Accessing rare activities from random RNA sequences: the importance of the length of molecules in the starting pool.

Abstract

In the past few years numerous binding and catalytic motifs have been isolated from pools of random nucleic acid sequences. To extend the utility of this approach it is important to learn how to design random-sequence pools that provide maximal access to rare activities. In an effort to better define the relative merits of longer and shorter pools (i.e. pools with longer or shorter random-sequence segments), we have examined the inhibitory effect of excess arbitrary sequence on ribozyme activity and have evaluated whether this inhibition overshadows the calculated advantage of longer pools. The calculated advantage of longer sequences was highly dependent on the size and complexity of the desired motif. Small, simple motifs were not much more abundant in longer molecules. In contrast, larger motifs, particularly the most complex (highly modular) motifs, were much more likely to be present in longer molecules. The experimentally determined inhibition of activity by excess sequence was moderate, with bulk effects among four libraries ranging from no effect to 18-fold inhibition. The median effect among 60 clones was fivefold inhibition. For accessing simple motifs (e.g. motifs at least as small and simple as the hammerhead ribozyme motif), longer pools have little if any advantage. For more complex motifs, the inhibitory effect of excess sequence does not approach the calculated advantage of pools of longer molecules. Thus, when seeking to access rare activities, the length of typical random-sequence pools (< or = 70 random positions) is shorter than optimal. As this conclusion holds over a range of incubation conditions, it may also be relevant when considering the emergence of new functional motifs during early evolution.