oligonucleotide  (New England Biolabs)

Journal: BMC Molecular Biology

Article Title: Structure-function analysis of Methanobacterium thermoautotrophicum RNA ligase - engineering a thermostable ATP independent enzyme

doi: 10.1186/1471-2199-13-24

Figure Lengend Snippet: Activity of the MthRnl and the K246A, K246Q and K97A mutants in different steps of the ligation reaction. (A) pH-dependence of self-adenylation activity of enzymes (step 1) in reactions with radioactive ATP. The reactions were carried out as described in the Methods and the products were resolved by an SDS-PAGE. Autoradiographs of the gels are shown. (B) pH-dependence of adenylation of 5’-phosphorylated, 3’-amino-blocked ssDNA (pDNA17-NH 2 ) (step 2) with wild type and the mutants of MthRnl under reaction conditions described in the Methods. The reaction products were separated by 15% urea-PAGE and visualized with SYBR Gold stain. (C) pH-dependence of deadenylation/hydrolysis of 5’ pre-adenylated DNA (AppDNA17-NH 2 ) with wild type and the mutants of MthRnl under reaction conditions described in the Methods. The products were analyzed on 15% urea-PAGE and stained with SYBR Gold. Enzymes used in each step of ligation reactions are indicated on the left. Position of DNA donor substrates and products are indicated on the right. The pH of the buffer is indicated at the bottom. The control (c) indicates the reaction without enzyme.

Article Snippet: Preparative adenylation of DNA and RNA linkers (donor substrates) were performed using 5’-DNA Adenylation kit (NEB) [ ] Table .

Techniques: Activity Assay, Ligation, SDS Page, Polyacrylamide Gel Electrophoresis, Staining

Journal: Nature Communications

Article Title: Ataluren binds to multiple protein synthesis apparatus sites and competitively inhibits release factor-dependent termination

doi: 10.1038/s41467-022-30080-6

Figure Lengend Snippet: Scatter plots of peptide and tRNA dissociation times. Each point represents dissociation times from an individual ribosome. CC is correlation coefficient. a 2 µM RFC. b 16 nM RFC. Source data are provided as a Source Data file.

Article Snippet: The miRNA cloning linker 2 (5´App/CACTCGGGCACCAAGGA/3´ddC, Integrated DNA Technologies) was used as the universal 3´ linker and pre-adenylated using the 5´ DNA Adenylation Kit (New England Biolab) with the provided protocol.

Techniques:

Journal: Nature Communications

Article Title: Ataluren binds to multiple protein synthesis apparatus sites and competitively inhibits release factor-dependent termination

doi: 10.1038/s41467-022-30080-6

Figure Lengend Snippet: Ataluren effects on RFC-dependent termination of polypeptide synthesis. Cumulative distributions of ( a ) peptide and ( b ) tRNA dissociation times at indicated RFC concentrations, one also with 1 mM ataluren. Each cumulative distribution is constructed from ≥300 kinetic traces. Rates of dissociation of ( c ). peptide and ( d ). tRNA as a function of RFC concentration at different fixed ataluren concentrations. Error bars are ± s.e.m. for n = 250–750 trials. Normalized plots of ensemble experiments showing single exponential fits (solid lines) of decimated smoothed raw data (points) of atto647 pentapeptide release reaction measured by fluorescence anisotropy decay vs. time at 25 °C. e At indicated RFC concentrations. The control shows the near constancy of observed anisotropy in the absence of added RFC or ataluren. f At an RFC concentration of 0.0625 µM and varying ataluren concentrations. g The rates of dissociation of atto647 pentapeptide in ensemble experiments as a function of free RFC concentration at varying ataluren concentrations. Error bars are average deviation (a. d.) for n = 2–6 independent determinations. Values of n for each point are presented in Supplementary Table 5 . h Ataluren inhibition of normalized rates of dissociation of atto647 pentapeptide as measured by single molecule (red) and plate reader (black) assays. [eRF1], 32 nM; [eRF3], 0.2 µM and 0.8 µM in the single molecule and ensemble assays, respectively. Error bars are ± s.e.m. n ≥ 250 trials (single molecule) and ± a.d., n = 2 independent determinations (plate reader). i Rates of dissociation of atto647 labeled and unlabeled pentapeptide as measured by millipore filtration, calculated using both filtrate and filter retained values. Error bars are ± s.d., n = 8 independent measurements for all points except for the 4 min measurements, for which n = 11. [eRF1], 0.2 µM; [eRF3], 0.8 µM; POST5, 0.05 µM. j Ataluren inhibition of peptide and tRNA release when added at different times (4–25 s) following RFC addition to Stop-POST5. [RFC], 0.08 µM; [Ataluren], 1 mM. Values at zero-time correspond to simultaneous addition of ataluren and RFC. Error bars are ±s.e.m. for n ≥ 200 trials. Source data for all panels are provided as a Source Data file.

Article Snippet: The miRNA cloning linker 2 (5´App/CACTCGGGCACCAAGGA/3´ddC, Integrated DNA Technologies) was used as the universal 3´ linker and pre-adenylated using the 5´ DNA Adenylation Kit (New England Biolab) with the provided protocol.

Techniques: Construct, Concentration Assay, Fluorescence, Inhibition, Labeling, Filtration

Journal: The Journal of Biological Chemistry

Article Title: Polynucleotide 3′-terminal Phosphate Modifications by RNA and DNA Ligases

doi: 10.1074/jbc.M114.612929

Figure Lengend Snippet: Modification of the 3′-phosphorylated nick in duplex DNA by various ligases. A , structure of the double-stranded substrate used in an assay and 3′-adenylation reaction. B , control for double-stranded DNA formation. After annealing of three

Article Snippet: Ligase from M. thermoautotrophicum (MthRnl), the 5′-DNA adenylation kit, and its K97A mutant thermostable 5′AppDNA/RNA ligase were from New England Biolabs ( , ).

Techniques: Modification