ribonuclease t1 Search Results


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  • 85
    Worthington Biochemical ribonuclease t1
    Ribonuclease T1, supplied by Worthington Biochemical, used in various techniques. Bioz Stars score: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Thermo Fisher ribonucleases a t1
    Ligand-induced conformational changes. In-line probing of a 5′-AlexaFluor 647-labeled Bs PreQ 1 -RS RNA and b 5′-AlexaFluor 647-labeled Tt PreQ 1 -RS RNA after treatment with 1 at increasing concentrations or a DMSO control in the absence (–) or presence (+) of 1 m m MgCl 2 . Treatment with PreQ 1 at a concentration of 10 μ m is used as a positive control. OH and T1 are a partial alkaline hydrolysis ladder and <t>ribonuclease</t> T1 digestion, respectively. Arrows designate nucleotide positions where the cleavage efficiency was significantly altered by compound treatment (blue) or preQ 1 treatment (red)
    Ribonucleases A T1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Roche ribonuclease rnase t1
    Ligand-induced conformational changes. In-line probing of a 5′-AlexaFluor 647-labeled Bs PreQ 1 -RS RNA and b 5′-AlexaFluor 647-labeled Tt PreQ 1 -RS RNA after treatment with 1 at increasing concentrations or a DMSO control in the absence (–) or presence (+) of 1 m m MgCl 2 . Treatment with PreQ 1 at a concentration of 10 μ m is used as a positive control. OH and T1 are a partial alkaline hydrolysis ladder and <t>ribonuclease</t> T1 digestion, respectively. Arrows designate nucleotide positions where the cleavage efficiency was significantly altered by compound treatment (blue) or preQ 1 treatment (red)
    Ribonuclease Rnase T1, supplied by Roche, used in various techniques. Bioz Stars score: 85/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Roche ribonuclease t1
    Ligand-induced conformational changes. In-line probing of a 5′-AlexaFluor 647-labeled Bs PreQ 1 -RS RNA and b 5′-AlexaFluor 647-labeled Tt PreQ 1 -RS RNA after treatment with 1 at increasing concentrations or a DMSO control in the absence (–) or presence (+) of 1 m m MgCl 2 . Treatment with PreQ 1 at a concentration of 10 μ m is used as a positive control. OH and T1 are a partial alkaline hydrolysis ladder and <t>ribonuclease</t> T1 digestion, respectively. Arrows designate nucleotide positions where the cleavage efficiency was significantly altered by compound treatment (blue) or preQ 1 treatment (red)
    Ribonuclease T1, supplied by Roche, used in various techniques. Bioz Stars score: 89/100, based on 49 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 89 stars, based on 49 article reviews
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    89
    Boehringer Mannheim ribonuclease t1
    Ligand-induced conformational changes. In-line probing of a 5′-AlexaFluor 647-labeled Bs PreQ 1 -RS RNA and b 5′-AlexaFluor 647-labeled Tt PreQ 1 -RS RNA after treatment with 1 at increasing concentrations or a DMSO control in the absence (–) or presence (+) of 1 m m MgCl 2 . Treatment with PreQ 1 at a concentration of 10 μ m is used as a positive control. OH and T1 are a partial alkaline hydrolysis ladder and <t>ribonuclease</t> T1 digestion, respectively. Arrows designate nucleotide positions where the cleavage efficiency was significantly altered by compound treatment (blue) or preQ 1 treatment (red)
    Ribonuclease T1, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 89/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 89 stars, based on 22 article reviews
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    95
    Millipore t1 rnase
    Mutational analysis of the HSUR 1 ARE. ( A ) Sequences of the six HSUR 1 point mutants, M1–M6, with the sites of U → G mutation underlined. ( B ) RNA levels of the HSUR 1 mutants. Wild-type HSUR 1 and mutants M1–M6, all driven by the same U1 promoter, were each transiently cotransfected with HSUR 3 into mouse L929 cells, and the RNA levels were assayed by <t>T1</t> RNase protection (lanes 1–7, respectively). The antisense HSUR 1 probe covered a 120-nucleotide region at the 3′ end, which is common to wild-type HSUR 1 and all six mutants. When quantitated and normalized to HSUR 3, the mutant M1–M6 levels were found to be 7.0-, 10.1-, 1.8-, 5.8-, 6.4-, and 6.1-fold that of wild-type HSUR 1.
    T1 Rnase, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 29 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Thermo Fisher a t1 ribonuclease mix
    Mutational analysis of the HSUR 1 ARE. ( A ) Sequences of the six HSUR 1 point mutants, M1–M6, with the sites of U → G mutation underlined. ( B ) RNA levels of the HSUR 1 mutants. Wild-type HSUR 1 and mutants M1–M6, all driven by the same U1 promoter, were each transiently cotransfected with HSUR 3 into mouse L929 cells, and the RNA levels were assayed by <t>T1</t> RNase protection (lanes 1–7, respectively). The antisense HSUR 1 probe covered a 120-nucleotide region at the 3′ end, which is common to wild-type HSUR 1 and all six mutants. When quantitated and normalized to HSUR 3, the mutant M1–M6 levels were found to be 7.0-, 10.1-, 1.8-, 5.8-, 6.4-, and 6.1-fold that of wild-type HSUR 1.
    A T1 Ribonuclease Mix, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher t1 rnase
    DPAGE analysis of Zn 2+ -specific 5′- 32 P-labeled cleavage after 24 h at 37°C, pH 8.6 in the absence (left) and presence (right) of non- cleavable substrate. Zn(OAc) 2 concentrations, from left to right in each set, are 0, 1, 10, 50, 100, 200, 500 and 1000 µM. ‘OH – ’ indicates a limited alkaline hydrolysis ladder of 5′- 32 P-labeled ribozyme and ‘T1’ indicates a <t>T1</t> RNase ladder of 5′- 32 P-labeled ribozyme.
    T1 Rnase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 173 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    GE Healthcare ribonuclease t1
    DPAGE analysis of Zn 2+ -specific 5′- 32 P-labeled cleavage after 24 h at 37°C, pH 8.6 in the absence (left) and presence (right) of non- cleavable substrate. Zn(OAc) 2 concentrations, from left to right in each set, are 0, 1, 10, 50, 100, 200, 500 and 1000 µM. ‘OH – ’ indicates a limited alkaline hydrolysis ladder of 5′- 32 P-labeled ribozyme and ‘T1’ indicates a <t>T1</t> RNase ladder of 5′- 32 P-labeled ribozyme.
    Ribonuclease T1, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 89/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Protein Sciences Inc ribonuclease t1
    DPAGE analysis of Zn 2+ -specific 5′- 32 P-labeled cleavage after 24 h at 37°C, pH 8.6 in the absence (left) and presence (right) of non- cleavable substrate. Zn(OAc) 2 concentrations, from left to right in each set, are 0, 1, 10, 50, 100, 200, 500 and 1000 µM. ‘OH – ’ indicates a limited alkaline hydrolysis ladder of 5′- 32 P-labeled ribozyme and ‘T1’ indicates a <t>T1</t> RNase ladder of 5′- 32 P-labeled ribozyme.
    Ribonuclease T1, supplied by Protein Sciences Inc, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher rnase
    Optimization of K8 intron 2 splice sites promotes recognition of K8 intron 2 by the <t>RNA</t> splicing machinery. (A) Maps of K8 pre-mRNAs containing part of exon 2, all of intron 2 (line), and part of exon 3 that were used for in vitro <t>RNase</t> H digestion. Wild-type
    Rnase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 9347 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Ligand-induced conformational changes. In-line probing of a 5′-AlexaFluor 647-labeled Bs PreQ 1 -RS RNA and b 5′-AlexaFluor 647-labeled Tt PreQ 1 -RS RNA after treatment with 1 at increasing concentrations or a DMSO control in the absence (–) or presence (+) of 1 m m MgCl 2 . Treatment with PreQ 1 at a concentration of 10 μ m is used as a positive control. OH and T1 are a partial alkaline hydrolysis ladder and ribonuclease T1 digestion, respectively. Arrows designate nucleotide positions where the cleavage efficiency was significantly altered by compound treatment (blue) or preQ 1 treatment (red)

    Journal: Nature Communications

    Article Title: Synthetic ligands for PreQ1 riboswitches provide structural and mechanistic insights into targeting RNA tertiary structure

    doi: 10.1038/s41467-019-09493-3

    Figure Lengend Snippet: Ligand-induced conformational changes. In-line probing of a 5′-AlexaFluor 647-labeled Bs PreQ 1 -RS RNA and b 5′-AlexaFluor 647-labeled Tt PreQ 1 -RS RNA after treatment with 1 at increasing concentrations or a DMSO control in the absence (–) or presence (+) of 1 m m MgCl 2 . Treatment with PreQ 1 at a concentration of 10 μ m is used as a positive control. OH and T1 are a partial alkaline hydrolysis ladder and ribonuclease T1 digestion, respectively. Arrows designate nucleotide positions where the cleavage efficiency was significantly altered by compound treatment (blue) or preQ 1 treatment (red)

    Article Snippet: Alkaline hydrolysis was performed in 50 mm NaHCO3 , pH 9.0 at 95 °C for 5 min. Ribonuclease T1 digestion was carried out with 0.1 U of ribonuclease T1 (Ambion) in 20 mm Tris, pH 7.5, 50 mm NaCl, 0.1 mm MgCl2 at room temperature for 20 min.

    Techniques: Labeling, Concentration Assay, Positive Control

    Mutational analysis of the HSUR 1 ARE. ( A ) Sequences of the six HSUR 1 point mutants, M1–M6, with the sites of U → G mutation underlined. ( B ) RNA levels of the HSUR 1 mutants. Wild-type HSUR 1 and mutants M1–M6, all driven by the same U1 promoter, were each transiently cotransfected with HSUR 3 into mouse L929 cells, and the RNA levels were assayed by T1 RNase protection (lanes 1–7, respectively). The antisense HSUR 1 probe covered a 120-nucleotide region at the 3′ end, which is common to wild-type HSUR 1 and all six mutants. When quantitated and normalized to HSUR 3, the mutant M1–M6 levels were found to be 7.0-, 10.1-, 1.8-, 5.8-, 6.4-, and 6.1-fold that of wild-type HSUR 1.

    Journal: Genes & Development

    Article Title: AU-rich elements target small nuclear RNAs as well as mRNAs for rapid degradation

    doi:

    Figure Lengend Snippet: Mutational analysis of the HSUR 1 ARE. ( A ) Sequences of the six HSUR 1 point mutants, M1–M6, with the sites of U → G mutation underlined. ( B ) RNA levels of the HSUR 1 mutants. Wild-type HSUR 1 and mutants M1–M6, all driven by the same U1 promoter, were each transiently cotransfected with HSUR 3 into mouse L929 cells, and the RNA levels were assayed by T1 RNase protection (lanes 1–7, respectively). The antisense HSUR 1 probe covered a 120-nucleotide region at the 3′ end, which is common to wild-type HSUR 1 and all six mutants. When quantitated and normalized to HSUR 3, the mutant M1–M6 levels were found to be 7.0-, 10.1-, 1.8-, 5.8-, 6.4-, and 6.1-fold that of wild-type HSUR 1.

    Article Snippet: T1 RNase protection assays were performed as described (S. ), with the following modifications: DNase-treated RNA (5–10 μg for snRNA, 20–30 μg for mRNA) was combined with 2 × 105 to 4 × 105 cpm of the appropriate [α-32 P]UTP-labeled antisense probe, heated at 85°C for 5 min, incubated at 45°C overnight to allow annealing, and then digested with T1 RNase (1 U/10 μg of RNA; Calbiochem) at 30°C for 1 hr.

    Techniques: Mutagenesis

    AUUUA repeats target other snRNAs for rapid decay. ( A ) The 5′ ARE of wild-type HSUR 2 and its mutants. The single guanosine that interrupts the AUUUA repeat sequence in HSUR 2 was mutated to UA, CC, and CA (underlined) in HSUR 2 M1, HSUR 2 M2, and HSUR 2 M3, respectively. ( B ) RNA levels of wild-type and mutant HSUR 2s. Wild-type HSUR 2 or mutant M1, M2, or M3, all controlled by the same U1 promoter, were each transiently cotransfected with HSUR 3 into mouse L929 cells and analyzed by T1 RNase protection. The level of mutant M1 (lane 2 ), which has four tandem copies of AUUU, is one-fifth that of wild-type HSUR 2 (lane 1 ), whereas controls M2 and M3 (lanes 3 and 4, respectively), which have been mutated at the same two positions as M1, have levels 1.3- and 1.1-fold of the wild-type HSUR 2, respectively. ( C ) The 5′-end sequences of wild-type U1 and two U1 mutants. The 5′ splice site recognition sequence of U1 and the sequences replacing it in the mutants are underlined. The AU3–U1 mutant has four tandem copies of AUUU, whereas in the AGU–U1 mutant, there are four AUUU repeats interrupted by 3 Gs (the same sequence as in HSUR 1 M2). ( D ) Levels of AU3–U1 and AGU–U1 in duplicate transfection experiments. Each U1 mutant was transiently cotransfected with HSUR 3 into mouse L929 cells. U1 RNA levels were analyzed by primer extension using an oligonucleotide complementary to the most 3′ 20 nucleotides of U1 (nucleotides 155–164), whereas HSUR 3 was assayed by T1 RNase protection assay as above. When normalized to HSUR 3 and averaged between the duplicate transfection experiments, the AU3–U1 level was found to be one-fourth that of AGU–U1.

    Journal: Genes & Development

    Article Title: AU-rich elements target small nuclear RNAs as well as mRNAs for rapid degradation

    doi:

    Figure Lengend Snippet: AUUUA repeats target other snRNAs for rapid decay. ( A ) The 5′ ARE of wild-type HSUR 2 and its mutants. The single guanosine that interrupts the AUUUA repeat sequence in HSUR 2 was mutated to UA, CC, and CA (underlined) in HSUR 2 M1, HSUR 2 M2, and HSUR 2 M3, respectively. ( B ) RNA levels of wild-type and mutant HSUR 2s. Wild-type HSUR 2 or mutant M1, M2, or M3, all controlled by the same U1 promoter, were each transiently cotransfected with HSUR 3 into mouse L929 cells and analyzed by T1 RNase protection. The level of mutant M1 (lane 2 ), which has four tandem copies of AUUU, is one-fifth that of wild-type HSUR 2 (lane 1 ), whereas controls M2 and M3 (lanes 3 and 4, respectively), which have been mutated at the same two positions as M1, have levels 1.3- and 1.1-fold of the wild-type HSUR 2, respectively. ( C ) The 5′-end sequences of wild-type U1 and two U1 mutants. The 5′ splice site recognition sequence of U1 and the sequences replacing it in the mutants are underlined. The AU3–U1 mutant has four tandem copies of AUUU, whereas in the AGU–U1 mutant, there are four AUUU repeats interrupted by 3 Gs (the same sequence as in HSUR 1 M2). ( D ) Levels of AU3–U1 and AGU–U1 in duplicate transfection experiments. Each U1 mutant was transiently cotransfected with HSUR 3 into mouse L929 cells. U1 RNA levels were analyzed by primer extension using an oligonucleotide complementary to the most 3′ 20 nucleotides of U1 (nucleotides 155–164), whereas HSUR 3 was assayed by T1 RNase protection assay as above. When normalized to HSUR 3 and averaged between the duplicate transfection experiments, the AU3–U1 level was found to be one-fourth that of AGU–U1.

    Article Snippet: T1 RNase protection assays were performed as described (S. ), with the following modifications: DNase-treated RNA (5–10 μg for snRNA, 20–30 μg for mRNA) was combined with 2 × 105 to 4 × 105 cpm of the appropriate [α-32 P]UTP-labeled antisense probe, heated at 85°C for 5 min, incubated at 45°C overnight to allow annealing, and then digested with T1 RNase (1 U/10 μg of RNA; Calbiochem) at 30°C for 1 hr.

    Techniques: Sequencing, Mutagenesis, Transfection, Rnase Protection Assay

    ARE-mediated HSUR 1 degradation in vivo. ( A ). ( B ) T1 RNase protection analysis of wild-type and mutant HSUR 1 levels in transient transfection assays. The pUC–U1–HSUR 1 constructs were transiently cotransfected with a pUC–U1–HSUR 3 plasmid into mouse L929 cells (see Materials and Methods). Total RNA collected 48 hr after transfection was subjected to RNase T1 protection assays with wild-type and mutant HSUR 1 antisense RNA (lanes 2 and 3, respectively), together with antisense HSUR 3 RNA as an internal control. One-fiftieth of the amount of the anti-wild-type and anti-mutant HSUR 1 RNA probes used is shown in lanes 4 and 5. The data were quantitated with a Molecular Dynamics PhosphorImager and normalized to HSUR 3. Wild-type HSUR 1 levels were reproducibly one-eighth of those of mutant HSUR 1. ( C ) Whole-cell run-on assays of wild-type and mutant HSUR 1 transcription. The pUC–U1–HSUR 1 construct containing wild-type or mutant HSUR 1 sequences was cotransfected with pUC–U1–HSUR 3 into L cells, and whole-cell run-on assays were performed (see Materials and Methods). Total RNA was hybridized to nylon membranes that had been dot-blotted with wild-type ( top ) or mutant ( bottom ) HSUR 1 and HSUR 3 DNA fragments. Untransfected HSUR 4 DNA was also dotted as a negative control. The patterns of dots are illustrated at right; hybridizations with the run-on RNAs are at left. After quantitation and normalization against the cotransfected positive control HSUR 3 (also subtracting the untransfected negative control HSUR 4), the wild-type HSUR 1 ( left dot, top ) and the mutant ( left dot, bottom ) were found to have similar transcription rates (wild type:mutant = 0.95). ( D ) Immunoprecipitation of wild-type and mutant HSUR 1 from transfected mouse L929 cells. L cells were transiently transfected with the pUC–U1 constructs containing wild-type or mutant HSUR 1 genes, and whole-cell extracts were prepared by sonication. Equal amounts of extract were precipitated with anti-Sm monoclonal antibody Y12 or anti-U1 70K monoclonal antibody H111 as a control. RNA was harvested from the immunoprecipitation pellets (lanes 1,3,5,7 ) and supernatants (lanes 2,4,6,8 ), and wild-type ( left ) and mutant ( right ) HSUR 1 were assayed by T1 RNase protection. For both wild-type and mutant HSUR 1s, > 90% of the RNA was in the anti-Sm precipitate (lanes 3,7 ), whereas > 99% of the HSUR 1s remained in the supernatant with the anti-U1 70K antibody (lanes 2,6 ).

    Journal: Genes & Development

    Article Title: AU-rich elements target small nuclear RNAs as well as mRNAs for rapid degradation

    doi:

    Figure Lengend Snippet: ARE-mediated HSUR 1 degradation in vivo. ( A ). ( B ) T1 RNase protection analysis of wild-type and mutant HSUR 1 levels in transient transfection assays. The pUC–U1–HSUR 1 constructs were transiently cotransfected with a pUC–U1–HSUR 3 plasmid into mouse L929 cells (see Materials and Methods). Total RNA collected 48 hr after transfection was subjected to RNase T1 protection assays with wild-type and mutant HSUR 1 antisense RNA (lanes 2 and 3, respectively), together with antisense HSUR 3 RNA as an internal control. One-fiftieth of the amount of the anti-wild-type and anti-mutant HSUR 1 RNA probes used is shown in lanes 4 and 5. The data were quantitated with a Molecular Dynamics PhosphorImager and normalized to HSUR 3. Wild-type HSUR 1 levels were reproducibly one-eighth of those of mutant HSUR 1. ( C ) Whole-cell run-on assays of wild-type and mutant HSUR 1 transcription. The pUC–U1–HSUR 1 construct containing wild-type or mutant HSUR 1 sequences was cotransfected with pUC–U1–HSUR 3 into L cells, and whole-cell run-on assays were performed (see Materials and Methods). Total RNA was hybridized to nylon membranes that had been dot-blotted with wild-type ( top ) or mutant ( bottom ) HSUR 1 and HSUR 3 DNA fragments. Untransfected HSUR 4 DNA was also dotted as a negative control. The patterns of dots are illustrated at right; hybridizations with the run-on RNAs are at left. After quantitation and normalization against the cotransfected positive control HSUR 3 (also subtracting the untransfected negative control HSUR 4), the wild-type HSUR 1 ( left dot, top ) and the mutant ( left dot, bottom ) were found to have similar transcription rates (wild type:mutant = 0.95). ( D ) Immunoprecipitation of wild-type and mutant HSUR 1 from transfected mouse L929 cells. L cells were transiently transfected with the pUC–U1 constructs containing wild-type or mutant HSUR 1 genes, and whole-cell extracts were prepared by sonication. Equal amounts of extract were precipitated with anti-Sm monoclonal antibody Y12 or anti-U1 70K monoclonal antibody H111 as a control. RNA was harvested from the immunoprecipitation pellets (lanes 1,3,5,7 ) and supernatants (lanes 2,4,6,8 ), and wild-type ( left ) and mutant ( right ) HSUR 1 were assayed by T1 RNase protection. For both wild-type and mutant HSUR 1s, > 90% of the RNA was in the anti-Sm precipitate (lanes 3,7 ), whereas > 99% of the HSUR 1s remained in the supernatant with the anti-U1 70K antibody (lanes 2,6 ).

    Article Snippet: T1 RNase protection assays were performed as described (S. ), with the following modifications: DNase-treated RNA (5–10 μg for snRNA, 20–30 μg for mRNA) was combined with 2 × 105 to 4 × 105 cpm of the appropriate [α-32 P]UTP-labeled antisense probe, heated at 85°C for 5 min, incubated at 45°C overnight to allow annealing, and then digested with T1 RNase (1 U/10 μg of RNA; Calbiochem) at 30°C for 1 hr.

    Techniques: In Vivo, Mutagenesis, Transfection, Construct, Plasmid Preparation, Negative Control, Quantitation Assay, Positive Control, Immunoprecipitation, Sonication

    Partial RNase digestion shows single- and double-stranded regions of RSE RNA. (A and B) The 5′-end-labeled RSE RNA (294 nt) was digested with increasing amounts of RNase T 1 . The resulting cleavage products were run on a 15% (A) or 8% (B) polyacrylamide-8 M urea gel. Note that in panel A the 294-nt RSE full-length (FL) RNA comigrates with a marker of approximately 400 nt. (C) The 5′-end-labeled RSE RNA was digested with increasing amounts of RNase A and resolved on an 8 M urea-15% polyacrylamide gel. (D) The 3′-end-labeled RSE RNA was digested with RNase V 1 and resolved on an 8 M urea-8% polyacrylamide gel. Numbers on the right correspond to the RSE nucleotide that is cleaved by RNase. Nucleotide position 4 of the RSE RNA corresponds to nt 2597 of the complete RSV genomic RNA sequence. The black triangle indicates increasing RNase concentrations. Lanes 0, RNA alone (no RNase added).

    Journal: Journal of Virology

    Article Title: Structural Characterization of the Rous Sarcoma Virus RNA Stability Element ▿Structural Characterization of the Rous Sarcoma Virus RNA Stability Element ▿ †

    doi: 10.1128/JVI.02113-08

    Figure Lengend Snippet: Partial RNase digestion shows single- and double-stranded regions of RSE RNA. (A and B) The 5′-end-labeled RSE RNA (294 nt) was digested with increasing amounts of RNase T 1 . The resulting cleavage products were run on a 15% (A) or 8% (B) polyacrylamide-8 M urea gel. Note that in panel A the 294-nt RSE full-length (FL) RNA comigrates with a marker of approximately 400 nt. (C) The 5′-end-labeled RSE RNA was digested with increasing amounts of RNase A and resolved on an 8 M urea-15% polyacrylamide gel. (D) The 3′-end-labeled RSE RNA was digested with RNase V 1 and resolved on an 8 M urea-8% polyacrylamide gel. Numbers on the right correspond to the RSE nucleotide that is cleaved by RNase. Nucleotide position 4 of the RSE RNA corresponds to nt 2597 of the complete RSV genomic RNA sequence. The black triangle indicates increasing RNase concentrations. Lanes 0, RNA alone (no RNase added).

    Article Snippet: The volume of each sample was brought to 40 μl and digested either without RNase or with the following amounts of RNase: RNase T1 (Calbiochem), 0.05, 0.005, or 0.0005 units; RNase A (Calbiochem), 1 μg/ml, 0.2 μg/ml, or 0.1 μg/ml; and RNase V1 (Pharmacia Biotech), 1.44, 0.72, or 0.36 units.

    Techniques: Labeling, Marker, Sequencing

    Determination of minimum RNA fragment bound by MSY2 and MSY4. (A) Schematic depiction of Prm1 1–37wt RNA and four mutant RNAs engineered such that RNase T1 treatment produces different-size RNA fragments containing the YRS. Arrows, RNase T1 cleavage sites. The single nucleotide substitution in T1.8m is underlined. (B) Urea gel analysis of RNase T1 precut RNAs. Top arrow, size of the RNAs prior to cutting (43 nt). Upon treatment with RNase T1, YRS-containing RNA fragments of 12, 10, and 8 nt are released. No uncut RNA of 43 nt is seen in the cut-RNA lanes. (C) UV cross-linking analysis of MSY2 and MSY4 binding of the RNAs depicted in panels A and B. MSY2 and MSY4 were able to bind the T1.12, T1.10, and T1.8 RNA substrates before and after treatment with RNase T1.

    Journal: Molecular and Cellular Biology

    Article Title: MSY2 and MSY4 Bind a Conserved Sequence in the 3? Untranslated Region of Protamine 1 mRNA In Vitro and In Vivo

    doi: 10.1128/MCB.21.20.7010-7019.2001

    Figure Lengend Snippet: Determination of minimum RNA fragment bound by MSY2 and MSY4. (A) Schematic depiction of Prm1 1–37wt RNA and four mutant RNAs engineered such that RNase T1 treatment produces different-size RNA fragments containing the YRS. Arrows, RNase T1 cleavage sites. The single nucleotide substitution in T1.8m is underlined. (B) Urea gel analysis of RNase T1 precut RNAs. Top arrow, size of the RNAs prior to cutting (43 nt). Upon treatment with RNase T1, YRS-containing RNA fragments of 12, 10, and 8 nt are released. No uncut RNA of 43 nt is seen in the cut-RNA lanes. (C) UV cross-linking analysis of MSY2 and MSY4 binding of the RNAs depicted in panels A and B. MSY2 and MSY4 were able to bind the T1.12, T1.10, and T1.8 RNA substrates before and after treatment with RNase T1.

    Article Snippet: Samples were then sequentially treated with 2 μl of RNase T1 (Calbiochem) at 2 U/μl and 4 μl of heparin (Sigma) at 5 mg/ml, each for 10 min at room temperature.

    Techniques: Mutagenesis, Binding Assay

    Mass analysis of platinated unmodified H69 is shown. Mass spectra of ( a ) unmodified H69 parent RNA (unplatinated H69); ( b ) RNase T1 digestion of unplatinated H69; ( c ) platinated unmodified H69; and ( d ) RNase T1 digestion of platinated unmodified H69 are given. Arrows indicate RNase T1 cleavage sites.

    Journal: International Journal of Molecular Sciences

    Article Title: Cisplatin Targeting of Bacterial Ribosomal RNA Hairpins

    doi: 10.3390/ijms160921392

    Figure Lengend Snippet: Mass analysis of platinated unmodified H69 is shown. Mass spectra of ( a ) unmodified H69 parent RNA (unplatinated H69); ( b ) RNase T1 digestion of unplatinated H69; ( c ) platinated unmodified H69; and ( d ) RNase T1 digestion of platinated unmodified H69 are given. Arrows indicate RNase T1 cleavage sites.

    Article Snippet: RNase T1 digestion of platinated and unplatinated RNAs was performed in water for 20 min at 37 °C using 1 unit of enzyme (Sigma).

    Techniques:

    Mass analysis of platinated modified H69 is shown. Mass spectra of ( a ) modified H69 parent RNA (unplatinated RNA); ( b ) RNase T1 digestion of unplatinated H69; ( c ) platinated modified H69; and ( d ) RNase T1 digestion of platinated modified H69 are given. Arrows indicate RNase T1 cleavage sites.

    Journal: International Journal of Molecular Sciences

    Article Title: Cisplatin Targeting of Bacterial Ribosomal RNA Hairpins

    doi: 10.3390/ijms160921392

    Figure Lengend Snippet: Mass analysis of platinated modified H69 is shown. Mass spectra of ( a ) modified H69 parent RNA (unplatinated RNA); ( b ) RNase T1 digestion of unplatinated H69; ( c ) platinated modified H69; and ( d ) RNase T1 digestion of platinated modified H69 are given. Arrows indicate RNase T1 cleavage sites.

    Article Snippet: RNase T1 digestion of platinated and unplatinated RNAs was performed in water for 20 min at 37 °C using 1 unit of enzyme (Sigma).

    Techniques: Modification

    Mass analysis of platinated 790 loop is shown. Mass spectra of ( a ) 790 loop parent strand (unplatinated 790 loop); ( b ) RNase T1 digestion of unplatinated 790 loop; ( c ) platinated 790 loop; and ( d ) RNase T1 digestion of platinated 790 loop are shown. Arrows indicate RNase T1 cleavage sites.

    Journal: International Journal of Molecular Sciences

    Article Title: Cisplatin Targeting of Bacterial Ribosomal RNA Hairpins

    doi: 10.3390/ijms160921392

    Figure Lengend Snippet: Mass analysis of platinated 790 loop is shown. Mass spectra of ( a ) 790 loop parent strand (unplatinated 790 loop); ( b ) RNase T1 digestion of unplatinated 790 loop; ( c ) platinated 790 loop; and ( d ) RNase T1 digestion of platinated 790 loop are shown. Arrows indicate RNase T1 cleavage sites.

    Article Snippet: RNase T1 digestion of platinated and unplatinated RNAs was performed in water for 20 min at 37 °C using 1 unit of enzyme (Sigma).

    Techniques:

    Ion dependence of B. stearothermophilus trp leader RNA structure formation. ( A ) 5′ 32 P-labeled RNA (1–232) was subjected to partial RNase T1 digestion to probe RNA structure under various salt conditions. C is the untreated RNA, OH − is a limited alkaline hydrolysis ladder, and T1 is a limited RNase T1 digest under RNA denaturing conditions. Lane 1 , RNA renatured in TK 0 M 0 ; lane 2 , RNA renatured in TK 100 M 0 ; lane 3 , RNA renatured in TK 100 M 4 . Important secondary structural features are given on the left side of the gel, including triplet repeat number. ( B ) Sites of RNase T1 cleavage superimposed on the secondary structure. Filled and open triangles indicate sites of strong and weak cleavage, respectively, in TK 100 M 0 . Asterisks (*) indicate sites of reduced cleavage upon the addition of 4 mM Mg 2+ . Numbering is from the start of transcription.

    Journal: RNA

    Article Title: Phylogenetic conservation of RNA secondary and tertiary structure in the trpEDCFBA operon leader transcript in Bacillus

    doi: 10.1261/rna.5149603

    Figure Lengend Snippet: Ion dependence of B. stearothermophilus trp leader RNA structure formation. ( A ) 5′ 32 P-labeled RNA (1–232) was subjected to partial RNase T1 digestion to probe RNA structure under various salt conditions. C is the untreated RNA, OH − is a limited alkaline hydrolysis ladder, and T1 is a limited RNase T1 digest under RNA denaturing conditions. Lane 1 , RNA renatured in TK 0 M 0 ; lane 2 , RNA renatured in TK 100 M 0 ; lane 3 , RNA renatured in TK 100 M 4 . Important secondary structural features are given on the left side of the gel, including triplet repeat number. ( B ) Sites of RNase T1 cleavage superimposed on the secondary structure. Filled and open triangles indicate sites of strong and weak cleavage, respectively, in TK 100 M 0 . Asterisks (*) indicate sites of reduced cleavage upon the addition of 4 mM Mg 2+ . Numbering is from the start of transcription.

    Article Snippet: Structure mapping experiments were carried out using 5′ end-labeled RNA (4 nM final concentration) and G-specific RNase T1 (Sigma).

    Techniques: Labeling

    DPAGE analysis of Zn 2+ -specific 5′- 32 P-labeled cleavage after 24 h at 37°C, pH 8.6 in the absence (left) and presence (right) of non- cleavable substrate. Zn(OAc) 2 concentrations, from left to right in each set, are 0, 1, 10, 50, 100, 200, 500 and 1000 µM. ‘OH – ’ indicates a limited alkaline hydrolysis ladder of 5′- 32 P-labeled ribozyme and ‘T1’ indicates a T1 RNase ladder of 5′- 32 P-labeled ribozyme.

    Journal: Nucleic Acids Research

    Article Title: Zinc-dependent cleavage in the catalytic core of the hammerhead ribozyme: evidence for a pH-dependent conformational change

    doi:

    Figure Lengend Snippet: DPAGE analysis of Zn 2+ -specific 5′- 32 P-labeled cleavage after 24 h at 37°C, pH 8.6 in the absence (left) and presence (right) of non- cleavable substrate. Zn(OAc) 2 concentrations, from left to right in each set, are 0, 1, 10, 50, 100, 200, 500 and 1000 µM. ‘OH – ’ indicates a limited alkaline hydrolysis ladder of 5′- 32 P-labeled ribozyme and ‘T1’ indicates a T1 RNase ladder of 5′- 32 P-labeled ribozyme.

    Article Snippet: T1 RNase digests were prepared by incubating 5′-32 P-labeled ribozyme (5 pmol) at 37°C for 30 min in the presence of 25 µg unlabeled Escherichia coli 5S ribosomal RNA, 7 M urea, 1 mM Na2 EDTA, 25 mM sodium citrate and 2.9 U T1 RNase (Invitrogen) (10 µl total).

    Techniques: Labeling

    Analysis of small molecule binding to miR-21-hp RNA by Mg 2+  induced in-line cleavage. (A) In-gel fluorescence of 5′-Cy5-labeled miR-21-hp RNA after treatment with  13  at concentrations of 3, 10, 30, 100, or 300  μ M or a DMSO control in the absence (−) or presence (+) of 1 mM MgCl 2 . OH and T1 are a partial alkaline hydrolysis ladder and ribonuclease T1 digestion, respectively. Arrows designate nucleotide positions where the cleavage efficiency was significantly altered by compound treatment. (B) Quantification of fluorescent band intensity from above gel, where band intensity from  13  or streptomycin treated samples was normalized to the average band intensity of DMSO control treated samples. (C) Structure of the 5′-Cy5-labeled miR-21-hp RNA used for the in-line probing. Significant changes to the cleavage pattern in the presence of small molecule are indicated in blue. The red line indicates the putative Dicer cleavage site on pre-miR-21.

    Journal: ACS chemical biology

    Article Title: Discovery of Inhibitors of MicroRNA-21 Processing Using Small Molecule Microarrays

    doi: 10.1021/acschembio.6b00945

    Figure Lengend Snippet: Analysis of small molecule binding to miR-21-hp RNA by Mg 2+ induced in-line cleavage. (A) In-gel fluorescence of 5′-Cy5-labeled miR-21-hp RNA after treatment with 13 at concentrations of 3, 10, 30, 100, or 300 μ M or a DMSO control in the absence (−) or presence (+) of 1 mM MgCl 2 . OH and T1 are a partial alkaline hydrolysis ladder and ribonuclease T1 digestion, respectively. Arrows designate nucleotide positions where the cleavage efficiency was significantly altered by compound treatment. (B) Quantification of fluorescent band intensity from above gel, where band intensity from 13 or streptomycin treated samples was normalized to the average band intensity of DMSO control treated samples. (C) Structure of the 5′-Cy5-labeled miR-21-hp RNA used for the in-line probing. Significant changes to the cleavage pattern in the presence of small molecule are indicated in blue. The red line indicates the putative Dicer cleavage site on pre-miR-21.

    Article Snippet: Alkaline hydrolysis was performed in 10 mM NaHCO3 at pH 9.0 and 95 °C for 5 min. Ribonuclease T1 digestion was carried out with 0.1 U of ribonuclease T1 (Ambion) in 20 mM Tris at pH 7.5, 50 mM NaCl, and 0.1 mM MgCl2 at RT for 20 min.

    Techniques: Binding Assay, Fluorescence, Labeling

    Optimization of K8 intron 2 splice sites promotes recognition of K8 intron 2 by the RNA splicing machinery. (A) Maps of K8 pre-mRNAs containing part of exon 2, all of intron 2 (line), and part of exon 3 that were used for in vitro RNase H digestion. Wild-type

    Journal:

    Article Title: Kaposi's Sarcoma-Associated Herpesvirus K8? Is Derived from a Spliced Intermediate of K8 Pre-mRNA and Antagonizes K8? (K-bZIP) To Induce p21 and p53 and Blocks K8?-CDK2 Interaction

    doi: 10.1128/JVI.79.22.14207-14221.2005

    Figure Lengend Snippet: Optimization of K8 intron 2 splice sites promotes recognition of K8 intron 2 by the RNA splicing machinery. (A) Maps of K8 pre-mRNAs containing part of exon 2, all of intron 2 (line), and part of exon 3 that were used for in vitro RNase H digestion. Wild-type

    Article Snippet: For RPA, 100 μg of total cell RNA was hybridized with 4 ng of an antisense RNA probe in hybridization buffer at 42°C overnight, followed by RNase (RNase A and T1) digestion as instructed by the manufacturer of the RPA III kit (Ambion).

    Techniques: In Vitro