Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Patent
1994-01-24
1998-10-27
LeGuyader, John L.
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
536 232, 536 245, 435 6, 435 9131, 4351721, C12N 1511, C07H 2104, C12Q 168
Patent
active
058279350
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
This invention relates to chimeric tRNA.sup.LYS ribozyme molecules which compete effectively with tRNA.sup.LYS for binding to HIV-1 reverse transcriptase. These chimeric molecules provide a mechanism for delivering inhibitors of HIV-1 transcriptase to the virion particle itself.
BACKGROUND OF THE INVENTION
It has been demonstrated that the entire tRNA.sup.LYS molecule as well as various segments of the tRNA per se are capable specifically of interacting with HIV-1 transcriptase. See Barat, et al. EMBO Journal 8:3279-3285 (1989); Khan, et al. J. Bio. Chem 267:6689-6695 (1992); Weiss, et al., Gene 111:183-197 (1992). Ben-Artzi, Proc. Natl. Acad. Soc. USA 89:927-931 (1992) reports an RNAse cleavage activity associated with HIV-1 reverse transcriptase. This activity is shown to cleave only HIV-1 RNA, not the primer.
Prior to this invention there has been no report of chimeric tRNA.sup.LYS -ribozyme molecules.
SUMMARY OF THE INVENTION
This invention provides novel chimeric tRNA.sup.LYS -ribozyme molecules that compete effectively with tRNA.sup.LYS for HIV-1 reverse transcriptase binding sites. The chimeric human tRNA.sup.LYS -ribozymes inhibit reverse HIV transcription by delivering inhibitors such as ribozymes of HIV-1 reverse transcriptase directly to the virion particle and render it non-functional. The chimeric molecules of this invention thus serve as highly specific non-toxic therapeutic agents.
These chimeric molecules also reveal a novel, site specific RNA cleaving activity of HIV-1.
EXAMPLES AND DESCRIPTION OF THE FIGURES
FIG. 1 shows the structure of one chimeric ribozyme. This tRNA.sup.LYS -ribozyme construct has been cloned into a Blue Script transcription vector using Sacll and Xhol restriction sites. Following linearization at the Sacll site the chimeric RNA can be transcribed in vitro using bacteriphage T-7 RNA polymerase. There is also a Mae I restriction site in between the tRNA and ribozyme moieties, allowing the tRNA to be transcribed independently of the ribozyme.
FIG. 2. This gel shift experiment shows binding of the chimeric tRNA.sup.LYS -ribozyme to HIV-1 reveres transcriptase. The eight lanes of the gel from left to right are:
1. Molecular weight marker;
2. tRNA.sup.LYS in vitro transcript which has extra bases at both the 5' and 3' ends. The extra 5' bases are from the Blue Script poly linker between the T-7 promoter and the Xhol site. There are six extra nucleotides at the 3' derived from the nucleotides after the CCA of the tRNA to the Mae I site which separates the tRNA from the ribozyme.
3. tRNA.sup.LYS -ribozyme in vitro transcript which has the same extra 5' bases as tRNA.sup.LYS, but terminates at Sacll site at the end of the ribozyme moiety.
4. tRNA.sup.LYS transcript incubated with HIV-1 reverse transcriptase.
5. tRNA.sup.LYS -ribozyme transcript incubated with HIV-1 reverse transcriptase.
6. tRNA.sup.LYS transcript incubated with AMV reverse transcriptase.
7. tRNA.sup.LYS -ribozyme incubated with AMV reverse transcriptase.
8. tRNA.sup.LYS with competing, non-radioactively labelled tRNA.sup.LYS -ribozyme incubated with HIV-1 reverse transcriptase.
This FIG. 2 shows that the chimeric tRNA.sup.LYS -ribozyme specifically binds to HIV-1 reverse transcriptase by a shift in radioactivity when HIV-1 reverse transcriptase is present. Cold tRNA.sup.LYS -ribozyme competes with tRNA.sup.LYS for binding to HIV-1 reverse transcriptase as indicated by the reduced radioactive shift in lane 8.
FIG. 3. This experiment demonstrates cleavage of a 162 nucleotide, radioactively labelled HIV-1 RNA containing the primer binding site plus sequences upstream of this and including the AUC cleavage signal for the ribozyme. The cleavage products are 101 and 61 bases. The extent of cleavage increases with increasing temperature.
GENERAL DESCRIPTION OF THE INVENTION
Genetic fusions consisting of the entire mature coding sequence or 18 bases of the 3' end of human tRNA.sup.LYS have been fused to hammerhead ribozyme containing RNAs with base pairing capabilities to the HIV-
REFERENCES:
Weiss et al., Gene 111:183 (1992).
Medicine Abstract 90060615.
Medline Abstract 00286389.
Taylor et al. Cell. Biochem. J. Suppl. 15D, 1991 p. 21,CD 217.
Rossi et al. Cell. Biochem. J. Suppl. 15D, 1991, pp. 374, D 429.
Rossi et al. Cell. Biochem J. Suppl. 15D, 1991, p. 7, CD 011.
Ruffner et al. Biochem. v. 29, No. 47:10695 (1990).
PNAS 88:7303 (1991) Siond et al.
Johnson et al. Science 260:1286-1292, May 1993.
Barigana Science 262:1512-1514, Dec. 1993.
Biotechnolog Abstracts Acc #: 95-00442.
Goodchild Arch. Biochem Biophys. 284:386 (1991).
Hampel et al. NAR 18:299 (1990).
Sarver et al. Science 247:1222 (1990).
Barat et al. EMBO J. 8:3279 (1989).
Wolfgang et al Science 253:314 (1991).
Larson Garry P.
Rossi John J.
City of Hope
LeGuyader John L.
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