Recombinant RNase H protein

• Cat.No.: RNASEH1-433H
• Product Overview: Recombinant RNase H was expressed in E. coli.

Specification

• Source: E.coli
• Tag: Non
• Description: Ribonuclease H (RNase H) is an RNA-specific endonuclease that hydrolyzes single-stranded RNA via a magnesium or manganese cofactor. As such, RNase H is often used to remove the RNA template following reverse transcription or cDNA generation.
• Form: Ribonuclease H (RNase H) is supplied in a buffer of 25 mM Tris-HCl (pH 7.5), 30 mM NaCl, 0.5 mM EDTA, 1 mM DTT and 50% glycerol.
• Unit Definition: One unit is defined as the amount of enzyme required to produce 1 nmol of acid-soluble product in 20 minutes at 30°C and pH 7.7 using poly(rA)-poly(dT) as a substrate.
• Applications: Removal of RNA template following second-strand cDNA synthesis;Removal of mRNA poly(A) sequence;Reverse transcription
• Storage: Store at -20 centigrade
• Concentration: 20–60 U/µL
• Publications:
  Characterization of Escherichia coli RNase H Discrimination of DNA Phosphorothioate Stereoisomers (2021)

Citation

RNase H sequence preferences influence antisense oligonucleotide efficiency

Journal: Nucleic Acids Research    PubMed ID: 29126318    Data: 2017/12/15

Authors: ?ukasz J. Kie?piński, Peter H. Hagedorn, Jeppe Vinther

Article Snippet:Hydrolysis reactions with recombinant human RNase H1 (Creative BioMart cat. RNASEH1-433H) were performed at 37°C in a buffer RNH1 (20 mM Tris–HCl pH 7.5, 20 mM KCl, 20 mM 2-mercaptoethanol, 2 mM MgCl 2 and 0.1 mM EDTA (modified from Lima et al. , 2001 ( )).. Preheated solution containing 0.3 pmol of prepared duplex in 16.5 μl was mixed with preheated 16.5 μl of 0.5 mU/μl RNase H1 dilution to initiate the hydrolysis reaction to give a final enzyme concentration of 0.25 mU/μl.Preheated solution containing 0.3 pmol of prepared duplex in 16.5 μl was mixed with preheated 16.5 μl of 0.5 mU/μl RNase H1 dilution to initiate the hydrolysis reaction to give a final enzyme concentration of 0.25 mU/μl.

Overview and validation of the H-SPA method. ( A ) Sequences of the cleavable randomized gaps used in this study. ( B ) Schematic representation of the experimental strategy. ( C ) Denaturing gel electrophoresis showing the limited digestion of double stranded substrates. The uncleaved duplexes are indicated with the asterisk and the cleaved fragment with the tilde. The reaction conditions are indicated: w: 37°C; c: 16°C and EDTA: 0.1 mM. ( D ) Correlation of heptamer counts for the construct R4b (positions 9–15) between two untreated controls (top left), two human RNase H1 treated samples (top right) and a control and a treated sample (bottom).

Sequence preferences of Escherichia coli, Homo sapiens and HIV-1 RNase H ( A ) The heatmaps display the changes in nucleotide composition at different positions for the R7 construct (left) and the R4b construct (right) after cleavage with the three different RNase H enzymes. The intensity of the red and blue color indicates the k rel of having given nucleotide at a given position fixed relative to the average hydrolysis rate of the randomized pool. The barplots below the heatmaps show the overall information content at each position and the sequence logos are based on the 1% most downregulated pentamers. Note that only the randomized parts of the probed duplexes is displayed. ( B ) Cleavage of sequences predicted to be preferred (‘P’), avoided (‘A’) and neutral (‘N’) with respect to cleavage with human RNase H1 compared to the cleavage of a reference substrate. With respect to the reference substrate, the k rel of the preferred substrate is 3.7, of the avoided is 0.26 and of the neutral it is 1.4. ( C ) The design of the dumbbell substrate mimics. The gray box indicates the region having either the preferred (‘P’) or avoided (‘A’) sequence. ( D ) The cleavage of a reference substrate in the presence of increasing concentrations of a preferred or avoided dumbbell substrate mimic.

RNase H Sequence Preferences correlate with gapmer efficiency. ( A ) Correlation between the log 2 fold changes of different hexamers observed for the R4b construct in the experiment and the corresponding log 2 fold changes as predicted by a single nucleotide model prepared from the data obtained in the R4a experiment. ( B ) As in (A), but with prediction with a dinucleotide model. ( C ) Prediction of RNase H1 mediated downregulation of the different 11-mers present in RNA sequence used for the RNase H RNA–DNA heteroduplex crystal structure [PDB: 2QK9]. Each bar corresponds to the cleavage site of a potential binding mode of RNase H1. The filled bar corresponds to the RNase H1 binding mode observed in the crystal structure and is also indicated in the drawing below the plot. ( D ) Correlation between the change of target RNA level for MAPT after treatment with 1518 different gapmers and the corresponding downregulation predicted by the dinucleotide model for the different binding modes of RNase H1 on each gapmer target duplex. The error bars show the 99% confidence intervals. The drawing below the plot indicates the RNase H1 binding mode associated with the best-observed correlation. ( E ) The same analysis as in (D), but with 1581 different gapmers targeted against ANGPTL3 .