Chemical Stability of mRNA/cDNA Complexes: Defining the Limits of mRNA Display

Abstract Messenger RNA (mRNA) display is being increasingly adopted for peptide drug candidate discovery. While many conditions have been reported for the affinity enrichment step and in some cases for peptide modification, there is still limited understanding about the versatility of peptide–puromycin–mRNA/cDNA (complementary DNA) complexes. This work explores the chemical stability of mRNA/cDNA hybrid complexes under a range of different fundamental chemical conditions as well as with peptide modification conditions reported in an mRNA display setting. We further compare the stability of full complexes originating from two different mRNA display systems (RaPID and cDNA‐TRAP). Overall, these complexes were found to be stable under a broad range of conditions, with some edge conditions benefitting from encoding directly in cDNA rather than mRNA. This should allow for more and broader exploitation of late‐stage peptide modification chemistry in mRNA display, with confidence regarding the stability of encoding, and potentially better hit‐finding campaigns as a result.