2452 Nucleic Acids Research, 2006, Vol. 34, No. 8 deoxyribozyme motif. Five of these classes were eliminated by generation 8, and another class was observed as a single clone in G13. Only one class (G15-30) survived the stringent 5 s selection pressure applied in G15, and represented 10% of the population (from a sample of 49 clones). G15-30 was therefore selected for further examination. It should be noted that this class does possess some of the characteristic sequence requirements of 8–17 near the 50 end, but lacks the strictly conserved G14 residue from the single-stranded turn region described in Figure 2A. Despite this discrepancy we originally suggested that catalysis in G15-30 [referred to previously as DZ4 in reference (33)], might still be supported by an unusual 8–17 variant. However, subsequent evidence from a more rigorous investigation supports the contrary. Mul- tiple truncation tests were carried out to determine the minimal sequence requirements and the effect of excess sequence elements on G15-30. Figure 5A shows the nucleotide sequence of G15-30 and the catalytic rate constant associated with different truncated versions. The full-length molecule pos- sesses a kobs of 0.48 min 1, but deleting 60 nt from the 30 end leads to a kobs of 1.4 min 1. Further deletion causes a reduction in activity, with no activity being observed after an additional 10 nt are removed. The relative activity of the various deletion constructs are illustrated in Figure 5B. Point mutations were also introduced into the 59 nt version at positions expected to be crucial for activity, if 8–17 was indeed responsible for catalysis. However, a 50 G22T mutant exhibited a kobs of 1.1 min 1, and a 50 C29T mutant exhibited a kobs of 0.28 min 1, indicating that G15-30 does not utilize an 8–17 motif. Remarkably, the 59 nt version of G15-30 in con- junction with a truncated RNA substrate (59SS) is about 6-fold more active than the original 119 nt version. We used mfold to facilitate the design of a trans-acting version, called G15-30T, that engages a shortened RNA substrate through the formation of two duplex stems ﬂanking the cleavage site (Figure 5C). The P1 primer-binding site is predicted to act as part of the 50 substrate-binding arm of G15-30T, thereby deﬁning the boundaries of the minimal catalytic core. Nevertheless, addi- tional conﬁrmatory deletion tests were conducted on the 50 end of this trans-acting deoxyribozyme, and the results were con- sistent with the proposed structure model (data not shown). Under single-turnover conditions, G15-30T exhibits biphasic kinetics with a kobs of 2.48 min 1 (Ymax ¼ 52%) in the ﬁrst phase, and 0.11 min 1 (Ymax ¼ 24%) in the second phase (Figure 5D). G15-30T is also capable of multiple substrate turnovers. Over a 60 min incubation period, in the presence of 10 nM deoxyribozyme and 1 mM substrate, 41% of substrate was cleaved corresponding to 41 turnovers (Figure 5E). Further characterization and optimization of G15-30T represents a future objective. DISCUSSION Herein, we have investigated the effects of excess sequence elements on a sample of RNA-cleaving deoxyribozymes isol- ated from a prior in vitro selection experiment. Despite an overall length of 119 nt, we have provided evidence indic- ating that catalysis in 10 of these long deoxyribozymes is supported by the short 8–17 motif. Presumably, this ﬁnding is also true for 94% of the 110 sequence classes previously isolated, which also contain variants of the 8–17 motif. More- over, we have demonstrated that excess sequence elements in both the deoxyribozyme and substrate domains have only a minimal effect on activity (positive or negative), suggesting that these 8–17 motifs survived increasing selection pressure despite the presence of excess sequence elements, rather than because of them. In contrast, only a few new deoxyribozymes were identiﬁed, and only one of these (G15-30) could compete with 8–17 under stringent time pressure. A minimized version of this deoxyribozyme is 2.5 times larger than 8–17, and experienced a 4-fold greater inhibitory effect from excess sequence elements than the average 8–17 motif. Collectively, these results suggest that 8–17 may be less susceptible to the potential inhibitory effect of excess sequence elements than larger motifs, which could help to account for its widespread recurrence. However, a more systematic study is necessary to conﬁrm this hypothesis. It remains possible that the minimal effects observed herein could be an artifact of the relative location of the 8–17 motifs, all of which were found at the 50 end of the DNA domain. Additional factors including a large catalytic rate, a compact size, loose sequence requirements, substrate cleavage versatility and metal ion cofactor versatil- ity, make 8–17 arguably one of the best solutions to deoxyri- bozyme mediated RNA-cleavage during in vitro selection. For these reasons, we suspect this motif will continue to ‘hijack’ future in vitro selection experiments, and hinder the discovery of novel RNA-cleaving deoxyribozymes. Not unlike 8–17, other simple functional motifs, such as the isoleucine RNA aptamer (42,43), ATP binding DNA aptamer (44), hammer- head ribozyme (45,46) and a small RNA ligase deoxyri- bozyme (13) have also been isolated on more than one occasion through in vitro selection. The general factors that inﬂuence the likelihood of recurrence have been addressed elsewhere (47). The work presented herein complements the results of a previous study that also sought to characterize the effect of excess arbitrary sequence, using RNA ligase ribozymes as a model system (6). Our results provide additional support to indicate that the potential inhibitory effect of excess arbitrary sequence does not represent a signiﬁcant deterrent against the use of longer random libraries. However, we were not able to reconcile the purported theoretical advantage of longer ran- dom libraries with our experimental outcome, in which a majority of small catalysts were isolated that did not signiﬁc- antly utilize additional nucleotides to their beneﬁt. To date, 18 other in vitro selection experiments have been conducted for the purpose of isolating RNA-cleaving deoxyribozymes, and the average length of the random-sequence domain has been 46 nt (10,11,27,28,30,32,38,48–58). By exploiting a library with 30 additional random-sequence nucleotides, we origin- ally expected a higher degree of structural diversity in the resulting population of catalysts. The recurrence of 8–17 in this study and elsewhere, alludes to the rarity of novel RNA- cleaving motifs (with comparable or higher activity) and/or reﬂects the limitations in our ability to access them through in vitro selection. A recent computational study that used graph theory to characterize the distribution of possible secondary structure motifs associated with various library lengths, has shown that typical library sizes strongly favour simple topo- logical structures (59). Although structural complexity was shown to increase with library length, the actual beneﬁt