R targeting CXCL12 for treatment of multiple myeloma and lymphoma are already in clinical trials [16,17]. One chief problem that arises in the therapeutic application of aptamers is their instability under in vitro and in vivo conditions [18]. They are susceptible to enzymatic nuclease attack in the cellular and serum fluids. To circumvent this problem, several chemical modification strategies have been employed to enhance their resistance against nucleases and to prolong their circulation half-life in the biological fluids. Such chemical modifications include incorporation of SIS3 site phosphorothioate linkages (PS-linkages) or DprE1-IN-2 locked nucleic acids (LNAs), addition of functional groups such as amino (-NH2), fluoro (-F), O-methyl (-OCH3) in 29-position of ribose sugar, and conjugation to high molecular mass polyethylene glycol (PEG) or cholesterol [19?5]. Studies have demonstrated that, compared to the unmodified version, the chemically modified aptamers exhibit not only longer lifetime in the biological milieu but sometimes also better binding affinity and specificity to their targets [21,26]. VEGF is a crucial angiogenic mitogen overexpressed in the tumor cells and induces their migration, excessive proliferation, invasion and metabolism inside the body. VEGF is considered to be the hallmark protein for tumor angiogenesis and has been associated with neoplastic transformation of cells inside the body [27]. It is generally thought to be secreted by endothelial cells to stimulate their proliferation and migration. Previous reports, however, indicate that different carcinoma and malignant mesothelioma cell lines also secrete this protein [28?1]. VEGFAntiproliferative Activity of Aptamer on Canceris the pre-dominant isoform of VEGF-A protein, one of the members of VEGF family, and primarily binds to its two tyrosinekinase receptors VEGFR-1/Flt-1 and VEGFR-2/KDR/Flk-1 with very high 1531364 affinity and to specific co-receptor neuropilins [27]. The mitogenic signaling and cell proliferation in tumor cells is induced by expression of VEGFR-2 [32,33]. In contrast, activation of VEGFR-1 results in cell invasion and cell migration but not cell proliferation [34?6]. In our previous study, a 26-mer DNA aptamer against heparin binding domain (HBD) of VEGF165 protein (referred to as SL2-B) was obtained using stem-loop truncation strategy [37]. Compared to the original untruncated aptamer, the SL2-B aptamer exhibited more than 200-fold increase in the binding affinity to VEGF165 protein. Herein, we modified the SL2-B aptamer by incorporating phosphorothioate (PS) linkages, tested its binding affinity, specificity, biostability, secondary structure and the potential feasibility of the PS-modified SL2-B aptamer as antagonist on the proliferation activity of cancer cells. We demonstrated that, compared to unmodified SL2-B aptamer, the PS-modified SL2-B aptamer is an improved sequence in terms of serum stability and antiproliferative activity without sacrificing the binding affinity and specificity for VEGF165 protein.(PVDF) membrane, wet pico chemiluminescence substrate and CL-exposure film were purchased from thermo scientific. The FITC annexin V apoptosis detection kit was purchased from BD Pharmingen, Germany. PMSF was purchased from CalBiochem.Surface Plasmon Resonance (SPR) SpectroscopyThe binding affinity and specificity of modified aptamer sequence was investigated using surface plasmon resonance (SPR) spectroscopy, where VEGF165 and VEGF121 acted as ligands and.R targeting CXCL12 for treatment of multiple myeloma and lymphoma are already in clinical trials [16,17]. One chief problem that arises in the therapeutic application of aptamers is their instability under in vitro and in vivo conditions [18]. They are susceptible to enzymatic nuclease attack in the cellular and serum fluids. To circumvent this problem, several chemical modification strategies have been employed to enhance their resistance against nucleases and to prolong their circulation half-life in the biological fluids. Such chemical modifications include incorporation of phosphorothioate linkages (PS-linkages) or locked nucleic acids (LNAs), addition of functional groups such as amino (-NH2), fluoro (-F), O-methyl (-OCH3) in 29-position of ribose sugar, and conjugation to high molecular mass polyethylene glycol (PEG) or cholesterol [19?5]. Studies have demonstrated that, compared to the unmodified version, the chemically modified aptamers exhibit not only longer lifetime in the biological milieu but sometimes also better binding affinity and specificity to their targets [21,26]. VEGF is a crucial angiogenic mitogen overexpressed in the tumor cells and induces their migration, excessive proliferation, invasion and metabolism inside the body. VEGF is considered to be the hallmark protein for tumor angiogenesis and has been associated with neoplastic transformation of cells inside the body [27]. It is generally thought to be secreted by endothelial cells to stimulate their proliferation and migration. Previous reports, however, indicate that different carcinoma and malignant mesothelioma cell lines also secrete this protein [28?1]. VEGFAntiproliferative Activity of Aptamer on Canceris the pre-dominant isoform of VEGF-A protein, one of the members of VEGF family, and primarily binds to its two tyrosinekinase receptors VEGFR-1/Flt-1 and VEGFR-2/KDR/Flk-1 with very high 1531364 affinity and to specific co-receptor neuropilins [27]. The mitogenic signaling and cell proliferation in tumor cells is induced by expression of VEGFR-2 [32,33]. In contrast, activation of VEGFR-1 results in cell invasion and cell migration but not cell proliferation [34?6]. In our previous study, a 26-mer DNA aptamer against heparin binding domain (HBD) of VEGF165 protein (referred to as SL2-B) was obtained using stem-loop truncation strategy [37]. Compared to the original untruncated aptamer, the SL2-B aptamer exhibited more than 200-fold increase in the binding affinity to VEGF165 protein. Herein, we modified the SL2-B aptamer by incorporating phosphorothioate (PS) linkages, tested its binding affinity, specificity, biostability, secondary structure and the potential feasibility of the PS-modified SL2-B aptamer as antagonist on the proliferation activity of cancer cells. We demonstrated that, compared to unmodified SL2-B aptamer, the PS-modified SL2-B aptamer is an improved sequence in terms of serum stability and antiproliferative activity without sacrificing the binding affinity and specificity for VEGF165 protein.(PVDF) membrane, wet pico chemiluminescence substrate and CL-exposure film were purchased from thermo scientific. The FITC annexin V apoptosis detection kit was purchased from BD Pharmingen, Germany. PMSF was purchased from CalBiochem.Surface Plasmon Resonance (SPR) SpectroscopyThe binding affinity and specificity of modified aptamer sequence was investigated using surface plasmon resonance (SPR) spectroscopy, where VEGF165 and VEGF121 acted as ligands and.
