1602 Nucleic Acids Research, 2010, Vol. 38, No. 5 Figure 3. Sequence-speciﬁc ss-DNA/RNA hydrolyzing assay of 3D8 VL variants selected against ss-DNA G18 (4MG3 and 4MG5) and Her218 (4MH2), compared with 3D8 VL WT and 4M. (A and B) Substrate-concentration dependent initial hydrolyzing velocity (V) of 3D8 VLs were plotted against various 18-bp ss-DNA substrate (S) concentrations (A) or 18-bp ss-RNA substrate (S) concentrations (B). Each protein (100 nM) was incubated at 37 C with the indicated substrates (16 nM to 2mM), which were double-labeled with 6-FAM at 50-terminus and its quencher BHQ-1 at 30-terminus and real-time ss-DNA/RNA hydrolyzing kinetic data were monitored by the ﬂuorescence intensity increase caused by the 6-FAM release from its quencher BHQ-1 due to the hydrolysis (7,18). Due to the diﬃculty in synthesizing double-labeled ss-DNA G18. 18-bp ss-DNA substrate of (G4T)3G3 was used as the G18 substrate. The detailed enzymatic kinetic parameters are shown in Table 2. All data points are represented as mean ± SD of three experiments. Lines through mean values represent a mathematical ﬁt of the data using the Michaelis–Menten equation. kinetics with respect to increasing substrate concen- tration, demonstrating that 3D8 VLs act as nucleic- acid-hydrolyzing enzymes with substrate preferences. Lineweaver–Burk plot analyses showed that 3D8 VL WT and 4M exhibited similar hydrolyzing eﬃciencies (kcat/Km 2.1–3.4 10 6 nM 1 s 1) with only slight dif- ferences in Km and kcat for all of the ss-DNA substrates tested (Table 2), conﬁrming their sequence non-speciﬁc hydrolyzing activity (12,13). In contrast, 3D8 VL variants hydrolyzed their respective target substrates 2–5-fold more eﬃciently (kcat/Km 7.1–7.8 10 6 nM 1 s 1) than the oﬀ-targets (kcat/Km 1.6– 4.1 10 6 nM 1 s 1) due to their improved substrate aﬃnity (Km) and catalytic rates (kcat) (Table 2), demonstrating that they preferentially recognized and degraded target substrates. Thus, we designated 4MG3 and 4MG5 as G18-selective and 4MH2 as Her218-selective 3D8 VL variants. When the sequence-selective hydrolyzing activity of 4MH2 was further evaluated for the substrates with the same sequence in ss-RNA form, 4MH2 exhibited 2-fold higher hydrolyzing eﬃ- ciency for the target ss-RNA Her218 (kcat/Km 10.0 10 6 nM 1 s 1) than the oﬀ-target ss-RNA N18 (kcat/Km 5.1 10 6 nM 1 s 1) (Table 2 and Figure 3B), like the cases for the ss-DNA Her218. However, the other 3D8 VLs showed indistinguishable catalytic eﬃciency for both ss-RNA Her218 and N18, indicative of their sequence-nonspeciﬁc hydrolyzing activities for even ss-RNAs. The catalytic eﬃciencies (kcat/Km) of 3D8 VLs for the ss-RNAs (Her218 and N18) were slightly higher than those for the corresponding ss-DNAs by the marginal improved substrate aﬃnity (Km) and turnover number (kcat) for the ss-RNAs, most likely due to the diﬀerence in metal ion-independent and -dependent catalytic mechanism for ss-RNAs and ss-DNAs, respec- tively (Figure 2). The Km values of the 3D8 VL variants