7172 Nucleic Acids Research, 2005, Vol. 33, No. 22 Engineering of a trans-acting deoxyribozyme Almost all the cis-acting RNA-cleaving DNA enzymes that operate under neutral pH conditions can be re-engineered into trans-acting deoxyribozymes with multiple-turnover capabil- ity (8,24–28). We therefore sought to design a trans system for pH4DZ1. From sequence truncation data presented in Figure 1B, it is apparent that nucleotides from G1 to G32 are completely dispensable. Based on this observation, we ﬁrst designed a trans substrate S2 by moving the ﬁrst 12 nt of the catalyst domain to the end of the S1 sequence. The newly designed 35 nt substrate S2 was used for all the trans experiments. Since all known trans deoxyribozymes interact with their RNA substrates through the formation of two Watson–Crick helices (one upstream and downstream of the cleavage site), we wondered whether we could design a trans deoxyribozyme that can engage S2 by forming a stable Watson–Crick duplex. For this consideration, ET1 (E: enzyme; T: trans-acting) was made that was able to form 17 bp duplex as shown in Figure 7A. Gel-shifting experiment conﬁrmed the binding of S2 and ET1 at neutral pH (data not shown). Under the single-turnover setting of ET1:S2 ¼ 50:1 ([ET1] ¼ 25 mM; [S2] ¼ 0.5 mM), ET1 exhibited a kobs of 0.40 min 1 (initial rate, see Figure 7B), which is almost identical to the catalytic rate constant displayed by the cis pH4DZ1 (a kobs of 1 min 1) (17). This ﬁnding indicates that the nucleotides added to the 50 end of the ET1 did not signiﬁcantly affect the folding and the catalytic function of the deoxyribozyme. We then sought to assess the dispensability of the artiﬁcially imposed duplex that engaged the deoxyribozyme to the substrate by assaying the relative activity of several deoxyri- bozyme constructs in which the nucleotides at the 50 end were progressively truncated (ET2–ET6). Removing up to 12 nt did not signiﬁcantly affect the deoxyribozyme activity in trans as ET2–ET4 exhibited an activity that was identical to ET1. Deletion of one more nucleotide (ET5) resulted in about 3- fold activity reduction. Further nucleotide truncation (ET6) essentially rendered the trans deoxyribozyme inactive. These results demonstrated that the formation of artiﬁcially engineering Watson–Crick duplex is not necessary to the func- tion of the trans deoxyribozyme. Consistently, ET1 and ET4 were fully functional when S1 was used instead of S2 (S1 does not contain the last 12 nucleotides of S2). Experiment shown in Figure 7C was then conducted to examine whether ET1 had multiple-turnover capability. When 100 nM of ET1 was incubated with 10 000 nM of S2 for 0.5, 1, 2, 4 and 8 h, 272, 535, 1157, 2067 and 3504 nM of the cleavage product P1 were produced (Figure 7C). Similar experiments were also conducted using other substrate con- centrations (500, 1000 and 5000 nM) and the calculated turn- over data are summarized in Supplementary Table 1. Overall, our results indicated that ET1 indeed had multiple-turnover capability. For instance, the deoxyribozyme produced 35 turn- overs in 8 h at the substrate concentration of 10 000 nM (100% cleavage under this setting can result in a total of 100 turn- overs). We noted that the rate of turnover appeared to be smaller than the observed rate constant for single-turnover rate. For example, 2.7 turnovers in 30 min at [S2] ¼ 10 000 nM (representing an excessive substrate concentration; [ET1] ¼ 100 nM) correspond to a rate of turnover of Figure 7. A trans-acting catalytic system. (A) Designing a trans-acting pH4DZ1 system. A 35 nt substrate S2 was designed that contained the sequence of S1 and the first 12 nt from the catalyst domain in the original pH4DZ1. The 72 nt trans-acting deoxyribozyme ET1 was constructed that contained the nucleotides from 34–92 of the original pH4DZ1 as well as 14 additional nu- cleotides (lower-case letters) introduced at the 50 end of the deoxyribozyme. The first 17 nucleotides were designed to form Watson–Crick base pairs with the last 17 nt of S2. Various ET1 mutants with nucleotides truncated from the 50 end were tested under single-turnover condition (S2 and each deoxyribozyme were set to be 0.5 and 25 mM, respectively). The activity of all deoxyribozymes was normalized to that of ET1. (B) Plot for deriving single-turnover kobs of S2/ ET1. Substrate S2 (0.5 mM) was incubated with ET1 (25 mM) in the selection/ assay buffer [50 mM Citric acid (pH 3.8), at 25 C, 400 mM NaCl and 10 mM CdCl2]. At indicated time points, an aliquot of the reaction was withdrawn, quenched and stored at 20 C. After collecting samples, the reaction mixture was analyzed by 10% denaturing PAGE. The formation of the cleavage product P1 (which is radioactive) was quantitated using ImageQuant Software. The x-axis represents reaction 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 represents the average value of two independent experiments. The ne- gative slope of the least-squares fit is taken as the first-order initial rate constant, which was found to be 0.40 min 1. (C) PAGE analysis of the multiple-turnover capability of S2/ET1 system. S2 (10 000 nM) (in the presence of trace amount of 50-32P-labeled S2) were incubated with 100 nM ET1 in the selection/assay buffer for 0, 30, 60, 120, 240 and 480 min and the reaction mixture was analyzed by 10% denaturing PAGE. The concentration of P1 was calculated using ImageQuant Software. The number of turnovers by ET2 was then computed as [P1]/[ET2].