Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-07-10
2002-07-16
Jones, W. Gary (Department: 1634)
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
06420115
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of Invention
The invention relates to nucleic acid triplexes, and more particularly to methods of accurately assaying triplex nucleic acid complexes employing fluorescent intensity measurements.
2. Description of Related Art
Fluorescent dyes have been used to detect and quantitate nucleic acids for decades. In their most basic form, fluorescent intensity-based assays have typically comprised contacting a target with a fluorophore-containing probe, removing any unbound probe from bound probe, and detecting fluorescence in the washed sample. Homogeneous assays improve upon such basic assays, in that the former do not require a washing step or the provision of a non-liquid phase support.
For example, U.S. Pat. No. 5,538,848 to Livak et al. and U.S. Pat. No. 4,220,450 to Maggio disclose homogeneous fluorescence-based assays of nucleotide sequences using oligonucleotide probes in solution. However, these patents require the use of a quenching agent in combination with a reporting agent, so as to distinguish between the signals generated by hybridized probes and unhybridized probes. Livak et al. also requires the use of enzymes in its disclosed method. Quenching agents and enzymes add complexity and expense to the methods.
U.S. Pat. No. 5,332,659 to Kidwell discloses a method for detecting nucleotide sequences in solution using probes comprising at least two fluorophore moieties. The fluorophores must be selected to electronically interact with each other when close enough to vary the wavelength dependence of their spectra. Unhybridized probes are much more flexible than probes hybridized to the target sequence, and consequently the two fluorophore moieties on each probe are more likely to be close to each other when the probe is unhybridized than when the probe is hybridized. Thus, a change in emission wavelength correlated with free probe can be monitored as an indication of the amount of free probe in the sample.
U.S. Pat. No. 5,846,729 to Wu et al. also discloses homogeneous fluorescence-based assays for detecting nucleic acid.
In addition to the aforementioned developments which detect fluorescent intensity, some have touted the advantages of fluorescent polarization assays. However, there are significant drawbacks to polarization-based assays. The degree of change in polarization as a function of binding can be unpredictable, and interpretation of data to conform inconsistent data to theoretical expectations can require more effort than is desirable in an analytical method, particularly when the method is to be automated. There are as well constraints arising from the molecular weight of the molecules whose motion is being evaluated in a fluorescent polarization assay.
Conventional assays for nucleic acids have generally been based on a duplex hybridization model, wherein a single-stranded probe specifically binds to a complementary single-stranded target sequence. Triplex hybridization of nucleic acids has been previously identified in the art; however, hybridization among three strands was largely 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, such triplex formation or hybridization was 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.
Despite the foregoing developments, a need has continued to exist in the art for additional simple, highly sensitive, effective and rapid methods for analyzing interaction between nucleic acids and/or nucleic acid analogs.
All references cited herein are incorporated herein by reference in their entireties.
SUMMARY OF THE INVENTION
The invention provides triplex complexes comprising a single-stranded probe bound to a double-stranded nucleic acid target, wherein the probe comprises a heteropolymeric nucleic acid or a heteropolymeric nucleic acid analog, and all base triplets of the complex are members selected from the group consisting of A-T-A, T-A-T, U-A-T, T-A-U, A-U-A, U-A-U, G-C-G and C-G-C.
Also provided is a method for assaying binding, said method comprising:
providing a double-stranded nucleic acid comprising a target sequence, wherein said target sequence contains at least one purine base and at least one pyrimidine base;
providing a probe comprising a nucleic acid sequence or a nucleic acid analog sequence;
providing a cation;
adding said probe, said target sequence and said cation to a medium to provide a test sample containing a triplex complex comprising said probe bound to said target sequence, wherein all base triplets of said complex are members selected from the group consisting of A-T-A, T-A-T, U-A-T, T-A-U, A-U-A, U-A-U, G-C-G and C-G-C;
irradiating said test sample with exciting radiation to cause test sample to emit fluorescent radiation;
detecting an intensity of said fluorescent radiation, wherein said intensity is correlated with a binding affinity between said probe and said target sequence; and
determining from said intensity an extent of matching between said probe and said target sequence.
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Sen
Daksis Jasmine I.
Erikson Glen H.
Picard Pierre
Caesar Rivise Bernstein Cohen & Pokotilow Ltd.
Forman B J
Ingeneus Corporation
Jones W. Gary
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