Our long-term goal is to achieve DNA-catalyzed covalent modification of large protein substrates. We previously reported that DNA catalysts (deoxyribozymes), identified by in vitro selection using tethered peptide substrates, can also modify the side chains of free peptides, although with rather poor efficiency. Here a new strategy was designed to select directly for free peptide reactivity. We used Cu(I)-catalyzed azide-alkyne cycloaddition with a 3'-alkyne-modified oligonucleotide as a capture step to separate deoxyribozyme-peptide conjugates that bear an azido group at the N-terminus of the peptide. This capture reaction provides a substantial difference in molecular weight and enables PAGE separation of the catalytically active DNA sequences. Via this selection strategy, several new deoxyribozymes were identified that catalyze the reaction between the side chain of a tyrosine or serine residue and 5'-triphosphorylated RNA or 5'-phosphorimidazolated DNA. Our results demonstrate the feasibility of direct selections with free peptide substrates and provide insight into achieving DNA-catalyzed covalent modification of large proteins.

DNA-catalyzed covalent modification of amino acid side chains in free peptide substrates

246th National Meeting (2013)

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