A: RNase R is a highly processive 3′ to 5′ exoribonuclease that degrades most linear RNAs while not degrading circular and lariat RNAs.
A: Yes, this reaction can be scaled up and optimized for your custom needs.
A: Yes, the final reaction product will be enriched for circular RNAs and highly structured RNAs.
A: RNase R displays reduced activity on highly structured RNAs. This class of RNAs includes tRNAs, rRNAs, small nuclear RNAs (snRNAs) and histone mRNAs among others.
A: Yes, RNase R requires very low magnesium concentrations for proper activity. Removal of prior reaction or preparation components is crucial. Do not resuspend your input RNA in an EDTA-containing buffer such as T10E1 Buffer.
A: Yes, the enzyme can be heat inactivated by incubation at 70°C for 10 mins. However, it is preferable to inactivate RNase R by the addition of EDTA to the reaction or proceeding directly to your preferred method of RNA cleanup due to integrity concerns for RNA subjected to elevated temperatures in the presence of magnesium ions.
A: Yes. RNase R specifically degrades linear RNA while leaving circular RNA intact due to its covalently closed loop structure, which makes it resistant to RNase R degradation. RNase R is used as a crucial component in in vitro circRNA preparation cleanup and enrichment with both RNA Ligase-based or self-splicing permutated intron–exon sequence (PIE method)-based methodologies.
A: Because poly(rA) is the preferred substrate for RNase R, the traditionally-reported RNase R units based on poly(A) digestion quantities do not accurately reflect the digestion rate on linear RNAs comprised of mixed nucleosides. Many commercially-available sources of RNase R provide the same mass concentration of the enzyme even when reporting poly(A) digestion units. However, different RNAs are digested at different rates based on RNA 3′ end availability and secondary structure content of the substrate RNA. Amounts of RNase R required for a complete digestion of some RNAs may need to be optimized if the standard reaction conditions do not result in complete digestion of the linear input RNA.
