RNase A

Ribonuclease A (EC 3.1.27.5) cleaves RNA 3′ to pyrimidines and actively cleaves RNA at every pyrimidine residue. Ribonuclease A catalyzes the hydrolysis of single stranded RNA in the absence of metal ions or cofactors.

A major application of RNase A is the removal of RNA from plasmid DNA preparations.

Preparation of RNase A stock solution
1. Use 10 mM sodium acetate (pH 5.2) to prepare 10 mg/mL RNase A stock solution;
2. Heat at 100 °C for 15 min;
3. Cool to room temperature, add 1/10 (v/v) of 1 M Tris-HCl (pH 7.4), adjust the pH to 7.4;
4. The solution can be stored at 20 °C for 2 years.
Note: When the RNase A solution is boiled under neutral conditions, RNase precipitate will form; if it is boiled at a lower pH, any precipitation may be caused by the presence of protein impurities. At this time, the precipitate can be removed by high-speed centrifugation (13 000 rpm).

[1]. Thakur P, et al. Locating chemical modifications in RNA sequences through ribonucleases and LC-MS based analysis. Methods Enzymol. 2021;658:1-24. 

[2]. Brown D, Pasloske BL. Ribonuclease-resistant RNA controls and standards. Methods Enzymol. 2001;341:648-54. 

[3]. Wallace JA, Shen JK. Predicting pKa values with continuous constant pH molecular dynamics. Methods Enzymol. 2009;466:455-75.