Background
As a major endoribonuclease, RNase A plays a key role in molecular biology. Its ability to specifically degrade RNA makes it widely used in experiments such as plasmid DNA and genomic DNA preparation, RNAse protection analysis and RNA interference. Yeasenbio's RNase A products, with their high activity, stability and wide applicability, are a reliable choice for researchers in related fields. With the continuous development of molecular biology technology, RNase A and related products will demonstrate their application value in more fields.
Picture 1. RNase A in Action: Breaking Down RNA Mysteries
Sources of RNase A
1. Animal origin
RNaseA, or ribonuclease A, is mainly derived from bovine pancreas. It is a single-chain polypeptide compound containing 4 disulfide bonds with a molecular weight of about 13.7 kDa. This enzyme has a high degree of stability and adaptability to a wide range of reaction conditions, allowing it to maintain its activity under a wide range of environmental conditions. Bovine pancrea-derived RNaseA powder does not completely remove DNase and protease, but it can be removed by heating RNaseA during the preparation of RNaseA into a solution Proteases, which can then be used to remove residual RNA from DNA samples, or nucleic acid residues from protein samples.
2. Sources of restructuring
Recombinant ribonuclease A, His-tagged (rRNase A, His-tagged) is the expression product of the bovine pancreas RNase A gene in E.coli obtained after inclusion body denaturation and multiple purification steps such as affinity chromatography and ion exchange chromatography. The purified RNaseA is free of DNase and protease contamination and is of non-animal origin. It can be used to remove residual RNA from DNA samples, or nucleic acid residues from protein samples.
RNase A: Mechanism and Reaction Conditions
(I) Substrate Specificity
RNase A specifically recognizes pyrimidine bases (cytosine and uracil) at the 3’-end positions of single-stranded RNA (ssRNA) and cleaves the phosphodiester bond between the pyrimidine nucleotide and the adjacent nucleotide.
(II) Catalytic Site Binding and Hydrolysis Reaction
Substrate Binding:
a. The catalytic residues His12 and His119 in RNase A coordinate with the phosphate group of the RNA backbone.
b. Lys41 stabilizes the transition state during hydrolysis.
Hydrolysis Mechanism:
- Step 1: His12 acts as a general base, deprotonating the 2’-OH group of the ribose.
- Step 2: The activated 2’-OH attacks the adjacent phosphorus atom, forming a pentacoordinate intermediate.
- Step 3: His119 donates a proton to the leaving group (5’-oxygen), resulting in cleavage and release of RNA fragments with 3’-phosphate termini.
Picture 2. RNase A Unveiled: Mechanism and Catalytic Insights
(III) Reaction Conditions
|
Parameterry |
Optimal Range |
Notes |
|
pH |
7.0–8.0 (neutral/weakly alkaline) |
Stable across pH 5.0–9.0; denatures reversibly at extreme pH. |
|
Temperature |
37°C |
Retains activity after heating to 95°C and cooling (renaturation). |
|
Ionic Strength |
Low [Na⁺/K⁺] (e.g., 50–150 mM) |
Enhances activity by stabilizing RNA-enzyme interactions. |
|
High [Na⁺/K⁺] (>300 mM) |
Inhibits activity due to competition for RNA binding. |
Application scenarios of RNaseA
1. Preparation of plasmid DNA and genomic DNA
During the preparation of plasmid DNA and genomic DNA, the presence of RNA may interfere with the results of subsequent experiments. Therefore, when extracting DNA, RNase A is often added to degrade the RNA molecules in the sample, resulting in a pure DNA sample. Since RNase A does not degrade DNA molecules, it ensures that the integrity and concentration of DNA molecules are not afected.
2. RNase protection assay
RNase protection assay is a hybridization technique for detecting RNA that has been developed in recent years. The basic principle is to use a single-stranded RNA probe to hybridize with the RNA sample to be tested to form RNA: RNA double-stranded molecules, because RNase A can specifically degrade unhybridized single-stranded RNA, while the double-stranded is protected from degradation, the length of the target RNA can be determined by gel electrophoresis. This method is more sensitive than the Northern hybridization method and can be quantified more accurately.
3. RNAi research
RNA interference (RNAi) is a technique that uses small RNA molecules (siRNA, miRNA, etc.) to regulate gene expression. In RNAi experiments, exogenous or endogenous RNA can interfere with experimental results. RNase A can degrade non-specific RNA and reduce background noise, thereby improving the specificity and efficiency of RNAi.
4.Protein-RNA interaction research
When studying the interaction between protein and RNA, unbound RNA needs to be removed. RNase A can specifically degrade free RNA and retain RNA bound to protein for subsequent analysis. For example, in the study of RNA-binding protein (RBP), the purity of RNA bound to protein increased by 50% after RNase A treatment, which helps to analyze the interaction mechanism of protein-RNA.
5. R&D of RNA vaccines
The preparation of RNA vaccines requires ensuring the integrity and purity of RNA. RNase A can be used to remove RNA impurities during the preparation process and improve the quality and safety of the vaccine. For example, in the development of the new crown vaccine, RNA vaccines treated with RNase A showed higher immunogenicity and lower side effects in animal experiments.
High-Quality & Diverse RNase by YeasenBio
|
Description |
Activity |
Cat. No. |
Size |
Application |
|
Ribonuclease A(RNase A),from bovine pancreas(Lyophilized powder) |
≥80 Kunitz units/mg |
10407ES60:100 mg 10407ES80:1 g 10407ES05:5 g 10407ES10:10 g |
Plasmid DNA and genomic DNA extraction, RNase protection analysis, RNA interference, etc. |
|
|
Ribonuclease A(RNase A),from bovine pancreas (Liquid form, 100 mg/mL) |
≥80 Kunitz units/mg |
10406ES03：1 mL |
||
|
Ribonuclease A(RNase A),from bovine pancreas(Liquid form, 100 mg/mL) |
≥80 Kunitz units/mg |
10405ES03：1 mL 10405ES10：10 mL |
References:
1.Raines, R. T. (1998). Ribonuclease A. Chemical Reviews, 98(3), 1045-1066.
2.Cuchillo, C. M., et al. (2011). Catalysis in RNase A. FEBS Journal, 278(17), 3166-3173.