CONTINUOUS IN-VITRO EVOLUTION OF BACTERIOPHAGE RNA-POLYMERASE PROMOTERS

Rapid in vitro evolution of bacteriophage T7, T3, and SP6 RNA polymera se promoters was achieved by a method that allows continuous enrichmen t of DNAs that contain functional promoter elements. This method explo its the ability of a special class of nucleic acid molecules to replic ate continuously in the presence of both a reverse transcriptase and a DNA-dependent RNA polymerase. Replication involves the synthesis of b oth RNA and cDNA intermediates. The cDNA strand contains an embedded p romoter sequence, which becomes converted to a functional double-stran ded promoter element, leading to the production of RNA transcripts. Sy nthetic cDNAs, including those that contain randomized promoter sequen ces, can be used to initiate the amplification cycle. However, only th ose cDNAs that contain functional promoter sequences are able to produ ce RNA transcripts. Furthermore, each RNA transcript encodes the RNA p olymerase promoter sequence that was responsible for initiation of its own transcription. Thus, the population of amplifying molecules quick ly becomes enriched for those templates that encode functional promote rs. Optimal promoter sequences for phage T7, T3, and SP6 RNA polymeras e were identified after a 2-h amplification reaction, initiated in eac h case with a pool of synthetic cDNAs encoding greater than 10(10) pro moter sequence variants.