RESULTS Selection, identification and confirmation of the 8–17 catalytic motif We previously conducted an in vitro selection experiment to isolate RNA-cleaving deoxyribozymes (33), using the library design illustrated in Figure 1A. In addition to 80 random deoxyribonucleotides, this library featured an RNA substrate composed of 50 ﬁxed-sequence ribonucleotides. After per- forming sixteen rounds of selection, 50 clones were sequenced from each of generations 7, 8, 10, 13 and 15, which were subjected to increasing selection pressure in the form of decreasing reaction times of 5 h, 30 min, 5 min, 30 s and 5 s, respectively. From a total sample set of 245 sequenced clones, 110 unique sequence classes were identiﬁed. On aver- age, different classes shared only 30% nucleotide identity, but clones within the same class boasted >90% identity. Further analysis revealed the presence of the 8–17 motif in 94% of all classes. For simplicity, we use the term 8–17 to describe both the canonical motif (32) and all related sequence variants. The discovery of a motif as simple and well deﬁned as 8–17, in so many different sequences, provided a unique opportunity to gain further insight into the role of excess sequence ele- ments during in vitro selection. Toward this end, 11 sequence classes were characterized in greater detail. All eight classes identiﬁed in the terminal G15 population were investigated, including one that does not contain 8–17 or any other previ- ously characterized motif. These deoxyribozymes presumably represent the best catalysts, because they survived under the most stringent selection time. For comparison, we also selec- ted three classes that were observed only transiently in the preceding generations 8, 10 or 13. Figure 1B shows the sequence of a speciﬁc clone from each of the ten 8–17 con- taining classes that were examined, as well as the frequency Nucleic Acids Research, 2006, Vol. 34, No. 8 2447 and generation from which they were isolated (the non-8–17 class is addressed separately in a subsequent section). The 8–17 motif has been studied extensively (27,30–32,34,35), and is readily identiﬁable near the 50 end by its characteristic sequence requirements. The secondary structure and sequence requirements of the 8–17 motif are summarized in Figure 2A. The nucleotide numbering system used in Figure 2A is analogous to the sys- tem originally proposed for the hammerhead ribozyme (36), and subsequently adopted for the 8–17 deoxyribozyme (31,34). The canonical 8–17 motif as ﬁrst described by Santoro and Joyce (32), was composed of 14–15 nt (plus 7–8 nt binding arms on either side) and characterized by a G-T wobble base pair at the cleavage site, a 3 bp stem of which at least two were G-C base pairs, an invariant A6G7C triloop, and a single- stranded region of sequence WC13G14R (W ¼ A or T; R ¼ A or G or AA). Subsequent studies have suggested that the sequence requirements of 8–17 are far more versatile. A study by Cruz et al. (30) reported that only four residues are strictly conserved (A6 and G7 in the triloop, C13 and G14 in the single- stranded turn region), the triloop region can also accept a tetraloop, the 3 bp stem can accommodate up to 1 or 2 mis- matches and/or single-nucleotide bulges, and the single- stranded region has a more relaxed sequence requirement for positions W and R. Most recently, Peracchi et al. (34) have conducted a comprehensive mutational analysis to show the effect of different mutations on the catalytic activity of 8–17. From the results of Peracchi’s study, the approximate relative activity at different nucleotide positions (in Mn2+ buffer) is predicted to be as follows: T2.1>A>>G,C; C8>T>>G,A; T12>G>A>>C; A15A15.0>A>T>C>G. The mag- nitude and relative order of activities vary depending on the type and concentration of metal ion cofactors used. Further- more, the composition of the 3 bp stem is also associated with varying levels of activity. The magnitudes of these effects are Figure 1. (A) Library design. Numbers indicate the nucleotide length of each section. A1 is a piece of DNA used to facilitate the separation of cleaved and non-cleaved molecules in our gel-based selection method. The total length of the RNA substrate is 50 nt (see Materials and Methods for nucleotide sequence), and cleavage occurs at the 50 G11G12 junction. P1, P2 and P3 represent primer binding sites. P1 was used from G0-G16, P2 was used from G0-G7, and P3 was used from G8-G16. The putative deoxyribozyme domain contains 80 random nucleotides. (B) Sequences of specific clones from selected deoxyribozymes classes. Only the random sequence domain is shown. Each sequence is preceded by a numeric designation that indicates the generation from which it was taken (i.e. G#), and the specific clone number. Bracketed numbers at the far right of each sequence indicate the number of clones observed in that generation.