RNA Hygiene (Part III) – The Mis-use of RNase Inhibitor Proteins
RNase Inhibitor proteins (RI) by their very nature have an unusual dichotomy. Although they can be quite effective in binding RNases and preventing RNA degradation, they can also be the cause of RNA degradation if they are mis-handled. Although this at first seems counter-intuitive, the explanation is very straight-forward.
Some RNases (RNase A and RNase T1, for example) are extremely heat resistant. These proteins can be heated to 100°C and still retain most or at least some of their activity upon cooling. They consist of multiple disulfide bonds which provide conformational stability and catalytic activity. It is these bonds that allow RNases to remain intact under high temperatures as well as other denaturing conditions.(1, 2)
RNase Inhibitors, however, are not as hearty. Most RIs are recommended for prolonged use below 50°C to avoid protein denaturation. Further, given the high affinity of RIs for their RNase targets, it is not difficult to imagine that some RIs are complexed with RNases even prior to addition to an RNA sample. If the RI denatures while it is complexed with a heat-stable RNase, it can release the active RNase into your sample. Paradoxically, the steps taken to ensure your RNA is protected end up being the steps that lead to its degradation!
There is some good news, however. Companies are selling thermally-resistant ribonuclease inhibitors that can withstand heating to 70˚C for up to 15 minutes. This allows protection of RNA all the way up to the reverse transcription protocol where the RNA is then used to make its stable DNA counterpart. And if protein denaturing conditions must be used on your RNA sample, there are even a few non-biological alternatives for inhibiting RNases (such as polyvinyl sulfonic acid). (3)
Use of RIs is not a ubiquitous answer to safeguarding your RNA, however. For one thing, not all RNases are bound by all RIs. For example, commercially available murine RIs are not effective against RNase 1, RNase T1, S1 Nuclease, RNase H, or RNase from Aspergillus. The use of a ribonuclease inhibitor is no replacement for good RNA handling techniques. Want to know more about manipulating your RNA safely and effectively? Look for the next part in our series!
- Klink TA, Woycechowsky KJ, Taylor KM, Raines RT. Contribution of disulfide bonds to the conformational stability and catalytic activity of ribonuclease A. Eur J Biochem. 2000 Jan;267(2):566-72.
- Pace CN, Grimsley GR, Thomson JA, Barnett BJ. Conformational stability and activity of ribonuclease T1 with zero, one, and two intact disulfide bonds. J Biol Chem. 1988 Aug 25;263(24):11820-5.
- Earl CC, Smith MT, Lease RA, Bundy BC. Polyvinylsulfonic acid: A Low-cost RNase inhibitor for enhanced RNA preservation and cell-free protein translation. Bioengineered. 2018 Jan 1;9(1):90-97. doi: 10.1080/21655979.2017.1313648. Epub 2017 Jun 29.