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T4 polynucleotide kinase is widely used for 5'-phosphorylation of DNA and RNA oligonucleotide termini, but no natural protein enzyme is capable of 3'-phosphorylation. Here, we report the in vitro selection of deoxyribozymes (DNA enzymes) capable of DNA oligonucleotide 3'-phosphorylation, using a 5'-triphosphorylated RNA transcript (pppRNA) as the phosphoryl donor. The basis of selection was the capture, during each selection round, of the 3'-phosphorylated DNA substrate terminus by 2-methylimidazole activation of the 3'-phosphate (forming 3'-MeImp) and subsequent splint ligation with a 5'-amino DNA oligonucleotide. Competing and precedented DNA-catalyzed reactions were DNA phosphodiester hydrolysis or deglycosylation, each also leading to a 3'-phosphate but at a different nucleotide position within the DNA substrate. One oligonucleotide 3'-kinase deoxyribozyme, obtained from an N40 random pool and named 3'Kin1, can 3'-phosphorylate nearly any DNA oligonucleotide substrate for which the 3'-terminus has the sequence motif 5'-NKR-3', where N denotes any oligonucleotide sequence, K = T or G, and R = A or G. These results establish the viabilty of in vitro selection for identifying DNA enzymes that 3'-phosphorylate DNA oligonucleotides.

Deoxyribozymes (DNA enzymes) have been developed for a growing variety of chemical reactions, including with peptide substrates. We recently described the first tyrosine kinase deoxyribozymes, which lacked the ability to discriminate among peptide substrates on the basis of the amino acids surrounding the tyrosine residue. Those deoxyribozymes were identified by in vitro selection using a DNA-anchored peptide substrate in which the residues neighboring tyrosine were all alanine. Here, we performed in vitro selection for tyrosine kinase activity using three peptide substrates in which the neighboring residues included a variety of side chains. For one of these three peptides, we found numerous deoxyribozymes that discriminate strongly in favor of phosphorylating tyrosine when the surrounding residues are specifically those used in the selection process. Three different short peptide sequence motifs of 2-4 amino acids were required for catalysis by three unique deoxyribozymes. For a second peptide substrate, the selection process led to one deoxyribozyme which exhibits partial discrimination among peptide sequences. These findings establish the feasibility of identifying DNA enzymes that catalyze sequence-selective tyrosine phosphorylation, which suggests the downstream practical utility of such deoxyribozymes. More broadly, this outcome reinforces the conclusion that nucleic acid catalysts can discriminate among peptide substrates in the context of biochemically relevant reactions.

We show that DNA catalysts (deoxyribozymes, DNA enzymes) can phosphorylate tyrosine residues of peptides. Using in vitro selection, we identified deoxyribozymes that transfer the γ-phosphoryl group from a 5'-triphosphorylated donor (a pppRNA oligonucleotide or GTP) to the tyrosine hydroxyl acceptor of a tethered hexapeptide. Tyrosine kinase deoxyribozymes that use pppRNA were identified from each of N30, N40, and N50 random-sequence pools. Each deoxyribozyme requires Zn(2+), and most additionally require Mn(2+). The deoxyribozymes have little or no selectivity for the amino acid identities near the tyrosine, but they are highly selective for phosphorylating tyrosine rather than serine. Analogous GTP-dependent DNA catalysts were identified and found to have apparent Km(GTP) as low as ∼20 μM. These findings establish that DNA has the fundamental catalytic ability to phosphorylate the tyrosine side chain of a peptide substrate.