Avoiding Misreading during Genetic Reprogramming in mRNA Display

mRNA display is a powerful and increasingly accessible peptide discovery technology. It takes advantage of a reconstituted in vitro transcription and translation system to generate highly diverse affinity screening libraries. However, this process relies on the faithful translation of genetically encoded peptides, a conversion that is imperfect. Errors in translational decoding of mRNA can occur, decoupling the produced library from its genetic code. Because mRNA display affinity selections are analyzed with sequencing of the encoding DNA, rather than direct detection of the peptides, misreading silently reduces library diversity and complicates analysis. Herein, the presence of significant translational misreading during the production of mRNA display libraries is confirmed, best practices for genetic reprogramming are developed, and those rules are deployed to minimize the disconnect between genotype and phenotype in peptide affinity selections.