Maximizing Output in RNA-Programmed Peptidyl-Transfer Reactions.

A chemical reaction that is triggered by a specific RNA molecule might provide opportunities for the design of artificial feedback loops. We envision a peptidyl transfer reaction in which mRNA encoding an antiapoptotic protein would instruct the synthesis of apoptosis-inducing peptides. In this study, we used the RNA-programmed synthesis of a 16-mer peptide derived from the BH3 domain of the protein Bak, which inhibits the antiapoptotic protein Bcl-x . The reaction involves the transfer of a thioester-linked donor peptide fragment from one PNA conjugate to an acceptor peptide-PNA conjugate. We asked two key questions. What are the chemical requirements that allow RNA-templated synthesis of a 16-mer peptide to proceed at lower (nanomolar) concentrations of RNA, that is, the concentration range found in cancer cells? Will such reactions provide sufficient amounts of peptide product and sufficient affinity to interfere with the targeted protein-protein interaction? Perhaps surprisingly, the lengths of the peptides involved in peptidyl transfer chemistry have little effect on the achievable rate enhancements. However, the nature of the thioester C terminus, the distance between the targeted template annealing sites, and template affinity play important roles. The investigation revealed guidelines for the reaction design for peptidyl transfer with low amounts (1-10 nm) of RNA, yet still provide sufficient product to antagonize a protein-protein interaction.