Single-turnover kinetic analysis of non-LTR retrotransposition defines the mechanism and rate constants governing each step

Abstract

Site specific retrotransposon-mediated gene therapy has the potential to revolutionize medicine by allowing insertion of large gene cargos. Despite decades of effort, the reaction sequence remains to be fully elucidated limiting the ability to engineer improved activity for gene insertion. Here we provide a kinetic/mechanistic framework for R2 non-LTR retrotransposition. Single turnover measurements and global data fitting defined the rate constants for each step in the pathway involving 1st-strand DNA cleavage to provide a DNA primer, reverse transcription to copy the RNA, 2nd-strand DNA cleavage to provide the second primer, and 2nd-strand synthesis to make duplex cDNA. Sequence analysis of the cDNA confirms accurate replication of the 1400 nt RNA used in this study. This represents the only complete analysis of the reaction sequence and first observation of 2nd-strand synthesis in vitro. We provide a kinetic framework to understand non-LTR retrotransposition, which provide a basis to engineer improved activity.