A densely modified M(2+)-independent DNAzyme that cleaves RNA efficiently with multiple catalytic turnover

Sequence-specific cleavage of RNA targets in the absence of a divalent metal cation (M(2+)) 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(im)TP), 5-guanidinoallyl-deoxyuridine (dU(ga)TP), and 5-aminoallyl-deoxycytidine (dC(aa)TP) along with dGTP. These modified dNTPs provide key functionalities reminiscent of the active sites of ribonucleases, notably RNase A. Of several such M(2+)-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 Mg(2+) 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 ∼10(6) M(–1) min(–1). Therefore, Dz7-38-32 represents a promising candidate towards the development of therapeutically efficient DNAzymes.