We report DNA catalysts (deoxyribozymes) that join tyrosine-containing peptides to RNA and DNA in one step and without requiring protecting groups on either peptide or nucleic acid. Our previous efforts towards this goal required tethering the peptide to a DNA anchor oligonucleotide. Here, we establish direct in vitro selection for deoxyribozymes that use untethered, free peptide substrates. This approach enables imposition of selection pressure via reduced peptide concentration and leads to preparatively useful lower apparent Km values of ∼100 µM peptide. Use of phosphorimidazolide (Imp) rather than triphosphate as the electrophile enables reactivity of either terminus (5′ or 3′) of both RNA and DNA. Our findings establish a generalizable means of joining unprotected peptide to nucleic acid in one step using DNA catalysts identified by in vitro selection.
We evaluate the ability of hexahistidine (His6) tags on peptide and protein substrates to recruit deoxyribozymes for modifying those substrates. For two different deoxyribozymes, one that creates tyrosine-RNA nucleopeptides and another that phosphorylates tyrosine side chains, we find substantial improvements in yield, kobs, and Km for peptide substrates due to recruiting by His6/Cu2+. However, the recruiting benefits of the histidine tag are not observed for larger protein substrates, likely because the tested deoxyribozymes either cannot access the target peptide segments or cannot function when these segments are presented in a structured protein context.
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