Table 1. Kinetic binding parameters for the interactions of 3D8 VLs with target and oﬀ-target 18-bp ss-DNAs or ss-RNAs, which were monitored by SPRa Nucleic Acids Research, 2010, Vol. 38, No. 5 1601 ss-DNA substrates (ss-RNA substrates)b Her218(ss-RNA) N18(ss-RNA) C18 0.78 ± 0.04 9.73 ± 0.88 126 ± 13 5.81 ± 0.25 5.88 ± 0.71 10.1 ± 1.6 3.07 ± 0.32 3.53 ± 0.41 115 ± 14 0.51 ± 0.03 1.92 ± 0.11 37.7 ± 2.9 0.43 ± 0.03 4.31 ± 0.87 100 ± 22 G18[ss-RNA (G4U)3G3]c 0.71 ± 0.01 (0.39 ± 0.02) 8.26 ± 0.62 (6.27 ± 0.15) 116 ± 15 (159 ± 12) 0.49 ± 0.13 (0.54 ± 0.09) 5.18 ± 0.10 (5.63 ± 0.45) 105 ± 23 (104 ± 19) 1.17 ± 0.3 (1.11 ± 0.08) 0.09 ± 0.01 (0.08 ± 0.01) 0.76 ± 0.04 (0.72 ± 0.05) 9.02 ± 0.77 (8.18 ± 0.08) 0.84 ± 0.05 (0.72 ± 0.04) 0.93 ± 0.03 (0.88 ± 0.07) 0.42 ± 0.07 (0.23 ± 0.01) 4.53 ± 0.82 (3.22 ± 0.17) 107 ± 3.4 (143 ± 11) 1.63 ± 0.30 (0.31 ± 0.02) 35.2 ± 2.3 (5.39 ± 0.24) 215 ± 5.3 (174 ± 15) 0.24 ± 0.02 (0.14 ± 0.01) 5.49 ± 0.47 (3.45 ± 0.98) 233 ± 2.7 (252 ± 19) 0.24 ± 0.04 (0.15 ± 0.02) 7.55 ± 0.89 (6.80 ± 0.55) 313 ± 24 (441 ± 23) 0.45 ± 0.05 (0.56 ± 0.07) 5.32 ± 0.82 (7.20 ± 0.31) 118 ± 29 (129 ± 28) 4.41 ± 0.13 (2.45 ± 0.10) 0.94 ± 0.07 (0.38 ± 0.02) 2.13 ± 0.13 (1.47 ± 0.27) 0.41 ± 0.03 (0.56 ± 0.02) 15.4 ± 2.7 (19.3 ± 1.3) 375 ± 4.2 (344 ± 16) 0.96 ± 0.02 (0.49 ± 0.13) 10.3 ± 1.8 (6.04 ± 0.28) 107 ± 3.5 (123 ± 27) 0.38 ± 0.02 (0.25 ± 0.03) 9.37 ± 0.97 (4.26 ± 0.36) 249 ± 31 (171 ± 12) 0.13 ± 0.03 (0.16 ± 0.03) 7.25 ± 0.87 (9.43 ± 0.22) 557 ± 54 (590 ± 22) 0.17 ± 0.01 (0.18 ± 0.02) 7.20 ± 0.66 (5.44 ± 0.90) 427 ± 29 (308 ± 21) 3D8 VLs WT 4M Kinetic parameters kon (M 1 s 1) ( 103) koﬀ (s 1) ( 10 3) KD (M) ( 10 7) kon (M 1 s 1) ( 103) koff (s 1) ( 10 3) KD (M) ( 10 7) koﬀ (s 1) ( 10 3) KD (M) ( 10 7) 4MG3 kon (M 1 s 1) ( 103) 4MG5 kon (M 1 s 1) ( 103) 4MH2 kon (M 1 s 1) ( 103) koﬀ (s 1) ( 10 3) KD (M) ( 10 7) koﬀ (s 1) ( 10 3) KD (M) ( 10 7) A18 0.13 ± 0.04 3.24 ± 0.21 239 ± 13 0.60 ± 0.02 7.32 ± 0.70 122 ± 12 0.14 ± 0.03 5.24 ± 0.95 371 ± 28 0.26 ± 0.02 7.92 ± 0.24 303 ± 19 0.13 ± 0.01 2.54 ± 0.21 194 ± 21 T18 0.67 ± 0.02 6.82 ± 0.13 102 ± 15 3.95 ± 0.21 5.80 ± 0.09 14.7 ± 1.6 0.25 ± 0.02 2.80 ± 0.19 112 ± 22 0.89 ± 0.04 3.13 ± 0.12 35.0 ± 3.6 0.25 ± 0.05 2.51 ± 0.61 103 ± 17 aEach value represents the mean ± SD of two independent experiments. In each experiment, at least ﬁve data sets were used in the determination of the kinetic constants. bThe values for ss-RNA substrates, G18, Her218 and N18, were presented in the parenthesis. cDue to the diﬃculty in synthesizing 50-biotinylated ss-RNA G18, ss-RNA (G4U)3G3 was used as the G18 substrate. molecular mass of each protein, demonstrating that they are existing in monomeric form in solution at concentra- tions higher than used in DNA- and RNA-hydrolyzing assays (Figure 2C). This result indicates that 3D8 VLs hydrolyze both DNA and RNA in monomeric form. Secondary structures of the 3D8 VL variants, 4MG3, 4MG5 and 4MH2, determined by far-UV circular dichroism (CD) spectroscopy exhibited very similar spectra to those of 3D8 VL WT and 4M, exhibiting a negative maximum of mean residue ellipticity 217 nm (Figure 2D), which is typical for the immunoglobulin fold (12). This data suggested that the extensive muta- tions incorporated into the inner b-strands of the 3D8 VL variants did not signiﬁcantly aﬀect the unique second- ary structure. Selected 3D8 VL variants preferentially bind to hydrolyze target substrates The speciﬁcity and aﬃnity of the selected 3D8 VLs for their respective target ss-DNA/RNA (G18 and Her218) and oﬀ- targets (A18, T18, C18 and N18) were evaluated using SPR technique (13). The kinetic binding parameters are summarized in Table 1. 3D8 VL WT and 4M exhibited indistinguishable binding aﬃnities (KD 10 5 M) for all tested ss-DNA/RNA substrates (12,13), except for the preferential bindings (KD 10 6 M) of 4M and 4MG5 for ss-DNA T18 and C18 substrates. In contrast, the 3D8 VL variants displayed 100–1000-fold tighter binding (KD 10 8 M of 4MG3 and 4MG5 against ss-DNA/RNA G18; KD 10 7 M of 4MH2 against ss-DNA/RNA Her218) to target substrates than to oﬀ-targets (KD 10 5 M), which was mainly due to the decreased dissociation rate constants (koﬀ) of the target substrates (Table 1). This result indicated that, even though 18-bp ss-DNAs were used as screening probes, the selected 3D8 VL variants maintained preferen- tial binding speciﬁcity for the target substrates with the same sequences in ss-RNA form. We next determined the target sequence-speciﬁc hydrolyzing activity of 3D8 VL variants by using a FRET-based cleavage assay with 18-bp target and oﬀ- target ss-DNAs as substrates, which were double-labeled with a ﬂuorophore (FAM) at 50-terminus and its quencher (BHQ -1) at 30-terminus (Figure 3) (7,18). The substrate- hydrolyzing can be monitored by following the ﬂuores- cence intensity increase caused by the 6-FAM release from its quencher BHQ -1 due to the hydrolysis (7,18). The reaction velocity of 3D8 VLs exhibited saturation