Nucleic Acids Research, 2005, Vol. 33, No. 22 7169 Figure 4. Sequence analysis of terminal pools from both 1 min and 5 h selection. The variation index (VI) complied for 1 min selection (A) and 5 h selection (B) upon sequencing 40 clones from each G4 population. The variation index was calculated by dividing the observed mutation rate at each position by 24% (the mutation rate of the starting library). The wild-type sequence is shown at the bottom of each chart (the last 22 nt at the 30 constant region is omitted for clarity). Only nucleotides shaded in the grey zones are partially mutagenized. The underlined nucleotides are the nucleotides either absolutely conserved (VI ¼ 0) or highly conserved (VI < 0.25). The letter or letters placed at the top of the VI bars that are significantly larger than 1 (meaning these nucleotides were hyper-mutagenized) indicated the dominant nucleotides in the selected deoxyribozyme clones at the given positions. The short black bars beneath certain nucleotides in (B) denote the nucleotides that are absolutely conserved in both selections. selection are given in Figure 3B. It is important to note that new primer-binding sequences were used in the reselection to avoid cross-contamination by the wild-type pH4DZ1 that exists in our lab as the possible contaminant. In particular, the last 19 nt of L1 sequence have never been used for any pH4DZ1 mutant construct, and this will certainly eliminate any chance for cross-contamination by any deoxyribozyme studied before the in vitro selection experiment. Two separate in vitro selection experiments were conducted under identical settings except that the permissive reaction times were different. In the ﬁrst selection, the reaction time for RNA cleavage was set at 1 min (denoted 1 min selection); in the second selection, a 5 h reaction time was used (denoted 5 h selection). Since 5 h selection is much less stringent than 1 min selection, we expected that the former should allow more base mutations than the later, which should help identify the nucleotides that are very crucial to the function of the deoxyribozyme. In contrast, 1 min selection should identify the nucleotides that are important to the optimal activity of the deoxyribozyme. Progress of each selection is shown in Figure 3C. After four rounds of selective ampliﬁcation, both populations reached 35% reaction completion. Approximately 40 clones were sequenced from both the cloned 1 min and 5 h pools. On average, each sequence acquired approximately ﬁve mutations as compared to the original sequence while only one sequence in each population contained all wild-type nucleotides (data not shown). A stat- istical analysis of the sequences of all isolated clones are given in Figure 4 where the original sequence of the shortened pH4DZ1 is shown with the variation index (V.I., a measure of variability) plotted for each base position (22). The vari- ation index was calculated by dividing the observed mutation rate (in percentage) at each position by 24%, which is the mutation rate of the starting library. A variation index of 0 indicates that the base is absolutely conserved in all the sequenced clones. In theory, if there are no constrictions on a base position, the variability observed within the sequenced population should be equivalent to the mutation rate (i.e. 24%); in such cases the variation index would be 1. Values greater than 1 could indicate that mutations confer a selective advantage over the original base. By far the majority of posi- tions from both reselections have variation indices between 0 and 1. We arbitrarily chose values of below 0.25 to indicate high-level conservation (underlined nucleotides in Figure 4). Three points are worth commenting. First, the overall vari- ation index pattern for both 1 min and 5 h populations is very similar. As expected, more mutations occurred in 1 min clones as compared to 5 h clones; however, 5 h selection only pro- duced mutations at four more nucleotide positions (mutations were observed in 18 positions in 5 h clones versus 14 in 1 min clones). The major difference between the two variation index plots is that 5 h clones had two more major mutation sites in G34 and A35. Interestingly, the variation index at A33 in the 1 min selection was very high (3.2) as compared to 5 h selec- tion (1.4). Furthermore, A33 was almost exclusively mutated to T in 1 min clones while the mutation at A33 in 5 h clones was variable. This suggests that A33T mutation conveys selective advantage for optimal catalytic function. Similarly, C56 was mainly mutated to T in 1 min clones whereas C56 was mutated either to A or T in 5 h clones. Second, this low-pH-functional deoxyribozyme could not tolerate a large number of base mutations in the three sequence elements. Twenty-one of the 30 nt exhibited a V.I. of 0.25 or lower in 1 min clones. The number of extremely conserved nucleotides only experi- enced a subtle drop to 19 nt in 5 h clones. Third, there are 10 nt that were absolutely conserved across all 1 min and 5 h clones (labeled with a black bar beneath each concerned nucleotide in Figure 4). Undoubtedly, these nucleotides are the residues that are most crucial to the structure and/or function of pH4DZ1.