5262 Nucleic Acids Research, 2008, Vol. 36, No. 16 (0.1 M triethylammonium acetate, 25% acetonitrile v/v) and the DNA Sep HPLC column were obtained from Transgenomic (Omaha, NE, USA). All oligonucleotide templates used in this study were synthesized by Integrated DNA Technologies, Inc. (Coralville, IA, USA). Each template was puriﬁed using urea–PAGE, UV-shadowing and elution of the oligonu- cleotide into Tris–EDTA buﬀer. The templates were quan- tiﬁed by spectrophotometry at 260 nm using the extinction coeﬃcients obtained for each oligonucleotide from the online OligoTools Calculator from Integrated DNA Technologies, Inc. Trinucleotide evaluation oligonucleotide sequence design Two oligonucleotide sequences that comprised all 64 potential trinucleotide sequences were designed to be between 20 and 40 nt in length since oligonucleotides with this length have been successfully used to study the yields of RNA polymers that are readily separable from the DNA templates by denaturing HPLC (25,28). The sequences incorporated features previously shown to be essential for activity. Since the shortest length of a nucleotide sequence containing two unique trinucleo- tides is 4, the shortest length of a sequence containing m unique trinucleotides is m + 2. In consideration of the overall length constriction, two oligonucleotides sequences were generated; the base element of each sequence con- taining 32 unique combinations of the 64 possible trinucleotides. A genetic algorithm (29,30) was used to generate a set of solutions under selection from the following ﬁtness cri- terion: fk = sk mk, where fk was the ﬁtness criterion, sk was the number of unique trinucleotides from the set of desired nucleotides in the sequence and mk was the number of bases in the ﬁnal oligonucleotide that can self-hybridize. The algorithm was initialized with a popu- lation of M = 4096 random solution vectors, each repro- duced through crossovers and modiﬁed through mutation at a rate of 5 10 3. Each derived population was culled using the ﬁtness criterion. The solution vectors were oli- gonucleotides of 34 nt comprised of four values corre- sponding to the four bases A, G, C and T. The set of desired nucleotides was selected to ensure that the trinucleotides represented in each of the two 34-nt sequences were distinct from each other, but that all 64 trinucleotides were present in the ﬁnal two oligonu- cleotides. The number of iterations required for conver- gence to the desired solution was approximately 10 000. For some solutions, the length constraint was relaxed to be 36, which substantially reduced the number of itera- tions. In those cases, the ﬁnal solution had a single repeated trinucleotide that was identiﬁed by inspection and removed. The two selected oligonucleotides were modiﬁed by adding d(CA) repeats at the 50- and 30-termini. The 30-terminal hexanucleotide CACACA was added to achieve optimal binding of primase. CAGACACACA was added to the 50-terminus to allow for elongation (31). Both oligonucleotides were terminated with a 30-propane- diol to prevent primase from elongating from the end Figure 1. Assessment of A. aeolicus primase activity. (A) Sequence of TEOS 1 and TEOS 2 with the 64 trinucleotides underlined and bacter- ial trinucleotide initiation sites denoted in bold. Small letters above the selected trinucleotides correspond to peaks in the chromatograms in (B). Numbers below the sequences correspond to the lengths of primers that terminate at the 50-end of TEOS 2. (B) Denaturing HPLC analysis of the resulting RNA polymers synthesized by A. aeolicus primase with TEOS 1 and TEOS 2. The peaks labeled a, b and c eluted at 10.6 and 13.1 min and corresponded to primers of 17 and 26 nt in length, respectively. of a stabilized 30-hairpin (31,32). These two selected oligo- nucleotides are hereafter referred to as trinucleotide eva- luation oligonucleotide sequence 1 (TEOS 1) and TEOS 2 (Figure 1A). TEOS 1 and TEOS 2 were each used in the primer synthesis assay to evaluate the activity of the pri- mase of interest, as well as to screen for trinucleotide speciﬁcity. Trinucleotide-specific templates and trinucleotide specificity confirmation To delineate the trinucleotide recognition sequence of A. aeolicus primase, the most likely trinucleotide initia- tion sequence was predicted by evaluation of the length of the primers obtained in the activity screen using TEOS 1 or TEOS 2. Aquifex aeolicus primase was then used in more detailed analyses using trinucleotide-speciﬁc templates to test the predicted recognition trinucleotides. The trinucleotide-speciﬁc DNA templates consisted of the sequence 50-CAGA(CA)5 XYZ (CA)3-1,3-propanediol-30, where XYZ was CAC, CCA, CCC, CCG, CGA, CGC, CGG, CTA, CTC, CTG, CTT, GCC, GCG, GGC, GGG, GGT, TTA or TTG. These 23-mer templates con- tained the minimal elements needed to support bacterial primase activity, as described previously (33), and were used in the primer synthesis assay with a high