7174 Nucleic Acids Research, 2005, Vol. 33, No. 22 DISCUSSION Undoubtedly, DNA, which is composed of only four chemic- ally analogous building blocks with extremely limited chemical functionalities, is and should be viewed as a polymer that is far less ﬁt for catalysis than protein molecules that are made of 20 functionality-diverse amino acids. However, it has been well demonstrated that catalytic DNAs can be conveni- ently derived in research laboratories by in vitro selection. In fact, human creativity has led to many surprising ﬁndings with regard to the catalytic aptitude of DNA. For example, the most well-known RNA-cleaving DNA enzyme 10–23 is not only small in size (which contains a catalytic core built of only 15 nt) but can also perform site-speciﬁc RNA cleavage with a kcat of 10 min 1, kcat/KM of 109 M 1 min 1 and a rate enhancement of 106 (8,14). Another example is a small DNA-adenylating deoxyribozyme known as the ‘class I DNA capase’ that exhibits a rate enhancement of 1010-fold (11). Accumulative evidence has convincingly shown that DNA, despite its simplicity in chemical functionality, still possesses considerable capacity for enzymatic function (1–5). Therefore, studying the catalytic ability of DNA can reveal useful information that will enhance our understanding of phenom- enon of catalysis itself. The experiments described in this study were designed to appreciate the catalytic and sequence properties of pH4DZ1, an unusual DNA catalyst that was speciﬁcally evolved to perform catalysis at pH 4—a rather challenging chemical condition. Our results have shown that pH4DZ1 can perform the given task with a surprising proﬁciency, reﬂected both by its catalytic rate constant of 1 min 1 and the rate enhance- ment of 106-fold. The fact that both parameters are compar- able to 10–23—the best deoxyribozyme known to date that operates at neutral pH range—strongly suggests that the cata- lytic power of DNA is retained at high acidity. There are some noticeable differences between neutral and acidic deoxyribozymes. First, neutral deoxyribozymes such as 10–23 and 8–17 are extremely small in size. Both 10–23 and 8–17 contain a catalytic core of about a dozen nucleotides and two substrate binding arms of about 6–8 nt. In other words, these two deoxyribozymes are made of less than 30 nt. Our sequence truncation experiments above have shown that pH4DZ1 is a much larger deoxyribozyme that requires 80 nt for the optimal catalytic activity. Second, all neutral RNA-cleaving deoxyribozymes (including 10–23 and 8–17) explore Watson–Crick base pairing interactions for substrate binding (8,14–16,23–28,30–33). It is expected that the Watson–Crick rules do not apply to DNA at acidic pH range because of extensive protonation at the N1 atom of adenosine and the N3 atom of cytosine (both of which are hydrogen-bonding sites in Watson–Crick base pairs). Indeed, we failed to conﬁrm the possible Watson–Crick helices pre- dicted by mfold (data not shown) (34). Therefore, we can conclude that Watson–Crick base pairing rules do not apply to pH4DZ1. The lack of simple yet effective Watson–Crick interactions may explain why pH4DZ1 is much a larger deoxyribozyme than 10–23 or 8–17: the tertiary structure of pH4DZ1 is likely constructed using much more complex inter- action network that involves several structural domains and many distal nucleotides. The third difference is that all the neutral trans-acting RNA-cleaving deoxyribozymes have excellent multiple-turnover capability while the engineered trans-acting pH4DZ1 has somewhat poorer ability in this regard. We have not determined the precise reasons for the smaller multiple-turnover rate observed for pH4DZ1. It is possible that the interactions that govern the substrate- deoxyribozyme recognition for pH4DZ1 are considerably dif- ferent from the ones exploited by the neutral deoxyribozymes, and as a result, the engineered trans-acting system of pH4DZ1 has a slow substrate binding or product release step that is rate limiting. Alternatively, the highly acidic medium and rather high ionic strength may cause severe DNA aggregation, such that only a fraction of the substrate exists in the cleavable form. Detailed kinetic analysis of the trans-acting system of pH4DZ1 will be carried out in the future and results will be reported elsewhere. The original pH4DZ1 contains 123 nt and Figure 9 sum- marizes the signiﬁcance of these nucleotides in relation to the catalytic function of the deoxyribozyme, based on the data obtained from both the sequence truncation and reselection experiments. Among the 75 nt required for the optimal func- tion of pH4DZ1 (nucleotides shown in black, blue and red), 53 of them appear to play crucial roles in the function of Figure 9. Summary of truncation, mutation and reselection data. The significance of each of the 123 nt within the original pH4DZ1 sequence is categorized using the following coloring scheme: grey: nonessential nucleotides (their truncation causes no activity loss); black: important nucleotides (activity drops by up to 100-fold upon truncation or mutation); blue: crucial nucleotides (activity drops by more than 100-fold upon truncation or mutation); red: essential nucleotides (absolutely conserved nucleotides revealed by the reselection data presented in Figure 4).