Photocleavable nucleoside base and nucleic acids including

Details Photocleavable nucleoside base and nucleic acids including Photocleavable nucleoside base and nucleic acids including

1999-07-15

2001-09-25

Geist, Gary (Department: 1623)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C536S025320, C536S025330, C536S029200

Reexamination Certificate

active

06294659

ABSTRACT:

TECHNICAL FIELD
The present invention relates, in general, to a nucleoside base and, in particular, to a universal, photocleavable nucleoside base. The invention further relates to oligonucleotides comprising such a base.
BACKGROUND
A variety of novel techniques for analysis of nucleic acids best utilize relatively short, single-stranded analytes. Many such techniques are based on the hybridization of analyte nucleic acids to miniaturized arrays of short (<25 nucleotides), single-stranded DNA probes (Ramsay, Nat. Biotechnol. 16:40 (1998); Marshall et al, J. Nat. Biotechnol. 16:27 (1998)). Samples of analyte DNA often arise by PCR amplification from a biological sample, range from hundreds to thousands of nucleotides and are obtained as double-stranded molecules. As hybridization requires single-stranded nucleic acids, various means have been devised to render amplification products single-stranded.
The stability of duplex nucleic acid is directly proportional to molecular length. Thus, it can be difficult to form single-stranded analytes from long, double-stranded amplicons. Further, it has been observed in some instances that the performance of various nucleic acid analysis techniques, such as the above-referenced array-based methods, is superior with shorter analyte nucleic acids. This has created a demand for methods of fragmenting analyte nucleic acid or amplification products thereof.
Available fragmentation methods include limited restriction digestion of the amplicon, as well as the incorporation of uridine during amplification followed by backbone cleavage using uracil-N-glycosylase. These methods, however, have drawbacks related to their a sequence bias and the additional processing steps needed to remove reagents/byproducts. Accordingly, there is a clear need for a nucleoside analog that can be enzymatically incorporated at random within a nucleic acid chain and subsequently activated for cleavage of the backbone.
A number of workers have developed analogs of the heterocyclic bases in nucleic acids for various purposes. For example, Bergstrom et al have advanced nitropyrrole and nitroindole as heterocycles that are accepted opposite any of the natural bases in double-stranded nucleic acid (Bergstrom et al, Nucleic Acids Res. 25:1935 (1997); Bergstrom et al, J. Am. Chem. Soc. 117:1201 (1995)). Such “universal” bases have uses including simplified syntheses of short oligonucleotides that are suitable for use as probes for hybridization, and as primers for DNA sequencing and nucleic acid amplification. On the other hand, Kool et al have shown that difluorotoluene specifically substitutes for the base thymidine (T) in duplex DNA with only a small loss in hybrid stability (Schweitzer et al, J. Org. Chem. 59:7238 (1994)). Further, difluorotoluene is accepted by DNA polymerases either in the template strand or as an incoming triphosphate (Moran et al, Proc. Natl. Acad. Sci. USA 94:10506 (1997); Moran et al, J. Am. Chem. Soc. 119:2056 (1997); Liu et al, Chem. Biol. 4:919 (1997)). As a result, high fidelity complementation of the unnatural base with a natural adenine (A) occurs. Finally, Ordoukhanian et al (J. Am. Chem. Soc. 117:9570 (1995)) have developed unnatural base analogs that can be cleaved into two portions (and thereby cleave the backbone of the nucleic acid) by irradiation. These compounds do not resemble natural nucleosides and are not accepted by nucleic acid modifying enzymes. Thus, they must be incorporated into oligonucleotides by chemical synthesis.
The present invention provides a nucleoside analog that can be enzymatically incorporated into a nucleic acid chain and then activated for cleavage of that chain.
SUMMARY OF THE INVENTION
The present invention relates to a nucleoside base that can be randomly incorporated into newly-synthesized nucleic acid strands by enzymes, and, in a subsequent step, cleaved by UV irradiation, thereby introducing single-strand nicks into double-stranded nucleic acid. Such nicks fragment larger nucleic acids into smaller ones. Because the nicks are random and, therefore, not opposed by nicks on the opposite strand, the average length of uninterrupted double stranded nucleic acid is shortened, facilitating conversion from double-stranded to single-stranded nucleic acid.
Objects and advantages of the invention will be clear from the description that follows.


REFERENCES:
patent: 5438131 (1995-08-01), Bergstrom et al.
patent: 5738993 (1998-04-01), Fugono et al.
patent: 6011143 (2000-01-01), Shionoya et al.
Liu et al., “The Structure of Chicanine, a New Lignan fromSchisandra sp.,”Canadian Journal of Chemistry, 59(11), 1680-1684 (1981);Chemical ABstracts, 95, Abstract No. 111711p (Sep. 28, 1981); only abstract supplied.*
Huang et al., “Structure of En Shi Zhi Su, a New Lignan fromSchisandra henryi Clarke, ”Zhongcaoyao,13(2), 22 (1982); 97, Abstract No. 107029w (Sep. 27, 1982); only abstract supplied.*
Kurihara et al., “Synthesis and Biological Activity of 3-Quinolineacetic acid Derivatives,”Yakugaku Zasshi, 98(6), 802-816 (1978);Chemical Abstracts, 90, Abstract No. 6216e (Jan. 1, 1979); only abstract supplied.

Affiliated with

Also associated with

Rating

Photocleavable nucleoside base and nucleic acids including does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Photocleavable nucleoside base and nucleic acids including, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Photocleavable nucleoside base and nucleic acids including will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-2462479