The phosphorothioate oligonucleotide T2G4T2 was identflied as an inhibitor of HIV infection in vitro by combinatorial screening of a library of phosphorothioate oligonucleotides that contained all possible octanucleotide sequences. The oligonucleotide forms a parallel-stranded tetrameric guanosine-quartet structure. Tetramer formation and the phosphorothioate backbone are essential for antiviral activity. The tetramer binds to the human immunodeficiency virus envelope protein gpl20 at the V3 loop and inhibits both cell-to-cell and virus-to-cell infection. Since functional inhibition of viral infection rather than nucleic acid binding is the selection criterion, the chances are enhanced that a useful compound will be identified.When the SURF strategy was used in a functional screen for an inhibitor of HIV infection, phosphorothioate oligonucleotides with four consecutive guanosines were found to have activity. Of the active compounds, T2G4T2 was chosen for further studies. Physical characterization of this oligonucleotide demonstrated that it formed a tetramer stabilized by G quartets. G-quartet structures are intra-or intermolecular four-stranded helices stabilized by planar Hoogsteen-paired quartets of guanosine (8, 9). The structures are stabilized by monovalent ions bound between two planes ofG quartets and coordinated to the carbonyl oxygens. The G-quartet structure and a phosphorothioate backbone were shown to be required for antiviral activity.Cell culture experiments revealed that the G-quartet structure blocks binding of HIV virions to cells and virus-mediated cell fusion. Virus and cell membrane fusion is initiated by binding of gp120, the major envelope protein of HIV, to its cellular receptor. The mechanism by which membrane attachment occurs is not well understood, but a particular region of gp120, the V3 loop, is essential. The V3 loop maintains a high percentage ofpositively charged amino acids across all HIV strains (10), and the extent of virus-mediated cell fusion and rapid viral replication have been correlated with increased cationic composition of the V3 loop (11). In vitro studies showed that the G-quartet structure bound directly to the V3 loop of gpl20. We hypothesize that the G-quartet structure provides a scaffold to present the phosphorothioate groups in a favorable orientation for binding to the highly cationic V3 loop. MATERIALS AND METHODSOligonucleotide Synthesis. Phosphorothioate oligonucleotides were synthesized by standard protocols (12). For oligonucleotides with randomized positions, the proportions of the individual amidites in a mixture were adjusted until equal amounts of the four dimers were obtained when coupled to dT on controlled-pore glass, as judged by reversed-phase HPLC. Oligonucleotides were purified by reversed-phase HPLC with a gradient of methanol in water. Several purified oligonucleotides were analyzed for base composition by total digestion with nuclease followed by reversed-phase HPLC (Len Cummins, personal communication) and yielded the expected rati...

The phosphorothioate and phosphodiester oligodeoxynucleotides d(TTGGGGTT) form parallel-stranded tetramer structures stabilized by guanosine quartets. The phosphorothioate tetramer has been shown to inhibit human immunodeficiency virus (HIV) in vitro. The kinetics of association and dissociation of both tetramers have been determined as a function of temperature using size exclusion chromatography to measure the ratio of single strand to tetramer. In phosphate buffered saline (pH 7.2) at 37 degrees C, the fourth-order association rate of the phosphorothioate tetramer was 6.1 (+/- 0.5) x 10(4) M-3 s-1; the dissociation rate was 8.2 (+/- 0.2) x 10(-6) min-1, resulting in a t(1/2) of about 60 days. The association rate of the phosphodiester was about one order of magnitude faster and the dissociation rate about one order of magnitude slower than that of the phosphorothioate tetramer. The association reaction had a negative energy of activation for both compounds. Despite thermodynamic instability of the tetramer at low concentrations, the extremely slow dissociation rate may allow use of the phosphorothioate tetramer for AIDS chemotherapy.

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Limited chemical bromination of poly[r(C-G)] (32% br8G, 26% br5C) results in partial modification of guanine C8 and cytosine C5, producing a mixture of A- and Z-RNA forms. The Z conformation in the brominated polynucleotide is stabilized at much lower ionic strength than in the unmodified polynucleotide. More extensive bromination of poly[r(C-G)] (greater than 49% br8G, 43% br5C) results in stabilization of a form of RNA having a Z-DNA-like (ZD) CD spectrum in low-salt, pH 7.0-7.5 buffers. Raising the ionic strength to 6 M NaBr or NaClO4 results in a transition in Br-poly[r(C-G)] to a Z-RNA (ZR) conformation as judged by CD spectroscopy. At lower ionic strength Z-DNA-like (ZD) and A-RNA conformations are also present. 1H NMR data demonstrate a 1/1 mixture of A- and Z-RNAs in 110 mM NaBr buffer at 37 degrees C. Nuclear Overhauser effect (NOE) experiments permit complete assignments of GH8, CH6, CH5, GH1', and CH1' resonances in both the A- and Z-forms. GH8----GH1' NOEs demonstrate the presence of both A- and Z-form GH8 resonances in slow exchange on the NMR time scale. The NMR results indicate that unbrominated guanine residues undergo transition to the syn conformation (Z-form). Raman scattering data are consistent with a mixture of A- and Z-RNAs in 110 mM NaCl buffer at 37 degrees C. Comparison with the spectrum of Z-DNA indicates that there may be different glycosidic torsion angles in Z-RNA and Z-DNA [Tinoco, I., Jr., Cruz, P., Davis, P., Hall, K., Hardin, C. C., Mathies, R. A., Puglisi, J. D., Trulson, M. O., Johnson, W. C., & Neilson, T. (1986) in Structure and Dynamics of RNA, pp 55-68, Plenum, New York].(ABSTRACT TRUNCATED AT 250 WORDS)

Left-handed double-helical Z-RNA has been studied using the ribohexanucleotide pentaphosphate r(CpGpCpGpCpG). One-dimensional and two-dimensional proton nmr experiments were used to probe the structural details of the left-handed helix in concentrated sodium perchlorate solution. In 1M NaClO4 the RNA adopts the normal A-form double helix, and in 6M NaClO4 it is nearly all in the Z form. In 4M NaClO4 it exists as nearly equal parts of A form and Z form. Resonances corresponding to both A and Z form appear in the nmr spectrum, indicating that the duplex exchanges slowly between forms. Spin-spin coupling constants between protons in the ribose rings were used to determine the sugar-pucker conformations of the individual nucleotides. Quantitative nuclear Overhauser experiments were used to determine proton-proton distances within the nucleoside, and from these distances values for the glycosidic torsion angle were determined. The results show that the cytidines adopt C2'-endo sugar puckers (S type) with pseudo-rotation phase values (P) of approximately 165 degrees. The bases are in the anti conformation, with chi values of approximately -140 degrees. The internal guanosines adopt C3'-endo sugar puckers (N type) with P approximately 18 degrees, while the 3'-terminal guanosine ribose exists in an equilibrium between S- and N-type conformations. All three guanosine bases adopt the syn conformation, with chi approximately 70 degrees. The results indicate that the solution structure of Z-RNA is very similar to that of Z-DNA.

An experimental evaluation of several different pooling strategies for combinatorial libraries was conducted using a library of 810 compounds and an enzyme inhibition assay (phospholipase A2). The library contained compounds with varying degrees of activity as well as inactive compounds. The compounds were synthesized in groups of three and pooled together in various formats to realize different pooling strategies. With one exception, all iterative deconvolution strategies and position scanning resulted in identification of the same compound. The results are in good agreement with the predicted outcome from theoretical and computational methods. These data support the tenet that active compounds for pharmaceutically relevant targets can be successfully identified from combinatorial libraries organized in mixtures.

The hairpin stem-loop form of the RNA oligonucleotide rCGC(UUU)GCG has been studied by NMR spectroscopy. In 10 mM phosphate buffer this RNA molecule forms a unimolecular hairpin with a stem of three base pairs and a loop of three uridines, as judged by both NMR and UV absorbance melting behavior. Distance and torsion angle restraints were determined using homonuclear proton-proton and heteronuclear proton-phosphorus 2-D NMR. These values were used in restrained molecular dynamics to determine the structure of the hairpin. The stem has characteristics of A-form geometry, although distortion from A-form occurs in the 3'-side of the stem, presumably to aid in accommodating the small loop. The loop nucleotides adopt C2'-endo conformations. NOE's strongly suggest stacking of the uracils with the stem, especially the first uracil on the 5'-side of the loop. The reversal of the chain direction in the loop seems to occur between U5 and U6. Loop structures produced by molecular dynamics simulations had a wide range of conformations and did not show stacking of the uracils. A flexible loop with significant dynamics is consistent with all the data.

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