Table 1. kobs and krel values of different truncation derivatives for each sequence class Table 2. Ymax and Ymax,rel values of different truncation derivatives for each sequence class Deoxyribozyme kobs (min 1) L S SS krel PBA PBAA S/L SS/S PBAA/S Deoxyribozyme SS PBA PBAA Ymax,rel S/L SS/S PBAA/S G15-1 G15-16 G15-39 G15-35* G15-47 G15-48* G15-49 G13-20* G10-47* G8-1 Average Median Maximum Minimum 1.0 0.83 0.76 0.10 0.65 2.1 1.5 2.7 3.3 — 1.7 2.1 1.8 2.0 — 0.87 0.90 1.2 0.06 0.72 0.76 0.38 0.46 0.69 — 0.61/0.024 0.73 0.90 0.06 0.72 0.56 0.57 0.92 1.2 — 0.36/0.025 0.31 0.86 0.06 0.72 0.24 0.36 0.45 0.06 0.72 0.14 0.20 0.20 0.24 — 0.8 0.7 1.3 1.0 0.5 1.2 1.0 0.9 1.5 1.4 1.0 1.0 1.5 0.5 0.9 1.8 0.8 1.3 1.2 1.2 1.6 2.8 1.2 1.0 1.4 1.2 2.8 0.8 0.8 2.2 0.9 0.8 1.8 1.0 2.1 2.3 2.0 1.2 1.5 1.5 2.3 0.8 G15-1 G15-16 G15-39 G15-35* G15-47 G15-48* G15-49 G13-20* G10-47* G8-1 Average Median Maximun Minimum Nucleic Acids Research, 2006, Vol. 34, No. 8 2449 Ymax (%) L S 72 66 45 78 75 60 74 76 62 70 66 61 55 62 58 26/34 58 59 65 21 20 35/32 67 76 66 66 69 59 58 57 49 83 85 42 83 42 76 42 42 55 66 — — 63 — 63 — 63 63 — 0.9 1.0 1.0 0.9 1.1 1.0 0.3 1.0 1.0 1.0 0.9 1.0 1.1 0.3 0.7 0.8 0.8 0.9 0.9 1.0 1.0 1.1 1.0 1.0 0.9 1.0 1.1 0.7 1.0 1.1 1.1 1.0 1.3 1.1 3.7 0.9 1.0 0.9 1.3 1.0 3.7 0.9 Data are the average of at least two independent trials, which typically differed by <20%. krel is defined as the activity of one construct (numerator) relative to another construct (denominator), as indicated in the table. All values have been rounded for clarity, with krel values calculated prior to rounding. G15-48 and G13-20 displayed biphasic kinetics and therefore have two different kobs values representing the first and second phase, respectively. Deoxyribozymes denoted with an asterisk contain the same 8–17 catalytic core. PBAA represents the same construct as PBA, but with an extra adenosine residue in the A15.0 position of the single-stranded turn region. Values in the PBAA/S column were determined from either the PBAA or PBA column. Reaction conditions: 400 mM NaCl, 100 mM KCl, 7.5 mM MnCl2, 7.5 mM MgCl2, 50 mM HEPES (pH 7.0, at 23 C) at room temperature. RNA substrate lacking 24 nt from the 30 end, denoted by ‘SS’ and (iv) a 30 truncated enzyme containing only the 8–17 motif and a perfect 30 binding arm containing 10 consecutive Watson–Crick base pairs, denoted by ‘PBA’. All rates were obtained using single-turnover cis constructs. However, each class is also active in a bimolecular trans format (Supplemen- tary Figure 1). Table 1 summarizes the results of these trun- cation experiments in terms of their effect on the catalytic rate constant, kobs. Since a deoxyribozyme’s ability to fold into an active conformation is critical for function, we have also sum- marized the results of the truncation experiments in terms of their effect on the maximum cleavage yield, denoted by Ymax in Table 2. Genotype to phenotype relationship The results shown in Table 1 indicate that the catalytic rate of each class closely parallels its abundance in the population. For instance, classes such as G15-1, G15-16 and G15-39 that boast the fastest rates also represent the largest fraction of the G15 population (see Figure 1B for class frequency). In con- trast, the classes that died out in earlier generations (i.e. G13-20, G10-47 and G8-1), not surprisingly, have the lowest rates. Similarly, the most abundant classes may beneﬁt from a slight folding advantage over others, as reﬂected by the higher Ymax values shown in Table 2. The relative activity of the different 8–17 motifs is also consistent with previous reports (34). The effect of excess sequence elements The average effect of the excess 30 sequence in the deoxyri- bozyme domain is neutral, although an even distribution of Data are the average of at least two independent trials, which typically differed by less than 5%. Ymax is defined as the maximum cleavage yield as determined from the best-fit curve using GraphPad Prism 4. Ymax,rel is defined as the maximum cleavage of one construct (numerator) relative to another construct (denominator), as indicated in the table. All values have been rounded for clarity, with Ymax,rel values calculated prior to rounding. G15-48 and G13-20 displayed biphasic kinetics and therefore have two different Ymax values representing the amplitude of the first and second phase, respectively. Deoxyribozymes denoted with an asterisk contain the same 8-17 catalytic core. both beneﬁcial and detrimental effects is observed (Table 1, column S/L). The magnitude of these effects, however, is relatively modest with a 50% increase or decrease in activity representing the maximum change. This modest change in activity suggests that the excess 30 sequence is acting only to mediate the stability of 8–17’s active fold, likely through the formation of the 30 substrate-binding arm. The average effect of deleting excess sequence from the RNA substrate domain is a 40% increase in activity (Table 1, column SS/S). The activity of seven classes were inhibited by the excess ribonucleotides in varying amounts, one was neutral (G8-1), and the remainder were associated with a slightly beneﬁcial effect (G15-1, G15- 39). This variation between classes is noteworthy, since the RNA sequence is constant and presumably can form the same 50 substrate-binding arm with P1 in each class. Therefore, the magnitude of the effect is likely inﬂuenced by downstream elements including the stability of the 30 substrate-binding arm and perhaps even the type of 8–17 motif. On average, the activity of the G15 classes appear to be less affected by the presence of excess sequence elements in both the deoxyri- bozyme and substrate domains, than classes that died out in earlier generations (krel,S/L,G15avg ¼ 0.9 vs. krel,S/L,<G15avg ¼ 1.3; krel,SS/S,G15avg ¼ 1.3 vs. krel,SS/S,<G15avg ¼ 1.7). This effect closely parallels the greater stability associated with the 30 substrate binding arm of G15 classes (krel,PBA/S,G15avg ¼ 1.4 versus krel,PBA/S,<G15avg ¼ 1.8). As expected, optimized 30 binding arms resulted in an aver- age increase in catalytic activity ( 50%), although three classes (G15-1, G15-39 and G15-35) actually decreased in activity ( 10–20%), contrary to expectations (Table 1, column PBAA/S). This decrease can probably be attributed in part to experimental error, but also to the peculiarities associated with G15-1 and G15-35 (and related classes G15-48, G13-20 and G10-47). These classes have only a sin- gle adenosine residue in the terminal A15 position of the