Optimal random libraries for the isolation of catalytic RNA.
The relationship between ribozyme size and catalytic activity is of fundamental importance for RNA catalysis and molecular evolution in the RNA world. We have performed a series of competitive in vitro selection experiments to probe the relationship using RNA libraries containing size-heterogeneous random regions. Our experiments have established an inverse correlation between RNA replication efficiency (the combined efficiency of PCR amplification, transcription, and reverse transcription) and RNA size. A number of ribozyme sequences have been isolated from different RNA size groups under competitive selection conditions. Comprehensive kinetic analysis on isolated ribozymes has revealed that large ribozymes do not confer a significant catalytic superiority over smaller ones under most selection conditions, and actually impose two significant problems of replication inefficiency and RNA misfolding into inactive conformations. The fraction of a misfolded ribozyme population is defined as alpha. Large ribozymes tend to possess high alpha values, which may significantly reduce ribozyme performance. Our results suggest that a random region of around 60 nucleotides represents the optimal balance between ribozyme catalytic activity, RNA misfolding (alpha), and replication efficiency, and may therefore constitute the most advantageous RNA libraries for successful isolation of functional RNA sequences.