing PAGE directly without enrichment. The 5′ duplex cleavage fragment can be enriched using streptavidin magnetic beads. The enriched RNase H cleavage products can be analyzed by LC-MS or capillary electrophoresis (if filled in with a fluorescent deoxynucleotide).
    Figure Legend Snippet: A general scheme of RNase H-based RNA cap analysis. A DNA–RNA or DNA-2′- O -methyl-ribonucleotide chimera is designed to be complementary to part of the 5′ end of the target RNA molecule such that the chimera stays annealed to the cleavage fragment after RNase H cleavage. The chimera (called targeting oligo or TO in this paper) contains a 3′-desthiobiotin group. After denaturation and annealing, RNase H cleaves at a predefined site within the RNA-TO duplex and generates a one-base recessive end at the 3′ end of the cleaved RNA. Because RNase H cleavage results in a 3′ hydroxyl group (24), this recessive 3′ end can be filled in with a fluorescently labeled deoxynucleotide using the Klenow fragment of DNA polymerase I. The fluorescently labeled 5′ cleavage fragment can be analyzed by denaturing PAGE directly without enrichment. The 5′ duplex cleavage fragment can be enriched using streptavidin magnetic beads. The enriched RNase H cleavage products can be analyzed by LC-MS or capillary electrophoresis (if filled in with a fluorescent deoxynucleotide).

    Techniques Used: Labeling, Polyacrylamide Gel Electrophoresis, Magnetic Beads, Liquid Chromatography with Mass Spectroscopy, Electrophoresis

    The effect of pseudouridine on RNase H cleavage. ( A ) A Cypridina luciferase transcript ( CLuc ; 1.8 kb) was cleaved using Tth RNase H in conjunction with CLuc TO-26, which directs RNase H, a cleavage site containing a uridine residue ( Supplemental Fig. 6 ). The size of the cleavage products and cleavage sites are indicated. ( B ) When unsubstituted, median cleavage frequency at the C|UC site was 73% (25 nt) and at the CU|C site was 27% (26 nt). When all uridines were substituted with pseudouridine, cleavage at the C|ΨC site decreased to a median frequency of 34%, with 66% cleavage at the CΨ|C site.
    Figure Legend Snippet: The effect of pseudouridine on RNase H cleavage. ( A ) A Cypridina luciferase transcript ( CLuc ; 1.8 kb) was cleaved using Tth RNase H in conjunction with CLuc TO-26, which directs RNase H, a cleavage site containing a uridine residue ( Supplemental Fig. 6 ). The size of the cleavage products and cleavage sites are indicated. ( B ) When unsubstituted, median cleavage frequency at the C|UC site was 73% (25 nt) and at the CU|C site was 27% (26 nt). When all uridines were substituted with pseudouridine, cleavage at the C|ΨC site decreased to a median frequency of 34%, with 66% cleavage at the CΨ|C site.

    Techniques Used: Luciferase

    Enrichment of RNase H cleavage products by size and affinity selection. The RNase H cleavage product (input) was first size-selected by two rounds of NEBNext magnetic beads. The clarified unbound fraction of the second round of size selection (ub2) was then added directly to streptavidin magnetic beads for affinity selection. After the first wash using a standard wash solution containing 1 M NaCl (W1), the resuspended bead slurry was divided into two fractions. One fraction was washed three more times using the standard wash buffer (W4_HS) and eluted using biotin (Eluate_biotin). The other fraction of the slurry was washed three more times using a low NaCl wash solution (W4_LS) followed by elution using the same volume of water (Eluate_water). Similar amounts of RNase H cleavage product and TO were eluted (also see Supplemental Fig. 4 ).
    Figure Legend Snippet: Enrichment of RNase H cleavage products by size and affinity selection. The RNase H cleavage product (input) was first size-selected by two rounds of NEBNext magnetic beads. The clarified unbound fraction of the second round of size selection (ub2) was then added directly to streptavidin magnetic beads for affinity selection. After the first wash using a standard wash solution containing 1 M NaCl (W1), the resuspended bead slurry was divided into two fractions. One fraction was washed three more times using the standard wash buffer (W4_HS) and eluted using biotin (Eluate_biotin). The other fraction of the slurry was washed three more times using a low NaCl wash solution (W4_LS) followed by elution using the same volume of water (Eluate_water). Similar amounts of RNase H cleavage product and TO were eluted (also see Supplemental Fig. 4 ).

    Techniques Used: Selection, Magnetic Beads

    Selection of targeting oligos for uniform RNase H cleavage. ( A ) In general, a surrogate RNA oligonucleotide containing the first 30–50 nt of the target transcript is chemically synthesized with a 5′ FAM group. A series of targeting oligos (TOs) are designed and chemically synthesized. The TOs used in the selection exercise did not require a desthiobiotin group, because unlike the long 3′ cleavage products of in vitro transcripts, the 3′ cleavage products of the surrogate RNA were short and did not interfere with LC-MS analysis. After RNase H cleavage, the fluorescently labeled 5′ cleavage fragments can be analyzed by urea PAGE and LC-MS intact mass analysis. ( B ) For consistency, the phosphodiester bonds are numbered around the nucleotide hybridized to the 5′ deoxynucleotide of the TO ( top panel; a cytidine in this case). Urea PAGE showed that RNase H cleavage is most efficient with FLuc TO-25, FLuc TO-26, and FLuc TO-27. Multiple cleavage products were observed with FLuc TO-24, FLuc TO-25, and FLuc TO-26 ( middle panel). LC-MS intact mass analysis of the cleavage products is shown in the lower panel. In the schematics, phosphodiester bonds are shown as “-”. Deoxynucleotides in the TOs are shown as gray bars; ribonucleotides are shown as yellow bars. Numbers represent frequency of cleavage detected at corresponding phosphodiester bonds obtained in triplicated experiments.
    Figure Legend Snippet: Selection of targeting oligos for uniform RNase H cleavage. ( A ) In general, a surrogate RNA oligonucleotide containing the first 30–50 nt of the target transcript is chemically synthesized with a 5′ FAM group. A series of targeting oligos (TOs) are designed and chemically synthesized. The TOs used in the selection exercise did not require a desthiobiotin group, because unlike the long 3′ cleavage products of in vitro transcripts, the 3′ cleavage products of the surrogate RNA were short and did not interfere with LC-MS analysis. After RNase H cleavage, the fluorescently labeled 5′ cleavage fragments can be analyzed by urea PAGE and LC-MS intact mass analysis. ( B ) For consistency, the phosphodiester bonds are numbered around the nucleotide hybridized to the 5′ deoxynucleotide of the TO ( top panel; a cytidine in this case). Urea PAGE showed that RNase H cleavage is most efficient with FLuc TO-25, FLuc TO-26, and FLuc TO-27. Multiple cleavage products were observed with FLuc TO-24, FLuc TO-25, and FLuc TO-26 ( middle panel). LC-MS intact mass analysis of the cleavage products is shown in the lower panel. In the schematics, phosphodiester bonds are shown as “-”. Deoxynucleotides in the TOs are shown as gray bars; ribonucleotides are shown as yellow bars. Numbers represent frequency of cleavage detected at corresponding phosphodiester bonds obtained in triplicated experiments.

    Techniques Used: Selection, Synthesized, In Vitro, Liquid Chromatography with Mass Spectroscopy, Labeling, Polyacrylamide Gel Electrophoresis

    Deconvoluted mass spectrums of capping analysis. An enzymatically capped Cap-1 CLuc ( A ) or FLuc transcript ( B ) was processed with RNase H and the single-step affinity enrichment and analyzed by LC-MS as described in the main text. The RNase H cleavage products with relevant 5′ groups were identified by their distinct deconvoluted mass values. The area under the identified mass peaks was used to calculate the relative percentage of each species in the sample. A mass corresponded to 24 nt + pG in the Cap-1 form was detected in the FLuc transcript. The addition of a pG was probably the result of T7 polymerase slippage at the 5′ end of the transcript, which is composed of three consecutive guanosine residues.
    Figure Legend Snippet: Deconvoluted mass spectrums of capping analysis. An enzymatically capped Cap-1 CLuc ( A ) or FLuc transcript ( B ) was processed with RNase H and the single-step affinity enrichment and analyzed by LC-MS as described in the main text. The RNase H cleavage products with relevant 5′ groups were identified by their distinct deconvoluted mass values. The area under the identified mass peaks was used to calculate the relative percentage of each species in the sample. A mass corresponded to 24 nt + pG in the Cap-1 form was detected in the FLuc transcript. The addition of a pG was probably the result of T7 polymerase slippage at the 5′ end of the transcript, which is composed of three consecutive guanosine residues.

    Techniques Used: Liquid Chromatography with Mass Spectroscopy

    4) Product Images from "RNase H-based analysis of synthetic mRNA 5’ cap incorporation"

    Article Title: RNase H-based analysis of synthetic mRNA 5’ cap incorporation

    Journal: bioRxiv

    doi: 10.1101/2022.02.02.478748

    The effect of pseudouridine on RNase H cleavage. A Cypridina luciferase transcript ( CLuc ; 1.8 kb) was cleaved using Tth RNase H in conjunction with clucTO-26, which was designed to include a uridine residue in the RNase H cleavage site. The expected size of the cleavage products and cleavage sites are indicated. When unsubstituted, median cleavage frequency at the C|UC site was 73% (25 nt) and at the CU|C site was 27% (26 nt). When all uridines were substituted with pseudouridine, cleavage at the C|ΨC site decreased to a median frequency of 34%, with 66% cleavage at the CΨ|C site.
    Figure Legend Snippet: The effect of pseudouridine on RNase H cleavage. A Cypridina luciferase transcript ( CLuc ; 1.8 kb) was cleaved using Tth RNase H in conjunction with clucTO-26, which was designed to include a uridine residue in the RNase H cleavage site. The expected size of the cleavage products and cleavage sites are indicated. When unsubstituted, median cleavage frequency at the C|UC site was 73% (25 nt) and at the CU|C site was 27% (26 nt). When all uridines were substituted with pseudouridine, cleavage at the C|ΨC site decreased to a median frequency of 34%, with 66% cleavage at the CΨ|C site.

    Techniques Used: Luciferase

    A general scheme of RNase H-based RNA cap analysis. A DNA-RNA or DNA-2’- O - methyl-ribonucleotide chimera is designed to be complementary to part of the 5’ end of the target RNA molecules, such that the chimera stays annealed to the cleavage fragment after RNase H cleavage. The chimera (called Targeting oligo or TO in this communication) contains a 3’-desthiobiotin group. After denaturation and annealing, RNase H cleaves at a pre-defined site within the RNA-TO duplex and generate a 1-base recessive end at the 3’ end of the cleaved RNA. Because RNase H cleavage results in a 3’ hydroxyl group ( 24 ), this recessive 3’ end can be filled in with a fluorescently-labeled deoxynucleotide using the Klenow fragment of DNA polymerase I. The fluorescently-labeled 5’ cleavage fragment can be analyzed by denaturing PAGE directly without enrichment. The 5’ duplex cleavage fragment can be enriched using streptavidin magnetic beads. The enriched RNase H cleavage products can be analyzed by LC/MS or capillary electrophoresis (if filled in with a fluorescent deoxynucleotide).
    Figure Legend Snippet: A general scheme of RNase H-based RNA cap analysis. A DNA-RNA or DNA-2’- O - methyl-ribonucleotide chimera is designed to be complementary to part of the 5’ end of the target RNA molecules, such that the chimera stays annealed to the cleavage fragment after RNase H cleavage. The chimera (called Targeting oligo or TO in this communication) contains a 3’-desthiobiotin group. After denaturation and annealing, RNase H cleaves at a pre-defined site within the RNA-TO duplex and generate a 1-base recessive end at the 3’ end of the cleaved RNA. Because RNase H cleavage results in a 3’ hydroxyl group ( 24 ), this recessive 3’ end can be filled in with a fluorescently-labeled deoxynucleotide using the Klenow fragment of DNA polymerase I. The fluorescently-labeled 5’ cleavage fragment can be analyzed by denaturing PAGE directly without enrichment. The 5’ duplex cleavage fragment can be enriched using streptavidin magnetic beads. The enriched RNase H cleavage products can be analyzed by LC/MS or capillary electrophoresis (if filled in with a fluorescent deoxynucleotide).

    Techniques Used: Labeling, Polyacrylamide Gel Electrophoresis, Magnetic Beads, Liquid Chromatography with Mass Spectroscopy, Electrophoresis

    Directed RNA cleavage with RNase H (A) Previously reported RNase H cleavage sites (17-19). (B) Synthetic RNA oligonucleotides with artificial sequences and corresponding Targeting Oligos (TOs) designed based on (A). RNase H cleavage sites are indicated by inverted triangles. The variable nucleotides of the RNA substrates are colored in red. Deoxynucleotides in TOs are colored in blue; Ribonucleotides are colored in green. The predicted loop region of the synthetic RNA oligonucleotides based on an RNA folding algorithm (RNAFold, University of Vienna) is indicated. (C) RNA cleavage using E. coli or Thermus thermophilus RNase H at 37°C. Both RNase H enzymes prefer A|C and A|U over A|G or A|A; the Tth enzyme is more efficient in cleavage in general.
    Figure Legend Snippet: Directed RNA cleavage with RNase H (A) Previously reported RNase H cleavage sites (17-19). (B) Synthetic RNA oligonucleotides with artificial sequences and corresponding Targeting Oligos (TOs) designed based on (A). RNase H cleavage sites are indicated by inverted triangles. The variable nucleotides of the RNA substrates are colored in red. Deoxynucleotides in TOs are colored in blue; Ribonucleotides are colored in green. The predicted loop region of the synthetic RNA oligonucleotides based on an RNA folding algorithm (RNAFold, University of Vienna) is indicated. (C) RNA cleavage using E. coli or Thermus thermophilus RNase H at 37°C. Both RNase H enzymes prefer A|C and A|U over A|G or A|A; the Tth enzyme is more efficient in cleavage in general.

    Techniques Used:

    Fluorescent labeling of RNase H 5’ cleavage products and analyses. (A) Schematic representation of targeting, cleavage, and labeling. The targeting oligo was designed to guide RNase H to generate a 1-nt 3’ recessive end, which can be filled in by a fluorescently labeled deoxynucleotide using the Klenow fragment. In this example, a FAM-labeled dCTP, was incorporated to the 3’ end of the FLuc RNase H cleavage fragment, which can be directly analyzed by denaturing PAGE (B) or capillary electrophoresis (C) after enrichment. (B) Polyacrylamide gel analysis of cleaved RNA fragments. The 1.7 kb FLuc transcript capped using Vaccinia RNA capping enzyme (VCE) was subjected to the RNase H/Klenow fill-in treatments. Reactions were analyzed directly by urea PAGE followed by laser scanning of total RNA stain using SYBR Gold (left panel) or fluorescent signal (right panel). The targeting oligo TO-1 is invisible when the gel was scanned using the FAM channel and did not interfere with quantitation of the 5’ cleavage products. (C) Resolution and quantification of capping and capping intermediates using capillary electrophoresis. The FLuc transcript was capped using a low concentration of VCE (10 nM) and subjected to the RNase H/Klenow fill-in reactions. After enrichment, the RNA was analyzed using capillary electrophoresis. In addition to substrate 5’ triphosphate (ppp-) and the product m7Gppp-capped forms, enzymatic intermediate products 5’ diphosphate (pp-) and the unmethyl-cap (Gppp-) can be resolved and quantified. (D) Synthetic mRNA cap analysis using gel electrophoresis, capillary electrophoresis, and LC- MS intact mass analysis produce comparable results. After RNase H/Klenow fill-in reactions and enrichment, an uncapped or partially capped FLuc transcript was analyzed using all three available methods. Capillary electrophoresis and LC-MS yielded comparable results in quantification of substrate (ppp-), product (m7Gppp-) and intermediate products (pp- and Gppp-). Urea-PAGE does not resolve pp- from ppp- or Gppp- from m7Gppp-. Considering ppp- and pp- as uncapped and Gppp- and m7Gppp- as capped species, quantitation of fluorescently labeled RNase H cleavage products using urea-PAGE generate results comparable to CE or LC-MS albeit the lack of resolution for intermediate products.
    Figure Legend Snippet: Fluorescent labeling of RNase H 5’ cleavage products and analyses. (A) Schematic representation of targeting, cleavage, and labeling. The targeting oligo was designed to guide RNase H to generate a 1-nt 3’ recessive end, which can be filled in by a fluorescently labeled deoxynucleotide using the Klenow fragment. In this example, a FAM-labeled dCTP, was incorporated to the 3’ end of the FLuc RNase H cleavage fragment, which can be directly analyzed by denaturing PAGE (B) or capillary electrophoresis (C) after enrichment. (B) Polyacrylamide gel analysis of cleaved RNA fragments. The 1.7 kb FLuc transcript capped using Vaccinia RNA capping enzyme (VCE) was subjected to the RNase H/Klenow fill-in treatments. Reactions were analyzed directly by urea PAGE followed by laser scanning of total RNA stain using SYBR Gold (left panel) or fluorescent signal (right panel). The targeting oligo TO-1 is invisible when the gel was scanned using the FAM channel and did not interfere with quantitation of the 5’ cleavage products. (C) Resolution and quantification of capping and capping intermediates using capillary electrophoresis. The FLuc transcript was capped using a low concentration of VCE (10 nM) and subjected to the RNase H/Klenow fill-in reactions. After enrichment, the RNA was analyzed using capillary electrophoresis. In addition to substrate 5’ triphosphate (ppp-) and the product m7Gppp-capped forms, enzymatic intermediate products 5’ diphosphate (pp-) and the unmethyl-cap (Gppp-) can be resolved and quantified. (D) Synthetic mRNA cap analysis using gel electrophoresis, capillary electrophoresis, and LC- MS intact mass analysis produce comparable results. After RNase H/Klenow fill-in reactions and enrichment, an uncapped or partially capped FLuc transcript was analyzed using all three available methods. Capillary electrophoresis and LC-MS yielded comparable results in quantification of substrate (ppp-), product (m7Gppp-) and intermediate products (pp- and Gppp-). Urea-PAGE does not resolve pp- from ppp- or Gppp- from m7Gppp-. Considering ppp- and pp- as uncapped and Gppp- and m7Gppp- as capped species, quantitation of fluorescently labeled RNase H cleavage products using urea-PAGE generate results comparable to CE or LC-MS albeit the lack of resolution for intermediate products.

    Techniques Used: Labeling, Polyacrylamide Gel Electrophoresis, Electrophoresis, Staining, Quantitation Assay, Concentration Assay, Nucleic Acid Electrophoresis, Liquid Chromatography with Mass Spectroscopy

    Uniform RNase H cleavage with designed targeting oligos (A) 5’ sequence of a 1.7 kb in vitro FLuc transcript containing an artificial 5’ UTR and the corresponding targeting oligo TO-1. The targeting oligo contains six deoxynucleotides (blue) at the 5’ end followed by 19 ribonucleotides (green) and a desthiobiotin (DTB) group at the 3’ end. The size of the expected RNase H cleavage products are shown. (B) Frequency of cleavage events expressed as percentage of detected cleavage product using LC- MS. Median of cleavage frequencies was 8%, 91% and 1% at (A|A), (A|C) and (C|U), respectively.
    Figure Legend Snippet: Uniform RNase H cleavage with designed targeting oligos (A) 5’ sequence of a 1.7 kb in vitro FLuc transcript containing an artificial 5’ UTR and the corresponding targeting oligo TO-1. The targeting oligo contains six deoxynucleotides (blue) at the 5’ end followed by 19 ribonucleotides (green) and a desthiobiotin (DTB) group at the 3’ end. The size of the expected RNase H cleavage products are shown. (B) Frequency of cleavage events expressed as percentage of detected cleavage product using LC- MS. Median of cleavage frequencies was 8%, 91% and 1% at (A|A), (A|C) and (C|U), respectively.

    Techniques Used: Sequencing, In Vitro, Liquid Chromatography with Mass Spectroscopy

    Tth RNase H cleavage frequency with respect to sequence context and TO design as analyzed by LC-MS. Within the sequence context and predicted structure of the artificial sequences in this study, high cleavage frequency was achieved at A|C, A|U, C|U or U|U sites. Variable nucleotides in the RNA substrate are colored in red; phosphodiester bonds are shown as “-”. Deoxynucleotides in the TOs are shown as grey bars; Ribonucleotides are shown as green dots. Numbers represent frequency of cleavage detected at corresponding phosphodiester bonds.
    Figure Legend Snippet: Tth RNase H cleavage frequency with respect to sequence context and TO design as analyzed by LC-MS. Within the sequence context and predicted structure of the artificial sequences in this study, high cleavage frequency was achieved at A|C, A|U, C|U or U|U sites. Variable nucleotides in the RNA substrate are colored in red; phosphodiester bonds are shown as “-”. Deoxynucleotides in the TOs are shown as grey bars; Ribonucleotides are shown as green dots. Numbers represent frequency of cleavage detected at corresponding phosphodiester bonds.

    Techniques Used: Sequencing, Liquid Chromatography with Mass Spectroscopy

    Enrichment of RNase H cleavage products by size and affinity selection. The RNase H cleavage products were first selected by size by two rounds of NEBNext magnetic beads. The clarified unbound fraction of the second round of size selection (ub2) was then added directly to streptavidin magnetic beads for affinity selection. After the first wash using a standard wash solution containing 1 M NaCl (W1), the resuspended bead slurry was divided into two fractions. One fraction was washed three more time using the standard wash buffer (W4_HS) and eluted using biotin (Eluate_biotin). The other fraction of the slurry was washed three more time using a low NaCl wash solution (W4_LS) followed by elution using the same volume of water (Eluate_water). Similar amount of RNase H cleavage product and TO were eluted (also see suppl. Figure. 4).
    Figure Legend Snippet: Enrichment of RNase H cleavage products by size and affinity selection. The RNase H cleavage products were first selected by size by two rounds of NEBNext magnetic beads. The clarified unbound fraction of the second round of size selection (ub2) was then added directly to streptavidin magnetic beads for affinity selection. After the first wash using a standard wash solution containing 1 M NaCl (W1), the resuspended bead slurry was divided into two fractions. One fraction was washed three more time using the standard wash buffer (W4_HS) and eluted using biotin (Eluate_biotin). The other fraction of the slurry was washed three more time using a low NaCl wash solution (W4_LS) followed by elution using the same volume of water (Eluate_water). Similar amount of RNase H cleavage product and TO were eluted (also see suppl. Figure. 4).

    Techniques Used: Selection, Magnetic Beads

    5) Product Images from "RNase H-based analysis of synthetic mRNA 5’ cap incorporation"

    Article Title: RNase H-based analysis of synthetic mRNA 5’ cap incorporation

    Journal: bioRxiv

    doi: 10.1101/2022.02.02.478748

    The effect of pseudouridine on RNase H cleavage. A Cypridina luciferase transcript ( CLuc ; 1.8 kb) was cleaved using Tth RNase H in conjunction with clucTO-26, which was designed to include a uridine residue in the RNase H cleavage site. The expected size of the cleavage products and cleavage sites are indicated. When unsubstituted, median cleavage frequency at the C|UC site was 73% (25 nt) and at the CU|C site was 27% (26 nt). When all uridines were substituted with pseudouridine, cleavage at the C|ΨC site decreased to a median frequency of 34%, with 66% cleavage at the CΨ|C site.
    Figure Legend Snippet: The effect of pseudouridine on RNase H cleavage. A Cypridina luciferase transcript ( CLuc ; 1.8 kb) was cleaved using Tth RNase H in conjunction with clucTO-26, which was designed to include a uridine residue in the RNase H cleavage site. The expected size of the cleavage products and cleavage sites are indicated. When unsubstituted, median cleavage frequency at the C|UC site was 73% (25 nt) and at the CU|C site was 27% (26 nt). When all uridines were substituted with pseudouridine, cleavage at the C|ΨC site decreased to a median frequency of 34%, with 66% cleavage at the CΨ|C site.

    Techniques Used: Luciferase

    A general scheme of RNase H-based RNA cap analysis. A DNA-RNA or DNA-2’- O - methyl-ribonucleotide chimera is designed to be complementary to part of the 5’ end of the target RNA molecules, such that the chimera stays annealed to the cleavage fragment after RNase H cleavage. The chimera (called Targeting oligo or TO in this communication) contains a 3’-desthiobiotin group. After denaturation and annealing, RNase H cleaves at a pre-defined site within the RNA-TO duplex and generate a 1-base recessive end at the 3’ end of the cleaved RNA. Because RNase H cleavage results in a 3’ hydroxyl group ( 24 ), this recessive 3’ end can be filled in with a fluorescently-labeled deoxynucleotide using the Klenow fragment of DNA polymerase I. The fluorescently-labeled 5’ cleavage fragment can be analyzed by denaturing PAGE directly without enrichment. The 5’ duplex cleavage fragment can be enriched using streptavidin magnetic beads. The enriched RNase H cleavage products can be analyzed by LC/MS or capillary electrophoresis (if filled in with a fluorescent deoxynucleotide).
    Figure Legend Snippet: A general scheme of RNase H-based RNA cap analysis. A DNA-RNA or DNA-2’- O - methyl-ribonucleotide chimera is designed to be complementary to part of the 5’ end of the target RNA molecules, such that the chimera stays annealed to the cleavage fragment after RNase H cleavage. The chimera (called Targeting oligo or TO in this communication) contains a 3’-desthiobiotin group. After denaturation and annealing, RNase H cleaves at a pre-defined site within the RNA-TO duplex and generate a 1-base recessive end at the 3’ end of the cleaved RNA. Because RNase H cleavage results in a 3’ hydroxyl group ( 24 ), this recessive 3’ end can be filled in with a fluorescently-labeled deoxynucleotide using the Klenow fragment of DNA polymerase I. The fluorescently-labeled 5’ cleavage fragment can be analyzed by denaturing PAGE directly without enrichment. The 5’ duplex cleavage fragment can be enriched using streptavidin magnetic beads. The enriched RNase H cleavage products can be analyzed by LC/MS or capillary electrophoresis (if filled in with a fluorescent deoxynucleotide).

    Techniques Used: Labeling, Polyacrylamide Gel Electrophoresis, Magnetic Beads, Liquid Chromatography with Mass Spectroscopy, Electrophoresis

    Directed RNA cleavage with RNase H (A) Previously reported RNase H cleavage sites (17-19). (B) Synthetic RNA oligonucleotides with artificial sequences and corresponding Targeting Oligos (TOs) designed based on (A). RNase H cleavage sites are indicated by inverted triangles. The variable nucleotides of the RNA substrates are colored in red. Deoxynucleotides in TOs are colored in blue; Ribonucleotides are colored in green. The predicted loop region of the synthetic RNA oligonucleotides based on an RNA folding algorithm (RNAFold, University of Vienna) is indicated. (C) RNA cleavage using E. coli or Thermus thermophilus RNase H at 37°C. Both RNase H enzymes prefer A|C and A|U over A|G or A|A; the Tth enzyme is more efficient in cleavage in general.
    Figure Legend Snippet: Directed RNA cleavage with RNase H (A) Previously reported RNase H cleavage sites (17-19). (B) Synthetic RNA oligonucleotides with artificial sequences and corresponding Targeting Oligos (TOs) designed based on (A). RNase H cleavage sites are indicated by inverted triangles. The variable nucleotides of the RNA substrates are colored in red. Deoxynucleotides in TOs are colored in blue; Ribonucleotides are colored in green. The predicted loop region of the synthetic RNA oligonucleotides based on an RNA folding algorithm (RNAFold, University of Vienna) is indicated. (C) RNA cleavage using E. coli or Thermus thermophilus RNase H at 37°C. Both RNase H enzymes prefer A|C and A|U over A|G or A|A; the Tth enzyme is more efficient in cleavage in general.

    Techniques Used:

    Fluorescent labeling of RNase H 5’ cleavage products and analyses. (A) Schematic representation of targeting, cleavage, and labeling. The targeting oligo was designed to guide RNase H to generate a 1-nt 3’ recessive end, which can be filled in by a fluorescently labeled deoxynucleotide using the Klenow fragment. In this example, a FAM-labeled dCTP, was incorporated to the 3’ end of the FLuc RNase H cleavage fragment, which can be directly analyzed by denaturing PAGE (B) or capillary electrophoresis (C) after enrichment. (B) Polyacrylamide gel analysis of cleaved RNA fragments. The 1.7 kb FLuc transcript capped using Vaccinia RNA capping enzyme (VCE) was subjected to the RNase H/Klenow fill-in treatments. Reactions were analyzed directly by urea PAGE followed by laser scanning of total RNA stain using SYBR Gold (left panel) or fluorescent signal (right panel). The targeting oligo TO-1 is invisible when the gel was scanned using the FAM channel and did not interfere with quantitation of the 5’ cleavage products. (C) Resolution and quantification of capping and capping intermediates using capillary electrophoresis. The FLuc transcript was capped using a low concentration of VCE (10 nM) and subjected to the RNase H/Klenow fill-in reactions. After enrichment, the RNA was analyzed using capillary electrophoresis. In addition to substrate 5’ triphosphate (ppp-) and the product m7Gppp-capped forms, enzymatic intermediate products 5’ diphosphate (pp-) and the unmethyl-cap (Gppp-) can be resolved and quantified. (D) Synthetic mRNA cap analysis using gel electrophoresis, capillary electrophoresis, and LC- MS intact mass analysis produce comparable results. After RNase H/Klenow fill-in reactions and enrichment, an uncapped or partially capped FLuc transcript was analyzed using all three available methods. Capillary electrophoresis and LC-MS yielded comparable results in quantification of substrate (ppp-), product (m7Gppp-) and intermediate products (pp- and Gppp-). Urea-PAGE does not resolve pp- from ppp- or Gppp- from m7Gppp-. Considering ppp- and pp- as uncapped and Gppp- and m7Gppp- as capped species, quantitation of fluorescently labeled RNase H cleavage products using urea-PAGE generate results comparable to CE or LC-MS albeit the lack of resolution for intermediate products.
    Figure Legend Snippet: Fluorescent labeling of RNase H 5’ cleavage products and analyses. (A) Schematic representation of targeting, cleavage, and labeling. The targeting oligo was designed to guide RNase H to generate a 1-nt 3’ recessive end, which can be filled in by a fluorescently labeled deoxynucleotide using the Klenow fragment. In this example, a FAM-labeled dCTP, was incorporated to the 3’ end of the FLuc RNase H cleavage fragment, which can be directly analyzed by denaturing PAGE (B) or capillary electrophoresis (C) after enrichment. (B) Polyacrylamide gel analysis of cleaved RNA fragments. The 1.7 kb FLuc transcript capped using Vaccinia RNA capping enzyme (VCE) was subjected to the RNase H/Klenow fill-in treatments. Reactions were analyzed directly by urea PAGE followed by laser scanning of total RNA stain using SYBR Gold (left panel) or fluorescent signal (right panel). The targeting oligo TO-1 is invisible when the gel was scanned using the FAM channel and did not interfere with quantitation of the 5’ cleavage products. (C) Resolution and quantification of capping and capping intermediates using capillary electrophoresis. The FLuc transcript was capped using a low concentration of VCE (10 nM) and subjected to the RNase H/Klenow fill-in reactions. After enrichment, the RNA was analyzed using capillary electrophoresis. In addition to substrate 5’ triphosphate (ppp-) and the product m7Gppp-capped forms, enzymatic intermediate products 5’ diphosphate (pp-) and the unmethyl-cap (Gppp-) can be resolved and quantified. (D) Synthetic mRNA cap analysis using gel electrophoresis, capillary electrophoresis, and LC- MS intact mass analysis produce comparable results. After RNase H/Klenow fill-in reactions and enrichment, an uncapped or partially capped FLuc transcript was analyzed using all three available methods. Capillary electrophoresis and LC-MS yielded comparable results in quantification of substrate (ppp-), product (m7Gppp-) and intermediate products (pp- and Gppp-). Urea-PAGE does not resolve pp- from ppp- or Gppp- from m7Gppp-. Considering ppp- and pp- as uncapped and Gppp- and m7Gppp- as capped species, quantitation of fluorescently labeled RNase H cleavage products using urea-PAGE generate results comparable to CE or LC-MS albeit the lack of resolution for intermediate products.

    Techniques Used: Labeling, Polyacrylamide Gel Electrophoresis, Electrophoresis, Staining, Quantitation Assay, Concentration Assay, Nucleic Acid Electrophoresis, Liquid Chromatography with Mass Spectroscopy

    Uniform RNase H cleavage with designed targeting oligos (A) 5’ sequence of a 1.7 kb in vitro FLuc transcript containing an artificial 5’ UTR and the corresponding targeting oligo TO-1. The targeting oligo contains six deoxynucleotides (blue) at the 5’ end followed by 19 ribonucleotides (green) and a desthiobiotin (DTB) group at the 3’ end. The size of the expected RNase H cleavage products are shown. (B) Frequency of cleavage events expressed as percentage of detected cleavage product using LC- MS. Median of cleavage frequencies was 8%, 91% and 1% at (A|A), (A|C) and (C|U), respectively.
    Figure Legend Snippet: Uniform RNase H cleavage with designed targeting oligos (A) 5’ sequence of a 1.7 kb in vitro FLuc transcript containing an artificial 5’ UTR and the corresponding targeting oligo TO-1. The targeting oligo contains six deoxynucleotides (blue) at the 5’ end followed by 19 ribonucleotides (green) and a desthiobiotin (DTB) group at the 3’ end. The size of the expected RNase H cleavage products are shown. (B) Frequency of cleavage events expressed as percentage of detected cleavage product using LC- MS. Median of cleavage frequencies was 8%, 91% and 1% at (A|A), (A|C) and (C|U), respectively.

    Techniques Used: Sequencing, In Vitro, Liquid Chromatography with Mass Spectroscopy

    Tth RNase H cleavage frequency with respect to sequence context and TO design as analyzed by LC-MS. Within the sequence context and predicted structure of the artificial sequences in this study, high cleavage frequency was achieved at A|C, A|U, C|U or U|U sites. Variable nucleotides in the RNA substrate are colored in red; phosphodiester bonds are shown as “-”. Deoxynucleotides in the TOs are shown as grey bars; Ribonucleotides are shown as green dots. Numbers represent frequency of cleavage detected at corresponding phosphodiester bonds.
    Figure Legend Snippet: Tth RNase H cleavage frequency with respect to sequence context and TO design as analyzed by LC-MS. Within the sequence context and predicted structure of the artificial sequences in this study, high cleavage frequency was achieved at A|C, A|U, C|U or U|U sites. Variable nucleotides in the RNA substrate are colored in red; phosphodiester bonds are shown as “-”. Deoxynucleotides in the TOs are shown as grey bars; Ribonucleotides are shown as green dots. Numbers represent frequency of cleavage detected at corresponding phosphodiester bonds.

    Techniques Used: Sequencing, Liquid Chromatography with Mass Spectroscopy

    Enrichment of RNase H cleavage products by size and affinity selection. The RNase H cleavage products were first selected by size by two rounds of NEBNext magnetic beads. The clarified unbound fraction of the second round of size selection (ub2) was then added directly to streptavidin magnetic beads for affinity selection. After the first wash using a standard wash solution containing 1 M NaCl (W1), the resuspended bead slurry was divided into two fractions. One fraction was washed three more time using the standard wash buffer (W4_HS) and eluted using biotin (Eluate_biotin). The other fraction of the slurry was washed three more time using a low NaCl wash solution (W4_LS) followed by elution using the same volume of water (Eluate_water). Similar amount of RNase H cleavage product and TO were eluted (also see suppl. Figure. 4).
    Figure Legend Snippet: Enrichment of RNase H cleavage products by size and affinity selection. The RNase H cleavage products were first selected by size by two rounds of NEBNext magnetic beads. The clarified unbound fraction of the second round of size selection (ub2) was then added directly to streptavidin magnetic beads for affinity selection. After the first wash using a standard wash solution containing 1 M NaCl (W1), the resuspended bead slurry was divided into two fractions. One fraction was washed three more time using the standard wash buffer (W4_HS) and eluted using biotin (Eluate_biotin). The other fraction of the slurry was washed three more time using a low NaCl wash solution (W4_LS) followed by elution using the same volume of water (Eluate_water). Similar amount of RNase H cleavage product and TO were eluted (also see suppl. Figure. 4).

    Techniques Used: Selection, Magnetic Beads

    6) Product Images from "Capillary Electrophoresis-Based Functional Genomics Screening to Discover Novel Archaeal DNA Modifying Enzymes"

    Article Title: Capillary Electrophoresis-Based Functional Genomics Screening to Discover Novel Archaeal DNA Modifying Enzymes

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.02137-21

    Functional genomics fosmid DNA library and RNA transcripts mapped to the T. kodakarensis genome. A typical functional genomics 384-well plate (Plate NEB260) was sequenced using the plexWell TM 384 Library Preparation kit and Illumina sequencing. Fosmid inserts (bottom black bars; average insert size of 35,233 ± 7,641 bp) were mapped to the T. kodakarensis genome using Geneious software. RNA-seq was conducted on a pooled 384-well plate (Plate NEB260) using NEBNext Ultra TM II RNA Library Prep kit and Illumina Sequencing. RNA transcripts were mapped to the T. kodakarensis genome using HISAT2 and htseq-count using the Galaxy server. Transcript coverage is plotted (log 2 ).
    Figure Legend Snippet: Functional genomics fosmid DNA library and RNA transcripts mapped to the T. kodakarensis genome. A typical functional genomics 384-well plate (Plate NEB260) was sequenced using the plexWell TM 384 Library Preparation kit and Illumina sequencing. Fosmid inserts (bottom black bars; average insert size of 35,233 ± 7,641 bp) were mapped to the T. kodakarensis genome using Geneious software. RNA-seq was conducted on a pooled 384-well plate (Plate NEB260) using NEBNext Ultra TM II RNA Library Prep kit and Illumina Sequencing. RNA transcripts were mapped to the T. kodakarensis genome using HISAT2 and htseq-count using the Galaxy server. Transcript coverage is plotted (log 2 ).

    Techniques Used: Functional Assay, Sequencing, Software, RNA Sequencing Assay

    7) Product Images from "Low-bias ncRNA libraries using ordered two-template relay: Serial template jumping by a modified retroelement reverse transcriptase"

    Article Title: Low-bias ncRNA libraries using ordered two-template relay: Serial template jumping by a modified retroelement reverse transcriptase

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

    doi: 10.1073/pnas.2107900118

    OTTR for NGS cDNA library generation. ( A ) Optimized workflow for single-tube synthesis of cDNA libraries. A pool of RNA and/or DNA input molecules (maroon) is first labeled by BoMoC with 3′ ddRTP. Buffer conditions are then toggled from Mn 2+ to Mg 2+ and any free ddRTPs are inactivated. Next, dNTPs, oligonucleotides, and BoMoC are added to initiate cDNA synthesis from the RNA-DNA primer duplex across the IT (maroon), ending after copying the AT (green). If desired, products can then be treated with RNase A and RNase H to remove RNA, yielding the desired cDNA. The illustrated blocking groups are detailed in SI Appendix , Table S1 . ( B ) Schematic of primers involved in Illumina Full-length ( Top ) or Universal ( Bottom ) adaptor addition and their respective cDNA library products. DNA primers were the complement of P7-i7-R2 or R2, while ATs were P5-i5-R1 or R1. In the Full-length adaptor strategy, only cDNA products elongated by copying the AT can bind to the flow cell. The covalently linked blocking group is indicated by a diamond. ( C and D ) Proof of principle for OTTR library generation using an RNA oligonucleotide template with Full-length ( C ) or Universal ( D ) adaptors. All reactions contained primer duplex. Only reactions containing primer duplex, RNA template, AT, and BoMoC (lanes 5 and 6) generate properly sized cDNA library product. Universal adaptor RT reactions required PCR amplification for P5 and P7 sequence fusion and indexing ( D , Bottom ). DAP, 2-amino-2′-deoxyadenosine triphosphate.
    Figure Legend Snippet: OTTR for NGS cDNA library generation. ( A ) Optimized workflow for single-tube synthesis of cDNA libraries. A pool of RNA and/or DNA input molecules (maroon) is first labeled by BoMoC with 3′ ddRTP. Buffer conditions are then toggled from Mn 2+ to Mg 2+ and any free ddRTPs are inactivated. Next, dNTPs, oligonucleotides, and BoMoC are added to initiate cDNA synthesis from the RNA-DNA primer duplex across the IT (maroon), ending after copying the AT (green). If desired, products can then be treated with RNase A and RNase H to remove RNA, yielding the desired cDNA. The illustrated blocking groups are detailed in SI Appendix , Table S1 . ( B ) Schematic of primers involved in Illumina Full-length ( Top ) or Universal ( Bottom ) adaptor addition and their respective cDNA library products. DNA primers were the complement of P7-i7-R2 or R2, while ATs were P5-i5-R1 or R1. In the Full-length adaptor strategy, only cDNA products elongated by copying the AT can bind to the flow cell. The covalently linked blocking group is indicated by a diamond. ( C and D ) Proof of principle for OTTR library generation using an RNA oligonucleotide template with Full-length ( C ) or Universal ( D ) adaptors. All reactions contained primer duplex. Only reactions containing primer duplex, RNA template, AT, and BoMoC (lanes 5 and 6) generate properly sized cDNA library product. Universal adaptor RT reactions required PCR amplification for P5 and P7 sequence fusion and indexing ( D , Bottom ). DAP, 2-amino-2′-deoxyadenosine triphosphate.

    Techniques Used: Next-Generation Sequencing, cDNA Library Assay, Labeling, Blocking Assay, Polymerase Chain Reaction, Amplification, Sequencing

    8) Product Images from "Selective Naked-Eye Detection of SARS-CoV-2 Mediated by N Gene Targeted Antisense Oligonucleotide Capped Plasmonic Nanoparticles"

    Article Title: Selective Naked-Eye Detection of SARS-CoV-2 Mediated by N Gene Targeted Antisense Oligonucleotide Capped Plasmonic Nanoparticles

    Journal: ACS Nano

    doi: 10.1021/acsnano.0c03822

    (a) Comparison of response of the Au-ASO mix nanoparticles toward the RNA (1 ng/μL) isolated from noninfected Vero cells, Vero cells infected with MERS-CoV, and Vero cells infected with SARS-CoV-2 virus. Relative change in absorbance at 660 nm wavelength for the Au-ASO mix nanoparticle treated with SARS-CoV-2 RNA (1 ng/μL) followed by the addition of RNase H has been plotted in (b) when the mixture was incubated at different temperatures for 5 min. The schematic representation for the visual naked-eye detection of SARS-CoV-2 with the treatment of RNase H at 65 °C for 5 min is shown in (c). The error bar indicates the average results obtained from three such independent experiments performed in triplicate.
    Figure Legend Snippet: (a) Comparison of response of the Au-ASO mix nanoparticles toward the RNA (1 ng/μL) isolated from noninfected Vero cells, Vero cells infected with MERS-CoV, and Vero cells infected with SARS-CoV-2 virus. Relative change in absorbance at 660 nm wavelength for the Au-ASO mix nanoparticle treated with SARS-CoV-2 RNA (1 ng/μL) followed by the addition of RNase H has been plotted in (b) when the mixture was incubated at different temperatures for 5 min. The schematic representation for the visual naked-eye detection of SARS-CoV-2 with the treatment of RNase H at 65 °C for 5 min is shown in (c). The error bar indicates the average results obtained from three such independent experiments performed in triplicate.

    Techniques Used: Allele-specific Oligonucleotide, Isolation, Infection, Incubation

    9) Product Images from "Control of ribosomal protein synthesis by Microprocessor complex"

    Article Title: Control of ribosomal protein synthesis by Microprocessor complex

    Journal: bioRxiv

    doi: 10.1101/2020.04.24.060236

    The microprocessor binds to the transcription start sites at RP gene loci. a. ChIP-seq profiles of RNAPII, H3K4me3, Drosha and Dgcr8 at the Rps15a, Rps24, Rpl4, and Rpl28 loci in mouse embryonic stem cells (mES). b. ChIP (IP: Drosha)-qPCR analysis of different RP genes as indicated with anti-Flag (M2) antibody or nonspecific IgG (control) in Flag tagged Drosha-expressing MEF (F-Drosha) or control (pBABE-MEF). Tuba1a and Tubb1 (negative control). Fold enrichment of anti-Flag IP over IgG IP is plotted as Mean ± SEM. n=3. c. ChIP (IP: Drosha)-qPCR analysis of different RP genes as indicated with anti-Flag (M2) antibody or nonspecific IgG (negative control) in Flag-Drosha-expressing MEF or control-MEF. Tuba1a and Tubb1 (negative control). Cells were treated with 1μg/ml actinomycin D (ActD) or vehicle (DMSO), followed by ChIP-qPCR analysis. Fold enrichment of anti-Flag IP over IgG IP is plotted as Mean ± SEM. n=3. d. ChIP (IP: Drosha)-qPCR analysis of different RP genes as indicated in MEF treated with 1μg/μl RNase A or vehicle (water) with anti-Drosha antibody or nonspecific IgG (control). Tuba1a and Tubb1 (negative control). Fold enrichment of Drosha IP over IgG IP is plotted as Mean ± SEM.n=3. e. ChIP (anti-Drosha antibody)-qPCR analysis of different RP genes in MEFs treated with 100U/ml RNase H or vehicle (water) with anti-Drosha antibody. Tuba1a and Tubb1 (negative control). Fold enrichment of Drosha IP over IgG IP is plotted as Mean ± SEM.n=3. f. ChIP (IP: Drosha)-qPCR analysis of different RP genes as indicated with anti-Drosha antibody or nonspecific IgG (control) in control MEFs (Ctrl) or MEFs deleted in Dgcr8 gene (Dgcr8-KO). Tuba1a (negative control). Fold enrichment of Drosha IP over IgG IP is plotted as Mean ± SEM.n=3. g. qRT-PCR analysis of different RP mRNAs and control mRNAs (Itgb1 and IL-6) (relative to GAPDH) as indicated in Ctrl or Dgcr8-KO MEFs. Result is plotted as Mean ± SEM. n=3.
    Figure Legend Snippet: The microprocessor binds to the transcription start sites at RP gene loci. a. ChIP-seq profiles of RNAPII, H3K4me3, Drosha and Dgcr8 at the Rps15a, Rps24, Rpl4, and Rpl28 loci in mouse embryonic stem cells (mES). b. ChIP (IP: Drosha)-qPCR analysis of different RP genes as indicated with anti-Flag (M2) antibody or nonspecific IgG (control) in Flag tagged Drosha-expressing MEF (F-Drosha) or control (pBABE-MEF). Tuba1a and Tubb1 (negative control). Fold enrichment of anti-Flag IP over IgG IP is plotted as Mean ± SEM. n=3. c. ChIP (IP: Drosha)-qPCR analysis of different RP genes as indicated with anti-Flag (M2) antibody or nonspecific IgG (negative control) in Flag-Drosha-expressing MEF or control-MEF. Tuba1a and Tubb1 (negative control). Cells were treated with 1μg/ml actinomycin D (ActD) or vehicle (DMSO), followed by ChIP-qPCR analysis. Fold enrichment of anti-Flag IP over IgG IP is plotted as Mean ± SEM. n=3. d. ChIP (IP: Drosha)-qPCR analysis of different RP genes as indicated in MEF treated with 1μg/μl RNase A or vehicle (water) with anti-Drosha antibody or nonspecific IgG (control). Tuba1a and Tubb1 (negative control). Fold enrichment of Drosha IP over IgG IP is plotted as Mean ± SEM.n=3. e. ChIP (anti-Drosha antibody)-qPCR analysis of different RP genes in MEFs treated with 100U/ml RNase H or vehicle (water) with anti-Drosha antibody. Tuba1a and Tubb1 (negative control). Fold enrichment of Drosha IP over IgG IP is plotted as Mean ± SEM.n=3. f. ChIP (IP: Drosha)-qPCR analysis of different RP genes as indicated with anti-Drosha antibody or nonspecific IgG (control) in control MEFs (Ctrl) or MEFs deleted in Dgcr8 gene (Dgcr8-KO). Tuba1a (negative control). Fold enrichment of Drosha IP over IgG IP is plotted as Mean ± SEM.n=3. g. qRT-PCR analysis of different RP mRNAs and control mRNAs (Itgb1 and IL-6) (relative to GAPDH) as indicated in Ctrl or Dgcr8-KO MEFs. Result is plotted as Mean ± SEM. n=3.

    Techniques Used: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Expressing, Negative Control, Quantitative RT-PCR

    The Ddx5 helicase reduces DNA/RNA hybrid and facilitates transcription elongation. A. DNA/RNA hybrid IP-sequencing (DRIP-seq) data indicate increased R-loops at the RPG loci (Rps2, Rps3A, Rpl28, and Rpl37A) and control loci (GAPDH and Tuba1a) in Drosha KD cells (red) compared to control U-2 OS cells (blue) (top). Quantitation of the DRIP-seq data is shown (bottom). b. DRIP analysis of RPG loci (Rps2, Rps5, Rpl14) and control locus (Tuba1a) locus in the presence or absence of RNase H in HCT116 cells expressing CRISPR/Cas9 against Drosha (KO) or non-specific control (Ctrl). Four sets of primers used for DRIP analysis are indicated as black boxes in the top panel (bottom). Signal relative to input is plotted as Mean ± SEM. n=3 c. ChIP-qPCR analysis using anti-RNAPII antibody was performed in K562 cells cells expressing CRISPR/Cas9 against Drosha (KO) or non-specific control (Ctrl). Primers for RPG loci (Rps2, Rps26, Rpl14, Rpl28) and control locus (Tuba1a) are shown. Mean ± SEM. n=3. d. ChIP-qPCR analysis using anti-Ddx5 antibody of RPG loci (Rps2, Rps5, Rps10, Rps12, Rps26, Rpl4, Rpl14, Rpl19, Rpl28) and control locus(Tuba1a) in control (Ctrl) or Drosha KO HCT116 cells. Fold enrichment of Ddx5 antibody pull-down against IgG pull-down is plotted as Mean ± SEM. n=3. e. Immunoprecipitation of DNA/RNA hybrids with the S9.6 antibody, followed by immunoblot analysis of Ddx5, Drosha, Dgcr8 and Lamin A/C (negative control) in Ctrl or Drosha KO K562 cells. f. DRIP analysis of RPG loci (Rps2, Rps5, Rpl4, Rpl28) and control loci (Tuba1a and Tubb1) locus in the presence or absence of RNase H in K562 cells targeting Ddx5 gene ( Ddx5 KO) by RNAi or non-specific control (Ctrl). Result is plotted as Mean ± SEM. n=3 g. qRT-PCR analysis of various RP mRNAs and Drosha mRNAs (relative to GAPDH) in Ctrl or Drosha KO HCT116 cells transfected with empty plasmid (mock), Ddx5 wild type (WT) or the RNA helicase dead (HD) mutant expression plasmid (left). Result is plotted as Mean ± SEM. n=3 Drosha, Ddx5 and GAPDH proteins were examined by western blot in total cell lysates from HCT116 cells (right). Relative protein amount normalized to GAPDH is shown below each blot.
    Figure Legend Snippet: The Ddx5 helicase reduces DNA/RNA hybrid and facilitates transcription elongation. A. DNA/RNA hybrid IP-sequencing (DRIP-seq) data indicate increased R-loops at the RPG loci (Rps2, Rps3A, Rpl28, and Rpl37A) and control loci (GAPDH and Tuba1a) in Drosha KD cells (red) compared to control U-2 OS cells (blue) (top). Quantitation of the DRIP-seq data is shown (bottom). b. DRIP analysis of RPG loci (Rps2, Rps5, Rpl14) and control locus (Tuba1a) locus in the presence or absence of RNase H in HCT116 cells expressing CRISPR/Cas9 against Drosha (KO) or non-specific control (Ctrl). Four sets of primers used for DRIP analysis are indicated as black boxes in the top panel (bottom). Signal relative to input is plotted as Mean ± SEM. n=3 c. ChIP-qPCR analysis using anti-RNAPII antibody was performed in K562 cells cells expressing CRISPR/Cas9 against Drosha (KO) or non-specific control (Ctrl). Primers for RPG loci (Rps2, Rps26, Rpl14, Rpl28) and control locus (Tuba1a) are shown. Mean ± SEM. n=3. d. ChIP-qPCR analysis using anti-Ddx5 antibody of RPG loci (Rps2, Rps5, Rps10, Rps12, Rps26, Rpl4, Rpl14, Rpl19, Rpl28) and control locus(Tuba1a) in control (Ctrl) or Drosha KO HCT116 cells. Fold enrichment of Ddx5 antibody pull-down against IgG pull-down is plotted as Mean ± SEM. n=3. e. Immunoprecipitation of DNA/RNA hybrids with the S9.6 antibody, followed by immunoblot analysis of Ddx5, Drosha, Dgcr8 and Lamin A/C (negative control) in Ctrl or Drosha KO K562 cells. f. DRIP analysis of RPG loci (Rps2, Rps5, Rpl4, Rpl28) and control loci (Tuba1a and Tubb1) locus in the presence or absence of RNase H in K562 cells targeting Ddx5 gene ( Ddx5 KO) by RNAi or non-specific control (Ctrl). Result is plotted as Mean ± SEM. n=3 g. qRT-PCR analysis of various RP mRNAs and Drosha mRNAs (relative to GAPDH) in Ctrl or Drosha KO HCT116 cells transfected with empty plasmid (mock), Ddx5 wild type (WT) or the RNA helicase dead (HD) mutant expression plasmid (left). Result is plotted as Mean ± SEM. n=3 Drosha, Ddx5 and GAPDH proteins were examined by western blot in total cell lysates from HCT116 cells (right). Relative protein amount normalized to GAPDH is shown below each blot.

    Techniques Used: Sequencing, Quantitation Assay, Expressing, CRISPR, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Immunoprecipitation, Negative Control, Quantitative RT-PCR, Transfection, Plasmid Preparation, Mutagenesis, Western Blot

    10) Product Images from "N6-methyladenosine in poly(A) tails stabilize VSG transcripts"

    Article Title: N6-methyladenosine in poly(A) tails stabilize VSG transcripts

    Journal: bioRxiv

    doi: 10.1101/2020.01.30.925776

    m 6 A is present in the poly(A) tail of VSG mRNA and other transcripts. a . Immunoblotting with anti-m 6 A antibody. RNA samples (from left to right): total RNA (Total), Poly(A)-enriched (A+) RNA and Poly(A)-depleted (A-) RNA from two life cycle stages (BSF and PCF). The last lane contains total mouse liver RNA (Mouse). 2 µ g of total RNA, 2 µ g of poly(A)-depleted RNA and 100 ng of poly(A)-enriched RNA was loaded per lane. rRNA was detected by staining RNA with methylene blue to confirm equal loading between total and poly(A)-depleted fractions. As expected the rRNA are undetectable in the poly(A)-enriched fraction. b . Intensity of the m 6 A signal in immunoblot, measured by Image J, in the whole lane containing the poly(A)-enriched RNA of bloodstream forms. The intensity of the ∼1.8 kb band was compared with the signal intensity of the entire lane, and averaged from 5 independent samples. c . Diagram displaying the location of the oligonucleotides used in RNase H digestion of VSG mRNA. The digestion products detected in the immunoblot (panel D) after incubation with each oligonucleotide (SL, A, B, C, dT) are also indicated. d . Immunoblotting with anti-m 6 A antibody of mammalian bloodstream forms total RNA pre-incubated with indicated oligonucleotides and digested with RNase H. 2 µ g of total RNA were loaded per lane. Staining of rRNA with Methylene Blue confirmed equal loading. SL: spliced leader, dT: poly deoxi-thymidines. Tub: α-Tubulin. (see also Extended Data Fig. S3)
    Figure Legend Snippet: m 6 A is present in the poly(A) tail of VSG mRNA and other transcripts. a . Immunoblotting with anti-m 6 A antibody. RNA samples (from left to right): total RNA (Total), Poly(A)-enriched (A+) RNA and Poly(A)-depleted (A-) RNA from two life cycle stages (BSF and PCF). The last lane contains total mouse liver RNA (Mouse). 2 µ g of total RNA, 2 µ g of poly(A)-depleted RNA and 100 ng of poly(A)-enriched RNA was loaded per lane. rRNA was detected by staining RNA with methylene blue to confirm equal loading between total and poly(A)-depleted fractions. As expected the rRNA are undetectable in the poly(A)-enriched fraction. b . Intensity of the m 6 A signal in immunoblot, measured by Image J, in the whole lane containing the poly(A)-enriched RNA of bloodstream forms. The intensity of the ∼1.8 kb band was compared with the signal intensity of the entire lane, and averaged from 5 independent samples. c . Diagram displaying the location of the oligonucleotides used in RNase H digestion of VSG mRNA. The digestion products detected in the immunoblot (panel D) after incubation with each oligonucleotide (SL, A, B, C, dT) are also indicated. d . Immunoblotting with anti-m 6 A antibody of mammalian bloodstream forms total RNA pre-incubated with indicated oligonucleotides and digested with RNase H. 2 µ g of total RNA were loaded per lane. Staining of rRNA with Methylene Blue confirmed equal loading. SL: spliced leader, dT: poly deoxi-thymidines. Tub: α-Tubulin. (see also Extended Data Fig. S3)

    Techniques Used: Staining, Incubation

    Conserved VSG 16-mer motif is required for inclusion of m6A in adjacent poly(A) tail. a . Diagram of 16-mer motif VSg double-expressor (DE) cell-lines. VSG117 was inserted immediately downstream of the promoter of the active bloodstream expression site, which naturally contains VSG 2 at the telomeric end. In VSG double expresor 16-mer WT cell-line, VSG117 contains its endogenous 3’UTR with the conserved 16-mer motif (sequence shown in blue). In VSG double expresor 16-mer MUT cell-line, the 16-mer motif was scrambled (sequence shown in orange). b . Transcript levels of VSG117 and VSG2 transcripts measured by qRT-PCR in both reporter cell-lines. Levels were normalized to transcript levels in cell-lines expressing only VSG2 or only VSG117 . c . Immunoblot with anti-m 6 A antibody of mRNA from VSG double-expressor cell-lines. RNase H digestion of VSG2 mRNA was used to resolve VSG2 and VSG117 transcripts. Different quantities of the same VSG double expresor 16-mer WT cell-line was loaded in two separet lanes (50ng and 12.5ng) to show that the VSG117 band is detectable in both conditions. d . m 6 A index calculated as the ratio of m 6 A intensity and mRNA levels, measured in panels c. and b., respectively. und., undetectable. # intensities measured in lane 3 of Figure 5C (see also Extended Data Fig. S5)
    Figure Legend Snippet: Conserved VSG 16-mer motif is required for inclusion of m6A in adjacent poly(A) tail. a . Diagram of 16-mer motif VSg double-expressor (DE) cell-lines. VSG117 was inserted immediately downstream of the promoter of the active bloodstream expression site, which naturally contains VSG 2 at the telomeric end. In VSG double expresor 16-mer WT cell-line, VSG117 contains its endogenous 3’UTR with the conserved 16-mer motif (sequence shown in blue). In VSG double expresor 16-mer MUT cell-line, the 16-mer motif was scrambled (sequence shown in orange). b . Transcript levels of VSG117 and VSG2 transcripts measured by qRT-PCR in both reporter cell-lines. Levels were normalized to transcript levels in cell-lines expressing only VSG2 or only VSG117 . c . Immunoblot with anti-m 6 A antibody of mRNA from VSG double-expressor cell-lines. RNase H digestion of VSG2 mRNA was used to resolve VSG2 and VSG117 transcripts. Different quantities of the same VSG double expresor 16-mer WT cell-line was loaded in two separet lanes (50ng and 12.5ng) to show that the VSG117 band is detectable in both conditions. d . m 6 A index calculated as the ratio of m 6 A intensity and mRNA levels, measured in panels c. and b., respectively. und., undetectable. # intensities measured in lane 3 of Figure 5C (see also Extended Data Fig. S5)

    Techniques Used: Expressing, Sequencing, Quantitative RT-PCR

    11) Product Images from "N6-methyladenosine in poly(A) tails stabilize VSG transcripts"

    Article Title: N6-methyladenosine in poly(A) tails stabilize VSG transcripts

    Journal: bioRxiv

    doi: 10.1101/2020.01.30.925776

    m 6 A is present in the poly(A) tail of VSG mRNA and other transcripts. a . Immunoblotting with anti-m 6 A antibody. RNA samples (from left to right): total RNA (Total), Poly(A)-enriched (A+) RNA and Poly(A)-depleted (A-) RNA from two life cycle stages (BSF and PCF). The last lane contains total mouse liver RNA (Mouse). 2 µ g of total RNA, 2 µ g of poly(A)-depleted RNA and 100 ng of poly(A)-enriched RNA was loaded per lane. rRNA was detected by staining RNA with methylene blue to confirm equal loading between total and poly(A)-depleted fractions. As expected the rRNA are undetectable in the poly(A)-enriched fraction. b . Intensity of the m 6 A signal in immunoblot, measured by Image J, in the whole lane containing the poly(A)-enriched RNA of bloodstream forms. The intensity of the ∼1.8 kb band was compared with the signal intensity of the entire lane, and averaged from 5 independent samples. c . Diagram displaying the location of the oligonucleotides used in RNase H digestion of VSG mRNA. The digestion products detected in the immunoblot (panel D) after incubation with each oligonucleotide (SL, A, B, C, dT) are also indicated. d . Immunoblotting with anti-m 6 A antibody of mammalian bloodstream forms total RNA pre-incubated with indicated oligonucleotides and digested with RNase H. 2 µ g of total RNA were loaded per lane. Staining of rRNA with Methylene Blue confirmed equal loading. SL: spliced leader, dT: poly deoxi-thymidines. Tub: α-Tubulin. (see also Extended Data Fig. S3)
    Figure Legend Snippet: m 6 A is present in the poly(A) tail of VSG mRNA and other transcripts. a . Immunoblotting with anti-m 6 A antibody. RNA samples (from left to right): total RNA (Total), Poly(A)-enriched (A+) RNA and Poly(A)-depleted (A-) RNA from two life cycle stages (BSF and PCF). The last lane contains total mouse liver RNA (Mouse). 2 µ g of total RNA, 2 µ g of poly(A)-depleted RNA and 100 ng of poly(A)-enriched RNA was loaded per lane. rRNA was detected by staining RNA with methylene blue to confirm equal loading between total and poly(A)-depleted fractions. As expected the rRNA are undetectable in the poly(A)-enriched fraction. b . Intensity of the m 6 A signal in immunoblot, measured by Image J, in the whole lane containing the poly(A)-enriched RNA of bloodstream forms. The intensity of the ∼1.8 kb band was compared with the signal intensity of the entire lane, and averaged from 5 independent samples. c . Diagram displaying the location of the oligonucleotides used in RNase H digestion of VSG mRNA. The digestion products detected in the immunoblot (panel D) after incubation with each oligonucleotide (SL, A, B, C, dT) are also indicated. d . Immunoblotting with anti-m 6 A antibody of mammalian bloodstream forms total RNA pre-incubated with indicated oligonucleotides and digested with RNase H. 2 µ g of total RNA were loaded per lane. Staining of rRNA with Methylene Blue confirmed equal loading. SL: spliced leader, dT: poly deoxi-thymidines. Tub: α-Tubulin. (see also Extended Data Fig. S3)

    Techniques Used: Staining, Incubation

    Conserved VSG 16-mer motif is required for inclusion of m6A in adjacent poly(A) tail. a . Diagram of 16-mer motif VSg double-expressor (DE) cell-lines. VSG117 was inserted immediately downstream of the promoter of the active bloodstream expression site, which naturally contains VSG 2 at the telomeric end. In VSG double expresor 16-mer WT cell-line, VSG117 contains its endogenous 3’UTR with the conserved 16-mer motif (sequence shown in blue). In VSG double expresor 16-mer MUT cell-line, the 16-mer motif was scrambled (sequence shown in orange). b . Transcript levels of VSG117 and VSG2 transcripts measured by qRT-PCR in both reporter cell-lines. Levels were normalized to transcript levels in cell-lines expressing only VSG2 or only VSG117 . c . Immunoblot with anti-m 6 A antibody of mRNA from VSG double-expressor cell-lines. RNase H digestion of VSG2 mRNA was used to resolve VSG2 and VSG117 transcripts. Different quantities of the same VSG double expresor 16-mer WT cell-line was loaded in two separet lanes (50ng and 12.5ng) to show that the VSG117 band is detectable in both conditions. d . m 6 A index calculated as the ratio of m 6 A intensity and mRNA levels, measured in panels c. and b., respectively. und., undetectable. # intensities measured in lane 3 of Figure 5C (see also Extended Data Fig. S5)
    Figure Legend Snippet: Conserved VSG 16-mer motif is required for inclusion of m6A in adjacent poly(A) tail. a . Diagram of 16-mer motif VSg double-expressor (DE) cell-lines. VSG117 was inserted immediately downstream of the promoter of the active bloodstream expression site, which naturally contains VSG 2 at the telomeric end. In VSG double expresor 16-mer WT cell-line, VSG117 contains its endogenous 3’UTR with the conserved 16-mer motif (sequence shown in blue). In VSG double expresor 16-mer MUT cell-line, the 16-mer motif was scrambled (sequence shown in orange). b . Transcript levels of VSG117 and VSG2 transcripts measured by qRT-PCR in both reporter cell-lines. Levels were normalized to transcript levels in cell-lines expressing only VSG2 or only VSG117 . c . Immunoblot with anti-m 6 A antibody of mRNA from VSG double-expressor cell-lines. RNase H digestion of VSG2 mRNA was used to resolve VSG2 and VSG117 transcripts. Different quantities of the same VSG double expresor 16-mer WT cell-line was loaded in two separet lanes (50ng and 12.5ng) to show that the VSG117 band is detectable in both conditions. d . m 6 A index calculated as the ratio of m 6 A intensity and mRNA levels, measured in panels c. and b., respectively. und., undetectable. # intensities measured in lane 3 of Figure 5C (see also Extended Data Fig. S5)

    Techniques Used: Expressing, Sequencing, Quantitative RT-PCR

    12) Product Images from "Simple and efficient synthesis of 5? pre-adenylated DNA using thermostable RNA ligase"

    Article Title: Simple and efficient synthesis of 5? pre-adenylated DNA using thermostable RNA ligase

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr544

    Comparison of different ligases in the efficiency of ssDNA adenylation. The RNA ligases used were MthRnl, CircLigase TM , T4Rnl1 and T4Rnl2 with two substrates, pDNA17-NH 2 ( a , c and f ) and pDNA21-3bioTEG ( b , d and e ) and analyzed by a 15% urea-PAGE as described in ‘Materials and Methods’ section. Single-stranded RNA size markers (Mr) are included for reference. ‘Input’ lanes are reactions without ligase. Above each gel, the molar ratio (S/E) of substrate to enzyme for each reaction is given. The molarity of MthRnl was calculated based on the molecular weight of a monomer.
    Figure Legend Snippet: Comparison of different ligases in the efficiency of ssDNA adenylation. The RNA ligases used were MthRnl, CircLigase TM , T4Rnl1 and T4Rnl2 with two substrates, pDNA17-NH 2 ( a , c and f ) and pDNA21-3bioTEG ( b , d and e ) and analyzed by a 15% urea-PAGE as described in ‘Materials and Methods’ section. Single-stranded RNA size markers (Mr) are included for reference. ‘Input’ lanes are reactions without ligase. Above each gel, the molar ratio (S/E) of substrate to enzyme for each reaction is given. The molarity of MthRnl was calculated based on the molecular weight of a monomer.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Molecular Weight

    pH dependence of adenylation of MthRnl and ssDNA. ( a ) Adenylation of non-adenylated MthRnl in the presence of Mg +2 or Mn +2 , ATP and α-[ 32 P]ATP with no DNA substrate. The products were analyzed by an SDS–PAGE and radioactive bands quantified as described in ‘Materials and Methods’ section. ( b ) pH dependence of pDNA17c-NH 2 adenylation by MthRnl under reaction conditions as described in ‘Material and Methods’ section in bis–Tris propane–HCl buffer. The products were analyzed by 15% urea–PAGE. The size markers at the left of the gel (Mr) are single-stranded RNA.
    Figure Legend Snippet: pH dependence of adenylation of MthRnl and ssDNA. ( a ) Adenylation of non-adenylated MthRnl in the presence of Mg +2 or Mn +2 , ATP and α-[ 32 P]ATP with no DNA substrate. The products were analyzed by an SDS–PAGE and radioactive bands quantified as described in ‘Materials and Methods’ section. ( b ) pH dependence of pDNA17c-NH 2 adenylation by MthRnl under reaction conditions as described in ‘Material and Methods’ section in bis–Tris propane–HCl buffer. The products were analyzed by 15% urea–PAGE. The size markers at the left of the gel (Mr) are single-stranded RNA.

    Techniques Used: SDS Page, Polyacrylamide Gel Electrophoresis

    Ligation of adenylated DNA linker made with MthRnl to RNA. AppDNA17c-NH 2 was ligated to RNA22 (lane 2) and FAM-RNA23 (lane 4) using T4 RNA ligase 2 truncated without ATP as described in ‘Materials and Methods’ section. Lanes 1 and 3 are controls without ligase. Single-stranded RNA size markers (Mr) are included for reference. The products were analyzed by a 15% urea–PAGE.
    Figure Legend Snippet: Ligation of adenylated DNA linker made with MthRnl to RNA. AppDNA17c-NH 2 was ligated to RNA22 (lane 2) and FAM-RNA23 (lane 4) using T4 RNA ligase 2 truncated without ATP as described in ‘Materials and Methods’ section. Lanes 1 and 3 are controls without ligase. Single-stranded RNA size markers (Mr) are included for reference. The products were analyzed by a 15% urea–PAGE.

    Techniques Used: Ligation, Polyacrylamide Gel Electrophoresis

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    New England Biolabs thermostable rnase h
    DDR at ALT telomeres induced by FANCM deficiency is mediated by XPF. a Representative images of TRF2 and XPF co-immunostaining in U2OS cells transfected with FANCM siRNAs for 3 days. Arrows indicate the colocalization events. b Quantification of colocalization events in ( a ). The number of XPF-TRF2 colocalization per nucleus (left). The intensity of XPF foci that are colocalized with TRF2 (right). P -values by two-sided Mann-Whitney test. Bars, mean ± SEM. n , cell number (left) or number of XPF foci (right). Data of three independent experiments. c DRIP-qPCR to detect telomeric R-loops in U2OS cells after transfection with control, XPF, or FANCM siRNAs for 3 days. Relative R-loop levels were normalized to the same amount of genomic DNA treated with <t>RNase</t> <t>H</t> prior to DRIP. P -values by two tailed Student’s t -test. Bars, mean ± SD. Representative of three independent experiments. Other replicates show similar trends and are provided in the Source Data file. d Quantification of APBs in U2OS cells transfected with siRNAs. APB foci were determined by large TRF2 foci with PML staining shown in Supplementary Fig. 5d . n , cell number. Bars, mean ± SEM. P -values by two-sided Mann-Whitney test. Data of three independent experiments. e Representative images of immuno-DNA FISH to detect the co-localization of γH2AX and telomeres in U2OS cells after transfection with control, XPF, or FANCM siRNAs for 3 days. Arrows indicate the colocalization events. f Quantification of γH2AX foci at telomeres in ( e ). n , cell number. Bars, mean ± SEM. P -values by two-sided Mann-Whitney test. Data of two independent experiments. g Quantification of top 5% telomere intensity foci in ( e ). n , cell number. Bars, medians. P -values by two-sided Mann-Whitney test. Representative of three independent experiments. Other replicates show similar trends and are provided in the Source Data file. a – g Mean or median values of each group shown on the top of figures.
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    DDR at ALT telomeres induced by FANCM deficiency is mediated by XPF. a Representative images of TRF2 and XPF co-immunostaining in U2OS cells transfected with FANCM siRNAs for 3 days. Arrows indicate the colocalization events. b Quantification of colocalization events in ( a ). The number of XPF-TRF2 colocalization per nucleus (left). The intensity of XPF foci that are colocalized with TRF2 (right). P -values by two-sided Mann-Whitney test. Bars, mean ± SEM. n , cell number (left) or number of XPF foci (right). Data of three independent experiments. c DRIP-qPCR to detect telomeric R-loops in U2OS cells after transfection with control, XPF, or FANCM siRNAs for 3 days. Relative R-loop levels were normalized to the same amount of genomic DNA treated with RNase H prior to DRIP. P -values by two tailed Student’s t -test. Bars, mean ± SD. Representative of three independent experiments. Other replicates show similar trends and are provided in the Source Data file. d Quantification of APBs in U2OS cells transfected with siRNAs. APB foci were determined by large TRF2 foci with PML staining shown in Supplementary Fig. 5d . n , cell number. Bars, mean ± SEM. P -values by two-sided Mann-Whitney test. Data of three independent experiments. e Representative images of immuno-DNA FISH to detect the co-localization of γH2AX and telomeres in U2OS cells after transfection with control, XPF, or FANCM siRNAs for 3 days. Arrows indicate the colocalization events. f Quantification of γH2AX foci at telomeres in ( e ). n , cell number. Bars, mean ± SEM. P -values by two-sided Mann-Whitney test. Data of two independent experiments. g Quantification of top 5% telomere intensity foci in ( e ). n , cell number. Bars, medians. P -values by two-sided Mann-Whitney test. Representative of three independent experiments. Other replicates show similar trends and are provided in the Source Data file. a – g Mean or median values of each group shown on the top of figures.

    Journal: Nature Communications

    Article Title: XPF activates break-induced telomere synthesis

    doi: 10.1038/s41467-022-33428-0

    Figure Lengend Snippet: DDR at ALT telomeres induced by FANCM deficiency is mediated by XPF. a Representative images of TRF2 and XPF co-immunostaining in U2OS cells transfected with FANCM siRNAs for 3 days. Arrows indicate the colocalization events. b Quantification of colocalization events in ( a ). The number of XPF-TRF2 colocalization per nucleus (left). The intensity of XPF foci that are colocalized with TRF2 (right). P -values by two-sided Mann-Whitney test. Bars, mean ± SEM. n , cell number (left) or number of XPF foci (right). Data of three independent experiments. c DRIP-qPCR to detect telomeric R-loops in U2OS cells after transfection with control, XPF, or FANCM siRNAs for 3 days. Relative R-loop levels were normalized to the same amount of genomic DNA treated with RNase H prior to DRIP. P -values by two tailed Student’s t -test. Bars, mean ± SD. Representative of three independent experiments. Other replicates show similar trends and are provided in the Source Data file. d Quantification of APBs in U2OS cells transfected with siRNAs. APB foci were determined by large TRF2 foci with PML staining shown in Supplementary Fig. 5d . n , cell number. Bars, mean ± SEM. P -values by two-sided Mann-Whitney test. Data of three independent experiments. e Representative images of immuno-DNA FISH to detect the co-localization of γH2AX and telomeres in U2OS cells after transfection with control, XPF, or FANCM siRNAs for 3 days. Arrows indicate the colocalization events. f Quantification of γH2AX foci at telomeres in ( e ). n , cell number. Bars, mean ± SEM. P -values by two-sided Mann-Whitney test. Data of two independent experiments. g Quantification of top 5% telomere intensity foci in ( e ). n , cell number. Bars, medians. P -values by two-sided Mann-Whitney test. Representative of three independent experiments. Other replicates show similar trends and are provided in the Source Data file. a – g Mean or median values of each group shown on the top of figures.

    Article Snippet: Genomic DNA was fragmented into 200~500 bp using Covaris S2 in microtubes with 10% duty cycle, 200 burst/cycles, intensity 3 for 60 s. For RNaseH controls, 8 μg of genomic DNA was treated with RNase H (5 U/μL, NEB, # M0523) at 37 °C overnight before immunoprecipitation, and purified by phenol-chloroform extraction.

    Techniques: Immunostaining, Transfection, MANN-WHITNEY, Real-time Polymerase Chain Reaction, Two Tailed Test, Staining, Fluorescence In Situ Hybridization

    Directed RNA cleavage with RNase H. ( A ) Previously reported RNase H cleavage sites (17–19). ( B ) Synthetic RNA oligonucleotide containing the first 33 nt of an artificial FLuc transcript (syn FLuc -AC) and targeting oligos FLuc TO-25 designed based on A . Inverted triangles represent RNase H cleavage sites reported in the literature ( A ) and in this study ( B ). Deoxynucleotides in TOs are colored in blue; ribonucleotides are colored in green. The predicted loop region of the synthetic RNA oligonucleotides based on an RNA folding algorithm (RNAFold, University of Vienna) is indicated. ( C ) RNA cleavage using E. coli or Thermus thermophilus RNase H at 37°C. The cleavage efficiency of both enzymes was similar but Tth RNase H generates more uniform cuts than the E. coli enzyme.

    Journal: RNA

    Article Title: RNase H-based analysis of synthetic mRNA 5′ cap incorporation

    doi: 10.1261/rna.079173.122

    Figure Lengend Snippet: Directed RNA cleavage with RNase H. ( A ) Previously reported RNase H cleavage sites (17–19). ( B ) Synthetic RNA oligonucleotide containing the first 33 nt of an artificial FLuc transcript (syn FLuc -AC) and targeting oligos FLuc TO-25 designed based on A . Inverted triangles represent RNase H cleavage sites reported in the literature ( A ) and in this study ( B ). Deoxynucleotides in TOs are colored in blue; ribonucleotides are colored in green. The predicted loop region of the synthetic RNA oligonucleotides based on an RNA folding algorithm (RNAFold, University of Vienna) is indicated. ( C ) RNA cleavage using E. coli or Thermus thermophilus RNase H at 37°C. The cleavage efficiency of both enzymes was similar but Tth RNase H generates more uniform cuts than the E. coli enzyme.

    Article Snippet: The mixtures were then incubated with E. coli RNase H (New England Biolabs) or T. thermophilus RNase H (Thermostable RNase H, New England Biolabs) at a final concentration of 5 U/μL at 37°C for 1 h. Reactions were quenched by addition of EDTA to a final concentration of 10 mM.

    Techniques:

    Fluorescent labeling of RNase H 5′ cleavage products and analyses. ( A ) Schematic representation of targeting, cleavage, and labeling. The targeting oligo was designed to guide RNase H to generate a 1-nt 3′ recessive end, which can be filled in by a fluorescently labeled deoxynucleotide using the Klenow fragment. In this example, a FAM-labeled dCTP was incorporated into the 3′ end of the FLuc RNase H cleavage fragment and directly analyzed by urea PAGE ( B ) or capillary electrophoresis ( C ) after enrichment. ( B ) Polyacrylamide gel analysis of cleaved RNA fragments. The 1.7 kb FLuc transcript capped using the vaccinia RNA capping enzyme (VCE) was subjected to the RNase H/Klenow fill-in treatments. Reactions were analyzed directly by urea PAGE followed by laser scanning of total RNA stain using SYBR Gold ( left panel) or fluorescent signal ( right panel). The targeting oligo TO-1 was invisible when the gel was scanned using the FAM channel and did not interfere with quantitation of the 5′ cleavage products. ( C ) Resolution and quantification of capping and capping intermediates using capillary electrophoresis. The FLuc transcript was capped using a low concentration of VCE (10 nM) and subjected to the RNase H/Klenow fill-in reactions. After enrichment, the RNA was analyzed using capillary electrophoresis. In addition to substrate 5′ triphosphate (ppp-) and the product m7Gppp-capped forms, enzymatic intermediate products 5′ diphosphate (pp-) and the unmethyl-cap (Gppp-) can be resolved and quantified. ( D ) Synthetic mRNA cap analysis using gel electrophoresis, capillary electrophoresis, and LC-MS intact mass analysis produce comparable results. After RNase H/Klenow fill-in reactions and enrichment, an uncapped or partially capped FLuc transcript was analyzed using all three available methods. Capillary electrophoresis and LC-MS yielded comparable results in quantification of substrate (ppp-), product (m7Gppp-), and intermediate products (pp- and Gppp-). Urea-PAGE does not resolve pp- from ppp- or Gppp- from m7Gppp-. Considering ppp- and pp- as uncapped and Gppp- and m7Gppp- as capped species, quantitation of fluorescently labeled RNase H cleavage products using urea-PAGE generate results comparable to CE or LC-MS, despite the lack of resolution for intermediate products.

    Journal: RNA

    Article Title: RNase H-based analysis of synthetic mRNA 5′ cap incorporation

    doi: 10.1261/rna.079173.122

    Figure Lengend Snippet: Fluorescent labeling of RNase H 5′ cleavage products and analyses. ( A ) Schematic representation of targeting, cleavage, and labeling. The targeting oligo was designed to guide RNase H to generate a 1-nt 3′ recessive end, which can be filled in by a fluorescently labeled deoxynucleotide using the Klenow fragment. In this example, a FAM-labeled dCTP was incorporated into the 3′ end of the FLuc RNase H cleavage fragment and directly analyzed by urea PAGE ( B ) or capillary electrophoresis ( C ) after enrichment. ( B ) Polyacrylamide gel analysis of cleaved RNA fragments. The 1.7 kb FLuc transcript capped using the vaccinia RNA capping enzyme (VCE) was subjected to the RNase H/Klenow fill-in treatments. Reactions were analyzed directly by urea PAGE followed by laser scanning of total RNA stain using SYBR Gold ( left panel) or fluorescent signal ( right panel). The targeting oligo TO-1 was invisible when the gel was scanned using the FAM channel and did not interfere with quantitation of the 5′ cleavage products. ( C ) Resolution and quantification of capping and capping intermediates using capillary electrophoresis. The FLuc transcript was capped using a low concentration of VCE (10 nM) and subjected to the RNase H/Klenow fill-in reactions. After enrichment, the RNA was analyzed using capillary electrophoresis. In addition to substrate 5′ triphosphate (ppp-) and the product m7Gppp-capped forms, enzymatic intermediate products 5′ diphosphate (pp-) and the unmethyl-cap (Gppp-) can be resolved and quantified. ( D ) Synthetic mRNA cap analysis using gel electrophoresis, capillary electrophoresis, and LC-MS intact mass analysis produce comparable results. After RNase H/Klenow fill-in reactions and enrichment, an uncapped or partially capped FLuc transcript was analyzed using all three available methods. Capillary electrophoresis and LC-MS yielded comparable results in quantification of substrate (ppp-), product (m7Gppp-), and intermediate products (pp- and Gppp-). Urea-PAGE does not resolve pp- from ppp- or Gppp- from m7Gppp-. Considering ppp- and pp- as uncapped and Gppp- and m7Gppp- as capped species, quantitation of fluorescently labeled RNase H cleavage products using urea-PAGE generate results comparable to CE or LC-MS, despite the lack of resolution for intermediate products.

    Article Snippet: The mixtures were then incubated with E. coli RNase H (New England Biolabs) or T. thermophilus RNase H (Thermostable RNase H, New England Biolabs) at a final concentration of 5 U/μL at 37°C for 1 h. Reactions were quenched by addition of EDTA to a final concentration of 10 mM.

    Techniques: Labeling, Polyacrylamide Gel Electrophoresis, Electrophoresis, Staining, Quantitation Assay, Concentration Assay, Nucleic Acid Electrophoresis, Liquid Chromatography with Mass Spectroscopy

    Uniform RNase H cleavage with designed targeting oligos. ( A ) 5′ sequence of a 1.7 kb in vitro FL uc transcript containing an artificial 5′ UTR and the corresponding targeting oligo TO-1. The targeting oligo contains six deoxynucleotides (blue) at the 5′ end followed by 19 ribonucleotides (green) and a desthiobiotin (DTB) group at the 3′ end. The size of the RNase H cleavage products is shown. ( B ) Frequency of cleavage events expressed as percentage of detected cleavage product using LC-MS. Median of cleavage frequencies was 8%, 91%, and 1% at (A|A), (A|C), and (C|U), respectively.

    Journal: RNA

    Article Title: RNase H-based analysis of synthetic mRNA 5′ cap incorporation

    doi: 10.1261/rna.079173.122

    Figure Lengend Snippet: Uniform RNase H cleavage with designed targeting oligos. ( A ) 5′ sequence of a 1.7 kb in vitro FL uc transcript containing an artificial 5′ UTR and the corresponding targeting oligo TO-1. The targeting oligo contains six deoxynucleotides (blue) at the 5′ end followed by 19 ribonucleotides (green) and a desthiobiotin (DTB) group at the 3′ end. The size of the RNase H cleavage products is shown. ( B ) Frequency of cleavage events expressed as percentage of detected cleavage product using LC-MS. Median of cleavage frequencies was 8%, 91%, and 1% at (A|A), (A|C), and (C|U), respectively.

    Article Snippet: The mixtures were then incubated with E. coli RNase H (New England Biolabs) or T. thermophilus RNase H (Thermostable RNase H, New England Biolabs) at a final concentration of 5 U/μL at 37°C for 1 h. Reactions were quenched by addition of EDTA to a final concentration of 10 mM.

    Techniques: Sequencing, In Vitro, Liquid Chromatography with Mass Spectroscopy

    A general scheme of RNase H-based RNA cap analysis. A DNA–RNA or DNA-2′- O -methyl-ribonucleotide chimera is designed to be complementary to part of the 5′ end of the target RNA molecule such that the chimera stays annealed to the cleavage fragment after RNase H cleavage. The chimera (called targeting oligo or TO in this paper) contains a 3′-desthiobiotin group. After denaturation and annealing, RNase H cleaves at a predefined site within the RNA-TO duplex and generates a one-base recessive end at the 3′ end of the cleaved RNA. Because RNase H cleavage results in a 3′ hydroxyl group (24), this recessive 3′ end can be filled in with a fluorescently labeled deoxynucleotide using the Klenow fragment of DNA polymerase I. The fluorescently labeled 5′ cleavage fragment can be analyzed by denaturing PAGE directly without enrichment. The 5′ duplex cleavage fragment can be enriched using streptavidin magnetic beads. The enriched RNase H cleavage products can be analyzed by LC-MS or capillary electrophoresis (if filled in with a fluorescent deoxynucleotide).

    Journal: RNA

    Article Title: RNase H-based analysis of synthetic mRNA 5′ cap incorporation

    doi: 10.1261/rna.079173.122

    Figure Lengend Snippet: A general scheme of RNase H-based RNA cap analysis. A DNA–RNA or DNA-2′- O -methyl-ribonucleotide chimera is designed to be complementary to part of the 5′ end of the target RNA molecule such that the chimera stays annealed to the cleavage fragment after RNase H cleavage. The chimera (called targeting oligo or TO in this paper) contains a 3′-desthiobiotin group. After denaturation and annealing, RNase H cleaves at a predefined site within the RNA-TO duplex and generates a one-base recessive end at the 3′ end of the cleaved RNA. Because RNase H cleavage results in a 3′ hydroxyl group (24), this recessive 3′ end can be filled in with a fluorescently labeled deoxynucleotide using the Klenow fragment of DNA polymerase I. The fluorescently labeled 5′ cleavage fragment can be analyzed by denaturing PAGE directly without enrichment. The 5′ duplex cleavage fragment can be enriched using streptavidin magnetic beads. The enriched RNase H cleavage products can be analyzed by LC-MS or capillary electrophoresis (if filled in with a fluorescent deoxynucleotide).

    Article Snippet: The mixtures were then incubated with E. coli RNase H (New England Biolabs) or T. thermophilus RNase H (Thermostable RNase H, New England Biolabs) at a final concentration of 5 U/μL at 37°C for 1 h. Reactions were quenched by addition of EDTA to a final concentration of 10 mM.

    Techniques: Labeling, Polyacrylamide Gel Electrophoresis, Magnetic Beads, Liquid Chromatography with Mass Spectroscopy, Electrophoresis

    The effect of pseudouridine on RNase H cleavage. ( A ) A Cypridina luciferase transcript ( CLuc ; 1.8 kb) was cleaved using Tth RNase H in conjunction with CLuc TO-26, which directs RNase H, a cleavage site containing a uridine residue ( Supplemental Fig. 6 ). The size of the cleavage products and cleavage sites are indicated. ( B ) When unsubstituted, median cleavage frequency at the C|UC site was 73% (25 nt) and at the CU|C site was 27% (26 nt). When all uridines were substituted with pseudouridine, cleavage at the C|ΨC site decreased to a median frequency of 34%, with 66% cleavage at the CΨ|C site.

    Journal: RNA

    Article Title: RNase H-based analysis of synthetic mRNA 5′ cap incorporation

    doi: 10.1261/rna.079173.122

    Figure Lengend Snippet: The effect of pseudouridine on RNase H cleavage. ( A ) A Cypridina luciferase transcript ( CLuc ; 1.8 kb) was cleaved using Tth RNase H in conjunction with CLuc TO-26, which directs RNase H, a cleavage site containing a uridine residue ( Supplemental Fig. 6 ). The size of the cleavage products and cleavage sites are indicated. ( B ) When unsubstituted, median cleavage frequency at the C|UC site was 73% (25 nt) and at the CU|C site was 27% (26 nt). When all uridines were substituted with pseudouridine, cleavage at the C|ΨC site decreased to a median frequency of 34%, with 66% cleavage at the CΨ|C site.

    Article Snippet: The mixtures were then incubated with E. coli RNase H (New England Biolabs) or T. thermophilus RNase H (Thermostable RNase H, New England Biolabs) at a final concentration of 5 U/μL at 37°C for 1 h. Reactions were quenched by addition of EDTA to a final concentration of 10 mM.

    Techniques: Luciferase

    Enrichment of RNase H cleavage products by size and affinity selection. The RNase H cleavage product (input) was first size-selected by two rounds of NEBNext magnetic beads. The clarified unbound fraction of the second round of size selection (ub2) was then added directly to streptavidin magnetic beads for affinity selection. After the first wash using a standard wash solution containing 1 M NaCl (W1), the resuspended bead slurry was divided into two fractions. One fraction was washed three more times using the standard wash buffer (W4_HS) and eluted using biotin (Eluate_biotin). The other fraction of the slurry was washed three more times using a low NaCl wash solution (W4_LS) followed by elution using the same volume of water (Eluate_water). Similar amounts of RNase H cleavage product and TO were eluted (also see Supplemental Fig. 4 ).

    Journal: RNA

    Article Title: RNase H-based analysis of synthetic mRNA 5′ cap incorporation

    doi: 10.1261/rna.079173.122

    Figure Lengend Snippet: Enrichment of RNase H cleavage products by size and affinity selection. The RNase H cleavage product (input) was first size-selected by two rounds of NEBNext magnetic beads. The clarified unbound fraction of the second round of size selection (ub2) was then added directly to streptavidin magnetic beads for affinity selection. After the first wash using a standard wash solution containing 1 M NaCl (W1), the resuspended bead slurry was divided into two fractions. One fraction was washed three more times using the standard wash buffer (W4_HS) and eluted using biotin (Eluate_biotin). The other fraction of the slurry was washed three more times using a low NaCl wash solution (W4_LS) followed by elution using the same volume of water (Eluate_water). Similar amounts of RNase H cleavage product and TO were eluted (also see Supplemental Fig. 4 ).

    Article Snippet: The mixtures were then incubated with E. coli RNase H (New England Biolabs) or T. thermophilus RNase H (Thermostable RNase H, New England Biolabs) at a final concentration of 5 U/μL at 37°C for 1 h. Reactions were quenched by addition of EDTA to a final concentration of 10 mM.

    Techniques: Selection, Magnetic Beads

    Selection of targeting oligos for uniform RNase H cleavage. ( A ) In general, a surrogate RNA oligonucleotide containing the first 30–50 nt of the target transcript is chemically synthesized with a 5′ FAM group. A series of targeting oligos (TOs) are designed and chemically synthesized. The TOs used in the selection exercise did not require a desthiobiotin group, because unlike the long 3′ cleavage products of in vitro transcripts, the 3′ cleavage products of the surrogate RNA were short and did not interfere with LC-MS analysis. After RNase H cleavage, the fluorescently labeled 5′ cleavage fragments can be analyzed by urea PAGE and LC-MS intact mass analysis. ( B ) For consistency, the phosphodiester bonds are numbered around the nucleotide hybridized to the 5′ deoxynucleotide of the TO ( top panel; a cytidine in this case). Urea PAGE showed that RNase H cleavage is most efficient with FLuc TO-25, FLuc TO-26, and FLuc TO-27. Multiple cleavage products were observed with FLuc TO-24, FLuc TO-25, and FLuc TO-26 ( middle panel). LC-MS intact mass analysis of the cleavage products is shown in the lower panel. In the schematics, phosphodiester bonds are shown as “-”. Deoxynucleotides in the TOs are shown as gray bars; ribonucleotides are shown as yellow bars. Numbers represent frequency of cleavage detected at corresponding phosphodiester bonds obtained in triplicated experiments.

    Journal: RNA

    Article Title: RNase H-based analysis of synthetic mRNA 5′ cap incorporation

    doi: 10.1261/rna.079173.122

    Figure Lengend Snippet: Selection of targeting oligos for uniform RNase H cleavage. ( A ) In general, a surrogate RNA oligonucleotide containing the first 30–50 nt of the target transcript is chemically synthesized with a 5′ FAM group. A series of targeting oligos (TOs) are designed and chemically synthesized. The TOs used in the selection exercise did not require a desthiobiotin group, because unlike the long 3′ cleavage products of in vitro transcripts, the 3′ cleavage products of the surrogate RNA were short and did not interfere with LC-MS analysis. After RNase H cleavage, the fluorescently labeled 5′ cleavage fragments can be analyzed by urea PAGE and LC-MS intact mass analysis. ( B ) For consistency, the phosphodiester bonds are numbered around the nucleotide hybridized to the 5′ deoxynucleotide of the TO ( top panel; a cytidine in this case). Urea PAGE showed that RNase H cleavage is most efficient with FLuc TO-25, FLuc TO-26, and FLuc TO-27. Multiple cleavage products were observed with FLuc TO-24, FLuc TO-25, and FLuc TO-26 ( middle panel). LC-MS intact mass analysis of the cleavage products is shown in the lower panel. In the schematics, phosphodiester bonds are shown as “-”. Deoxynucleotides in the TOs are shown as gray bars; ribonucleotides are shown as yellow bars. Numbers represent frequency of cleavage detected at corresponding phosphodiester bonds obtained in triplicated experiments.

    Article Snippet: The mixtures were then incubated with E. coli RNase H (New England Biolabs) or T. thermophilus RNase H (Thermostable RNase H, New England Biolabs) at a final concentration of 5 U/μL at 37°C for 1 h. Reactions were quenched by addition of EDTA to a final concentration of 10 mM.

    Techniques: Selection, Synthesized, In Vitro, Liquid Chromatography with Mass Spectroscopy, Labeling, Polyacrylamide Gel Electrophoresis

    Deconvoluted mass spectrums of capping analysis. An enzymatically capped Cap-1 CLuc ( A ) or FLuc transcript ( B ) was processed with RNase H and the single-step affinity enrichment and analyzed by LC-MS as described in the main text. The RNase H cleavage products with relevant 5′ groups were identified by their distinct deconvoluted mass values. The area under the identified mass peaks was used to calculate the relative percentage of each species in the sample. A mass corresponded to 24 nt + pG in the Cap-1 form was detected in the FLuc transcript. The addition of a pG was probably the result of T7 polymerase slippage at the 5′ end of the transcript, which is composed of three consecutive guanosine residues.

    Journal: RNA

    Article Title: RNase H-based analysis of synthetic mRNA 5′ cap incorporation

    doi: 10.1261/rna.079173.122

    Figure Lengend Snippet: Deconvoluted mass spectrums of capping analysis. An enzymatically capped Cap-1 CLuc ( A ) or FLuc transcript ( B ) was processed with RNase H and the single-step affinity enrichment and analyzed by LC-MS as described in the main text. The RNase H cleavage products with relevant 5′ groups were identified by their distinct deconvoluted mass values. The area under the identified mass peaks was used to calculate the relative percentage of each species in the sample. A mass corresponded to 24 nt + pG in the Cap-1 form was detected in the FLuc transcript. The addition of a pG was probably the result of T7 polymerase slippage at the 5′ end of the transcript, which is composed of three consecutive guanosine residues.

    Article Snippet: The mixtures were then incubated with E. coli RNase H (New England Biolabs) or T. thermophilus RNase H (Thermostable RNase H, New England Biolabs) at a final concentration of 5 U/μL at 37°C for 1 h. Reactions were quenched by addition of EDTA to a final concentration of 10 mM.

    Techniques: Liquid Chromatography with Mass Spectroscopy