2007;8:603C606

2007;8:603C606. binding region of the target molecule. Aptamers have inherent advantages that merit application as therapeutic agents (10): (i) the ability to withstand high heat and denaturants, (ii) rapid chemical synthesis, (iii) small size (10C20?000?Da versus 150?000?Da for antibodies) and (iv) non-immunogenicity (11). In therapeutic applications, antibodies are limited by large size and the consequent inability to easily diffuse extravascularly or to penetrate large solid tumors (12). Typical monovalent aptamers are potentially limited by reduced retention times Decursin on the target cell and lack of crosslinking and subsequent activation of targets. Aptamer-based bivalent ligands, however, have been demonstrated to increase affinity and function compared to the monovalent versions; for example, bivalent aptamers were used to activate thrombin and T cells (13C15). Recently, selection of a high-affinity DNA aptamer (TD05) reactive with Burkitts lymphoma was reported (16). At 4C, TD05 binds to an epitope on B-cell surface mIgM BCR, exclusively expressed on B cells and most B-cell lymphomas (17). Aptamer TD05 is not useful for diagnostic and therapeutic applications if the epitope was also present on circulating IgM, which is found in the plasma at 450C1500?mg/l (18). To address these issues and to produce aptamers with potential medical applications, we first truncated TD05, and further optimized it by introducing locked nucleic acids Decursin (LNA) to increase nuclease resistance and conformational stability. The construct was additionally redesigned into bivalent, trivalent and tetravalent scaffolds in order to improve the affinity, and possibly to create an agent that could crosslink the BCR, which might have the biological effect of modulating the cell surface expression of the BCR, internalizing the complex, or activating or deactivating signaling pathways (19,20). We report the rational engineering of multivalent aptamer scaffolds that show higher thermal and nuclease stability, conformational stability, improved kinetic and biochemical properties at physiological temperatures. MATERIALS AND METHODS Cell lines, Ramos (Burkitts lymphoma), Daudi (Burkitts lymphoma), Raji (Burkitts lymphoma), Jeko (mantle cell lymphoma), SKLY-16 (B-cell lymphoma), CRW22R (Prostate cancer), H5V (Endothelial cells), HCT116 (Colorectal carcinoma), HEK293 (human embryonic kidney), HeLa (human adenocarcinoma cervical), K562 (leukemia chronic myelogenous), MOLT (acute lymphoblastic leukemia), SKOV-3 (human adenocarcinoma ovarian), HL60 (acute myelocytic leukemia), Jurkat (T lymphocyte) and SKLY-18 (B-cell lymphoma) were purchased from ATCC except for SKLY16 and 18. All of the cells were cultured in RPMI 1640 medium supplemented with 100?U/ml penicillinCstreptomycin and 10% fetal bovine serum (heat-inactivated; Invitrogen). Clinical samples were obtained from patients at Memorial Sloan Kettering Cancer Center CXCL5 or from healthy donors, on IRB approved protocols. Phosphoramidites including spacer phosphoramidite 18, amino Decursin modifier C6-dT, 5-fluorescein phosphoramidite, Cy3? phosphoramidite, and all the DNA reagents that are needed for DNA synthesis were purchased from Glen Research. LNA dT and dC were purchased from Exiqon, TetVA.8?S, L-TetVA.8?S were purchased from Trilink Biotechnologies Inc. All the DNA oligo sequences were chemically synthesized attaching a fluorophore at the 5 end using standard solid phase phosphoramidite chemistry on an ABI394 DNA synthesizer using either a 0.2?mol or 1?mol scale. The completed DNA sequences were de-protected. The crude product was purified using HPLC (Beckman Coulter System Gold Bioessential 125/168 diode-array detection instrument) equipped with a C-18 column (Dyanamax 250??10?mm, Varian) using 0.1M TEAA as the mobile phase. The length of each DNA construct was confirmed using 10%-TBE urea polyacrylamide gel electrophoresis. Full-length DNA was quantified by measuring the absorbance at 260?nm and absorbance of the corresponding dye at the 5 position using a Cary Bio-100 UV-Visible spectrophotometer (Varian). Sequences used in nuclear Decursin magnetic resonance (NMR) experiments were further dialyzed overnight with 0.5?mM NaHPO4 buffer using a MWCO 1000-Da dialysis bag. All the experiments were done using a binding buffer composed of RPMI 1640 and 4.5?g/l glucose (Sigma-Aldrich) and 5?mM MgCl2 for monomers, 20?mM MgCl2 for multimers (Sigma-Aldrich), 100?mg/l tRNA (Sigma-Aldrich), 100?mg/l single-stranded DNA (Sigma-Aldrich),.