1600 Nucleic Acids Research, 2010, Vol. 38, No. 5 Figure 2. Biochemical characterization of representative 3D8 VL variants selected against ss-DNA G18 (4MG3 and 4MG5) and Her218 ss-DNA (4MH2), compared with 3D8 VL WT and 4M. (A) DNA hydrolyzing activity analyses of primarily isolated 3D8 VLs selected against ss-DNA G18 (4MG1-4MG6) and Her218 ss-DNA (4MH1-4MH5) by agarose gel electrophoresis, compared with 3D8 VL WT and 4 M. The supercoiled plasmid of pUC19 (2.2 nM) was incubated with 3D8 VLs (5 mM) for 1 h at 37 C in the TBS buﬀer, pH 7.4, containing 2 mM MgCl2 (indicated as ‘Mg’) or 50 mM EDTA (indicated as ‘E’). The reaction mixtures were analyzed by electrophoresis on 0.7% agarose gels, and then stained with ethidium bromide. The arrows indicate supercoiled (sc), linear (lin) and relaxed circular (rc) DNAs. The samples incubated with only buﬀer alone and molecular mass markers were designated as ‘B’ and ‘M’, respectively. (B) RNA-hydrolyzing activity of 3D8 VLs, including WT, 4M, G18-selective 4MG3 and 4MG5, and Her218-selective 4MH2. Total cellular RNA (1 mg) extracted from HeLa cells were incubated at 37 C for 2 h in TBS buﬀer, pH 7.4, containing 2 mM MgCl2 (indicated as ‘Mg’) or 50 mM EDTA (indicated as ‘E’) with 3D8 VLs (0.1 mM), RNase A (1 U), irrelevant HW1 scFv protein (0.1 mM) as a negative control prior to gel electrophoresis. The bands corresponding to rRNAs of 28S and 18S are indicated. (C) SEC elution proﬁles of representative 3D8 VLs ( 12 kDa). Each protein (20 mM 260 mg/ml in TBS, pH7.4) indicated in diﬀerent lines was injected and chromatograms were obtained by absorbance at 280 nm. Arrows indicate the elution positions of mass standard markers (Sigma) [BSA (66 kDa), ovalbumin (45 kDa), chymotrypsinogen A (25 kDa), ribonuclease A (13.7 kDa)]. (D) Far-UV CD spectra of representative 3D8 VLs (100 mg/ml in TBS, pH7.4) to monitor the secondary structure are shown. S4). First, we investigated whether the isolated 3D8 VL variants maintained DNA-hydrolyzing catalytic activity by agarose gel electrophoresis using the supercoiled plasmid of pUC19 as a substrate. Most of the variants eﬃciently hydrolyzed the substrate in the presence of Mg2+, but not EDTA, demonstrating that they retain DNA-hydrolyzing activity in a Mg2+-dependent manner (Figure 2A), similar to the parents of 3D8 VL WT and 4M (12,13). Among them, we focused further investigation on 4MG3 and 4MG5 isolated against G18 as well as 4MH2 isolated against Her218, which showed the most eﬃcient DNA-hydrolyzing activities. The chosen 3D8 VL variants, including 3D8 VL WT and 4 M, also eﬃciently hydrolyzed total cellular RNAs extracted from HeLa cells even in the presence of EDTA (Figure 2B), demonstrating that 3D8 VLs possess RNase activity without requirement of divalent metal ions, like RNase A (24). However, an irrelevant HW1 scFv (15) puriﬁed from bacterial cells using the same procedure with that of 3D8 VLs did not show the RNA- hydrolyzing activity. This demonstrated that the RNA- hydrolyzing activity of 3D8 VLs was their intrinsic property, but not due to co-puriﬁed bacterial nucleases. Taken altogether, the 3D8 VLs have both DNase and RNase activity in vitro, like 3D8 scFv (7,12,13). Size exclusion chromatography (SEC) analyses demonstrated that 3D8 VLs (20 mM 260 mg/ml) were eluted as single peaks that correspond to an apparent