For lanes 8C10, T1 was incubated with concentrations of 20, 100, and 200?M, respectively. inhibitors that display an anti-HIV-1 fusion activity . Among these inhibitors, the most representative structure is Hotoda’s sequence, d(TGGGAG), which interacts with the V3 loop or CD4-binding site of viral glycoprotein 120 (gp120) [3C13]. Recently, we designed a novel class of DNA duplex-based HIV-1 fusion inhibitors, which have hydrophobic groups at several selected positions [14,15]. A fluorescent resonance energy transfer (FRET)-based inhibitory assay showed that these duplex inhibitors interact with the primary pocket at the glycoprotein 41 (gp41) N-terminal heptad repeat (NHR). No specific requirement for sequence composition was observed; however, a thermal stability (Tm) above the physiological heat (37C) was crucial for the activity. In structure, the quadruplex- and duplex-based inhibitors have aromatic substituents, as well as a rigid and negatively charged DNA helical skeleton. We hypothesized that DNA triplexes with (24S)-24,25-Dihydroxyvitamin D3 hydrophobic modifications at suitable Gja5 positions would display inhibitory activities against the fusion of HIV-1 to the cell membrane. This hypothesis was made because an aromatic substituent and a negatively charged helical skeleton can also be achieved on DNA triplex-based molecules. DNA triplex-based inhibitors may represent a novel category of HIV-1 inhibitors in anti-HIV-1 drug discovery and may be as important as the previously explained quadruplex- and duplex-based inhibitors. To the best of our knowledge, no DNA triplex-based HIV-1 inhibitor has been reported to date. Furthermore, because they are distinguished from quadruplex- and duplex-based inhibitors by their oligonucleotide assembly pattern, charge density, and molecular length, DNA triplex-based inhibitors provide structural diversity. Analysis of these DNA helix-based inhibitors will improve understanding of the structureCactivity relationship among them. Materials and Methods Oligodeoxynucleotides and formation of triplexes Oligodeoxynucleotides (ODNs) were synthesized in an ABI 392 DNA/RNA synthesizer (Applied Biosystems) on a 0.2-mol scale. Universal CPG (SU3010; Beijing DNAchem Biotechnology Co. Ltd.; Supplementary Fig. S1; Supplementary Data are available online at www.liebertpub.com/nat) was used. All ODNs (24S)-24,25-Dihydroxyvitamin D3 were deprotected by incubation in concentrated ammonia for 3?h at 55C. ODNs were purified by reverse-phase high-performance liquid chromatography in a C-10AT system (Shimadzu) equipped with a Diamonsil C-18 column (4.6250?mm; 5-m particle size; Dikma). Mobile phone phase A consisted of 0.07?M triethylammonium acetate (pH 7.0) and 5% acetonitrile. Mobile phone phase B consisted of acetonitrile, which was diluted in A from 5% to 60% for 30?min at 1?mL/min. ODNs were desalted with SEP-PAK cartridges (Oasis MCX, C18; Waters), lyophilized, and stored at ?18C Supplementary Fig. S2. For characterization of the ODNs, matrix-assisted laser desorptionCionization time-of-flight mass spectrometry (MS) (KRATOS Analytical, Shimadzu Group Organization), with 2,4,6-trihydroxyacetophenone (THAP) as the matrix, and electrospray ionization MS (380?V; Thermo LCQ DECA) were used. Triplexes were formed in a phosphate-buffered saline (PBS) answer made up of 0.1?M Na+ and 0.05?M Mg2+. The corresponding ODNs were mixed at molar ratios of 1 1:1:1 (for triplexes T1 and T2, Table 1) or 1:1 (for triplexes T3CT5). Triplexes were annealed by heating at 90C for 5?min and then cooling to 20C at a rate of 0.3C/min, followed by overnight incubation at 20C. The stock (24S)-24,25-Dihydroxyvitamin D3 concentration of triplex was 100?M. Table 1. Anticell Fusion Activity and Tm Associated with DNA Molecules represents the nucleoside analogs in Fig. 1. aIC50 is the concentration of inhibitor required for 50% inhibition of fusion in Tzm-bl cells and HL2/3 cells. Data were derived from the results of three separate experiments and are expressed as the meanstandard deviation. // represents the 3 end of each oligonucleotide strand. T, triplex; D, duplex; S, single strand of oligonucleotide. NA, not applicable; ND, not detected; ODN, oligodeoxynucleotide. Molecular modeling The molecular model of triplex T3 was initially built as two separate complexes (O7 and O8) in HyperChem Professional 7.0 with O8 base-pairing to the corresponding part of O7 in the duplex model. Then, the two separate complexes were manually assembled into the triplex (24S)-24,25-Dihydroxyvitamin D3 T3 in Accelrys DS ViewerPro 5.0, according to the structural information from a similar PDB file of triplex DNA (PDB ID: 1D3R). Finally, the complex was energy minimized in HyperChem. CellCcell fusion assay The cellCcell fusion assay was performed as described [14,15]. HIV-1 envelope glycoprotein (Env)-mediated cellCcell fusion was used to determine the inhibitory activity of ODNs in HL2/3 and TZM-bl cells [gifts from the AIDS Reference and Reagent Program, NIH, from Drs. B.K. Felber, G.N. Pavlakis (for HL2/3), J.C. Kappes, X. Wu, and Tranzyme, Inc. (for TZM-bl)]. HL2/3 is an effector cell line expressing HIV-1 Gag, Env, and Tat proteins at the cell surface . TZM-bl.