rna substrates  (Integrated DNA Technologies)


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    Name:
    DNA oligo
    Description:
    Single stranded pooled or duplexed DNA synthesized to customer specifications Sspecialized platforms with industry leading synthesis capabilities deliver the purest primers for PCR dual labelled probes for qPCR indexed adapters and fusion primers for sequencing and a variety of advanced and custom products
    Catalog Number:
    do-577595
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    Category:
    Nucleic acids
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    Structured Review

    Integrated DNA Technologies rna substrates
    Primer extension inhibition analyses of mRNA cleavage by GB1_HP0315. Lanes 1–4 are <t>DNA</t> sequence ladders. Lane 5–9 is primer extension products of mRNA of HP0315 after digestion with various amounts of GB1_HP0315. Cleavage sites are indicated by sequential numbers on right side of the images. The <t>RNA</t> recognition sequences analyzed by the DNA sequencing ladder are on bottom side of the images. Preferential cleavage sites are mainly before the bases A and G.
    Single stranded pooled or duplexed DNA synthesized to customer specifications Sspecialized platforms with industry leading synthesis capabilities deliver the purest primers for PCR dual labelled probes for qPCR indexed adapters and fusion primers for sequencing and a variety of advanced and custom products
    https://www.bioz.com/result/rna substrates/product/Integrated DNA Technologies
    Average 99 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    rna substrates - by Bioz Stars, 2020-09
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    Images

    1) Product Images from "Structural and biochemical characterization of HP0315 from Helicobacter pylori as a VapD protein with an endoribonuclease activity"

    Article Title: Structural and biochemical characterization of HP0315 from Helicobacter pylori as a VapD protein with an endoribonuclease activity

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr1305

    Primer extension inhibition analyses of mRNA cleavage by GB1_HP0315. Lanes 1–4 are DNA sequence ladders. Lane 5–9 is primer extension products of mRNA of HP0315 after digestion with various amounts of GB1_HP0315. Cleavage sites are indicated by sequential numbers on right side of the images. The RNA recognition sequences analyzed by the DNA sequencing ladder are on bottom side of the images. Preferential cleavage sites are mainly before the bases A and G.
    Figure Legend Snippet: Primer extension inhibition analyses of mRNA cleavage by GB1_HP0315. Lanes 1–4 are DNA sequence ladders. Lane 5–9 is primer extension products of mRNA of HP0315 after digestion with various amounts of GB1_HP0315. Cleavage sites are indicated by sequential numbers on right side of the images. The RNA recognition sequences analyzed by the DNA sequencing ladder are on bottom side of the images. Preferential cleavage sites are mainly before the bases A and G.

    Techniques Used: Inhibition, Sequencing, DNA Sequencing

    2) Product Images from "Mitochondrial Transcription Factor A (TFAM) Binds to RNA Containing 4-Way Junctions and Mitochondrial tRNA"

    Article Title: Mitochondrial Transcription Factor A (TFAM) Binds to RNA Containing 4-Way Junctions and Mitochondrial tRNA

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0142436

    TFAM binding of linear DNA and RNA substrates by EMSA. Increasing amounts of TFAM were bound to 20 fM of each biotinylated substrate as follows; (A) dsDNA control sequence LSP, 0–1.44 μM TFAM with five-fold serial dilutions, apparent K d of 6 nM, (B) dsDNA control sequence scrambled LSP, with serial dilutions of TFAM as in (A), apparent K d of 100 nM, (C) dsRNA:DNA hybrid with 0–3.5 μM TFAM with two-fold serial dilutions, (D) ss poly [rArC] 12 , with TFAM dilutions as in panel (C), (E) poly [rU] 20 , with TFAM dilutions as in (C), (F) dsRNA 5681 , with TFAM dilutions as in panel (C).
    Figure Legend Snippet: TFAM binding of linear DNA and RNA substrates by EMSA. Increasing amounts of TFAM were bound to 20 fM of each biotinylated substrate as follows; (A) dsDNA control sequence LSP, 0–1.44 μM TFAM with five-fold serial dilutions, apparent K d of 6 nM, (B) dsDNA control sequence scrambled LSP, with serial dilutions of TFAM as in (A), apparent K d of 100 nM, (C) dsRNA:DNA hybrid with 0–3.5 μM TFAM with two-fold serial dilutions, (D) ss poly [rArC] 12 , with TFAM dilutions as in panel (C), (E) poly [rU] 20 , with TFAM dilutions as in (C), (F) dsRNA 5681 , with TFAM dilutions as in panel (C).

    Techniques Used: Binding Assay, Sequencing

    Binding kinetics of TFAM to RNA and DNA substrates using surface plasmon resonance. (A-D) Sensograms displaying the TFAM binding and dissociation rates of (A) a DNA 4-way junction, (B) an RNA 4-way junction, (C) linear double-stranded DNA, and (D) purified mitochondrial tRNAs. Individual tracings represent a single value in a range of TFAM concentrations in each of the experiments. (E) Kinetic data derived from these tracings include associate rate constant ( k a ), dissociate rate constant ( k d ) and the apparent dissociation constant ( K d ) for each of these substrates.
    Figure Legend Snippet: Binding kinetics of TFAM to RNA and DNA substrates using surface plasmon resonance. (A-D) Sensograms displaying the TFAM binding and dissociation rates of (A) a DNA 4-way junction, (B) an RNA 4-way junction, (C) linear double-stranded DNA, and (D) purified mitochondrial tRNAs. Individual tracings represent a single value in a range of TFAM concentrations in each of the experiments. (E) Kinetic data derived from these tracings include associate rate constant ( k a ), dissociate rate constant ( k d ) and the apparent dissociation constant ( K d ) for each of these substrates.

    Techniques Used: Binding Assay, SPR Assay, Purification, Derivative Assay

    TFAM-bound mitochondrial tRNAs are processed and have mature ends. (A) Schematic for PCR detection of unprocessed tRNAs showing a tRNA flanked by putative RNA sequences from adjacent genes. PCR primer positions used on cDNAs are shown as arrows. Expected PCR fragments from unprocessed 5’ ends (i), unprocessed 3’ ends (ii) and internal tRNA (iii) are displayed. (B and C) PCR templates in lane 1 from total cellular cDNA, lane 2 templates made excluding reverse transcriptase, lane 3 templates are from TFAM-RNA IP, and lane 4 from TFAM-DNA IP. Samples for lanes 2, 3, and 4 are identical to those used for data obtained in Fig 4 , which further controls for the sample preparation and PCR procedures. (B) PCR amplicons detecting 5’ flanking regions from each tRNA as in (Ai). V (internal) serves as a control for the TFAM-RIP reaction using tRNA internal primers as in (Aiii). (C) PCR amplicons detecting 3’ flanking regions from each tRNA as in (Aii). P(internal) serves as a control for RT-PCR using tRNA internal primers as in (Aiii). (D and E) 3’-end sequence frequency of multiple clones isolated from tRNA Val RNA circularization is pie-graph displayed. (D) tRNA Val sequences isolated from total cellular RNA, n = 104. (E) TFAM-RIP isolated tRNA Val sequences, n = 67.
    Figure Legend Snippet: TFAM-bound mitochondrial tRNAs are processed and have mature ends. (A) Schematic for PCR detection of unprocessed tRNAs showing a tRNA flanked by putative RNA sequences from adjacent genes. PCR primer positions used on cDNAs are shown as arrows. Expected PCR fragments from unprocessed 5’ ends (i), unprocessed 3’ ends (ii) and internal tRNA (iii) are displayed. (B and C) PCR templates in lane 1 from total cellular cDNA, lane 2 templates made excluding reverse transcriptase, lane 3 templates are from TFAM-RNA IP, and lane 4 from TFAM-DNA IP. Samples for lanes 2, 3, and 4 are identical to those used for data obtained in Fig 4 , which further controls for the sample preparation and PCR procedures. (B) PCR amplicons detecting 5’ flanking regions from each tRNA as in (Ai). V (internal) serves as a control for the TFAM-RIP reaction using tRNA internal primers as in (Aiii). (C) PCR amplicons detecting 3’ flanking regions from each tRNA as in (Aii). P(internal) serves as a control for RT-PCR using tRNA internal primers as in (Aiii). (D and E) 3’-end sequence frequency of multiple clones isolated from tRNA Val RNA circularization is pie-graph displayed. (D) tRNA Val sequences isolated from total cellular RNA, n = 104. (E) TFAM-RIP isolated tRNA Val sequences, n = 67.

    Techniques Used: Polymerase Chain Reaction, Sample Prep, Reverse Transcription Polymerase Chain Reaction, Sequencing, Clone Assay, Isolation

    TFAM bound RNA represents a lesser fraction of TFAM bound DNA. (A) Immunofluorescence images of TFAM in untreated cells (i), cells treated with RNase (ii), DNase (iii) or both DNase and RNase (iv). (B) Relative mean fluorescence of TFAM retained during nuclease treatments shown in (A). (C) Levels of TFAM-bound tRNA relative to TFAM-bound to the corresponding mtDNA. Parallel RNA and DNA immunoprecipitations from the same samples were quantified by RT-PCR and PCR, respectively. Relative bound tRNA level is expressed as a percentage of bound mtDNA.
    Figure Legend Snippet: TFAM bound RNA represents a lesser fraction of TFAM bound DNA. (A) Immunofluorescence images of TFAM in untreated cells (i), cells treated with RNase (ii), DNase (iii) or both DNase and RNase (iv). (B) Relative mean fluorescence of TFAM retained during nuclease treatments shown in (A). (C) Levels of TFAM-bound tRNA relative to TFAM-bound to the corresponding mtDNA. Parallel RNA and DNA immunoprecipitations from the same samples were quantified by RT-PCR and PCR, respectively. Relative bound tRNA level is expressed as a percentage of bound mtDNA.

    Techniques Used: Immunofluorescence, Fluorescence, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction

    TFAM binding of complex DNA and RNA substrates by EMSA. Varying amounts of TFAM were bound to 20 fM of each biotinylated substrate with as follows; (A) Stem-loop RNA, 2.4–0.0375 μM TFAM with two-fold serial dilutions, (B) dsRNA with internal 8 nucleotide mismatch loop, TFAM dilutions as in (A), apparent K d of 2.04 μM, (C) Alternating, four arm RNA:DNA 4-way junction, TFAM dilutions as in (A), apparent K d of 299 nM, (D) RNA 4-way junction, 600–9.375 nM TFAM with two-fold serial dilutions, apparent K d of 270 nM, (E) Mixed pairing RNA and DNA 4-way junction, TFAM dilutions as in (A), apparent K d of 63 nM, (F) DNA 4-way junction, TFAM dilutions as in (D), apparent K d of 63 nM. Left lane in each panel is free template without TFAM. Substrate diagrams appear to the right of each panel with RNA depicted in red and DNA in blue.
    Figure Legend Snippet: TFAM binding of complex DNA and RNA substrates by EMSA. Varying amounts of TFAM were bound to 20 fM of each biotinylated substrate with as follows; (A) Stem-loop RNA, 2.4–0.0375 μM TFAM with two-fold serial dilutions, (B) dsRNA with internal 8 nucleotide mismatch loop, TFAM dilutions as in (A), apparent K d of 2.04 μM, (C) Alternating, four arm RNA:DNA 4-way junction, TFAM dilutions as in (A), apparent K d of 299 nM, (D) RNA 4-way junction, 600–9.375 nM TFAM with two-fold serial dilutions, apparent K d of 270 nM, (E) Mixed pairing RNA and DNA 4-way junction, TFAM dilutions as in (A), apparent K d of 63 nM, (F) DNA 4-way junction, TFAM dilutions as in (D), apparent K d of 63 nM. Left lane in each panel is free template without TFAM. Substrate diagrams appear to the right of each panel with RNA depicted in red and DNA in blue.

    Techniques Used: Binding Assay

    Relative levels of TFAM-immunoprecipitated and total cellular mitochondrial tRNAs determined by RT-PCR. (A) Ranked relative levels of mitochondrial tRNAs purified with TFAM in RNA immunoprecipitations. (B) Relative detectable levels of mitochondrial tRNAs obtained from 1x10 6 3T3sw cells.
    Figure Legend Snippet: Relative levels of TFAM-immunoprecipitated and total cellular mitochondrial tRNAs determined by RT-PCR. (A) Ranked relative levels of mitochondrial tRNAs purified with TFAM in RNA immunoprecipitations. (B) Relative detectable levels of mitochondrial tRNAs obtained from 1x10 6 3T3sw cells.

    Techniques Used: Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction, Purification

    3) Product Images from "Cohesin SA1 and SA2 are RNA binding proteins that localize to RNA containing regions on DNA"

    Article Title: Cohesin SA1 and SA2 are RNA binding proteins that localize to RNA containing regions on DNA

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkaa284

    Cohesin SA1 and SA2 bind to the model R-loop substrate. ( A ) Native gel showing 25-nt RNA, the RNA:DNA hybrid, and the gel-purified three-stranded model R-loop substrate. In the schematic illustrations of the substrates, the star represents the 5′ fluorescein label. The sequences of RNA and DNA oligos for making the model R-loop substrate are shown in Supplementary Table S1 . ( B ) EMSA showing the S9.6 antibody binding to the model R-loop substrate (left panel), and no significant binding of the S9.6 antibody to the control 69-bp dsDNA (right panel). ( C ) EMSA showing EWSR1 binding to the model R-loop substrate, and no significant stable binding of EWSR1 to the control 66-bp dsDNA. ( D ) Fluorescence anisotropy showing concentration-dependent binding of SA1 and SA2 to the model R-loop substrate. The data were fitted to the law of mass action ( R 2 > 0.99). The error bars (standard deviations) are from three measurements. The equilibrium dissociation constants ( K d ) were calculated from at least two independent experiments (Table 1 ).
    Figure Legend Snippet: Cohesin SA1 and SA2 bind to the model R-loop substrate. ( A ) Native gel showing 25-nt RNA, the RNA:DNA hybrid, and the gel-purified three-stranded model R-loop substrate. In the schematic illustrations of the substrates, the star represents the 5′ fluorescein label. The sequences of RNA and DNA oligos for making the model R-loop substrate are shown in Supplementary Table S1 . ( B ) EMSA showing the S9.6 antibody binding to the model R-loop substrate (left panel), and no significant binding of the S9.6 antibody to the control 69-bp dsDNA (right panel). ( C ) EMSA showing EWSR1 binding to the model R-loop substrate, and no significant stable binding of EWSR1 to the control 66-bp dsDNA. ( D ) Fluorescence anisotropy showing concentration-dependent binding of SA1 and SA2 to the model R-loop substrate. The data were fitted to the law of mass action ( R 2 > 0.99). The error bars (standard deviations) are from three measurements. The equilibrium dissociation constants ( K d ) were calculated from at least two independent experiments (Table 1 ).

    Techniques Used: Purification, Binding Assay, Fluorescence, Concentration Assay

    SA1 and SA2 bind to double-stranded substrates containing RNA. ( A ) Schematic illustration of double-stranded substrates containing RNA that were used for fluorescence anisotropy experiments. The green star represents the 5′ fluorescein label. The RNA oligo sequences are shown in Supplementary Table S1 . (B and C) Concentration-dependent binding of SA1 ( B ) and SA2 ( C ) to dsRNA, dsRNA with an overhang, and RNA:DNA hybrids with or without overhangs. The data were fitted to the law of mass action ( R 2 > 0.96). The error bars (standard deviations) are from three measurements. The equilibrium dissociation constants ( K d ) were calculated from at least two independent experiments (Table 1 ).
    Figure Legend Snippet: SA1 and SA2 bind to double-stranded substrates containing RNA. ( A ) Schematic illustration of double-stranded substrates containing RNA that were used for fluorescence anisotropy experiments. The green star represents the 5′ fluorescein label. The RNA oligo sequences are shown in Supplementary Table S1 . (B and C) Concentration-dependent binding of SA1 ( B ) and SA2 ( C ) to dsRNA, dsRNA with an overhang, and RNA:DNA hybrids with or without overhangs. The data were fitted to the law of mass action ( R 2 > 0.96). The error bars (standard deviations) are from three measurements. The equilibrium dissociation constants ( K d ) were calculated from at least two independent experiments (Table 1 ).

    Techniques Used: Fluorescence, Concentration Assay, Binding Assay

    SA1 and SA2 localize to regions containing RNA on DNA tightropes. ( A ) Schematics of the DNA tightrope assay showing QD-labeled proteins (red) loading onto DNA (green) anchored be tween micron-sized beads. ( B ) QD conjugation strategy: His-NTA-biotin/streptavidin-QD sandwich method for His-tagged SA1 and SA2 proteins. ( C ) Schematics of ligated DNA tightropes containing R-loops formed through in vitro transcription. (D and E) Images (top panels) and kymographs (bottom panels) of red (655 nm) QD-labeled SA1 ( D ) and SA2 ( E ) on the ligated DNA tightropes containing R-loops (left panels) and control DNA tightropes (middle panels), and measurements of the distance between adjacent protein pairs on DNA tightropes (right panels). The dotted white lines mark the contour of the beads. SA1: N = 136 pairs on the control DNA; N = 175 pairs on the R-loop DNA. SA2: N = 116 pairs on the control DNA; N = 257 pairs on the R-loop DNA. Length scale bar: 5 μm; Time scale bar: 1 s.
    Figure Legend Snippet: SA1 and SA2 localize to regions containing RNA on DNA tightropes. ( A ) Schematics of the DNA tightrope assay showing QD-labeled proteins (red) loading onto DNA (green) anchored be tween micron-sized beads. ( B ) QD conjugation strategy: His-NTA-biotin/streptavidin-QD sandwich method for His-tagged SA1 and SA2 proteins. ( C ) Schematics of ligated DNA tightropes containing R-loops formed through in vitro transcription. (D and E) Images (top panels) and kymographs (bottom panels) of red (655 nm) QD-labeled SA1 ( D ) and SA2 ( E ) on the ligated DNA tightropes containing R-loops (left panels) and control DNA tightropes (middle panels), and measurements of the distance between adjacent protein pairs on DNA tightropes (right panels). The dotted white lines mark the contour of the beads. SA1: N = 136 pairs on the control DNA; N = 175 pairs on the R-loop DNA. SA2: N = 116 pairs on the control DNA; N = 257 pairs on the R-loop DNA. Length scale bar: 5 μm; Time scale bar: 1 s.

    Techniques Used: Labeling, Conjugation Assay, In Vitro

    4) Product Images from "Characterization of a novel type III CRISPR-Cas effector provides new insights into the allosteric activation and suppression of the Cas10 DNase"

    Article Title: Characterization of a novel type III CRISPR-Cas effector provides new insights into the allosteric activation and suppression of the Cas10 DNase

    Journal: Cell Discovery

    doi: 10.1038/s41421-020-0160-4

    Effect of LdCsm3 mutations on the ssDNA cleavage and binding of LdCsm. a Target RNA cleavage of LdCsm3 mutated derivatives. Fifty nM of S1–46 RNA were incubated with 50 nM of LdCsm or the indicated mutant derivatives for 10 min, and the samples were analyzed by denaturing PAGE. Duplex: Duplex of crRNA and substrate. b RNA-activated ssDNA cleavage by effectors carrying one of the constructed LdCsm3 mutants. Reaction conditions were the same as in Fig. 4b . c ssDNA binding by effectors carrying each of the constructed LdCsm3 mutants. Reaction conditions were the same as in Fig. 4c . d Relative ssDNA binding between the wild-type LdCsm effector and its LdCsm3 mutated derivatives. The ssDNA activity of LdCsm in non-homologous RNA was used as the standard and set up as 1. Results shown are average of three independent assays; bars represent the mean standard deviation (±SD). The red arrow indicates the ssDNA-LdCsm complex.
    Figure Legend Snippet: Effect of LdCsm3 mutations on the ssDNA cleavage and binding of LdCsm. a Target RNA cleavage of LdCsm3 mutated derivatives. Fifty nM of S1–46 RNA were incubated with 50 nM of LdCsm or the indicated mutant derivatives for 10 min, and the samples were analyzed by denaturing PAGE. Duplex: Duplex of crRNA and substrate. b RNA-activated ssDNA cleavage by effectors carrying one of the constructed LdCsm3 mutants. Reaction conditions were the same as in Fig. 4b . c ssDNA binding by effectors carrying each of the constructed LdCsm3 mutants. Reaction conditions were the same as in Fig. 4c . d Relative ssDNA binding between the wild-type LdCsm effector and its LdCsm3 mutated derivatives. The ssDNA activity of LdCsm in non-homologous RNA was used as the standard and set up as 1. Results shown are average of three independent assays; bars represent the mean standard deviation (±SD). The red arrow indicates the ssDNA-LdCsm complex.

    Techniques Used: Binding Assay, Incubation, Mutagenesis, Polyacrylamide Gel Electrophoresis, Construct, Activity Assay, Standard Deviation

    Target RNA-directed allosteric regulation of LdCsm involves activation and deactivation mechanisms. a CTR activates the LdCsm DNase. b NTR mediates autoimmunity avoidance by deactivation. Reactions were set up with 5 nM S10–60 ssDNA, 100 nM of LdCsm, and 400 nM non-homologous RNA (non-h. RNA) or 500 nM target RNA. After addition of one of the target RNAs, the mixture was incubated at 37 °C for 3 min. Samples were then analyzed by non-denaturing PAGE (in this page) or denaturing PAGE (Supplementary Fig. S8 ). Red arrowheads indicate the LdCsm–ssDNA complex. Relative ssDNA binding and percentage ssDNA cleaved of LdCsm facilitated by each of these target RNAs were estimated by image quantification of bands on non-denaturing PAGE and denaturing PAGE, using the accessory analysis tool equipped with a Typhoon FLA 7000. For the quantification of the substrate binding, the amount of ssDNA-LdCsm-CTR +6 complex is arbitrarily defined as 1. Results of average of three independent assays are shown with bars representing the standard deviation (±SD).
    Figure Legend Snippet: Target RNA-directed allosteric regulation of LdCsm involves activation and deactivation mechanisms. a CTR activates the LdCsm DNase. b NTR mediates autoimmunity avoidance by deactivation. Reactions were set up with 5 nM S10–60 ssDNA, 100 nM of LdCsm, and 400 nM non-homologous RNA (non-h. RNA) or 500 nM target RNA. After addition of one of the target RNAs, the mixture was incubated at 37 °C for 3 min. Samples were then analyzed by non-denaturing PAGE (in this page) or denaturing PAGE (Supplementary Fig. S8 ). Red arrowheads indicate the LdCsm–ssDNA complex. Relative ssDNA binding and percentage ssDNA cleaved of LdCsm facilitated by each of these target RNAs were estimated by image quantification of bands on non-denaturing PAGE and denaturing PAGE, using the accessory analysis tool equipped with a Typhoon FLA 7000. For the quantification of the substrate binding, the amount of ssDNA-LdCsm-CTR +6 complex is arbitrarily defined as 1. Results of average of three independent assays are shown with bars representing the standard deviation (±SD).

    Techniques Used: Activation Assay, Incubation, Polyacrylamide Gel Electrophoresis, Binding Assay, Standard Deviation

    Model of allosteric activation and repression of the LdCsm DNase. The previous works have proposed the initial recognition of nascent transcript at the 5ʹ end of target RNA for type III complex, since both of Csm5 subunit in Csm complex and Cmr1 subunit in Cmr complex are crucial for target RNA binding 36 , 37 , 68 . These suggested that the binary LdCsm effector complex interacts with target transcript initially at the 5ʹ end of target RNA and further via sequence complementarity between the protospacer and the corresponding crRNA, leading to the formation of a ternary effector complex with a major conformational change. Addition of a single nucleotide at the 3ʹ-end of protospacer RNA results in an important allosteric change in the LdCsm DNase, giving an active enzyme. CTR-bound LdCsm exhibits the full level of substrate binding and DNA cleavage, whereas NTR-bound LdCsm closes the substrate-binding pocket, which deactivates the DNase. Finally, multiple Csm3 subunits cleave the target transcripts, and release of target RNA cleavage products restores the binary conformation, completing the spatiotemporal regulation of LdCsm systems.
    Figure Legend Snippet: Model of allosteric activation and repression of the LdCsm DNase. The previous works have proposed the initial recognition of nascent transcript at the 5ʹ end of target RNA for type III complex, since both of Csm5 subunit in Csm complex and Cmr1 subunit in Cmr complex are crucial for target RNA binding 36 , 37 , 68 . These suggested that the binary LdCsm effector complex interacts with target transcript initially at the 5ʹ end of target RNA and further via sequence complementarity between the protospacer and the corresponding crRNA, leading to the formation of a ternary effector complex with a major conformational change. Addition of a single nucleotide at the 3ʹ-end of protospacer RNA results in an important allosteric change in the LdCsm DNase, giving an active enzyme. CTR-bound LdCsm exhibits the full level of substrate binding and DNA cleavage, whereas NTR-bound LdCsm closes the substrate-binding pocket, which deactivates the DNase. Finally, multiple Csm3 subunits cleave the target transcripts, and release of target RNA cleavage products restores the binary conformation, completing the spatiotemporal regulation of LdCsm systems.

    Techniques Used: Activation Assay, RNA Binding Assay, Sequencing, Binding Assay

    Cloning, expression, and purification of the L. delbrueckii subsp. bulgaricus Csm complex in E. coli . a Schematic of the LdCsm system. LdCsm genes and the adjacent CRISPR assay are indicated with filled large arrows and small rectangles, respectively. Line with an arrowhead denotes the promoter of the csm gene cassette and the direction of transcription. b Strategy for reconstitution of the LdCsm effector in E. coli . LdCsm genes ( cas6 + csm1–5 genes) were cloned into p15AIE, yielding p15AIE-cas (Supplementary Fig. S2 ). A CRISPR array carrying 10 copies of S1 spacer was generated and inserted into pUCE, giving pUCE-S1 (Supplementary Fig. S2 ). LdCsm2 was cloned into pET30a, giving pET30a-Csm2 that yields the His-tagged Csm2 upon plasmid-born gene expression in the cell. All three plasmids were introduced into E. coli BL21(DE3) by electroporation. c UV spectrum of SEC purification of LdCsm effector complex. E. coli cell extracts were employed for Nickel-His tag affinity purification of LdCsm2 by which LdCsm effector complexes were copurified. The resulting protein samples were further purified by SEC. Blue: UV absorbance at 280 nm; red: UV absorbance at 254 nm. d SDS-PAGE analysis of SEC samples collected in the peak region. M: protein mass marker; Input: proteins purified by nickel Csm2-His affinity chromatography. e Denaturing gel electrophoresis of 5ʹ-labeled RNAs from LdCsm samples. RNAs were extracted from the SEC-purified LdCsm samples. M: RNA size ladder.
    Figure Legend Snippet: Cloning, expression, and purification of the L. delbrueckii subsp. bulgaricus Csm complex in E. coli . a Schematic of the LdCsm system. LdCsm genes and the adjacent CRISPR assay are indicated with filled large arrows and small rectangles, respectively. Line with an arrowhead denotes the promoter of the csm gene cassette and the direction of transcription. b Strategy for reconstitution of the LdCsm effector in E. coli . LdCsm genes ( cas6 + csm1–5 genes) were cloned into p15AIE, yielding p15AIE-cas (Supplementary Fig. S2 ). A CRISPR array carrying 10 copies of S1 spacer was generated and inserted into pUCE, giving pUCE-S1 (Supplementary Fig. S2 ). LdCsm2 was cloned into pET30a, giving pET30a-Csm2 that yields the His-tagged Csm2 upon plasmid-born gene expression in the cell. All three plasmids were introduced into E. coli BL21(DE3) by electroporation. c UV spectrum of SEC purification of LdCsm effector complex. E. coli cell extracts were employed for Nickel-His tag affinity purification of LdCsm2 by which LdCsm effector complexes were copurified. The resulting protein samples were further purified by SEC. Blue: UV absorbance at 280 nm; red: UV absorbance at 254 nm. d SDS-PAGE analysis of SEC samples collected in the peak region. M: protein mass marker; Input: proteins purified by nickel Csm2-His affinity chromatography. e Denaturing gel electrophoresis of 5ʹ-labeled RNAs from LdCsm samples. RNAs were extracted from the SEC-purified LdCsm samples. M: RNA size ladder.

    Techniques Used: Clone Assay, Expressing, Purification, CRISPR, Generated, Plasmid Preparation, Electroporation, Affinity Purification, SDS Page, Marker, Affinity Chromatography, Nucleic Acid Electrophoresis, Labeling

    Biochemical characterization of the LdCsm effector complex. a Schematic of three different homologous target RNAs: CTR cognate target RNA carrying 6-nt 3ʹ anti-tag with mismatch to the 5ʹ tag of the corresponding crRNA, NTR noncognate target RNA containing 8-nt 3ʹ anti-tag that is complementary to the 5ʹ tag of the corresponding crRNA, PTR 40 nt protospacer target RNA completely lacking 3ʹ anti-tag. b Analysis of target RNA cleavage by LdCsm. Different target RNAs (50 nM) were individually mixed with 50 nM LdCsm and incubated for 10 min. The resulting samples were analyzed by denaturing PAGE. Duplex: Duplex of crRNA and substrate. c Analysis of RNA-activated ssDNA cleavage by LdCsm. 50 nM S10–60 ssDNA substrate was mixed with 50 nM LdCsm and 500 nM of each of the target RNA and incubated for 10 min. Samples were analyzed by denaturing PAGE. d Analysis of cOA synthesis by LdCsm. Approximately 2 nM [α- 32 P]-ATP was mixed with a range of cold ATP (48 nM–1 mM) and incubated with 50 nM LdCsm in the presence of 500 nM CTR for 120 min; the S. islandicus Cmr-α complex was used as the positive reference.
    Figure Legend Snippet: Biochemical characterization of the LdCsm effector complex. a Schematic of three different homologous target RNAs: CTR cognate target RNA carrying 6-nt 3ʹ anti-tag with mismatch to the 5ʹ tag of the corresponding crRNA, NTR noncognate target RNA containing 8-nt 3ʹ anti-tag that is complementary to the 5ʹ tag of the corresponding crRNA, PTR 40 nt protospacer target RNA completely lacking 3ʹ anti-tag. b Analysis of target RNA cleavage by LdCsm. Different target RNAs (50 nM) were individually mixed with 50 nM LdCsm and incubated for 10 min. The resulting samples were analyzed by denaturing PAGE. Duplex: Duplex of crRNA and substrate. c Analysis of RNA-activated ssDNA cleavage by LdCsm. 50 nM S10–60 ssDNA substrate was mixed with 50 nM LdCsm and 500 nM of each of the target RNA and incubated for 10 min. Samples were analyzed by denaturing PAGE. d Analysis of cOA synthesis by LdCsm. Approximately 2 nM [α- 32 P]-ATP was mixed with a range of cold ATP (48 nM–1 mM) and incubated with 50 nM LdCsm in the presence of 500 nM CTR for 120 min; the S. islandicus Cmr-α complex was used as the positive reference.

    Techniques Used: Incubation, Polyacrylamide Gel Electrophoresis

    Effect of LdCsm1 mutations on ssDNA binding and cleavage by the LdCsm effector complex. a Domain architecture of the LdCsm1 protein. HD represents the HD-type nuclease domain; Palm 1 and Palm 2 denote the two cyclase domains; Linker is a domain that adjoins the Palm1 and Palm2 domains, consisting of four cysteine residues; D4 is located in the C-terminus rich in α-helices. Amino acid residues selected for alanine substitution mutagenesis are indicated with their names and positions. b RNA-activated ssDNA cleavage by effectors carrying one of the constructed LdCsm1 mutants. Fifty nM S10–60 ssDNA substrates were mixed with 50 nM mutated LdCsm carrying each of LdCsm1 mutant proteins and 500 nM CTR and incubated for 10 min. Samples were analyzed by denaturing PAGE. c ssDNA binding by effectors carrying each of the constructed LdCsm1 mutants. Five nM labeled S10–60 ssDNA were incubated for 3 min with 100 nM of LdCsm effectors in the presence of 400 nM of non-homologous RNA (S10 RNA) or 500 nM of one of the target RNAs, PTR or CTR or NTR. Samples were analyzed by non-denaturing PAGE. Red arrowheads indicate the Csm–ssDNA complex. d Relative ssDNA binding between the wild-type LdCsm effector and its LdCsm1 mutated derivatives. The relative ssDNA-binding activities were estimated by image quantification of the non-denaturing PAGE in c by the accessory analysis tool in Typhoon FLA 7000, the ssDNA activity of LdCsm in non-homologous RNA was used as the standard and set up as 1. Results shown are average of three independent assays; bars represent the mean standard deviation (±SD).
    Figure Legend Snippet: Effect of LdCsm1 mutations on ssDNA binding and cleavage by the LdCsm effector complex. a Domain architecture of the LdCsm1 protein. HD represents the HD-type nuclease domain; Palm 1 and Palm 2 denote the two cyclase domains; Linker is a domain that adjoins the Palm1 and Palm2 domains, consisting of four cysteine residues; D4 is located in the C-terminus rich in α-helices. Amino acid residues selected for alanine substitution mutagenesis are indicated with their names and positions. b RNA-activated ssDNA cleavage by effectors carrying one of the constructed LdCsm1 mutants. Fifty nM S10–60 ssDNA substrates were mixed with 50 nM mutated LdCsm carrying each of LdCsm1 mutant proteins and 500 nM CTR and incubated for 10 min. Samples were analyzed by denaturing PAGE. c ssDNA binding by effectors carrying each of the constructed LdCsm1 mutants. Five nM labeled S10–60 ssDNA were incubated for 3 min with 100 nM of LdCsm effectors in the presence of 400 nM of non-homologous RNA (S10 RNA) or 500 nM of one of the target RNAs, PTR or CTR or NTR. Samples were analyzed by non-denaturing PAGE. Red arrowheads indicate the Csm–ssDNA complex. d Relative ssDNA binding between the wild-type LdCsm effector and its LdCsm1 mutated derivatives. The relative ssDNA-binding activities were estimated by image quantification of the non-denaturing PAGE in c by the accessory analysis tool in Typhoon FLA 7000, the ssDNA activity of LdCsm in non-homologous RNA was used as the standard and set up as 1. Results shown are average of three independent assays; bars represent the mean standard deviation (±SD).

    Techniques Used: Binding Assay, Mutagenesis, Construct, Incubation, Polyacrylamide Gel Electrophoresis, Labeling, Activity Assay, Standard Deviation

    5) Product Images from "Force-dependent stimulation of RNA unwinding by SARS-CoV-2 nsp13 helicase"

    Article Title: Force-dependent stimulation of RNA unwinding by SARS-CoV-2 nsp13 helicase

    Journal: bioRxiv

    doi: 10.1101/2020.07.31.231274

    Single-molecule measurement of nsp13 helicase activity. a. Assay geometry showing dsRNA hairpin complex. dsDNA handles separate the hairpin from the beads (connected by biotin-streptavidin, B-S). A 20 nucleotide (nt) region allows single nsp13 molecules to bind and begin 5’-3’ helicase activity. Motion of the optically-trapped bead (top) under force-feedback enables a set force to be applied to the dsRNA hairpin substrate. Diagram not to scale. b. Representative force-extension curve of a single DNA/RNA tether (no nsp13 present). Above 20 pN, external force begins to unwind the RNA hairpin. c. Representative example trace at 4 pN. Only one event is observed in ~250 s. d. Zoomed-in blue region from panel c. e. Representative example trace at 18 pN. Numerous events are observed in ~250 s. f. Zoomed-in orange region from panel e. Arrows denote slippage events. Red dotted line denotes 180 bp, the total length of the RNA hairpin. g. Zoomed-in purple region from panel d. Red dotted line denotes 180 bp, the total length of the RNA hairpin.
    Figure Legend Snippet: Single-molecule measurement of nsp13 helicase activity. a. Assay geometry showing dsRNA hairpin complex. dsDNA handles separate the hairpin from the beads (connected by biotin-streptavidin, B-S). A 20 nucleotide (nt) region allows single nsp13 molecules to bind and begin 5’-3’ helicase activity. Motion of the optically-trapped bead (top) under force-feedback enables a set force to be applied to the dsRNA hairpin substrate. Diagram not to scale. b. Representative force-extension curve of a single DNA/RNA tether (no nsp13 present). Above 20 pN, external force begins to unwind the RNA hairpin. c. Representative example trace at 4 pN. Only one event is observed in ~250 s. d. Zoomed-in blue region from panel c. e. Representative example trace at 18 pN. Numerous events are observed in ~250 s. f. Zoomed-in orange region from panel e. Arrows denote slippage events. Red dotted line denotes 180 bp, the total length of the RNA hairpin. g. Zoomed-in purple region from panel d. Red dotted line denotes 180 bp, the total length of the RNA hairpin.

    Techniques Used: Activity Assay

    Biochemical characterization of nsp13 a. Diagram showing the domain architecture of SARS-CoV-2 nsp13. b. Size exclusion chromatography (Superdex 200 Increase) of purified nsp13. c. SDS-PAGE gel (Coomassie stain) showing nsp13 purity. d. Differential scanning fluorimetry (DSF) of SARS-CoV-2 nsp13 in the absence and presence of ATP analogs (1 mM). Melting temperatures are: APO- 40.5 °C, ATPγS- 45.5 °C, ADP:AlF 3 - 52.5 °C, and ADP- 41.0 °C for n=2 independent measurements from two separate protein preparations. e. Fluorescence anisotropy measurements for fluorescence DNA and RNA partial duplexes (10 nM) with and without nsp13 (3.4 μM) added. Data shown as mean standard ± deviation (SD) for n=2 independent measurements from two separate protein preparations.
    Figure Legend Snippet: Biochemical characterization of nsp13 a. Diagram showing the domain architecture of SARS-CoV-2 nsp13. b. Size exclusion chromatography (Superdex 200 Increase) of purified nsp13. c. SDS-PAGE gel (Coomassie stain) showing nsp13 purity. d. Differential scanning fluorimetry (DSF) of SARS-CoV-2 nsp13 in the absence and presence of ATP analogs (1 mM). Melting temperatures are: APO- 40.5 °C, ATPγS- 45.5 °C, ADP:AlF 3 - 52.5 °C, and ADP- 41.0 °C for n=2 independent measurements from two separate protein preparations. e. Fluorescence anisotropy measurements for fluorescence DNA and RNA partial duplexes (10 nM) with and without nsp13 (3.4 μM) added. Data shown as mean standard ± deviation (SD) for n=2 independent measurements from two separate protein preparations.

    Techniques Used: Size-exclusion Chromatography, Purification, SDS Page, Staining, Fluorescence, Standard Deviation

    Nsp13 is a DNA and RNA unwindase a. Diagram of fluorescence-based helicase assay using a partial duplex oligonucleotide substrate with 10-nt overhang. Unwinding of the duplex results in a fluorescent signal. b. Nsp13 helicase activity (10 nM) as a function of time with increasing DNA substrate concentration. c. Nsp13 helicase activity as a function of time with a constant concentration of DNA (1 μM) and increasing nsp13 concentrations. d. Plot of initial substrate unwinding velocities versus substrate concentration (0-4 μM for DNA, 0-6 μM for RNA). Data points show the mean ± SD for n = 2 or 3 measurements. Fitting the data points to the Michaelis-Menten model revealed K M and k cat values for DNA unwinding (inset; fit ±95% confidence intervals). e. Nsp13 helicase activity (10 nM) as a function of time with increasing RNA substrate concentration. f. Initial DNA unwinding rates at varying KCl concentration for DNA and RNA substrates. Data points show the mean ± SD for n = 2 measurements. g. Dose-response curves of nsp13 helicase activity in the presence of increasing concentration of ADP:AlF 3 (0-500 μM). Data points show the mean ± SD for n = 3 measurements. IC 50 values were determined by fitting the data to the Hill equation (inset).
    Figure Legend Snippet: Nsp13 is a DNA and RNA unwindase a. Diagram of fluorescence-based helicase assay using a partial duplex oligonucleotide substrate with 10-nt overhang. Unwinding of the duplex results in a fluorescent signal. b. Nsp13 helicase activity (10 nM) as a function of time with increasing DNA substrate concentration. c. Nsp13 helicase activity as a function of time with a constant concentration of DNA (1 μM) and increasing nsp13 concentrations. d. Plot of initial substrate unwinding velocities versus substrate concentration (0-4 μM for DNA, 0-6 μM for RNA). Data points show the mean ± SD for n = 2 or 3 measurements. Fitting the data points to the Michaelis-Menten model revealed K M and k cat values for DNA unwinding (inset; fit ±95% confidence intervals). e. Nsp13 helicase activity (10 nM) as a function of time with increasing RNA substrate concentration. f. Initial DNA unwinding rates at varying KCl concentration for DNA and RNA substrates. Data points show the mean ± SD for n = 2 measurements. g. Dose-response curves of nsp13 helicase activity in the presence of increasing concentration of ADP:AlF 3 (0-500 μM). Data points show the mean ± SD for n = 3 measurements. IC 50 values were determined by fitting the data to the Hill equation (inset).

    Techniques Used: Fluorescence, Helicase Assay, Activity Assay, Concentration Assay

    6) Product Images from "Evolutionarily Conserved Roles of the Dicer Helicase Domain in Regulating RNA Interference Processing"

    Article Title: Evolutionarily Conserved Roles of the Dicer Helicase Domain in Regulating RNA Interference Processing

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M114.589051

    DNA and RNA Substrates
    Figure Legend Snippet: DNA and RNA Substrates

    Techniques Used:

    7) Product Images from "Characterization of a novel type III CRISPR-Cas effector provides new insights into the allosteric activation and suppression of the Cas10 DNase"

    Article Title: Characterization of a novel type III CRISPR-Cas effector provides new insights into the allosteric activation and suppression of the Cas10 DNase

    Journal: bioRxiv

    doi: 10.1101/2019.12.17.879585

    Effect of LdCsm3 mutations on the ssDNA cleavage and binding of LdCsm. ( a ) Target RNA cleavage of LdCsm3 mutated derivatives. 50 nM of S1-46 RNA were incubated with 50 nM of LdCsm or the indicated mutant derivatives for 10 min and the samples were analyzed by denaturing PAGE. Duplex: Duplex of crRNA and substrate. ( b ) RNA-activated ssDNA cleavage by effectors carrying one of the constructed LdCsm3 mutants. Reaction conditions were the same as in Fig. 4b . ( c ) ssDNA binding by effectors carrying each of the constructed LdCsm3 mutants. Reaction conditions were the same as in Fig. 4c . ( d ) Relative ssDNA binding between the wild-type LdCsm effector and its LdCsm3 mutated derivatives. The ssDNA activity of LdCsm in non-homologous RNA was used as the standard and set up as 1. Results shown are average of three independent assays, bars represent the mean standard deviation (± SD).
    Figure Legend Snippet: Effect of LdCsm3 mutations on the ssDNA cleavage and binding of LdCsm. ( a ) Target RNA cleavage of LdCsm3 mutated derivatives. 50 nM of S1-46 RNA were incubated with 50 nM of LdCsm or the indicated mutant derivatives for 10 min and the samples were analyzed by denaturing PAGE. Duplex: Duplex of crRNA and substrate. ( b ) RNA-activated ssDNA cleavage by effectors carrying one of the constructed LdCsm3 mutants. Reaction conditions were the same as in Fig. 4b . ( c ) ssDNA binding by effectors carrying each of the constructed LdCsm3 mutants. Reaction conditions were the same as in Fig. 4c . ( d ) Relative ssDNA binding between the wild-type LdCsm effector and its LdCsm3 mutated derivatives. The ssDNA activity of LdCsm in non-homologous RNA was used as the standard and set up as 1. Results shown are average of three independent assays, bars represent the mean standard deviation (± SD).

    Techniques Used: Binding Assay, Incubation, Mutagenesis, Polyacrylamide Gel Electrophoresis, Construct, Activity Assay, Standard Deviation

    Target RNA-directed allosteric regulation of LdCsm involves activation and deactivation mechanisms. ( a ) CTR activates the LdCsm DNase. ( b ) NTR mediates autoimmunity avoidance by deactivation. Reactions were set up with 5 nM S10-60 ssDNA, 100 nM of LdCsm and 400 nM non-homologous RNA (non-h. RNA) or 500 nM target RNA. After addition of one of target RNAs, the mixture was incubated at 37 °C for 3 min. Samples were then analyzed by non-denaturing PAGE (in this page) or denaturing PAGE (Supplementary Fig. S8). Red arrowheads indicate the LdCsm-ssDNA complex. Relative ssDNA binding and percentage ssDNA cleaved of LdCsm facilitated by each of these target RNAs were estimated by image quantification of bands on non-denaturing PAGE and denaturing PAGE, using the accessory analysis tool equipped with a Typhoon FLA 7000. For the quantification of the substrate binding, the amount of ssDNA-LdCsm-CTR +6 complex is arbitrarily defined as 1. Results of average of three independent assays are shown with bars representing the standard deviation (± SD).
    Figure Legend Snippet: Target RNA-directed allosteric regulation of LdCsm involves activation and deactivation mechanisms. ( a ) CTR activates the LdCsm DNase. ( b ) NTR mediates autoimmunity avoidance by deactivation. Reactions were set up with 5 nM S10-60 ssDNA, 100 nM of LdCsm and 400 nM non-homologous RNA (non-h. RNA) or 500 nM target RNA. After addition of one of target RNAs, the mixture was incubated at 37 °C for 3 min. Samples were then analyzed by non-denaturing PAGE (in this page) or denaturing PAGE (Supplementary Fig. S8). Red arrowheads indicate the LdCsm-ssDNA complex. Relative ssDNA binding and percentage ssDNA cleaved of LdCsm facilitated by each of these target RNAs were estimated by image quantification of bands on non-denaturing PAGE and denaturing PAGE, using the accessory analysis tool equipped with a Typhoon FLA 7000. For the quantification of the substrate binding, the amount of ssDNA-LdCsm-CTR +6 complex is arbitrarily defined as 1. Results of average of three independent assays are shown with bars representing the standard deviation (± SD).

    Techniques Used: Activation Assay, Incubation, Polyacrylamide Gel Electrophoresis, Binding Assay, Standard Deviation

    Model of allosteric activation and repression of the LdCsm DNase. The previous works have proposed that the initial recognition of nascent transcript at the 5′ end of target RNA for type III complex, since both of Csm5 subunit in Csm complex and Cmr1 subunit in Cmr complex are crucial for target RNA binding 36 , 37 , 68 . These suggested that the binary LdCsm effector complex interacts with target transcript initially at the 5′ end of target RNA and further via sequence complementarity between the protospacer and the corresponding crRNA, leading to the formation of a ternary effector complex with a major conformational change. Addition of a single nucleotide at the 3′-end of protospacer RNA results in an important allosteric change in the LdCsm DNase, giving an active enzyme. CTR-bound LdCsm exhibits the full level of substrate binding and DNA cleavage whereas NTR-bound LdCsm closes the substrate-binding pocket, which deactivates the DNase. Finally, multiple Csm3 subunits cleave the target transcripts, and release of target RNA cleavage products restores the binary conformation, completing the spatiotemporal regulation of LdCsm systems.
    Figure Legend Snippet: Model of allosteric activation and repression of the LdCsm DNase. The previous works have proposed that the initial recognition of nascent transcript at the 5′ end of target RNA for type III complex, since both of Csm5 subunit in Csm complex and Cmr1 subunit in Cmr complex are crucial for target RNA binding 36 , 37 , 68 . These suggested that the binary LdCsm effector complex interacts with target transcript initially at the 5′ end of target RNA and further via sequence complementarity between the protospacer and the corresponding crRNA, leading to the formation of a ternary effector complex with a major conformational change. Addition of a single nucleotide at the 3′-end of protospacer RNA results in an important allosteric change in the LdCsm DNase, giving an active enzyme. CTR-bound LdCsm exhibits the full level of substrate binding and DNA cleavage whereas NTR-bound LdCsm closes the substrate-binding pocket, which deactivates the DNase. Finally, multiple Csm3 subunits cleave the target transcripts, and release of target RNA cleavage products restores the binary conformation, completing the spatiotemporal regulation of LdCsm systems.

    Techniques Used: Activation Assay, RNA Binding Assay, Sequencing, Binding Assay

    Cloning, expression and purification of the L. delbrueckii subsp. bulgaricus Csm complex in E. coli . ( a ) Schematic of the LdCsm system. LdCsm genes and the adjacent CRISPR assay are indicated with filled large arrows and small rectangles, respectively. Line with an arrowhead denotes the promoter of the csm gene cassette and the direction of transcription. ( b ) Strategy for reconstitution of the LdCsm effector in E. coli . LdCsm genes ( cas6 + csm1-5 genes) were cloned into p15AIE, yielding p15AIE-cas (Supplementary Fig. S2). A CRISPR array carrying 10 copies of S1 spacer was generated and inserted into pUCE, giving pUCE-S1 (Supplementary Fig. S2). LdCsm2 was cloned into pET30a, giving pET30a-Csm2 that yields the His-tagged Csm2 upon plasmid-born gene expression in the cell. All three plasmids were introduced into E. coli BL21(DE3) by electroporation. ( c ) UV spectrum of SEC purification of LdCsm effector complex. E. coli cell extracts were employed for Nickel-His tag affinity purification of LdCsm2 by which LdCsm effector complexes were copurified. The resulting protein samples were further purified by SEC. Blue: UV absorbance at 280 nm; red: UV absorbance at 254 nm. ( d ) SDS-PAGE analysis of SEC samples collected in the peak region. M: protein mass marker; Input: proteins purified by nickel Csm2-His affinity chromatography. ( e ) Denaturing gel electrophoresis of 5′-labeled RNAs from LdCsm samples. RNAs were extracted from the SEC-purified LdCsm samples. M: RNA size ladder.
    Figure Legend Snippet: Cloning, expression and purification of the L. delbrueckii subsp. bulgaricus Csm complex in E. coli . ( a ) Schematic of the LdCsm system. LdCsm genes and the adjacent CRISPR assay are indicated with filled large arrows and small rectangles, respectively. Line with an arrowhead denotes the promoter of the csm gene cassette and the direction of transcription. ( b ) Strategy for reconstitution of the LdCsm effector in E. coli . LdCsm genes ( cas6 + csm1-5 genes) were cloned into p15AIE, yielding p15AIE-cas (Supplementary Fig. S2). A CRISPR array carrying 10 copies of S1 spacer was generated and inserted into pUCE, giving pUCE-S1 (Supplementary Fig. S2). LdCsm2 was cloned into pET30a, giving pET30a-Csm2 that yields the His-tagged Csm2 upon plasmid-born gene expression in the cell. All three plasmids were introduced into E. coli BL21(DE3) by electroporation. ( c ) UV spectrum of SEC purification of LdCsm effector complex. E. coli cell extracts were employed for Nickel-His tag affinity purification of LdCsm2 by which LdCsm effector complexes were copurified. The resulting protein samples were further purified by SEC. Blue: UV absorbance at 280 nm; red: UV absorbance at 254 nm. ( d ) SDS-PAGE analysis of SEC samples collected in the peak region. M: protein mass marker; Input: proteins purified by nickel Csm2-His affinity chromatography. ( e ) Denaturing gel electrophoresis of 5′-labeled RNAs from LdCsm samples. RNAs were extracted from the SEC-purified LdCsm samples. M: RNA size ladder.

    Techniques Used: Clone Assay, Expressing, Purification, CRISPR, Generated, Plasmid Preparation, Electroporation, Affinity Purification, SDS Page, Marker, Affinity Chromatography, Nucleic Acid Electrophoresis, Labeling

    Biochemical characterization of the LdCsm effector complex. ( a ) Schematic of three different homologous target RNAs: CTR, cognate target RNA carrying 6-nt 3′ anti-tag with mismatch to the 5′ tag of the corresponding crRNA; NTR, noncognate target RNA containing 8-nt 3′ anti-tag that is complementary to the 5′ tag of the corresponding crRNA, and PTR, 40 nt protospacer target RNA completely lacking 3′ anti-tag. ( b ) Analysis of target RNA cleavage by LdCsm. Different target RNAs (50 nM) were individually mixed with 50 nM LdCsm and incubated for 10 min. The resulting samples were analyzed by denaturing PAGE. Duplex: Duplex of crRNA and substrate. ( c ) Analysis of RNA-activated ssDNA cleavage by LdCsm. 50 nM S10-60 ssDNA substrate was mixed with 50 nM LdCsm and 500 nM of each of the target RNA and incubated for 10 min. Samples were analyzed by denaturing PAGE. ( d ) Analysis of cOA synthesis by LdCsm. ~2 nM [α- 32 P]-ATP was mixed with a range of cold ATP (48 nM – 1 mM) and incubated with 50 nM LdCsm in the presence of 500 nM CTR for 120 min, the S. islandicus Cmr-α complex was used as the positive reference.
    Figure Legend Snippet: Biochemical characterization of the LdCsm effector complex. ( a ) Schematic of three different homologous target RNAs: CTR, cognate target RNA carrying 6-nt 3′ anti-tag with mismatch to the 5′ tag of the corresponding crRNA; NTR, noncognate target RNA containing 8-nt 3′ anti-tag that is complementary to the 5′ tag of the corresponding crRNA, and PTR, 40 nt protospacer target RNA completely lacking 3′ anti-tag. ( b ) Analysis of target RNA cleavage by LdCsm. Different target RNAs (50 nM) were individually mixed with 50 nM LdCsm and incubated for 10 min. The resulting samples were analyzed by denaturing PAGE. Duplex: Duplex of crRNA and substrate. ( c ) Analysis of RNA-activated ssDNA cleavage by LdCsm. 50 nM S10-60 ssDNA substrate was mixed with 50 nM LdCsm and 500 nM of each of the target RNA and incubated for 10 min. Samples were analyzed by denaturing PAGE. ( d ) Analysis of cOA synthesis by LdCsm. ~2 nM [α- 32 P]-ATP was mixed with a range of cold ATP (48 nM – 1 mM) and incubated with 50 nM LdCsm in the presence of 500 nM CTR for 120 min, the S. islandicus Cmr-α complex was used as the positive reference.

    Techniques Used: Incubation, Polyacrylamide Gel Electrophoresis

    Effect of LdCsm1 mutations on ssDNA binding and cleavage by the LdCsm effector complex. ( a ) Domain architecture of the LdCsm1 protein. HD represents the HD-type nuclease domain; Palm 1 and Palm 2 denote the two cyclase domains; Linker is a domain that adjoins the Palm1 and Palm2 domains, consisting 4 cysteine residues, D4 is located in the C-terminus rich in α-helices. Amino acid residues selected for alanine substitution mutagenesis are indicated with their names and positions. ( b ) RNA-activated ssDNA cleavage by effectors carrying one of the constructed LdCsm1 mutants. 50 nM S10-60 ssDNA substrates were mixed with 50 nM mutated LdCsm carrying each of LdCsm1 mutant proteins and 500 nM CTR, and incubated for 10 min. Samples were analyzed by denaturing PAGE. ( c ) ssDNA binding by effectors carrying each of the constructed LdCsm1 mutants. 5 nM labeled S10-60 ssDNA were incubated with 100 nM of LdCsm effectors indicated in each experiment. 400 nM of non-homologous RNA (S10 RNA) or in the presence of 500 nM of one of the target RNAs, PTR or CTR or NTR for 3 min. Samples were analyzed by non-denaturing PAGE. Red arrowheads indicate the Csm-ssDNA complex. ( d ) Relative ssDNA binding between the wild-type LdCsm effector and its LdCsm1 mutated derivatives. The relative ssDNA binding activities were estimated by image quantification of the non-denaturing PAGE in (c) by the accessory analysis tool in Typhoon FLA 7000, the ssDNA activity of LdCsm in non-homologous RNA was used as the standard and set up as 1. Results shown are average of three independent assays, bars represent the mean standard deviation (± SD).
    Figure Legend Snippet: Effect of LdCsm1 mutations on ssDNA binding and cleavage by the LdCsm effector complex. ( a ) Domain architecture of the LdCsm1 protein. HD represents the HD-type nuclease domain; Palm 1 and Palm 2 denote the two cyclase domains; Linker is a domain that adjoins the Palm1 and Palm2 domains, consisting 4 cysteine residues, D4 is located in the C-terminus rich in α-helices. Amino acid residues selected for alanine substitution mutagenesis are indicated with their names and positions. ( b ) RNA-activated ssDNA cleavage by effectors carrying one of the constructed LdCsm1 mutants. 50 nM S10-60 ssDNA substrates were mixed with 50 nM mutated LdCsm carrying each of LdCsm1 mutant proteins and 500 nM CTR, and incubated for 10 min. Samples were analyzed by denaturing PAGE. ( c ) ssDNA binding by effectors carrying each of the constructed LdCsm1 mutants. 5 nM labeled S10-60 ssDNA were incubated with 100 nM of LdCsm effectors indicated in each experiment. 400 nM of non-homologous RNA (S10 RNA) or in the presence of 500 nM of one of the target RNAs, PTR or CTR or NTR for 3 min. Samples were analyzed by non-denaturing PAGE. Red arrowheads indicate the Csm-ssDNA complex. ( d ) Relative ssDNA binding between the wild-type LdCsm effector and its LdCsm1 mutated derivatives. The relative ssDNA binding activities were estimated by image quantification of the non-denaturing PAGE in (c) by the accessory analysis tool in Typhoon FLA 7000, the ssDNA activity of LdCsm in non-homologous RNA was used as the standard and set up as 1. Results shown are average of three independent assays, bars represent the mean standard deviation (± SD).

    Techniques Used: Binding Assay, Mutagenesis, Construct, Incubation, Polyacrylamide Gel Electrophoresis, Labeling, Activity Assay, Standard Deviation

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    Integrated DNA Technologies ssdna rna substrates
    Purification and biochemical characterization of rA3G-CD1. ( a ) Phylogenetic tree of A3 domains, with rA3G-CD1 in red. ( b ) Sequence alignments of loop-8 region for rA3G-CD1 (used for crystallization), rA3G-CD1 (WT), hA3G-CD1 (WT), hA3G-CD2 and A3G-sNTD. The four residues in red are the only mutated sequence in the rA3G-CD1 construct. ( c ) Elution profile of rA3G-CD1 analyzed by size-exclusion chromatography (Suprose 6 10/300 GL) before (red) and after (blue) PEI treatment. Inset: SDS–PAGE shows the purified rA3G-CD1 from LMWt that was used for biochemical studies and crystallization. ( d ) Deamination assays of rA3G-CD1, hA3G-CD2, hA3F-CD2 and rA3G-CD2 with a 5′-FAM-labelled 30 nt <t>ssDNA.</t> 17 nt and 15 nt are the products by deamination of the 3rd and 1st C from 5′ end, respectively. ( e ) Electrophoresis mobility shift assays (EMSA) of purified rA3G-CD1 binding to 5′-FAM-labelled 50 nt ssDNA (top panel) and <t>RNA</t> (bottom panel). ( f ) ITC of rA3G-CD1 binding to short 10 nt ssDNA (poly-dT, left panel) and 10 nt RNA (right panel).
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    Integrated DNA Technologies rna substrates
    Primer extension inhibition analyses of mRNA cleavage by GB1_HP0315. Lanes 1–4 are <t>DNA</t> sequence ladders. Lane 5–9 is primer extension products of mRNA of HP0315 after digestion with various amounts of GB1_HP0315. Cleavage sites are indicated by sequential numbers on right side of the images. The <t>RNA</t> recognition sequences analyzed by the DNA sequencing ladder are on bottom side of the images. Preferential cleavage sites are mainly before the bases A and G.
    Rna Substrates, supplied by Integrated DNA Technologies, used in various techniques. Bioz Stars score: 91/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Integrated DNA Technologies rna substrates synthetic rnas
    The 3′-end of deadenylated <t>RNA</t> is a 2′,3′-cyclic phosphate group. ( A ) Radiolabeled 21-mer <t>RNAs</t> used in experiments. 21-mer with 5′-OH and 3′-PO 4 ( OH RNAp) was prepared by ligating [α- 32 P]pAp to 3′-end of 20-mer synthetic RNA (Supplementary Fig. S5 ). Other RNAs ( OH RNA OH , pRNA OH and pRNAp) were prepared from radiolabeled OH RNAp by enzymatic modification. Position of radiolabled phosphate is underlined. ( B ) 5′-phosphate is not required for RNA 3′-deadenylation. Standard ligation reaction mixture (20 µl) containing 0.45 µg of MthRnl with 1 pmol of either pRNA OH , OH RNAp, pRNAp, or OH RNA OH . MthRnl was omitted from a control reaction (-). Positions of 20-mer pRNA(-3′A), OH RNA(-3′A) and cRNA (cRNA 20 ) are indicated. ( C ) 2′,3′-cyclic phosphate is present at the 3′-end of deadenylated RNA. The 3′-deadenylated OH RNA OH was generated by MthRnl and was purified by PAGE (lane 5). Purified 3′-deadenylated OH RNA OH ( OH RNA OH + MthRnl) was treated with 0.1 M HCl for 17 hrs at 4 °C, recovered by ethanol precipitation, and then incubated with or without AP (lanes 8 and 7, respectively). ( D ) 3′-deadenylated RNA can be circularized by RtcB. Same as ( C ) except that AP was replaced with E. coli RtcB (0.2 µg) in a reaction mixture (20 µl) containing 50 mM Tris-HCl (pH 8.0), 2 mM MnCl 2 and 100 µM GTP. Positions of 21-mer cRNA (cRNA 21 ), 20-mer cRNA (cRNA 20 ), and a 20-mer deadenylated OH RNA OH [ OH RNAp(-3′A)] are indicated. ( E ) Proposed mechanism of 3′-deadenylation by archaeal ATP-dependent RNA ligase. Functional groups on the RNA that are required for the 3′-deadenylation reaction are boxed in red. See text for details.
    Rna Substrates Synthetic Rnas, supplied by Integrated DNA Technologies, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Purification and biochemical characterization of rA3G-CD1. ( a ) Phylogenetic tree of A3 domains, with rA3G-CD1 in red. ( b ) Sequence alignments of loop-8 region for rA3G-CD1 (used for crystallization), rA3G-CD1 (WT), hA3G-CD1 (WT), hA3G-CD2 and A3G-sNTD. The four residues in red are the only mutated sequence in the rA3G-CD1 construct. ( c ) Elution profile of rA3G-CD1 analyzed by size-exclusion chromatography (Suprose 6 10/300 GL) before (red) and after (blue) PEI treatment. Inset: SDS–PAGE shows the purified rA3G-CD1 from LMWt that was used for biochemical studies and crystallization. ( d ) Deamination assays of rA3G-CD1, hA3G-CD2, hA3F-CD2 and rA3G-CD2 with a 5′-FAM-labelled 30 nt ssDNA. 17 nt and 15 nt are the products by deamination of the 3rd and 1st C from 5′ end, respectively. ( e ) Electrophoresis mobility shift assays (EMSA) of purified rA3G-CD1 binding to 5′-FAM-labelled 50 nt ssDNA (top panel) and RNA (bottom panel). ( f ) ITC of rA3G-CD1 binding to short 10 nt ssDNA (poly-dT, left panel) and 10 nt RNA (right panel).

    Journal: Nature Communications

    Article Title: Crystal structures of APOBEC3G N-domain alone and its complex with DNA

    doi: 10.1038/ncomms12193

    Figure Lengend Snippet: Purification and biochemical characterization of rA3G-CD1. ( a ) Phylogenetic tree of A3 domains, with rA3G-CD1 in red. ( b ) Sequence alignments of loop-8 region for rA3G-CD1 (used for crystallization), rA3G-CD1 (WT), hA3G-CD1 (WT), hA3G-CD2 and A3G-sNTD. The four residues in red are the only mutated sequence in the rA3G-CD1 construct. ( c ) Elution profile of rA3G-CD1 analyzed by size-exclusion chromatography (Suprose 6 10/300 GL) before (red) and after (blue) PEI treatment. Inset: SDS–PAGE shows the purified rA3G-CD1 from LMWt that was used for biochemical studies and crystallization. ( d ) Deamination assays of rA3G-CD1, hA3G-CD2, hA3F-CD2 and rA3G-CD2 with a 5′-FAM-labelled 30 nt ssDNA. 17 nt and 15 nt are the products by deamination of the 3rd and 1st C from 5′ end, respectively. ( e ) Electrophoresis mobility shift assays (EMSA) of purified rA3G-CD1 binding to 5′-FAM-labelled 50 nt ssDNA (top panel) and RNA (bottom panel). ( f ) ITC of rA3G-CD1 binding to short 10 nt ssDNA (poly-dT, left panel) and 10 nt RNA (right panel).

    Article Snippet: Protein samples of rA3G-CD1, FWKL and Y124A mutants in buffer (100 mM NaCl, 50 mM HEPES pH 7.5) were filled in the sample cell (280 μl volume) and titrated with ssDNA/RNA substrates (40 μl, synthesized and purified by Integrated DNA Technologies, ), which were dialyzed (for ssDNA) or dissolved (for RNA) in the same buffer.

    Techniques: Purification, Sequencing, Crystallization Assay, Construct, Size-exclusion Chromatography, SDS Page, Electrophoresis, Mobility Shift, Binding Assay

    Primer extension inhibition analyses of mRNA cleavage by GB1_HP0315. Lanes 1–4 are DNA sequence ladders. Lane 5–9 is primer extension products of mRNA of HP0315 after digestion with various amounts of GB1_HP0315. Cleavage sites are indicated by sequential numbers on right side of the images. The RNA recognition sequences analyzed by the DNA sequencing ladder are on bottom side of the images. Preferential cleavage sites are mainly before the bases A and G.

    Journal: Nucleic Acids Research

    Article Title: Structural and biochemical characterization of HP0315 from Helicobacter pylori as a VapD protein with an endoribonuclease activity

    doi: 10.1093/nar/gkr1305

    Figure Lengend Snippet: Primer extension inhibition analyses of mRNA cleavage by GB1_HP0315. Lanes 1–4 are DNA sequence ladders. Lane 5–9 is primer extension products of mRNA of HP0315 after digestion with various amounts of GB1_HP0315. Cleavage sites are indicated by sequential numbers on right side of the images. The RNA recognition sequences analyzed by the DNA sequencing ladder are on bottom side of the images. Preferential cleavage sites are mainly before the bases A and G.

    Article Snippet: The short RNA substrates were synthesized by Integrated DNA Technologies (IDT, Coralville, IA, USA).

    Techniques: Inhibition, Sequencing, DNA Sequencing

    SeCsm1 cleaves single-stranded (ss)DNA and RNA. The sequences and length of the DNA and RNA substrates used are shown in Table 1 . Lane 1 (labeled “Marker”) is an RNA marker in both panels. ( A ) The ssDNA cleavage activity of SeCsm1 and its metal dependence. SeCsm1 cleaves ssDNA in the presence of Mg 2+ and Mn 2+ but not other divalent cations tested. The metal-dependent cleavage activity is inhibited by the addition of ethylenediaminetetraacetic acid (EDTA). The concentration of the enzyme used in all reactions is 1 µM, that of each divalent metal is 2 mM and that of EDTA is 10 mM. ( B ) The ssRNA cleavage activity of SeCsm1 and its metal dependence. Cleavage of ssRNA by SeCsm1 also depends on Mg 2+ and Mn 2+ and is inhibited by EDTA. Same concentrations are used as in (A).

    Journal: Nucleic Acids Research

    Article Title: Staphylococcus epidermidis Csm1 is a 3?-5? exonuclease

    doi: 10.1093/nar/gkt914

    Figure Lengend Snippet: SeCsm1 cleaves single-stranded (ss)DNA and RNA. The sequences and length of the DNA and RNA substrates used are shown in Table 1 . Lane 1 (labeled “Marker”) is an RNA marker in both panels. ( A ) The ssDNA cleavage activity of SeCsm1 and its metal dependence. SeCsm1 cleaves ssDNA in the presence of Mg 2+ and Mn 2+ but not other divalent cations tested. The metal-dependent cleavage activity is inhibited by the addition of ethylenediaminetetraacetic acid (EDTA). The concentration of the enzyme used in all reactions is 1 µM, that of each divalent metal is 2 mM and that of EDTA is 10 mM. ( B ) The ssRNA cleavage activity of SeCsm1 and its metal dependence. Cleavage of ssRNA by SeCsm1 also depends on Mg 2+ and Mn 2+ and is inhibited by EDTA. Same concentrations are used as in (A).

    Article Snippet: DNA and RNA cleavage assays Single-stranded DNA and RNA substrates were purchased from Integrated DNA Technology (see for sequence and length information).

    Techniques: Labeling, Marker, Activity Assay, Concentration Assay

    SeCsm1 does not have a DNA endonuclease cleavage activity. Sequences of the DNA substrates used are shown in Table 1 . The first lane is a 5′-labeled RNA marker. ( A ) SeCsm1 cleaves single-stranded DNA1 and DNA3 (lanes 3 and 5) but not the DNA1:DNA3 duplex (lanes 7 and 9). ( B ). SeCsm1 does not cleave the bulged duplex DNA mimicking an artificial R-loop. The DNA bulge is formed by annealing DNA 10 and DNA11, where either DNA10 or DNA11 is radiolabeled.

    Journal: Nucleic Acids Research

    Article Title: Staphylococcus epidermidis Csm1 is a 3?-5? exonuclease

    doi: 10.1093/nar/gkt914

    Figure Lengend Snippet: SeCsm1 does not have a DNA endonuclease cleavage activity. Sequences of the DNA substrates used are shown in Table 1 . The first lane is a 5′-labeled RNA marker. ( A ) SeCsm1 cleaves single-stranded DNA1 and DNA3 (lanes 3 and 5) but not the DNA1:DNA3 duplex (lanes 7 and 9). ( B ). SeCsm1 does not cleave the bulged duplex DNA mimicking an artificial R-loop. The DNA bulge is formed by annealing DNA 10 and DNA11, where either DNA10 or DNA11 is radiolabeled.

    Article Snippet: DNA and RNA cleavage assays Single-stranded DNA and RNA substrates were purchased from Integrated DNA Technology (see for sequence and length information).

    Techniques: Activity Assay, Labeling, Marker

    SeCsm1 is a 3′–5′ exonuclease. DNA strands are annealed to form duplexes with either a 3′ (DNA3:DNA5, DNA7:DNA8) or 5′ overhang (DNA3:DNA6, DNA7:DNA9). The duplexes with a 3′ overhang, but not 5′ overhang, resulted in a major cleavage product consistent with the base-paired region (denoted with a star). Both types of duplexes exhibited shorter cleavage products that are believed to be due to cleavage of some unannealed ssDNA. The sequences and length of the DNA substrates used are shown in Table 1 . RNA substrates have been labeled on either 5′- or 3′-ends, and tested for ssRNA cleavage in a time-dependent manner, or annealed to form duplexes ( A ) Cleavage of duplexes formed by DNA3, DNA5 and DNA6 ( Table 1 ). ( B ) Cleavage of duplexes formed by DNA7, DNA8 and DNA9. ( C ) Time-course study of the cleavage of SeCsm1 on RNA1 labeled on the 5′- or 3′-end. ( D ) Cleavage results of SeCsm1 on RNA strands with either 3′ or 5′ overhangs and a bulged structure. The RNA2 strand has been labeled on either the 3′- or 5′-end. RNA oligos used to form these substrates are described in Table 1 . The first lane is a 5′-labeled RNA marker.

    Journal: Nucleic Acids Research

    Article Title: Staphylococcus epidermidis Csm1 is a 3?-5? exonuclease

    doi: 10.1093/nar/gkt914

    Figure Lengend Snippet: SeCsm1 is a 3′–5′ exonuclease. DNA strands are annealed to form duplexes with either a 3′ (DNA3:DNA5, DNA7:DNA8) or 5′ overhang (DNA3:DNA6, DNA7:DNA9). The duplexes with a 3′ overhang, but not 5′ overhang, resulted in a major cleavage product consistent with the base-paired region (denoted with a star). Both types of duplexes exhibited shorter cleavage products that are believed to be due to cleavage of some unannealed ssDNA. The sequences and length of the DNA substrates used are shown in Table 1 . RNA substrates have been labeled on either 5′- or 3′-ends, and tested for ssRNA cleavage in a time-dependent manner, or annealed to form duplexes ( A ) Cleavage of duplexes formed by DNA3, DNA5 and DNA6 ( Table 1 ). ( B ) Cleavage of duplexes formed by DNA7, DNA8 and DNA9. ( C ) Time-course study of the cleavage of SeCsm1 on RNA1 labeled on the 5′- or 3′-end. ( D ) Cleavage results of SeCsm1 on RNA strands with either 3′ or 5′ overhangs and a bulged structure. The RNA2 strand has been labeled on either the 3′- or 5′-end. RNA oligos used to form these substrates are described in Table 1 . The first lane is a 5′-labeled RNA marker.

    Article Snippet: DNA and RNA cleavage assays Single-stranded DNA and RNA substrates were purchased from Integrated DNA Technology (see for sequence and length information).

    Techniques: Labeling, Marker

    The 3′-end of deadenylated RNA is a 2′,3′-cyclic phosphate group. ( A ) Radiolabeled 21-mer RNAs used in experiments. 21-mer with 5′-OH and 3′-PO 4 ( OH RNAp) was prepared by ligating [α- 32 P]pAp to 3′-end of 20-mer synthetic RNA (Supplementary Fig. S5 ). Other RNAs ( OH RNA OH , pRNA OH and pRNAp) were prepared from radiolabeled OH RNAp by enzymatic modification. Position of radiolabled phosphate is underlined. ( B ) 5′-phosphate is not required for RNA 3′-deadenylation. Standard ligation reaction mixture (20 µl) containing 0.45 µg of MthRnl with 1 pmol of either pRNA OH , OH RNAp, pRNAp, or OH RNA OH . MthRnl was omitted from a control reaction (-). Positions of 20-mer pRNA(-3′A), OH RNA(-3′A) and cRNA (cRNA 20 ) are indicated. ( C ) 2′,3′-cyclic phosphate is present at the 3′-end of deadenylated RNA. The 3′-deadenylated OH RNA OH was generated by MthRnl and was purified by PAGE (lane 5). Purified 3′-deadenylated OH RNA OH ( OH RNA OH + MthRnl) was treated with 0.1 M HCl for 17 hrs at 4 °C, recovered by ethanol precipitation, and then incubated with or without AP (lanes 8 and 7, respectively). ( D ) 3′-deadenylated RNA can be circularized by RtcB. Same as ( C ) except that AP was replaced with E. coli RtcB (0.2 µg) in a reaction mixture (20 µl) containing 50 mM Tris-HCl (pH 8.0), 2 mM MnCl 2 and 100 µM GTP. Positions of 21-mer cRNA (cRNA 21 ), 20-mer cRNA (cRNA 20 ), and a 20-mer deadenylated OH RNA OH [ OH RNAp(-3′A)] are indicated. ( E ) Proposed mechanism of 3′-deadenylation by archaeal ATP-dependent RNA ligase. Functional groups on the RNA that are required for the 3′-deadenylation reaction are boxed in red. See text for details.

    Journal: Scientific Reports

    Article Title: Cleavage of 3′-terminal adenosine by archaeal ATP-dependent RNA ligase

    doi: 10.1038/s41598-017-11693-0

    Figure Lengend Snippet: The 3′-end of deadenylated RNA is a 2′,3′-cyclic phosphate group. ( A ) Radiolabeled 21-mer RNAs used in experiments. 21-mer with 5′-OH and 3′-PO 4 ( OH RNAp) was prepared by ligating [α- 32 P]pAp to 3′-end of 20-mer synthetic RNA (Supplementary Fig. S5 ). Other RNAs ( OH RNA OH , pRNA OH and pRNAp) were prepared from radiolabeled OH RNAp by enzymatic modification. Position of radiolabled phosphate is underlined. ( B ) 5′-phosphate is not required for RNA 3′-deadenylation. Standard ligation reaction mixture (20 µl) containing 0.45 µg of MthRnl with 1 pmol of either pRNA OH , OH RNAp, pRNAp, or OH RNA OH . MthRnl was omitted from a control reaction (-). Positions of 20-mer pRNA(-3′A), OH RNA(-3′A) and cRNA (cRNA 20 ) are indicated. ( C ) 2′,3′-cyclic phosphate is present at the 3′-end of deadenylated RNA. The 3′-deadenylated OH RNA OH was generated by MthRnl and was purified by PAGE (lane 5). Purified 3′-deadenylated OH RNA OH ( OH RNA OH + MthRnl) was treated with 0.1 M HCl for 17 hrs at 4 °C, recovered by ethanol precipitation, and then incubated with or without AP (lanes 8 and 7, respectively). ( D ) 3′-deadenylated RNA can be circularized by RtcB. Same as ( C ) except that AP was replaced with E. coli RtcB (0.2 µg) in a reaction mixture (20 µl) containing 50 mM Tris-HCl (pH 8.0), 2 mM MnCl 2 and 100 µM GTP. Positions of 21-mer cRNA (cRNA 21 ), 20-mer cRNA (cRNA 20 ), and a 20-mer deadenylated OH RNA OH [ OH RNAp(-3′A)] are indicated. ( E ) Proposed mechanism of 3′-deadenylation by archaeal ATP-dependent RNA ligase. Functional groups on the RNA that are required for the 3′-deadenylation reaction are boxed in red. See text for details.

    Article Snippet: RNA substrates Synthetic RNAs were purchased from Integrated DNA Technologies, Inc. (Coralville, IA, USA).

    Techniques: Modification, Ligation, Generated, Purification, Polyacrylamide Gel Electrophoresis, Ethanol Precipitation, Incubation, Functional Assay