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  • 95
    Integrated DNA Technologies oligos
    Enhancement of T4 DNA ligase activity by supplemental <t>oligonucleotides.</t> (a) Unsuccessful 4-bp duplex reactions could be salvaged by utilizing a supplementary oligonucleotide, designed to complement the first <t>oligonucleotide-dsDNA</t> duplex but is unphosphorylated to prevent ligation of itself. Two hour ligation of the 4-bp reaction at 16°C supplemented with 3.33 μM of the hexamer, shows successful ligation (■) while reactions without the supplementary hexamer show no activity (◆). (b) Ligation reaction of an octamer supplemented with a second octamer in which one is used for ligation and the other is used to extend the duplex. A two hour ligation at 16°C of serial concentrations of the octamer with 3.33 μM of the supplementary octamer shows significant ligation (■) compared to reactions without the supplemental octamer (◆). (c) Unsuccessful 3-bp duplex reactions could be salvaged by utilizing a supplementary hexamer that hybridized at all six positions. A two hour ligation of the 3-bp reaction at 16°C with 3.33 μM supplementary hexamer shows successful ligation (■) while reactions without the supplementary hexamer show no activity (◆). (d) Ligation using a hexamer pair at 4°C for 16 hours shows limited improvement (■) compared to the unsupplemented (◆) control.
    Oligos, supplied by Integrated DNA Technologies, used in various techniques. Bioz Stars score: 95/100, based on 1114 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Millipore biotinylated oligos
    Enhancement of T4 DNA ligase activity by supplemental <t>oligonucleotides.</t> (a) Unsuccessful 4-bp duplex reactions could be salvaged by utilizing a supplementary oligonucleotide, designed to complement the first <t>oligonucleotide-dsDNA</t> duplex but is unphosphorylated to prevent ligation of itself. Two hour ligation of the 4-bp reaction at 16°C supplemented with 3.33 μM of the hexamer, shows successful ligation (■) while reactions without the supplementary hexamer show no activity (◆). (b) Ligation reaction of an octamer supplemented with a second octamer in which one is used for ligation and the other is used to extend the duplex. A two hour ligation at 16°C of serial concentrations of the octamer with 3.33 μM of the supplementary octamer shows significant ligation (■) compared to reactions without the supplemental octamer (◆). (c) Unsuccessful 3-bp duplex reactions could be salvaged by utilizing a supplementary hexamer that hybridized at all six positions. A two hour ligation of the 3-bp reaction at 16°C with 3.33 μM supplementary hexamer shows successful ligation (■) while reactions without the supplementary hexamer show no activity (◆). (d) Ligation using a hexamer pair at 4°C for 16 hours shows limited improvement (■) compared to the unsupplemented (◆) control.
    Biotinylated Oligos, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 44 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    82
    Integrated DNA Technologies idt oligos
    Enhancement of T4 DNA ligase activity by supplemental <t>oligonucleotides.</t> (a) Unsuccessful 4-bp duplex reactions could be salvaged by utilizing a supplementary oligonucleotide, designed to complement the first <t>oligonucleotide-dsDNA</t> duplex but is unphosphorylated to prevent ligation of itself. Two hour ligation of the 4-bp reaction at 16°C supplemented with 3.33 μM of the hexamer, shows successful ligation (■) while reactions without the supplementary hexamer show no activity (◆). (b) Ligation reaction of an octamer supplemented with a second octamer in which one is used for ligation and the other is used to extend the duplex. A two hour ligation at 16°C of serial concentrations of the octamer with 3.33 μM of the supplementary octamer shows significant ligation (■) compared to reactions without the supplemental octamer (◆). (c) Unsuccessful 3-bp duplex reactions could be salvaged by utilizing a supplementary hexamer that hybridized at all six positions. A two hour ligation of the 3-bp reaction at 16°C with 3.33 μM supplementary hexamer shows successful ligation (■) while reactions without the supplementary hexamer show no activity (◆). (d) Ligation using a hexamer pair at 4°C for 16 hours shows limited improvement (■) compared to the unsupplemented (◆) control.
    Idt Oligos, supplied by Integrated DNA Technologies, used in various techniques. Bioz Stars score: 82/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Integrated DNA Technologies antisense oligonucleotides
    miR-4728-3p IS regulates ESR1. A. qRT-PCR analysis of ESR1 and HER2 transcripts and miR-4728-3p among a panel of 38 breast cancer tumors (19 HER2+, 19 HER2-). Calibrated Normalized Relative Quantity (CNRQ) of miR-4728-3p (left) and HER2 (right) is plotted against expression levels of ESR1. Tumors classified as HER2+ by ISH are shown in red, HER2- in grey. Expression was normalized to a panel of reference genes. For details see text and material and methods. B. Luciferase assay in BT-474 with ESR1 3′UTR constructs carrying either wild type target site of miR-4728-3p internal seed (WT) or mutated internal seed site (MUT). Firefly luciferase activity was normalized against Renilla luciferase. Reporter activity is given as % of WT in respective experiment. Repression of WT ESR1 construct by endogenous miR-4728-3p (left) is alleviated by an <t>antisense</t> oligo (AS) against endogenous miRNA (right) but not by a non-targeting control (middle). C. Western blot (left) and protein quantification (right) of ESR1 in MCF7. The two main isoforms of ESR1 (47 and 66 kDa), plotted as percentage of control signal of matching size, are down regulated upon transfection of miR-4728-3p mimics. Levels of HER2, (p)MAPK and (p)AKT remain largely unchanged. D. MCF7 cells were transfected with indicated concentrations of miR-4728-3p mimic. ESR1 levels show a concentration-dependent down-regulation that is most pronounced at highest tested concentration (25 nM). E. Western blot (left) and protein quantification (right) of ESR1 in BT474. ESR1 is up regulated when blocking endogenous miR-4728-3p with AS-oligonucleotides, while pMAPK and pAKT remain largely unchanged. F. Western blot (left) and protein quantification (right) of ESR1 in HCC1954 cells. ESR1 isoform of 47 kDa is up regulated under miR-4728-3p blocking. The main 66 kDa isoform is not detectable in this ER- cell line. Signals were quantified with ImageJ and normalized to total protein by Coomassie stain. Tubulin was used as a loading control. Asterisks denote p-values of
    Antisense Oligonucleotides, supplied by Integrated DNA Technologies, used in various techniques. Bioz Stars score: 95/100, based on 246 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Integrated DNA Technologies trugrade oligonucleotides
    miR-4728-3p IS regulates ESR1. A. qRT-PCR analysis of ESR1 and HER2 transcripts and miR-4728-3p among a panel of 38 breast cancer tumors (19 HER2+, 19 HER2-). Calibrated Normalized Relative Quantity (CNRQ) of miR-4728-3p (left) and HER2 (right) is plotted against expression levels of ESR1. Tumors classified as HER2+ by ISH are shown in red, HER2- in grey. Expression was normalized to a panel of reference genes. For details see text and material and methods. B. Luciferase assay in BT-474 with ESR1 3′UTR constructs carrying either wild type target site of miR-4728-3p internal seed (WT) or mutated internal seed site (MUT). Firefly luciferase activity was normalized against Renilla luciferase. Reporter activity is given as % of WT in respective experiment. Repression of WT ESR1 construct by endogenous miR-4728-3p (left) is alleviated by an <t>antisense</t> oligo (AS) against endogenous miRNA (right) but not by a non-targeting control (middle). C. Western blot (left) and protein quantification (right) of ESR1 in MCF7. The two main isoforms of ESR1 (47 and 66 kDa), plotted as percentage of control signal of matching size, are down regulated upon transfection of miR-4728-3p mimics. Levels of HER2, (p)MAPK and (p)AKT remain largely unchanged. D. MCF7 cells were transfected with indicated concentrations of miR-4728-3p mimic. ESR1 levels show a concentration-dependent down-regulation that is most pronounced at highest tested concentration (25 nM). E. Western blot (left) and protein quantification (right) of ESR1 in BT474. ESR1 is up regulated when blocking endogenous miR-4728-3p with AS-oligonucleotides, while pMAPK and pAKT remain largely unchanged. F. Western blot (left) and protein quantification (right) of ESR1 in HCC1954 cells. ESR1 isoform of 47 kDa is up regulated under miR-4728-3p blocking. The main 66 kDa isoform is not detectable in this ER- cell line. Signals were quantified with ImageJ and normalized to total protein by Coomassie stain. Tubulin was used as a loading control. Asterisks denote p-values of
    Trugrade Oligonucleotides, supplied by Integrated DNA Technologies, used in various techniques. Bioz Stars score: 92/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Integrated DNA Technologies ultramer oligonucleotides
    Endpoint fluorescence scatter plot containing 4 in-run controls and 40 clinical specimens. Control reactions using <t>Ultramer</t> oligonucleotides for wild type (Wt) (A), A2058G (B), and A2059G (C) are shown. (D) No template control. The gray box indicates
    Ultramer Oligonucleotides, supplied by Integrated DNA Technologies, used in various techniques. Bioz Stars score: 95/100, based on 71 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Integrated DNA Technologies rna oligos
    Differential expression of <t>RNA:m</t> 5 C, RCMTs and hnRNPK in 5-AZA-sensitive and 5-AZA-resistant leukaemia cells and the binding of hnRNPK to unmethylated and cytosine-methylated RNA. a Western blot analysis of expression of RCMTs, hnRNPK and other proteins in the 5-AZA-sensitive OCI-M2 and SC leukaemia cells and the 5-AZA-resistant M2AR and SCAR leukaemia cells. b Dot blot analysis of 5-methylcytosine (m 5 C) in RNA and DNA from OCI-M2 and M2AR cells. c Dot blot analysis of 5-methylcytosine (m 5 C) in RNA and DNA from SC and SCAR cells. d , e Visualization and measurement of the binding of purified recombinant hnRNPK to the unmethylated and cytosine-methylated fluorescein (FAM)-labelled RNA <t>oligos</t> by an antibody-coupled bead assay
    Rna Oligos, supplied by Integrated DNA Technologies, used in various techniques. Bioz Stars score: 95/100, based on 141 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    agena bioscience control oligos
    Differential expression of <t>RNA:m</t> 5 C, RCMTs and hnRNPK in 5-AZA-sensitive and 5-AZA-resistant leukaemia cells and the binding of hnRNPK to unmethylated and cytosine-methylated RNA. a Western blot analysis of expression of RCMTs, hnRNPK and other proteins in the 5-AZA-sensitive OCI-M2 and SC leukaemia cells and the 5-AZA-resistant M2AR and SCAR leukaemia cells. b Dot blot analysis of 5-methylcytosine (m 5 C) in RNA and DNA from OCI-M2 and M2AR cells. c Dot blot analysis of 5-methylcytosine (m 5 C) in RNA and DNA from SC and SCAR cells. d , e Visualization and measurement of the binding of purified recombinant hnRNPK to the unmethylated and cytosine-methylated fluorescein (FAM)-labelled RNA <t>oligos</t> by an antibody-coupled bead assay
    Control Oligos, supplied by agena bioscience, used in various techniques. Bioz Stars score: 99/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    87
    biomers.net detection oligos
    Differential expression of <t>RNA:m</t> 5 C, RCMTs and hnRNPK in 5-AZA-sensitive and 5-AZA-resistant leukaemia cells and the binding of hnRNPK to unmethylated and cytosine-methylated RNA. a Western blot analysis of expression of RCMTs, hnRNPK and other proteins in the 5-AZA-sensitive OCI-M2 and SC leukaemia cells and the 5-AZA-resistant M2AR and SCAR leukaemia cells. b Dot blot analysis of 5-methylcytosine (m 5 C) in RNA and DNA from OCI-M2 and M2AR cells. c Dot blot analysis of 5-methylcytosine (m 5 C) in RNA and DNA from SC and SCAR cells. d , e Visualization and measurement of the binding of purified recombinant hnRNPK to the unmethylated and cytosine-methylated fluorescein (FAM)-labelled RNA <t>oligos</t> by an antibody-coupled bead assay
    Detection Oligos, supplied by biomers.net, used in various techniques. Bioz Stars score: 87/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Integrated DNA Technologies idt oligo analyzer
    Differential expression of <t>RNA:m</t> 5 C, RCMTs and hnRNPK in 5-AZA-sensitive and 5-AZA-resistant leukaemia cells and the binding of hnRNPK to unmethylated and cytosine-methylated RNA. a Western blot analysis of expression of RCMTs, hnRNPK and other proteins in the 5-AZA-sensitive OCI-M2 and SC leukaemia cells and the 5-AZA-resistant M2AR and SCAR leukaemia cells. b Dot blot analysis of 5-methylcytosine (m 5 C) in RNA and DNA from OCI-M2 and M2AR cells. c Dot blot analysis of 5-methylcytosine (m 5 C) in RNA and DNA from SC and SCAR cells. d , e Visualization and measurement of the binding of purified recombinant hnRNPK to the unmethylated and cytosine-methylated fluorescein (FAM)-labelled RNA <t>oligos</t> by an antibody-coupled bead assay
    Idt Oligo Analyzer, supplied by Integrated DNA Technologies, used in various techniques. Bioz Stars score: 99/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Integrated DNA Technologies dna oligonucleotides
    Differential expression of <t>RNA:m</t> 5 C, RCMTs and hnRNPK in 5-AZA-sensitive and 5-AZA-resistant leukaemia cells and the binding of hnRNPK to unmethylated and cytosine-methylated RNA. a Western blot analysis of expression of RCMTs, hnRNPK and other proteins in the 5-AZA-sensitive OCI-M2 and SC leukaemia cells and the 5-AZA-resistant M2AR and SCAR leukaemia cells. b Dot blot analysis of 5-methylcytosine (m 5 C) in RNA and DNA from OCI-M2 and M2AR cells. c Dot blot analysis of 5-methylcytosine (m 5 C) in RNA and DNA from SC and SCAR cells. d , e Visualization and measurement of the binding of purified recombinant hnRNPK to the unmethylated and cytosine-methylated fluorescein (FAM)-labelled RNA <t>oligos</t> by an antibody-coupled bead assay
    Dna Oligonucleotides, supplied by Integrated DNA Technologies, used in various techniques. Bioz Stars score: 99/100, based on 2158 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    77
    Elim Bio 71 nt oligos
    Schematic of miRNA-based RNAi vector—shmiRs (A) Standard method for cloning miRNA-based RNAi vectors, left, is compared to miRNA-based vectors described in this study, right. (B) Structure of shmiR- dpp 2 in pHB scaffold. Annealed <t>71-nt</t> DNA <t>oligos</t> are inserted into the open-loop region with the specified restriction sequences, HindIII-BamHI in this example. Resultant siRNAs will be excised, like miR-1 , from the 3′ arm of the stem-loop (orange highlighted region). This construct is flanked by 5′ and 3′ cloning sites (M.C.S.), enabling the creation of polycistronic hairpins. (C) shmiR- dpp 2 knockdown oligos inserted into the HindIII-BamHI site of pHB. The siRNA sequence is highlighted in red, while the blue nucleotides represent the presumptive star sequence of the dpp 2 siRNA. Highlighted in yellow are two specific mismatched bases necessary to maintain the endogenous pre- miR-1 stem-loop structure.
    71 Nt Oligos, supplied by Elim Bio, used in various techniques. Bioz Stars score: 77/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Integrated DNA Technologies biotinylated oligonucleotides
    EMSA analysis using phosphorylated or non-phosphorylated AtcR. <t>Biotinylated</t> DNA duplexes derived from several genes (indicated at the top) were incubated with increasing concentrations (μM) of either non-phosphorylated AtcR (AtcR) or phosphorylated
    Biotinylated Oligonucleotides, supplied by Integrated DNA Technologies, used in various techniques. Bioz Stars score: 96/100, based on 111 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    Millipore p m 32 p labeled oligonucleotides idt
    EMSA analysis using phosphorylated or non-phosphorylated AtcR. <t>Biotinylated</t> DNA duplexes derived from several genes (indicated at the top) were incubated with increasing concentrations (μM) of either non-phosphorylated AtcR (AtcR) or phosphorylated
    P M 32 P Labeled Oligonucleotides Idt, supplied by Millipore, used in various techniques. Bioz Stars score: 78/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    87
    Promega oligo dt idt
    EMSA analysis using phosphorylated or non-phosphorylated AtcR. <t>Biotinylated</t> DNA duplexes derived from several genes (indicated at the top) were incubated with increasing concentrations (μM) of either non-phosphorylated AtcR (AtcR) or phosphorylated
    Oligo Dt Idt, supplied by Promega, used in various techniques. Bioz Stars score: 87/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    84
    Integrated DNA Technologies crrna oligonucleotides
    EMSA analysis using phosphorylated or non-phosphorylated AtcR. <t>Biotinylated</t> DNA duplexes derived from several genes (indicated at the top) were incubated with increasing concentrations (μM) of either non-phosphorylated AtcR (AtcR) or phosphorylated
    Crrna Oligonucleotides, supplied by Integrated DNA Technologies, used in various techniques. Bioz Stars score: 84/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Integrated DNA Technologies ribo oligonucleotides
    EMSA analysis using phosphorylated or non-phosphorylated AtcR. <t>Biotinylated</t> DNA duplexes derived from several genes (indicated at the top) were incubated with increasing concentrations (μM) of either non-phosphorylated AtcR (AtcR) or phosphorylated
    Ribo Oligonucleotides, supplied by Integrated DNA Technologies, used in various techniques. Bioz Stars score: 99/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    75
    Integrated DNA Technologies nontemplate oligonucleotides
    The 2′–5′ phosphodiester linkage in RNA primer end reduces RNA pol II transcriptional efficiency but does not affect fidelity. ( A ) Scaffold of RNA, template DNA, and <t>nontemplate</t> DNA for runoff elongation from the 2′–5′–linked
    Nontemplate Oligonucleotides, supplied by Integrated DNA Technologies, used in various techniques. Bioz Stars score: 75/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Integrated DNA Technologies dsirna oligonucleotides
    The 2′–5′ phosphodiester linkage in RNA primer end reduces RNA pol II transcriptional efficiency but does not affect fidelity. ( A ) Scaffold of RNA, template DNA, and <t>nontemplate</t> DNA for runoff elongation from the 2′–5′–linked
    Dsirna Oligonucleotides, supplied by Integrated DNA Technologies, used in various techniques. Bioz Stars score: 93/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    80
    Integrated DNA Technologies thiolated oligonucleotides
    The 2′–5′ phosphodiester linkage in RNA primer end reduces RNA pol II transcriptional efficiency but does not affect fidelity. ( A ) Scaffold of RNA, template DNA, and <t>nontemplate</t> DNA for runoff elongation from the 2′–5′–linked
    Thiolated Oligonucleotides, supplied by Integrated DNA Technologies, used in various techniques. Bioz Stars score: 80/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    80
    Integrated DNA Technologies ssrna oligonucleotides
    Effects of magnesium on the endoribonuclease, RNA binding, <t>AP-ssDNA,</t> and <t>AP-ssRNA</t> endonuclease activities of APE1. (a) 1.4 μM of APE1 (lanes 2 – 9) was tested against 25 nM of 5′-γ- 32 P-radiolabeled c- myc CRD RNA in a total
    Ssrna Oligonucleotides, supplied by Integrated DNA Technologies, used in various techniques. Bioz Stars score: 80/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Integrated DNA Technologies sgrna oligos
    Effects of magnesium on the endoribonuclease, RNA binding, <t>AP-ssDNA,</t> and <t>AP-ssRNA</t> endonuclease activities of APE1. (a) 1.4 μM of APE1 (lanes 2 – 9) was tested against 25 nM of 5′-γ- 32 P-radiolabeled c- myc CRD RNA in a total
    Sgrna Oligos, supplied by Integrated DNA Technologies, used in various techniques. Bioz Stars score: 99/100, based on 35 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Integrated DNA Technologies dsrna oligonucleotides
    Effects of magnesium on the endoribonuclease, RNA binding, <t>AP-ssDNA,</t> and <t>AP-ssRNA</t> endonuclease activities of APE1. (a) 1.4 μM of APE1 (lanes 2 – 9) was tested against 25 nM of 5′-γ- 32 P-radiolabeled c- myc CRD RNA in a total
    Dsrna Oligonucleotides, supplied by Integrated DNA Technologies, used in various techniques. Bioz Stars score: 88/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Integrated DNA Technologies ssdna oligonucleotides
    Cdc13 proteins exhibit cooperativity when binding adjacent sites. ( A ) The two-site ligand Tel11-A 6 -Tel11 was incubated with titrations of Cdc13, Cdc13-L91R or Cdc13-DBD under low-salt conditions (75 mM KCl + 75 mM NaCl) and analyzed by EMSA. Three species are formed, as indicated by the inset schematics. The signal of each species is plotted as a fraction of total <t>ssDNA</t> signal: closed circle, free oligo; closed triangle, singly bound oligo; closed square, doubly bound oligo. Three independent data sets were collected and used to solve the cooperativity constants listed in Table 2 . Dashed lines are the least-squares fits of the binding isotherm for each set of data. ( B ) Representative EMSA gels for the data plotted in (A). Asterisks indicate a third binding event to a low-affinity cryptic binding site that occurred at high concentrations of protein. Gray arrowheads indicate lower-mobility <t>DNA</t> conformations.
    Ssdna Oligonucleotides, supplied by Integrated DNA Technologies, used in various techniques. Bioz Stars score: 99/100, based on 68 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    79
    Integrated DNA Technologies mutagenized oligonucleotides
    Cdc13 proteins exhibit cooperativity when binding adjacent sites. ( A ) The two-site ligand Tel11-A 6 -Tel11 was incubated with titrations of Cdc13, Cdc13-L91R or Cdc13-DBD under low-salt conditions (75 mM KCl + 75 mM NaCl) and analyzed by EMSA. Three species are formed, as indicated by the inset schematics. The signal of each species is plotted as a fraction of total <t>ssDNA</t> signal: closed circle, free oligo; closed triangle, singly bound oligo; closed square, doubly bound oligo. Three independent data sets were collected and used to solve the cooperativity constants listed in Table 2 . Dashed lines are the least-squares fits of the binding isotherm for each set of data. ( B ) Representative EMSA gels for the data plotted in (A). Asterisks indicate a third binding event to a low-affinity cryptic binding site that occurred at high concentrations of protein. Gray arrowheads indicate lower-mobility <t>DNA</t> conformations.
    Mutagenized Oligonucleotides, supplied by Integrated DNA Technologies, used in various techniques. Bioz Stars score: 79/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Integrated DNA Technologies degenerate oligos
    Cdc13 proteins exhibit cooperativity when binding adjacent sites. ( A ) The two-site ligand Tel11-A 6 -Tel11 was incubated with titrations of Cdc13, Cdc13-L91R or Cdc13-DBD under low-salt conditions (75 mM KCl + 75 mM NaCl) and analyzed by EMSA. Three species are formed, as indicated by the inset schematics. The signal of each species is plotted as a fraction of total <t>ssDNA</t> signal: closed circle, free oligo; closed triangle, singly bound oligo; closed square, doubly bound oligo. Three independent data sets were collected and used to solve the cooperativity constants listed in Table 2 . Dashed lines are the least-squares fits of the binding isotherm for each set of data. ( B ) Representative EMSA gels for the data plotted in (A). Asterisks indicate a third binding event to a low-affinity cryptic binding site that occurred at high concentrations of protein. Gray arrowheads indicate lower-mobility <t>DNA</t> conformations.
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    Integrated DNA Technologies control oligos
    Cdc13 proteins exhibit cooperativity when binding adjacent sites. ( A ) The two-site ligand Tel11-A 6 -Tel11 was incubated with titrations of Cdc13, Cdc13-L91R or Cdc13-DBD under low-salt conditions (75 mM KCl + 75 mM NaCl) and analyzed by EMSA. Three species are formed, as indicated by the inset schematics. The signal of each species is plotted as a fraction of total <t>ssDNA</t> signal: closed circle, free oligo; closed triangle, singly bound oligo; closed square, doubly bound oligo. Three independent data sets were collected and used to solve the cooperativity constants listed in Table 2 . Dashed lines are the least-squares fits of the binding isotherm for each set of data. ( B ) Representative EMSA gels for the data plotted in (A). Asterisks indicate a third binding event to a low-affinity cryptic binding site that occurred at high concentrations of protein. Gray arrowheads indicate lower-mobility <t>DNA</t> conformations.
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    Integrated DNA Technologies decamer oligonucleotides
    Cdc13 proteins exhibit cooperativity when binding adjacent sites. ( A ) The two-site ligand Tel11-A 6 -Tel11 was incubated with titrations of Cdc13, Cdc13-L91R or Cdc13-DBD under low-salt conditions (75 mM KCl + 75 mM NaCl) and analyzed by EMSA. Three species are formed, as indicated by the inset schematics. The signal of each species is plotted as a fraction of total <t>ssDNA</t> signal: closed circle, free oligo; closed triangle, singly bound oligo; closed square, doubly bound oligo. Three independent data sets were collected and used to solve the cooperativity constants listed in Table 2 . Dashed lines are the least-squares fits of the binding isotherm for each set of data. ( B ) Representative EMSA gels for the data plotted in (A). Asterisks indicate a third binding event to a low-affinity cryptic binding site that occurred at high concentrations of protein. Gray arrowheads indicate lower-mobility <t>DNA</t> conformations.
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    90
    Thermo Fisher oligo
    Cdc13 proteins exhibit cooperativity when binding adjacent sites. ( A ) The two-site ligand Tel11-A 6 -Tel11 was incubated with titrations of Cdc13, Cdc13-L91R or Cdc13-DBD under low-salt conditions (75 mM KCl + 75 mM NaCl) and analyzed by EMSA. Three species are formed, as indicated by the inset schematics. The signal of each species is plotted as a fraction of total <t>ssDNA</t> signal: closed circle, free oligo; closed triangle, singly bound oligo; closed square, doubly bound oligo. Three independent data sets were collected and used to solve the cooperativity constants listed in Table 2 . Dashed lines are the least-squares fits of the binding isotherm for each set of data. ( B ) Representative EMSA gels for the data plotted in (A). Asterisks indicate a third binding event to a low-affinity cryptic binding site that occurred at high concentrations of protein. Gray arrowheads indicate lower-mobility <t>DNA</t> conformations.
    Oligo, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 26222 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Integrated DNA Technologies primer oligonucleotides
    Cdc13 proteins exhibit cooperativity when binding adjacent sites. ( A ) The two-site ligand Tel11-A 6 -Tel11 was incubated with titrations of Cdc13, Cdc13-L91R or Cdc13-DBD under low-salt conditions (75 mM KCl + 75 mM NaCl) and analyzed by EMSA. Three species are formed, as indicated by the inset schematics. The signal of each species is plotted as a fraction of total <t>ssDNA</t> signal: closed circle, free oligo; closed triangle, singly bound oligo; closed square, doubly bound oligo. Three independent data sets were collected and used to solve the cooperativity constants listed in Table 2 . Dashed lines are the least-squares fits of the binding isotherm for each set of data. ( B ) Representative EMSA gels for the data plotted in (A). Asterisks indicate a third binding event to a low-affinity cryptic binding site that occurred at high concentrations of protein. Gray arrowheads indicate lower-mobility <t>DNA</t> conformations.
    Primer Oligonucleotides, supplied by Integrated DNA Technologies, used in various techniques. Bioz Stars score: 99/100, based on 65 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Enhancement of T4 DNA ligase activity by supplemental oligonucleotides. (a) Unsuccessful 4-bp duplex reactions could be salvaged by utilizing a supplementary oligonucleotide, designed to complement the first oligonucleotide-dsDNA duplex but is unphosphorylated to prevent ligation of itself. Two hour ligation of the 4-bp reaction at 16°C supplemented with 3.33 μM of the hexamer, shows successful ligation (■) while reactions without the supplementary hexamer show no activity (◆). (b) Ligation reaction of an octamer supplemented with a second octamer in which one is used for ligation and the other is used to extend the duplex. A two hour ligation at 16°C of serial concentrations of the octamer with 3.33 μM of the supplementary octamer shows significant ligation (■) compared to reactions without the supplemental octamer (◆). (c) Unsuccessful 3-bp duplex reactions could be salvaged by utilizing a supplementary hexamer that hybridized at all six positions. A two hour ligation of the 3-bp reaction at 16°C with 3.33 μM supplementary hexamer shows successful ligation (■) while reactions without the supplementary hexamer show no activity (◆). (d) Ligation using a hexamer pair at 4°C for 16 hours shows limited improvement (■) compared to the unsupplemented (◆) control.

    Journal: BMC Research Notes

    Article Title: Efficient assembly of very short oligonucleotides using T4 DNA Ligase

    doi: 10.1186/1756-0500-3-291

    Figure Lengend Snippet: Enhancement of T4 DNA ligase activity by supplemental oligonucleotides. (a) Unsuccessful 4-bp duplex reactions could be salvaged by utilizing a supplementary oligonucleotide, designed to complement the first oligonucleotide-dsDNA duplex but is unphosphorylated to prevent ligation of itself. Two hour ligation of the 4-bp reaction at 16°C supplemented with 3.33 μM of the hexamer, shows successful ligation (■) while reactions without the supplementary hexamer show no activity (◆). (b) Ligation reaction of an octamer supplemented with a second octamer in which one is used for ligation and the other is used to extend the duplex. A two hour ligation at 16°C of serial concentrations of the octamer with 3.33 μM of the supplementary octamer shows significant ligation (■) compared to reactions without the supplemental octamer (◆). (c) Unsuccessful 3-bp duplex reactions could be salvaged by utilizing a supplementary hexamer that hybridized at all six positions. A two hour ligation of the 3-bp reaction at 16°C with 3.33 μM supplementary hexamer shows successful ligation (■) while reactions without the supplementary hexamer show no activity (◆). (d) Ligation using a hexamer pair at 4°C for 16 hours shows limited improvement (■) compared to the unsupplemented (◆) control.

    Article Snippet: Preparation of immobilized dsDNA All oligos, including those 5'-biotinylated, 3'-FAM6, and 5'-phosphorylated were synthesized by Integrated DNA Technologies (IDT Inc., IA, USA).

    Techniques: Activity Assay, Ligation

    Evaluation of minimal oligonucleotide substrate requirements for T4 DNA ligase. (a) Schematic diagram of an immobilized DNA strand used in ligation assays and DNA construction. M-270 Dynabeads (Invitrogen) are attached through a streptavidin-biotin linkage to the 5' end of a double stranded DNA. The free end is designed with a variable 5' overhang, complementary to labeled oligonucleotides used in ligation. An additional BbsI restriction site and a forward primer site are included in the case of DNA construction. (b) Increasing concentrations of 5'-phosphorylated, 3'-fluorescently labeled oligonucleotide are ligated to 5 pmoles of immobilized dsDNA with a complementary overhang. Reactions were performed for one hour at 16°C and washed with TE to remove unligated substrate. Successful ligation kinetics are observed at the 5-bp duplex length (▲), but no significant ligation occurs at lengths of 4-bp (■) or 3-bp (◆).

    Journal: BMC Research Notes

    Article Title: Efficient assembly of very short oligonucleotides using T4 DNA Ligase

    doi: 10.1186/1756-0500-3-291

    Figure Lengend Snippet: Evaluation of minimal oligonucleotide substrate requirements for T4 DNA ligase. (a) Schematic diagram of an immobilized DNA strand used in ligation assays and DNA construction. M-270 Dynabeads (Invitrogen) are attached through a streptavidin-biotin linkage to the 5' end of a double stranded DNA. The free end is designed with a variable 5' overhang, complementary to labeled oligonucleotides used in ligation. An additional BbsI restriction site and a forward primer site are included in the case of DNA construction. (b) Increasing concentrations of 5'-phosphorylated, 3'-fluorescently labeled oligonucleotide are ligated to 5 pmoles of immobilized dsDNA with a complementary overhang. Reactions were performed for one hour at 16°C and washed with TE to remove unligated substrate. Successful ligation kinetics are observed at the 5-bp duplex length (▲), but no significant ligation occurs at lengths of 4-bp (■) or 3-bp (◆).

    Article Snippet: Preparation of immobilized dsDNA All oligos, including those 5'-biotinylated, 3'-FAM6, and 5'-phosphorylated were synthesized by Integrated DNA Technologies (IDT Inc., IA, USA).

    Techniques: Ligation, Labeling

    DNA unzipping force measured using DNA nanoswitches on the CFM. ( a ,i) Schematic of the unzipping construct. Two complementary oligos (red and purple strands) hybridize to form a looped DNA nanoswitch. Force can unzip the two complementary strands, resulting in a measurable increase in tether length, providing a signature of DNA unzipping. ( a ,ii) Images of a bead tethered to the surface via a DNA nanoswitch showing the looped and unlooped states. The scale bar, 1-μm long. ( a ,iii) In one example of rupture-force measurement, we identified 381 tethers with the DNA nanoswitch transitions signature to collect rupture forces while the remaining 673 transitions that corresponded to bead detachment and improper transitions were omitted (see also Supplementary Fig. 5 for detail). ( b ) Unzipping force histograms of 29 bp dsDNA measured with the DNA nanoswitch under two different buffer conditions. ( c ) Average unzipping force of 29 bp dsDNA under different temperatures with PBS buffer (Total n =306), with histograms of rupture forces shown as an inset. The theoretical line is calculated using a previously described thermodynamic model 30 .

    Journal: Nature Communications

    Article Title: Multiplexed single-molecule force spectroscopy using a centrifuge

    doi: 10.1038/ncomms11026

    Figure Lengend Snippet: DNA unzipping force measured using DNA nanoswitches on the CFM. ( a ,i) Schematic of the unzipping construct. Two complementary oligos (red and purple strands) hybridize to form a looped DNA nanoswitch. Force can unzip the two complementary strands, resulting in a measurable increase in tether length, providing a signature of DNA unzipping. ( a ,ii) Images of a bead tethered to the surface via a DNA nanoswitch showing the looped and unlooped states. The scale bar, 1-μm long. ( a ,iii) In one example of rupture-force measurement, we identified 381 tethers with the DNA nanoswitch transitions signature to collect rupture forces while the remaining 673 transitions that corresponded to bead detachment and improper transitions were omitted (see also Supplementary Fig. 5 for detail). ( b ) Unzipping force histograms of 29 bp dsDNA measured with the DNA nanoswitch under two different buffer conditions. ( c ) Average unzipping force of 29 bp dsDNA under different temperatures with PBS buffer (Total n =306), with histograms of rupture forces shown as an inset. The theoretical line is calculated using a previously described thermodynamic model 30 .

    Article Snippet: Subsequently, a set of complementary oligos (Integrated DNA Technologies) was hybridized onto the linear ssDNA.

    Techniques: Construct

    miR-4728-3p IS regulates ESR1. A. qRT-PCR analysis of ESR1 and HER2 transcripts and miR-4728-3p among a panel of 38 breast cancer tumors (19 HER2+, 19 HER2-). Calibrated Normalized Relative Quantity (CNRQ) of miR-4728-3p (left) and HER2 (right) is plotted against expression levels of ESR1. Tumors classified as HER2+ by ISH are shown in red, HER2- in grey. Expression was normalized to a panel of reference genes. For details see text and material and methods. B. Luciferase assay in BT-474 with ESR1 3′UTR constructs carrying either wild type target site of miR-4728-3p internal seed (WT) or mutated internal seed site (MUT). Firefly luciferase activity was normalized against Renilla luciferase. Reporter activity is given as % of WT in respective experiment. Repression of WT ESR1 construct by endogenous miR-4728-3p (left) is alleviated by an antisense oligo (AS) against endogenous miRNA (right) but not by a non-targeting control (middle). C. Western blot (left) and protein quantification (right) of ESR1 in MCF7. The two main isoforms of ESR1 (47 and 66 kDa), plotted as percentage of control signal of matching size, are down regulated upon transfection of miR-4728-3p mimics. Levels of HER2, (p)MAPK and (p)AKT remain largely unchanged. D. MCF7 cells were transfected with indicated concentrations of miR-4728-3p mimic. ESR1 levels show a concentration-dependent down-regulation that is most pronounced at highest tested concentration (25 nM). E. Western blot (left) and protein quantification (right) of ESR1 in BT474. ESR1 is up regulated when blocking endogenous miR-4728-3p with AS-oligonucleotides, while pMAPK and pAKT remain largely unchanged. F. Western blot (left) and protein quantification (right) of ESR1 in HCC1954 cells. ESR1 isoform of 47 kDa is up regulated under miR-4728-3p blocking. The main 66 kDa isoform is not detectable in this ER- cell line. Signals were quantified with ImageJ and normalized to total protein by Coomassie stain. Tubulin was used as a loading control. Asterisks denote p-values of

    Journal: PLoS ONE

    Article Title: The HER2-Encoded miR-4728-3p Regulates ESR1 through a Non-Canonical Internal Seed Interaction

    doi: 10.1371/journal.pone.0097200

    Figure Lengend Snippet: miR-4728-3p IS regulates ESR1. A. qRT-PCR analysis of ESR1 and HER2 transcripts and miR-4728-3p among a panel of 38 breast cancer tumors (19 HER2+, 19 HER2-). Calibrated Normalized Relative Quantity (CNRQ) of miR-4728-3p (left) and HER2 (right) is plotted against expression levels of ESR1. Tumors classified as HER2+ by ISH are shown in red, HER2- in grey. Expression was normalized to a panel of reference genes. For details see text and material and methods. B. Luciferase assay in BT-474 with ESR1 3′UTR constructs carrying either wild type target site of miR-4728-3p internal seed (WT) or mutated internal seed site (MUT). Firefly luciferase activity was normalized against Renilla luciferase. Reporter activity is given as % of WT in respective experiment. Repression of WT ESR1 construct by endogenous miR-4728-3p (left) is alleviated by an antisense oligo (AS) against endogenous miRNA (right) but not by a non-targeting control (middle). C. Western blot (left) and protein quantification (right) of ESR1 in MCF7. The two main isoforms of ESR1 (47 and 66 kDa), plotted as percentage of control signal of matching size, are down regulated upon transfection of miR-4728-3p mimics. Levels of HER2, (p)MAPK and (p)AKT remain largely unchanged. D. MCF7 cells were transfected with indicated concentrations of miR-4728-3p mimic. ESR1 levels show a concentration-dependent down-regulation that is most pronounced at highest tested concentration (25 nM). E. Western blot (left) and protein quantification (right) of ESR1 in BT474. ESR1 is up regulated when blocking endogenous miR-4728-3p with AS-oligonucleotides, while pMAPK and pAKT remain largely unchanged. F. Western blot (left) and protein quantification (right) of ESR1 in HCC1954 cells. ESR1 isoform of 47 kDa is up regulated under miR-4728-3p blocking. The main 66 kDa isoform is not detectable in this ER- cell line. Signals were quantified with ImageJ and normalized to total protein by Coomassie stain. Tubulin was used as a loading control. Asterisks denote p-values of

    Article Snippet: Antisense oligonucleotides contained 2′ O-methyl modifications and were from IDT DNA Technologies.

    Techniques: Quantitative RT-PCR, Expressing, In Situ Hybridization, Luciferase, Construct, Activity Assay, Western Blot, Transfection, Concentration Assay, Blocking Assay, Staining

    Endpoint fluorescence scatter plot containing 4 in-run controls and 40 clinical specimens. Control reactions using Ultramer oligonucleotides for wild type (Wt) (A), A2058G (B), and A2059G (C) are shown. (D) No template control. The gray box indicates

    Journal: Journal of Clinical Microbiology

    Article Title: A 5′ Nuclease Genotyping Assay for Identification of Macrolide-Resistant Mycoplasma genitalium in Clinical Specimens

    doi: 10.1128/JCM.00012-16

    Figure Lengend Snippet: Endpoint fluorescence scatter plot containing 4 in-run controls and 40 clinical specimens. Control reactions using Ultramer oligonucleotides for wild type (Wt) (A), A2058G (B), and A2059G (C) are shown. (D) No template control. The gray box indicates

    Article Snippet: As in-run controls, synthesized Ultramer oligonucleotides (Integrated DNA Technologies, Leuven, Belgium) comprising the entire PCR product were used.

    Techniques: Fluorescence

    Nucleotide sequences of the in-run controls used for the 5′ nuclease genotyping assay. The nucleotide sequences of wild-type Ultramer oligonucleotide (Wt) and Ultramer oligonucleotides containing the two most frequent macrolide resistance mutations,

    Journal: Journal of Clinical Microbiology

    Article Title: A 5′ Nuclease Genotyping Assay for Identification of Macrolide-Resistant Mycoplasma genitalium in Clinical Specimens

    doi: 10.1128/JCM.00012-16

    Figure Lengend Snippet: Nucleotide sequences of the in-run controls used for the 5′ nuclease genotyping assay. The nucleotide sequences of wild-type Ultramer oligonucleotide (Wt) and Ultramer oligonucleotides containing the two most frequent macrolide resistance mutations,

    Article Snippet: As in-run controls, synthesized Ultramer oligonucleotides (Integrated DNA Technologies, Leuven, Belgium) comprising the entire PCR product were used.

    Techniques: Genotyping Assay

    Differential expression of RNA:m 5 C, RCMTs and hnRNPK in 5-AZA-sensitive and 5-AZA-resistant leukaemia cells and the binding of hnRNPK to unmethylated and cytosine-methylated RNA. a Western blot analysis of expression of RCMTs, hnRNPK and other proteins in the 5-AZA-sensitive OCI-M2 and SC leukaemia cells and the 5-AZA-resistant M2AR and SCAR leukaemia cells. b Dot blot analysis of 5-methylcytosine (m 5 C) in RNA and DNA from OCI-M2 and M2AR cells. c Dot blot analysis of 5-methylcytosine (m 5 C) in RNA and DNA from SC and SCAR cells. d , e Visualization and measurement of the binding of purified recombinant hnRNPK to the unmethylated and cytosine-methylated fluorescein (FAM)-labelled RNA oligos by an antibody-coupled bead assay

    Journal: Nature Communications

    Article Title: RNA cytosine methylation and methyltransferases mediate chromatin organization and 5-azacytidine response and resistance in leukaemia

    doi: 10.1038/s41467-018-03513-4

    Figure Lengend Snippet: Differential expression of RNA:m 5 C, RCMTs and hnRNPK in 5-AZA-sensitive and 5-AZA-resistant leukaemia cells and the binding of hnRNPK to unmethylated and cytosine-methylated RNA. a Western blot analysis of expression of RCMTs, hnRNPK and other proteins in the 5-AZA-sensitive OCI-M2 and SC leukaemia cells and the 5-AZA-resistant M2AR and SCAR leukaemia cells. b Dot blot analysis of 5-methylcytosine (m 5 C) in RNA and DNA from OCI-M2 and M2AR cells. c Dot blot analysis of 5-methylcytosine (m 5 C) in RNA and DNA from SC and SCAR cells. d , e Visualization and measurement of the binding of purified recombinant hnRNPK to the unmethylated and cytosine-methylated fluorescein (FAM)-labelled RNA oligos by an antibody-coupled bead assay

    Article Snippet: Visualize and quantify the binding of hnRNPK to RNA The 6-FAM (Fluorescein)-labelled methylated and unmethylated RNA oligos were purchased from Integrated DNA Technologies (Skokie, IL 60076).

    Techniques: Expressing, Binding Assay, Methylation, Western Blot, Dot Blot, Purification, Recombinant

    Structural relationships between NOCT and related phosphodiesterases. ( A ) Structures, sequence identity and function summary for NOCT, PDE12, CNOT6L and TDP2. ( B ) Active site superposition for NOCT, PDE12, CNOT6L and TDP2. ( C ) Catalytic residues in the active site of human NOCT. Residues are numbered using human NOCT as a reference. ( D ) Superposition of the catalytic residues in RNA and DNA hydrolases.

    Journal: Scientific Reports

    Article Title: Crystal Structure of Human Nocturnin Catalytic Domain

    doi: 10.1038/s41598-018-34615-0

    Figure Lengend Snippet: Structural relationships between NOCT and related phosphodiesterases. ( A ) Structures, sequence identity and function summary for NOCT, PDE12, CNOT6L and TDP2. ( B ) Active site superposition for NOCT, PDE12, CNOT6L and TDP2. ( C ) Catalytic residues in the active site of human NOCT. Residues are numbered using human NOCT as a reference. ( D ) Superposition of the catalytic residues in RNA and DNA hydrolases.

    Article Snippet: Preparation of labeled oligonucleotides RNA oligonucleotides were purchased from Integrated DNA Technologies.

    Techniques: Sequencing

    Small RNA injection leads to heritable somatic fusion of 2 complex loci. (a) Fusion of 2 highly scrambled genes, contig9.1 and contig310.1 , whose precursor MDS segments are intertwined in the germline on a 54 kb MIC contig (ctg7180000089708). Partial germline and somatic reference maps are shown, with segment numbers for contig9.1 in blue and contig310.1 in orange; other nomenclature as in Fig. 2 (full germline and somatic maps available: accession numbers given in Data Deposition section); PCR primers used to detect chromosomal fusion are indicated by small arrows above blue MDS 3 (inverted) and orange MDS 2; thick black bars denote Southern hybridization probes for contig9.1 , spanning DNA segments 4–15, and for contig310.1 (MDS 2). (b) Southern analysis provides direct evidence for the presence of the full-length chromosome fusion induced by small RNA injection. Quantitative assessment of the phosphorimager signal in lane 4 (injected line 2 probed with contig310.1 ) suggests that the fusion chromosome is present at roughly half the levels of wild-type contig310.1 . “Strip” is the signal remaining before hybridization to the Contig9.1 probe (exposure length and settings the same for each panel); asterisk indicates an aberrant band containing contig9.1 but not contig310.1 . JRB310 and JRB510 are compatible WT mating strains of O. trifallax . (c) Transgenerational inheritance of the fusion chromosome revealed by PCR of a backcross to JRB510 cells (BC1, backcross generation 1). (d) Detailed map (not to scale) of the micronuclear locus containing intertwined precursor segments for these genes and 4 others. The upward pointing triangle represents 18 MDSs for 2 other genes (Contig15950 and Contig211.1), and the downward pointing triangle represents 20 MDSs for 3 other genes: Contig13252, Contig15950, and Contig7005.

    Journal: RNA Biology

    Article Title: Chromosome fusions triggered by noncoding RNA

    doi: 10.1080/15476286.2016.1195940

    Figure Lengend Snippet: Small RNA injection leads to heritable somatic fusion of 2 complex loci. (a) Fusion of 2 highly scrambled genes, contig9.1 and contig310.1 , whose precursor MDS segments are intertwined in the germline on a 54 kb MIC contig (ctg7180000089708). Partial germline and somatic reference maps are shown, with segment numbers for contig9.1 in blue and contig310.1 in orange; other nomenclature as in Fig. 2 (full germline and somatic maps available: accession numbers given in Data Deposition section); PCR primers used to detect chromosomal fusion are indicated by small arrows above blue MDS 3 (inverted) and orange MDS 2; thick black bars denote Southern hybridization probes for contig9.1 , spanning DNA segments 4–15, and for contig310.1 (MDS 2). (b) Southern analysis provides direct evidence for the presence of the full-length chromosome fusion induced by small RNA injection. Quantitative assessment of the phosphorimager signal in lane 4 (injected line 2 probed with contig310.1 ) suggests that the fusion chromosome is present at roughly half the levels of wild-type contig310.1 . “Strip” is the signal remaining before hybridization to the Contig9.1 probe (exposure length and settings the same for each panel); asterisk indicates an aberrant band containing contig9.1 but not contig310.1 . JRB310 and JRB510 are compatible WT mating strains of O. trifallax . (c) Transgenerational inheritance of the fusion chromosome revealed by PCR of a backcross to JRB510 cells (BC1, backcross generation 1). (d) Detailed map (not to scale) of the micronuclear locus containing intertwined precursor segments for these genes and 4 others. The upward pointing triangle represents 18 MDSs for 2 other genes (Contig15950 and Contig211.1), and the downward pointing triangle represents 20 MDSs for 3 other genes: Contig13252, Contig15950, and Contig7005.

    Article Snippet: Microinjection of synthetic oligonucleotides 27 nt DNA and RNA oligonucleotides were purchased from IDT DNA with standard desalting and used without further purification.

    Techniques: Injection, Polymerase Chain Reaction, Hybridization

    Small RNA injection leads to formation of a putative chromosome circle or dimer. (a) Schematic germline and somatic map of scrambled contig7005.0 and the injected sRNA (purple) that spans the end of the last MDS 6 and beginning of MDS 1, as well as a 4bp sequence that separates them; other nomenclature as in Fig. 2 . The pair of inverse PCR primers used to detect the chromosome fusion are shown as small arrows; the thick black bar in MDS 1 denotes the Southern hybridization probe. (b) Inverse PCR confirms the formation and epigenetic inheritance of a fused chromosome in clonal lines derived from F1 and F2 cells. (c) Southern analysis of both undigested and Hin dIII digested total DNA provides direct evidence for the formation of the fusion in F1 progeny of sRNA-injected cells, as well as the offspring of a mixed mating between F1 lines #1-#5 (F2).

    Journal: RNA Biology

    Article Title: Chromosome fusions triggered by noncoding RNA

    doi: 10.1080/15476286.2016.1195940

    Figure Lengend Snippet: Small RNA injection leads to formation of a putative chromosome circle or dimer. (a) Schematic germline and somatic map of scrambled contig7005.0 and the injected sRNA (purple) that spans the end of the last MDS 6 and beginning of MDS 1, as well as a 4bp sequence that separates them; other nomenclature as in Fig. 2 . The pair of inverse PCR primers used to detect the chromosome fusion are shown as small arrows; the thick black bar in MDS 1 denotes the Southern hybridization probe. (b) Inverse PCR confirms the formation and epigenetic inheritance of a fused chromosome in clonal lines derived from F1 and F2 cells. (c) Southern analysis of both undigested and Hin dIII digested total DNA provides direct evidence for the formation of the fusion in F1 progeny of sRNA-injected cells, as well as the offspring of a mixed mating between F1 lines #1-#5 (F2).

    Article Snippet: Microinjection of synthetic oligonucleotides 27 nt DNA and RNA oligonucleotides were purchased from IDT DNA with standard desalting and used without further purification.

    Techniques: Injection, Sequencing, Inverse PCR, Hybridization, Derivative Assay

    Small RNA injection leads to heritable fusion of 2 somatic chromosomes encoding the non-scrambled genes, contig11396.0 and TEBP β. (a) Schematic germline micronuclear (MIC, top) and somatic macronuclear (MAC, middle) maps are shown for each gene. The injected sRNA (purple bar), MDSs (numbered white boxes; TEBP β MDS 1–7 are in the inverse orientation, indicated by a bar), IESs (gray boxes), somatic telomeres (black vertical rectangles), and a 4 bp overlap (CATG) between the 2 loci are not to scale. Locations of PCR primers are shown as small colored arrows; hybridization probes as thick black lines. (b) Southern analysis provides direct evidence for the presence of full-length somatic chromosome fusions in sRNA-injected but not DNA oligonucleotide-injected cells. “Strip” indicates an image of the stripped membrane before hybridizing to the TEBP β probe. The full length WT TEBP β chromosome is 1,858 bp, contig11396 is 1,635 bp and the fusion is predicted 3,493bp; each panel exposed to X-ray film for an equal time (24 hrs). (c) Transgenerational inheritance of the DNA fusion revealed by PCR analysis (with green primers) of a backcross (BC1) to WT strain JRB510. (d) RT-PCR with the same primers using oligo-dT (dT) or random hexamer (hex) primed cDNA from WT and fusion cells reveals conjugation-specific transcription (at 8–10 hrs) across the chromosomal fusion site, relative to asexually growing offspring of injected cells (Veg). (e) Mapping of piRNAs indicates an absence of piRNAs near the chromosomal fusion site in WT cells, but (f) the presence in BC1 cells of newly-produced piRNAs that bridge the normal chromosome ends but are distinct from the injected 27 nt sRNA (shown in purple). To normalize sequencing depth across libraries, the same number of raw, uncompressed reads (35 million) from each library were mapped onto the MIC contig containing TEBP β and contig11396 . Asterisks mark the 5′ ends of novel piRNAs and the injected sRNA (purple); contig11396 , green; TEBP β, blue.

    Journal: RNA Biology

    Article Title: Chromosome fusions triggered by noncoding RNA

    doi: 10.1080/15476286.2016.1195940

    Figure Lengend Snippet: Small RNA injection leads to heritable fusion of 2 somatic chromosomes encoding the non-scrambled genes, contig11396.0 and TEBP β. (a) Schematic germline micronuclear (MIC, top) and somatic macronuclear (MAC, middle) maps are shown for each gene. The injected sRNA (purple bar), MDSs (numbered white boxes; TEBP β MDS 1–7 are in the inverse orientation, indicated by a bar), IESs (gray boxes), somatic telomeres (black vertical rectangles), and a 4 bp overlap (CATG) between the 2 loci are not to scale. Locations of PCR primers are shown as small colored arrows; hybridization probes as thick black lines. (b) Southern analysis provides direct evidence for the presence of full-length somatic chromosome fusions in sRNA-injected but not DNA oligonucleotide-injected cells. “Strip” indicates an image of the stripped membrane before hybridizing to the TEBP β probe. The full length WT TEBP β chromosome is 1,858 bp, contig11396 is 1,635 bp and the fusion is predicted 3,493bp; each panel exposed to X-ray film for an equal time (24 hrs). (c) Transgenerational inheritance of the DNA fusion revealed by PCR analysis (with green primers) of a backcross (BC1) to WT strain JRB510. (d) RT-PCR with the same primers using oligo-dT (dT) or random hexamer (hex) primed cDNA from WT and fusion cells reveals conjugation-specific transcription (at 8–10 hrs) across the chromosomal fusion site, relative to asexually growing offspring of injected cells (Veg). (e) Mapping of piRNAs indicates an absence of piRNAs near the chromosomal fusion site in WT cells, but (f) the presence in BC1 cells of newly-produced piRNAs that bridge the normal chromosome ends but are distinct from the injected 27 nt sRNA (shown in purple). To normalize sequencing depth across libraries, the same number of raw, uncompressed reads (35 million) from each library were mapped onto the MIC contig containing TEBP β and contig11396 . Asterisks mark the 5′ ends of novel piRNAs and the injected sRNA (purple); contig11396 , green; TEBP β, blue.

    Article Snippet: Microinjection of synthetic oligonucleotides 27 nt DNA and RNA oligonucleotides were purchased from IDT DNA with standard desalting and used without further purification.

    Techniques: Injection, Polymerase Chain Reaction, Hybridization, Reverse Transcription Polymerase Chain Reaction, Random Hexamer Labeling, Conjugation Assay, Produced, Sequencing

    Microinjection of a long chimeric RNA leads to somatic formation of a hybrid TEBP β/α chromosome. (a) Schematic map of injected RNA (1.375 kb): Gray and black horizontal bars denote Southern hybridization probes for TEBP β and TEBP α, respectively; Numbered boxes are MDSs (not to scale); terminal black rectangles indicate telomeres; Locations of PCR primers to detect chimeric products are shown as colored arrows. (b) PCR confirms the formation of hybrid TEBP β/α molecules in the progeny of sense (s) or antisense (as) RNA-injected cells but not uninjected cells (ctrl) (all primer and PCR sequences provided in Supplementary Information). (c) Southern analysis provides direct evidence for the presence of TEBP β/α chimeric DNA molecules in the same cells used in (b). (d) Oligo-dT primed RT-PCR using either pair of primers detects chimeric RNA transcripts in the progeny of injected cells. Sequencing of the larger band confirmed that these RNA molecules do not contain the point substitution in the injected RNA. NT, no template control, RT+/− indicates the presence of reverse transcriptase enzyme; M, marker.

    Journal: RNA Biology

    Article Title: Chromosome fusions triggered by noncoding RNA

    doi: 10.1080/15476286.2016.1195940

    Figure Lengend Snippet: Microinjection of a long chimeric RNA leads to somatic formation of a hybrid TEBP β/α chromosome. (a) Schematic map of injected RNA (1.375 kb): Gray and black horizontal bars denote Southern hybridization probes for TEBP β and TEBP α, respectively; Numbered boxes are MDSs (not to scale); terminal black rectangles indicate telomeres; Locations of PCR primers to detect chimeric products are shown as colored arrows. (b) PCR confirms the formation of hybrid TEBP β/α molecules in the progeny of sense (s) or antisense (as) RNA-injected cells but not uninjected cells (ctrl) (all primer and PCR sequences provided in Supplementary Information). (c) Southern analysis provides direct evidence for the presence of TEBP β/α chimeric DNA molecules in the same cells used in (b). (d) Oligo-dT primed RT-PCR using either pair of primers detects chimeric RNA transcripts in the progeny of injected cells. Sequencing of the larger band confirmed that these RNA molecules do not contain the point substitution in the injected RNA. NT, no template control, RT+/− indicates the presence of reverse transcriptase enzyme; M, marker.

    Article Snippet: Microinjection of synthetic oligonucleotides 27 nt DNA and RNA oligonucleotides were purchased from IDT DNA with standard desalting and used without further purification.

    Techniques: Injection, Hybridization, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Sequencing, Marker

    Modest inhibition of RNA:RNA duplex formation by RNAP ( a ) The ePEC nucleic-acid scaffold ( Table S1 ). DNA strands are shown in black, RNA in red, Pyrrolo-C (PC) in blue, and the 8-mer asRNA oligo in purple. The position of the pause (U19; −1 from 3′ end) is indicated by an arrow. ( b ) Models of E. coli RNAP EC and PEC. Left, closed-clamp, active EC model (based on PDB 2o5i and 3lu0 35 , 52 ) showing clamp (light pink), flap (light blue), bridge helix (BH; cyan), and location of major E.coli sequence insertions (SI1, SI2, SI3) 52 , 53 . The RNA:DNA hybrid (red and black), the exiting RNA (red), and asRNA (purple) are indicated. Right, model of open-clamp, duplex-stabilized PEC containing E. coli RNAP (based on PDB 4gzy 16 ) with arrows indicating clamp and flap movement upon duplex formation. The blow-up shows the location of the lid (pink), flap-tip, and PC (blue), which is quenched by duplex formation with asRNA in the PEC. ( c ) An averaged time trace of PC fluorescence ( n = 6) after mixing 1 µM asRNA with 250 nM ePECs. ( d ) asRNA concentration dependence of PC-quenching for ePECs. The slope of the linear regression line yields the average on-rate ( k on,ensemble ) of asRNA binding to ePECs (~80%) and free scaffold (~20%). ( e ) asRNA dissociation as monitored by PC fluorescence increase after addition of 100 µM competitor RNA to 500 nM preformed duplex-stabilized PEC (averaged trace; n=3). ( f ) The bimolecular rate constants of RNA duplex formation and asRNA dissociation in ePECs or free scaffold. Error bars, s.d. ( n = 3).

    Journal: Nature structural & molecular biology

    Article Title: RNA polymerase pausing and nascent RNA structure formation are linked through clamp domain movement

    doi: 10.1038/nsmb.2867

    Figure Lengend Snippet: Modest inhibition of RNA:RNA duplex formation by RNAP ( a ) The ePEC nucleic-acid scaffold ( Table S1 ). DNA strands are shown in black, RNA in red, Pyrrolo-C (PC) in blue, and the 8-mer asRNA oligo in purple. The position of the pause (U19; −1 from 3′ end) is indicated by an arrow. ( b ) Models of E. coli RNAP EC and PEC. Left, closed-clamp, active EC model (based on PDB 2o5i and 3lu0 35 , 52 ) showing clamp (light pink), flap (light blue), bridge helix (BH; cyan), and location of major E.coli sequence insertions (SI1, SI2, SI3) 52 , 53 . The RNA:DNA hybrid (red and black), the exiting RNA (red), and asRNA (purple) are indicated. Right, model of open-clamp, duplex-stabilized PEC containing E. coli RNAP (based on PDB 4gzy 16 ) with arrows indicating clamp and flap movement upon duplex formation. The blow-up shows the location of the lid (pink), flap-tip, and PC (blue), which is quenched by duplex formation with asRNA in the PEC. ( c ) An averaged time trace of PC fluorescence ( n = 6) after mixing 1 µM asRNA with 250 nM ePECs. ( d ) asRNA concentration dependence of PC-quenching for ePECs. The slope of the linear regression line yields the average on-rate ( k on,ensemble ) of asRNA binding to ePECs (~80%) and free scaffold (~20%). ( e ) asRNA dissociation as monitored by PC fluorescence increase after addition of 100 µM competitor RNA to 500 nM preformed duplex-stabilized PEC (averaged trace; n=3). ( f ) The bimolecular rate constants of RNA duplex formation and asRNA dissociation in ePECs or free scaffold. Error bars, s.d. ( n = 3).

    Article Snippet: Materials DNA and RNA oligonucleotides ( ) were obtained from IDT (Corvalville, IA); PC-containing oligonucleotides, from TriLink Biotechnologies (San Diego, CA); and 6-MI-containing oligonucleotides, from Fidelity Systems (Gaithersburg, MD).

    Techniques: Inhibition, Sequencing, Fluorescence, Concentration Assay, Binding Assay

    Schematic of miRNA-based RNAi vector—shmiRs (A) Standard method for cloning miRNA-based RNAi vectors, left, is compared to miRNA-based vectors described in this study, right. (B) Structure of shmiR- dpp 2 in pHB scaffold. Annealed 71-nt DNA oligos are inserted into the open-loop region with the specified restriction sequences, HindIII-BamHI in this example. Resultant siRNAs will be excised, like miR-1 , from the 3′ arm of the stem-loop (orange highlighted region). This construct is flanked by 5′ and 3′ cloning sites (M.C.S.), enabling the creation of polycistronic hairpins. (C) shmiR- dpp 2 knockdown oligos inserted into the HindIII-BamHI site of pHB. The siRNA sequence is highlighted in red, while the blue nucleotides represent the presumptive star sequence of the dpp 2 siRNA. Highlighted in yellow are two specific mismatched bases necessary to maintain the endogenous pre- miR-1 stem-loop structure.

    Journal: Developmental biology

    Article Title: A simplified miRNA-based gene silencing method for Drosophila melanogaster

    doi: 10.1016/j.ydbio.2008.06.015

    Figure Lengend Snippet: Schematic of miRNA-based RNAi vector—shmiRs (A) Standard method for cloning miRNA-based RNAi vectors, left, is compared to miRNA-based vectors described in this study, right. (B) Structure of shmiR- dpp 2 in pHB scaffold. Annealed 71-nt DNA oligos are inserted into the open-loop region with the specified restriction sequences, HindIII-BamHI in this example. Resultant siRNAs will be excised, like miR-1 , from the 3′ arm of the stem-loop (orange highlighted region). This construct is flanked by 5′ and 3′ cloning sites (M.C.S.), enabling the creation of polycistronic hairpins. (C) shmiR- dpp 2 knockdown oligos inserted into the HindIII-BamHI site of pHB. The siRNA sequence is highlighted in red, while the blue nucleotides represent the presumptive star sequence of the dpp 2 siRNA. Highlighted in yellow are two specific mismatched bases necessary to maintain the endogenous pre- miR-1 stem-loop structure.

    Article Snippet: 71-nt oligos (Elim Biopharmaceuticals or IDT) were annealed at a final concentration of 50 µM in 1X annealing buffer (75 mM KCl, 20 mM Tris [pH 8.0]), boiled for ~2 minutes, and then cooled to room temperature for ~30 minutes.

    Techniques: Plasmid Preparation, Clone Assay, Construct, Sequencing

    EMSA analysis using phosphorylated or non-phosphorylated AtcR. Biotinylated DNA duplexes derived from several genes (indicated at the top) were incubated with increasing concentrations (μM) of either non-phosphorylated AtcR (AtcR) or phosphorylated

    Journal: Molecular oral microbiology

    Article Title: The Treponema denticola AtcR LytTR domain-containing response regulator interacts with three architecturally distinct promoter elements: implications for understanding the molecular signaling mechanisms that drive the progression of periodontal disease

    doi: 10.1111/omi.12059

    Figure Lengend Snippet: EMSA analysis using phosphorylated or non-phosphorylated AtcR. Biotinylated DNA duplexes derived from several genes (indicated at the top) were incubated with increasing concentrations (μM) of either non-phosphorylated AtcR (AtcR) or phosphorylated

    Article Snippet: Biotinylated-oligonucleotides ( ) (Integrated DNA Technologies) were annealed by heating to 80 °C followed by cooling to 25 °C (1 °C min−1 ).

    Techniques: Derivative Assay, Incubation

    EMSA analysis of AtcR binding to 60 base pair DNA duplexes. Biotinylated DNA duplexes that have potential LytTR recognition sequences (identified as described in the text) were incubated with or without purified AtcR for EMSA analyses. The T. denticola

    Journal: Molecular oral microbiology

    Article Title: The Treponema denticola AtcR LytTR domain-containing response regulator interacts with three architecturally distinct promoter elements: implications for understanding the molecular signaling mechanisms that drive the progression of periodontal disease

    doi: 10.1111/omi.12059

    Figure Lengend Snippet: EMSA analysis of AtcR binding to 60 base pair DNA duplexes. Biotinylated DNA duplexes that have potential LytTR recognition sequences (identified as described in the text) were incubated with or without purified AtcR for EMSA analyses. The T. denticola

    Article Snippet: Biotinylated-oligonucleotides ( ) (Integrated DNA Technologies) were annealed by heating to 80 °C followed by cooling to 25 °C (1 °C min−1 ).

    Techniques: Binding Assay, Incubation, Purification

    The 2′–5′ phosphodiester linkage in RNA primer end reduces RNA pol II transcriptional efficiency but does not affect fidelity. ( A ) Scaffold of RNA, template DNA, and nontemplate DNA for runoff elongation from the 2′–5′–linked

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

    Article Title: Strand-specific (asymmetric) contribution of phosphodiester linkages on RNA polymerase II transcriptional efficiency and fidelity

    doi: 10.1073/pnas.1406234111

    Figure Lengend Snippet: The 2′–5′ phosphodiester linkage in RNA primer end reduces RNA pol II transcriptional efficiency but does not affect fidelity. ( A ) Scaffold of RNA, template DNA, and nontemplate DNA for runoff elongation from the 2′–5′–linked

    Article Snippet: The DNA template and nontemplate oligonucleotides were purchased from Integrated DNA Technologies.

    Techniques:

    Transcriptional efficiency of RNA pol II is greatly reduced by the linkage alteration in the DNA template. ( A ) Scaffold of RNA, template DNA, and nontemplate DNA for runoff elongation through the 2′–5′ linkage site in the template.

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

    Article Title: Strand-specific (asymmetric) contribution of phosphodiester linkages on RNA polymerase II transcriptional efficiency and fidelity

    doi: 10.1073/pnas.1406234111

    Figure Lengend Snippet: Transcriptional efficiency of RNA pol II is greatly reduced by the linkage alteration in the DNA template. ( A ) Scaffold of RNA, template DNA, and nontemplate DNA for runoff elongation through the 2′–5′ linkage site in the template.

    Article Snippet: The DNA template and nontemplate oligonucleotides were purchased from Integrated DNA Technologies.

    Techniques:

    Effects of magnesium on the endoribonuclease, RNA binding, AP-ssDNA, and AP-ssRNA endonuclease activities of APE1. (a) 1.4 μM of APE1 (lanes 2 – 9) was tested against 25 nM of 5′-γ- 32 P-radiolabeled c- myc CRD RNA in a total

    Journal: Journal of molecular biology

    Article Title: Characterization of Endoribonuclease Active Site of Human Apurinic/Apyrimidinic Endonuclease 1

    doi: 10.1016/j.jmb.2011.06.050

    Figure Lengend Snippet: Effects of magnesium on the endoribonuclease, RNA binding, AP-ssDNA, and AP-ssRNA endonuclease activities of APE1. (a) 1.4 μM of APE1 (lanes 2 – 9) was tested against 25 nM of 5′-γ- 32 P-radiolabeled c- myc CRD RNA in a total

    Article Snippet: Tetrahydrofuran (abasic site analog, as depicted as F) containing ssDNA and ssRNA oligonucleotides were purchased from IDT Inc., and radiolabeled at the 5′ end using [γ-32 P] ATP and T4 polynucleotide kinase (New England Biolabs, Ipswich, MA).

    Techniques: RNA Binding Assay

    Cdc13 proteins exhibit cooperativity when binding adjacent sites. ( A ) The two-site ligand Tel11-A 6 -Tel11 was incubated with titrations of Cdc13, Cdc13-L91R or Cdc13-DBD under low-salt conditions (75 mM KCl + 75 mM NaCl) and analyzed by EMSA. Three species are formed, as indicated by the inset schematics. The signal of each species is plotted as a fraction of total ssDNA signal: closed circle, free oligo; closed triangle, singly bound oligo; closed square, doubly bound oligo. Three independent data sets were collected and used to solve the cooperativity constants listed in Table 2 . Dashed lines are the least-squares fits of the binding isotherm for each set of data. ( B ) Representative EMSA gels for the data plotted in (A). Asterisks indicate a third binding event to a low-affinity cryptic binding site that occurred at high concentrations of protein. Gray arrowheads indicate lower-mobility DNA conformations.

    Journal: Nucleic Acids Research

    Article Title: The tenacious recognition of yeast telomere sequence by Cdc13 is fully exerted by a single OB-fold domain

    doi: 10.1093/nar/gkt843

    Figure Lengend Snippet: Cdc13 proteins exhibit cooperativity when binding adjacent sites. ( A ) The two-site ligand Tel11-A 6 -Tel11 was incubated with titrations of Cdc13, Cdc13-L91R or Cdc13-DBD under low-salt conditions (75 mM KCl + 75 mM NaCl) and analyzed by EMSA. Three species are formed, as indicated by the inset schematics. The signal of each species is plotted as a fraction of total ssDNA signal: closed circle, free oligo; closed triangle, singly bound oligo; closed square, doubly bound oligo. Three independent data sets were collected and used to solve the cooperativity constants listed in Table 2 . Dashed lines are the least-squares fits of the binding isotherm for each set of data. ( B ) Representative EMSA gels for the data plotted in (A). Asterisks indicate a third binding event to a low-affinity cryptic binding site that occurred at high concentrations of protein. Gray arrowheads indicate lower-mobility DNA conformations.

    Article Snippet: For EMSA, all Cdc13 proteins were assayed in 50 mM Tris–HCl, pH 7.8 at 4°C, 75 mM KCl, 75 mM NaCl, 1 mM dithiothreitol (DTT), 0.1 mM EDTA, 0.1 mg/ml bovine serum albumin and 15% glycerol. ssDNA oligonucleotides (Integrated DNA Technologies) were resuspended in nuclease-free water and 5′-end-labeled using γ-32 P-ATP with T4 polynucleotide kinase (New England Biolabs).

    Techniques: Binding Assay, Incubation