Ication involves the substitution of unbridged phosphoryl oxygen inAntiproliferative Activity of Aptamer on CancerFigure 3. Nuclease-resistance stability of unmodified and modified SL2-B aptamer sequence in 10 FBS. Aptamers were incubated with 10 FBS dissolved in DMEM media at 37uC for different time points and percentage of intact aptamer was determined by measuring the band density after running denaturing PAGE. Filled columns are PS-modified SL2-B, while open columns are unmodified SL2-B. doi:10.1371/GHRH (1-29) journal.pone.0050964.gphosphodiester linkage by sulfur atom. Since the excess incorporation of 22948146 PS-linkages leads to non-specific binding and can perturb the aptamer conformation and its interaction with the target, the modification was introduced only at aptamer termini [38]. The Kd value for PS-modified SL2-B aptamer was determined using SPR technique at different aptamer concentrations (Figure 1 and Table 1). The Kd value for the PS-modified SL2-B was found to be 0.56 nM, which is similar to the Kd for unmodified SL2-B. Introducing PS-modification does not appear to affect the binding affinity of the SL2-B aptamer. Moreover, the affinity of PSmodified SL2-B is similar to the FDA approved humanized antiVEGF monoclonal antibody “bevacizumab” (Kd , 0.5 nM) used for cancer treatment [4].Specificity of PS-modified SL2-B Aptamer SequenceVEGF165 as well as 
other VEGF isoforms, such as VEGF189 and VEGF206, are generated from splicing of a single VEGF gene that shares a carboxyl-terminal heparin-binding domain (HBD) of 50-residues and binds to heparin with different binding affinities [27,39,40]. HBD is responsible for enhancing the interaction of VEGF with its receptors (VEGFR-1/Flt-1 and 
VEGFR-2/KDR/Flk-1) and the specific co-receptor neuropilins to trigger the angiogenic response in malignant cells [41]. VEGF121, however, does not share the HBD as other VEGF isoforms and can be used as a control for HBD binding specificity study. The SPR sensorgram in Figure 2 shows that compared to VEGF165 protein at same aptamer concentration (80 nM), the response signal of PS-modified SL2-B binding to VEGF121 protein was weak and displayed a high Kd 23727046 value of 17 mM. This indicates that PS modification does not reduce the binding specificity of SL2-B aptamer towards HBD significantly (Kd = 17 mM for PSmodified SL2-B towards VEGF121, Kd = 10 mM for unmodified SL2-B towards VEGF121). Compared to the “bevacizumab” monoclonal antibody that binds to all isoforms of VEGF, the PS-modified SL2-B is specific to HBD of VEGF165 protein [4]. Since VEGF-A is involved in normal physiological processes, such as formation of new blood vessels and wound healing process, the complete inhibition of VEGF protein can affect the maintenance of the normal vascular system inside the body [42,43]. Therefore, inhibition of specific VEGF protein (for example, VEGF165 in this case) may be a better therapeutic approach.Antiproliferative Activity of Aptamer on CancerFigure 4. CD spectra of 10 mM PS-modified SL2-B aptamer in phosphate buffer saline (PBS) buffer, pH-7.2. Spectra were measured at 256C (solid line) and 376C (dotted line). doi:10.1371/journal.pone.0050964.gFigure 5. Relative proliferation of Hep G2 cells (compared to control) after treating with unmodified and PS-modified SL2-B aptamers at different concentrations in hypoxia conditions. The sequence specificity was determined using (-)-Calyculin A manufacturer scrambled sequence for PSmodified SL2-B for each data point at same concentration to t.Ication involves the substitution of unbridged phosphoryl oxygen inAntiproliferative Activity of Aptamer on CancerFigure 3. Nuclease-resistance stability of unmodified and modified SL2-B aptamer sequence in 10 FBS. Aptamers were incubated with 10 FBS dissolved in DMEM media at 37uC for different time points and percentage of intact aptamer was determined by measuring the band density after running denaturing PAGE. Filled columns are PS-modified SL2-B, while open columns are unmodified SL2-B. doi:10.1371/journal.pone.0050964.gphosphodiester linkage by sulfur atom. Since the excess incorporation of 22948146 PS-linkages leads to non-specific binding and can perturb the aptamer conformation and its interaction with the target, the modification was introduced only at aptamer termini [38]. The Kd value for PS-modified SL2-B aptamer was determined using SPR technique at different aptamer concentrations (Figure 1 and Table 1). The Kd value for the PS-modified SL2-B was found to be 0.56 nM, which is similar to the Kd for unmodified SL2-B. Introducing PS-modification does not appear to affect the binding affinity of the SL2-B aptamer. Moreover, the affinity of PSmodified SL2-B is similar to the FDA approved humanized antiVEGF monoclonal antibody “bevacizumab” (Kd , 0.5 nM) used for cancer treatment [4].Specificity of PS-modified SL2-B Aptamer SequenceVEGF165 as well as other VEGF isoforms, such as VEGF189 and VEGF206, are generated from splicing of a single VEGF gene that shares a carboxyl-terminal heparin-binding domain (HBD) of 50-residues and binds to heparin with different binding affinities [27,39,40]. HBD is responsible for enhancing the interaction of VEGF with its receptors (VEGFR-1/Flt-1 and VEGFR-2/KDR/Flk-1) and the specific co-receptor neuropilins to trigger the angiogenic response in malignant cells [41]. VEGF121, however, does not share the HBD as other VEGF isoforms and can be used as a control for HBD binding specificity study. The SPR sensorgram in Figure 2 shows that compared to VEGF165 protein at same aptamer concentration (80 nM), the response signal of PS-modified SL2-B binding to VEGF121 protein was weak and displayed a high Kd 23727046 value of 17 mM. This indicates that PS modification does not reduce the binding specificity of SL2-B aptamer towards HBD significantly (Kd = 17 mM for PSmodified SL2-B towards VEGF121, Kd = 10 mM for unmodified SL2-B towards VEGF121). Compared to the “bevacizumab” monoclonal antibody that binds to all isoforms of VEGF, the PS-modified SL2-B is specific to HBD of VEGF165 protein [4]. Since VEGF-A is involved in normal physiological processes, such as formation of new blood vessels and wound healing process, the complete inhibition of VEGF protein can affect the maintenance of the normal vascular system inside the body [42,43]. Therefore, inhibition of specific VEGF protein (for example, VEGF165 in this case) may be a better therapeutic approach.Antiproliferative Activity of Aptamer on CancerFigure 4. CD spectra of 10 mM PS-modified SL2-B aptamer in phosphate buffer saline (PBS) buffer, pH-7.2. Spectra were measured at 256C (solid line) and 376C (dotted line). doi:10.1371/journal.pone.0050964.gFigure 5. Relative proliferation of Hep G2 cells (compared to control) after treating with unmodified and PS-modified SL2-B aptamers at different concentrations in hypoxia conditions. The sequence specificity was determined using scrambled sequence for PSmodified SL2-B for each data point at same concentration to t.
