rnase  (Worthington Biochemical)

Journal: Nucleic Acids Research

Article Title: An analytical platform for mass spectrometry-based identification and chemical analysis of RNA in ribonucleoprotein complexes

doi: 10.1093/nar/gkp732

Figure Lengend Snippet: LC-MS analysis of RNaseT1 digests of yeast tRNA Phe−1 . ( a ) Base peak chromatogram of the RNase T1 digest of the yeast tRNA Phe−1 preparation (200 fmol). Chromatography was performed as described in the ‘Materials and Methods’ section. Sixteen major oligoribonucleotide peaks, indicated by arrows with peak numbers, are assigned to the fragments of yeast tRNA Phe−1 , tRNA Phe−2 , tRNA Lys−2 , or tRNA Tyr (see Table 1 ). ( b and c ) A typical MS spectrum of the RNase T1 digest of tRNA Phe−1 ; (b) [AUUUAm 2 G > p] 2− and (c) [ACmUGmAAyWAΨUm 5 CUG > p] 3− .

Article Snippet: RNase T1 was purchased from Worthington (Lakewood, NJ) and further purified by reversed-phase liquid chromatography (RPLC) before use.

Techniques: Liquid Chromatography with Mass Spectroscopy, Chromatography, Mass Spectrometry

Journal: Nucleic Acids Research

Article Title: An analytical platform for mass spectrometry-based identification and chemical analysis of RNA in ribonucleoprotein complexes

doi: 10.1093/nar/gkp732

Figure Lengend Snippet: CID-MS/MS spectrum of [AUUUAm 2 G] 2− derived from RNase T1 digestion of yeast tRNA Phe−1 . The doubly charged ion with m / z = 966.6 was analyzed by CID. The nucleotide sequence was verified by manual interpretation of the a- and c-type (normal text) and w - and y -type ( italic text ) product ion series as indicated in the figure. The parent ion losing methyl guanine [P−B(mG) 2− ], the parent ion losing adenine [P−B(A) 2− ], the y5 ion losing methyl guanine [y5−B(mG) 2− ], the y5 ion losing adenine [y5−B(A) 2− ], y5 2− and c5 2− were doubly charged products. All other assigned signals were singly charged products, unless indicated otherwise. The asterisks indicate hydrated or dehydrated ions of a-, c-, w- or y-type products.

Article Snippet: RNase T1 was purchased from Worthington (Lakewood, NJ) and further purified by reversed-phase liquid chromatography (RPLC) before use.

Techniques: Mass Spectrometry, Derivative Assay, Sequencing

Journal: Nucleic Acids Research

Article Title: An analytical platform for mass spectrometry-based identification and chemical analysis of RNA in ribonucleoprotein complexes

doi: 10.1093/nar/gkp732

Figure Lengend Snippet: Base peak chromatogram of the RNaseT1 digest of yeast U4 snRNA isolated from the Lsm3-associated RNP complex. The gel piece containing U4 snRNA was in-gel digested with RNaseT1 and subjected to the LC-MS analysis. Major oligoribonucleotide peaks assigned as RNase T1 fragments of yeast U4 snRNA are indicated by arrows with the corresponding sequence. Detailed data for MS/MS-based assignment of each fragment are given in Supplementary Table S3 .

Article Snippet: RNase T1 was purchased from Worthington (Lakewood, NJ) and further purified by reversed-phase liquid chromatography (RPLC) before use.

Techniques: Isolation, Liquid Chromatography with Mass Spectroscopy, Sequencing, Mass Spectrometry

Journal: Nucleic Acids Research

Article Title: A mass spectrometry-based method for direct determination of pseudouridine in RNA

doi: 10.1093/nar/gkv1462

Figure Lengend Snippet: Identification of four Ψ-containing fragments in human U6 snRNA. The RNase T1 digest of human U6 snRNA (100 fmol) was analyzed by LC-MS and MS 2 . The base peak chromatogram (BPC) of MS, EPIC of MS 2 and four EICs of MS are shown. The major peaks in the BPC, indicated by the arrow or arrowheads with peak numbers, were assigned to the fragments of U6 snRNA (see Supplementary Table S1). The peaks in the EPIC indicated by asterisks were assigned to the Ψ-containing fragments of U6 snRNA by Ariadne, whereas the peaks with numbered closed circles indicate fragments from contaminating RNAs in our U6 snRNA preparation and the U6 snRNA fragment with the 2′,3′-cyclic-phosphate terminus assigned as follows: 1, CΨUCG > p (U6 snRNA); 2, TΨCGp (tRNA); 3, AUΨUCCGp (U5 snRNA); 4, A(Cm)UAAAGp (U5 snRNA); 5, CAUAAAUCUUUC(Gm)CCUUU(Um)A(Cm)ΨAAAGp (U5 snRNA); 6, UAUAAAUCUUUC(Gm)CCUUU(Um)A(Cm)ΨAAAGp (U5 snRNA). The oligoribonucleotide with a sequence UUCCGp detected in the EIC with m/z 791.58 was derived from U5 snRNA contaminated in our U6 snRNA preparation. Note that contaminated RNA fragments can easily be distinguished those derived from a sample RNA by comparing the identified sequence with its genomic sequence using Ariadne. The peaks of the Ψ-containing fragments of U6 snRNA reappear in the EIC. The sequence of each RNase T1 fragment and its m/z value (±15 ppm mass tolerance) for extraction are indicated on each EIC. Corresponding peaks on the EPIC and EIC are linked with dotted lines.

Article Snippet: RNase T1 and A were purchased from Worthington (Lakewood, NJ, USA) and further purified by reversed-phase liquid chromatography (LC) before use.

Techniques: Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Sequencing, Derivative Assay

Journal: Nucleic Acids Research

Article Title: A mass spectrometry-based method for direct determination of pseudouridine in RNA

doi: 10.1093/nar/gkv1462

Figure Lengend Snippet: Identification of four Ψ-containing fragments in human U5 snRNA. The RNase T1 digest of human U5 snRNA (100 fmol mixture of U5A and U5B) was analyzed by LC-MS and MS 2 . The BPC of MS, EPIC of MS 2 and four EICs of MS are shown. The major peaks in the BPC, indicated by the arrow or arrowheads with peak numbers, were assigned to the fragments of U5A or U5B snRNA (see Supplementary Table S1). The major peaks in EPIC indicated by asterisks were assigned to the Ψ-containing fragments of U5A/U5B snRNA. The peaks with numbered closed circles in the EPIC were fragments of contaminating RNA in our U5A/B preparation assigned as follows: 1, CΨUCGp (U6 snRNA); 2, TΨCGp (tRNA); 3, ΨAC(mA)Gp (U6 snRNA). The peaks of Ψ-containing fragments of U5A or U5B snRNA reappear in the EIC. The sequence of each RNase T1 fragment and its m/z value (±15 ppm mass tolerance) are indicated. Corresponding peaks on EPIC and EIC are linked with dotted lines.

Article Snippet: RNase T1 and A were purchased from Worthington (Lakewood, NJ, USA) and further purified by reversed-phase liquid chromatography (LC) before use.

Techniques: Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Sequencing

Journal: Genes & Development

Article Title: Localization-dependent translation requires a functional interaction between the 5? and 3? ends of oskar mRNA

doi:

Figure Lengend Snippet: p50 binds both to the 5′ end of osk ) was incubated and UV cross-linked to proteins in the oocyte extract in the absence of competitor RNA (lane 1 ). Competitions were carried out with a 100-fold excess of either repressor fragment (rep, lane 2 ), Eco RI– Bgl II fragment containing the 5′ activator in sense orientation (5′/act-s, lane 3), or the same Eco RI– Bgl II fragment in antisense orientation (5′/act-as, lane 4 ). Immunoprecipitation of proteins UV cross-linked to radiolabeled repressor fragment (lane 5 ) by anti-Bruno antiserum (lane 6 ) or preimmune serum (lane 7 ). Simultaneous binding of p50 and Bruno was tested by treating with RNase A only after the immunoprecipitation. (Lane 8 ) Anti-Bruno antiserum; (lane 9 ) preimmune serum. We noted that in some cases (as shown here) the intensity of the upper band of the p50 doublet was reduced after coprecipitation. The locations of the 5′ and 3′ competitors in the osk transcript are indicated below. The radiolabeled probe is indicated by an asterisk. The reverse experiment, with activator element RNA as a radioactive probe, and cold activator and repressor RNA elements as unlabeled competitors, yielded the same result with respect to p50 (data not shown).

Article Snippet: Fifty micrograms of RNase A and 4 units of RNase T1 (Worthington Biochemical) were used.

Techniques: Incubation, Activated Clotting Time Assay, Immunoprecipitation, Binding Assay

Journal: BMC Infectious Diseases

Article Title: Abolishing HIV-1 infectivity using a polypurine tract-specific G-quadruplex-forming oligonucleotide

doi: 10.1186/s12879-016-1713-x

Figure Lengend Snippet: Antiviral mechanism of ODN sequences. a Schematic representation of reverse transcription of HIV and mechanism of ODN A action. The HIV-1 extended polypurine tract (PPT) is indicated as black box with the RT/RNase H cleavage side depicted in white. ODN A interacts with the highly conserved PPT, mimicking the natural replication intermediate RNA-DNA hybrid, which results in premature activation of reverse transcriptase (RT)/RNase H hydroloysis of the viral RNA genome. b Sequences of the ODNs used. All ODNs form hairpin-like structures with an antisense ( lower ) and passenger ( upper ) strand linked by four thymidines. Watson-Crick bonds are shown as vertical bars . Phosphorothioate-modified nucleotides are shown in bold and marked by a star . The ODN A sequence is complementary to the extended PPT, and ODN Co targets an HIV-1 RNA region outside of the PPT. ODN G serves as a further control, with a similar passenger strand sequence compared to ODN A but a non-complementary HIV-1 PPT sequence. c Sequence of the extended polypurine tract of HIV-1 recognized by the viral RNase H, whose specific cleavage site is indicated by an arrow

Article Snippet: For annealing, purified RNA2 transcripts were mixed with 50 nM of ODNs in hybridization buffer (50 mM NaCl, 10 mM MgCl2 , 1 mM DTT, 0.4 mM spermine hydrochloride, 25 mM Tris-acetate, pH 6.8), heat-treated for 3 min at 90 °C, cooled, and incubated at 37 °C for 30 min. After annealing, samples were incubated with 0.05 units/μl of HIV RT/RNase H (Worthington, USA) for a further 30 min at 37 °C.

Techniques: Activation Assay, Modification, Sequencing

Journal: BMC Infectious Diseases

Article Title: Abolishing HIV-1 infectivity using a polypurine tract-specific G-quadruplex-forming oligonucleotide

doi: 10.1186/s12879-016-1713-x

Figure Lengend Snippet: ODN A is active in vitro and in cell culture infection assays after long-term storage at 37 °C. a ODN A (8 μM) was stored for the indicated duration in PBS or ( b ) in H 2 O at 37 °C. Samples were analyzed by non-denaturing 10 % PAGE. c ODN A (50 nM), either freshly thawed or stored for the indicated time periods at 37 °C in PBS or H 2 O was hybridized to 50 nM in vitro transcribed γ-32-ATP 5′-labeled PPT-containing RNA in the presence of HIV-1 RT/RNase H. The cleavage products were analyzed by denaturing polyacrylamide/8 M urea gel electrophoresis and are presented schematically on the right . Cleavage sites are indicated by arrowheads and labeled products are shown in black . ODN Co, annealing to sequences downstream of the PPT, served as a control. d Following the experimental procedure shown at the top : Freshly thawed ODN A or ODN Co (250 nM), or ODN A stored for 102 days at 37 °C in PBS were incubated with replication-competent HIV-1 particles (1 × 10 9 ) at 37 °C for 6 h in cell culture medium. Jurkat 1G5 T cells were infected with the mixtures overnight and HIV-1 p24 antigen in the supernatant was detected at 3–14 days post infection. Two-way ANOVA followed by Bonferroni posttest was used for statistical evaluation. ODN A-mediated inhibition (as compared to buffer alone) was highly significant ( p

Article Snippet: For annealing, purified RNA2 transcripts were mixed with 50 nM of ODNs in hybridization buffer (50 mM NaCl, 10 mM MgCl2 , 1 mM DTT, 0.4 mM spermine hydrochloride, 25 mM Tris-acetate, pH 6.8), heat-treated for 3 min at 90 °C, cooled, and incubated at 37 °C for 30 min. After annealing, samples were incubated with 0.05 units/μl of HIV RT/RNase H (Worthington, USA) for a further 30 min at 37 °C.

Techniques: In Vitro, Cell Culture, Infection, Polyacrylamide Gel Electrophoresis, Labeling, Nucleic Acid Electrophoresis, Incubation, Inhibition

Journal: BMC Infectious Diseases

Article Title: Abolishing HIV-1 infectivity using a polypurine tract-specific G-quadruplex-forming oligonucleotide

doi: 10.1186/s12879-016-1713-x

Figure Lengend Snippet: The lubricant CMC (K-Y Jelly) does not influence the stability and antiviral activity of ODN A. a ODN A (8 μM) was incubated for the indicated days in 25 % K-Y Jelly (in PBS) at 37 °C. The samples were analyzed by 10 % non-denaturing polyacrylamide gel electrophoresis. b Analysis of ODN A-triggered, RNase H-mediated cleavage of PPT RNA in vitro. Assays were performed as described in Fig. 2c . Cleavage sites are indicated by arrowheads and the labeled products are indicated in black . c Following the experimental procedure shown at the top : ODN A pre-incubated in 25 % K-Y Jelly/PBS for 57 days, freshly thawed ODN A, or ODN Co (both 250 nM) were incubated with replication-competent HIV-1 virions (1 × 10 9 ) at 37 °C for 6 h in cell culture medium. Jurkat 1G5 T cells were subsequently infected overnight. Virus replication was monitored by HIV-1 p24 antigen ELISA of culture supernatants at day 3–14 post infection. Two-way ANOVA followed by Bonferroni posttest was used for statistical evaluation. ODN A-mediated inhibition (as compared to buffer alone) was significant (day 7, p

Article Snippet: For annealing, purified RNA2 transcripts were mixed with 50 nM of ODNs in hybridization buffer (50 mM NaCl, 10 mM MgCl2 , 1 mM DTT, 0.4 mM spermine hydrochloride, 25 mM Tris-acetate, pH 6.8), heat-treated for 3 min at 90 °C, cooled, and incubated at 37 °C for 30 min. After annealing, samples were incubated with 0.05 units/μl of HIV RT/RNase H (Worthington, USA) for a further 30 min at 37 °C.

Techniques: Activity Assay, Incubation, Polyacrylamide Gel Electrophoresis, In Vitro, Labeling, Cell Culture, Infection, Enzyme-linked Immunosorbent Assay, Inhibition