A Lead-Dependent DNAzyme with a Two-Step Mechanism

Abstract

A detailed biochemical and mechanistic study of in vitro selected variants of 8-17 DNAzymes is presented. Even though the 8-17 DNAzyme motif has been obtained through in vitro selection under three different conditions involving 10 mM Mg(2+) (called 8-17), 0.5 mM Mg(2+)/50 mM histidine (called Mg5), or 100 microM Zn(2+) (called 17E), all variants are shown to be the most active with Pb(2+) (8-17: k(obs) approximately 0.5 min(-1); Mg5: k(obs) approximately 2 min(-1); 17E: k(obs) approximately 1 min(-1) with 200 microM Pb(2+) at pH 5.0). For the 17E variant of the 8-17 DNAzyme, the single-turnover rate constants followed the order of Pb(2+) >> Zn(2+) >> Mn(2+) approximately Co(2+) > Ni(2+) > Mg(2+) approximately Ca(2+) > Sr(2+) approximately Ba(2+). The catalytic rate is half-maximal at 13.5 microM Pb(2+), 0.97 mM Zn(2+), or 10.5 mM Mg(2+), suggesting that the metal-binding affinity of the DNAzymes is in the order of Pb(2+) > Zn(2+) > Mg(2+). The Pb(2+)-dependent activity increases linearly with pH and the slope of the plot of log k(obs) versus pH is approximately 1, suggesting a single deprotonation in the rate-limiting step of the reaction. Sequence variations of the DNAzyme confirm the importance of the G*T wobble pair, the two loops and the intervening stem in maintaining the active conformation of the system. While Mg(2+) and Zn(2+) catalyze only a transesterification reaction with formation of a product containing a 2',3'-cyclic phosphate, Pb(2+) catalyzes a transesterification reaction followed by hydrolysis of the 2',3'-cyclic phosphate. Although this two-step mechanism has shown to be operative in protein ribonucleases and in the leadzyme RNAzyme, it is now demonstrated for the first time that this DNAzyme may also use the same mechanism. Therefore, the two-step mechanism is observed in metalloenzymes of all classes, and this 8-17 DNAzyme provides a simple, stable, and cost-effective model system for understanding the structure of Pb(2+)-binding sites and their roles in the two-step mechanism.

Tandem DNAzymes for mRNA cleavage: Choice of enzyme, metal ions and the antisense effect

“…19 At the same time, it is also studied as a model DNAzyme due to its recurrence in many different in vitro selections and high catalytic efficiency. [20][21][22][23][24][25] Examples of using the 8-17 DNAzyme for gene silencing are also known. 18,26 While many examples of DNAzyme-based intracellular mRNA cleavage are reported, 13 concerns have also been raised that the 10-23 DNAzyme is basically inactive with physiological concentrations of free Mg 2+ .…”

“…Pb 2+ induced the most cleavage, which was the same as the 8-17 DNAzyme. 23 To have a quantitative comparison, we further measured the enzyme rate in the presence of these metal ions ( Figure 1E). Pb 2+ has the highest activity followed by transition metals such as Mn 2+ and Zn 2+ (note that Mg 2+ and Ca 2+ were used at a much higher concentration).…”

“…For example, when an rAC junction was used, barely any cleavage was observed even with 500 mM Mg 2+ ( Figure S1, ESI). Therefore, it is also true for the [10][11][12][13][14][15][16][17][18][19][20][21][22][23] DNAzyme that its cleavage activity for every junction is not the same.…”

The group trend of lanthanides binding to DNA and DNAzymes with a complex but symmetric pattern

“…Since the difference is only a single oxygen atom, the perturbation on metal binding should be minimal. For comparison, we also included a classic DNAzyme, 17E ( Figure 1C), 7,27,28 which has a lanthanide binding site as well but lanthanides inhibit its Pb 2+ -dependent activity. 17 Finally, we hybridized the substrate strand with its cDNA forming a duplex, where all the bases are paired and 2 | J.…”

Characterization of Metal Ion-Nucleic Acid Interactions in Solution

“…Qualitative and quantitative assessment of sequence specific ion binding to DNA Atracts has been undertaken based on the reduced effective charge that can be detected by free solution capillary chromatography [271,272]. Apparent metal affinity constants have been determined from observed rate constants of ribozyme catalysis in many cases (e.g., [154,157,[273][274][275]). An approach based on the gas-phase fragmentation of metal-nucleic acid complexes in ESI-MS (electrospray ionization mass spectrometry) was recently applied to determine binding affinities to the thrombin binding aptamer [276].…”