2446 Nucleic Acids Research, 2006, Vol. 34, No. 8 as 5 h to as short as 5 s) to promote phenotypic heterogeneity across different generations, and sequenced many clones from these different generations in an effort to thoroughly assess the population diversity. Ultimately, 110 different sequence classes were identiﬁed, but >90% of these contained variants of the 8–17 motif, which we suspected might be responsible for the observed catalytic activity. Peracchi (31) has previ- ously demonstrated that catalysis in the Mg5 RNA-cleaving deoxyribozyme is likely supported by an 8–17 motif, contrary to the much larger secondary structure originally proposed by Famulok and colleagues (29). While the mere presence of the 8–17 motif is highly sug- gestive given its recurrence in other studies, we wondered if it might represent just one module within a larger, more soph- isticated structure. For instance, Silverman and colleagues (13) re-isolated the catalytic core of a short RNA ligase deoxyri- bozyme whose activity beneﬁted by more than an order of magnitude from the presence of 20 additional nucleotides. Our objective in this study was 3-fold. We wanted to (i) sub- stantiate the suspected catalytic role played by the 8–17 motifs, (ii) determine how their activity was affected by excess sequence elements and (iii) determine what kind of impact this had on the outcome of selection. This knowledge could have general implications for the effective design and implementa- tion of future in vitro selection efforts. MATERIALS AND METHODS Oligonucleotides and reagents Oligonucleotides were prepared by automated DNA synthesis using cyanoethylphosphoramidite chemistry (Keck Biotech- nology Resource Laboratory, Yale University; Mobix Central Facility, McMaster University). DNA and RNA oligonuc- leotides were puriﬁed by 10% preparative denaturing (8 M urea) PAGE and their concentrations were determined by spectroscopic methods. Nucleoside 50-triphosphates and [g-32P]ATP were pur- chased from Amersham Pharmacia. T4 DNA ligase, T4 poly- nucleotide kinase (PNK), calf intestine alkaline phosphatase and T7 RNA polymerase were purchased from MBI Fer- mentas. All chemical reagents were purchased from Sigma. The 50 nt RNA substrate (S) was produced by in vitro tran- scription using T7 RNA polymerase and an appropriate double-stranded DNA template generated by PCR as described previously (33), and standard in vitro transcription protocols were used with pairs of complementary synthetic DNA oligos to produce the 26 nt SS substrate, and 18 nt ST substrate. Nucleotide sequences of RNA substrates are as follows: 50 nt S ¼ 50-ggagagagaugggugcguuacguaaac- uuacaucuacgaaucagguucga; 26 nt SS ¼ 50-ggagagagaugggug- cguuacguaaa; 18 nt ST substrate ¼ 50-gggagagaugggugcguu. Construction of substrate-deoxyribozyme cis constructs The 50 nt or 26 nt RNA substrates were ﬁrst ligated to 14 nt A1 (50-ggaaactagacaga) in a separate large-scale ligation reaction. Typically, 2500 pmol of RNA substrate was combined with 2750 pmol ligation template (50-tctctctcctctgtctag) and 3000 pmol of A1, heated for 30 s at 90 C and allowed to cool at room temperature for 10 min. After cooling, 10 ml of 10· ligase buffer (supplied by Fermentas) and 10 ml of T4 DNA ligase (5 Weiss U/ml) were added to initiate the ligation reac- tion. The reaction mixture (100 ml, total volume) was incub- ated at room temperature for 4 h or overnight. The ligated A1-RNA substrate was recovered by standard ethanol precip- itation and puriﬁed by 10% denaturing PAGE. The A1-RNA substrate construct was 50-32P-labelled with PNK, extracted twice with phenol/chloroform/isoamyl alcohol (25:24:1), and ethanol precipitated before being used in separate small-scale ligation reactions with speciﬁc deoxyribozymes. Thirty pmol of A1-substrate was combined with 90 pmol of 50-phos- phorylated deoxyribozyme, 36 pmol of ligation template (50-gcgtacgtgtcgaacctgattcg), and 45 pmol of substrate cleav- age blocker (50-tttacgtaacgcacccatctctctcctctgtctag), heated at 90 C for 30 s and allowed to cool at room temperature for 5–10 min. The substrate cleavage blocker is synthetic DNA complementary to a section of the RNA substrate surrounding the cleavage site, and was used to prevent deoxyribozyme- mediated cleavage during ligation. After cooling, 1.25 ml of 10· ligase buffer (supplied by Fermentas), 4 ml of T4 DNA ligase (5 Weiss U/ml) and DEPC treated H2O were added to a ﬁnal volume of 25 ml. The reaction was incubated at 37 C for 1 h, then ethanol precipitated and puriﬁed by 10% denaturing PAGE. Kinetic analyses of cis-cleaving deoxyribozymes A typical intramolecular self-cleavage assay consisted of the following steps: (i) heat denaturation of the deoxyribozyme- substrate construct in water for 30 s at 90 C, followed by slow cooling at room temperature for 10 min, (ii) the addition of selection buffer (ﬁnal concentration, 400 mM NaCl, 100 mM KCl, 7.5 mM MgCl2, 7.5 mM MnCl2, 50 mM HEPES, pH 7.0 at 23 C) and incubation at room temperature for a designated period of time, (iii) addition of EDTA to 30 mM to stop the reaction, (iv) separation of cleavage products by denaturing 10% PAGE and (v) quantitation using a PhosphorImager and ImageQuant software. Time courses for each deoxyribozyme were conducted at least twice using 13–16 different time points for each. The experimental data was ﬁt to either a single, Y ¼ Ymax(1 e( kobst)), or double, Y ¼ Ymax1(1 e( kobs1t)) + Ymax2(1 e( kobs2t)), exponential equation using non-linear regression analysis in GraphPad Prism 4, from which the observed rate constant (kobs) and maximum cleavage yield (Ymax) were determined. Kinetic analyses of trans-cleaving deoxyribozymes For single turnover experiments, a 100-fold excess of deoxyri- bozyme was mixed with 50-32P-labelled substrate. Substrate and deoxyribozyme were heated together at 90 C for 30 s, and allowed to cool at room temperature for 10 min. A 2· selection buffer was added to initiate the reaction giving a ﬁnal concentration of 1.66 mM deoxyribozyme to 0.0166 mM substrate. The reaction was terminated after a designated period of time by the addition of EDTA to 30 mM. The rate constant was determined from at least two independent experi- ments that differed by <10%. Each time course consisted of 16 time points. A graph of fraction cleaved versus time was plotted, and the experimental data ﬁt to a single or double exponential equation using non-linear regression analysis in GraphPad Prism 4.