Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-04-19
2002-06-04
Zitomer, Stephanie W. (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S287100, C435S287200, C435S288300, C435S288400, C435S288700, C536S024300
Reexamination Certificate
active
06399302
ABSTRACT:
FIELD OF THE INVENTION
The present invention is directed to a new class of signal generating high-affinity nucleic acid ligands that specifically bind a desired target molecule and produce a detectable signal upon binding to the target molecule. A method is described for creating these signal-generating ligands, and for their use in assay methods, diagnostic procedures, cell sorting, and for other analytical procedures including their use as probes.
BACKGROUND OF THE INVENTION
The current methods of detecting specific analytes in complex mixtures (for example detecting complex organic compounds such as pesticides in a given sample) requires the extraction of the sample into organic solvents, followed by analysis using gas-liquid chromatography and/or mass spectroscopy. These methods are slow (several days, typically) and expensive ($100-$300 per single analysis, from a survey of commercial laboratories that perform these assays, in mid-1999). The development of a biosensor device that could detect/screen for a dozen or more different analytes within minutes would therefore provide a significant cost and time benefit.
Despite the widespread interest in the development of biosensors, little progress has been made, except on a case-by-case basis for single targets. One problem has been the lack of a general method for developing specific receptors for diverse types of targets. Several approaches to resolve this problem have been developed (see Turk, C, gold, L Science 249: 505-510 (1990); U.S. Pat. Nos. 5,843,653, and 5,843,701). The other, more intractable problem, has been signal generation upon binding of the target to the biosensor.
The present invention is directed to the development of a multi-target biosensor through the use of nucleic acid based, signal-generating ligands that can be used for detecting analytes. The signal-generating ligands of the present invention are derived from oligonucleotides (single-stranded RNA, DNA or modified variants of these structures). These oligonucleotides are refer to as aptamers. Aptamers are used as the basis for the signal-generating ligands because they have the capacity for forming a virtually limitless array of shapes, sizes and configurations and thus are capable of forming specific binding pairs with virtually any chemical compound, whether monomeric or polymeric. A procedure for the selection of aptamers that bind a desired target has been disclosed in U.S. Pat. No. 5,843,653, and is named SELEX. However, this method does not provide for a signal-generating ligand. Advantageously, the present invention provides a ligand complex with enhanced target selectivity as well as a means for the complex to produce a detectable signal upon binding to its specific target analyte.
The present invention uses two aptamers that recognize different epitopes of the same target molecule. The two aptamers are coupled together to create a new structure, termed a biaptamer. The construction of the biaptamer is based on the inventors' insight that the coupling together of two aptamers dramatically increases the affinity and selectivity of the ligand complex (biaptamer) for the target analyte, because binding becomes cooperative. Furthermore, this enhanced specificity and affinity for the target analyte reduces the need to exhaustively select for highly specific, high affinity aptamers for each target. In addition, a biaptamer will undergo a conformational change when it binds to the target analyte. The conformational change is a change in the separation and/or the angle between the two aptamer components of the biaptamer. This change is exploited in the present invention to create a signal-generating mechanism. The biaptamer is altered by the addition of fluorophores (defined here as fluorescent or luminescent entities, or chromophores). Interaction of the target analyte with this modified biaptamer changes the characteristics of the photon absorption or emission from the modified biaptamer. These changes provide a real time detection of the binding event. The prior art has failed to teach or suggest the fusion of multiple aptamers to create biaptamers with intrinsic signaling properties.
Several properties of the photon absorption or emission of paired fluorophores change with changes in their spatial arrangement. For example, if two fluorophores are used that possess a substantial overlap between the emission spectrum of one of the pair (the donor) and the excitation spectrum of the other (the acceptor), then the excitation of the donor can result in transfer of energy to the acceptor. This process is termed resonance energy transfer (RET). RET is very sensitive to the distance and angle between the fluorophores. It can be detected as a decrease in donor emission or fluorescence lifetime, or by an increase in acceptor emission or lifetime. Conformational changes can also lead to changes in the anisotropy properties of the fluorophores.
In accordance with one embodiment of the invention the signal-generating ligands of the present invention are bound to a solid surface. In this embodiment, fluorescence energy transfer in combination with established DNA chip technology allows the system to be easily multiplexed.
SUMMARY OF THE INVENTION
The present invention provides oligonucleotide-based biosensors and a general method for their production and use. Specifically, the invention describes a novel use for oligonucleotide aptamers that have been selected against target molecules of interest. The invention describes the combination of these aptamers into “biaptamers” that show co-operative binding to the target, and which have an enhanced probability of undergoing a conformational change on binding to the target molecule. The biaptamers are further provided with a signaling system that provides a detectable signal upon binding of the biaptamer to its specific analyte. Energy transfer is induced by the conformational change in the biaptamer caused by the interaction of its two distinct binding sites with the target.
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Lannigan Deborah A.
Macara Ian G.
Breen John P.
University of Virginia Patent Foundation
Zitomer Stephanie W.
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