DNA-catalyzed sequence-specific hydrolysis of DNA

Abstract

Deoxyribozymes (DNA catalysts) have been reported for cleavage of RNA phosphodiester linkages, but cleaving peptide or DNA phosphodiester linkages is much more challenging. Using in vitro selection, here we identified deoxyribozymes that sequence-specifically hydrolyze DNA with multiple turnover and with a rate enhancement of 108 (possibly as high as 1014). The new DNA catalysts require both Mn2+ and Zn2+, which is noteworthy because many natural DNA nucleases are bimetallic protein enzymes.

Figure 1

In vitro selection of deoxyribozymes that cleave a substrate with both amide and DNA phosphodiester linkages. (a) Key step of the selection strategy. The loop on the right enables PAGE separation during selection of the DNA sequences that cleave the substrate, but this loop is dispensable for catalysis. (b) Chemical composition of the substrate between the two unpaired T DNA nucleotides at the ends of the binding arms. (c) Observed cleavage sites within the substrate for nine unique new deoxyribozymes. All four sites correspond to hydrolysis at DNA phosphodiester linkages. 10MD30, 36, and 41 cleave at the same site as 10MD9; 10MD4 and 13 cleave at the same site as 10MD1. Sequences of all nine deoxyribozymes are provided in Supplementary Table 1.

Figure 2

Hydrolysis of DNA phosphodiester bonds by the new deoxyribozymes. (a) Single-turnover data under standard incubation conditions (including Mn²⁺ and Zn²⁺) for each of 10MD9 (■), 10MD1 (▲), 10MD14 (▼;), and 10MD5 (●), with either the original tripeptide-containing substrate (open symbols) or the all-DNA analogue in which the tripeptide portion is replaced by deoxyadenosine (filled symbols). k_obs values are in Supplementary Table 3. The PAGE image shows cleavage of the all-DNA substrate by 10MD5. (b) Summary of DNA substrate sequence requirements for 10MD5, which efficiently cleaves an all-DNA substrate with a modest recognition site.