A general purpose RNA-cleaving DNA enzyme

Abstract

An in vitro selection procedure was used to develop a DNA enzyme that can be made to cleave almost any targeted RNA substrate under simulated physiological conditions. The enzyme is comprised of a catalytic domain of 15 deoxynucleotides, flanked by two substrate-recognition domains of seven to eight deoxynucleotides each. The RNA substrate is bound through Watson-Crick base pairing and is cleaved at a particular phosphodiester located between an unpaired purine and a paired pyrimidine residue. Despite its small size, the DNA enzyme has a catalytic efficiency (kcat/Km) of approximately 10(9) M-1.min-1 under multiple turnover conditions, exceeding that of any other known nucleic acid enzyme. Its activity is dependent on the presence of Mg2+ ion. By changing the sequence of the substrate-recognition domains, the DNA enzyme can be made to target different RNA substrates. In this study, for example, it was directed to cleave synthetic RNAs corresponding to the start codon region of HIV-1 gag/pol, env, vpr, tat, and nef mRNAs.

Figure 1

In vitro selection of RNA-cleaving DNAs. A library of 10¹⁴ chimeric molecules was constructed (Inset), each containing a 5′ biotin (encircled B), 12 target ribonucleotides (sequence shown), and 50 random sequence deoxynucleotides (N₅₀). Ten rounds of selective amplification yielded molecules that underwent phosphoester cleavage after either the first (dark arrow) or seventh (light arrow) target ribonucleotide. Self-cleavage activity was measured for each successive population. Dark and light bars correspond to cleavage at positions indicated by the dark and light arrows, respectively. Reaction conditions: 1 nM 5′-³²P-labeled precursor, 10 mM MgCl₂, and 1 M NaCl (pH 7.5) at 37°C for 2 h.

Figure 2

Composition of the 8-17 and 10-23 catalytic motifs. The DNA enzyme (bottom strand) binds the RNA substrate (top strand) through Watson–Crick pairing. Cleavage occurs at the position indicated by the arrow. R = A or G; Y = U or C.

Figure 3

Catalytic activity of the 10-23 DNA enzyme under multiple turnover conditions. (A) Initial velocities were measured over the first 10% of the reaction, using a fixed concentration of enzyme (0.004 nM) and varying concentrations of substrate (0.02–4 nM). The 17-mer RNA substrate, corresponding to the start codon region of HIV-1 gag/pol mRNA, was prepared by in vitro transcription. Reaction conditions: 2 mM MgCl₂ and 150 mM NaCl (pH 7.5) at 37°C. Data from two independent experiments are shown and were fit to the Michaelis–Menten equation: v = k_cat [E]/(K_m + [S]). (B) Catalytic rates were determined in the presence of saturating substrate (100 nM) and varying concentrations of MgCl₂ (2–300 mM). Reaction conditions were otherwise as above. Data from two independent experiments are shown and were fit to the Michaelis–Menten equation to obtain the apparent K_m for Mg²⁺.

Figure 4

DNA-catalyzed cleavage of RNA under laboratory conditions. 5′-³²P-labeled RNA substrate (S), having the sequence 5′-GGAGAGAGA⋅UGGGUGCG-3′, was cleaved by the corresponding 10-23 DNA enzyme to generate a single-labeled product (P). Reaction conditions: 1 μM substrate, 10 nM enzyme, and 50 mM MgCl₂ (pH 8.0) at 37°C; sampled at 0, 1, 2, 5, 10, and 20 min. Reaction products were separated by electrophoresis in a denaturing 20% polyacrylamide gel, an autoradiogram of which is shown. The ladder at the right was produced by partial alkaline hydrolysis of the substrate; at short oligonucleotide lengths, products terminated by a 2′ or 3′ phosphate have slightly faster mobility compared with those with a 2′,3′-cyclic phosphate.