0.09 min 1, which is about 23% of the single-turnover rate of 0.4 min 1 (Figure 7B). We also assessed the pH dependence and metal speciﬁcity of ET1/S2 under single-turnover condition and found that the trans-acting system exhibited essentially identical properties described previously for pH4DZ1 (data not shown) (17). This result shows that truncation of nonessential nucleo- tides did not alter these two key properties of the deoxyri- bozyme. Initial rate constant and rate enhancement of representative deoxyribozymes Up to this point, the activities of all mutant deoxyribozymes were compared using the semi-quantitative scoring scheme given in Figure 1B. To assess the accuracy of the method, we sought to determine the ﬁrst-order initial rate constant for the 13 representative deoxyribozymes listed in Table 1. The chosen deoxyribozymes cover all activity classes: six from ‘+++++’ class (pH4DZ1, EC1, EC2, EC6, EC12 and EC39), one each from ‘++++’ and ‘+++’ classes (EC21 and EC29, respectively), two from ‘++’ class (EC46 and EC48) and two from ‘+’ class (EC9 and EC14). The plot for deriving kobs of EC6 (the most efﬁcient mutant pH4DZ1) is shown as Figure 8A. All the catalytic rate constants, in duplicate (shown as the second and third columns in Table 1, with the fourth column being the average value), were derived in the same fashion. The data given in Table 1 are consistent with the activities of the concerned deoxyribozymes assigned by the qualitative scoring method, with no exception. For example, the deoxyribozyme variants scored with a ‘+++++’ activity indeed exhibit a catalytic rate constant of 0.1 min 1 or above, with EC6 and EC12 at the opposite ends with a kobs of 1.2 min 1 and 0.12 min 1, respectively. Table 1. Initial rate constants for various mutants of pH4DZ1 DNAzyme name pH4DZ1 EC1 EC2 EC6 EC9 EC12 EC14 EC21 EC29 EC39 EC46 EC48 ET1/S2 S1 only kobs, min 1 repeat 1 1.1 0.16 0.96 1.1 0.000031 0.10 0.000030 0.020 0.0026 0.84 0.00086 0.00046 0.40 0.00000048 kobs, min 1, repeat 2 0.95 0.15 0.90 1.2 0.000028 0.14 0.000017 0.016 0.0027 0.77 0.00088 0.00048 0.41 0.00000071 kobs, min 1, average 1.0 0.16 0.93 1.2 0.000030 0.12 0.000024 0.018 0.0027 0.81 0.00087 0.00047 0.40 0.00000060 Rate enhancement 1.7 · 106 2.6 · 105 1.6 · 106 1.9 · 106 4.9 · 10 2.0 · 105 3.9 · 10 3.0 · 104 4.4 · 103 1.3 · 106 1.5 · 103 7.8 · 102 6.8 · 105 Each deoxyribozyme was allowed to cleave in the selection/assay buffer. The reaction was stopped at certain time intervals to obtain data points under 20% reaction completion. The rate constants (kobs) were then determined by plotting the natural logarithm of the fraction of DNA that remains uncleaved versus the reaction time. The negative slope of the line produced by a least-squares fit to the data was taken as the rate constant. ‘ET1/S2’ represents the trans deoxyribozyme at ET1/S2 ratio of 50:1. Two independent experiments were carried out for each deoxyribozyme construct (shown as the second and third columns; the fourth column is the average value. ‘S1 only’ represents the rate constant of the uncatalyzed reaction (kbackgroud). The rate enhancement (the last column) was calculated by dividing kobs of a concerned deoxyribozyme with kbackgroud. Nucleic Acids Research, 2005, Vol. 33, No. 22 7173 We also measured the rate constant for the spontaneous cleavage of the substrate S1 as an approximation of the uncatalyzed reaction rate under our experimental conditions (kuncatalyzed). The plot for the rate determination is shown in Figure 8B. It is worth commenting that we used a highly radioactive, 50-32P-phosphorylated S1 so that the cleavage product could still be detected by a Phosphorimager after several weeks of decay. The analysis of the cleavage products from two independent reactions incubated for up to 28 days revealed that the spontaneous cleavage rate of S1 was 6.0 · 10 7 min 1. As a comparison, the spontaneous RNA cleavage at pH 7 and 23 C under similar salt concen- trations was previously reported to have a rate constant of 1 · 10 8 min 1 (29). The rate enhancements of the deoxyribozymes featured in Table 1 were estimated by kobs/kuncatalyzed and are listed as the last column of the table. The original pH4DZ1 and several members of ‘+++++’ class exhibit a rate enhancement of 106-fold, equaling the performance by the well-known 10–23 deoxyribozyme that is capable of cleaving RNA in the neutral pH range (8,14). This analysis clearly indicates that the catalytic power of DNA is fully retained even in a highly acidic solution. Figure 8. Determining catalyzed and uncatalyzed RNA cleavage reaction rates at pH 3.8. (A) Plot for deriving the initial rate constant of EC6 as a representative deoxyribozyme. The cis deoxyribozyme was allowed to cleave in the selection/ assay buffer. The reaction was stopped at certain time intervals to obtain data points under 20% reaction completion. The natural logarithm of the fraction of DNA that remains cleaved (y-axis) is then plotted against the reaction time (x-axis). (B) Plot for deriving the background rate. The x-axis represents reac- tion time in min and y-axis plots natural logarithm of the fraction of S1 that remained uncleaved at the various reaction times. Each data point in both plots represents the average value of two independent experiments. The negative slope of the least-squares fit is taken as the first-order initial rate constant.