7170 Nucleic Acids Research, 2005, Vol. 33, No. 22 Chemical probing using dimethyl sulfate As discussed above, the deletion walking and reselection experiments identiﬁed many highly conserved nucleotides that must be important to the structure and function of pH4DZ1. Noticeably, many of these nucleobases are guanines. Therefore, it is certain that some of these guanine residues are critically involved in the structural folding and catalytic func- tion of this deoxyribozyme. We sought to use the methylation interference approach to obtain additional evidence that can further implicate the func- tional roles of the crucial guanine residues in pH4DZ1. More speciﬁcally, we wanted to assess the level of tolerance by a fully functional deoxyribozyme construct EC2 (+++++) towards the methylation of the N7 atom of each of its component guanine residues. Hypothetically, the methyl- tion of N7 of a guanine residue is prohibited if any one of the following three scenarios applies. First, the N7 atom is directly involved in a tertiary interaction that is essential for structure folding or catalytic function. Second, the N7 atom is located in a deﬁned structural arrangement that cannot tolerate the bulky methyl group. Third, the positive charge introduced by the methylation disrupts the vital electronic landscape where the guanine residue is located. In contrast, the guanine residues located in functionally insigniﬁcant sequence motifs should not produce signiﬁcant methylation interference effect. For each methylation interference experiment, we used a set of two DNA samples, which are denoted ‘control’ and ‘test’ (22,23). The control sample was subjected to a procedure illustrated in Figure 5A. For illustration purpose, only two identical deoxyribozyme molecules, EC2A and EC2B each containing two hypothetical guanine residues shown as ‘g’ and ‘G’, were drawn. When these two molecules were allowed to cleave under permissive reaction conditions (step 1), the 30 cleavage product (P2A and P2B from EC2A and EC2B, respectively) and 50-cleavage fragment (P1) of the cis deoxyri- bozyme would be produced. The resulted DNA fragments were then subjected to treatment with DMS in water (step 2). Under this condition single-stranded DNA should exist in unfolded random-coil state and the ‘g’ and ‘G’ residues should be methylated with approximately equal probability. The methylation was purposely carried out at a DMS concen- tration that should only allow the methylation of one guanine per DNA molecule. P2A and P2B, isolated from the reaction mixture by denaturing PAGE following the phosphorylation with [g -32P]ATP/PNK (step 3), were then allowed to cleave in the presence of piperidine (step 4, which cleaves the DNA strand at all methylated guanines). Thus generated DNA frag- ments were ﬁnally analyzed by denaturing PAGE. Because methylation was performed after the catalysis step, radioactive DNA fragment P2A1 and P2B1 (corresponding to the respect- ive cleavage product at the ‘g’ and ‘G’ residues) should be observed at relatively equal intensity. The test sample was treated according to the scheme shown in Figure 5B. The procedural difference for the two samples was the order of the ﬁrst two steps: methylation with DMS was carried out with the deoxyribozyme construct before the self- cleavage reaction for the test sample (while for the control sample, the self-cleavage reaction was conducted ﬁrst). If the methylation at a particular guanine residue (represented Figure 5. Methylation interference. (A) and (B) outline the experimental scheme for obtaining DNA ladders to be analyzed as ‘Lane C’ and ‘Lane T’ in (C). Briefly, the 30-cleavage fragment obtained from EC2 treated with DMS before (Test-T) or after (Control-C) the cleavage reaction was performed and labeled at the 50 end with 32P. Under our reaction condition, DMS only methy- lated the N7 atom of one guanine per deoxyribozyme molecule on average. Methylated guanines were cleaved by piperidine at 90 C and cleaved fragments were resolved by 10% denaturing PAGE. Methylated guanines that disrupt deoxyribozyme activity appear missing or lighter in lane T than lane C. Filled black circles at the left side of the gel (and next to a concerned nucleotide) indicate the DNA bands with significantly reduced intensity in T lanes while the black square labels the DNA band that had a significantly enhanced intensity in the T lane. EC2 was used for this experiment. The sequence of the cleavage fragment P2 in (C) was drawn using the same labeling scheme given in Figure 4. herein by the ‘g’ residue) hindered the self-cleaving activity of the deoxyribozyme, no RNA cleavage or signiﬁcantly reduced RNA cleavage would occur. As a result, no cleaved product from the ‘g’ location would be obtained. This would eventually lead to a missing DNA band or a band with signi- ﬁcantly reduced intensity on denaturing PAGE. Similarly, methylated guanines that enhanced the activity of the deoxyri- bozyme would be identiﬁed by their increased band visibility. The PAGE analysis result of methylation interference experiment is given in Figure 5C. Nucleotides with noticeably reduced band intensity were labeled with a ﬁlled circle; those with enhanced intensity were labeled with a ﬁlled square. No cleavage band was observed in T lane for the following four guanine residues: G45, G61, G65 and G84. This ﬁnding indicates that pH4DZ1 cannot tolerate the methylation of the N7 atom of these guanines. The intensity of the DNA band corresponding to G80 was visible but signiﬁcantly reduced, suggesting that the methylation of this guanine residue considerably reduced