Nucleic acid ligand interaction assays

Details Nucleic acid ligand interaction assays Nucleic acid ligand interaction assays

1999-09-10

2002-03-12

Allen, Marianne P. (Department: 1631)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C436S501000

Reexamination Certificate

active

06355428

ABSTRACT:

FIELD OF THE INVENTION
The invention is directed to the determination of relative binding affinities of various ligands to various nucleic acid sequences, particularly double stranded nucleic acid sequences, and in particular to the determination of binding specificities and base pair determinants of particular ligands via a competitive binding assay.
REFERENCES
Brophy, G. P. et al., U.S. Pat. No. 5,789,179 (1998).
Campbell, A. K.,
Chemiluminescence: Principles and Applications in Biology and Medicine
; VCH, Ellis Horwood Ltd.: New York, 1988.
Cook, N. D., U.S. Pat. No. 5,665,562 (1997).
Haugland, R. P. (1996)
Handbook of Fluorescent Probes and Research Chemicals
(Spenze, M. T. Z., ed.) Molecular Probes, Eugene, Oreg.
Nieman, T., “Chemiluminescence: Techniques, Liquid-Phase Chemiluminescence”, in
Encyclopedia of Analytical Science
, pp 613-621; Academic Press; Orlando, Fla., 1995.
Cantor, C. R., “Lighting Up Hybridization”,
Nature Biotechnology
14:264 (1996).
Chen, Q., Shafer, R. H., and Kuntz, I. D.,
Biochemistry
36:11402-7 (1997).
Hart, H., U.S. Pat. Nos. 4,271,139 (1978) and 4,382,074 (1983).
Morrison, L. E. and Stols, L. M., “Sensitive Fluorescence-Based Thermodynamic and Kinetic Measurements of DNA Hybridization in Solution”,
Biochemistry
32: 3095-3104 (1993).
Morrison, L. E., Halder, T. C., and Stols, L. M., “Solution Phase Detection of Polynucleotides Using Interacting Fluorescent Labels and Competitive Hybridization”,
Anal. Biochem.
183:231-244 (1989).
Saenger, W., in
Principles of Nucleic Acid Structure
(Cantor, C. R., ed.), Springer-Verlag, New York, 1984.
Tyagi, S. and Kramer, F. R., “Molecular Beacons: Probes that Fluoresce upon Hybridization”,
Nature Biotech.
14:303-306 (1996).
Tyagi, S., Bratu, D. P., and Kramer, F. R., “Multicolor Molecular Beacons for Allele Discrimination”,
Nature Biotech.
16:49-53 (1998).
Wilson, W. D., Tanious, F. A., Fernandez-Saiz, M., and Rigl, C. T., “Evaluation of Drug-Nucleic Acid Interactions by Thermal Melting Curves” in
Methods in Molecular Biology
, Vol. 90:
Drug-DNA Interaction Protocols
(Fox, K. R. ed.), Humana Press, Totawa, N.J., pp. 219-240.
BACKGROUND OF THE INVENTION
The specific molecular recognition of nucleic acids is fundamental to essential processes in molecular biology, including replication, transcription and translation. It has been shown that, in the majority of cases, binding of ligands to double-stranded nucleic acids stabilizes the duplex, or helical, form of DNA or RNA. (See, for example, Wilson et al.) The current understanding of the interactions between DNA or RNA and bound ligands is largely based on information obtained via biochemical and biophysical methods such as chemical and nuclease footprinting, affinity probing, UV, CD, fluorescent, and NMR spectroscopy, calorimetry, gel electrophoresis, and x-ray crystallography.
In a typical application of DNA footprinting, for example, a labeled oligonucleotide is digested with a DNA nuclease to the extent necessary to create an average of one cut per chain, producing a series of fragments differing by one base pair in length. A similar operation is performed on the oligonucleotide having a bound ligand. The ligand protects the oligonucleotide, at and around its binding site, from nuclease activity, creating a characteristic pattern of “missing” fragments at this site on a polyacrylamide gel following electrophoresis. This method suffers from the disadvantages of being very time and labor intensive, and in revealing not necessarily the critical molecular determinants for the ligand binding, but rather the area of the oligonucleotide that is shielded by the bulk of the ligand.
The most widely used method for studying nucleic acid hybridization is thermal denaturation, or melting, of duplex nucleic acids. Ligand binding has also been studied using thermal denaturation, since binding of ligands to duplex DNA or RNA tends to stabilize the helix against melting. Techniques used to observe this change include UV, fluorescent, CD and NMR spectroscopy, electrophoresis, and calorimetry.
Certain disadvantages are inherent in ligand binding studies based on observation of duplex denaturation, or melting. The methods provide information about binding only at or near the T
m
of the system, rather than at standard (25° C.) or physiological (37° C.) temperatures. Because the presence of the ligand generally raises the T
m
of the duplex, it is necessary that the ligand, e.g. the drug, be stable at this higher temperature. In addition, these methods do not routinely provide information about the binding site of the ligand (see, for example, Chen et al., 1997; Wilson et al., 1997). Therefore, the need exists for assays which are sensitive, are rapidly and simply carried out, and provide precise binding site information.
SUMMARY OF THE INVENTION
In one aspect, the invention provides methods of determining the binding affinity of a ligand to an oligonucleotide sequence. The methods are particularly useful for determining relative binding affinities of various ligands to various oligonucleotide sequences, particularly double stranded oligonucleotide sequences. One such method, described herein as a “direct” assay, comprises the following steps:
(i) providing first and second oligonucleotides, which are effective to hybridize by Watson-Crick base pairing to form a duplex;
wherein the first oligonucleotide comprises a first group effective to produce a detectable signal, and the second oligonucleotide comprises a second group, such that in the presence of the second group the signal is detectably altered upon hybridization of the first and second oligonucleotides, and in the absence of the second group the signal would not be detectably altered upon such hybridization;
(ii) forming a mixture of the oligonucleotides under conditions such that, in the absence of the ligand, the oligonucleotides exist primarily in single-stranded form;
(iii) observing the signal from the mixture in the absence of the ligand;
(iv) adding the ligand to the mixture; and
(v) observing the signal from the mixture in the presence of the ligand.
By carrying out steps (i)-(v) for each of a plurality of pairs of such first and second oligonucleotides, the relative binding affinity of the plurality of oligonucleotide pairs for the ligand may be determined. Similarly, by carrying out steps (iv)-(v) for each of a plurality of ligands, whereby the relative binding affinity of the plurality of ligands for the oligonucleotide pair may be determined.
The ligand is typically a metal ion, a small organic or inorganic molecule, a protein, or a multi-protein complex, as defined herein. Preferably, the ligand is added to the mixture in increasing concentrations, and in step (v) above, the signal is thus observed in the presence of increasing concentrations of the ligand. The mixture may be held at a substantially constant temperature, e.g. at or near room temperature, as the ligand is added.
With respect to the oligonucleotide pair, the first group above is preferably attached at the 5′-end or 3′-end of the first oligonucleotide, while the second group is attached at the 3′-end or 5′-end, respectively, of the second oligonucleotide. In various embodiments, the first and second groups may be, respectively: a radiation emitting group and a group effective to absorb the emitted radiation; a group comprising a scintillant, and a radioactive group; or, a chemiluminescent group, and a group which participates in a chemiluminescent reaction with the first group. In another embodiment, the first group is effective to produce a detectable signal, as recited above, and the second group is effective to alter the proximity of the oligonucleotide duplex to a source which is effective to stimulate or modulate the production of the signal from the first group, where this stimulation or modulation is proximity-dependent. For example, the second group may be a binding group effective to bind to a surface. In one embodiment, the first group is an electrochemiluminescent group; in this case, the

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