The tridecamer d(A-A-T-G-G-T-A-A-A-A-T-G-G), which is complementary to 13 nucleotides of the 3'- and 5'-reiterated terminal sequences of Rous sarcoma virus 35S RNA, was added to chick embryo fibroblast tissue cultures infected with Rous sarcoma virus. Inhibition of virus production resulted. The inference emerges that the tridecamer and its counterpart with blocked 3'- and 5'-hydroxyl termini enter the chick fibroblast cells, hybridize with the terminal reiterated sequences at the 3' and 5' ends of the 35S RNA, and interfere with one or more steps involved in viral production and cell transformation. Likely sites of action are (i) the circularization step of the proviral DNA intermediate, and (ii) the initiation of translation, the latter being described in the following communication [Stephenson, M. L. & Zamecnik, P. C. (1978) Proc. Natl. Acad. Sci. USA 75, 285--288].

A tridecamer oligodeoxynucleotide, d(A-A-T-G-G-T-A-A-A-A-T-G-G), which is complementary to reiterated 3'- and 5'-terminal nucleotides of Rous sarcoma virus 35S RNA, is an efficient inhibitor of the translation of proteins specified by the viral RNA in the wheat embryo cell-free system. The inhibition specificity for oncornavirus RNA is greater than for rabbit reticulocyte mRNA or brome mosaic virus RNA. Other oligodeoxynucleotides of similar size have little or no specific effect on the RNA-directed translation. The tridecamer acts as a primer for the avian myeloblastosis virus DNA polymerase when Rous sarcoma virus heated 70S RNA is used as a template, offering evidence that it can hybridize to the RNA. The possible use of such an oligodeoxynucleotide hybridization competitor to inhibit Rous sarcoma virus replication is described in the preceding paper [Zamecnik, P. C. & Stephenson, M. L. (1978) Proc. Natl. Acad. Sci. USA. 75, 280--284].

Three approaches were used to study hybridization of complementary oligodeoxynucleotides by nonradiative fluorescence resonance energy transfer. (i) Fluorescein (donor) and rhodamine (acceptor) were covalently attached to the 5' ends of complementary oligodeoxynucleotides of various lengths. Upon hybridization of the complementary oligodeoxynucleotides, energy transfer was detected by both a decrease in fluorescein emission intensity and an enhancement in rhodamine emission intensity. In all cases, fluorescein emission intensity was quenched by about 26% in the presence of unlabeled complement. Transfer efficiency at 50C decreased from 0.50 to 0.22 to 0.04 as the distance between donor and acceptor fluorophores in the hybrid increased from 8 to 12 to 16 nucleotides. Modeling of these hybrids as double helices showed that transfer efficiency decreased as the reciprocal of the sixth power of the donor-acceptor separation R, as predicted by theory with a corresponding Ro of 49 A. (u) Fluorescence resonance energy transfer was used to study hybridization of two fluorophore-labeled oligonucleotides to a longer, unlabeled oligodeoxynucleotide. Two 12-mers were prepared that were complementary to two adjacent sequences separated by four bases on a 29-mer. The adjacent 5' and 3' ends of the two 12-mers labeled with fluorescein and rhodamine exhibited a transfer efficiency of -0.60 at 50C when they both hybridized to the unlabeled 29-mer. (Wi) An intercalating dye, acridine orange, was used as the donor fluorophore to a single rhodamine covalently attached to the 5' end of one oligodeoxynucleotide in a 12-base-pair hybrid. Under these conditions, the transfer efficiency was =0.47 at 50C. These results establish that fluorescence modulation and nonradiative fluorescence resonance energy transfer can detect nucleic acid hybridization in solution. These techniques, with further development, may also prove useful for detecting and quantifying nucleic acid hybridization in living cells.In this paper we describe how fluorescently labeled oligodeoxynucleotides (ODNTs) and the process of nonradiative fluorescence resonance energy transfer (FRET) can be used to study nucleic acid hybridization. When two fluorophores whose excitation and emission spectra overlap are in sufficiently close proximity, the excited-state energy of the donor molecule is transferred by a resonance dipole-induced dipole interaction to the neighboring acceptor fluorophore. The results are a decrease in donor lifetime, a quenching of donor fluorescence, an enhancement of acceptor fluorescence intensity, and a depolarization of fluorescence intensity. The efficiency of energy transfer, Et, falls off rapidly with the distance between donor and acceptor molecule, R, and is expressed as Et = 1/[1 + (R/R0)6], [1] where Ro is a value that depends upon the overlap integral of the donor emission spectrum and the acceptor excitation spectrum, the index of refraction, the quantum yield of the donor, and the orientation of the donor emission and the acce...

The possibility ofusing oligodeoxynucleotides complementary to viral RNA or proviral DNA to inhibit the replication of human T-cell lymphotropic virus type III (HTLV-Ill) [the etiological agent of acquired imnunodeficiency syndrome (AIDS)] in cultured human cells was addressed by studying the association of 32P-labeled oligodeoxynucleotides with mammalian cellular components. The results indicated that exogenous oligodeoxynudeotides at 20 pM became associated with the membrane/cytosol fractions of the cell in amounts approximating 1.5 pM. Oligodeoynudeotides complementary to a region close to the tRNAL9 primer binding site on HTLV-M RNA and others complementary to HTLV-I mRNA donor or acceptor splice sites inhibited viral replication (assayed as reverse transcriptase) and gene expression (assayed as virus-encoded proteins p15 and p24) by as much as 95%. Use of control (random) oligodeoxynudeotides suggests that the antiviral effects were specific. Although these esults pertain to HTLV-I-infected cells in tissue culture, rather than to AIDS patients, they nevertheless point to a therapeutic potential of the complementary oligodeoxynudeotide ("hybridization competition" or "hybridon") approach in the treatment of patients with AIDS and AIDS-related complex.

Modified oligodeoxynucleotides complementary to RNA of human immunodeficiency virus 1 (HIV-1) were tested for their ability to inhibit virally induced syncytium formation and expression of viral p24 protein. The Fig. 1. All the modified oligonucleotides were found to be resistant to nucleases such as snake venom or spleen phosphodiesterases. amounts of material for toxicity studies were made by a manual solid-support method on an Omnifit assembly (13). Unmodified oligomers were made and purified by methods described previously (2). Phosphorothioates and phosphoramidates were synthesized by a modification of the Hphosphonate procedure (14,15). To generate phosphorothioate linkages, oxidation following the final coupling and detritylation was replaced by treatment with 0.1 M sulfur in carbon disulfide/triethylamine (9:1, vol/vol) at room temperature for up to 2 hr, depending on chain length (16). For the phosphoramidates, this step was replaced by treatment with a 10% solution of the appropriate amine in carbon tetrachloride for up to 1.5 hr, depending on chain length (16).After deblocking in concentrated ammonium hydroxide (550C for 51/2 hr or room temperature for >2 days), products were purified on 2-mm-thick preparative layer plates (Merck silica gel 60) in propanol/water/concentrated ammonium hydroxide (55:35:10, vol/vol). Phosphorothioates were further purified on DEAE-cellulose and C18 silica (details will be published elsewhere). Phosphoramidates from preparative layer plates were passed through Sephadex G-25 in aqueous 30% (vol/vol) ethanol and then through C18 silica and dialyzed. In all cases, small amounts of oligomers attached to the solid support were taken before the final treatment with sulfur or amine and were oxidized with 2% iodine in pyridine/water (98:2, vol/vol) (15) to phosphodiesters. These were used for determination of base composition by enzymatic degradation to nucleosides followed by HPLC. The phosphorothioates were also characterized by polyacrylamide gel electrophoresis, where they had similar mobility to the corresponding diesters.Phosphoramidates were further characterized by hydrolysis with formic acid to the phosphodiesters (16) for comparison with authentic samples on HPLC and degradation to nucleosides for determination of nucleoside composition and integrity by HPLC. A useful indicator of oligonucleotide purity and integrity is the melting curve of its duplex with a complementary DNA Abbreviations: HIV, human immunodeficiency virus; AIDS, acquired immunodeficiency syndrome. 7079The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.