2450 Nucleic Acids Research, 2006, Vol. 34, No. 8 Figure 4. Kinetic analysis of G13-20 and G15-48. The fraction of substrate cleaved was plotted versus time, and the experimental data fit to either single or double exponential equations using non-linear regression analysis in GraphPad Prism 4. Two independent trials were conducted (trial 1, white circles; trial 2, black triangles), and the correlation coefficient (R2) for the average of both trials is indicated. (A) G13-20L. The data are best described by a curve fit to a double exponential equation (solid black line) rather than a single exponential equation (dotted black line). (B) G13-20S. The data are best described by a curve fit to a single exponential equation. (C) G15-48L. The data are best described by a curve fit to a double exponential equation (solid black line) rather than a single exponential equation (dotted black line). (D) G15-48S. The data are best described by a curve fit to a single exponential equation. single-stranded bulge region, which may base pair with the uridine residue immediately 50 to the GG cleavage site. The extra stability conferred by the additional downstream base pairs in the perfect binding arm (PBA construct), may promote the A-U base pair to form and thereby constrain the 8–17 motif into a sub-optimal fold. Consistent with this theory, the ori- ginal weaker binding arm (S construct) is associated with higher activity in these classes. Moreover, the insertion of another A residue adjacent to the existing one in the perfect binding arm (PBAA column in Tables 1 and 2) nearly restores activity to wild-type level. Interestingly, the excess sequence elements in both the DNA and RNA domains do not seem to have a signiﬁcant impact on the maximum cleavage yield. The general effect appears to be neutral, and the minor changes that are observed are likely due to inherent experimental variation. However, one class (G15-49) experiences a 70% decrease in Ymax after deletion of its excess 30 DNA sequence, the magnitude of which cannot be explained by simple experimental variation. Instead, we suspect the disruptive effect of the two T bulges in the 50 and 30 binding arms is attenuated by the presence of the downstream DNA sequence, and intensiﬁed in its absence. The downstream sequence elements may form additional stabiliz- ing contacts with A1. Consistent with this theory, Ymax is 3.7-fold larger when both T bulges are removed in G15-49 PBA. Characterization of the global 8–17 population We wondered if the results of our 10 deoxyribozyme sample set were representative of the global 8–17 population from our selection experiment. On average, a neutral or modest inhib- itory effect was observed with excess sequence elements in the deoxyribozyme and substrate domains, respectively. However, we might expect that the G15 classes would have been selected, in part, for their ability to minimize any neg- ative effect associated with excess sequence elements. To address this issue, we looked for the six different 8–17 variants from G15 (G15-35 and G15-48 have the same 8–17 core motif) in the other 100 sequence classes that were eliminated during earlier generations under more permissive selection conditions. Most of these fast 8–17 variants were unique, and not observed in other generations. However, the 8–17 catalytic core from G15-1 was also present in G13-E78, and G15-35 shares the same core motif with G15-48, G13- 20, G10-47 and G7-E33. We determined that the full-length G13-E78 deoxyribozyme has a kobs of 1.09 min 1, and a Ymax of 66%. Therefore, G13-E78 was not eliminated because of an uncharacteristically large inhibitory effect from excess sequence elements. We suspect G13-E78’s absence from the generation 15 population may be due to sampling error, PCR bias, sub-sampling effects or some combination thereof. Classes G15-35 and G15-48 are 2–3 times faster than G13-20 and G10-47 even though they contain the same 8–17 catalytic core. The disparity between their rates, how- ever, can be explained by the difference in their 30 substrate binding arms. Both G13-20 and G10-47 have relatively poor 30 substrate binding arms as reﬂected by the 2-fold higher activity observed upon optimization (Table 1, PBAA/S col- umn). This contrasts with G15-35 and G15-48, which experi- ence an approximately neutral effect upon optimization of their 30 substrate binding arms. It should be noted that G15-48 and G13-20 appear to follow biphasic kinetics, which are characterized by a fast initial phase followed by a slower second phase. Though not particularly common, biphasic kinetics have been observed previously in other deoxyribozymes (38), and ribozymes like the hairpin (39,40) in which it was attributed to the formation of