Reactions of phosphoryl donor substrates (5′-triphosphorylated RNA or GTP, where X = RNA or G). (A) Reaction of a nucleophile at the α-phosphorus, (leaving group = pyrophosphate). In RNA ligation (top), the nucleophile is an RNA hydroxyl group (could also be an internal 2′-OH group). In nucleopeptide formation as illustrated with tyrosine (bottom), the nucleophile is an amino acid hydroxyl group. (B) Reaction of a tyrosine nucleophile at the γ-phosphorus, phosphorylating the tyrosine (leaving group = oligonucleotide 5′-diphosphate or GDP).

Identifying DNA catalysts with Tyr kinase activity. (A) Key in vitro selection step in which DNA catalyst sequences transfer the γ-phosphoryl group from pppRNA to the tyrosine hydroxyl group of a tethered peptide. Analogously (not depicted), free GTP can be used as the phosphoryl donor, in which case the remainder of the RNA oligonucleotide is absent. See for structure and synthesis of the peptide substrate and for nucleotide details. (B) Use of 8VP1 as a “capture deoxyribozyme” that selectively attaches an RNA strand to Tyr^P (phosphorylated) but not Tyr^OH (unphosphorylated), thereby enabling PAGE-shift separation of DNA catalyst sequences that phosphorylate Tyr.

Characterization of individual tyrosine kinase deoxyribozymes that use pppRNA as the phosphoryl donor. All assays were performed in trans, i.e., without the dispensable loop of . (A) Kinetic analysis. The PAGE image shows three representative time points (t = 30 s, 5 h, and 24 h) for each of six new DNA catalysts. Y^OH = substrate; Y^P = product. The kinetic plots are for the same six deoxyribozymes. Incubation conditions: 70 mM HEPES, pH 7.5, 0.5 mM ZnCl₂, 20 mM MnCl₂, 40 mM MgCl₂, and 150 mM NaCl at 37 °C. Kinetic plots for all 17 new deoxyribozymes are in . (B) Metal dependence. Each deoxyribozyme was assayed in the presence of the indicated combinations of 0.5 mM Zn²⁺, 20 mM Mn²⁺, and 40 mM Mg²⁺. Shown are time points at t = 24 h; full kinetic plots along with data for the other eleven deoxyribozymes are in . The combination Mn/Mg had no activity in all cases (<0.5%; data not shown). Reproducibility of such measurements is conservatively estimated as ±10% yield.

Dependence of the tyrosine kinase deoxyribozymes on peptide sequence and length. All data were acquired using the incubation conditions of (A) Peptide sequence dependence. The substrate included the indicated peptide on a HEG tether (t = 24 h). Two behaviors were observed, represented by 6CF134 and 7CH102; data for the other deoxyribozymes is in . 6CF134 was tolerant of replacing either of the flanking Ala (A) with any of Phe (F), Glu (E), or Lys (K). 7CH102 was tolerant of all of these substitutions except for Glu to the N-terminal side of the Tyr. (B) Peptide length dependence. The substrate included the indicated peptide on a HEG tether, or the substrate provided only a DNA 3′-hydroxyl group for phosphorylation (t = 12 h). Kinetic plots are in .

Activities of 8EA101 and 16EC103, the N₃₀ and N₅₀ deoxyribozymes that use free GTP as the phosphoryl donor for Tyr phosphorylation. Kinetic analysis as in (+ 100 μM GTP). k_obs values (n = 3): 8EA101 0.23 ± 0.01 h⁻¹; 16EC103 0.23 ± 0.04 h⁻¹. See for apparent K_m analysis, which used initial-rate kinetics (0 to 2 h).