Improved RNA Diels-Alderase by Mutation

Abstract

DA-22 is a well-studied Diels-Alderase with good catalysis. DA-2 and DA-22 share a highly conserved sequence. The RNA selection would have converged on a local catalytic maximum and that further mutation might lead to better catalysis. My research was to mutagenize DA-2 by mutagenic PCR to diversify the number of sequences and to determine if better Diels-Alderases could be found. RNA Diels-Alderase DA-2 was selected through in vitro evolution cycles. The strategy is to transcribe mutagenized DNA to RNA with T7 RNA polymerase, conjugate RNA with a diene via a PEG linker in a DNA bridge-mediated ligation, incubate RNA-diene conjugate with dienophile, then use streptavidin to separate the reacted and unreacted RNA. After the separation, RNA can be reverse transcribed to DNA and then the cycles can be repeated.