A densely modified M2+-independent DNAzyme that cleaves RNA efficiently with multiple catalytic turnover

Abstract

Sequence-specific cleavage of RNA targets in the absence of a divalent metal cation (M²⁺) has been a long-standing goal in bioorganic chemistry. Herein, we report the in vitro selection of novel RNA cleaving DNAzymes that are selected using 8-histaminyl-deoxyadenosine (dA^imTP), 5-guanidinoallyl-deoxyuridine (dU^gaTP), and 5-aminoallyl-deoxycytidine (dC^aaTP) along with dGTP. These modified dNTPs provide key functionalities reminiscent of the active sites of ribonucleases, notably RNase A. Of several such M²⁺-free DNAymes, DNAzyme 7-38-32 cleaves a 19 nt all-RNA substrate with multiple-turnover, under simulated physiological conditions wherein only 0.5 mM Mg²⁺ was present, attaining values of k_cat of 1.06 min^-1 and a K_M of 1.37 μM corresponding to a catalytic efficiency of ∼10⁶ M^-1 min^-1. Therefore, Dz7-38-32 represents a promising candidate towards the development of therapeutically efficient DNAzymes.

Fig. 1. (A) Chemical structures of modified deoxynucleotides for RNase A mimic: dA^imTP 1, dU^gaTP 2, dC^aaTP 3. (B) Unimolecular construct for in vitro selection of all-RNA cleaving DNAzymes. The 17 nt all-RNA substrate is depicted in red and is taken from the HIV-LTR untranslated message; the potential cleavage was directed at either of the four unpaired RNA linkages (indicated by arrows) opposite the initial random region (N40); the two fixed sequence regions (P1 and P2) flanking the N40 random region ensuring PCR amplification are depicted in black, with the nucleosides in the substrate recognition arms (arm I and arm II) underlined.

Fig. 2. cis-Cleavage activity of Dz7-38-32 at 25 °C. (A) Individual sequences of the initially random (N40) region of two families of highly active DNAzymes (T is noted from the sequencing data however is replaced with dU^ga in the modified sequences). (B) Autoradiograph of a representative cis-cleavage reaction. (C) Biphasic kinetic plot of cis-cleaving Dz7-38-32 (R² = 0.98).

Fig. 3. trans-Cleavage activity of Dz7-38-32t. (A) Hypothetical 2D structures of Dz7-38-32t (bottom sequence: 3′–5′) in complex with the 19 nt HIV-1 LTR-promoter mRNA (top sequence in red: 5′–3′). The cleavage site is indicated by the arrow. All the three modified nucleosides (A, C, and U) are in blue. (B and C) Multiple-turnover activities at 30 °C (left panel) and 37 °C (right panel), respectively. (B) Representative autoradiographic images of trans-cleavage activity ([D7-38-32t] = 5 nM, [substrate] = 500 nM); (C) Multiple-turnover profiles generated by fitting obtained k_obs values and substrate concentrations in Michaelis–Menten equation. Data are the mean of three replicative experiments, with R² ≥ 0.98 in both cases.