Enhancement of deoxyribozyme activity by cationic copolymers

Abstract

Deoxyribozymes, or DNAzymes, are DNA molecules with enzymatic activity. DNAzymes with ribonuclease activity have various potential applications in biomedical and bioanalytical fields; however, most constructs have limited turnover despite optimization of reaction conditions and DNAzyme structures. A cationic comb-type copolymer accelerates DNA hybridization and strand exchange rates, and we hypothesized that the copolymer would enhance deoxyribozyme activity by promoting turnover. The copolymer did not change DNAzyme activity under single-turnover conditions, suggesting that the copolymer affects neither the folding structure of DNAzyme nor the association of a divalent cation, a catalytic cofactor, to DNAzyme. The copolymer enhanced activity of the evaluated DNAzyme over a wide temperature range under multiple-turnover conditions. The copolymer increased the DNAzyme kcat/KM by fifty-fold at 50 °C, the optimal temperature for the DNAzyme in the absence of the copolymer. The acceleration effect was most significant when the reaction temperature was slightly higher than the melting temperature of the enzyme/substrate complex; acceleration of two orders of magnitude was observed. We concluded that the copolymer accelerated the turnover step without influencing the chemical cleavage step. In contrast to the copolymer, a cationic surfactant, CTAB, strongly inhibited the DNAzyme activity under either single- or multiple-turnover conditions.

MNAzyme-catalyzed nucleic acid detection enhanced by a cationic copolymer

“…The signal results from substrate cleavage, generally upon release of a fluorophore from proximity to a quencher. 23 Here, we show that the cationic copolymer also enhances activity of an MNAzyme derived from the 10-23 DNAzyme and increased MNAzyme sensitivity. The MNAzyme integrated with various detection methods has been widely investigated.…”

“…MNAzyme activity did not significantly change in 25 mM Mg 2+ and 5 mM Mn 2+ in the absence of the copolymer (Fig. 23 The target concentration dependences in the presence of 5 mM Mn 2+ were determined in the presence and absence of PLL-g-Dex. Considerably higher MNAzyme activity was observed in 5 mM Mn 2+ than in 25 mM Mg 2+ when the copolymer was present (Fig.…”

“…When the part-zymes are assembled in the presence of a target nucleic acid, they form an active MNAzyme assembly that catalytically produces signals. 23 The copolymer facilitated turnover of the DNAzyme and stabilized the DNAzyme over a wide range of temperature. As multiple signals can be generated from a single target molecule, isothermal signal amplification is possible.…”

Cooperative enhancement of deoxyribozyme activity by chemical modification and added cationic copolymer

“…The copolymer increased more than 17-fold the catalytic activity of DNAzymes at the optimum temperature, in accordance with our previous observation. [18] The copolymer increased T m of the ES complex by 11.5°C. The added PLL- g -Dex increased T m of the ES complex similarly to LNA-modification, indicating both the added PLL- g -Dex and the LNA modification contributed to the stabilization of ES complex.…”

“…[13–17] We recently reported that the copolymer accelerated multiple-turnover reactivity of 10–23 DNAzyme [18] and its derivative, multicomponent nucleotide enzyme (MNAzyme). [19]…”