the activity of the deoxyribozyme. Interestingly, there are four cleaved DNA bands in T lane that showed a noticeable enhancement in intensity over the same band in C lane and they corresponded to G29, G34, G69 and G70. G34 in particular had the most drastic intensity enhancement. As a control experiment, we also treated the cleavage fragment directly with piperidine without a DMS methylation step and found no breakage at any of these guanosine residues (data not shown). This analysis indicates that each of these residues was not selectively depurinated at pH 3.8. Since methylation of N7 atom of a guanine residue introduces both a positive charge and a relatively bulky methyl group on the guanine, the intensity enhancement may have two possible reasons. One is that the introduction of the methyl group brings in a better spatial arrangement of the guanine in the folded deoxyri- bozyme structure, and the other is that the positive charge introduced strengthens an interaction that is important to the structure and function of the deoxyribozyme. We favor the second option based on the following three considera- tions. First, there are a total of four guanines with intensity enhancement. It is hard to believe that pH4DZ1, by chance, explores the better spatial arrangement introduced by the methyl group at four different locations. Second, the same hyper-methylation has not been observed for the two self- phosphorylating deoxyribozymes recently characterized by our lab (22), suggesting that this phenomenon may be an exclusive property of this low-pH-functional deoxyribozyme. Third, at pH 3.8, the extensive protonations at adenines and cytosines may make the catalytic core of the deoxyribozyme highly positively charged and the methylation at a relevant guanine could signiﬁcantly strengthen this property. However, the precise reason for the enhancement could not be pin- pointed at this moment and will be investigated in greater detail in our future research efforts. Not surprisingly, most of methylation-interfering guanines reside in the three crucial sequence elements identiﬁed by the deletion walking experiment. The only exception was G45 which is located 1 nt away from the ﬁrst such sequence element. Taken together, the methylation interference experi- ment provided strong evidence that further implicated the functional involvement of many important guanine residues identiﬁed by mutational analysis and reselection. Sequence truncation and nucleotide permutation of the substrate domain We also examined functional relevance of nucleotides in the substrate region and the data are shown in Figure 6. This region contains two specially modiﬁed deoxyribonucleotides Q 8 (DABCYL-dT) and F 10 (ﬂuorescein-dT) as well as the lone ribonucleotide A 9 (ribo-A). Deoxyribozyme constructs in which either ﬂuorescein (EC34) or dabcyl (EC35) or both (data not shown) were removed exhibited no self-cleaving activity. These results indicate that both the ﬂuorescein and DABCYL labels are crucial to the deoxyribozyme function. Similarly, we found that A 9 is also a nucleotide speciﬁcally required for the deoxyribozyme function, reﬂected by the inability to self-cleave seen with the three constructs in which the cleavage site was changed to cytidine ribonucleotide (EC36), guanosine ribonucleotide (EC37) and uridine ribonuc- leotide (EC38). These ﬁndings imply that the adenine attached Nucleic Acids Research, 2005, Vol. 33, No. 22 7171 Figure 6. Nucleotide alteration within the substrate domain. Different nucleo- tides from both the 50 and 30 ends of the substrate were altered (deletion or mutation) to identify the catalytically important nucleotides. H ¼ equimolar mixture of A, C and T; D ¼ equimolar mixture of A, G and T; and V ¼ equimolar mixture of A, G and C. A ¼ Adenine ribonucleotide, G ¼ Guanine ribonucleotide, C ¼ Cytosine ribonucleotide and U ¼ Uracil ribonucleotide. Nucleotides shaded in grey are catalytically important. to ribose is involved in highly speciﬁc interactions that are part of the active deoxyribozyme structure. Several deoxyribozyme constructs with sequential nucleot- ide truncations from either 50 or 30 end of the substrate domain were next examined. Removing the ﬁrst 6 nt from the 50 end 3 nt each time (EC39 and EC40, both +++++) produced no effect on the catalytic activity. The construct with three more nuc- leotides deleted from the same end (EC41, ++) exhibited an activity reduction by 1000-fold. Further truncations of the remaining nucleotides two at each time completely deactiv- ated the deoxyribozyme (EC42 and EC43, both). Interestingly, all nucleotides downstream of the cleavage site in the substrate domain were required for the optimal function of the deoxyribozyme, as the deletion of even 2 nt from the 30 end of the substrate domain (EC44, +++) lowered the catalytic activity by 100-fold. Removal of three more nucleotides from the same end (EC45, ++) led to a new con- struct whose activity was weakened by 1000-fold. Finally, we examined the functional roles of the ﬁve stand- ard nucleotides in the region shaded in grey, G 7, C 11, G 12, T 13 and G 14, by nucleotide permutations. All these nucle- otides appear to play important roles in the catalytic function of the deoxyribozyme, because mutating any of these nucleotides to the mixture of the three remaining residues (G 7 to H 7, EC50, +; C 11 to D 11, EC49, ++; G 12 to H 12, EC48, ++; T 13 to V 13, EC47, ++; G 14 to H 14, EC48, ++; H ¼ ACT, D ¼ AGT) weakened the RNA- cleaving activity by at least 1000-fold. Together, these results indicate that a majority of nucleotides in the substrate domain are required for the optimal function of the deoxyri- bozyme. It is possible that these nucleotides are engaged in extensive tertiary interactions responsible for substrate– catalyst recognition.