10-23 DNAzyme has the potential to suppress gene expressions through sequence-specific mRNA cleavage. However, the dependence on exogenous delivery limits its applications. The objective of this work is to establish a replicating DNAzyme in bacteria using a single-stranded DNA vector. By cloning the 10-23 DNAzyme into the M13mp18 vector, we constructed two circular DNAzymes, C-Dz(7) and C-Dz(482), targeting the beta-lactamase mRNA. These circular DNAzymes showed in vitro catalytic efficiencies (k(cat)/K-M) of 7.82 x 10(6) and 1.36 x 10(7) M-1.min(-1), respectively. Their dependence on divalent metal ions is similar to that found with linear 10-23 DNAzyme. Importantly, the circular DNAzymes were not only capable of replicating in bacteria but also exhibited high activities in inhibiting beta-lactamase and bacterial growth. This study thus provides a novel strategy to produce replicating DNAzymes which may find widespread applications.

In view of the weakness of antibiotics and the properties of antisense drugs, we applied DNAzymes to the field of drug resistance in bacteria. Two 10-23 mono-DNAzymes (Dz(1), Dz(2)) and a di-DNAzyme (Dz(1-2)) targeted to beta-lactamase mRNA were designed to determine to what degree the growth of ampicillin-resistant bacteria (TEM-1, TEM-3) was inhibited. All three DNAzymes can play a role both in vitro and in vivo. In vitro, they exhibited high catalytic efficiency (k(cat)/K-M) of 63.5, 91.1, and 30.8 pM(-1) (.) min(-1), respectively, under multiple-turnover conditions. In vivo, after 9 hours' incubation, the degree of inhibition of Dz(1), Dz(2), and Dz(1-2) for TEM-1 bacteria was 27.2%, 39.6%, and 57.7%, respectively, and that for TEM-3 bacteria was 39.1%, 44%, and 62.6%, respectively. Dz(1-2) showed the greatest inhibiting effect, demonstrating in vivo activity may be increased by constructing multiple-target DNAzymes. The results indicated a potential possibility for DNAzymes to act as a new type of antibacterial or a tool of gene functional analysis for prokaryocytes.