As noted in our previous post, RNases are the number one enemy of RNA stability. So how do you keep your RNA intact when these ubiquitous molecules infiltrate your precious sample?
Enter the Ribonuclease inhibitors (RI). These proteins are offered commercially under names like RNaseOUT and RNasin. They originate from different animal sources (mouse, pig, and human are among the most common) and are sold as recombinant proteins. They are fairly large in size and structure but with a fairly average molecular weight (around 50kDa). This is much larger than that of the RNAses to which they bind (e.g., RNase A is only 12.6 kDa). They have a unique arrangement of a-helix / b-sheet repeating structures, as shown in the figure below left. Shown at right is a picture of the inhibitor : RNase A complex.
Ribonuclease Inhibitors function by binding RNases almost irreversibly. The affinity of their bond is among the highest for any protein-protein interaction with dissociation constants in the femtomolar range, comparable to the biotin–avidin protein interaction used as the basis for so many immunological and molecular diagnostic assays. This effectively prevents RNases from being available to degrade your RNA sample. As little as a microliter of a commercially available RI prep in an RNA sample or RT cocktail is all that is required. RNA can even be eluted from extraction columns into a solution containing RI to protect the RNA as soon as it is isolated (but more about that in a later post).
Ironically, however, use of RIs can be a double-edged sword. Under certain conditions addition of RI into your sample can be worse than not adding it at all. Click here to find out why in our next segment.
- Protein DataBank, PORCINE RIBONUCLEASE INHIBITOR http://www.rcsb.org/structure/2BNH
- Protein DataBank, RIBONUCLEASE INHIBITOR COMPLEXED WITH RIBONUCLEASE A, http://www.rcsb.org/structure/1DFJ