7166 Nucleic Acids Research, 2005, Vol. 33, No. 22 DMS methylation interference assay The method to be described below was adapted from a pre- viously published protocol (22,23). A truncated pH4DZ1, named EC2 (see the Results section for its sequence), was used for this experiment. The cis deoxyribozyme was con- structed by ligating a DNA oligonucleotide denoted donor EC2 (its sequence is 50-GGGGA GAAAC ATCTT TGCGG GATAA GGCCG CCGAT AGAGC GGAAG CGACT TGGTT GTAGC TGAT-30) with the substrate S1. The ligation reaction was performed as follows. Donor EC2 (500 pmol) was ﬁrst phosphorylated with a trace amount of [g-32P]ATP and PNK (0.1 U/ml, ﬁnal concentration; total reaction volume is 50 ml) for 20 min at 37 C, followed by the addition of non-radioactive ATP to a ﬁnal concentration of 1 mM. The mixture was further incubated for 20 min to ensure complete phosphorylation. PNK was then inactivated by heating the reaction mixture at 90 C for 5 min. This mixture was directly added to a 200 ml ligation solution containing 600 pmol of the DNA template T2 (50-GTTTC TCCCC TCGAA CCATA) and 550 pmol of S1 (see the Results section for its sequence). The ligation was initiated by the addition of T4 DNA ligase (0.2 U/ ml, ﬁnal concentration) and continued at room temperature for 5 h. The ligated DNA was subsequently puriﬁed by denaturing 10% PAGE. The above ligated EC2 (100 pmol) in water was heated at 90 C for 30 s and cooled to room temperature for 5 min. The cleavage reaction was initiated by the addition of the reaction buffer, followed by incubation at room temperature for 5 min. The reaction was then quenched by EDTA (30 mM, ﬁnal concentration). The DNA in the solution was recovered by ethanol precipitation, resuspended in water (500 ml), heated at 90 C for 1 min and cooled to room temperature quickly. An equal volume of 0.4% (v/v) DMS (freshly made) was added and the resultant mixture was incubated at room temperature for 40 min. Thus treated DNA was recovered by ethanol pre- cipitation. This DNA sample was labeled as the ‘control’. Another 100 pmol of uncleaved EC2 was methylated with DMS in the same fashion. After the recovery by ethanol pre- cipitation, the methylated DNA was then allowed to cleave for 5 min with the addition of the 2· reaction/assay buffer. After quenching with EDTA (30 mM, ﬁnal concentration), the DNA was recovered by ethanol precipitation. This DNA sample was labeled as the ‘test’. Both the control and test DNA samples were phosphorylated with 20 mCi of [g-32P]ATP and PNK at 37 C for 20 min. After puriﬁcation by 10% denaturing PAGE, both the control DNA and the test DNA were dissolved in 10% (v/v) piperidine (100 ml, freshly made) and heated at 90 C for 30 min. The resultant cleavage products were dried under vacuum. The dried pellet was resuspended in 25 ml of H2O and evaporated to dryness under vacuum. This step was repeated one more time. Both the control and test samples were then subjected to denaturing 10% PAGE analysis to reveal piperidine-mediated cleavage fragments. The kobs value of the trans deoxyribozyme under a single-turnover condition 50-32P-phosphorylated S2 (0.5 mM, 35 nt) and ET1 (25 mM; for the sequences of S2 and ET1, see the Results section) in H2O were heated at 90 C for 30 s, cooled to room temperature, followed by the addition of the 2· selection/assay buffer. After pre-determined time intervals, a fraction of the reaction solu- tion was taken, quenched with the stop solution and stored at 20 C. These mixtures were ﬁnally analyzed by 10% dena- turing PAGE. The rate constant (kobs) was determined by plotting the natural logarithm of the fraction of S2 that remained uncleaved versus the reaction time (the negative slope of the least-squares ﬁt was taken as the initial rate constant). Assessing the multiple-turnover capability of the trans deoxyribozyme The chimeric DNA/RNA substrate S2 used for the multiple- turnover experiment was double puriﬁed by PAGE. After gel elution and ethanol precipitation steps, the potential contam- inants (such as salts and EDTA) were removed from the sub- strate by centrifugation using a spin column (Nanosep 3K Omega, Pall Corp., Ann Arbor, Michigan). The ﬁnal concen- tration of the substrate S2 was varied from 500, 1000, 5000 and 10 000 nM (plus trace amount of 50-32P-labeled S2) at ﬁxed ET1 (trans-acting deoxyribozyme) concentration (100 nM). In a typical reaction, S2 and ET1 in H2O were heated at 90 C for 30 s, cooled to room temperature before the addition of the 2· reaction/assay buffer. After incubation at room temperature for 0, 0.5, 1, 2, 4 and 8 h, the reaction was quenched with the 2· stop solution and stored at 20 C. The reaction mixtures were ﬁnally analyzed by 10% denaturing PAGE. RESULTS Scoring catalytic activity of mutant deoxyribozyme constructs We purposely divide the sequence of pH4DZ1 into two domains labeled in Figure 1A as ‘substrate’ (italicized letters) and ‘catalyst’ (normal letters), to simply reﬂect the fact that this catalytic DNA was originally derived using a library of 100 nt single-stranded DNA molecules covalently joined to the 23 nt substrate (denoted S1). The two sequence stretches shown in lower-case letters were used as the primer-binding sites for the PCR during the original in vitro selection. For convenience, we have marked the nucleotides in the sub- strate domain from 1 to 23 in the 30-to-50 direction and the ones in the catalyst domain from 1 to 100 in the 50-to-30 direction. The speciﬁc number assigned to each nucleotide remains unchanged throughout this report, even though many mutant deoxyribozyme constructs contained one or more truncated nucleotides. It should be noted that the substrate domain contains three special nucleotides: the DABCYL-containing deoxyribothymidine (Q; DABCYL: 4- (4-dimethylaminophenylazo)benzoic acid), adenine ribonuc- leotide (A) and the ﬂuorescein-containing deoxyribothymidine (F) at the 8th, 9th and 10th positions, respectively. The ribonucleotide is the cleavage site while F and Q provide a setting for ﬂuorescence signaling upon catalysis for future biosensing applications. The activity of all mutant deoxyribozymes, analyzed using synthetic DNA oligonucleotides containing an internal 32P- labeled phosphodiester, was scored as described in ‘Materials and Methods’ (see the insert in Figure 1B). Generally speak- ing, a mutant deoxyribozyme with a score of +++++ or ++++ is