Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-07-20
2003-12-02
Horlick, Kenneth R. (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091200, C536S023100, C536S024300, C536S024330, C536S025320, C536S025400
Reexamination Certificate
active
06656692
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of Invention
The invention relates to nucleobase binding in complexes, such as duplexes, triplexes and quadruplexes, and more particularly to methods wherein such complexes are formed by specific binding between single-stranded or double-stranded nucleobase-containing probes and single-stranded or double-stranded nucleobase-containing target sequences.
2. Description of Related Art
The Watson-Crick model of nucleic acids has been the accepted standard in molecular biology for nearly fifty years. As recounted by James Watson in his book entitled “A Personal Account of the Discovery of the Structure of DNA,” (1968), the Watson-Crick model, which won Watson and Crick the Nobel Prize, arose from the ashes of their abandoned theory that bases bind to like bases on opposing strands (Watson at p.125). Watson described how he abandoned his “briefly considered like-with-like pairing” model when he realized the advantages of a model based on A:T and G:C binding. Id.
Although antiparallel nucleic acid duplexes first suggested by Watson and Crick are the most widely studied type of multiple-strand nucleic acid structures, it has been discovered that nucleic acids also form triplex structures and quadruplex structures under certain conditions.
Until recently, binding among three nucleic acid strands to form a triplex was widely believed to be confined to very limited species of nucleic acids (e.g., polypurine or polypyrimidine sequences). See, e.g., Floris et al., “Effect of cations on purine-purine-pyrimidine triple helix formation in mixed-valence salt solutions,” 260 Eur. J. Biochem. 801-809 (1999). Moreover, canonical triplex binding or hybridization was thought to be based on Hoogsteen binding between limited varieties of adjacent nucleobases, rather than Watson-Crick base pairing. See, e.g., Floris et al. and U.S. Pat. No. 5,874,555 to Dervan et al. However, the inventors have recently disclosed in several patent applications that specifically bound mixed base sequence triplex nucleic acids based on Watson-Crick base pairing can be created and used as the basis for a highly accurate and sensitive assay for specific binding. See U.S. patent applications Ser. Nos. 09/613,263 and 09/468,679, respectively filed Jul. 10, 2000 and Dec. 21, 1999.
Zhurkin et al., 239 J. Mol. Biol. 181 (1994) discloses the possibility of parallel DNA triplexes; however, these triplexes are said to be created by the third strand binding in the major groove of the duplex in the presence of recombination proteins, such as RecA protein.
As has been the case with triplex nucleic acids, the conventional wisdom regarding quadruplex nucleic acids has been that such peculiar structures only exist under relatively extreme conditions for a narrow class of nucleic acids. In particular, Sen et al. (Nature 334:364-366 (1988)) disclosed that guanine-rich oligonucleotides can spontaneously self-assemble into four-stranded helices in vitro. Sen et al. (Biochemistry 31:65-70 (1992)) disclosed that these four-stranded complexes can further associate into superstructures composed of 8, 12, or 16 oligomers.
Marsh et al. (Biochemistry 33:10718-10724 (1994), and Nucleic Acids Research 23:696-700 (1995)) disclosed that some guanine-rich oligonucleotides can also assemble in an offset, parallel alignment, forming long “G-wires”. These higher-order structures are stabilized by G-quartets that consist of four guanosine residues arranged in a plane and held together through Hoogsteen base pairings. According to Sen et al. (Biochemistry 31:65-70 (1992)), at least three contiguous guanines within the oligomer are critical for the formation of these higher order structures.
It has been suggested that four-stranded DNAs play a role in a variety of biological processes, such as inhibition of HIV-1 integrase (Mazumder et al., Biochemistry 35:13762-13771 (1996)), formation of synapsis during meiosis (Sen et al., Nature 334:364-366 (1988)), and telomere maintenance (Williamson et al., Cell 59:871-880 (1989)); Baran et al., Nucleic Acids Research 25:297-303 (1997)). It has been further suggested that controlling the production of guanine-rich quadruplexes might be the key to controlling such biological processes. For example, U.S. Pat. No. 6,017,709 to Hardin et al. suggests that telomerase activity might be controlled through drugs that inhibit the formation of guanine quartets.
U.S. Pat. No. 5,888,739 to Pitner et al. discloses that G-quartet based quadruplexes can be employed in an assay for detecting nucleic acids. Upon hybridization to a complementary oligonucleotide, the G-quartet structure unfolds or linearizes, thereby increasing the distance between donor and acceptor moieties on different parts of the G-quartet structure, resulting in a decrease in their interaction and a detectable change in a signal (e.g., fluorescence) emitted from the structure.
U.S. Pat. No. 5,912,332 to Agrawal et al. discloses a method for the purification of synthetic oligonucleotides, wherein the synthetic oligonucleotides hybridize specifically with a desired, full-length oligonucleotide and concomitantly form a multimer aggregate, such as quadruplex DNA. The multimer aggregate containing the oligonucleotide to be purified is then isolated using size-exclusion techniques.
Despite the foregoing developments, a need has continued to exist to systematically investigate and catalogue all specific interactions between mixed base sequence nucleic acids and to create new, effective and rapid methods for producing and analyzing specific interaction between nucleic acids and/or nucleic acid analogues.
All references cited herein are incorporated herein by reference in their entireties.
BRIEF SUMMARY OF THE INVENTION
The invention provides a complex comprising: (1) a probe containing a heteropolymeric probe sequence of nucleic acids or nucleic acid analogues; and (2) a target containing a heteropolymeric target sequence of nucleic acids or nucleic acid analogues, wherein: (a) at least one of the probe and the target is purified or synthetic; and (b) the heteropolymeric probe sequence is bonded to the heteropolymeric target sequence by Watson-Crick complementary base interaction or by homologous base interaction, provided that when the complex is a duplex and the heteropolymeric probe sequence is antiparallel to the heteropolymeric target sequence, the heteropolymeric probe sequence is bonded to the heteropolymeric target sequence by homologous base interaction, and provided that when the complex is a triplex, the complex is free of recombination proteins.
Also provided is a method for assaying a target, the method comprising: (1) providing a sample comprising the target containing a heteropolymeric target sequence of nucleic acids or nucleic acid analogues; (2) providing a probe containing a heteropolymeric probe sequence of nucleic acids or nucleic acid analogues; (3) providing a hybridization mixture comprising the target, the probe, water, and a buffer; (4) incubating the hybridization mixture for an incubation time effective to bind the heteropolymeric target sequence to the heteropolymeric probe sequence to provide a complex; and (5) detecting a signal correlated with binding affinity between the probe and the target to assay the target, wherein the heteropolymeric probe sequence is bonded to the heteropolymeric target sequence by Watson-Crick complementary base interaction or by homologous base interaction, provided that when the complex is a duplex and the heteropolymeric probe sequence is antiparallel to the heteropolymeric target sequence, the heteropolymeric probe sequence is bonded to the heteropolymeric target sequence by homologous base interaction, and provided that when the complex is a triplex, the complex is free of recombination proteins.
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Daksis Jasmine I.
Erikson Glen H.
Kandic Ivana
Picard Pierre
Caesar Rivise Bernstein Cohen & Pokotilow Ltd.
Horlick Kenneth R.
Ingeneus Corporation
Wilder Cynthia
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