Table 2. Kinetic parameters of 3D8 VLs hydrolyzing activity with various 18-bp ss-DNAs and ss-RNAs, derived from the data given in Figure 3a Nucleic Acids Research, 2010, Vol. 38, No. 5 1603 ss-DNA substrates (ss-RNA substrates)b Her218(ss-RNA) N18(ss-RNA) 3D8 VLs WT 4M Kinetic parameters Km (nM) kcat (s 1) ( 10 3) kcat/Km (nM 1 s 1) ( 10 6) Km (nM) kcat (s 1) ( 10 3) kcat/Km (nM 1 s 1) ( 10 6) 4MG3 Km (nM) 4MG5 Km (nM) 4MH2 Km (nM) kcat (s 1) ( 10 3) kcat/Km (nM 1 s 1) ( 10 6) kcat (s 1) ( 10 3) kcat/Km (nM 1 s 1) ( 10 6) kcat (s 1) ( 10 3) kcat/Km (nM 1 s 1) ( 10 6) A18 644 ± 18 1.6 ± 0.1 2.5 ± 0.4 614 ± 16 1.3 ± 0.2 2.1 ± 0.1 684 ± 21 1.5 ± 0.1 2.2 ± 0.1 643 ± 16 1.4 ± 0.2 2.2 ± 0.3 758 ± 14* 1.2 ± 0.1* 1.6 ± 0.1* T18 568 ± 14 1.8 ± 0.1 3.2 ± 0.2 576 ± 15 1.2 ± 0.1* 2.1 ± 0.1* 829 ± 14* 1.9 ± 0.1 2.3 ± 0.2* 493 ± 11 1.7 ± 0.1 3.3 ± 0.1 558 ± 13 1.8 ± 0.13 3.3 ± 0.2 C18 522 ± 18 1.7 ± 0.1 3.3 ± 0.1 597 ± 17 1.9 ± 0.1* 3.2 ± 0.2* 512 ± 12 1.3 ± 0.1 2.6 ± 0.1 522 ± 13 2.0 ± 0.1 3.8 ± 0.3 527 ± 11 1.6 ± 0.1 3.0 ± 0.2 (G4T)3G3c 543 ± 12 1.9 ± 0.1 3.4 ± 0.2 525 ± 13* 1.5 ± 0.1 2.9 ± 0.1 297 ± 11* 2.1 ± 0.1* 7.1 ± 0.3* 352 ± 13* 2.8 ± 0.1* 7.8 ± 0.2* 459 ± 14 1.7 ± 0.2 3.7 ± 0.3 549 ± 13 (421 ± 19) 1.7 ± 0.1 (1.8 ± 0.1) 3.0 ± 0.2 (4.2 ± 0.3) 584 ± 15 (394 ± 12) 1.5 ± 0.1 (1.8 ± 0.1) 2.5 ± 0.2 (4.4 ± 0.3) 507 ± 14 (397 ± 14) 1.7 ± 0.1 (1.8 ± 0.1) 3.3 ± 0.2 (4.6 ± 0.2) 643 ± 12 (422 ± 8.6) 1.7 ± 0.1 (2.1 ± 0.1) 2.7 ± 0.1 (4.9 ± 0.2) 300 ± 14* (245 ± 12*) 2.2 ± 0.1* (2.4 ± 0.1*) 7.3 ± 0.6* (10.0 ± 0.3*) 587 ± 11 (441 ± 17) 1.7 ± 0.1 (1.7 ± 0.1) 2.8 ± 0.1 (4.0 ± 0.1) 576 ± 10 (404 ± 15) 1.5 ± 0.1 (1.6 ± 0.1) 2.6 ± 0.2 (4.0 ± 0.1) 559 ± 10 (427 ± 10) 1.6 ± 0.1 (1.9 ± 0.1) 2.8 ± 0.1 (4.5 ± 0.2) 601 ± 14 (415 ± 17) 1.5 ± 0.1 (2.0 ± 0.1) 2.5 ± 0.2 (4.9 ± 0.2) 459 ± 19 (395 ± 7) 1.9 ± 0.1 (2.0 ± 0.1) 4.1 ± 0.2 (5.1 ± 0.4) aEnzyme kinetic parameters were obtained by incubating each protein (100 nM) with various substrate concentrations (16 nM–2 mM) at 37 C, as described in Figure 3. All experiments were performed in triplicate and the results are represented as mean ± SD. Signiﬁcant diﬀerence of each kinetic parameter was determined using two-tailed Student’s t-test versus the control N18 substrate (*P < 0.01). bThe values for ss-RNA substrates, Her218 and N18, were presented in the parenthesis. cDue to the diﬃculty in synthesizing the double labeled ss-DNA G18 FRET substrate, ss-DNA (G4T)3G3 was used as the G18 substrate. for each substrate were not strictly correlated with the substrate aﬃnity (KD) obtained by SPR (Table 1), indicating a complicated catalytic mechanism in solution. Compared with endonuclease of RNA-induced silencing complex (RISC) involved in RNAi (25), the Km and kcat values of 3D8 VL variants were 10-fold higher and slightly lower, respectively, resulting in overally 10- fold lower catalytic eﬃciency for target substrates. 3D8 VLs eﬃciently internalize into living cells and accumulate in the cytosol The recently identiﬁed ability of 3D8 scFv to penetrate into living cells and accumulate in the cytosol without translocating to the nucleus (7) led us to investigate whether 3D8 VLs act similarly. Indeed, human cervical carcinoma HeLa and breast carcinoma SK-BR-3 living cells treated with 3D8 VLs (WT, 4MG3, 4MG5 and 4MH2) exhibited strong 3D8 VL-speciﬁc ﬂuorescence signal, compared with untreated cells (Figure 4A). Confocal ﬂuorescence microscopic analysis revealed that all of 3D8 VL proteins dominantly accumulated in the cytosol with little further traﬃcking into the nucleus (Figure 4B and Supplementary Figure S5), like 3D8 scFv (7). To our best knowledge, this is the ﬁrst report of single-domain antibodies with the ability to internalize into living cells. Soluble heparin substantially blocked the internaliza- tion of 3D8 VLs (Figure 4C), indicating that 3D8 VLs interact with negatively charged cell surface proteoglycans prior to internalization, like 3D8 scFv (7). Thus, the cell- penetrating ability of 3D8 VLs seems not to be cell type- speciﬁc, like other cell-penetrating anti-DNA antibodies and peptides (6,26). To elucidate the speciﬁc internal- ization mechanism, HeLa cells were pre-treated with the following pharmacological inhibitors prior to the addition of 3D8 VLs to interfere with the three major endocytic pathways: chlorpromazine (CPZ) to inhibit clathrin- dependent endocytosis, methyl-b-cyclodextrin (MbCD) to inhibit caveolae/lipid raft endocytosis, and cytochalasin D (Cyt-D) to inhibit macropinocycosis [please refer to the refs. (7,26) for details]. Flow cytometric analyses revealed that pre-incubation of cells with MbCD (5 mM), but neither with CPZ (10 mg/ml) nor Cyt-D (1 mg/ml), led to