oligo  (New England Biolabs)


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    Name:
    Oligo d T 18 no 5 Phosphate
    Description:
    Oligo d T 18 no 5 Phosphate 5 0 A260 units
    Catalog Number:
    s1316s
    Price:
    93
    Size:
    5 0 A260 units
    Category:
    Probes and Primers
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    Structured Review

    New England Biolabs oligo
    Oligo d T 18 no 5 Phosphate
    Oligo d T 18 no 5 Phosphate 5 0 A260 units
    https://www.bioz.com/result/oligo/product/New England Biolabs
    Average 99 stars, based on 368 article reviews
    Price from $9.99 to $1999.99
    oligo - by Bioz Stars, 2020-09
    99/100 stars

    Images

    1) Product Images from "An mRNA-binding channel in the ES6S region of the translation 48S-PIC promotes RNA unwinding and scanning"

    Article Title: An mRNA-binding channel in the ES6S region of the translation 48S-PIC promotes RNA unwinding and scanning

    Journal: eLife

    doi: 10.7554/eLife.48246

    Quality and reproducibility of the experiment shown in Figure 5 . ( a ) Polysome profile of HEK293T cells transfected with the indicated oligonucleotides. This experiment corresponds to the replica_1 used for RNA-seq, indicating fractions pooled as monosomes or polysomes. The bottom panel shows a western blot analysis of the resulting fractions probed with anti-eS6 antibody. ( b ) Analysis of correlation between replicas using a normalized number of reads for every mRNA in the monosomal and polysomal samples. ( c ) Changes in mRNA abundance between oligo 4 and VIC–oligo-4-transfected cells. Data are the average from two replicates. ( d ) Enrichment in KEGG pathways detected in the ‘TE down’ group of mRNAs. The p-value and enrichment factor for each term are indicated.
    Figure Legend Snippet: Quality and reproducibility of the experiment shown in Figure 5 . ( a ) Polysome profile of HEK293T cells transfected with the indicated oligonucleotides. This experiment corresponds to the replica_1 used for RNA-seq, indicating fractions pooled as monosomes or polysomes. The bottom panel shows a western blot analysis of the resulting fractions probed with anti-eS6 antibody. ( b ) Analysis of correlation between replicas using a normalized number of reads for every mRNA in the monosomal and polysomal samples. ( c ) Changes in mRNA abundance between oligo 4 and VIC–oligo-4-transfected cells. Data are the average from two replicates. ( d ) Enrichment in KEGG pathways detected in the ‘TE down’ group of mRNAs. The p-value and enrichment factor for each term are indicated.

    Techniques Used: Transfection, RNA Sequencing Assay, Western Blot

    Effect of VIC–oligo 4 on protein crosslinking in 48S complexes assembled with unstructured or SV-DLP n27 mRNAs. The amount of [ 32 P]-mRNA bound to WRF is indicated (upper panel), showing the pattern of protein crosslinking with the indicated mRNAs in the absence and presence of VIC–oligo 4. The unresolved eIF3g and eIF4A bands are shown (lower panel).
    Figure Legend Snippet: Effect of VIC–oligo 4 on protein crosslinking in 48S complexes assembled with unstructured or SV-DLP n27 mRNAs. The amount of [ 32 P]-mRNA bound to WRF is indicated (upper panel), showing the pattern of protein crosslinking with the indicated mRNAs in the absence and presence of VIC–oligo 4. The unresolved eIF3g and eIF4A bands are shown (lower panel).

    Techniques Used:

    mRNA threading into the ES6S region slows down scanning but makes it more processive. ( a ) Effect of FITC-oligo 4 on the translation of Luc mRNAs with different 5′ UTRs in RRL. Translation mixtures were incubated for 90 min, which represented the endpoint measurement because no further increase in luc activity was detected. Data are represented as the mean ± SD from at least three independent experiments. ( b ) Luc activity accumulation in continuously recording experiments programmed with the indicated mRNAs. Measurements were taken every 3 min: gray line, no oligo; black line, +FITC–oligo 4. Hipp was added to the indicated samples at a concentration of 2 μM (dashed line). ( c ) Estimates of full translation time (FTT) for 5′ UTR G-less and 5′ UTR-SL20 mRNAs, and the effect of FITC–oligo 4 and hipp on FTT. Data from panel (B) were processed as described before ( Vassilenko et al., 2011 ). The determined FTT values were: 5′ UTR G-less = 9.53 min; 5′ UTR G-less+FITC–oligo 4 = 7.89 min; 5′ UTR-SL20 = 17.57 min; 5′ UTR-SL20+FITC–oligo 4 = 17.56 min; 5′ UTR-SL20+hipp = 19.44 min. ( d ) Synergistic inhibitory effect of VIC–oligo 4 and hipp on translation of 5′UTR SL30-Luc and 5′ UTR G4-1-Luc mRNAs in RRL. Translation mixtures were preincubated with 6 μM of VIC–oligo 4 and with increasing concentrations of hipp for 5 min. Then, mRNAs were added and measurements were taken 90 min later; the calculated combination index (CI) for each mRNA is indicated.
    Figure Legend Snippet: mRNA threading into the ES6S region slows down scanning but makes it more processive. ( a ) Effect of FITC-oligo 4 on the translation of Luc mRNAs with different 5′ UTRs in RRL. Translation mixtures were incubated for 90 min, which represented the endpoint measurement because no further increase in luc activity was detected. Data are represented as the mean ± SD from at least three independent experiments. ( b ) Luc activity accumulation in continuously recording experiments programmed with the indicated mRNAs. Measurements were taken every 3 min: gray line, no oligo; black line, +FITC–oligo 4. Hipp was added to the indicated samples at a concentration of 2 μM (dashed line). ( c ) Estimates of full translation time (FTT) for 5′ UTR G-less and 5′ UTR-SL20 mRNAs, and the effect of FITC–oligo 4 and hipp on FTT. Data from panel (B) were processed as described before ( Vassilenko et al., 2011 ). The determined FTT values were: 5′ UTR G-less = 9.53 min; 5′ UTR G-less+FITC–oligo 4 = 7.89 min; 5′ UTR-SL20 = 17.57 min; 5′ UTR-SL20+FITC–oligo 4 = 17.56 min; 5′ UTR-SL20+hipp = 19.44 min. ( d ) Synergistic inhibitory effect of VIC–oligo 4 and hipp on translation of 5′UTR SL30-Luc and 5′ UTR G4-1-Luc mRNAs in RRL. Translation mixtures were preincubated with 6 μM of VIC–oligo 4 and with increasing concentrations of hipp for 5 min. Then, mRNAs were added and measurements were taken 90 min later; the calculated combination index (CI) for each mRNA is indicated.

    Techniques Used: Incubation, Activity Assay, Concentration Assay

    Effect of 5' UTR length of luc mRNAs on the sensitivity to VIC-oligo 4-mediated translational block. ( a ) Effect of 5′ UTR length on cap-dependent translation ofluc mRNAs in the presence of VIC-oligo 4. Data are the mean of two independent experiments performed in MEF cells as described in Figure 4a . ( b ) Comparative analysis of the effect of oligo 4, VIC–oligo C and VIC–oligo 4 on the translation of the indicated mRNAs. Data are the mean ± SD from at least four independent experiments in MEF cells. ( c ) Effect of VIC–oligo 4 on the translation of poliovirus (PV) mRNA. HeLa cells were transfected with the indicated oligonucleotides, and infected 12 hr later with PV1 (Mahoney strain) at a multiplicity of infection (MOI) of 10 pfu/cell. The cultures were metabolically labeled with [ 35 S]-Met at 6 hr post-infection and analyzed as described in the Materials and methods; SYPRO staining was included as the loading control.
    Figure Legend Snippet: Effect of 5' UTR length of luc mRNAs on the sensitivity to VIC-oligo 4-mediated translational block. ( a ) Effect of 5′ UTR length on cap-dependent translation ofluc mRNAs in the presence of VIC-oligo 4. Data are the mean of two independent experiments performed in MEF cells as described in Figure 4a . ( b ) Comparative analysis of the effect of oligo 4, VIC–oligo C and VIC–oligo 4 on the translation of the indicated mRNAs. Data are the mean ± SD from at least four independent experiments in MEF cells. ( c ) Effect of VIC–oligo 4 on the translation of poliovirus (PV) mRNA. HeLa cells were transfected with the indicated oligonucleotides, and infected 12 hr later with PV1 (Mahoney strain) at a multiplicity of infection (MOI) of 10 pfu/cell. The cultures were metabolically labeled with [ 35 S]-Met at 6 hr post-infection and analyzed as described in the Materials and methods; SYPRO staining was included as the loading control.

    Techniques Used: Blocking Assay, Transfection, Infection, Metabolic Labelling, Labeling, Staining

    2) Product Images from "An RNA trapping mechanism in Alphavirus mRNA promotes ribosome stalling and translation initiation"

    Article Title: An RNA trapping mechanism in Alphavirus mRNA promotes ribosome stalling and translation initiation

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw172

    Effect of VIC oligo 4 on translation of alphavirus 26S mRNA. ( A ) Bottom view of rabbit 40S subunit crystal structure showing the region ES6S C-D where oligo 4 hybridizes (marked in green). ( B ) Binding of VIC oligo 4 to rabbit 40S subunit. Approximately 50 pmol of purified rabbit 40S subunit was incubated with 200 pmol of VIC oligo 4 or VIC SVcapside (negative control, ( 20 )) for 20 min at 30°C and centrifuged in a 10–30% sucrose gradient at 45 000 rpm for 3 h. The fluorescence of VIC fluorochrome in each fraction was measured in a FLUOstar OPTIMA apparatus. ( C ) Effect of VIC oligo 4 on accumulation of SFV C protein analyzed by immunofluorescence using a polyclonal antibody raised against SFV C protein. Note that those cells showing strong VIC fluorescence (orange-red) showed little SFV C staining (green). ( D ) Effect of VIC oligo 4 on translation of 26S mRNA in SFV-infected cells. Cells were transfected with the indicated oligonucleotide, and 15 h later infected with SFV at a moi of 25 pfu/cell. The cells were metabolically labeled with [ 35 S]-Met/Cys at 6 hpi. Labeled proteins were analyzed by autoradiography. p62, E1 and C are the structural proteins of the virus encoded by the 26S mRNA.
    Figure Legend Snippet: Effect of VIC oligo 4 on translation of alphavirus 26S mRNA. ( A ) Bottom view of rabbit 40S subunit crystal structure showing the region ES6S C-D where oligo 4 hybridizes (marked in green). ( B ) Binding of VIC oligo 4 to rabbit 40S subunit. Approximately 50 pmol of purified rabbit 40S subunit was incubated with 200 pmol of VIC oligo 4 or VIC SVcapside (negative control, ( 20 )) for 20 min at 30°C and centrifuged in a 10–30% sucrose gradient at 45 000 rpm for 3 h. The fluorescence of VIC fluorochrome in each fraction was measured in a FLUOstar OPTIMA apparatus. ( C ) Effect of VIC oligo 4 on accumulation of SFV C protein analyzed by immunofluorescence using a polyclonal antibody raised against SFV C protein. Note that those cells showing strong VIC fluorescence (orange-red) showed little SFV C staining (green). ( D ) Effect of VIC oligo 4 on translation of 26S mRNA in SFV-infected cells. Cells were transfected with the indicated oligonucleotide, and 15 h later infected with SFV at a moi of 25 pfu/cell. The cells were metabolically labeled with [ 35 S]-Met/Cys at 6 hpi. Labeled proteins were analyzed by autoradiography. p62, E1 and C are the structural proteins of the virus encoded by the 26S mRNA.

    Techniques Used: Binding Assay, Purification, Incubation, Negative Control, Fluorescence, Immunofluorescence, Staining, Infection, Transfection, Metabolic Labelling, Labeling, Autoradiography

    3) Product Images from "Multimerization Domains are Associated with Apparent Strand Exchange Activity in BLM and WRN DNA helicases"

    Article Title: Multimerization Domains are Associated with Apparent Strand Exchange Activity in BLM and WRN DNA helicases

    Journal: DNA repair

    doi: 10.1016/j.dnarep.2014.07.015

    Characterization of apparent DNA strand exchange activity in SA proteins. (A) The reaction illustrated at the top of the panel was performed using a 32 bp radiolabeled dsDNA with flush ends as donor (oligos #4*/#5) and an unlabeled 57 nt oligo (#6) as recipient. Reactions containing 5 nM donor and 20 nM recipient were initiated by addition of the indicated concentrations of Rad52N, Sgs1 103–322 , or Rad59. Following incubation at 37°C for 30 min, the reactions were terminated and the products analyzed by 10% native PAGE and phosphorimaging. ( B ) SE reactions were carried out as in (A) except that aliquots of the reactions were removed at the indicated times and treated as above. ( C ) SE reactions were performed with Rad52 mutants. ( D ) SE assays were performed with sub-domains of WRN 235–526 .
    Figure Legend Snippet: Characterization of apparent DNA strand exchange activity in SA proteins. (A) The reaction illustrated at the top of the panel was performed using a 32 bp radiolabeled dsDNA with flush ends as donor (oligos #4*/#5) and an unlabeled 57 nt oligo (#6) as recipient. Reactions containing 5 nM donor and 20 nM recipient were initiated by addition of the indicated concentrations of Rad52N, Sgs1 103–322 , or Rad59. Following incubation at 37°C for 30 min, the reactions were terminated and the products analyzed by 10% native PAGE and phosphorimaging. ( B ) SE reactions were carried out as in (A) except that aliquots of the reactions were removed at the indicated times and treated as above. ( C ) SE reactions were performed with Rad52 mutants. ( D ) SE assays were performed with sub-domains of WRN 235–526 .

    Techniques Used: Activity Assay, Incubation, Clear Native PAGE

    4) Product Images from "Rapid and dynamic nucleic acid hybridization enables enzymatic oligonucleotide synthesis by cyclic reversible termination: A novel mechanism for enzymatic DNA synthesis"

    Article Title: Rapid and dynamic nucleic acid hybridization enables enzymatic oligonucleotide synthesis by cyclic reversible termination: A novel mechanism for enzymatic DNA synthesis

    Journal: bioRxiv

    doi: 10.1101/561092

    Dynamic hybridization of DNA enables EOS. (a) Extension of single-stranded DNA by DNA polymerases and reverse transcriptases. Denaturing PAGE analysis of a 20 base single-stranded sequence, self-priming oligo-1 aka SPO-1, using different enzymes and dGTP. Despite a maximum of two bases of hybridization, some enzymes are able to extend this solid-phase oligonucleotide. None of these enzymes have previously been reported to have nucleotidyl transferase activity on single-stranded DNA. (b) Sequence-specific extension of single-stranded DNA. Duplase extension of four different 20 base oligos proceeds in a sequence-specific manner, and only two bases of hybridization are required for extension. * indicates unextended control. Extension with non-templated bases can be explained by misincorporation that occurs at long reaction times . (c) Extension of solid-phase oligos through intermolecular reactions. Magnetic beads were conjugated with either a 20 base poly-T oligo as illustrated in i, or with that oligo plus a 30 base poly-T oligo with an internal 5’-CAA-3’ sequence as illustrated in ii . Beads were extended using Duplase-3 and Cy5-ddGTP (* indicates unextended control during this step), after which, all samples were labeled with TdT and fluorescein-12-ddUTP. Oligos appear blue if extended with Duplase-3 and green if extended wtih TdT.
    Figure Legend Snippet: Dynamic hybridization of DNA enables EOS. (a) Extension of single-stranded DNA by DNA polymerases and reverse transcriptases. Denaturing PAGE analysis of a 20 base single-stranded sequence, self-priming oligo-1 aka SPO-1, using different enzymes and dGTP. Despite a maximum of two bases of hybridization, some enzymes are able to extend this solid-phase oligonucleotide. None of these enzymes have previously been reported to have nucleotidyl transferase activity on single-stranded DNA. (b) Sequence-specific extension of single-stranded DNA. Duplase extension of four different 20 base oligos proceeds in a sequence-specific manner, and only two bases of hybridization are required for extension. * indicates unextended control. Extension with non-templated bases can be explained by misincorporation that occurs at long reaction times . (c) Extension of solid-phase oligos through intermolecular reactions. Magnetic beads were conjugated with either a 20 base poly-T oligo as illustrated in i, or with that oligo plus a 30 base poly-T oligo with an internal 5’-CAA-3’ sequence as illustrated in ii . Beads were extended using Duplase-3 and Cy5-ddGTP (* indicates unextended control during this step), after which, all samples were labeled with TdT and fluorescein-12-ddUTP. Oligos appear blue if extended with Duplase-3 and green if extended wtih TdT.

    Techniques Used: Hybridization, Polyacrylamide Gel Electrophoresis, Sequencing, Activity Assay, Magnetic Beads, Labeling

    5) Product Images from "Molecular Factors Affecting the Accumulation of Recombinant Proteins in the Chlamydomonas reinhardtii Chloroplast"

    Article Title: Molecular Factors Affecting the Accumulation of Recombinant Proteins in the Chlamydomonas reinhardtii Chloroplast

    Journal: Molecular Biotechnology

    doi: 10.1007/s12033-010-9348-4

    Toeprint analyses of translation initiation complexes on atpA and atpA – gfp RNAs. In vitro transcribed atpA – atpA ( left panels ) and atpA – gfp ( right panels ) RNAs (20 nM) were extended from [γ- 32 P] ATP-labeled oligos that anneal 120-nt downstream of the initiator AUGs, either alone or in the presence of tRNA Met , 30S subunit or both. Sequencing reactions were run alongside. The treatments are indicated at the top of each panel. The positions of the toeprints respective to the initiator AUG (+1) are indicated with lines on the side. a Toeprint reactions using 100-nM 30S subunit, b Inset showing the toeprint area of experiments similar to ( a ) with 200-nM 30S subunit. The figure shows representative autoradiographs obtained from three independent experiments
    Figure Legend Snippet: Toeprint analyses of translation initiation complexes on atpA and atpA – gfp RNAs. In vitro transcribed atpA – atpA ( left panels ) and atpA – gfp ( right panels ) RNAs (20 nM) were extended from [γ- 32 P] ATP-labeled oligos that anneal 120-nt downstream of the initiator AUGs, either alone or in the presence of tRNA Met , 30S subunit or both. Sequencing reactions were run alongside. The treatments are indicated at the top of each panel. The positions of the toeprints respective to the initiator AUG (+1) are indicated with lines on the side. a Toeprint reactions using 100-nM 30S subunit, b Inset showing the toeprint area of experiments similar to ( a ) with 200-nM 30S subunit. The figure shows representative autoradiographs obtained from three independent experiments

    Techniques Used: In Vitro, Labeling, Sequencing

    6) Product Images from "Local regulation of gene expression by lncRNA promoters, transcription, and splicing"

    Article Title: Local regulation of gene expression by lncRNA promoters, transcription, and splicing

    Journal: Nature

    doi: 10.1038/nature20149

    Characterization of genetic modifications in the Blustr locus. (a) Allele-specific GRO-seq signal for clones with the indicated modifications at the Blustr locus. Only reads specifically mapping to one of the two alleles are shown. Y -axis scale represents normalized read count and is the same for all tracks, and is magnified 5 times at the indicated location to better visualize the reads in the Sfmbt2 locus. (b) Quantification of allele-specific GRO-seq signal in the Sfmbt2 locus on alleles modified as indicated. TSS: region including the two alternative TSSs of Sfmbt2 and 2 kb downstream. Gene body: region containing the remainder of the Sfmbt2 gene locus. Pause index: ratio of TSS to gene body. Dashed gray lines indicate the 95% CI for the mean of 8 wild-type clones. (c) Schematic of the 5’ end of the Blustr locus and genotypes of two knockout clones. The 5’ splice site is located 78 bp downstream of the Blustr transcription start site (in this panel, Blustr is transcribed from left to right). One of the alleles from the two clones contains insertion of the oligo mediated by homologous recombination; the remaining three alleles contain insertions or deletions resulting from non-homologous end joining repair of sgRNA-mediated double-strand breaks, some of which also disrupt the 5’ splice site. Barplots show allele-specific RNA expression for knockout clones and control clones (+/+). (d) Schematic of the observed splice structures of Blustr RNA transcripts in p(A)+ RNA sequencing of the exon deletion clones. Each deletion removes a region including ~50–200 bp on either side of the exon, thereby removing both the exon and its splice sites. The Exon 4 deletion removes the endogenous pAS, leading to new isoforms of the lncRNA transcript that splice into two cryptic splice acceptors downstream. (e) GRO-Seq, H3K4me3 ChIP-Seq, and chromatin accessibility (ATAC-Seq FPKM) at the Blustr and Sfmbt2 promoters in cell lines with the indicated genotypes. Deletion of the first 5’ splice site leads to a significant reduction in H3K4me3, RNA polymerase occupancy, and chromatin accessibility at the Blustr promoter, as well as H3K4me3 and RNA polymerase occupancy (but not accessibility) at the Sfmbt2 promoter. ( f ) H3K27me3 ChIP-seq at the Blustr and Sfmbt2 loci in cell lines with the indicated genotypes. Deletion of the Blustr promoter or 5’ splice site leads to spreading of the repression-associated H3K27me3 modification across a ~30 kb region.
    Figure Legend Snippet: Characterization of genetic modifications in the Blustr locus. (a) Allele-specific GRO-seq signal for clones with the indicated modifications at the Blustr locus. Only reads specifically mapping to one of the two alleles are shown. Y -axis scale represents normalized read count and is the same for all tracks, and is magnified 5 times at the indicated location to better visualize the reads in the Sfmbt2 locus. (b) Quantification of allele-specific GRO-seq signal in the Sfmbt2 locus on alleles modified as indicated. TSS: region including the two alternative TSSs of Sfmbt2 and 2 kb downstream. Gene body: region containing the remainder of the Sfmbt2 gene locus. Pause index: ratio of TSS to gene body. Dashed gray lines indicate the 95% CI for the mean of 8 wild-type clones. (c) Schematic of the 5’ end of the Blustr locus and genotypes of two knockout clones. The 5’ splice site is located 78 bp downstream of the Blustr transcription start site (in this panel, Blustr is transcribed from left to right). One of the alleles from the two clones contains insertion of the oligo mediated by homologous recombination; the remaining three alleles contain insertions or deletions resulting from non-homologous end joining repair of sgRNA-mediated double-strand breaks, some of which also disrupt the 5’ splice site. Barplots show allele-specific RNA expression for knockout clones and control clones (+/+). (d) Schematic of the observed splice structures of Blustr RNA transcripts in p(A)+ RNA sequencing of the exon deletion clones. Each deletion removes a region including ~50–200 bp on either side of the exon, thereby removing both the exon and its splice sites. The Exon 4 deletion removes the endogenous pAS, leading to new isoforms of the lncRNA transcript that splice into two cryptic splice acceptors downstream. (e) GRO-Seq, H3K4me3 ChIP-Seq, and chromatin accessibility (ATAC-Seq FPKM) at the Blustr and Sfmbt2 promoters in cell lines with the indicated genotypes. Deletion of the first 5’ splice site leads to a significant reduction in H3K4me3, RNA polymerase occupancy, and chromatin accessibility at the Blustr promoter, as well as H3K4me3 and RNA polymerase occupancy (but not accessibility) at the Sfmbt2 promoter. ( f ) H3K27me3 ChIP-seq at the Blustr and Sfmbt2 loci in cell lines with the indicated genotypes. Deletion of the Blustr promoter or 5’ splice site leads to spreading of the repression-associated H3K27me3 modification across a ~30 kb region.

    Techniques Used: Clone Assay, Modification, Knock-Out, Homologous Recombination, Non-Homologous End Joining, RNA Expression, RNA Sequencing Assay, Chromatin Immunoprecipitation

    7) Product Images from "An mRNA-binding channel in the ES6S region of the translation 48S-PIC promotes RNA unwinding and scanning"

    Article Title: An mRNA-binding channel in the ES6S region of the translation 48S-PIC promotes RNA unwinding and scanning

    Journal: eLife

    doi: 10.7554/eLife.48246

    Quality and reproducibility of the experiment shown in Figure 5 . ( a ) Polysome profile of HEK293T cells transfected with the indicated oligonucleotides. This experiment corresponds to the replica_1 used for RNA-seq, indicating fractions pooled as monosomes or polysomes. The bottom panel shows a western blot analysis of the resulting fractions probed with anti-eS6 antibody. ( b ) Analysis of correlation between replicas using a normalized number of reads for every mRNA in the monosomal and polysomal samples. ( c ) Changes in mRNA abundance between oligo 4 and VIC–oligo-4-transfected cells. Data are the average from two replicates. ( d ) Enrichment in KEGG pathways detected in the ‘TE down’ group of mRNAs. The p-value and enrichment factor for each term are indicated.
    Figure Legend Snippet: Quality and reproducibility of the experiment shown in Figure 5 . ( a ) Polysome profile of HEK293T cells transfected with the indicated oligonucleotides. This experiment corresponds to the replica_1 used for RNA-seq, indicating fractions pooled as monosomes or polysomes. The bottom panel shows a western blot analysis of the resulting fractions probed with anti-eS6 antibody. ( b ) Analysis of correlation between replicas using a normalized number of reads for every mRNA in the monosomal and polysomal samples. ( c ) Changes in mRNA abundance between oligo 4 and VIC–oligo-4-transfected cells. Data are the average from two replicates. ( d ) Enrichment in KEGG pathways detected in the ‘TE down’ group of mRNAs. The p-value and enrichment factor for each term are indicated.

    Techniques Used: Transfection, RNA Sequencing Assay, Western Blot

    Effect of VIC–oligo 4 on protein crosslinking in 48S complexes assembled with unstructured or SV-DLP n27 mRNAs. The amount of [ 32 P]-mRNA bound to WRF is indicated (upper panel), showing the pattern of protein crosslinking with the indicated mRNAs in the absence and presence of VIC–oligo 4. The unresolved eIF3g and eIF4A bands are shown (lower panel).
    Figure Legend Snippet: Effect of VIC–oligo 4 on protein crosslinking in 48S complexes assembled with unstructured or SV-DLP n27 mRNAs. The amount of [ 32 P]-mRNA bound to WRF is indicated (upper panel), showing the pattern of protein crosslinking with the indicated mRNAs in the absence and presence of VIC–oligo 4. The unresolved eIF3g and eIF4A bands are shown (lower panel).

    Techniques Used:

    mRNA threading into the ES6S region slows down scanning but makes it more processive. ( a ) Effect of FITC-oligo 4 on the translation of Luc mRNAs with different 5′ UTRs in RRL. Translation mixtures were incubated for 90 min, which represented the endpoint measurement because no further increase in luc activity was detected. Data are represented as the mean ± SD from at least three independent experiments. ( b ) Luc activity accumulation in continuously recording experiments programmed with the indicated mRNAs. Measurements were taken every 3 min: gray line, no oligo; black line, +FITC–oligo 4. Hipp was added to the indicated samples at a concentration of 2 μM (dashed line). ( c ) Estimates of full translation time (FTT) for 5′ UTR G-less and 5′ UTR-SL20 mRNAs, and the effect of FITC–oligo 4 and hipp on FTT. Data from panel (B) were processed as described before ( Vassilenko et al., 2011 ). The determined FTT values were: 5′ UTR G-less = 9.53 min; 5′ UTR G-less+FITC–oligo 4 = 7.89 min; 5′ UTR-SL20 = 17.57 min; 5′ UTR-SL20+FITC–oligo 4 = 17.56 min; 5′ UTR-SL20+hipp = 19.44 min. ( d ) Synergistic inhibitory effect of VIC–oligo 4 and hipp on translation of 5′UTR SL30-Luc and 5′ UTR G4-1-Luc mRNAs in RRL. Translation mixtures were preincubated with 6 μM of VIC–oligo 4 and with increasing concentrations of hipp for 5 min. Then, mRNAs were added and measurements were taken 90 min later; the calculated combination index (CI) for each mRNA is indicated.
    Figure Legend Snippet: mRNA threading into the ES6S region slows down scanning but makes it more processive. ( a ) Effect of FITC-oligo 4 on the translation of Luc mRNAs with different 5′ UTRs in RRL. Translation mixtures were incubated for 90 min, which represented the endpoint measurement because no further increase in luc activity was detected. Data are represented as the mean ± SD from at least three independent experiments. ( b ) Luc activity accumulation in continuously recording experiments programmed with the indicated mRNAs. Measurements were taken every 3 min: gray line, no oligo; black line, +FITC–oligo 4. Hipp was added to the indicated samples at a concentration of 2 μM (dashed line). ( c ) Estimates of full translation time (FTT) for 5′ UTR G-less and 5′ UTR-SL20 mRNAs, and the effect of FITC–oligo 4 and hipp on FTT. Data from panel (B) were processed as described before ( Vassilenko et al., 2011 ). The determined FTT values were: 5′ UTR G-less = 9.53 min; 5′ UTR G-less+FITC–oligo 4 = 7.89 min; 5′ UTR-SL20 = 17.57 min; 5′ UTR-SL20+FITC–oligo 4 = 17.56 min; 5′ UTR-SL20+hipp = 19.44 min. ( d ) Synergistic inhibitory effect of VIC–oligo 4 and hipp on translation of 5′UTR SL30-Luc and 5′ UTR G4-1-Luc mRNAs in RRL. Translation mixtures were preincubated with 6 μM of VIC–oligo 4 and with increasing concentrations of hipp for 5 min. Then, mRNAs were added and measurements were taken 90 min later; the calculated combination index (CI) for each mRNA is indicated.

    Techniques Used: Incubation, Activity Assay, Concentration Assay

    Effect of 5' UTR length of luc mRNAs on the sensitivity to VIC-oligo 4-mediated translational block. ( a ) Effect of 5′ UTR length on cap-dependent translation ofluc mRNAs in the presence of VIC-oligo 4. Data are the mean of two independent experiments performed in MEF cells as described in Figure 4a . ( b ) Comparative analysis of the effect of oligo 4, VIC–oligo C and VIC–oligo 4 on the translation of the indicated mRNAs. Data are the mean ± SD from at least four independent experiments in MEF cells. ( c ) Effect of VIC–oligo 4 on the translation of poliovirus (PV) mRNA. HeLa cells were transfected with the indicated oligonucleotides, and infected 12 hr later with PV1 (Mahoney strain) at a multiplicity of infection (MOI) of 10 pfu/cell. The cultures were metabolically labeled with [ 35 S]-Met at 6 hr post-infection and analyzed as described in the Materials and methods; SYPRO staining was included as the loading control.
    Figure Legend Snippet: Effect of 5' UTR length of luc mRNAs on the sensitivity to VIC-oligo 4-mediated translational block. ( a ) Effect of 5′ UTR length on cap-dependent translation ofluc mRNAs in the presence of VIC-oligo 4. Data are the mean of two independent experiments performed in MEF cells as described in Figure 4a . ( b ) Comparative analysis of the effect of oligo 4, VIC–oligo C and VIC–oligo 4 on the translation of the indicated mRNAs. Data are the mean ± SD from at least four independent experiments in MEF cells. ( c ) Effect of VIC–oligo 4 on the translation of poliovirus (PV) mRNA. HeLa cells were transfected with the indicated oligonucleotides, and infected 12 hr later with PV1 (Mahoney strain) at a multiplicity of infection (MOI) of 10 pfu/cell. The cultures were metabolically labeled with [ 35 S]-Met at 6 hr post-infection and analyzed as described in the Materials and methods; SYPRO staining was included as the loading control.

    Techniques Used: Blocking Assay, Transfection, Infection, Metabolic Labelling, Labeling, Staining

    8) Product Images from "Action of CMG with strand-specific DNA blocks supports an internal unwinding mode for the eukaryotic replicative helicase"

    Article Title: Action of CMG with strand-specific DNA blocks supports an internal unwinding mode for the eukaryotic replicative helicase

    Journal: eLife

    doi: 10.7554/eLife.23449

    Substrate single strands do not spontaneously reanneal at 30˚C. To determine whether unwound substrate oligos spontaneously reanneal under our reaction conditions, we mixed 0.5 nM final concentration of radiolabeled 50duplex LAG oligo ( Table 1 ) with 0.5 nM unlabeled 50 duplex LEAD oligo under reaction conditions identical to those of Figure 3 in a total reaction volume of 55 μl. Complete reactions were mixed on ice and started by incubating at 30°C. At the indicated time points, 10 μl aliquots were removed and flash frozen in liquid nitrogen. Lack of re-annealing of the unwound strands allowed us to perform unwinding reactions in Figure 3 and subsequent Figures in the absence of a trap, eliminating potential side interactions between CMG and the unlabeled trap DNA. DOI: http://dx.doi.org/10.7554/eLife.23449.010
    Figure Legend Snippet: Substrate single strands do not spontaneously reanneal at 30˚C. To determine whether unwound substrate oligos spontaneously reanneal under our reaction conditions, we mixed 0.5 nM final concentration of radiolabeled 50duplex LAG oligo ( Table 1 ) with 0.5 nM unlabeled 50 duplex LEAD oligo under reaction conditions identical to those of Figure 3 in a total reaction volume of 55 μl. Complete reactions were mixed on ice and started by incubating at 30°C. At the indicated time points, 10 μl aliquots were removed and flash frozen in liquid nitrogen. Lack of re-annealing of the unwound strands allowed us to perform unwinding reactions in Figure 3 and subsequent Figures in the absence of a trap, eliminating potential side interactions between CMG and the unlabeled trap DNA. DOI: http://dx.doi.org/10.7554/eLife.23449.010

    Techniques Used: Concentration Assay

    CMG requires a 3’ dT 40 /ssDNA tail for loading. . ( A ) Schematic of the substrates used in Figure 2 and in part ( B ) of this Figure. Further details on the oligos used are in Table 1 . The flush duplex oligo is named ‘flush duplex LAG’. The 5’ tailed oligo is ‘50duplex LAG’. The leading strand template is either ‘Paired duplex LEAD + 3’ tail’ or ‘Paired duplex LEAD no 3’ tail’. ( B ) Control experiments to show the requirement for the 3’ dT 40 tail in CMG loading. At left are two helicase assays like those described in Figure 2 but using the substrate containing a 3’ dT 40 tail with the radiolabel on the 5’ tailed duplex. Lanes 1–4 show unwinding in the absence of the flush duplex oligo and lanes 5–8 in the presence of the flush duplex. The % of 5’ tailed oligo unwound is quantified in the graph below the gels, showing that the presence of the flush duplex oligo does not affect CMG loading or unwinding. At right is an identical pair of experiments but with a substrate that does not contain the 3’ dT 40 tail. Lanes 9–12 and the quantitation in the graph below show that unwinding is greatly reduced in the absence of the 3’ tail (compare lanes 9–12 with lanes 1–4), and in the presence of the flush duplex oligo (lanes 13–16) CMG does not load/unwind at all. These experiments support the conclusion of Figure 2 that CMG translocates over flush duplex without unwinding and requires a 3’ dT 40 tail for efficient loading. DOI: http://dx.doi.org/10.7554/eLife.23449.004
    Figure Legend Snippet: CMG requires a 3’ dT 40 /ssDNA tail for loading. . ( A ) Schematic of the substrates used in Figure 2 and in part ( B ) of this Figure. Further details on the oligos used are in Table 1 . The flush duplex oligo is named ‘flush duplex LAG’. The 5’ tailed oligo is ‘50duplex LAG’. The leading strand template is either ‘Paired duplex LEAD + 3’ tail’ or ‘Paired duplex LEAD no 3’ tail’. ( B ) Control experiments to show the requirement for the 3’ dT 40 tail in CMG loading. At left are two helicase assays like those described in Figure 2 but using the substrate containing a 3’ dT 40 tail with the radiolabel on the 5’ tailed duplex. Lanes 1–4 show unwinding in the absence of the flush duplex oligo and lanes 5–8 in the presence of the flush duplex. The % of 5’ tailed oligo unwound is quantified in the graph below the gels, showing that the presence of the flush duplex oligo does not affect CMG loading or unwinding. At right is an identical pair of experiments but with a substrate that does not contain the 3’ dT 40 tail. Lanes 9–12 and the quantitation in the graph below show that unwinding is greatly reduced in the absence of the 3’ tail (compare lanes 9–12 with lanes 1–4), and in the presence of the flush duplex oligo (lanes 13–16) CMG does not load/unwind at all. These experiments support the conclusion of Figure 2 that CMG translocates over flush duplex without unwinding and requires a 3’ dT 40 tail for efficient loading. DOI: http://dx.doi.org/10.7554/eLife.23449.004

    Techniques Used: Quantitation Assay

    Schematics of biotinylated DNA fork substrates. The fork substrates used in the experiments in Figures 3 – 6 are shown including the location of the biotinylated dT nucleotides in either the leading or lagging strand template. The oligos used to make the forked duplex substrates are indicated to the right of the schematics and the oligo sequences and modifications are in Table 1 . DOI: http://dx.doi.org/10.7554/eLife.23449.008
    Figure Legend Snippet: Schematics of biotinylated DNA fork substrates. The fork substrates used in the experiments in Figures 3 – 6 are shown including the location of the biotinylated dT nucleotides in either the leading or lagging strand template. The oligos used to make the forked duplex substrates are indicated to the right of the schematics and the oligo sequences and modifications are in Table 1 . DOI: http://dx.doi.org/10.7554/eLife.23449.008

    Techniques Used:

    9) Product Images from "The Annealing Helicase and Branch Migration Activities of Drosophila HARP"

    Article Title: The Annealing Helicase and Branch Migration Activities of Drosophila HARP

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0098173

    The branch migration activities of dm HARP and hs HARP differ. ( A ) dm HARP is competent for catalyzing Holliday junction migration but not replication fork regression. The reaction scheme is shown above the figure along with whether dm HARP was expressed in E. coli [E] or baculovirus-infected insect cells [b]. The gel migration of the replication fork and Holliday junction substrates is shown at the left along with the dsDNA products. The red dot signifies the common 5′- 32 P-labeled oligo (A60; Material and Methods ). Lanes dealing with an unrelated helicase were removed between lanes 4 and 5. ( B ) dm HARP is competent in disrupting D-loops. D-loops were formed with a supercoiled plasmid and a labeled 90mer DNA oligonucleotide, chromatographically purified and incubated with dm HARP or hs HARP expressed in E. coli or baculovirus-infected cells as indicated above the gel image. The D-loop and 90mer are identified at the left.
    Figure Legend Snippet: The branch migration activities of dm HARP and hs HARP differ. ( A ) dm HARP is competent for catalyzing Holliday junction migration but not replication fork regression. The reaction scheme is shown above the figure along with whether dm HARP was expressed in E. coli [E] or baculovirus-infected insect cells [b]. The gel migration of the replication fork and Holliday junction substrates is shown at the left along with the dsDNA products. The red dot signifies the common 5′- 32 P-labeled oligo (A60; Material and Methods ). Lanes dealing with an unrelated helicase were removed between lanes 4 and 5. ( B ) dm HARP is competent in disrupting D-loops. D-loops were formed with a supercoiled plasmid and a labeled 90mer DNA oligonucleotide, chromatographically purified and incubated with dm HARP or hs HARP expressed in E. coli or baculovirus-infected cells as indicated above the gel image. The D-loop and 90mer are identified at the left.

    Techniques Used: Migration, Infection, Labeling, Plasmid Preparation, Purification, Incubation

    10) Product Images from "Epitope mapping using mRNA display and a unidirectional nested deletion library"

    Article Title: Epitope mapping using mRNA display and a unidirectional nested deletion library

    Journal:

    doi: 10.1093/protein/gzi038

    In vitro selection scheme using mRNA display. The starting dsDNA pool (top, center) which encodes the peptide library is transcribed in vitro . Purified mRNA is enzymatically ligated to a puromycin-DNA oligo prior to RNA-peptide fusion formation via in
    Figure Legend Snippet: In vitro selection scheme using mRNA display. The starting dsDNA pool (top, center) which encodes the peptide library is transcribed in vitro . Purified mRNA is enzymatically ligated to a puromycin-DNA oligo prior to RNA-peptide fusion formation via in

    Techniques Used: In Vitro, Selection, Purification

    11) Product Images from "Somatic Hypermutation at A/T-Rich Oligonucleotide Substrates Shows Different Strand Polarities in Ung-Deficient or -Proficient Backgrounds"

    Article Title: Somatic Hypermutation at A/T-Rich Oligonucleotide Substrates Shows Different Strand Polarities in Ung-Deficient or -Proficient Backgrounds

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.01452-13

    Knock-in strategy for the generation of A/T-rich oligonucleotide mutation substrates. Oligonucleotide mutation substrates were inserted by knock-in at the heavy chain locus, 43 bp downstream of the J H 4 segment. A schematized view of the mutation distribution within 490 bp of the J H 4 intron highlights the mutation density around the oligonucleotide insertion site. The oligonucleotides consisted of 10 repeats of a 9-mer sequence, TATTATTAA, with 4 repeats flanking 3 G's separated by the same 9-mer sequence (oligo-G). Oligo-C represents the same sequence inserted in reverse orientation. Oligo-noG and -noC consisted of the same A/T backbone, with G's and C's, respectively, omitted. The 3 segments of the oligonucleotide transgene used in subsequent mutation analysis are represented as the 5′ segment, core segment, and 3′ segment.
    Figure Legend Snippet: Knock-in strategy for the generation of A/T-rich oligonucleotide mutation substrates. Oligonucleotide mutation substrates were inserted by knock-in at the heavy chain locus, 43 bp downstream of the J H 4 segment. A schematized view of the mutation distribution within 490 bp of the J H 4 intron highlights the mutation density around the oligonucleotide insertion site. The oligonucleotides consisted of 10 repeats of a 9-mer sequence, TATTATTAA, with 4 repeats flanking 3 G's separated by the same 9-mer sequence (oligo-G). Oligo-C represents the same sequence inserted in reverse orientation. Oligo-noG and -noC consisted of the same A/T backbone, with G's and C's, respectively, omitted. The 3 segments of the oligonucleotide transgene used in subsequent mutation analysis are represented as the 5′ segment, core segment, and 3′ segment.

    Techniques Used: Knock-In, Mutagenesis, Sequencing

    12) Product Images from "Depletion of erythropoietic miR-486-5p and miR-451a improves detectability of rare microRNAs in peripheral blood-derived small RNA sequencing libraries"

    Article Title: Depletion of erythropoietic miR-486-5p and miR-451a improves detectability of rare microRNAs in peripheral blood-derived small RNA sequencing libraries

    Journal: bioRxiv

    doi: 10.1101/789891

    Blocking oligonucleotides efficiently suppress miR-486-5p and miR-451a, and increase detectability of other miRNA species in small RNA libraries. ( A ) A bar chart represents quantitative estimates of miR-486-5p and miR-451a in blocked (red) and unblocked (blue) libraries prepared by NEXTflex and TruSeq protocols. The y-axis depicts counts per million (CPM) and it is scaled by square root. The error bars indicate min and max values obtained from replicates. The graph illustrates a high degree suppression of miR-451a and miR-486-5p sequences in blocked NEXTflex and TruSeq libraries; ( B ) A dot plot represents blocking efficiencies (y-axis) of miR-486-5p and miR-451a in NEXTflex and TruSeq libraries. The data points depict mean values, whereas the error bars indicate min and max values obtained from replicates. The overall blocking efficiency is observed to be slightly better in the NEXTflex than in the TruSeq small RNA libraries (Wilcoxon P-value = 0.012); ( C ) A bar chart shows number of detected miRNA species (y-axis) in blocked (red) and unblocked (blue) libraries prepared by NEXTflex and TruSeq protocols. The detectability of miRNAs is increased in both blocked NEXTflex and TruSeq libraries; ( D ) An upset plot representing intersection of uniquely detected miRNA species amongst the set of the four protocols. The highest numbers of uniquely detected miRNAs are found in blocked libraries; ( E ) A line chart illustrates number of detected miRNAs (y-axis) in subsamples (x-axis; scaled by square root) of down-sampled libraries prepared by different protocols. The data points represent mean values, whereas the error bars depict standard errors of the mean. The graph displays a steady increase of detected miRNAs over the increasing size of subsampled miRNA counts.
    Figure Legend Snippet: Blocking oligonucleotides efficiently suppress miR-486-5p and miR-451a, and increase detectability of other miRNA species in small RNA libraries. ( A ) A bar chart represents quantitative estimates of miR-486-5p and miR-451a in blocked (red) and unblocked (blue) libraries prepared by NEXTflex and TruSeq protocols. The y-axis depicts counts per million (CPM) and it is scaled by square root. The error bars indicate min and max values obtained from replicates. The graph illustrates a high degree suppression of miR-451a and miR-486-5p sequences in blocked NEXTflex and TruSeq libraries; ( B ) A dot plot represents blocking efficiencies (y-axis) of miR-486-5p and miR-451a in NEXTflex and TruSeq libraries. The data points depict mean values, whereas the error bars indicate min and max values obtained from replicates. The overall blocking efficiency is observed to be slightly better in the NEXTflex than in the TruSeq small RNA libraries (Wilcoxon P-value = 0.012); ( C ) A bar chart shows number of detected miRNA species (y-axis) in blocked (red) and unblocked (blue) libraries prepared by NEXTflex and TruSeq protocols. The detectability of miRNAs is increased in both blocked NEXTflex and TruSeq libraries; ( D ) An upset plot representing intersection of uniquely detected miRNA species amongst the set of the four protocols. The highest numbers of uniquely detected miRNAs are found in blocked libraries; ( E ) A line chart illustrates number of detected miRNAs (y-axis) in subsamples (x-axis; scaled by square root) of down-sampled libraries prepared by different protocols. The data points represent mean values, whereas the error bars depict standard errors of the mean. The graph displays a steady increase of detected miRNAs over the increasing size of subsampled miRNA counts.

    Techniques Used: Blocking Assay

    Blocking oligonucleotides for miR-486-5p and miR-451a have positive and some negative effects on measured expression of non-targeted miRNAs. ( A ) A density chart shows distributions of averaged log2-transformed CPM values of blocked (red) and unblocked (blue) libraries prepared by NEXTflex and TruSeq protocols. The expression values of each miRNA were averaged per protocol (x-axis). The graph illustrates a proportional global shift of log2-transformed CPM values towards higher expression estimates in both blocked NEXTflex and TruSeq libraries; ( B ) A scatter plot represents correlation of log2-transformed CPM values of paired blocked (y-axis) and unblocked (x-axis) exemplary libraries generated by NEXTflex and TruSeq protocols. The red dashed line divides panel in two equal parts, whereas the grey dashed line displays a linear regression curve. The chart illustrates a high concordance of miRNA expression values between blocked and unblocked paired libraries; ( C ) An MA plot shows paired-differential expression analysis results of blocked versus unblocked libraries, where log2 fold changes are presented on the y-axis and averaged normalized counts on the x-axis. The red colored dots indicate significantly differentially expressed miRNAs (corrected P-value
    Figure Legend Snippet: Blocking oligonucleotides for miR-486-5p and miR-451a have positive and some negative effects on measured expression of non-targeted miRNAs. ( A ) A density chart shows distributions of averaged log2-transformed CPM values of blocked (red) and unblocked (blue) libraries prepared by NEXTflex and TruSeq protocols. The expression values of each miRNA were averaged per protocol (x-axis). The graph illustrates a proportional global shift of log2-transformed CPM values towards higher expression estimates in both blocked NEXTflex and TruSeq libraries; ( B ) A scatter plot represents correlation of log2-transformed CPM values of paired blocked (y-axis) and unblocked (x-axis) exemplary libraries generated by NEXTflex and TruSeq protocols. The red dashed line divides panel in two equal parts, whereas the grey dashed line displays a linear regression curve. The chart illustrates a high concordance of miRNA expression values between blocked and unblocked paired libraries; ( C ) An MA plot shows paired-differential expression analysis results of blocked versus unblocked libraries, where log2 fold changes are presented on the y-axis and averaged normalized counts on the x-axis. The red colored dots indicate significantly differentially expressed miRNAs (corrected P-value

    Techniques Used: Blocking Assay, Expressing, Transformation Assay, Generated

    Blocking oligo design and application workflows using modified TruSeq and NEXTflex small RNA library preparation protocols. ( A ) Design principle of miR-486-5p and miR-451a blocking oligonucleotides. Briefly, unique pooled-sample sequences which mapped to precursors of miR-486-5p and miR-451a were used to retrieve the most frequent nucleotide found at each position in a sequence alignment. The most stable consensus sequences were used to generate reverse complement DNA oligonucleotides of targeted miRNAs. The C3 spacer (propyl group) modification was added to the 3’ ends of the synthetic oligonucleotides to avoid self-ligation. Whole blood smRNA-seq data used for oligo design was obtained from GSE100467; ( B ) A schematic representation of modified Illumina’s TruSeq small RNA library preparation protocol. The modified protocol involves an additional step, where synthetic blocking oligonucleotides are introduced right before the 5’ adapter ligation reaction. In this step, the blocking oligonucleotides are annealed to target miRNAs, which results in double-stranded RNA:DNA hybrid formation. These blunt-ended or slight 3’ DNA overhang-having double-stranded hybrids are not suitable substrates for T4 RNA ligase-mediated addition of adapter oligonucleotide to the 5’ end of RNA strand in the hybrid. As a consequence, blocked RNA:DNA hybrids without 5’ adapter sequences cannot be amplified and therefore are depleted from final small RNA library; ( C ) A schematic workflow of modified Perkin Elmer’s NEXTflex small RNA library preparation protocol. In comparison to TruSeq, the standard NEXTflex protocol includes an extra step called 3’ adapter inactivation, where end-filling is performed to fill the gaps of random nucleotides bearing 5’ overhang portions of 3’ adapter duplexes. Because of this step, in order to avoid denaturation of the 3’ adapter duplexes, blocking oligos for miR-486-5p and miR-451a were introduced directly to total RNA sample.
    Figure Legend Snippet: Blocking oligo design and application workflows using modified TruSeq and NEXTflex small RNA library preparation protocols. ( A ) Design principle of miR-486-5p and miR-451a blocking oligonucleotides. Briefly, unique pooled-sample sequences which mapped to precursors of miR-486-5p and miR-451a were used to retrieve the most frequent nucleotide found at each position in a sequence alignment. The most stable consensus sequences were used to generate reverse complement DNA oligonucleotides of targeted miRNAs. The C3 spacer (propyl group) modification was added to the 3’ ends of the synthetic oligonucleotides to avoid self-ligation. Whole blood smRNA-seq data used for oligo design was obtained from GSE100467; ( B ) A schematic representation of modified Illumina’s TruSeq small RNA library preparation protocol. The modified protocol involves an additional step, where synthetic blocking oligonucleotides are introduced right before the 5’ adapter ligation reaction. In this step, the blocking oligonucleotides are annealed to target miRNAs, which results in double-stranded RNA:DNA hybrid formation. These blunt-ended or slight 3’ DNA overhang-having double-stranded hybrids are not suitable substrates for T4 RNA ligase-mediated addition of adapter oligonucleotide to the 5’ end of RNA strand in the hybrid. As a consequence, blocked RNA:DNA hybrids without 5’ adapter sequences cannot be amplified and therefore are depleted from final small RNA library; ( C ) A schematic workflow of modified Perkin Elmer’s NEXTflex small RNA library preparation protocol. In comparison to TruSeq, the standard NEXTflex protocol includes an extra step called 3’ adapter inactivation, where end-filling is performed to fill the gaps of random nucleotides bearing 5’ overhang portions of 3’ adapter duplexes. Because of this step, in order to avoid denaturation of the 3’ adapter duplexes, blocking oligos for miR-486-5p and miR-451a were introduced directly to total RNA sample.

    Techniques Used: Blocking Assay, Modification, Sequencing, Ligation, Amplification

    13) Product Images from "Transcriptomic and proteomic analysis reveals wall-associated and glucan-degrading proteins with potential roles in Phytophthora infestans sexual spore development"

    Article Title: Transcriptomic and proteomic analysis reveals wall-associated and glucan-degrading proteins with potential roles in Phytophthora infestans sexual spore development

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0198186

    Polyadenylated RNA fraction of P . infestans tissues. Values are based on two biological replicates, using an oligo-dT binding assay.
    Figure Legend Snippet: Polyadenylated RNA fraction of P . infestans tissues. Values are based on two biological replicates, using an oligo-dT binding assay.

    Techniques Used: Binding Assay

    14) Product Images from "High Glucose–Induced Hypomethylation Promotes Binding of Sp-1 to Myo-Inositol Oxygenase"

    Article Title: High Glucose–Induced Hypomethylation Promotes Binding of Sp-1 to Myo-Inositol Oxygenase

    Journal: The American Journal of Pathology

    doi: 10.1016/j.ajpath.2016.12.011

    Identification of transcription factor specificity protein (Sp)-1–binding site in the human MIOX promoter and effect of in vitro methylation on the binding with nucleoproteins. Eletrophoretic mobility shift assays using Sp-1 and differentially methylated segment (DMS) oligos (DMS1 includes CpG sites -784 and -787 and DMS2 includes -581). A: An increased binding of Sp-1 is observed under high-glucose ambience (30 mmol/L) when the Sp-1 oligo is differentially demethylated or unmethylated ( arrowhead ). No binding of methylated or demethylated oligos is observed under low-glucose (5 mmol/L) ambience. B and C: Likewise an increased binding of unmethylated DMS1 and DMS2 oligos is observed under high-glucose ambience ( arrowhead ). The binding is also seen under low-glucose ambience; nevertheless, binding with unmethylated oligos (Sp-1, DMS1, and DMS2) under high-glucose ambience is much stronger. The arrows and asterisks indicate the nonspecific bands in the autoradiograms.
    Figure Legend Snippet: Identification of transcription factor specificity protein (Sp)-1–binding site in the human MIOX promoter and effect of in vitro methylation on the binding with nucleoproteins. Eletrophoretic mobility shift assays using Sp-1 and differentially methylated segment (DMS) oligos (DMS1 includes CpG sites -784 and -787 and DMS2 includes -581). A: An increased binding of Sp-1 is observed under high-glucose ambience (30 mmol/L) when the Sp-1 oligo is differentially demethylated or unmethylated ( arrowhead ). No binding of methylated or demethylated oligos is observed under low-glucose (5 mmol/L) ambience. B and C: Likewise an increased binding of unmethylated DMS1 and DMS2 oligos is observed under high-glucose ambience ( arrowhead ). The binding is also seen under low-glucose ambience; nevertheless, binding with unmethylated oligos (Sp-1, DMS1, and DMS2) under high-glucose ambience is much stronger. The arrows and asterisks indicate the nonspecific bands in the autoradiograms.

    Techniques Used: Binding Assay, In Vitro, Methylation, Mobility Shift

    15) Product Images from "Rapid and dynamic nucleic acid hybridization enables enzymatic oligonucleotide synthesis by cyclic reversible termination: A novel mechanism for enzymatic DNA synthesis"

    Article Title: Rapid and dynamic nucleic acid hybridization enables enzymatic oligonucleotide synthesis by cyclic reversible termination: A novel mechanism for enzymatic DNA synthesis

    Journal: bioRxiv

    doi: 10.1101/561092

    Dynamic hybridization of DNA enables EOS. (a) Extension of single-stranded DNA by DNA polymerases and reverse transcriptases. Denaturing PAGE analysis of a 20 base single-stranded sequence, self-priming oligo-1 aka SPO-1, using different enzymes and dGTP. Despite a maximum of two bases of hybridization, some enzymes are able to extend this solid-phase oligonucleotide. None of these enzymes have previously been reported to have nucleotidyl transferase activity on single-stranded DNA. (b) Sequence-specific extension of single-stranded DNA. Duplase extension of four different 20 base oligos proceeds in a sequence-specific manner, and only two bases of hybridization are required for extension. * indicates unextended control. Extension with non-templated bases can be explained by misincorporation that occurs at long reaction times . (c) Extension of solid-phase oligos through intermolecular reactions. Magnetic beads were conjugated with either a 20 base poly-T oligo as illustrated in i, or with that oligo plus a 30 base poly-T oligo with an internal 5’-CAA-3’ sequence as illustrated in ii . Beads were extended using Duplase-3 and Cy5-ddGTP (* indicates unextended control during this step), after which, all samples were labeled with TdT and fluorescein-12-ddUTP. Oligos appear blue if extended with Duplase-3 and green if extended wtih TdT.
    Figure Legend Snippet: Dynamic hybridization of DNA enables EOS. (a) Extension of single-stranded DNA by DNA polymerases and reverse transcriptases. Denaturing PAGE analysis of a 20 base single-stranded sequence, self-priming oligo-1 aka SPO-1, using different enzymes and dGTP. Despite a maximum of two bases of hybridization, some enzymes are able to extend this solid-phase oligonucleotide. None of these enzymes have previously been reported to have nucleotidyl transferase activity on single-stranded DNA. (b) Sequence-specific extension of single-stranded DNA. Duplase extension of four different 20 base oligos proceeds in a sequence-specific manner, and only two bases of hybridization are required for extension. * indicates unextended control. Extension with non-templated bases can be explained by misincorporation that occurs at long reaction times . (c) Extension of solid-phase oligos through intermolecular reactions. Magnetic beads were conjugated with either a 20 base poly-T oligo as illustrated in i, or with that oligo plus a 30 base poly-T oligo with an internal 5’-CAA-3’ sequence as illustrated in ii . Beads were extended using Duplase-3 and Cy5-ddGTP (* indicates unextended control during this step), after which, all samples were labeled with TdT and fluorescein-12-ddUTP. Oligos appear blue if extended with Duplase-3 and green if extended wtih TdT.

    Techniques Used: Hybridization, Polyacrylamide Gel Electrophoresis, Sequencing, Activity Assay, Magnetic Beads, Labeling

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    Staining:

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    Article Snippet: .. Fluorescent labeling of oligos with terminal deoxynucleotidyl transferase (TdT) Labeling of poly-T sequences, which do not stain well with SYBR Gold, was accomplished by end-labeling oligos with TdT (New England Biolabs, Ipswich, MA) and fluorescein-12-ddUTP (Perkin Elmer, San Jose, CA) by incubating 0.1mg of beads with 5μM nucleotide and 20U of enzyme in 20mM Tris-HCl pH7.5, 10mM ammonium sulfate, 10mM KCl, 0.1% Triton X-100, and 0.5mM MnCl2. ..

    Magnetic Beads:

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    Article Title: Local regulation of gene expression by lncRNA promoters, transcription, and splicing
    Article Snippet: .. We enriched for poly(A)+ RNA using oligo d(T)25 magnetic beads (NEB) and eluted in 18 μl H2 O. .. We fragmented RNA to an average of ~150-nt by adding 2 μl Ambion Fragmentation Buffer and incubating at 70°C for exactly 2.5 minutes.

    Article Title: An mRNA-binding channel in the ES6S region of the translation 48S-PIC promotes RNA unwinding and scanning
    Article Snippet: .. After UV crosslinking at 360 nm, the WRF was denatured in 500 μl buffer D (Tris-HCl 30 mM [pH 7.5], 0.5 M LiCl, 0.5% LiDS, 0.5 mM EDTA and 1 mM DTT) and poly(A)+ mRNA was captured with oligo(dT) magnetic beads (NEB) under denaturing conditions. .. After extensive washing, RNA was eluted with 100 μl H2 O and concentrated by ethanol precipitation in the presence of glycogen.

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    Helicase Assay:

    Article Title: Action of CMG with strand-specific DNA blocks supports an internal unwinding mode for the eukaryotic replicative helicase
    Article Snippet: .. Helicase assay substrates For all radiolabeled oligos, 10 pmols of oligo were labeled at the 5’ terminus with 0.05 mCi [γ-32 P]-ATP using T4 Polynucleotide Kinase (New England Biolabs) in a 25 μl reaction for 30’ @ 37°C according to the manufacturer’s instructions. .. For annealing, 4 pmols of the radiolabeled strand were mixed with 6 pmols of the unlabeled complementary strand, NaCl was added to a final concentration of 200 mM, and the mixture was heated to 90°C and cooled slowly to room temperature.

    Labeling:

    Article Title: Action of CMG with strand-specific DNA blocks supports an internal unwinding mode for the eukaryotic replicative helicase
    Article Snippet: .. Helicase assay substrates For all radiolabeled oligos, 10 pmols of oligo were labeled at the 5’ terminus with 0.05 mCi [γ-32 P]-ATP using T4 Polynucleotide Kinase (New England Biolabs) in a 25 μl reaction for 30’ @ 37°C according to the manufacturer’s instructions. .. For annealing, 4 pmols of the radiolabeled strand were mixed with 6 pmols of the unlabeled complementary strand, NaCl was added to a final concentration of 200 mM, and the mixture was heated to 90°C and cooled slowly to room temperature.

    Article Title: Rapid and dynamic nucleic acid hybridization enables enzymatic oligonucleotide synthesis by cyclic reversible termination: A novel mechanism for enzymatic DNA synthesis
    Article Snippet: .. Fluorescent labeling of oligos with terminal deoxynucleotidyl transferase (TdT) Labeling of poly-T sequences, which do not stain well with SYBR Gold, was accomplished by end-labeling oligos with TdT (New England Biolabs, Ipswich, MA) and fluorescein-12-ddUTP (Perkin Elmer, San Jose, CA) by incubating 0.1mg of beads with 5μM nucleotide and 20U of enzyme in 20mM Tris-HCl pH7.5, 10mM ammonium sulfate, 10mM KCl, 0.1% Triton X-100, and 0.5mM MnCl2. ..

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    End Labeling:

    Article Title: Rapid and dynamic nucleic acid hybridization enables enzymatic oligonucleotide synthesis by cyclic reversible termination: A novel mechanism for enzymatic DNA synthesis
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    New England Biolabs oligo
    Quality and reproducibility of the experiment shown in Figure 5 . ( a ) Polysome profile of HEK293T cells transfected with the indicated oligonucleotides. This experiment corresponds to the replica_1 used for RNA-seq, indicating fractions pooled as monosomes or polysomes. The bottom panel shows a western blot analysis of the resulting fractions probed with anti-eS6 antibody. ( b ) Analysis of correlation between replicas using a normalized number of reads for every <t>mRNA</t> in the monosomal and polysomal samples. ( c ) Changes in mRNA abundance between <t>oligo</t> 4 and VIC–oligo-4-transfected cells. Data are the average from two replicates. ( d ) Enrichment in KEGG pathways detected in the ‘TE down’ group of mRNAs. The p-value and enrichment factor for each term are indicated.
    Oligo, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 212 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Quality and reproducibility of the experiment shown in Figure 5 . ( a ) Polysome profile of HEK293T cells transfected with the indicated oligonucleotides. This experiment corresponds to the replica_1 used for RNA-seq, indicating fractions pooled as monosomes or polysomes. The bottom panel shows a western blot analysis of the resulting fractions probed with anti-eS6 antibody. ( b ) Analysis of correlation between replicas using a normalized number of reads for every mRNA in the monosomal and polysomal samples. ( c ) Changes in mRNA abundance between oligo 4 and VIC–oligo-4-transfected cells. Data are the average from two replicates. ( d ) Enrichment in KEGG pathways detected in the ‘TE down’ group of mRNAs. The p-value and enrichment factor for each term are indicated.

    Journal: eLife

    Article Title: An mRNA-binding channel in the ES6S region of the translation 48S-PIC promotes RNA unwinding and scanning

    doi: 10.7554/eLife.48246

    Figure Lengend Snippet: Quality and reproducibility of the experiment shown in Figure 5 . ( a ) Polysome profile of HEK293T cells transfected with the indicated oligonucleotides. This experiment corresponds to the replica_1 used for RNA-seq, indicating fractions pooled as monosomes or polysomes. The bottom panel shows a western blot analysis of the resulting fractions probed with anti-eS6 antibody. ( b ) Analysis of correlation between replicas using a normalized number of reads for every mRNA in the monosomal and polysomal samples. ( c ) Changes in mRNA abundance between oligo 4 and VIC–oligo-4-transfected cells. Data are the average from two replicates. ( d ) Enrichment in KEGG pathways detected in the ‘TE down’ group of mRNAs. The p-value and enrichment factor for each term are indicated.

    Article Snippet: After UV crosslinking at 360 nm, the WRF was denatured in 500 μl buffer D (Tris-HCl 30 mM [pH 7.5], 0.5 M LiCl, 0.5% LiDS, 0.5 mM EDTA and 1 mM DTT) and poly(A)+ mRNA was captured with oligo(dT) magnetic beads (NEB) under denaturing conditions.

    Techniques: Transfection, RNA Sequencing Assay, Western Blot

    Effect of VIC–oligo 4 on protein crosslinking in 48S complexes assembled with unstructured or SV-DLP n27 mRNAs. The amount of [ 32 P]-mRNA bound to WRF is indicated (upper panel), showing the pattern of protein crosslinking with the indicated mRNAs in the absence and presence of VIC–oligo 4. The unresolved eIF3g and eIF4A bands are shown (lower panel).

    Journal: eLife

    Article Title: An mRNA-binding channel in the ES6S region of the translation 48S-PIC promotes RNA unwinding and scanning

    doi: 10.7554/eLife.48246

    Figure Lengend Snippet: Effect of VIC–oligo 4 on protein crosslinking in 48S complexes assembled with unstructured or SV-DLP n27 mRNAs. The amount of [ 32 P]-mRNA bound to WRF is indicated (upper panel), showing the pattern of protein crosslinking with the indicated mRNAs in the absence and presence of VIC–oligo 4. The unresolved eIF3g and eIF4A bands are shown (lower panel).

    Article Snippet: After UV crosslinking at 360 nm, the WRF was denatured in 500 μl buffer D (Tris-HCl 30 mM [pH 7.5], 0.5 M LiCl, 0.5% LiDS, 0.5 mM EDTA and 1 mM DTT) and poly(A)+ mRNA was captured with oligo(dT) magnetic beads (NEB) under denaturing conditions.

    Techniques:

    mRNA threading into the ES6S region slows down scanning but makes it more processive. ( a ) Effect of FITC-oligo 4 on the translation of Luc mRNAs with different 5′ UTRs in RRL. Translation mixtures were incubated for 90 min, which represented the endpoint measurement because no further increase in luc activity was detected. Data are represented as the mean ± SD from at least three independent experiments. ( b ) Luc activity accumulation in continuously recording experiments programmed with the indicated mRNAs. Measurements were taken every 3 min: gray line, no oligo; black line, +FITC–oligo 4. Hipp was added to the indicated samples at a concentration of 2 μM (dashed line). ( c ) Estimates of full translation time (FTT) for 5′ UTR G-less and 5′ UTR-SL20 mRNAs, and the effect of FITC–oligo 4 and hipp on FTT. Data from panel (B) were processed as described before ( Vassilenko et al., 2011 ). The determined FTT values were: 5′ UTR G-less = 9.53 min; 5′ UTR G-less+FITC–oligo 4 = 7.89 min; 5′ UTR-SL20 = 17.57 min; 5′ UTR-SL20+FITC–oligo 4 = 17.56 min; 5′ UTR-SL20+hipp = 19.44 min. ( d ) Synergistic inhibitory effect of VIC–oligo 4 and hipp on translation of 5′UTR SL30-Luc and 5′ UTR G4-1-Luc mRNAs in RRL. Translation mixtures were preincubated with 6 μM of VIC–oligo 4 and with increasing concentrations of hipp for 5 min. Then, mRNAs were added and measurements were taken 90 min later; the calculated combination index (CI) for each mRNA is indicated.

    Journal: eLife

    Article Title: An mRNA-binding channel in the ES6S region of the translation 48S-PIC promotes RNA unwinding and scanning

    doi: 10.7554/eLife.48246

    Figure Lengend Snippet: mRNA threading into the ES6S region slows down scanning but makes it more processive. ( a ) Effect of FITC-oligo 4 on the translation of Luc mRNAs with different 5′ UTRs in RRL. Translation mixtures were incubated for 90 min, which represented the endpoint measurement because no further increase in luc activity was detected. Data are represented as the mean ± SD from at least three independent experiments. ( b ) Luc activity accumulation in continuously recording experiments programmed with the indicated mRNAs. Measurements were taken every 3 min: gray line, no oligo; black line, +FITC–oligo 4. Hipp was added to the indicated samples at a concentration of 2 μM (dashed line). ( c ) Estimates of full translation time (FTT) for 5′ UTR G-less and 5′ UTR-SL20 mRNAs, and the effect of FITC–oligo 4 and hipp on FTT. Data from panel (B) were processed as described before ( Vassilenko et al., 2011 ). The determined FTT values were: 5′ UTR G-less = 9.53 min; 5′ UTR G-less+FITC–oligo 4 = 7.89 min; 5′ UTR-SL20 = 17.57 min; 5′ UTR-SL20+FITC–oligo 4 = 17.56 min; 5′ UTR-SL20+hipp = 19.44 min. ( d ) Synergistic inhibitory effect of VIC–oligo 4 and hipp on translation of 5′UTR SL30-Luc and 5′ UTR G4-1-Luc mRNAs in RRL. Translation mixtures were preincubated with 6 μM of VIC–oligo 4 and with increasing concentrations of hipp for 5 min. Then, mRNAs were added and measurements were taken 90 min later; the calculated combination index (CI) for each mRNA is indicated.

    Article Snippet: After UV crosslinking at 360 nm, the WRF was denatured in 500 μl buffer D (Tris-HCl 30 mM [pH 7.5], 0.5 M LiCl, 0.5% LiDS, 0.5 mM EDTA and 1 mM DTT) and poly(A)+ mRNA was captured with oligo(dT) magnetic beads (NEB) under denaturing conditions.

    Techniques: Incubation, Activity Assay, Concentration Assay

    Effect of 5' UTR length of luc mRNAs on the sensitivity to VIC-oligo 4-mediated translational block. ( a ) Effect of 5′ UTR length on cap-dependent translation ofluc mRNAs in the presence of VIC-oligo 4. Data are the mean of two independent experiments performed in MEF cells as described in Figure 4a . ( b ) Comparative analysis of the effect of oligo 4, VIC–oligo C and VIC–oligo 4 on the translation of the indicated mRNAs. Data are the mean ± SD from at least four independent experiments in MEF cells. ( c ) Effect of VIC–oligo 4 on the translation of poliovirus (PV) mRNA. HeLa cells were transfected with the indicated oligonucleotides, and infected 12 hr later with PV1 (Mahoney strain) at a multiplicity of infection (MOI) of 10 pfu/cell. The cultures were metabolically labeled with [ 35 S]-Met at 6 hr post-infection and analyzed as described in the Materials and methods; SYPRO staining was included as the loading control.

    Journal: eLife

    Article Title: An mRNA-binding channel in the ES6S region of the translation 48S-PIC promotes RNA unwinding and scanning

    doi: 10.7554/eLife.48246

    Figure Lengend Snippet: Effect of 5' UTR length of luc mRNAs on the sensitivity to VIC-oligo 4-mediated translational block. ( a ) Effect of 5′ UTR length on cap-dependent translation ofluc mRNAs in the presence of VIC-oligo 4. Data are the mean of two independent experiments performed in MEF cells as described in Figure 4a . ( b ) Comparative analysis of the effect of oligo 4, VIC–oligo C and VIC–oligo 4 on the translation of the indicated mRNAs. Data are the mean ± SD from at least four independent experiments in MEF cells. ( c ) Effect of VIC–oligo 4 on the translation of poliovirus (PV) mRNA. HeLa cells were transfected with the indicated oligonucleotides, and infected 12 hr later with PV1 (Mahoney strain) at a multiplicity of infection (MOI) of 10 pfu/cell. The cultures were metabolically labeled with [ 35 S]-Met at 6 hr post-infection and analyzed as described in the Materials and methods; SYPRO staining was included as the loading control.

    Article Snippet: After UV crosslinking at 360 nm, the WRF was denatured in 500 μl buffer D (Tris-HCl 30 mM [pH 7.5], 0.5 M LiCl, 0.5% LiDS, 0.5 mM EDTA and 1 mM DTT) and poly(A)+ mRNA was captured with oligo(dT) magnetic beads (NEB) under denaturing conditions.

    Techniques: Blocking Assay, Transfection, Infection, Metabolic Labelling, Labeling, Staining

    Effect of VIC oligo 4 on translation of alphavirus 26S mRNA. ( A ) Bottom view of rabbit 40S subunit crystal structure showing the region ES6S C-D where oligo 4 hybridizes (marked in green). ( B ) Binding of VIC oligo 4 to rabbit 40S subunit. Approximately 50 pmol of purified rabbit 40S subunit was incubated with 200 pmol of VIC oligo 4 or VIC SVcapside (negative control, ( 20 )) for 20 min at 30°C and centrifuged in a 10–30% sucrose gradient at 45 000 rpm for 3 h. The fluorescence of VIC fluorochrome in each fraction was measured in a FLUOstar OPTIMA apparatus. ( C ) Effect of VIC oligo 4 on accumulation of SFV C protein analyzed by immunofluorescence using a polyclonal antibody raised against SFV C protein. Note that those cells showing strong VIC fluorescence (orange-red) showed little SFV C staining (green). ( D ) Effect of VIC oligo 4 on translation of 26S mRNA in SFV-infected cells. Cells were transfected with the indicated oligonucleotide, and 15 h later infected with SFV at a moi of 25 pfu/cell. The cells were metabolically labeled with [ 35 S]-Met/Cys at 6 hpi. Labeled proteins were analyzed by autoradiography. p62, E1 and C are the structural proteins of the virus encoded by the 26S mRNA.

    Journal: Nucleic Acids Research

    Article Title: An RNA trapping mechanism in Alphavirus mRNA promotes ribosome stalling and translation initiation

    doi: 10.1093/nar/gkw172

    Figure Lengend Snippet: Effect of VIC oligo 4 on translation of alphavirus 26S mRNA. ( A ) Bottom view of rabbit 40S subunit crystal structure showing the region ES6S C-D where oligo 4 hybridizes (marked in green). ( B ) Binding of VIC oligo 4 to rabbit 40S subunit. Approximately 50 pmol of purified rabbit 40S subunit was incubated with 200 pmol of VIC oligo 4 or VIC SVcapside (negative control, ( 20 )) for 20 min at 30°C and centrifuged in a 10–30% sucrose gradient at 45 000 rpm for 3 h. The fluorescence of VIC fluorochrome in each fraction was measured in a FLUOstar OPTIMA apparatus. ( C ) Effect of VIC oligo 4 on accumulation of SFV C protein analyzed by immunofluorescence using a polyclonal antibody raised against SFV C protein. Note that those cells showing strong VIC fluorescence (orange-red) showed little SFV C staining (green). ( D ) Effect of VIC oligo 4 on translation of 26S mRNA in SFV-infected cells. Cells were transfected with the indicated oligonucleotide, and 15 h later infected with SFV at a moi of 25 pfu/cell. The cells were metabolically labeled with [ 35 S]-Met/Cys at 6 hpi. Labeled proteins were analyzed by autoradiography. p62, E1 and C are the structural proteins of the virus encoded by the 26S mRNA.

    Article Snippet: The complexes were crosslinked, centrifuged at high speed and ribosomal pellets were resuspended in cracking buffer (25 mM Tris-HCl pH 7.5, 0.5 M LiCl, 0.5% LiDS, 1 mM EDTA and 5 mM DTT). mRNA poly(A)+ was bound to oligo (dT)25 magnetic beads (NEB) at RT for 20 min. Beads were extensively washed according to the manufacturer's recommendations and eluted in TE buffer by heating the samples at 60°C for 10 min. RNA was extracted with phenol, precipitated with ethanol and resuspended in 15 μl of TE buffer.

    Techniques: Binding Assay, Purification, Incubation, Negative Control, Fluorescence, Immunofluorescence, Staining, Infection, Transfection, Metabolic Labelling, Labeling, Autoradiography

    Characterization of apparent DNA strand exchange activity in SA proteins. (A) The reaction illustrated at the top of the panel was performed using a 32 bp radiolabeled dsDNA with flush ends as donor (oligos #4*/#5) and an unlabeled 57 nt oligo (#6) as recipient. Reactions containing 5 nM donor and 20 nM recipient were initiated by addition of the indicated concentrations of Rad52N, Sgs1 103–322 , or Rad59. Following incubation at 37°C for 30 min, the reactions were terminated and the products analyzed by 10% native PAGE and phosphorimaging. ( B ) SE reactions were carried out as in (A) except that aliquots of the reactions were removed at the indicated times and treated as above. ( C ) SE reactions were performed with Rad52 mutants. ( D ) SE assays were performed with sub-domains of WRN 235–526 .

    Journal: DNA repair

    Article Title: Multimerization Domains are Associated with Apparent Strand Exchange Activity in BLM and WRN DNA helicases

    doi: 10.1016/j.dnarep.2014.07.015

    Figure Lengend Snippet: Characterization of apparent DNA strand exchange activity in SA proteins. (A) The reaction illustrated at the top of the panel was performed using a 32 bp radiolabeled dsDNA with flush ends as donor (oligos #4*/#5) and an unlabeled 57 nt oligo (#6) as recipient. Reactions containing 5 nM donor and 20 nM recipient were initiated by addition of the indicated concentrations of Rad52N, Sgs1 103–322 , or Rad59. Following incubation at 37°C for 30 min, the reactions were terminated and the products analyzed by 10% native PAGE and phosphorimaging. ( B ) SE reactions were carried out as in (A) except that aliquots of the reactions were removed at the indicated times and treated as above. ( C ) SE reactions were performed with Rad52 mutants. ( D ) SE assays were performed with sub-domains of WRN 235–526 .

    Article Snippet: The oligos were radiolabeled at their 5’-end with γ-32 P-ATP by T4 polynucleotide kinase (New England BioLabs).

    Techniques: Activity Assay, Incubation, Clear Native PAGE

    Dynamic hybridization of DNA enables EOS. (a) Extension of single-stranded DNA by DNA polymerases and reverse transcriptases. Denaturing PAGE analysis of a 20 base single-stranded sequence, self-priming oligo-1 aka SPO-1, using different enzymes and dGTP. Despite a maximum of two bases of hybridization, some enzymes are able to extend this solid-phase oligonucleotide. None of these enzymes have previously been reported to have nucleotidyl transferase activity on single-stranded DNA. (b) Sequence-specific extension of single-stranded DNA. Duplase extension of four different 20 base oligos proceeds in a sequence-specific manner, and only two bases of hybridization are required for extension. * indicates unextended control. Extension with non-templated bases can be explained by misincorporation that occurs at long reaction times . (c) Extension of solid-phase oligos through intermolecular reactions. Magnetic beads were conjugated with either a 20 base poly-T oligo as illustrated in i, or with that oligo plus a 30 base poly-T oligo with an internal 5’-CAA-3’ sequence as illustrated in ii . Beads were extended using Duplase-3 and Cy5-ddGTP (* indicates unextended control during this step), after which, all samples were labeled with TdT and fluorescein-12-ddUTP. Oligos appear blue if extended with Duplase-3 and green if extended wtih TdT.

    Journal: bioRxiv

    Article Title: Rapid and dynamic nucleic acid hybridization enables enzymatic oligonucleotide synthesis by cyclic reversible termination: A novel mechanism for enzymatic DNA synthesis

    doi: 10.1101/561092

    Figure Lengend Snippet: Dynamic hybridization of DNA enables EOS. (a) Extension of single-stranded DNA by DNA polymerases and reverse transcriptases. Denaturing PAGE analysis of a 20 base single-stranded sequence, self-priming oligo-1 aka SPO-1, using different enzymes and dGTP. Despite a maximum of two bases of hybridization, some enzymes are able to extend this solid-phase oligonucleotide. None of these enzymes have previously been reported to have nucleotidyl transferase activity on single-stranded DNA. (b) Sequence-specific extension of single-stranded DNA. Duplase extension of four different 20 base oligos proceeds in a sequence-specific manner, and only two bases of hybridization are required for extension. * indicates unextended control. Extension with non-templated bases can be explained by misincorporation that occurs at long reaction times . (c) Extension of solid-phase oligos through intermolecular reactions. Magnetic beads were conjugated with either a 20 base poly-T oligo as illustrated in i, or with that oligo plus a 30 base poly-T oligo with an internal 5’-CAA-3’ sequence as illustrated in ii . Beads were extended using Duplase-3 and Cy5-ddGTP (* indicates unextended control during this step), after which, all samples were labeled with TdT and fluorescein-12-ddUTP. Oligos appear blue if extended with Duplase-3 and green if extended wtih TdT.

    Article Snippet: Fluorescent labeling of oligos with terminal deoxynucleotidyl transferase (TdT) Labeling of poly-T sequences, which do not stain well with SYBR Gold, was accomplished by end-labeling oligos with TdT (New England Biolabs, Ipswich, MA) and fluorescein-12-ddUTP (Perkin Elmer, San Jose, CA) by incubating 0.1mg of beads with 5μM nucleotide and 20U of enzyme in 20mM Tris-HCl pH7.5, 10mM ammonium sulfate, 10mM KCl, 0.1% Triton X-100, and 0.5mM MnCl2.

    Techniques: Hybridization, Polyacrylamide Gel Electrophoresis, Sequencing, Activity Assay, Magnetic Beads, Labeling