t4 pnk  (New England Biolabs)


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    Name:
    T4 Polynucleotide Kinase - 2
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    M0201L
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    Structured Review

    New England Biolabs t4 pnk
    The antigenomic HDV small RNA is 2′-3′ hydroxylated and has an mRNA-like 5′ cap (Northern Blot, 293 cells, RNA induction). ( a ) 3′ end by β-elimination. The mobility of the HDV small RNA is increased following β-elimination. miR-15a: 2′-3′ hydroxylated positive control; +β: +β-elimination; -β: untreated RNA. ( b ) 5′ end by enzymatic analysis. 1: mock-treated (+HDV); 2: mock-treated (no HDV); 3: PNK (+HDV); 4: Decapping enzyme (TAP; +HDV); 5: T4 RNA Ligase (+HDV); 6: Terminator Exonuclease (+HDV). The size of the HDV small RNA was estimated to be ∼24nt based on the largely 22nt, 5′ phosphorylated miR15-a shown in the inset (IS). ( c ) Confirmation that the 5′ end of the HDV small RNA is capped, not triphosphorylated (enlarged image to emphasize changes in gel mobility for miR-15a, but not HDV small RNA). 1: mock-treated (+HDV); 2: mock-treated (no HDV); 3: Antarctic Phosphatase (+HDV); 4: Antarctic Phosphatase followed by T4 PNK (+HDV). ( d ) RNA immunoprecipitation with anti-2,2,7-trimethylguanosine antibody K121. The immunoprecipitation efficiency of the HDV small RNA, U5 snRNA (positive control) and microRNAs miR-15a and let-7a (negative controls) was analysed by Northern blot. ‘S’: supernatant; ‘I’: IP fraction. ( e ) Predicted structure of the HDV small RNA. The various RNAs in  a - d  were detected after stripping and rehybridisation to the same blot. M: RNA marker.

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    Images

    1) Product Images from "Capped small RNAs and MOV10 in Human Hepatitis Delta Virus replication"

    Article Title: Capped small RNAs and MOV10 in Human Hepatitis Delta Virus replication

    Journal: Nature structural & molecular biology

    doi: 10.1038/nsmb.1440

    The antigenomic HDV small RNA is 2′-3′ hydroxylated and has an mRNA-like 5′ cap (Northern Blot, 293 cells, RNA induction). ( a ) 3′ end by β-elimination. The mobility of the HDV small RNA is increased following β-elimination. miR-15a: 2′-3′ hydroxylated positive control; +β: +β-elimination; -β: untreated RNA. ( b ) 5′ end by enzymatic analysis. 1: mock-treated (+HDV); 2: mock-treated (no HDV); 3: PNK (+HDV); 4: Decapping enzyme (TAP; +HDV); 5: T4 RNA Ligase (+HDV); 6: Terminator Exonuclease (+HDV). The size of the HDV small RNA was estimated to be ∼24nt based on the largely 22nt, 5′ phosphorylated miR15-a shown in the inset (IS). ( c ) Confirmation that the 5′ end of the HDV small RNA is capped, not triphosphorylated (enlarged image to emphasize changes in gel mobility for miR-15a, but not HDV small RNA). 1: mock-treated (+HDV); 2: mock-treated (no HDV); 3: Antarctic Phosphatase (+HDV); 4: Antarctic Phosphatase followed by T4 PNK (+HDV). ( d ) RNA immunoprecipitation with anti-2,2,7-trimethylguanosine antibody K121. The immunoprecipitation efficiency of the HDV small RNA, U5 snRNA (positive control) and microRNAs miR-15a and let-7a (negative controls) was analysed by Northern blot. ‘S’: supernatant; ‘I’: IP fraction. ( e ) Predicted structure of the HDV small RNA. The various RNAs in  a - d  were detected after stripping and rehybridisation to the same blot. M: RNA marker.
    Figure Legend Snippet: The antigenomic HDV small RNA is 2′-3′ hydroxylated and has an mRNA-like 5′ cap (Northern Blot, 293 cells, RNA induction). ( a ) 3′ end by β-elimination. The mobility of the HDV small RNA is increased following β-elimination. miR-15a: 2′-3′ hydroxylated positive control; +β: +β-elimination; -β: untreated RNA. ( b ) 5′ end by enzymatic analysis. 1: mock-treated (+HDV); 2: mock-treated (no HDV); 3: PNK (+HDV); 4: Decapping enzyme (TAP; +HDV); 5: T4 RNA Ligase (+HDV); 6: Terminator Exonuclease (+HDV). The size of the HDV small RNA was estimated to be ∼24nt based on the largely 22nt, 5′ phosphorylated miR15-a shown in the inset (IS). ( c ) Confirmation that the 5′ end of the HDV small RNA is capped, not triphosphorylated (enlarged image to emphasize changes in gel mobility for miR-15a, but not HDV small RNA). 1: mock-treated (+HDV); 2: mock-treated (no HDV); 3: Antarctic Phosphatase (+HDV); 4: Antarctic Phosphatase followed by T4 PNK (+HDV). ( d ) RNA immunoprecipitation with anti-2,2,7-trimethylguanosine antibody K121. The immunoprecipitation efficiency of the HDV small RNA, U5 snRNA (positive control) and microRNAs miR-15a and let-7a (negative controls) was analysed by Northern blot. ‘S’: supernatant; ‘I’: IP fraction. ( e ) Predicted structure of the HDV small RNA. The various RNAs in a - d were detected after stripping and rehybridisation to the same blot. M: RNA marker.

    Techniques Used: Northern Blot, Positive Control, Immunoprecipitation, Stripping Membranes, Marker

    Cloning and characterization of an HDV small RNA of genomic polarity. ( a ) Relative location and cloning frequency of sequenced HDV small RNAs derived from the genomic and antigenomic pode hairpins (main species highlighted in red). ( b ) Detection of genomic HDV small RNA by Northern Blot (293 cells, day 5). 1: DNA induction, wt HDAg; 2: DNA induction, mutant HDAg. ( c ) Enzymatic analysis of genomic small RNA 5′ end. 1: mock-treated (+HDV); 2: mock-treated (no HDV); 3: PNK (+HDV); 4: Antarctic Phosphatase (+HDV); 5: Antarctic Phosphatase followed by T4 PNK (+HDV); 6: Decapping enzyme (TAP; +HDV); 7: T4 RNA Ligase (+HDV); 8: Terminator Exonuclease (+HDV). Note that unlike the antigenomic small RNA, a minor fraction of the genomic small RNA does not appear to be shifted following TAP treatment. ( d - f ) Localization of the HDV small RNAs. ( d ) Nuclear-cytoplasmic fractionation of antigenomic HDV small RNA (polyacrylamide gel) and full-length antigenomic and genomic HDV RNA (denaturing agarose gel). The main species in the full-length genomic/antigenomic RNA blot corresponds to the monomer, the higher molecular weight species to dimer, trimer etc. 1: DNA induction, mutant HDAg; 2: DNA induction, wt HDAg; 3: untransfected. ( e ) Genomic small RNA is restricted to the nucleus (nuclear-cytoplasmic fractionation). 1: DNA induction, wt HDAg; 2: DNA induction, mutant HDAg. miR-15a and U6 snRNA chosen as largely cytoplasmic and nuclear RNA controls, respectively. ( f ) The HDV small RNA can be found in the HDV virion. 1: RNA induction (same RNA as in  Fig. 2c ); 2: virion RNA isolated from tissue culture media (∼1.25×10 9  particles). MR: RNA Marker. The various RNAs in  c-f  were detected after stripping and re-hybridization to the same blot.
    Figure Legend Snippet: Cloning and characterization of an HDV small RNA of genomic polarity. ( a ) Relative location and cloning frequency of sequenced HDV small RNAs derived from the genomic and antigenomic pode hairpins (main species highlighted in red). ( b ) Detection of genomic HDV small RNA by Northern Blot (293 cells, day 5). 1: DNA induction, wt HDAg; 2: DNA induction, mutant HDAg. ( c ) Enzymatic analysis of genomic small RNA 5′ end. 1: mock-treated (+HDV); 2: mock-treated (no HDV); 3: PNK (+HDV); 4: Antarctic Phosphatase (+HDV); 5: Antarctic Phosphatase followed by T4 PNK (+HDV); 6: Decapping enzyme (TAP; +HDV); 7: T4 RNA Ligase (+HDV); 8: Terminator Exonuclease (+HDV). Note that unlike the antigenomic small RNA, a minor fraction of the genomic small RNA does not appear to be shifted following TAP treatment. ( d - f ) Localization of the HDV small RNAs. ( d ) Nuclear-cytoplasmic fractionation of antigenomic HDV small RNA (polyacrylamide gel) and full-length antigenomic and genomic HDV RNA (denaturing agarose gel). The main species in the full-length genomic/antigenomic RNA blot corresponds to the monomer, the higher molecular weight species to dimer, trimer etc. 1: DNA induction, mutant HDAg; 2: DNA induction, wt HDAg; 3: untransfected. ( e ) Genomic small RNA is restricted to the nucleus (nuclear-cytoplasmic fractionation). 1: DNA induction, wt HDAg; 2: DNA induction, mutant HDAg. miR-15a and U6 snRNA chosen as largely cytoplasmic and nuclear RNA controls, respectively. ( f ) The HDV small RNA can be found in the HDV virion. 1: RNA induction (same RNA as in Fig. 2c ); 2: virion RNA isolated from tissue culture media (∼1.25×10 9 particles). MR: RNA Marker. The various RNAs in c-f were detected after stripping and re-hybridization to the same blot.

    Techniques Used: Clone Assay, Derivative Assay, Northern Blot, Mutagenesis, Fractionation, Agarose Gel Electrophoresis, Northern blot, Molecular Weight, Isolation, Marker, Stripping Membranes, Hybridization

    2) Product Images from "Capped small RNAs and MOV10 in Human Hepatitis Delta Virus replication"

    Article Title: Capped small RNAs and MOV10 in Human Hepatitis Delta Virus replication

    Journal: Nature structural & molecular biology

    doi: 10.1038/nsmb.1440

    The antigenomic HDV small RNA is 2′-3′ hydroxylated and has an mRNA-like 5′ cap (Northern Blot, 293 cells, RNA induction). ( a ) 3′ end by β-elimination. The mobility of the HDV small RNA is increased following β-elimination. miR-15a: 2′-3′ hydroxylated positive control; +β: +β-elimination; -β: untreated RNA. ( b ) 5′ end by enzymatic analysis. 1: mock-treated (+HDV); 2: mock-treated (no HDV); 3: PNK (+HDV); 4: Decapping enzyme (TAP; +HDV); 5: T4 RNA Ligase (+HDV); 6: Terminator Exonuclease (+HDV). The size of the HDV small RNA was estimated to be ∼24nt based on the largely 22nt, 5′ phosphorylated miR15-a shown in the inset (IS). ( c ) Confirmation that the 5′ end of the HDV small RNA is capped, not triphosphorylated (enlarged image to emphasize changes in gel mobility for miR-15a, but not HDV small RNA). 1: mock-treated (+HDV); 2: mock-treated (no HDV); 3: Antarctic Phosphatase (+HDV); 4: Antarctic Phosphatase followed by T4 PNK (+HDV). ( d ) RNA immunoprecipitation with anti-2,2,7-trimethylguanosine antibody K121. The immunoprecipitation efficiency of the HDV small RNA, U5 snRNA (positive control) and microRNAs miR-15a and let-7a (negative controls) was analysed by Northern blot. ‘S’: supernatant; ‘I’: IP fraction. ( e ) Predicted structure of the HDV small RNA. The various RNAs in  a - d  were detected after stripping and rehybridisation to the same blot. M: RNA marker.
    Figure Legend Snippet: The antigenomic HDV small RNA is 2′-3′ hydroxylated and has an mRNA-like 5′ cap (Northern Blot, 293 cells, RNA induction). ( a ) 3′ end by β-elimination. The mobility of the HDV small RNA is increased following β-elimination. miR-15a: 2′-3′ hydroxylated positive control; +β: +β-elimination; -β: untreated RNA. ( b ) 5′ end by enzymatic analysis. 1: mock-treated (+HDV); 2: mock-treated (no HDV); 3: PNK (+HDV); 4: Decapping enzyme (TAP; +HDV); 5: T4 RNA Ligase (+HDV); 6: Terminator Exonuclease (+HDV). The size of the HDV small RNA was estimated to be ∼24nt based on the largely 22nt, 5′ phosphorylated miR15-a shown in the inset (IS). ( c ) Confirmation that the 5′ end of the HDV small RNA is capped, not triphosphorylated (enlarged image to emphasize changes in gel mobility for miR-15a, but not HDV small RNA). 1: mock-treated (+HDV); 2: mock-treated (no HDV); 3: Antarctic Phosphatase (+HDV); 4: Antarctic Phosphatase followed by T4 PNK (+HDV). ( d ) RNA immunoprecipitation with anti-2,2,7-trimethylguanosine antibody K121. The immunoprecipitation efficiency of the HDV small RNA, U5 snRNA (positive control) and microRNAs miR-15a and let-7a (negative controls) was analysed by Northern blot. ‘S’: supernatant; ‘I’: IP fraction. ( e ) Predicted structure of the HDV small RNA. The various RNAs in a - d were detected after stripping and rehybridisation to the same blot. M: RNA marker.

    Techniques Used: Northern Blot, Positive Control, Immunoprecipitation, Stripping Membranes, Marker

    Cloning and characterization of an HDV small RNA of genomic polarity. ( a ) Relative location and cloning frequency of sequenced HDV small RNAs derived from the genomic and antigenomic pode hairpins (main species highlighted in red). ( b ) Detection of genomic HDV small RNA by Northern Blot (293 cells, day 5). 1: DNA induction, wt HDAg; 2: DNA induction, mutant HDAg. ( c ) Enzymatic analysis of genomic small RNA 5′ end. 1: mock-treated (+HDV); 2: mock-treated (no HDV); 3: PNK (+HDV); 4: Antarctic Phosphatase (+HDV); 5: Antarctic Phosphatase followed by T4 PNK (+HDV); 6: Decapping enzyme (TAP; +HDV); 7: T4 RNA Ligase (+HDV); 8: Terminator Exonuclease (+HDV). Note that unlike the antigenomic small RNA, a minor fraction of the genomic small RNA does not appear to be shifted following TAP treatment. ( d - f ) Localization of the HDV small RNAs. ( d ) Nuclear-cytoplasmic fractionation of antigenomic HDV small RNA (polyacrylamide gel) and full-length antigenomic and genomic HDV RNA (denaturing agarose gel). The main species in the full-length genomic/antigenomic RNA blot corresponds to the monomer, the higher molecular weight species to dimer, trimer etc. 1: DNA induction, mutant HDAg; 2: DNA induction, wt HDAg; 3: untransfected. ( e ) Genomic small RNA is restricted to the nucleus (nuclear-cytoplasmic fractionation). 1: DNA induction, wt HDAg; 2: DNA induction, mutant HDAg. miR-15a and U6 snRNA chosen as largely cytoplasmic and nuclear RNA controls, respectively. ( f ) The HDV small RNA can be found in the HDV virion. 1: RNA induction (same RNA as in  Fig. 2c ); 2: virion RNA isolated from tissue culture media (∼1.25×10 9  particles). MR: RNA Marker. The various RNAs in  c-f  were detected after stripping and re-hybridization to the same blot.
    Figure Legend Snippet: Cloning and characterization of an HDV small RNA of genomic polarity. ( a ) Relative location and cloning frequency of sequenced HDV small RNAs derived from the genomic and antigenomic pode hairpins (main species highlighted in red). ( b ) Detection of genomic HDV small RNA by Northern Blot (293 cells, day 5). 1: DNA induction, wt HDAg; 2: DNA induction, mutant HDAg. ( c ) Enzymatic analysis of genomic small RNA 5′ end. 1: mock-treated (+HDV); 2: mock-treated (no HDV); 3: PNK (+HDV); 4: Antarctic Phosphatase (+HDV); 5: Antarctic Phosphatase followed by T4 PNK (+HDV); 6: Decapping enzyme (TAP; +HDV); 7: T4 RNA Ligase (+HDV); 8: Terminator Exonuclease (+HDV). Note that unlike the antigenomic small RNA, a minor fraction of the genomic small RNA does not appear to be shifted following TAP treatment. ( d - f ) Localization of the HDV small RNAs. ( d ) Nuclear-cytoplasmic fractionation of antigenomic HDV small RNA (polyacrylamide gel) and full-length antigenomic and genomic HDV RNA (denaturing agarose gel). The main species in the full-length genomic/antigenomic RNA blot corresponds to the monomer, the higher molecular weight species to dimer, trimer etc. 1: DNA induction, mutant HDAg; 2: DNA induction, wt HDAg; 3: untransfected. ( e ) Genomic small RNA is restricted to the nucleus (nuclear-cytoplasmic fractionation). 1: DNA induction, wt HDAg; 2: DNA induction, mutant HDAg. miR-15a and U6 snRNA chosen as largely cytoplasmic and nuclear RNA controls, respectively. ( f ) The HDV small RNA can be found in the HDV virion. 1: RNA induction (same RNA as in Fig. 2c ); 2: virion RNA isolated from tissue culture media (∼1.25×10 9 particles). MR: RNA Marker. The various RNAs in c-f were detected after stripping and re-hybridization to the same blot.

    Techniques Used: Clone Assay, Derivative Assay, Northern Blot, Mutagenesis, Fractionation, Agarose Gel Electrophoresis, Northern blot, Molecular Weight, Isolation, Marker, Stripping Membranes, Hybridization

    3) Product Images from "Circadian and feeding rhythms differentially affect rhythmic mRNA transcription and translation in mouse liver"

    Article Title: Circadian and feeding rhythms differentially affect rhythmic mRNA transcription and translation in mouse liver

    Journal:

    doi: 10.1073/pnas.1515308112

    Technical validation of ribosome profiling experiments. ( A ) Simplified representation of the modified ribosome profiling method. RNase I digestion leaves a 5′-OH and a 3′-cyclophosphate. T4 polynucleotide kinase treatment performed in
    Figure Legend Snippet: Technical validation of ribosome profiling experiments. ( A ) Simplified representation of the modified ribosome profiling method. RNase I digestion leaves a 5′-OH and a 3′-cyclophosphate. T4 polynucleotide kinase treatment performed in

    Techniques Used: Modification

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

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

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gks593

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

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

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

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

    Journal: Nature Communications

    doi: 10.1038/s41467-017-00484-w

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

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

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

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

    Journal: Nature biotechnology

    doi: 10.1038/nbt.3289

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

    Techniques Used: Ligation, In Situ, RNA Sequencing Assay

    7) Product Images from "The RNA Binding Specificity of Human APOBEC3 Proteins Resembles That of HIV-1 Nucleocapsid"

    Article Title: The RNA Binding Specificity of Human APOBEC3 Proteins Resembles That of HIV-1 Nucleocapsid

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1005833

    Flow diagram of CLIP-seq. Cells are fed with the ribonucleoside analog 4-thiouridine (4SU) which is incorporated into nascent RNA. Live cells are then irradiated with ultraviolet (UV) light, which induces covalent cross-links between proteins and RNA at sites of contact and 4SU incorporation. Cells are then lysed and treated with RNase A to generate oligonucleotides crosslinked to proteins of interest. Protein-RNA complexes are then immunopurified, and then the RNA is dephosphorylated at the 3'-end with alkaline phosphatase. The RNA is subsequently radiolabeled with  32 P using T4 polynucleotide kinase (PNK) for detection by autoradiography. Protein-RNA complexes are then separated by SDS-PAGE, transferred to nitrocellulose and a region corresponding to protein-RNA adducts is excised. Cross-linked RNA is isolated by proteinase K treatment and phenol:chloroform extraction. After sequential adapter ligations, the RNA library is reverse transcribed. Reverse transcriptase often misincorporates a G opposite the 4SU cross-linking site, which leads to T to C substitutions in positive-strand of the cDNA, enabling the precise mapping of protein-RNA interaction sites. After PCR amplification, the cDNA library is sequenced by Illumina sequencing.
    Figure Legend Snippet: Flow diagram of CLIP-seq. Cells are fed with the ribonucleoside analog 4-thiouridine (4SU) which is incorporated into nascent RNA. Live cells are then irradiated with ultraviolet (UV) light, which induces covalent cross-links between proteins and RNA at sites of contact and 4SU incorporation. Cells are then lysed and treated with RNase A to generate oligonucleotides crosslinked to proteins of interest. Protein-RNA complexes are then immunopurified, and then the RNA is dephosphorylated at the 3'-end with alkaline phosphatase. The RNA is subsequently radiolabeled with 32 P using T4 polynucleotide kinase (PNK) for detection by autoradiography. Protein-RNA complexes are then separated by SDS-PAGE, transferred to nitrocellulose and a region corresponding to protein-RNA adducts is excised. Cross-linked RNA is isolated by proteinase K treatment and phenol:chloroform extraction. After sequential adapter ligations, the RNA library is reverse transcribed. Reverse transcriptase often misincorporates a G opposite the 4SU cross-linking site, which leads to T to C substitutions in positive-strand of the cDNA, enabling the precise mapping of protein-RNA interaction sites. After PCR amplification, the cDNA library is sequenced by Illumina sequencing.

    Techniques Used: Flow Cytometry, Cross-linking Immunoprecipitation, Irradiation, Autoradiography, SDS Page, Isolation, Polymerase Chain Reaction, Amplification, cDNA Library Assay, Sequencing

    8) Product Images from "Purification of cross-linked RNA-protein complexes by phenol-toluol extraction"

    Article Title: Purification of cross-linked RNA-protein complexes by phenol-toluol extraction

    Journal: Nature Communications

    doi: 10.1038/s41467-019-08942-3

    PTex recovers bacterial RNPs.  a Salmonella  Typhimurium SL1344 Hfq-FLAG was UV-cross-linked and HOT-PTex was performed to purify bacterial RNPs.  b  Western blot using an anti-FLAG antibody demonstrates recovery of Hfq monomers linked to RNA. Note that the physiologically active Hfq hexamer partially withstands SDS-PAGE conditions  49  and that this complex is also enriched after PTex.  c  RNPs in  Salmonella  were purified by PTex globally. 172 Proteins enriched after UV-cross-linking (PTex CL) contain ribosomal proteins (transparent red), known RBPs (red) and DNA-binders (orange). Individual enriched proteins not known to associate with RNA before were used for validation (in parentheses).  d  Validation of PTex-enriched RNA-interactors:  Salmonella  strains expressing FLAG-tagged proteins were immunoprecipitated  ±UV irradiation. RNA-association is confirmed by radioactive labelling of RNA 5′ ends by polynucleotide kinase (T4 PNK) using autoradiography; a signal is exclusively detectable after UV-cross-linking and radiolabelling of precipitated RNA. CsrA-FLAG (pos. ctr.), YigA-FLAG (neg. ctr.), AhpC-FLAG, SipA-FLAG and YihI-FLAG are bound to RNA in vivo.  e  GO terms significantly enriched among the RNA-associated proteins;  p -value derived from a one-tail Fisher Exact test  75 . For full gels/blots see Supplementary Figures   25 ,   26
    Figure Legend Snippet: PTex recovers bacterial RNPs. a Salmonella Typhimurium SL1344 Hfq-FLAG was UV-cross-linked and HOT-PTex was performed to purify bacterial RNPs. b Western blot using an anti-FLAG antibody demonstrates recovery of Hfq monomers linked to RNA. Note that the physiologically active Hfq hexamer partially withstands SDS-PAGE conditions 49 and that this complex is also enriched after PTex. c RNPs in Salmonella were purified by PTex globally. 172 Proteins enriched after UV-cross-linking (PTex CL) contain ribosomal proteins (transparent red), known RBPs (red) and DNA-binders (orange). Individual enriched proteins not known to associate with RNA before were used for validation (in parentheses). d Validation of PTex-enriched RNA-interactors: Salmonella strains expressing FLAG-tagged proteins were immunoprecipitated  ±UV irradiation. RNA-association is confirmed by radioactive labelling of RNA 5′ ends by polynucleotide kinase (T4 PNK) using autoradiography; a signal is exclusively detectable after UV-cross-linking and radiolabelling of precipitated RNA. CsrA-FLAG (pos. ctr.), YigA-FLAG (neg. ctr.), AhpC-FLAG, SipA-FLAG and YihI-FLAG are bound to RNA in vivo. e GO terms significantly enriched among the RNA-associated proteins; p -value derived from a one-tail Fisher Exact test 75 . For full gels/blots see Supplementary Figures  25 , 26

    Techniques Used: Western Blot, Recovery, SDS Page, Purification, Expressing, Immunoprecipitation, Irradiation, Autoradiography, In Vivo, Derivative Assay

    9) Product Images from "Circadian and feeding rhythms differentially affect rhythmic mRNA transcription and translation in mouse liver"

    Article Title: Circadian and feeding rhythms differentially affect rhythmic mRNA transcription and translation in mouse liver

    Journal:

    doi: 10.1073/pnas.1515308112

    Technical validation of ribosome profiling experiments. ( A ) Simplified representation of the modified ribosome profiling method. RNase I digestion leaves a 5′-OH and a 3′-cyclophosphate. T4 polynucleotide kinase treatment performed in
    Figure Legend Snippet: Technical validation of ribosome profiling experiments. ( A ) Simplified representation of the modified ribosome profiling method. RNase I digestion leaves a 5′-OH and a 3′-cyclophosphate. T4 polynucleotide kinase treatment performed in

    Techniques Used: Modification

    10) Product Images from "Life without tRNAIle-lysidine synthetase: translation of the isoleucine codon AUA in Bacillus subtilis lacking the canonical tRNA2Ile"

    Article Title: Life without tRNAIle-lysidine synthetase: translation of the isoleucine codon AUA in Bacillus subtilis lacking the canonical tRNA2Ile

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkt1009

    Lysidine is absent in tRNA 2 Ile from Bacillus subtilis JJS80 lacking tilS . ( A ) 1D TLC analysis of the wobble position 34 in tRNA 2 Ile purified from B. subtilis wild-type and JJS80. Purified wild-type tRNA 2 and mutant tRNA 2 were partially hydrolyzed by alkali, the 5′ termini of the fragments were 32 P-labeled using T4-PNK. 32 P-labeled fragments were subsequently digested with nuclease P1 and the nature of the 5′ terminal nucleotide was determined by TLC. The solvent used was isobutyric acid:concentrated ammonia:water (66:1:33) ( v : v : v ). The mobility of each nucleotide (pA, pC, pG, pU) was confirmed with non-radiolabeled standards used as internal markers and visualized by UV shadowing. ( B and C ) Template-dependent binding of purified wild-type 3 H-Ile-tRNA 2 (B) and mutant 35 S-Met-tRNA 2 (C) to ribosomes isolated from B. subtilis . Oligonucleotides used were AUG AUA, AUG AUC, AUG AUG, AUG AUU and AUG UUU; the oligonucleotide concentration was 200 μM.
    Figure Legend Snippet: Lysidine is absent in tRNA 2 Ile from Bacillus subtilis JJS80 lacking tilS . ( A ) 1D TLC analysis of the wobble position 34 in tRNA 2 Ile purified from B. subtilis wild-type and JJS80. Purified wild-type tRNA 2 and mutant tRNA 2 were partially hydrolyzed by alkali, the 5′ termini of the fragments were 32 P-labeled using T4-PNK. 32 P-labeled fragments were subsequently digested with nuclease P1 and the nature of the 5′ terminal nucleotide was determined by TLC. The solvent used was isobutyric acid:concentrated ammonia:water (66:1:33) ( v : v : v ). The mobility of each nucleotide (pA, pC, pG, pU) was confirmed with non-radiolabeled standards used as internal markers and visualized by UV shadowing. ( B and C ) Template-dependent binding of purified wild-type 3 H-Ile-tRNA 2 (B) and mutant 35 S-Met-tRNA 2 (C) to ribosomes isolated from B. subtilis . Oligonucleotides used were AUG AUA, AUG AUC, AUG AUG, AUG AUU and AUG UUU; the oligonucleotide concentration was 200 μM.

    Techniques Used: Thin Layer Chromatography, Purification, Mutagenesis, Labeling, Binding Assay, Isolation, Concentration Assay

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

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

    Journal: Nature biotechnology

    doi: 10.1038/nbt.3289

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

    Techniques Used: Ligation, In Situ, RNA Sequencing Assay

    12) Product Images from "Evidence that base stacking potential in annealed 3' overhangs determines polymerase utilization in yeast nonhomologous end joining"

    Article Title: Evidence that base stacking potential in annealed 3' overhangs determines polymerase utilization in yeast nonhomologous end joining

    Journal:

    doi: 10.1016/j.dnarep.2007.07.018

    5’ dRP lesions demand gap filling by Pol4, but do not require the Pol4 lyase activity or Rad27. (A) Schematic for construction of OMPs with 5’ dRP termini. Deoxyuracil residues are indicated in gray. Treatment with T4 PNK followed by UDG
    Figure Legend Snippet: 5’ dRP lesions demand gap filling by Pol4, but do not require the Pol4 lyase activity or Rad27. (A) Schematic for construction of OMPs with 5’ dRP termini. Deoxyuracil residues are indicated in gray. Treatment with T4 PNK followed by UDG

    Techniques Used: Activity Assay

    13) Product Images from "Evidence that base stacking potential in annealed 3' overhangs determines polymerase utilization in yeast nonhomologous end joining"

    Article Title: Evidence that base stacking potential in annealed 3' overhangs determines polymerase utilization in yeast nonhomologous end joining

    Journal:

    doi: 10.1016/j.dnarep.2007.07.018

    5’ dRP lesions demand gap filling by Pol4, but do not require the Pol4 lyase activity or Rad27. (A) Schematic for construction of OMPs with 5’ dRP termini. Deoxyuracil residues are indicated in gray. Treatment with T4 PNK followed by UDG
    Figure Legend Snippet: 5’ dRP lesions demand gap filling by Pol4, but do not require the Pol4 lyase activity or Rad27. (A) Schematic for construction of OMPs with 5’ dRP termini. Deoxyuracil residues are indicated in gray. Treatment with T4 PNK followed by UDG

    Techniques Used: Activity Assay

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

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

    Journal: Nature Communications

    doi: 10.1038/ncomms9430

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

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

    15) Product Images from "Life without tRNAIle-lysidine synthetase: translation of the isoleucine codon AUA in Bacillus subtilis lacking the canonical tRNA2Ile"

    Article Title: Life without tRNAIle-lysidine synthetase: translation of the isoleucine codon AUA in Bacillus subtilis lacking the canonical tRNA2Ile

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkt1009

    Lysidine is absent in tRNA 2 Ile from Bacillus subtilis JJS80 lacking tilS . ( A ) 1D TLC analysis of the wobble position 34 in tRNA 2 Ile purified from B. subtilis wild-type and JJS80. Purified wild-type tRNA 2 and mutant tRNA 2 were partially hydrolyzed by alkali, the 5′ termini of the fragments were 32 P-labeled using T4-PNK. 32 P-labeled fragments were subsequently digested with nuclease P1 and the nature of the 5′ terminal nucleotide was determined by TLC. The solvent used was isobutyric acid:concentrated ammonia:water (66:1:33) ( v : v : v ). The mobility of each nucleotide (pA, pC, pG, pU) was confirmed with non-radiolabeled standards used as internal markers and visualized by UV shadowing. ( B and C ) Template-dependent binding of purified wild-type 3 H-Ile-tRNA 2 (B) and mutant 35 S-Met-tRNA 2 (C) to ribosomes isolated from B. subtilis . Oligonucleotides used were AUG AUA, AUG AUC, AUG AUG, AUG AUU and AUG UUU; the oligonucleotide concentration was 200 μM.
    Figure Legend Snippet: Lysidine is absent in tRNA 2 Ile from Bacillus subtilis JJS80 lacking tilS . ( A ) 1D TLC analysis of the wobble position 34 in tRNA 2 Ile purified from B. subtilis wild-type and JJS80. Purified wild-type tRNA 2 and mutant tRNA 2 were partially hydrolyzed by alkali, the 5′ termini of the fragments were 32 P-labeled using T4-PNK. 32 P-labeled fragments were subsequently digested with nuclease P1 and the nature of the 5′ terminal nucleotide was determined by TLC. The solvent used was isobutyric acid:concentrated ammonia:water (66:1:33) ( v : v : v ). The mobility of each nucleotide (pA, pC, pG, pU) was confirmed with non-radiolabeled standards used as internal markers and visualized by UV shadowing. ( B and C ) Template-dependent binding of purified wild-type 3 H-Ile-tRNA 2 (B) and mutant 35 S-Met-tRNA 2 (C) to ribosomes isolated from B. subtilis . Oligonucleotides used were AUG AUA, AUG AUC, AUG AUG, AUG AUU and AUG UUU; the oligonucleotide concentration was 200 μM.

    Techniques Used: Thin Layer Chromatography, Purification, Mutagenesis, Labeling, Binding Assay, Isolation, Concentration Assay

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

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

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gks593

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

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

    Related Articles

    Transduction:

    Article Title: G-quadruplex recognition activities of E. Coli MutS
    Article Snippet: End labeling used T4 PNK (New England Biolabs (NEB), Ipswich, MA) and 32 PγATP (from either MP biomedicals or Perkin Elmer). .. End labeling used T4 PNK (New England Biolabs (NEB), Ipswich, MA) and 32 PγATP (from either MP biomedicals or Perkin Elmer).

    Centrifugation:

    Article Title: Structural requirements for protein-catalyzed annealing of U4 and U6 RNAs during di-snRNP assembly
    Article Snippet: After precipitation, RNAs were pelleted by centrifugation and resuspended in the 5' end labeling reaction consisting of 10 units of T4 PNK, 70 mM Tris-HCl pH 7.6, 10 mM MgCl2 , 5 mM DTT and 0.03 μCi of [γ-32 P] ATP (3000 Ci/mmol). .. After in vitro transcription, U4(14–160) was modified as previously described using CIP and T4 PNK (NEB).

    Amplification:

    Article Title: Dissecting neural differentiation regulatory networks through epigenetic footprinting
    Article Snippet: Fragments smaller than 200 bps were eliminated (Zymo, R1016) and the remaining fraction was treated with FastAP Thermosensitive Alkaline Phosphatase (Thermo Scientific, EF0652) and T4 Polynucleotide Kinase (NEB, M0201L). .. Fragments smaller than 200 bps were eliminated (Zymo, R1016) and the remaining fraction was treated with FastAP Thermosensitive Alkaline Phosphatase (Thermo Scientific, EF0652) and T4 Polynucleotide Kinase (NEB, M0201L).

    Filtration:

    Article Title: Identification of nucleotides and amino acids that mediate the interaction between ribosomal protein L30 and the SECIS element
    Article Snippet: Transcripts were purified using organic extraction followed by gel filtration using Micro Bio-Spin 30 Columns (BioRad). .. Dephosphorylated transcripts were end-labeled in the presence of [γ32 P] ATP (3000 Ci/mmole; Perkin Elmer Easy Tides) and T4 PNK (NEB) at 20 units/pmole RNA.

    Binding Assay:

    Article Title: Genetic and Molecular Functional Characterization of Variants within TNFSF13B, a Positional Candidate Preeclampsia Susceptibility Gene on 13q
    Article Snippet: All oligonucleotides were 5′ end-labeled using T4 polynucleotide kinase (PNK) (New England Biolabs) and [γ33P] ATP (3000 Ci/mmol) (PerkinElmer) and annealed to their complementary unlabeled oligonucleotides as previously described . .. The samples were purified according to manufacturers' instructions through G25 Microspin™ columns (GE Healthcare).

    Stable Transfection:

    Article Title: The TEL patch of telomere protein TPP1 mediates telomerase recruitment and processivity
    Article Snippet: Genomic DNA was isolated from confluent 10 cm culture dishes of the original HeLa-EM2-11ht cells and FLAG-TPP1 expressing stable cell lines using the GenElute kit (Sigma). .. A 5' 32 P-labeled (with T4 PNK; NEB) λ DNA-HindIII digest ladder (10,000 cpm) was run as a marker on a separate lane on the gel.

    Synthesized:

    Article Title: Identification of nucleotides and amino acids that mediate the interaction between ribosomal protein L30 and the SECIS element
    Article Snippet: For UV cross-linking and REMSA analyses, RNAs were synthesized in the presence of [α32 P] UTP (800 Ci/mmole; Perkin Elmer Easy Tides) using the RiboMAX™ Large Scale RNA Production Systems (T7) (Promega). .. Dephosphorylated transcripts were end-labeled in the presence of [γ32 P] ATP (3000 Ci/mmole; Perkin Elmer Easy Tides) and T4 PNK (NEB) at 20 units/pmole RNA.

    Article Title: Structural requirements for protein-catalyzed annealing of U4 and U6 RNAs during di-snRNP assembly
    Article Snippet: Fluorescently-labeled U4 and U6 were made via ligation of an in vitro transcribed RNA to a synthesized fluorescently labeled RNA (IDT) using T4 RNA ligase 2. .. After in vitro transcription, U4(14–160) was modified as previously described using CIP and T4 PNK (NEB).

    Autoradiography:

    Article Title: Plasmodium falciparum UvrD activities are downregulated by DNA-interacting compounds and its dsRNA inhibits malaria parasite growth
    Article Snippet: Oligodeoxynucleotide was labeled at 5′-end with T4 polynucleotide kinase (PNK) (5U) (New England Biolabs) and 1.85 MBq of [γ-32 P] ATP (specific activity 222 TBq/mmol) at 37°C for one hour and then annealed using standard annealing buffer (20 mM Tris-HCl, pH 7.5, 10 mM MgCl2 , 100 mM NaCl, 1 mM DTT) with 0.5 μg of single-stranded circular M13mp19 (+) phage DNA by heating at 95°C for 1 min and then transferring immediately to 65°C for 2 min and then slow cooling to room temperature. .. The reaction volume of 10 μl containing the 32 P-labeled helicase substrate (1000 cpm/10 μl) in appropriate buffer (20 mMTris-HCl, pH 8.0, 8 mM DTT, 1.0 mM MgCl2 , 20 mMKCl and 16 μg/ml BSA) and PfUDN was incubated at 37°C for 60 min.

    Construct:

    Article Title: A highly conserved Tyrosine residue of family B DNA polymerases contributes to dictate translesion synthesis past 8-oxo-7,8-dihydro-2?-deoxyguanosine
    Article Snippet: Hybridizations were performed in the presence of 0.2 M NaCl and 50 mM Tris-HCl (pH 7.5), resulting in primer/template structures. .. Phage T4 polynucleotide kinase was obtained from New England Biolabs. ϕ29 DNA polymerase variants at Tyr390 residue Y390F and Y390S were constructed by J. Saturno in an exonuclease deficient background (D12A/D66A) (hereafter PolY390F Exo− and PolY390S Exo− , respectively (unpublished data) and further purified from Escherichia coli BL21(DE3) cells harbouring the corresponding recombinant plasmid ( ). .. The hybrid molecules Pber-2/T4 and Pber-2/8oxodG, containing the unmodified dG and an 8oxodG lesion at the +3 position of the template, respectively, can be used both as substrate for the 3′–5′ exonuclease activity and for DNA-dependent DNA polymerization.

    Electrophoresis:

    Article Title: Plasmodium falciparum UvrD activities are downregulated by DNA-interacting compounds and its dsRNA inhibits malaria parasite growth
    Article Snippet: Oligodeoxynucleotide was labeled at 5′-end with T4 polynucleotide kinase (PNK) (5U) (New England Biolabs) and 1.85 MBq of [γ-32 P] ATP (specific activity 222 TBq/mmol) at 37°C for one hour and then annealed using standard annealing buffer (20 mM Tris-HCl, pH 7.5, 10 mM MgCl2 , 100 mM NaCl, 1 mM DTT) with 0.5 μg of single-stranded circular M13mp19 (+) phage DNA by heating at 95°C for 1 min and then transferring immediately to 65°C for 2 min and then slow cooling to room temperature. .. The reaction volume of 10 μl containing the 32 P-labeled helicase substrate (1000 cpm/10 μl) in appropriate buffer (20 mMTris-HCl, pH 8.0, 8 mM DTT, 1.0 mM MgCl2 , 20 mMKCl and 16 μg/ml BSA) and PfUDN was incubated at 37°C for 60 min.

    Microarray:

    Article Title: Sequencing by Cyclic Ligation and Cleavage (CycLiC) directly on a microarray captured template
    Article Snippet: The surface of the microarray slide, containing surface attached primers, was conditioned by incubation with 10 μl of 1 × T4 ligase buffer (50 mM Tris–HCl, 10 mM MgCl2 , 10 mM DTT, 1 mM ATP, 25 ug/ml BSA, pH 7.5, New England Biolabs, Beverly, MA, USA) at 37°C for 1 h with shaking at 300 rpm on a slide incubator (Thermomixer Comfort, Eppendorf, Hamburg, Germany). .. A 10 μl ligation mix containing the following was prepared: 1 × T4 ligase buffer, 20 pmol β-globin template, 1 μl of 50% PEG 4000 (Fermentas, Vilnius, Lithuania), 1 μl (400 U = 6 Weiss U) of T4 DNA ligase (NEB), 1 μl (10 U) of T4 PNK kinase (NEB) and 50 pmol of 10mer degenerate ON library for each of the four colors (Cy3, Cy5, Fluorescein, Alexa594) ( Supplementary Table 1 ).

    Incubation:

    Article Title: Unconventional miR-122 binding stabilizes the HCV genome by forming a trimolecular RNA structure
    Article Snippet: MiR-122 RNAs (IDT) were 5′32 P-labelled using [γ-32 P]ATP (PerkinElmer) and T4 PNK (NEB) [10 µL of reaction; 1× T4 PNK buffer, 50 pmol miR-122 RNA, 50 µCi [γ-32 P]ATP, 10 U T4 PNK] at 37°C for 1 h and subsequently diluted with nuclease-free water (40 µL). .. MiR-122 RNAs (IDT) were 5′32 P-labelled using [γ-32 P]ATP (PerkinElmer) and T4 PNK (NEB) [10 µL of reaction; 1× T4 PNK buffer, 50 pmol miR-122 RNA, 50 µCi [γ-32 P]ATP, 10 U T4 PNK] at 37°C for 1 h and subsequently diluted with nuclease-free water (40 µL).

    Article Title: RNAi triggered by specialized machinery silences developmental genes and retrotransposons
    Article Snippet: Adapters were in molar excess of RNA, and reactions contained 200 units of T4 Rnl2 truncated enzyme and 20 units of murine RNase inhibitor in a total volume of 20 μl. .. 10μl of the 3′-ligated RNA was phosphorylated by adding 3 μl of water, 0.5 μl of T4 Rnl1 buffer and 0.5 μl (20 U) of murine RNase inhibitor, and 1 μl (10 units) of T4 PNK (NEB) followed by incubation at 37°C for 30 min. .. Final buffer composition for this reaction was 50 mM Tris pH 7.5, 10 mM MgCl2 , 1mM DTT, 8.25% PEG, 0.33 mM ATP in a 15 μl reaction volume.

    Article Title: Functional characterization of C. elegans Y-box-binding proteins reveals tissue-specific functions and a critical role in the formation of polysomes
    Article Snippet: RNAse I (200 U/110 OD, Ambion) was added and the mixture was incubated at 23°C for 1 h. The remaining extract was used for total RNA extraction for subsequent total RNA sequencing. .. RNA was first dephosphorylated using T4 PNK (NEB), followed by 3′ adapter ligation (T4 RNA ligase 2 truncated, NEB).

    Article Title: Totipotent Embryonic Stem Cells Arise in Ground-State Culture Conditions
    Article Snippet: RNA was fragmented by chemical hydrolysis; heating to 95°C, 10 min in 1× RNase III buffer (AM2290, Life Technologies), and snap cooled on ice. .. ATP (0.83 mM, 11140965001, Roche) and 10 U of T4 PNK (M0201L, NEB) were added and incubated at 37°C for 30 min. RNA was purified using Purelink RNA Micro Kit (12183-016, Life Technologies). .. Equimolar pools of RNA-seq libraries were made following quantitative PCR quantification using a Kapa Library Quantification kit (KK4823, Kapa Biosystems).

    Article Title: Genetic and Molecular Functional Characterization of Variants within TNFSF13B, a Positional Candidate Preeclampsia Susceptibility Gene on 13q
    Article Snippet: All oligonucleotides were 5′ end-labeled using T4 polynucleotide kinase (PNK) (New England Biolabs) and [γ33P] ATP (3000 Ci/mmol) (PerkinElmer) and annealed to their complementary unlabeled oligonucleotides as previously described . .. The EMSA reactions were carried out in binding buffer (4% glycerol, 1 mM MgCl2, 0.5 mM EDTA, 0.5 mM DTT, 50 mM NaCl, 10 mM Tris–HCl (pH 7.5), 0.25 mg/ml poly(dI-dC)) in a final volume of 10 µl.

    Article Title: Dissecting domains necessary for activation and repression of splicing by muscleblind-like protein 1
    Article Snippet: For the PCR reaction, the 3’ primer was 5’ end labeled with [32 P]-ATP,. .. Oligo primer (4 pmoles per PCR reaction) was incubated at 37°C for 60 minutes in 50 mM Tris 10 mM MgCl2 T4 polynucleotide kinase enzyme (NEB) and 0.1 μl [α-32 P]-UTP per PCR reaction. .. After incubation the solution was phenol extracted, and purified on a G-50 spin column (GE Healthcare).

    Article Title: Sequencing by Cyclic Ligation and Cleavage (CycLiC) directly on a microarray captured template
    Article Snippet: The surface of the microarray slide, containing surface attached primers, was conditioned by incubation with 10 μl of 1 × T4 ligase buffer (50 mM Tris–HCl, 10 mM MgCl2 , 10 mM DTT, 1 mM ATP, 25 ug/ml BSA, pH 7.5, New England Biolabs, Beverly, MA, USA) at 37°C for 1 h with shaking at 300 rpm on a slide incubator (Thermomixer Comfort, Eppendorf, Hamburg, Germany). .. A 10 μl ligation mix containing the following was prepared: 1 × T4 ligase buffer, 20 pmol β-globin template, 1 μl of 50% PEG 4000 (Fermentas, Vilnius, Lithuania), 1 μl (400 U = 6 Weiss U) of T4 DNA ligase (NEB), 1 μl (10 U) of T4 PNK kinase (NEB) and 50 pmol of 10mer degenerate ON library for each of the four colors (Cy3, Cy5, Fluorescein, Alexa594) ( Supplementary Table 1 ).

    Article Title: Plasmodium falciparum UvrD activities are downregulated by DNA-interacting compounds and its dsRNA inhibits malaria parasite growth
    Article Snippet: Oligodeoxynucleotide was labeled at 5′-end with T4 polynucleotide kinase (PNK) (5U) (New England Biolabs) and 1.85 MBq of [γ-32 P] ATP (specific activity 222 TBq/mmol) at 37°C for one hour and then annealed using standard annealing buffer (20 mM Tris-HCl, pH 7.5, 10 mM MgCl2 , 100 mM NaCl, 1 mM DTT) with 0.5 μg of single-stranded circular M13mp19 (+) phage DNA by heating at 95°C for 1 min and then transferring immediately to 65°C for 2 min and then slow cooling to room temperature. .. Using gel filtration through a Sepharose 4B column (Pharmacia, Sweden) the non-hybridized oligodeoxynucleotide was removed [ ].

    Article Title: A novel enrichment strategy reveals unprecedented number of novel transcription start sites at single base resolution in a model prokaryote and the gut microbiome
    Article Snippet: The RNA was eluted from the beads in 100 μl of low TE. .. 3′ phosphates were removed from the RNA by addition 8.2 μl of 10× T4 polynucleotide buffer to 75 μl of the RNA solution and 4 μl of ATP-free T4 polynucleotide kinase (NEB) was added and incubated for 15 min. Hydrophilic streptavidin magnetic beads (NEB) were prepared by washing 2 times with 400 μl of 10 mM Tris–HCl pH 7.5, 50 mM NaCl, 1 mM EDTA and 2 times with 400 μl of 10 mM Tris–HCl pH 7.5, 500 mM NaCl, 1 mM EDTA and suspended in their original suspension concentration of 4 mg/ml in wash buffer A. .. 50 μl of the kinase treated RNA was added to 30 μl of the prewashed streptavidin beads at room temperature with occasional resuspension for 20 min.

    Diffusion-based Assay:

    Article Title: Structural requirements for protein-catalyzed annealing of U4 and U6 RNAs during di-snRNP assembly
    Article Snippet: RNAs were extracted from the gel by passive diffusion into 300 mM sodium acetate pH 5.2 and ethanol precipitated. .. After in vitro transcription, U4(14–160) was modified as previously described using CIP and T4 PNK (NEB).

    Activity Assay:

    Article Title: Plasmodium falciparum UvrD activities are downregulated by DNA-interacting compounds and its dsRNA inhibits malaria parasite growth
    Article Snippet: This oligodeoxynucleotide of the nucleotide sequence 5'- (T)15 GTTTTCCCAGTCACGAC(T)15 -3' contains 15 base-pairs of non-complementary region (T)15 at both the 5’ and 3’ ends. .. Oligodeoxynucleotide was labeled at 5′-end with T4 polynucleotide kinase (PNK) (5U) (New England Biolabs) and 1.85 MBq of [γ-32 P] ATP (specific activity 222 TBq/mmol) at 37°C for one hour and then annealed using standard annealing buffer (20 mM Tris-HCl, pH 7.5, 10 mM MgCl2 , 100 mM NaCl, 1 mM DTT) with 0.5 μg of single-stranded circular M13mp19 (+) phage DNA by heating at 95°C for 1 min and then transferring immediately to 65°C for 2 min and then slow cooling to room temperature. .. Using gel filtration through a Sepharose 4B column (Pharmacia, Sweden) the non-hybridized oligodeoxynucleotide was removed [ ].

    Expressing:

    Article Title: The TEL patch of telomere protein TPP1 mediates telomerase recruitment and processivity
    Article Snippet: Genomic DNA was isolated from confluent 10 cm culture dishes of the original HeLa-EM2-11ht cells and FLAG-TPP1 expressing stable cell lines using the GenElute kit (Sigma). .. A 5' 32 P-labeled (with T4 PNK; NEB) λ DNA-HindIII digest ladder (10,000 cpm) was run as a marker on a separate lane on the gel.

    Modification:

    Article Title: Structural requirements for protein-catalyzed annealing of U4 and U6 RNAs during di-snRNP assembly
    Article Snippet: Fluorescent U4 was created by ligation of a 5′-Cy5 labeled RNA consisting of U4 nucleotides 1–13 to an in vitro transcribed U4 containing nucleotides 14–160 (modified from the pUC118 plasmid described previously). .. After in vitro transcription, U4(14–160) was modified as previously described using CIP and T4 PNK (NEB). .. U6(13–112) was modified using only T4 PNK due to the presence of a 5'-OH group in the original transcript.

    Gel Purification:

    Article Title: RNAi triggered by specialized machinery silences developmental genes and retrotransposons
    Article Snippet: Small RNA fractions < 40 nt were prepared by gel purification from denaturing acrylamide gels, or by using flashPAGE™ fractionation (Life Technologies) according to the manufacturer’s recommendation . .. 10μl of the 3′-ligated RNA was phosphorylated by adding 3 μl of water, 0.5 μl of T4 Rnl1 buffer and 0.5 μl (20 U) of murine RNase inhibitor, and 1 μl (10 units) of T4 PNK (NEB) followed by incubation at 37°C for 30 min.

    Transfection:

    Article Title: Dissecting domains necessary for activation and repression of splicing by muscleblind-like protein 1
    Article Snippet: Paragraph title: RNA and protein analysis of transfections ... Oligo primer (4 pmoles per PCR reaction) was incubated at 37°C for 60 minutes in 50 mM Tris 10 mM MgCl2 T4 polynucleotide kinase enzyme (NEB) and 0.1 μl [α-32 P]-UTP per PCR reaction.

    Ligation:

    Article Title: RNAi triggered by specialized machinery silences developmental genes and retrotransposons
    Article Snippet: 10μl of the 3′-ligated RNA was phosphorylated by adding 3 μl of water, 0.5 μl of T4 Rnl1 buffer and 0.5 μl (20 U) of murine RNase inhibitor, and 1 μl (10 units) of T4 PNK (NEB) followed by incubation at 37°C for 30 min. .. 10μl of the 3′-ligated RNA was phosphorylated by adding 3 μl of water, 0.5 μl of T4 Rnl1 buffer and 0.5 μl (20 U) of murine RNase inhibitor, and 1 μl (10 units) of T4 PNK (NEB) followed by incubation at 37°C for 30 min.

    Article Title: Functional characterization of C. elegans Y-box-binding proteins reveals tissue-specific functions and a critical role in the formation of polysomes
    Article Snippet: The library was then prepared using the TruSeq Small RNA kit (Illumina), whereby the RNA was precipitated for at least 4 h in between each of the following steps. .. RNA was first dephosphorylated using T4 PNK (NEB), followed by 3′ adapter ligation (T4 RNA ligase 2 truncated, NEB). .. The 5′ end was then re-phosphorylated using T4 PNK (NEB) supplied with ATP, followed by 5′ adapter ligation, cDNA synthesis and PCR.

    Article Title: Structural requirements for protein-catalyzed annealing of U4 and U6 RNAs during di-snRNP assembly
    Article Snippet: Fluorescent U4 was created by ligation of a 5′-Cy5 labeled RNA consisting of U4 nucleotides 1–13 to an in vitro transcribed U4 containing nucleotides 14–160 (modified from the pUC118 plasmid described previously). .. After in vitro transcription, U4(14–160) was modified as previously described using CIP and T4 PNK (NEB).

    Article Title: Sequencing by Cyclic Ligation and Cleavage (CycLiC) directly on a microarray captured template
    Article Snippet: The surface of the microarray slide, containing surface attached primers, was conditioned by incubation with 10 μl of 1 × T4 ligase buffer (50 mM Tris–HCl, 10 mM MgCl2 , 10 mM DTT, 1 mM ATP, 25 ug/ml BSA, pH 7.5, New England Biolabs, Beverly, MA, USA) at 37°C for 1 h with shaking at 300 rpm on a slide incubator (Thermomixer Comfort, Eppendorf, Hamburg, Germany). .. A 10 μl ligation mix containing the following was prepared: 1 × T4 ligase buffer, 20 pmol β-globin template, 1 μl of 50% PEG 4000 (Fermentas, Vilnius, Lithuania), 1 μl (400 U = 6 Weiss U) of T4 DNA ligase (NEB), 1 μl (10 U) of T4 PNK kinase (NEB) and 50 pmol of 10mer degenerate ON library for each of the four colors (Cy3, Cy5, Fluorescein, Alexa594) ( Supplementary Table 1 ). .. The mix was placed on the slide under a coverslip and incubated at 46°C for 1 h with acoustic wave mixing (ArrayBooster, Implen, Munich, Germany).

    Footprinting:

    Article Title: Identification of nucleotides and amino acids that mediate the interaction between ribosomal protein L30 and the SECIS element
    Article Snippet: For RNase footprinting experiments, cold synthetic transcripts were dephosphorylated with SuperSAP (Affymetrix), purified, and resuspended in nuclease-free water. .. Dephosphorylated transcripts were end-labeled in the presence of [γ32 P] ATP (3000 Ci/mmole; Perkin Elmer Easy Tides) and T4 PNK (NEB) at 20 units/pmole RNA.

    Hemagglutination Assay:

    Article Title: Identification of Novel RNA-Protein Contact in Complex of Ribosomal Protein S7 and 3'-Terminal Fragment of 16S rRNA in E. coli
    Article Snippet: It can be hypothesized that the structure of the binary rRNA–protein complex that is formed during the initial stages of the small ribosomal subunit assembly must undergo rearrangement during the formation of an intact subunit. .. T4 polynucleotide kinase (PNK) and PNK buffer (New England Biolabs, USA), reverse transcriptase of the avian myeloblastosis virus (RT-AMV), Taq DNA polymerase, RNase inhibitor, proteinase K, nucleoside triphosphate and its dideoxy derivatives (Roche, Germany), [γ- 32 Р]АТР (Amersham, Germany), bovine serum albumin (BSА, MBI, Fermentas, Lithuania), 0.45 µm nitrocellulose filters (Millipore HA, USA;Schleicher & Schuell BA85, Germany), Ni-NTA-agarose (QIAGEN, Germany), phenylmethylsulfonyl fluoride (PMSF, Merk, Germany) were used. .. The рFD3LH plasmid was kindly provided by L. Brakier-Gingras (University of Montreal, Canada).

    other:

    Article Title: Involvement of the TPR2 subdomain movement in the activities of ?29 DNA polymerase
    Article Snippet: Phage T4 polynucleotide kinase was obtained from New England Biolabs.

    DNA Sequencing:

    Article Title: G-quadruplex recognition activities of E. Coli MutS
    Article Snippet: End labeling used T4 PNK (New England Biolabs (NEB), Ipswich, MA) and 32 PγATP (from either MP biomedicals or Perkin Elmer). .. The mutS:Tn10 allele from FC1124 (provided by Dr. Pat Foster, Indiana University, Bloomington, IN) was transferred to NM522 (NEB) by P1 transduction to create JW1 then tested for mutator phenotype by nalidixic acid screening.

    Polymerase Chain Reaction:

    Article Title: G-quadruplex recognition activities of E. Coli MutS
    Article Snippet: End labeling used T4 PNK (New England Biolabs (NEB), Ipswich, MA) and 32 PγATP (from either MP biomedicals or Perkin Elmer). .. The mutS:Tn10 allele from FC1124 (provided by Dr. Pat Foster, Indiana University, Bloomington, IN) was transferred to NM522 (NEB) by P1 transduction to create JW1 then tested for mutator phenotype by nalidixic acid screening.

    Article Title: Functional characterization of C. elegans Y-box-binding proteins reveals tissue-specific functions and a critical role in the formation of polysomes
    Article Snippet: RNA was first dephosphorylated using T4 PNK (NEB), followed by 3′ adapter ligation (T4 RNA ligase 2 truncated, NEB). .. The 5′ end was then re-phosphorylated using T4 PNK (NEB) supplied with ATP, followed by 5′ adapter ligation, cDNA synthesis and PCR.

    Article Title: Totipotent Embryonic Stem Cells Arise in Ground-State Culture Conditions
    Article Snippet: ATP (0.83 mM, 11140965001, Roche) and 10 U of T4 PNK (M0201L, NEB) were added and incubated at 37°C for 30 min. RNA was purified using Purelink RNA Micro Kit (12183-016, Life Technologies). .. Equimolar pools of RNA-seq libraries were made following quantitative PCR quantification using a Kapa Library Quantification kit (KK4823, Kapa Biosystems).

    Article Title: Dissecting neural differentiation regulatory networks through epigenetic footprinting
    Article Snippet: Fragments smaller than 200 bps were eliminated (Zymo, R1016) and the remaining fraction was treated with FastAP Thermosensitive Alkaline Phosphatase (Thermo Scientific, EF0652) and T4 Polynucleotide Kinase (NEB, M0201L). .. Fragments smaller than 200 bps were eliminated (Zymo, R1016) and the remaining fraction was treated with FastAP Thermosensitive Alkaline Phosphatase (Thermo Scientific, EF0652) and T4 Polynucleotide Kinase (NEB, M0201L).

    Article Title: Dissecting domains necessary for activation and repression of splicing by muscleblind-like protein 1
    Article Snippet: For the PCR reaction, the 3’ primer was 5’ end labeled with [32 P]-ATP,. .. Oligo primer (4 pmoles per PCR reaction) was incubated at 37°C for 60 minutes in 50 mM Tris 10 mM MgCl2 T4 polynucleotide kinase enzyme (NEB) and 0.1 μl [α-32 P]-UTP per PCR reaction. .. After incubation the solution was phenol extracted, and purified on a G-50 spin column (GE Healthcare).

    Hybridization:

    Article Title: The TEL patch of telomere protein TPP1 mediates telomerase recruitment and processivity
    Article Snippet: A 5' 32 P-labeled (with T4 PNK; NEB) λ DNA-HindIII digest ladder (10,000 cpm) was run as a marker on a separate lane on the gel. .. A 5' 32 P-labeled (with T4 PNK; NEB) λ DNA-HindIII digest ladder (10,000 cpm) was run as a marker on a separate lane on the gel.

    Nucleic Acid Electrophoresis:

    Article Title: Unconventional miR-122 binding stabilizes the HCV genome by forming a trimolecular RNA structure
    Article Snippet: MiR-122 RNAs (IDT) were 5′32 P-labelled using [γ-32 P]ATP (PerkinElmer) and T4 PNK (NEB) [10 µL of reaction; 1× T4 PNK buffer, 50 pmol miR-122 RNA, 50 µCi [γ-32 P]ATP, 10 U T4 PNK] at 37°C for 1 h and subsequently diluted with nuclease-free water (40 µL). .. The pre-folded s1 and s2 RNAs were serial diluted by 2-fold in folding buffer to obtain desired concentration range before incubation with ∼3000 cpm/µL (∼0.5–1 pmol) in a 5 µL of reaction for 20–30 min at 37°C.

    RNA Sequencing Assay:

    Article Title: Functional characterization of C. elegans Y-box-binding proteins reveals tissue-specific functions and a critical role in the formation of polysomes
    Article Snippet: RNAse I (200 U/110 OD, Ambion) was added and the mixture was incubated at 23°C for 1 h. The remaining extract was used for total RNA extraction for subsequent total RNA sequencing. .. RNA was first dephosphorylated using T4 PNK (NEB), followed by 3′ adapter ligation (T4 RNA ligase 2 truncated, NEB).

    Article Title: Totipotent Embryonic Stem Cells Arise in Ground-State Culture Conditions
    Article Snippet: Paragraph title: RNA-Seq ... ATP (0.83 mM, 11140965001, Roche) and 10 U of T4 PNK (M0201L, NEB) were added and incubated at 37°C for 30 min. RNA was purified using Purelink RNA Micro Kit (12183-016, Life Technologies).

    Article Title: Dissecting neural differentiation regulatory networks through epigenetic footprinting
    Article Snippet: Paragraph title: Strand Specific RNA-Sequencing Library Construction ... Fragments smaller than 200 bps were eliminated (Zymo, R1016) and the remaining fraction was treated with FastAP Thermosensitive Alkaline Phosphatase (Thermo Scientific, EF0652) and T4 Polynucleotide Kinase (NEB, M0201L).

    Magnetic Beads:

    Article Title: A novel enrichment strategy reveals unprecedented number of novel transcription start sites at single base resolution in a model prokaryote and the gut microbiome
    Article Snippet: The RNA was eluted from the beads in 100 μl of low TE. .. 3′ phosphates were removed from the RNA by addition 8.2 μl of 10× T4 polynucleotide buffer to 75 μl of the RNA solution and 4 μl of ATP-free T4 polynucleotide kinase (NEB) was added and incubated for 15 min. Hydrophilic streptavidin magnetic beads (NEB) were prepared by washing 2 times with 400 μl of 10 mM Tris–HCl pH 7.5, 50 mM NaCl, 1 mM EDTA and 2 times with 400 μl of 10 mM Tris–HCl pH 7.5, 500 mM NaCl, 1 mM EDTA and suspended in their original suspension concentration of 4 mg/ml in wash buffer A. .. 50 μl of the kinase treated RNA was added to 30 μl of the prewashed streptavidin beads at room temperature with occasional resuspension for 20 min.

    Isolation:

    Article Title: The TEL patch of telomere protein TPP1 mediates telomerase recruitment and processivity
    Article Snippet: Genomic DNA was isolated from confluent 10 cm culture dishes of the original HeLa-EM2-11ht cells and FLAG-TPP1 expressing stable cell lines using the GenElute kit (Sigma). .. A 5' 32 P-labeled (with T4 PNK; NEB) λ DNA-HindIII digest ladder (10,000 cpm) was run as a marker on a separate lane on the gel.

    Article Title: Functional characterization of C. elegans Y-box-binding proteins reveals tissue-specific functions and a critical role in the formation of polysomes
    Article Snippet: Ribosome-protected fragments (RPFs) were then isolated as described above for total RNA extraction. .. RNA was first dephosphorylated using T4 PNK (NEB), followed by 3′ adapter ligation (T4 RNA ligase 2 truncated, NEB).

    Article Title: Dissecting neural differentiation regulatory networks through epigenetic footprinting
    Article Snippet: Poly(A) RNA was isolated using Oligo d (T25 ) beads (NEB, E7490L). .. Fragments smaller than 200 bps were eliminated (Zymo, R1016) and the remaining fraction was treated with FastAP Thermosensitive Alkaline Phosphatase (Thermo Scientific, EF0652) and T4 Polynucleotide Kinase (NEB, M0201L).

    Transferring:

    Article Title: Plasmodium falciparum UvrD activities are downregulated by DNA-interacting compounds and its dsRNA inhibits malaria parasite growth
    Article Snippet: This oligodeoxynucleotide of the nucleotide sequence 5'- (T)15 GTTTTCCCAGTCACGAC(T)15 -3' contains 15 base-pairs of non-complementary region (T)15 at both the 5’ and 3’ ends. .. Oligodeoxynucleotide was labeled at 5′-end with T4 polynucleotide kinase (PNK) (5U) (New England Biolabs) and 1.85 MBq of [γ-32 P] ATP (specific activity 222 TBq/mmol) at 37°C for one hour and then annealed using standard annealing buffer (20 mM Tris-HCl, pH 7.5, 10 mM MgCl2 , 100 mM NaCl, 1 mM DTT) with 0.5 μg of single-stranded circular M13mp19 (+) phage DNA by heating at 95°C for 1 min and then transferring immediately to 65°C for 2 min and then slow cooling to room temperature. .. Using gel filtration through a Sepharose 4B column (Pharmacia, Sweden) the non-hybridized oligodeoxynucleotide was removed [ ].

    Electrophoretic Mobility Shift Assay:

    Article Title: Unconventional miR-122 binding stabilizes the HCV genome by forming a trimolecular RNA structure
    Article Snippet: Paragraph title: Electrophoretic mobility shift assays with HCV s1 and s2 RNAs ... MiR-122 RNAs (IDT) were 5′32 P-labelled using [γ-32 P]ATP (PerkinElmer) and T4 PNK (NEB) [10 µL of reaction; 1× T4 PNK buffer, 50 pmol miR-122 RNA, 50 µCi [γ-32 P]ATP, 10 U T4 PNK] at 37°C for 1 h and subsequently diluted with nuclease-free water (40 µL).

    Article Title: Genetic and Molecular Functional Characterization of Variants within TNFSF13B, a Positional Candidate Preeclampsia Susceptibility Gene on 13q
    Article Snippet: Paragraph title: Electrophoretic Mobility Shift Assays (EMSA) ... All oligonucleotides were 5′ end-labeled using T4 polynucleotide kinase (PNK) (New England Biolabs) and [γ33P] ATP (3000 Ci/mmol) (PerkinElmer) and annealed to their complementary unlabeled oligonucleotides as previously described .

    Purification:

    Article Title: Identification of nucleotides and amino acids that mediate the interaction between ribosomal protein L30 and the SECIS element
    Article Snippet: For RNase footprinting experiments, cold synthetic transcripts were dephosphorylated with SuperSAP (Affymetrix), purified, and resuspended in nuclease-free water. .. Dephosphorylated transcripts were end-labeled in the presence of [γ32 P] ATP (3000 Ci/mmole; Perkin Elmer Easy Tides) and T4 PNK (NEB) at 20 units/pmole RNA.

    Article Title: Structural requirements for protein-catalyzed annealing of U4 and U6 RNAs during di-snRNP assembly
    Article Snippet: After in vitro transcription, U4(14–160) was modified as previously described using CIP and T4 PNK (NEB). .. U6(13–112) was modified using only T4 PNK due to the presence of a 5'-OH group in the original transcript.

    Article Title: Totipotent Embryonic Stem Cells Arise in Ground-State Culture Conditions
    Article Snippet: RNA was fragmented by chemical hydrolysis; heating to 95°C, 10 min in 1× RNase III buffer (AM2290, Life Technologies), and snap cooled on ice. .. ATP (0.83 mM, 11140965001, Roche) and 10 U of T4 PNK (M0201L, NEB) were added and incubated at 37°C for 30 min. RNA was purified using Purelink RNA Micro Kit (12183-016, Life Technologies). .. Equimolar pools of RNA-seq libraries were made following quantitative PCR quantification using a Kapa Library Quantification kit (KK4823, Kapa Biosystems).

    Article Title: The classification of mRNA expression levels by the phosphorylation state of RNAPII CTD based on a combined genome-wide approach
    Article Snippet: The ChIP DNA and the Input DNA ends were repaired using T4 DNA polymerase, Klenow enzyme, and T4 polynucleotide kinase (PNK) (New England Biolabs), followed by treatment with Klenow exo- to add an A base to the 3' end. .. The ChIP DNA and the Input DNA ends were repaired using T4 DNA polymerase, Klenow enzyme, and T4 polynucleotide kinase (PNK) (New England Biolabs), followed by treatment with Klenow exo- to add an A base to the 3' end.

    Article Title: Dissecting domains necessary for activation and repression of splicing by muscleblind-like protein 1
    Article Snippet: For RNA analysis, cells in 6 well plates were washed using twice with 2 ml PBS, then to each well 1 ml tri-reagent (Sigma-Aldrich) was added, and purified according to the manufacturers’ protocols. .. Oligo primer (4 pmoles per PCR reaction) was incubated at 37°C for 60 minutes in 50 mM Tris 10 mM MgCl2 T4 polynucleotide kinase enzyme (NEB) and 0.1 μl [α-32 P]-UTP per PCR reaction.

    Article Title: A highly conserved Tyrosine residue of family B DNA polymerases contributes to dictate translesion synthesis past 8-oxo-7,8-dihydro-2?-deoxyguanosine
    Article Snippet: Hybridizations were performed in the presence of 0.2 M NaCl and 50 mM Tris-HCl (pH 7.5), resulting in primer/template structures. .. Phage T4 polynucleotide kinase was obtained from New England Biolabs. ϕ29 DNA polymerase variants at Tyr390 residue Y390F and Y390S were constructed by J. Saturno in an exonuclease deficient background (D12A/D66A) (hereafter PolY390F Exo− and PolY390S Exo− , respectively (unpublished data) and further purified from Escherichia coli BL21(DE3) cells harbouring the corresponding recombinant plasmid ( ). .. The hybrid molecules Pber-2/T4 and Pber-2/8oxodG, containing the unmodified dG and an 8oxodG lesion at the +3 position of the template, respectively, can be used both as substrate for the 3′–5′ exonuclease activity and for DNA-dependent DNA polymerization.

    Article Title: Plasmodium falciparum UvrD activities are downregulated by DNA-interacting compounds and its dsRNA inhibits malaria parasite growth
    Article Snippet: Helicase assay was demonstrated using the purified fraction of PfUDN. .. Oligodeoxynucleotide was labeled at 5′-end with T4 polynucleotide kinase (PNK) (5U) (New England Biolabs) and 1.85 MBq of [γ-32 P] ATP (specific activity 222 TBq/mmol) at 37°C for one hour and then annealed using standard annealing buffer (20 mM Tris-HCl, pH 7.5, 10 mM MgCl2 , 100 mM NaCl, 1 mM DTT) with 0.5 μg of single-stranded circular M13mp19 (+) phage DNA by heating at 95°C for 1 min and then transferring immediately to 65°C for 2 min and then slow cooling to room temperature.

    Article Title: Purification and Characterization of the RecA Protein from Neisseria gonorrhoeae
    Article Snippet: The E. coli RecA, SSB , and LexA proteins were purified as described. .. Restriction enzymes, Klenow, polynucleotide kinase (PNK), T4 DNA ligase, and M13K07 Helper Phage were purchased from New England Biolabs.

    Sequencing:

    Article Title: Functional characterization of C. elegans Y-box-binding proteins reveals tissue-specific functions and a critical role in the formation of polysomes
    Article Snippet: RNA was first dephosphorylated using T4 PNK (NEB), followed by 3′ adapter ligation (T4 RNA ligase 2 truncated, NEB). .. The PCR product was then loaded on a Novex 6% TBE-Urea gel (Life Technologies) and the band around 150 bp (5′ adapter + 30 nt RPF + 3′ adapter) was excised from the gel.

    Article Title: The classification of mRNA expression levels by the phosphorylation state of RNAPII CTD based on a combined genome-wide approach
    Article Snippet: The ChIP DNA and the Input DNA ends were repaired using T4 DNA polymerase, Klenow enzyme, and T4 polynucleotide kinase (PNK) (New England Biolabs), followed by treatment with Klenow exo- to add an A base to the 3' end. .. The samples were purified using the QIAquick MinElute kit (Qiagen) at each preparation step.

    Article Title: Plasmodium falciparum UvrD activities are downregulated by DNA-interacting compounds and its dsRNA inhibits malaria parasite growth
    Article Snippet: This oligodeoxynucleotide of the nucleotide sequence 5'- (T)15 GTTTTCCCAGTCACGAC(T)15 -3' contains 15 base-pairs of non-complementary region (T)15 at both the 5’ and 3’ ends. .. Oligodeoxynucleotide was labeled at 5′-end with T4 polynucleotide kinase (PNK) (5U) (New England Biolabs) and 1.85 MBq of [γ-32 P] ATP (specific activity 222 TBq/mmol) at 37°C for one hour and then annealed using standard annealing buffer (20 mM Tris-HCl, pH 7.5, 10 mM MgCl2 , 100 mM NaCl, 1 mM DTT) with 0.5 μg of single-stranded circular M13mp19 (+) phage DNA by heating at 95°C for 1 min and then transferring immediately to 65°C for 2 min and then slow cooling to room temperature.

    Recombinant:

    Article Title: A highly conserved Tyrosine residue of family B DNA polymerases contributes to dictate translesion synthesis past 8-oxo-7,8-dihydro-2?-deoxyguanosine
    Article Snippet: Hybridizations were performed in the presence of 0.2 M NaCl and 50 mM Tris-HCl (pH 7.5), resulting in primer/template structures. .. Phage T4 polynucleotide kinase was obtained from New England Biolabs. ϕ29 DNA polymerase variants at Tyr390 residue Y390F and Y390S were constructed by J. Saturno in an exonuclease deficient background (D12A/D66A) (hereafter PolY390F Exo− and PolY390S Exo− , respectively (unpublished data) and further purified from Escherichia coli BL21(DE3) cells harbouring the corresponding recombinant plasmid ( ). .. The hybrid molecules Pber-2/T4 and Pber-2/8oxodG, containing the unmodified dG and an 8oxodG lesion at the +3 position of the template, respectively, can be used both as substrate for the 3′–5′ exonuclease activity and for DNA-dependent DNA polymerization.

    Labeling:

    Article Title: Structural requirements for protein-catalyzed annealing of U4 and U6 RNAs during di-snRNP assembly
    Article Snippet: Paragraph title: RNA labeling ... After in vitro transcription, U4(14–160) was modified as previously described using CIP and T4 PNK (NEB).

    Article Title: Genetic and Molecular Functional Characterization of Variants within TNFSF13B, a Positional Candidate Preeclampsia Susceptibility Gene on 13q
    Article Snippet: All oligonucleotides were 5′ end-labeled using T4 polynucleotide kinase (PNK) (New England Biolabs) and [γ33P] ATP (3000 Ci/mmol) (PerkinElmer) and annealed to their complementary unlabeled oligonucleotides as previously described . .. The EMSA reactions were carried out in binding buffer (4% glycerol, 1 mM MgCl2, 0.5 mM EDTA, 0.5 mM DTT, 50 mM NaCl, 10 mM Tris–HCl (pH 7.5), 0.25 mg/ml poly(dI-dC)) in a final volume of 10 µl.

    Article Title: Dissecting domains necessary for activation and repression of splicing by muscleblind-like protein 1
    Article Snippet: For the PCR reaction, the 3’ primer was 5’ end labeled with [32 P]-ATP,. .. Oligo primer (4 pmoles per PCR reaction) was incubated at 37°C for 60 minutes in 50 mM Tris 10 mM MgCl2 T4 polynucleotide kinase enzyme (NEB) and 0.1 μl [α-32 P]-UTP per PCR reaction.

    Article Title: Plasmodium falciparum UvrD activities are downregulated by DNA-interacting compounds and its dsRNA inhibits malaria parasite growth
    Article Snippet: This oligodeoxynucleotide of the nucleotide sequence 5'- (T)15 GTTTTCCCAGTCACGAC(T)15 -3' contains 15 base-pairs of non-complementary region (T)15 at both the 5’ and 3’ ends. .. Oligodeoxynucleotide was labeled at 5′-end with T4 polynucleotide kinase (PNK) (5U) (New England Biolabs) and 1.85 MBq of [γ-32 P] ATP (specific activity 222 TBq/mmol) at 37°C for one hour and then annealed using standard annealing buffer (20 mM Tris-HCl, pH 7.5, 10 mM MgCl2 , 100 mM NaCl, 1 mM DTT) with 0.5 μg of single-stranded circular M13mp19 (+) phage DNA by heating at 95°C for 1 min and then transferring immediately to 65°C for 2 min and then slow cooling to room temperature. .. Using gel filtration through a Sepharose 4B column (Pharmacia, Sweden) the non-hybridized oligodeoxynucleotide was removed [ ].

    Article Title: A novel enrichment strategy reveals unprecedented number of novel transcription start sites at single base resolution in a model prokaryote and the gut microbiome
    Article Snippet: The desthiobiotin-GTP labeled RNA was fragmented by adding 2.5 μl of NEB 10× T4 polynucleotide kinase buffer to a 100 μl volume of capped RNA and incubated for 5 min at 94 °C. .. 3′ phosphates were removed from the RNA by addition 8.2 μl of 10× T4 polynucleotide buffer to 75 μl of the RNA solution and 4 μl of ATP-free T4 polynucleotide kinase (NEB) was added and incubated for 15 min. Hydrophilic streptavidin magnetic beads (NEB) were prepared by washing 2 times with 400 μl of 10 mM Tris–HCl pH 7.5, 50 mM NaCl, 1 mM EDTA and 2 times with 400 μl of 10 mM Tris–HCl pH 7.5, 500 mM NaCl, 1 mM EDTA and suspended in their original suspension concentration of 4 mg/ml in wash buffer A.

    Polyacrylamide Gel Electrophoresis:

    Article Title: Plasmodium falciparum UvrD activities are downregulated by DNA-interacting compounds and its dsRNA inhibits malaria parasite growth
    Article Snippet: Oligodeoxynucleotide was labeled at 5′-end with T4 polynucleotide kinase (PNK) (5U) (New England Biolabs) and 1.85 MBq of [γ-32 P] ATP (specific activity 222 TBq/mmol) at 37°C for one hour and then annealed using standard annealing buffer (20 mM Tris-HCl, pH 7.5, 10 mM MgCl2 , 100 mM NaCl, 1 mM DTT) with 0.5 μg of single-stranded circular M13mp19 (+) phage DNA by heating at 95°C for 1 min and then transferring immediately to 65°C for 2 min and then slow cooling to room temperature. .. The reaction volume of 10 μl containing the 32 P-labeled helicase substrate (1000 cpm/10 μl) in appropriate buffer (20 mMTris-HCl, pH 8.0, 8 mM DTT, 1.0 mM MgCl2 , 20 mMKCl and 16 μg/ml BSA) and PfUDN was incubated at 37°C for 60 min.

    Chromatin Immunoprecipitation:

    Article Title: The classification of mRNA expression levels by the phosphorylation state of RNAPII CTD based on a combined genome-wide approach
    Article Snippet: For ChIPseq, sample preparation was performed using the ChIP protocol described above. .. The ChIP DNA and the Input DNA ends were repaired using T4 DNA polymerase, Klenow enzyme, and T4 polynucleotide kinase (PNK) (New England Biolabs), followed by treatment with Klenow exo- to add an A base to the 3' end. .. After ligation of the Solexa adaptor using TaKaRa ligation Mix (TaKaRa), the adaptor-ligated DNAs were amplified using Solexa PCR primers for 18 cycles, and the amplified library was isolated from an agarose gel.

    Plasmid Preparation:

    Article Title: Structural requirements for protein-catalyzed annealing of U4 and U6 RNAs during di-snRNP assembly
    Article Snippet: Fluorescent U4 was created by ligation of a 5′-Cy5 labeled RNA consisting of U4 nucleotides 1–13 to an in vitro transcribed U4 containing nucleotides 14–160 (modified from the pUC118 plasmid described previously). .. After in vitro transcription, U4(14–160) was modified as previously described using CIP and T4 PNK (NEB).

    Article Title: A highly conserved Tyrosine residue of family B DNA polymerases contributes to dictate translesion synthesis past 8-oxo-7,8-dihydro-2?-deoxyguanosine
    Article Snippet: Hybridizations were performed in the presence of 0.2 M NaCl and 50 mM Tris-HCl (pH 7.5), resulting in primer/template structures. .. Phage T4 polynucleotide kinase was obtained from New England Biolabs. ϕ29 DNA polymerase variants at Tyr390 residue Y390F and Y390S were constructed by J. Saturno in an exonuclease deficient background (D12A/D66A) (hereafter PolY390F Exo− and PolY390S Exo− , respectively (unpublished data) and further purified from Escherichia coli BL21(DE3) cells harbouring the corresponding recombinant plasmid ( ). .. The hybrid molecules Pber-2/T4 and Pber-2/8oxodG, containing the unmodified dG and an 8oxodG lesion at the +3 position of the template, respectively, can be used both as substrate for the 3′–5′ exonuclease activity and for DNA-dependent DNA polymerization.

    Article Title: Purification and Characterization of the RecA Protein from Neisseria gonorrhoeae
    Article Snippet: Restriction enzymes, Klenow, polynucleotide kinase (PNK), T4 DNA ligase, and M13K07 Helper Phage were purchased from New England Biolabs. .. Restriction enzymes, Klenow, polynucleotide kinase (PNK), T4 DNA ligase, and M13K07 Helper Phage were purchased from New England Biolabs.

    Irradiation:

    Article Title: The TEL patch of telomere protein TPP1 mediates telomerase recruitment and processivity
    Article Snippet: A 5' 32 P-labeled (with T4 PNK; NEB) λ DNA-HindIII digest ladder (10,000 cpm) was run as a marker on a separate lane on the gel. .. A 5' 32 P-labeled (with T4 PNK; NEB) λ DNA-HindIII digest ladder (10,000 cpm) was run as a marker on a separate lane on the gel.

    RNA Extraction:

    Article Title: Functional characterization of C. elegans Y-box-binding proteins reveals tissue-specific functions and a critical role in the formation of polysomes
    Article Snippet: Ribosome-protected fragments (RPFs) were then isolated as described above for total RNA extraction. .. RNA was first dephosphorylated using T4 PNK (NEB), followed by 3′ adapter ligation (T4 RNA ligase 2 truncated, NEB).

    Helicase Assay:

    Article Title: Plasmodium falciparum UvrD activities are downregulated by DNA-interacting compounds and its dsRNA inhibits malaria parasite growth
    Article Snippet: Paragraph title: Preparation of substrate and DNA helicase assay ... Oligodeoxynucleotide was labeled at 5′-end with T4 polynucleotide kinase (PNK) (5U) (New England Biolabs) and 1.85 MBq of [γ-32 P] ATP (specific activity 222 TBq/mmol) at 37°C for one hour and then annealed using standard annealing buffer (20 mM Tris-HCl, pH 7.5, 10 mM MgCl2 , 100 mM NaCl, 1 mM DTT) with 0.5 μg of single-stranded circular M13mp19 (+) phage DNA by heating at 95°C for 1 min and then transferring immediately to 65°C for 2 min and then slow cooling to room temperature.

    Sample Prep:

    Article Title: The classification of mRNA expression levels by the phosphorylation state of RNAPII CTD based on a combined genome-wide approach
    Article Snippet: For ChIPseq, sample preparation was performed using the ChIP protocol described above. .. The ChIP DNA and the Input DNA ends were repaired using T4 DNA polymerase, Klenow enzyme, and T4 polynucleotide kinase (PNK) (New England Biolabs), followed by treatment with Klenow exo- to add an A base to the 3' end.

    In Vitro:

    Article Title: Structural requirements for protein-catalyzed annealing of U4 and U6 RNAs during di-snRNP assembly
    Article Snippet: Fluorescent U4 was created by ligation of a 5′-Cy5 labeled RNA consisting of U4 nucleotides 1–13 to an in vitro transcribed U4 containing nucleotides 14–160 (modified from the pUC118 plasmid described previously). .. After in vitro transcription, U4(14–160) was modified as previously described using CIP and T4 PNK (NEB). .. U6(13–112) was modified using only T4 PNK due to the presence of a 5'-OH group in the original transcript.

    Ethanol Precipitation:

    Article Title: RNAi triggered by specialized machinery silences developmental genes and retrotransposons
    Article Snippet: After phenol /chloroform/isoamyl alcohol extraction and ethanol precipitation, 3′-adapters (5′-App TCG TAT GCC GTC TTC TGC TTG T NH2 -3′) were ligated overnight at 16°C under conditions that are insensitive to 2′-O-methylation of the terminal nucleotide (50 mM Tris pH 7.5, 10 mM MgCl2, 1mM DTT, 12.5% PEG.) .. 10μl of the 3′-ligated RNA was phosphorylated by adding 3 μl of water, 0.5 μl of T4 Rnl1 buffer and 0.5 μl (20 U) of murine RNase inhibitor, and 1 μl (10 units) of T4 PNK (NEB) followed by incubation at 37°C for 30 min.

    Concentration Assay:

    Article Title: Unconventional miR-122 binding stabilizes the HCV genome by forming a trimolecular RNA structure
    Article Snippet: MiR-122 RNAs (IDT) were 5′32 P-labelled using [γ-32 P]ATP (PerkinElmer) and T4 PNK (NEB) [10 µL of reaction; 1× T4 PNK buffer, 50 pmol miR-122 RNA, 50 µCi [γ-32 P]ATP, 10 U T4 PNK] at 37°C for 1 h and subsequently diluted with nuclease-free water (40 µL). .. MiR-122 RNAs (IDT) were 5′32 P-labelled using [γ-32 P]ATP (PerkinElmer) and T4 PNK (NEB) [10 µL of reaction; 1× T4 PNK buffer, 50 pmol miR-122 RNA, 50 µCi [γ-32 P]ATP, 10 U T4 PNK] at 37°C for 1 h and subsequently diluted with nuclease-free water (40 µL).

    Article Title: Dissecting domains necessary for activation and repression of splicing by muscleblind-like protein 1
    Article Snippet: Oligo primer (4 pmoles per PCR reaction) was incubated at 37°C for 60 minutes in 50 mM Tris 10 mM MgCl2 T4 polynucleotide kinase enzyme (NEB) and 0.1 μl [α-32 P]-UTP per PCR reaction. .. After incubation the solution was phenol extracted, and purified on a G-50 spin column (GE Healthcare).

    Article Title: A novel enrichment strategy reveals unprecedented number of novel transcription start sites at single base resolution in a model prokaryote and the gut microbiome
    Article Snippet: The RNA was eluted from the beads in 100 μl of low TE. .. 3′ phosphates were removed from the RNA by addition 8.2 μl of 10× T4 polynucleotide buffer to 75 μl of the RNA solution and 4 μl of ATP-free T4 polynucleotide kinase (NEB) was added and incubated for 15 min. Hydrophilic streptavidin magnetic beads (NEB) were prepared by washing 2 times with 400 μl of 10 mM Tris–HCl pH 7.5, 50 mM NaCl, 1 mM EDTA and 2 times with 400 μl of 10 mM Tris–HCl pH 7.5, 500 mM NaCl, 1 mM EDTA and suspended in their original suspension concentration of 4 mg/ml in wash buffer A. .. 50 μl of the kinase treated RNA was added to 30 μl of the prewashed streptavidin beads at room temperature with occasional resuspension for 20 min.

    Fractionation:

    Article Title: RNAi triggered by specialized machinery silences developmental genes and retrotransposons
    Article Snippet: Small RNA fractions < 40 nt were prepared by gel purification from denaturing acrylamide gels, or by using flashPAGE™ fractionation (Life Technologies) according to the manufacturer’s recommendation . .. 10μl of the 3′-ligated RNA was phosphorylated by adding 3 μl of water, 0.5 μl of T4 Rnl1 buffer and 0.5 μl (20 U) of murine RNase inhibitor, and 1 μl (10 units) of T4 PNK (NEB) followed by incubation at 37°C for 30 min.

    DNA Purification:

    Article Title: Purification and Characterization of the RecA Protein from Neisseria gonorrhoeae
    Article Snippet: Restriction enzymes, Klenow, polynucleotide kinase (PNK), T4 DNA ligase, and M13K07 Helper Phage were purchased from New England Biolabs. .. Plasmid pGEM-3Zf(+) was purchased from Promega.

    End Labeling:

    Article Title: G-quadruplex recognition activities of E. Coli MutS
    Article Snippet: All oligonucleotides (detailed in Additional file ) were purchased from Fisher-Operon. .. End labeling used T4 PNK (New England Biolabs (NEB), Ipswich, MA) and 32 PγATP (from either MP biomedicals or Perkin Elmer). .. ATPγS was purchased from MP biomedicals.

    Article Title: Structural requirements for protein-catalyzed annealing of U4 and U6 RNAs during di-snRNP assembly
    Article Snippet: After precipitation, RNAs were pelleted by centrifugation and resuspended in the 5' end labeling reaction consisting of 10 units of T4 PNK, 70 mM Tris-HCl pH 7.6, 10 mM MgCl2 , 5 mM DTT and 0.03 μCi of [γ-32 P] ATP (3000 Ci/mmol). .. After in vitro transcription, U4(14–160) was modified as previously described using CIP and T4 PNK (NEB).

    Marker:

    Article Title: The TEL patch of telomere protein TPP1 mediates telomerase recruitment and processivity
    Article Snippet: The DNA digests were run on a 0.8% Agarose-1X TBE gel at 50 V for a total of 1100 volt-hours. .. A 5' 32 P-labeled (with T4 PNK; NEB) λ DNA-HindIII digest ladder (10,000 cpm) was run as a marker on a separate lane on the gel. .. Next morning, the gel was shaken in 0.25 M HCl for 15 min followed by two rounds (15 min each) of shaking in solution containing 0.5 M NaOH and 1.5 M NaCl.

    Gel Extraction:

    Article Title: Purification and Characterization of the RecA Protein from Neisseria gonorrhoeae
    Article Snippet: Restriction enzymes, Klenow, polynucleotide kinase (PNK), T4 DNA ligase, and M13K07 Helper Phage were purchased from New England Biolabs. .. Plasmid pGEM-3Zf(+) was purchased from Promega.

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  • 99
    New England Biolabs 5 phosphorylated
    Structure of a ssDNA molecule bound within the active site of LHK-Exo. ( A ) The structure of LHK-Exo complexed with <t>5′-phosphorylated</t> pentamer of oligothymidine [5′-PO 4 -(dT) 5 ]. The oligonucleotide is shown in yellow-colored stick form and is covered by a 2 F o – F c electron density map contoured at 1σ (calculated at 2.80 Å). Bound Mg 2+ ions are shown as gray spheres, and the residues that interact with the ssDNA molecule are labeled and shown in stick form. dAMP ligand (green) is superposed on the ssDNA molecule and the 5′-phosphate groups of dAMP and ssDNA bind in identical positions. ( B ) After mapping the electrostatic potential onto the protein surface, four residues of the ssDNA ligand (green) can be seen to fit comfortably into a binding channel within one of the LHK-Exo monomers. The 5′- and 3′-termini of the complexed ssDNA molecule are indicated. The trimer is shown with the wide-end of the tapered central channel facing the front, i.e. DNA enters from the front face. ( C and D ) Surface representations of the native structures of λ-exonuclease (PDB code: 1AVQ) and the HS_1420 exonuclease from Haemophilus somnus 129PT (PDB code: 3K93), respectively. Both exonucleases contain similar channels (one is indicated with a yellow arrow) that may accommodate the 5′-end of the DNA strand to be hydrolyzed; with a more ‘closed’ channel observable for λ-exonuclease (C). Both protein trimers are shown orientated in a manner analogous to that of LHK-Exo in B, revealing an equivalent positioning of the ssDNA-binding channels adjacent to the central tapered channel.
    5 Phosphorylated, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    New England Biolabs 5 ends
    Northern analysis of the ( A ) RyfB, RyfC and ( B ) RyfD RNAs derived from intergenic sequences. The samples were the same as in   Figure 1 , and the RNA marker lane similarly corresponds to one of the panels. Arrows indicate predominant bands. The arrangement of adjacent genes is shown below each panel as in   Figure 2 . Sequences capable of base pairing and diagrams of possible pairing arrangements are given below the gene arrangements. Bases that differ between  E.coli  0157 and  E.coli  K12 are indicated in red. The 5′ ends of the RyfB, RyfC and RyfD RNAs (indicated by balls) all correspond to the actual transcription start.
    5 Ends, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 78/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Structure of a ssDNA molecule bound within the active site of LHK-Exo. ( A ) The structure of LHK-Exo complexed with 5′-phosphorylated pentamer of oligothymidine [5′-PO 4 -(dT) 5 ]. The oligonucleotide is shown in yellow-colored stick form and is covered by a 2 F o – F c electron density map contoured at 1σ (calculated at 2.80 Å). Bound Mg 2+ ions are shown as gray spheres, and the residues that interact with the ssDNA molecule are labeled and shown in stick form. dAMP ligand (green) is superposed on the ssDNA molecule and the 5′-phosphate groups of dAMP and ssDNA bind in identical positions. ( B ) After mapping the electrostatic potential onto the protein surface, four residues of the ssDNA ligand (green) can be seen to fit comfortably into a binding channel within one of the LHK-Exo monomers. The 5′- and 3′-termini of the complexed ssDNA molecule are indicated. The trimer is shown with the wide-end of the tapered central channel facing the front, i.e. DNA enters from the front face. ( C and D ) Surface representations of the native structures of λ-exonuclease (PDB code: 1AVQ) and the HS_1420 exonuclease from Haemophilus somnus 129PT (PDB code: 3K93), respectively. Both exonucleases contain similar channels (one is indicated with a yellow arrow) that may accommodate the 5′-end of the DNA strand to be hydrolyzed; with a more ‘closed’ channel observable for λ-exonuclease (C). Both protein trimers are shown orientated in a manner analogous to that of LHK-Exo in B, revealing an equivalent positioning of the ssDNA-binding channels adjacent to the central tapered channel.

    Journal: Nucleic Acids Research

    Article Title: Structural and functional insight into the mechanism of an alkaline exonuclease from Laribacter hongkongensis

    doi: 10.1093/nar/gkr660

    Figure Lengend Snippet: Structure of a ssDNA molecule bound within the active site of LHK-Exo. ( A ) The structure of LHK-Exo complexed with 5′-phosphorylated pentamer of oligothymidine [5′-PO 4 -(dT) 5 ]. The oligonucleotide is shown in yellow-colored stick form and is covered by a 2 F o – F c electron density map contoured at 1σ (calculated at 2.80 Å). Bound Mg 2+ ions are shown as gray spheres, and the residues that interact with the ssDNA molecule are labeled and shown in stick form. dAMP ligand (green) is superposed on the ssDNA molecule and the 5′-phosphate groups of dAMP and ssDNA bind in identical positions. ( B ) After mapping the electrostatic potential onto the protein surface, four residues of the ssDNA ligand (green) can be seen to fit comfortably into a binding channel within one of the LHK-Exo monomers. The 5′- and 3′-termini of the complexed ssDNA molecule are indicated. The trimer is shown with the wide-end of the tapered central channel facing the front, i.e. DNA enters from the front face. ( C and D ) Surface representations of the native structures of λ-exonuclease (PDB code: 1AVQ) and the HS_1420 exonuclease from Haemophilus somnus 129PT (PDB code: 3K93), respectively. Both exonucleases contain similar channels (one is indicated with a yellow arrow) that may accommodate the 5′-end of the DNA strand to be hydrolyzed; with a more ‘closed’ channel observable for λ-exonuclease (C). Both protein trimers are shown orientated in a manner analogous to that of LHK-Exo in B, revealing an equivalent positioning of the ssDNA-binding channels adjacent to the central tapered channel.

    Article Snippet: Both PCR-amplified substrates were 5′-phosphorylated (T4 polynucleotide kinase, NEB) and purified (QIAquick PCR purification kit, QIAgen) prior to use.

    Techniques: Labeling, Binding Assay

    Qualitative analysis of ssDNA and dsDNA hydrolysis activities of LHK-Exo. ( A ) dsDNA exonuclease activities. Agarose gel showing aliquots taken (0–15 min) from an incubation of LHK-Exo (30 µg, 0.41 nmol of trimers) and BamHI-linearized pET28a (1.8 µg, 0.54 pmol) in Tris–HCl (pH 8.0, 50 mM), 50 mM NaCl, 7.5 mM MgCl 2 at 37°C. ( B ) Polarity of dsDNA digestion. A total of 6 µg of LHK-Exo (82 pmol of trimers, lanes 2–5) or λ-exonuclease (74 pmol of trimers, lanes 6–9) protein was incubated with 0.1 µg (0.23 pmol) of a 712-bp linear 5′-phosphorylated dsDNA substrate (‘unmodified’; lanes 2, 3, 6 and 7), or an analogous 5′-phosphorylated linear dsDNA substrate containing three consecutive ‘nuclease-resistant’ phosphorothioate linkages at its 5′-termini (‘PT-modified’; lanes 4, 5, 8, 9). Assays were quenched immediately (0 min) or incubated at 37°C for 20 min, before analysis of digestion products on 1% agarose gels. ( C ) Digestion of 5′-phosphorylated ssDNA. Reaction mixtures (80 µl) containing LHK-Exo (4.5 µg, 61.4 pmol of trimers) and 5′-PO 4 -(dT) 50 (0.4 nmol) in 25 mM Tris–HCl (pH 8.0), 7.5 mM MgCl 2 , 1 mM DTT were incubated at 37°C. Aliquots (20 µl) were withdrawn after 0, 0.5, 5 and 20 min, and immediately quenched. Reaction products were analyzed by denaturing gel electrophoresis. ( D ) Digestion of non-phosphorylated ssDNA. Analogous sets of assays were performed using non-phosphorylated 50-mers of oligothymidine [5′-OH-(dT) 50 ]. Fluorescent gel images were scanned after SYBR Gold staining. A ssDNA ladder [Oligo Length Standards 20/100 Ladder (IDT)] is included in lane 1.

    Journal: Nucleic Acids Research

    Article Title: Structural and functional insight into the mechanism of an alkaline exonuclease from Laribacter hongkongensis

    doi: 10.1093/nar/gkr660

    Figure Lengend Snippet: Qualitative analysis of ssDNA and dsDNA hydrolysis activities of LHK-Exo. ( A ) dsDNA exonuclease activities. Agarose gel showing aliquots taken (0–15 min) from an incubation of LHK-Exo (30 µg, 0.41 nmol of trimers) and BamHI-linearized pET28a (1.8 µg, 0.54 pmol) in Tris–HCl (pH 8.0, 50 mM), 50 mM NaCl, 7.5 mM MgCl 2 at 37°C. ( B ) Polarity of dsDNA digestion. A total of 6 µg of LHK-Exo (82 pmol of trimers, lanes 2–5) or λ-exonuclease (74 pmol of trimers, lanes 6–9) protein was incubated with 0.1 µg (0.23 pmol) of a 712-bp linear 5′-phosphorylated dsDNA substrate (‘unmodified’; lanes 2, 3, 6 and 7), or an analogous 5′-phosphorylated linear dsDNA substrate containing three consecutive ‘nuclease-resistant’ phosphorothioate linkages at its 5′-termini (‘PT-modified’; lanes 4, 5, 8, 9). Assays were quenched immediately (0 min) or incubated at 37°C for 20 min, before analysis of digestion products on 1% agarose gels. ( C ) Digestion of 5′-phosphorylated ssDNA. Reaction mixtures (80 µl) containing LHK-Exo (4.5 µg, 61.4 pmol of trimers) and 5′-PO 4 -(dT) 50 (0.4 nmol) in 25 mM Tris–HCl (pH 8.0), 7.5 mM MgCl 2 , 1 mM DTT were incubated at 37°C. Aliquots (20 µl) were withdrawn after 0, 0.5, 5 and 20 min, and immediately quenched. Reaction products were analyzed by denaturing gel electrophoresis. ( D ) Digestion of non-phosphorylated ssDNA. Analogous sets of assays were performed using non-phosphorylated 50-mers of oligothymidine [5′-OH-(dT) 50 ]. Fluorescent gel images were scanned after SYBR Gold staining. A ssDNA ladder [Oligo Length Standards 20/100 Ladder (IDT)] is included in lane 1.

    Article Snippet: Both PCR-amplified substrates were 5′-phosphorylated (T4 polynucleotide kinase, NEB) and purified (QIAquick PCR purification kit, QIAgen) prior to use.

    Techniques: Agarose Gel Electrophoresis, Incubation, Modification, Nucleic Acid Electrophoresis, Staining

    Processivity of double strand DNA digestion by wild-type LHK-Exo and Arg12Ala mutant. Time course analysis of the digestion of 5′-phosphorylated double strand DNA (5′-PO 4 -dsDNA: EcoRV-linearized pMal-c2) and 5′-dephosphorylated double strand DNA (5′-OH-dsDNA: 5′dephosphorylated EcoRV-linearized pMal-c2) substrates by wild-type LHK-Exo and the Arg12Ala mutant form using a ‘heparin trap’ approach. A total of 6 µg (82 pmol of trimers) of LHK-Exo or Arg12Ala mutant protein was incubated at 25°C with 60 ng (0.015 pmol) of 5′-PO 4 -dsDNA or 5′-OH-dsDNA in Tris–HCl (25 mM, pH 8.0), 1 mM DTT, 7.5 mM MgCl 2 . After 15 s, excess heparin was added to sequester all unbound protein, and to prevent disassociated protein from re-binding. Aliquots were removed at various time points (0–20 min), and dsDNA levels were determined using fluorescent PicoGreen assays, to enable the extent of DNA digestion to be calculated. In one set of assays, heparin was added to LHK-Exo prior to the addition of dsDNA substrate, to confirm the efficacy of the heparin trap method (filled black squares, green line). Graphs show the mean number of nucleotides digested from each terminus (±SD; y -axis) plotted against the time of analysis (in minutes; x -axis).

    Journal: Nucleic Acids Research

    Article Title: Structural and functional insight into the mechanism of an alkaline exonuclease from Laribacter hongkongensis

    doi: 10.1093/nar/gkr660

    Figure Lengend Snippet: Processivity of double strand DNA digestion by wild-type LHK-Exo and Arg12Ala mutant. Time course analysis of the digestion of 5′-phosphorylated double strand DNA (5′-PO 4 -dsDNA: EcoRV-linearized pMal-c2) and 5′-dephosphorylated double strand DNA (5′-OH-dsDNA: 5′dephosphorylated EcoRV-linearized pMal-c2) substrates by wild-type LHK-Exo and the Arg12Ala mutant form using a ‘heparin trap’ approach. A total of 6 µg (82 pmol of trimers) of LHK-Exo or Arg12Ala mutant protein was incubated at 25°C with 60 ng (0.015 pmol) of 5′-PO 4 -dsDNA or 5′-OH-dsDNA in Tris–HCl (25 mM, pH 8.0), 1 mM DTT, 7.5 mM MgCl 2 . After 15 s, excess heparin was added to sequester all unbound protein, and to prevent disassociated protein from re-binding. Aliquots were removed at various time points (0–20 min), and dsDNA levels were determined using fluorescent PicoGreen assays, to enable the extent of DNA digestion to be calculated. In one set of assays, heparin was added to LHK-Exo prior to the addition of dsDNA substrate, to confirm the efficacy of the heparin trap method (filled black squares, green line). Graphs show the mean number of nucleotides digested from each terminus (±SD; y -axis) plotted against the time of analysis (in minutes; x -axis).

    Article Snippet: Both PCR-amplified substrates were 5′-phosphorylated (T4 polynucleotide kinase, NEB) and purified (QIAquick PCR purification kit, QIAgen) prior to use.

    Techniques: Mutagenesis, Incubation, Binding Assay

    The antigenomic HDV small RNA is 2′-3′ hydroxylated and has an mRNA-like 5′ cap (Northern Blot, 293 cells, RNA induction). ( a ) 3′ end by β-elimination. The mobility of the HDV small RNA is increased following β-elimination. miR-15a: 2′-3′ hydroxylated positive control; +β: +β-elimination; -β: untreated RNA. ( b ) 5′ end by enzymatic analysis. 1: mock-treated (+HDV); 2: mock-treated (no HDV); 3: PNK (+HDV); 4: Decapping enzyme (TAP; +HDV); 5: T4 RNA Ligase (+HDV); 6: Terminator Exonuclease (+HDV). The size of the HDV small RNA was estimated to be ∼24nt based on the largely 22nt, 5′ phosphorylated miR15-a shown in the inset (IS). ( c ) Confirmation that the 5′ end of the HDV small RNA is capped, not triphosphorylated (enlarged image to emphasize changes in gel mobility for miR-15a, but not HDV small RNA). 1: mock-treated (+HDV); 2: mock-treated (no HDV); 3: Antarctic Phosphatase (+HDV); 4: Antarctic Phosphatase followed by T4 PNK (+HDV). ( d ) RNA immunoprecipitation with anti-2,2,7-trimethylguanosine antibody K121. The immunoprecipitation efficiency of the HDV small RNA, U5 snRNA (positive control) and microRNAs miR-15a and let-7a (negative controls) was analysed by Northern blot. ‘S’: supernatant; ‘I’: IP fraction. ( e ) Predicted structure of the HDV small RNA. The various RNAs in  a - d  were detected after stripping and rehybridisation to the same blot. M: RNA marker.

    Journal: Nature structural & molecular biology

    Article Title: Capped small RNAs and MOV10 in Human Hepatitis Delta Virus replication

    doi: 10.1038/nsmb.1440

    Figure Lengend Snippet: The antigenomic HDV small RNA is 2′-3′ hydroxylated and has an mRNA-like 5′ cap (Northern Blot, 293 cells, RNA induction). ( a ) 3′ end by β-elimination. The mobility of the HDV small RNA is increased following β-elimination. miR-15a: 2′-3′ hydroxylated positive control; +β: +β-elimination; -β: untreated RNA. ( b ) 5′ end by enzymatic analysis. 1: mock-treated (+HDV); 2: mock-treated (no HDV); 3: PNK (+HDV); 4: Decapping enzyme (TAP; +HDV); 5: T4 RNA Ligase (+HDV); 6: Terminator Exonuclease (+HDV). The size of the HDV small RNA was estimated to be ∼24nt based on the largely 22nt, 5′ phosphorylated miR15-a shown in the inset (IS). ( c ) Confirmation that the 5′ end of the HDV small RNA is capped, not triphosphorylated (enlarged image to emphasize changes in gel mobility for miR-15a, but not HDV small RNA). 1: mock-treated (+HDV); 2: mock-treated (no HDV); 3: Antarctic Phosphatase (+HDV); 4: Antarctic Phosphatase followed by T4 PNK (+HDV). ( d ) RNA immunoprecipitation with anti-2,2,7-trimethylguanosine antibody K121. The immunoprecipitation efficiency of the HDV small RNA, U5 snRNA (positive control) and microRNAs miR-15a and let-7a (negative controls) was analysed by Northern blot. ‘S’: supernatant; ‘I’: IP fraction. ( e ) Predicted structure of the HDV small RNA. The various RNAs in a - d were detected after stripping and rehybridisation to the same blot. M: RNA marker.

    Article Snippet: The primer extension oligo was end-labeled with T4 PNK (NEB) and γ-32 P ATP (PerkinElmer).

    Techniques: Northern Blot, Positive Control, Immunoprecipitation, Stripping Membranes, Marker

    Cloning and characterization of an HDV small RNA of genomic polarity. ( a ) Relative location and cloning frequency of sequenced HDV small RNAs derived from the genomic and antigenomic pode hairpins (main species highlighted in red). ( b ) Detection of genomic HDV small RNA by Northern Blot (293 cells, day 5). 1: DNA induction, wt HDAg; 2: DNA induction, mutant HDAg. ( c ) Enzymatic analysis of genomic small RNA 5′ end. 1: mock-treated (+HDV); 2: mock-treated (no HDV); 3: PNK (+HDV); 4: Antarctic Phosphatase (+HDV); 5: Antarctic Phosphatase followed by T4 PNK (+HDV); 6: Decapping enzyme (TAP; +HDV); 7: T4 RNA Ligase (+HDV); 8: Terminator Exonuclease (+HDV). Note that unlike the antigenomic small RNA, a minor fraction of the genomic small RNA does not appear to be shifted following TAP treatment. ( d - f ) Localization of the HDV small RNAs. ( d ) Nuclear-cytoplasmic fractionation of antigenomic HDV small RNA (polyacrylamide gel) and full-length antigenomic and genomic HDV RNA (denaturing agarose gel). The main species in the full-length genomic/antigenomic RNA blot corresponds to the monomer, the higher molecular weight species to dimer, trimer etc. 1: DNA induction, mutant HDAg; 2: DNA induction, wt HDAg; 3: untransfected. ( e ) Genomic small RNA is restricted to the nucleus (nuclear-cytoplasmic fractionation). 1: DNA induction, wt HDAg; 2: DNA induction, mutant HDAg. miR-15a and U6 snRNA chosen as largely cytoplasmic and nuclear RNA controls, respectively. ( f ) The HDV small RNA can be found in the HDV virion. 1: RNA induction (same RNA as in  Fig. 2c ); 2: virion RNA isolated from tissue culture media (∼1.25×10 9  particles). MR: RNA Marker. The various RNAs in  c-f  were detected after stripping and re-hybridization to the same blot.

    Journal: Nature structural & molecular biology

    Article Title: Capped small RNAs and MOV10 in Human Hepatitis Delta Virus replication

    doi: 10.1038/nsmb.1440

    Figure Lengend Snippet: Cloning and characterization of an HDV small RNA of genomic polarity. ( a ) Relative location and cloning frequency of sequenced HDV small RNAs derived from the genomic and antigenomic pode hairpins (main species highlighted in red). ( b ) Detection of genomic HDV small RNA by Northern Blot (293 cells, day 5). 1: DNA induction, wt HDAg; 2: DNA induction, mutant HDAg. ( c ) Enzymatic analysis of genomic small RNA 5′ end. 1: mock-treated (+HDV); 2: mock-treated (no HDV); 3: PNK (+HDV); 4: Antarctic Phosphatase (+HDV); 5: Antarctic Phosphatase followed by T4 PNK (+HDV); 6: Decapping enzyme (TAP; +HDV); 7: T4 RNA Ligase (+HDV); 8: Terminator Exonuclease (+HDV). Note that unlike the antigenomic small RNA, a minor fraction of the genomic small RNA does not appear to be shifted following TAP treatment. ( d - f ) Localization of the HDV small RNAs. ( d ) Nuclear-cytoplasmic fractionation of antigenomic HDV small RNA (polyacrylamide gel) and full-length antigenomic and genomic HDV RNA (denaturing agarose gel). The main species in the full-length genomic/antigenomic RNA blot corresponds to the monomer, the higher molecular weight species to dimer, trimer etc. 1: DNA induction, mutant HDAg; 2: DNA induction, wt HDAg; 3: untransfected. ( e ) Genomic small RNA is restricted to the nucleus (nuclear-cytoplasmic fractionation). 1: DNA induction, wt HDAg; 2: DNA induction, mutant HDAg. miR-15a and U6 snRNA chosen as largely cytoplasmic and nuclear RNA controls, respectively. ( f ) The HDV small RNA can be found in the HDV virion. 1: RNA induction (same RNA as in Fig. 2c ); 2: virion RNA isolated from tissue culture media (∼1.25×10 9 particles). MR: RNA Marker. The various RNAs in c-f were detected after stripping and re-hybridization to the same blot.

    Article Snippet: The primer extension oligo was end-labeled with T4 PNK (NEB) and γ-32 P ATP (PerkinElmer).

    Techniques: Clone Assay, Derivative Assay, Northern Blot, Mutagenesis, Fractionation, Agarose Gel Electrophoresis, Northern blot, Molecular Weight, Isolation, Marker, Stripping Membranes, Hybridization

    Northern analysis of the ( A ) RyfB, RyfC and ( B ) RyfD RNAs derived from intergenic sequences. The samples were the same as in   Figure 1 , and the RNA marker lane similarly corresponds to one of the panels. Arrows indicate predominant bands. The arrangement of adjacent genes is shown below each panel as in   Figure 2 . Sequences capable of base pairing and diagrams of possible pairing arrangements are given below the gene arrangements. Bases that differ between  E.coli  0157 and  E.coli  K12 are indicated in red. The 5′ ends of the RyfB, RyfC and RyfD RNAs (indicated by balls) all correspond to the actual transcription start.

    Journal: Nucleic Acids Research

    Article Title: Detection of 5?- and 3?-UTR-derived small RNAs and cis-encoded antisense RNAs in Escherichia coli

    doi: 10.1093/nar/gki256

    Figure Lengend Snippet: Northern analysis of the ( A ) RyfB, RyfC and ( B ) RyfD RNAs derived from intergenic sequences. The samples were the same as in Figure 1 , and the RNA marker lane similarly corresponds to one of the panels. Arrows indicate predominant bands. The arrangement of adjacent genes is shown below each panel as in Figure 2 . Sequences capable of base pairing and diagrams of possible pairing arrangements are given below the gene arrangements. Bases that differ between E.coli 0157 and E.coli K12 are indicated in red. The 5′ ends of the RyfB, RyfC and RyfD RNAs (indicated by balls) all correspond to the actual transcription start.

    Article Snippet: The ligation products were phosphorylated at their 5′ ends (30 μl reaction, 37°C, 1 h, 2 mM ATP and 5 U T4 polynucleotide kinase; New England Biolabs Inc., Beverly MA).

    Techniques: Northern Blot, Derivative Assay, Marker

    Northern analysis of antisense RNAs expressed near the ( A ) 5′ end or ( B ) 3′ end of the mRNA encoded on the opposing strand. The samples were the same as in   Figure 1 , and the RNA marker lane similarly corresponds to one of the panels. Small arrows indicate the full-length mRNA and large arrows indicate processed transcripts. The arrangement of adjacent genes is shown below each panel as in   Figure 2 . The 5′ ends of the RyjB and RyjC (indicated by balls) correspond to the actual transcription start.

    Journal: Nucleic Acids Research

    Article Title: Detection of 5?- and 3?-UTR-derived small RNAs and cis-encoded antisense RNAs in Escherichia coli

    doi: 10.1093/nar/gki256

    Figure Lengend Snippet: Northern analysis of antisense RNAs expressed near the ( A ) 5′ end or ( B ) 3′ end of the mRNA encoded on the opposing strand. The samples were the same as in Figure 1 , and the RNA marker lane similarly corresponds to one of the panels. Small arrows indicate the full-length mRNA and large arrows indicate processed transcripts. The arrangement of adjacent genes is shown below each panel as in Figure 2 . The 5′ ends of the RyjB and RyjC (indicated by balls) correspond to the actual transcription start.

    Article Snippet: The ligation products were phosphorylated at their 5′ ends (30 μl reaction, 37°C, 1 h, 2 mM ATP and 5 U T4 polynucleotide kinase; New England Biolabs Inc., Beverly MA).

    Techniques: Northern Blot, Marker