We have recently shown that phosphorothioate (PS) oligodeoxynucleotide (ODN) analogs, unlike their normal congeners, exhibit significant anti-HIV activity (Matsukura et al., (1987) Proc. Natl. Acad. Sci. USA 84, 7706-7710). We now report the syntheses, melting temperatures (Tm), and nuclease susceptibilities of a series of phosphorothioate ODN analogs. These include all-PS duplexes, duplexes with one normal chain and the other chain either all-PS, or end-capped with several PS groups at both 3' and 5' ends. The DNase susceptibilities of the S-ODNs are much less than the normal phosphodiesters, but by contrast duplexes of poly-rA with S-dT40 are much more susceptible to RNase H digestion. The Tm's for AT base pairs of S-ODNs are significantly depressed relative to normals, while GC base pairs show much less Tm depression. The Tm's of S-dT oligomers with poly-rA are reduced relative to the duplexes with normal dA oligomers. These results have significance for the biological properties of these analogs as anti-message inhibitors of gene expression, and provide a rational basis for the S-dC/G sequences as potential effective anti-AIDS agents.

ABSTRACT- Nuclease-resistant 25 jaM. An N3-methylthymidine-containing phosphorothioate analog, which does not hybridize efficiently in vitro to complementary normal DNA, showed no antiviral activity. A 14-mer phosphorothioate oligodeoxycytidine (S-dC14) synergistically enhanced the antiviral activity of 2',3'-dideoxyadenosine, an anti-HIV nucleoside. Therefore, phosphorothioate analogs of oligodeoxynucleotides could represent a unique class of experimental therapeutic agents against the acquired immunodeficiency syndrome and related diseases. However, their mechanisnm of action is likely to be complex.

Rapid atropisomerization of cisplatin-DNA cross-link models, cis-PtA(2)G(2) (A(2) = two amines or a diamine, G = guanine derivative, bold font indicating a guanine not linked to another guanine), makes their NMR spectra uninformative. The conformers [two head-to-tail (DeltaHT and LambdaHT) conformers, one head-to-head (HH) conformer] are detected in (CCC)PtG(2) retro models (CCC = chirality-controlling chelates designed to reduce rotation around the G N7 to Pt bond by destabilizing the transition state). Clear trends are found with four CCC ligands, 2,2'-bipiperidine (Bip) and N,N'-dimethyl-2,3-diaminobutane (each with S,R,R,S and R,S,S,R configurations at the chelate ring N, C, C, and N atoms, respectively). S,R,R,S ligands favor left-handed G base canting and the LambdaHT form; R,S,S,R ligands favor right-handed canting and the DeltaHT form. The HH conformer is normally negligible unless G = 5'-GMP. However, understanding this 5'-phosphate effect is complicated by possible interligand interactions of the 5'-phosphate with the N1H of the cis-5'-GMP and a CCC NH; these interactions are referred to as second-sphere (SSC) and first-to-second-sphere (FSC) communication, respectively. We now investigate the four (CCC)PtG(2) models with 1-Me-5'-GMP, a G lacking the N1H needed for SSC. The phosphate location makes FSC possible in the major but not the minor HT form. The major form should increase from pH 3 to pH 7 because the phosphate is deprotonated at pH 7. However, the major DeltaHT form for the R,S,S,R pair did not change in abundance, and the major LambdaHT form for the S,R,R,S pair actually decreased. Thus, FSC is weak. At pH approximately 7 the HH conformer of the S,R,R,S pair had an abundance (40-44%) higher than that in any reported cis-PtA(2)G(2) adduct. FSC involving one 1-Me-5'-GMP could play a role. The high HH abundance and use of a pH jump experiment with (S,R,R,S)-BipPt(1-Me-5'-GMP)(2) allowed us to obtain the first deconvoluted CD spectrum for a cis-PtA(2)G(2) HH conformer. The CD features for the HH conformer are much weaker than for the HT conformers. Our findings are best interpreted to indicate that FSC is not important in aqueous solution, especially for the HT form. Weak FSC is consistent with recent models of the cross-link in duplexes. In contrast, crystals of fluxional models often reveal FSC, but not the more important SSC. SSC was unrecognized until our retro model studies, and the new results reinforce the value of studying retro models for identifying interactions in solution.

In this report, we demonstrate the sequencespecific suppression of viral expression in T cells chronically infected with human immunodeficiency virus 1 (HIV-1), using antisense phosphorothioate oligodeoxynucleotides. As a target for antisense intervention, we used the HIV-1 gene rev, which is essential for viral replication and regulates the expression of virion proteins, in part, by affecting the splicing of the viral mRNA. A phosphorothioate oligomer complementary to the initiation sequence of HIV-1 rev had a significant and selective inhibitory effect on the production of several viral proteins in chronically HIV-1-infected T cells and drastically reduced the unspliced (genomic) viral mRNA transcripts, with relative sparing of smaller (spliced) METHODS AND MATERIALSSynthesis and Purification of Phosphorothioate and Unmodified Normal Oligo(dN). The phosphorothioate oligomers were synthesized by the method previously reported (6). Unmodified normal oligomers were synthesized by the standard method. All syntheses were performed with an automated DNA synthesizer (Applied Biosystems; model 380-B).Sequences of the Target Region in the HIV Genome and Oligomers Used. We used the rev gene (formerly called art/trs) as a target for antisense intervention of viral gene expression. The rev gene is well conserved among HIV-1 clones. To clarify the sequence specificity, we tested an unmodified antisense oligomer of rev (normal phosphodiester linkages; n-arev) and phosphorothioate oligomers containing rev sense sequence (S-sense-rev), rev antisense sequence (S-arev), random sequence with the same base composition as S-arev (S-random-arev), 28-mer homooligomer oligo(dC)28 (S-dC28), rev antisense sequence containing four N3-methylthymidine (N-MedThd) residues (S-N-Me-arev), and an antisense sequence against the initiation site of gag, the gene encoding the group-specific antigen gag (S-agag) (Fig. 1). Viral Gene Expression Inhibition Assay. To test whether an agent has regulatory activity on HIV viral expression, we used chronically HIV-1-infected H9 cells, which were exposed to the virus isolate HTLV-IIIB, kept in culture for several months or more in complete medium (RPMI 1640 supplemented with 15% fetal calf serum, 4 mM L-glutamine, 50 nM 2-mercaptoethanol, and 50 units of penicillin, and 50 ,ug of streptomycin per ml N3-methylthymidine; n-arev, nor-mal (unmodified) antisense oligo(dN) against rev; S-dC28, 28-mer phosphorothioate oligo(dC); S-agag, antisense phosphorothioate oligo(dN) against gag; S-arev, antisense phosphorothioate oligo(dN) against rev; S-N-Me-arev, antisense phosphorothioate oligo(dN) against rev containing four N-MedThd residues in the sequence; S-random-arev, phosphorothioate random oligo(dN) with the same base composition as S-arev; S-sense-rev, phosphorothioate sense oligo(dN) of rev; RIPA, radioimmunoprecipitation assay.