Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1995-05-04
1998-02-03
Campbell, Eggerton A.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
435 912, C12Q 168, C12P 1934
Patent
active
057143234
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
The invention concerns the field of recombinant DNA. More particularly, the invention relates to a method of synthesis of new and useful single-stranded DNA which have a stem-loop configuration (ss-slDNA). The invention relates to an in vitro and in vivo method of synthesis. Further, the invention relates to the replicating vehicle which produce these ss-slDNAs. Moreover, the invention relates these novel structures and discloses uses for these structures. There is described a method for amplifying ss-slDNAs with or without genes encoding a target protein. Moreover, the invention discloses a method for regulating gene function by the use of ss-slDNA.
BACKGROUND
Duplication of part of the genome is known to occur via an RNA intermediate which is reverse-transcribed into complementary DNA (cDNA) by reverse transcriptase. For a review, see Weiner et al., Ann. Rev. Biochem., 55, 631 (1986). The consequential reverse flow of genetic information is considered to have played a major role in the evolutionary diversification of eukaryotic genomes. A similar mechanism may very well have been responsible for genomic evolution in procaryotes in the light of the recent discoveries of bacterial transcriptases. See Inouye and Inouye, TIBS, 16, 18 (1991a) and Inouye and Inouye, Ann. Rev. Microbiol., 45, 163 (1991b). Gene duplication through cDNA synthesis by reverse transcriptase is believed to have played an important role in diversification of genomes during evolution.
The invention arose in connection with basic research related to genome evolution. The synthesis of a unique ss-slDNA during plasmid DNA replication was demonstrated. It may be speculated that slDNA production may be widely prevalent during both procaryotic as well as eucaryotic chromosomal DNA replication. The chromosomal genetic elements followed by IR structures may always be subject to duplication into slDNA at a frequency depending on the stability of the IR structure and the property of the polymerase(s).
It has been shown that there are many inverted repeat (IR) structures (approximately 1,000 copies in E. coli), known as REPs for repetitive extragenic palindromic sequences or PUs for palindromic units. Higgins et al., Nature, 298, 760 (1982), Gilson et al., EMBO. J., 3, 1417 (1984) and Gilson et al., Nucl. Acids Res., 19, 1375 (1991). These structures appear to be associated with specific cellular components including DNA polymerase I, and may be playing a significant role in chromosomal organization. Gilson et al., Nucl. Acids Res., 18, 3941 (1990) and Gilson et al., EMBO. J., 3, 1417 (1984). It should also be noted that approximately 6% of the human genome are occupied with elements called Alu whose transcriptional products have been shown to contain substantial secondary structures. Simmett et al., J. Biol. Chem., 266, 8675 (1991).
Since slDNA synthesis does not require RNA intermediates nor reverse transcriptase activity in contrast to that of cDNA synthesis, slDNA may be more frequently produced than cDNA. Thus, slDNAs might have played a major role similar to cDNA in the genomic evolution of both procaryotes and eucaryotes by duplicating genetic elements which then were dispersed or rearranged within the genome.
SUMMARY OF THE INVENTION
The parent applications relate to processes for synthesizing a novel single-stranded DNA structure, slDNA, in vivo or in vitro, to the slDNA structures and to various other genetic constructs. The parent applications also relate to slDNAs which carry an antigene in the single-stranded portion of the slDNA, which can be an antisense fragment which binds to a target mRNA and inhibits mRNA translation to protein or binds to double-stranded (ds)DNA, thereby forming a triple helix which inhibits the expression of target DNA.
The present invention relates to the over-expression of single-stranded DNA molecules, more particularly slDNAs in yields heretofore never accomplished, to the method of such production and to the genetic constructs to achieve these objectives.
The slDNA structure
REFERENCES:
patent: 4792523 (1988-12-01), Wong et al.
patent: 4910141 (1990-03-01), Wong et al.
patent: 5128256 (1992-07-01), Huse et al.
Maniatis et al "Molecular Cloning" 1982 pp. 405-406.
Inouye Masayori
Inouye Sumiko
Ohshima Atushi
Campbell Eggerton A.
The University of Medecine and Dentistry of New Jersey
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