Nucleic Acids Research, 2006, Vol. 34, No. 8 2451 Figure 5. (A) Sequence of G15-30 and the corresponding kobs values for various truncation derivatives. The numbers on top indicate the nucleotide length of various truncated versions, and the corresponding kobs value is depicted below the sequence. Each kobs value is the average of two independent trials, which typically differed by <20%. For simplicity, primer-binding sites are depicted as boxes. The underlined region shows some resemblance to an 8–17 motif, but does not satisfy all sequence requirements. (B) Relative activity of truncated deoxyribozymes. The activity of the 119 nt deoxyribozyme is taken as 1. Construct 59SS represents the 59 nt deoxyribozyme attached to the same truncated RNA substrate described previously. (C) Design of a shorter, trans-acting version of the G15-30 deoxyribozyme (G15-30T), and a shorter 18 nt RNA substrate (ST). (D) Kinetic analysis of G15-30T under single-turnover conditions (deoxyribozyme:substrate ratio of 100:1). The black line represents the non-linear least-squares fit to the experimental data using a double exponential equation (R2 ¼ 0.997). Two independent trials were conducted (trial 1, black circles; trial 2, white triangles), which differed by <10%. The rate constants kobs1 and kobs2 refer to the first and second phases, respectively. (E) Autoradiogram of trans cleavage assay for G15-30T conducted under multiple turnover conditions. A 100 fold excess of 50-32P-labelled substrate was incubated with G15-30T. Reaction conditions; 1 mM substrate, 10 nM enzyme and 50 mM HEPES (pH 7.0, at 23 C), 400 mM NaCl, 100 mM KCl, 7.5 mM MnCl2, 7.5 mM MgCl2, at room temperature; sampled at 0, 1, 2, 5, 10, 30 and 60 min. Cleavage products (Clv) were separated from the uncleaved precursor substrate (Pre) by electrophoresis in a denaturing 10% polyacrylamide gel. alternative conformations. Interestingly, the 30-truncated ver- sions of each deoxyribozyme exhibit monophasic kinetics, which suggests that the extra nucleotides may facilitate the formation of alternative structures and the observed biphasic kinetics (Figure 4). The 8–17 motifs of all sequence classes are listed in Supplementary Figure 2, and are grouped into four primary phenotypic classes (based on the relative activity scheme described in Figure 2A). Although we have listed 22 other 8–17 variants in the same general phenotypic class as G15-1, G15-16 and G15-39, it should be noted that these classes are still likely to be diverse in activity, and further subdivision has not been attempted due to insufﬁcient mutational data. How- ever, we do not expect that any of these classes died out due to an uncharacteristically large inhibitory effect from excess sequence elements. We identiﬁed two classes (G7-E129 and G10-E36.2) that closely resemble the fastest characterized 8–17 variant (i.e. identical to G15-16 but lacking one of the terminal adenosine residues and containing a different 30 substrate-binding arm), yet died out in generation 7 and 10, respectively. The full-length version of G7-E129 was assayed for self-cleavage yielding a kobs of 0.58 min 1 and Ymax of 61%, which provides further evidence that the results from our sample set are likely representative of the general population. At this time, we can only speculate as to why G7-E129 was eliminated so early in the selection, despite hav- ing catalytic properties comparable to G15-49 (kobs ¼ 0.56 min 1, Ymax ¼ 65%). Other factors, such as PCR ampliﬁcation bias, random genetic drift, and sampling error may have contributed to the loss of this sequence class. We have previ- ously initiated an investigation into the population dynamics of in vitro selection, and expect future reports to yield even greater insight into the signiﬁcance of these factors on the outcome of selection (41). Characterization of a non-8–17 motif From a total of 110 sequence classes isolated in our previous selection experiment, only seven did not appear to contain the 8–17 motif or any other previously reported RNA-cleaving