Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-07-29
2003-05-27
Nashed, Nashaat T. (Department: 1652)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S007100, C435S018000, C435S195000
Reexamination Certificate
active
06569619
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates generally to high-throughput in vitro screening assays for identifying modulators of helicase activity. New solid phase assays, related compositions, apparatus and integrated systems are provided.
BACKGROUND OF THE INVENTION
The unwinding of duplex DNA is a prerequisite for DNA relication and repair, providing the single-stranded DNA (ssDNA) template for DNA polymerase to copy. Duplex DNA must also be unwound to generate the ssDNA required in recombination pathways or transferred during bacterial conjugation. A class of enzymes, the DNA helicase, enzymatically unwind DNA to facilitate this strand separation reaction. These enzymes are distinct from topoisomerases, which alter the linking number of the duplex DNA molecule through phosphodiester bond breakage and reunion. The helicases, on the other hand, simply disrupt the hydrogen bonds that hold the two strands of duplex DNA together. This is accomplished in a reaction that is coupled with the hydrolysis of a nucleoside 5′-triphosphate (NTP) and, thus, all helicases described to date are also DNA-dependent nucleoside 5′-triphosphatases (NTPases).
Examples of DNA helicase enzymes are now numerous in phage, bacteria, viruses, and in eukaryotic cells suggesting that these enzymes are ubiquitous in nature. Individual cells contain multiple DNA helicases; each helicase having a unique biochemical role in the cell. In fact, it is now known that helicases are involved in a wide variety of cellular functions, including DNA replication, recombination and repair, and RNA transcription, translation and processing. As a result of the critical functions played by helicases, they provide promising targets for therapeutic intervention, e.g., in pathogenic infection.
The standard assay currently used for measuring helicase activity employs gel electrophoresis to monitor the unwinding of double-stranded DNA or RNA. Because this assay is time-consuming and cumbersome, several additional types of assays for measuring helicase activity have been developed. Such assays involve measuring the sensitization of labeled duplex DNA to single-strand specific nucleases, electron microscopy, displacement of a labeled fragment that is annealed to a single-stranded DNA or RNA molecule and, more recently, spectrophotometric assays utilizing a dye or ssDNA binding protein as the reporter molecule (see, e.g., Matson, et al.,
Annu. Rev. Biochem.,
59:289-329 (1990); Houston and Kodadek,
Proc. Natl. Acad. Sci. USA,
91:5471-6474 (1994); Raney, et al.,
Proc. Natl. Acad. Sci. USA,
91:6644-6648 (1994); and Roman and Kowalczykowski,
Biochemistry
28: 2863-2873 (1989)).
Shortcomings associated with these previously used assays have hampered the search for novel modulators of helicase activity. For example, many of these assays are slow, expensive, and insensitive, subject to interference and/or require considerable manipulation. Moreover, these assays are not amenable to high-throughput screening methods such as are needed to screen large libraries or groups of potential modulators.
Giordano, et al. have recently developed a high-throughput screening assay for inhibitors of nucleic acid helicases that overcomes the shortcomings of the previously used assays (see, U.S. Pat. No. 5,705,344). In this assay, helicase activity is detected by the solid-phase, preferential capture of retained (non-liberated) single-stranded nucleic acid comprising a detectable label. Although this assay is invaluable, it would be advantageous to have additional high-throughput screening assays for identifying modulators of helicase activity. The present invention provides such assays.
SUMMARY OF THE INVENTION
High-throughput assays for identifying modulators of helicase activity are provided. Both inhibitors and activators of helicase activity can be screened using the assays of the present invention. Solid phase throughput assays are provided, as are related assay compositions, integrated systems for assay screening and other features that will be evident upon review.
In one aspect, high-throughput in vitro screening assays are provided for helicase activity. In one such assay, a reaction mixture comprising a first nucleic acid hybridized to a complementary unlabeled second nucleic acid, a helicase polypeptide and a nucleoside triphosphate is incubated under conditions suitable for the helicase polypeptide to unhybridize, i.e., unwind, the first nucleic acid and the second nucleic acid. An excess amount of a third nucleic acid comprising an immobilizable tag is added to the reaction mixture, the third nucleic acid having a region that is complementary to the first nucleic acid. The reaction mixture is incubated under conditions suitable for the third nucleic acid to hybridize to the first nucleic acid to form an immobilizable hybridization complex, whereby the immobilizable hybridization complex becomes immobilized on a solid suppot to which the immobilizable tag binds. The solid support is washed and, thereafter, the presence, absence or amount of the immobilizable hybridization complex bound to the solid support is determined directly or indirectly.
In a presently preferred embodiment, a potential modulator of helicase activity is also added to the reaction mixture and the effect of the modulator on the helicase activity is determined. As such, the present invention includes methods of screening for helicase activity by comparing candidate helicase activities with one or more defined control helicase activities or, alternatively, methods of screening for modulators of one or more defined helicase activities by comparing helicase activities in the presence and absence of a candidate modulator.
In another aspect, the present invention provides kits, compositions and integrated systems for performing the assays disclosed herein.
Other features objects and advantages of the invention and its preferred embodiments will become apparent from the detailed description that follows.
REFERENCES:
patent: 4203848 (1980-05-01), Grandine, II
patent: 5217864 (1993-06-01), Heintz et al.
patent: 5273881 (1993-12-01), Sena et al.
patent: 5705344 (1998-01-01), Giordano et al.
patent: 5747247 (1998-05-01), Kowalczykowski et al.
patent: 5958696 (1999-09-01), Crute
patent: 2311068 (1997-09-01), None
patent: WO 97/36006 (1997-10-01), None
Bachur, et al., “Helicase Inhibition by Anthracycline Anticancer Angents,”Molecular Pharmacology, 41:993-998 (1992).
Crute, et al., “Inhibition of Herpes Simplex Virus Type 1 Helicase—Primase by (Dichloroanilino) purines and -pyrimidines,”J. Med. Chem., 38(10):1820-1825.
Kyono, et al., “Detection of Hepatitis C Virus Helicase Activity Using the Scintillation Proximity Assay System,”Analytical Biochemistry, 257:120-126.
Maine, et al., “The Antitumor Agent CC-1065 Inhibits Helicase-Catalyzed Unwinding of Duplex DNA+,”Biochemistry, 31(16):3968-3975 (1992).
Naegeli, et al., “Inhibition of Rad3 DNA Helicase Activity by DNA Adducts and Abasic Sites: Implications for the Role of a DNA Helicase in Damage-Specific Incision of DNA+,”Biochemistry, 32(2) :613-621 (1993).
Matson, et al., “DNA Helicases,”Annu. Rev. Biochemistry, 59:289-329 (1990).
Seki, et al., “DNA Helicase and Nucleoside-5′-triphosphatase Activities of Polyoma Virus Large Tumor Antigen,”Biochemistry, 29(4):1003-1009 (1990).
Tuteja, et al., “DNA helicase III from HeLa cells: an enzyme that acts preferentially on partially unwound DNA duplexes,” 20(20):5329-5337 (1992).
Roman, et al.,Biochemistry, 28:2863-2873 (1989).
Nashed Nashaat T.
Townsend & Townsend & Crew LLP
Tularik Inc.
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