- Ribonuclease A (RNase A) is a highly stable and well-characterized pancreatic endoribonuclease that specifically cleaves single-stranded RNA at the 3′-side of pyrimidine residues—cytosine (C) and uracil (U). It catalyzes the hydrolysis of the phosphodiester bonds, producing RNA fragments with 3′-phosphates and 5′-hydroxyl termini. RNase A is one of the most studied enzymes in biochemistry and molecular biology due to its robust activity and resistance to denaturation.
- RNase A is particularly notable for its high thermal stability, lack of dependence on cofactors, and resistance to proteolysis, making it ideal for laboratory use. It is naturally secreted by the pancreas of mammals, including bovines (the common source for commercial RNase A), where it functions in digesting RNA in the small intestine.
- In molecular biology, RNase A is commonly used for the removal of RNA contaminants during DNA purification protocols, particularly in plasmid DNA isolation and genomic DNA extraction. It is also used in nuclease protection assays, ribosomal RNA analysis, and to selectively degrade RNA in hybridization studies. RNase A is not active on double-stranded RNA or RNA-DNA hybrids under standard conditions, making it selective for single-stranded RNA.
- Because of its potent and irreversible activity, RNase A contamination in RNA work can be highly problematic. It is extremely difficult to inactivate, as it refolds spontaneously after denaturation, even with heat. Therefore, laboratories working with RNA must use RNase-free reagents, dedicated equipment, and RNase inhibitors to avoid degradation of RNA samples.
- In summary, RNase A is a robust and specific ribonuclease essential for routine nucleic acid work, particularly for eliminating unwanted RNA and studying RNA structure and stability, while also representing a key cautionary challenge in RNA-based applications due to its resilience.
