DNA Catalysts with Tyrosine Kinase Activity

Abstract

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 Zn2+, and most additionally require Mn2+. 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 ca. 20 μM. These findings establish that DNA has the fundamental catalytic ability to phosphorylate the tyrosine side chain of a peptide substrate.

The Expanding View of RNA and DNA Function

“…DNA has its place as well, now with more than 20 examples of DNA enzymes that catalyze diverse chemical transformations. These include the phosphorylation (Li and Breaker, 1999), ligation (Sreedhara et al, 2004), deglycosylation (Sheppard et al, 2000), and hydrolytic cleavage (Chandra et al, 2009) of DNA substrates, as well as reactions involving non-nucleic-acid substrates, such as porphyrin metallation (Li and Sen, 1996), Diels-Alder cycloaddition (Chandra and Silverman, 2008), and tyrosine phosphorylation (Walsh et al, 2013). …”

Assessing histidine tags for recruiting deoxyribozymes to catalyze peptide and protein modification reactions

“…4A). 53 However, 6CF134 does not catalyze detectable phosphorylation of a discrete, untethered peptide at any peptide concentration tested. Here we sought to recruit 6CF134 to an untethered 24-mer peptide substrate via a His 6 tag.…”

“…From these observations, we conclude that 6CF134 is inherently capable of modifying an untethered peptide, despite its strict tether requirement found in our original report. 53 The His 6 tag and tris(NTA) interaction functionally replaces the covalent tether and enables 6CF134 catalysis with the formally untethered peptide substrate.…”

“…(A) Originally reported arrangement of 6CF134 and disulfide-tethered peptide substrate. No phosphorylation is observed without the tether; 53 ~50% phosphorylation yield is observed with the tether. Conditions: 20 nM 5′- 32 P-radiolabeled DNA-anchored CAAYAA peptide, 0.5 μM 6CF134 deoxyribozyme, 5 μM 5′-pppRNA, 70 mM HEPES, pH 7.5, 40 mM MgCl 2 , 20 mM MnCl 2 , 0.5 mM ZnCl 2 , and 150 mM NaCl at room temperature ( t = 30 s, 30 min, 2 h, 5 h, and 16 h; Y = substrate, Y P = product).…”

A modular tyrosine kinase deoxyribozyme with discrete aptamer and catalyst domains

“…10 We recently reported the first tyrosine kinase deoxyribozymes, which phosphorylate the tyrosine side chain of a peptide substrate and were identified from N 30 –N 50 random regions that lack any predefined aptamer domain. 11 Our current experiments were performed analogously, using the previously identified 8VP1 deoxyribozyme 12 as the “capture” catalyst to enable the selection process (Fig. 2).…”