MATERIALS AND METHODS Oligonucleotides and chemical reagents Standard and modiﬁed DNA oligonucleotides were prepared by automated chemical synthesis (HHMI-Keck Biotechnology Resource Laboratory, Yale University; Central Facility, McMaster University) using general phosphoramidite chem- istry. The partially randomized libraries used for reselection were synthesized using the following mixture of the four standard phosphoramidites (produced by manual mixing): 76% of the wild-type nucleotide, 8% of each of the remaining 3 nt using protocol described previously (10). DNA oligonuc- leotides were puriﬁed by 10% denaturing (8 M urea) PAGE. The triisopropylsilyloxymethyl (TOM) group of the RNA linkage was removed using the previously reported method (18). Nucleoside 50-triphosphates, [g-32P]ATP and [a-32P]dGTP were purchased from Amersham Pharmacia. T4 DNA ligase, T4 polynucleotide kinase (PNK) and Taq DNA polymerase were purchased from MBI Fermentas. All other chemical reagents were purchased from Sigma and used without further puriﬁcation. Water used throughout the study was deionized. All catalytic assays and the reselection experiment were carried out in the selection/assay buffer that contained a ﬁnal concentration of 50 mM citric acid (pH 3.8, 25 C), 400 mM NaCl and 10 mM CdCl2 (2· stock solution was used). The stop solution contains 40 mM EDTA (pH 8.0, 25 C), 8 M urea, 90 mM Tris, 90 mM boric acid, 10% sucrose (w/v), 0.025% xylene cyanol and 0.025% bromophenol blue (stored as 2· stock solution). The DNA phosphorylation reaction was performed in 50 mM Tris–HCl (pH 7.6, 25 C), 10 mM MgCl2, 5 mM DTT, 0.1 mM spermidine and 0.1 mM EDTA (pH 8.0, 25 C) (supplied by the manu- facturer as 10· stock solution). The DNA ligation reaction was carried out in 40 mM Tris–HCl (pH 7.8, 25 C), 0.5 mM ATP, 10 mM MgCl2 and 10 mM DTT (also supplied by the manu- facturer as 10· stock solution). Assessing the activity of cis deoxyribozymes All the cis pH4DZ1 variants (the substrate was covalently attached to each deoxyribozyme) required for the sequence truncation and mutation studies in discussed in the Results section were constructed in the following way. A synthetic oligonucleotide as the donor DNA (at ﬁnal concentration of 4 mM) was labeled at the 50 end with 10 mCi of [g-32P]ATP and PNK (0.1 U/ml, ﬁnal concentration; total reaction volume is 50 ml). Thus phosphorylated DNA was ligated with a required substrate in the presence of appropriate DNA template (acceptor:donor:template ¼ 1:1:1) and T4 DNA ligase at room temperature for 1 h. The ligated deoxyribozyme con- struct was puriﬁed by 10% denaturing PAGE. The deoxyri- bozyme (1 pmol) in water was heated at 90 C for 30 s and then cooled to room temperature for 5 min. The RNA cleavage reaction was initiated by the addition of the 2· selection/ assay buffer. The ﬁnal DNA concentration was set at 0.1 mM. At 0.1, 1, 10, 100 and 1000 min, an aliquot of the reaction mixture was withdrawn and quenched with the 2· stop solution, followed by freezing at 20 C. Cleavage products were separated by 10% denaturing PAGE and product yields were determined by quantitating product Nucleic Acids Research, 2005, Vol. 33, No. 22 7165 bands using a PhosphoImager and ImageQuant software (Molcular Dynamics). The catalytic ability of each deoxyri- bozyme was scored using the following scheme. Any deoxyri- bozyme construct exhibiting a kobs of 0.1 min 1 (10% cleavage in 1 min) or above was given the highest score at ‘+++++’; mutant deoxyribozymes with a kobs at or above 0.01 (>10% cleavage in 10 min), 0.001 (>10% cleavage in 100 min), 0.0001 min 1 (>10% cleavage in 1000 min) but below the threshold kobs value set for the next upper level were scored ‘++++’, ‘+++’ and ‘++’, respectively; those with a kobs below 0.0001 min 1 (<10% cleavage in 1000 min) were given a ‘+’; molecules that produced no cleav- age bands after incubation for 1000 min were denoted by a ‘ ’. At least two independent assays were conducted for each deoxyribozyme construct to ensure that each scoring was accurate. Selective deoxyribozymes were chosen for determination of accurate initial rate constants. Each deoxyribozyme was allowed to cleave in the selection/assay buffer. The reaction was stopped at some time intervals chosen to obtain data points that were under 20% completion of the reaction. The rate constants (kobs) were then calculated by plotting the nat- ural logarithm of the fraction of DNA that remained uncleaved versus the reaction time. The negative slope of the line pro- duced by a least-squares ﬁt to the data was taken as the rate constant. Reselection protocol Reselection of mutant deoxyribozymes from a partially ran- domized, pH4DZ1-based library was carried out largely according to the previously reported selection protocol (17) with some variations. Brieﬂy, for the ﬁrst round of selection, 500 pmol of gel-puriﬁed library was phosphorylated at the 50 end with 20 mCi of [g-32P]ATP and PNK at 37 C for 30 min. Non-radioactive ATP was then added into the solution (to a ﬁnal concentration of 1 mM) and the mixture was further incubated for 20 min at 37 C. The phosphorylated DNA lib- rary was ligated to a substrate named S3 (see the Results section) using T4 DNA ligase. The ligated DNA was puriﬁed by denaturing 10% PAGE and was resuspended in 100 ml of water. Two parallel selections were conducted, one with 1 min incubation time and the other with 5 h incubation time. In each selection, 100 pmol of ligated DNA in 40 ml was heated at 90 C for 30 s and cooled to room temperature for 5 min. After the addition of 40 ml of 2· selection/assay buffer and incuba- tion for either 1 min or 5 h at room temperature, the reaction was quenched by the addition of the 2· stop solution. The 30 cleavage fragment was isolated by 10% denaturing PAGE, ampliﬁed by PCR and then made into single-stranded DNA, all according to the previous protocol (17). For the subsequent rounds, the cleavage reaction was per- formed as described above. The DNA populations from both 1 min and 5 h selections after the 4th round were ampliﬁed by PCR and cloned into the plasmid pUC57 using the T/A cloning method. Plasmids containing individual catalysts were isol- ated using the 96-well PerfectPrep kit (Eppendorf ). Deoxyri- bozyme inserts were sequenced on a CEQ 2000XL DNA sequencer (Beckman-Coulter) using the CEQ Quick Start Kit (Beckman-Coulter) according to the protocol provided by the manufacturer.