Optimal Random Libraries for the Isolation of Catalytic RNA

Abstract

The relationship between ribozyme size and catalytic activity is of fundamental importance forRNA catalysis and molecular evolution in the RNA world. We have performed a series ofcompetitive in vitro selection experiments to probe the relationship using RNA librariescontaining size-heterogeneous random regions. Our experiments have established an inversecorrelation between RNA replication efficiency (the combined efficiency of PCR amplification,transcription, and reverse transcription) and RNA size. A number of ribozyme sequences havebeen isolated from different RNA size groups under competitive selection conditions.Comprehensive kinetic analysis on isolated ribozymes has revealed that large ribozymes do notconfer a significant catalytic superiority over smaller ones under most selection conditions, andactually impose two significant problems of replication inefficiency and RNA misfolding intoinactive conformations. The fraction of a misfolded ribozyme population is defined as alpha.Large ribozymes tend to possess high alpha values, which may significantly reduce ribozymeperformance. Our results suggest that a random region of around 60 nucleotides represents theoptimal balance between ribozyme catalytic activity, RNA misfolding (alpha), and replicationefficiency, and may therefore constitute the most advantageous RNA libraries for successfulisolation of functional RNA sequences.