DNA-catalyzed glycosylation using aryl glycoside donors

Abstract

We report the identification by in vitro selection of Zn(2+)/Mn(2+)-dependent deoxyribozymes that glycosylate the 3'-OH of a DNA oligonucleotide. Both β and α anomers of aryl glycosides can be used as the glycosyl donors. Individual deoxyribozymes are each specific for a particular donor anomer.

Fig. 1

Structures of glycosyl donors. Each is an aryl glycoside of D-glucose (1, β-D-Glc; 2, α-D-Glc). Compounds 1a, 1b, 1c, and 2b were evaluated in this study.

Fig. 2

Key step of in vitro selection, in which DNA catalyzes glycosylation of a 3′-OH using an aryl glycoside donor, illustrated with a β-D-Glc donor. Glycosylation leads to a PAGE shift; the catalytically active DNA sequences are separated and PCR-amplified to enable the next selection round. The product is depicted here as the α-anomer, but the configurations for individual deoxyribozyme products in this study have not been assigned experimentally. See Fig. S1 and Table S1 (ESI) for nucleotide details.

Fig. 3

Glycosylation of a 3′-OH acceptor using the 11GV112 deoxyribozyme. (A) Metal ion dependence when using the 2-chloro-4-nitrophenyl β-D-Glc glycosyl donor 1a. In the PAGE image, representative timepoints are shown at t = 0, 12, and 48 h for the indicated metal ion combinations. The metal ion combinations not shown, including Mn²⁺ or Mg²⁺ alone, had no activity. S = substrate, P = product. Incubation conditions: 70 mM HEPES, pH 7.5, 150 mM NaCl, combinations of 1 mM ZnCl₂, 20 mM MnCl₂, and 40 mM MgCl₂ as indicated, at 37 °C. The Zn²⁺ concentration was optimized; with 1a, the yield was lower at either 0.8 or 1.2 mM Zn²⁺ (data not shown). (B) Glycosyl donor dependence (in presence of all of Zn²⁺, Mn²⁺, and Mg²⁺).

Fig. 4

Glycosylation of a 3′-OH acceptor using the 16MJ132 deoxyribozyme. Shown are the metal ion dependence using the 4-nitrophenyl α-D-Glc glycosyl donor 2b, as well as assay with the 4-nitrophenyl β-D-Glc glycosyl donor 1b. Incubation conditions: 70 mM HEPES, pH 7.5, 150 mM NaCl, combinations of 0.4 mM ZnCl₂, 20 mM MnCl₂, and 40 mM MgCl₂ as indicated, at 37 °C. The Zn²⁺ concentration was optimized; with 2b, similar k_obs and yield were observed with 0.2–0.5 mM Zn²⁺, whereas the yield was lower at or above 0.6 mM Zn²⁺ (data not shown). The metal ion combinations not shown, including Mn²⁺ or Mg²⁺ alone, had no activity.