Less isn't always more.

Abstract

A favorite experiment of RNA scientists is the “cutdown” or “boundary” experiment where an RNA of interest is truncated from its 5′ or 3′ end and the function of the smaller molecules evaluated. Such experiments often reveal that only a small part of a large RNA molecule is needed for the function of interest. In addition, by reducing the number of alternative conformational states that can plague RNA preparations, truncated RNAs that emerge from boundary experiments are often more active than the original molecule. A common complication in the interpretation of boundary experiments arises from the general way in which RNAs fold into their active structures. As we all know, RNA secondary structure elements often form between two or more parts of the molecule that are quite distant on the primary sequence. A boundary experiment performed on an RNA containing a critical long-range secondary structure element will identify as essential all of the RNA between the 3′-most and 5′-most parts of the element, even if much of the internal sequence is not important for function. This complication can be resolved by comparing several active sequences, deducing a consensus secondary structure, and preparing an appropriate internal deletion. This classic combination of cutdowns and internal deletions has been used to define the minimal RNA binding sequences of many different proteins and small molecules, establish RNA regulatory regions and define catalytic RNA motifs.