Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1999-11-23
2001-01-23
Horlick, Kenneth R. (Department: 1656)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S024300, C435S006120, C435S007100
Reexamination Certificate
active
06177555
ABSTRACT:
FIELD OF THE INVENTION
This invention is directed to a novel method for the highly selective detection of specific target molecules. The binding of a nucleic acid ligand to a target molecule is accompanied by a change in the fluorescence spectrum of the assay solution. The subject invention will be useful in any application where it is desired to detect a target molecule.
BACKGROUND OF THE INVENTION
The ability to detect the presence of a specific target molecule, such as a nucleic acid or a protein, has proved to have increasing importance in a large number of applications. One of the most significant applications utilizing sensitive and selective detection of such target molecules is in diagnostic assays. In these assays, measurement of the concentration of a target molecule is used to yield diagnostic or prognostic medical information.
A recently described reagent for nucleic acid detection is the “molecular beacon”. A molecular beacon is a unimolecular nucleic acid molecule comprising a stem-loop structure (FIG.
1
). The stem is formed by intramolecular base pairing of two complementary sequences such that the 5′ and 3′ ends of the nucleic acid are at the base of the stem. The loop links the two strands of the stem, and is comprised of sequences complementary to those to be detected. A fluorescent group (star in
FIG. 1
) is covalently attached to one end of the molecule, and a fluorescent quenching group (filled circle in
FIG. 1
) is attached to the other end. In the stem-loop configuration, these two moieties are physically adjacent to one another. When the molecular beacon is illuminated with light corresponding to the excitation wavelength of the fluorescent group, no fluorescence is observed. This is because energy transfer occurs between the fluorescent group and the quenching group such that light emitted from the fluorescent group upon excitation is absorbed by the quenching group.
When the molecular beacon is contacted with sequences complementary to the loop, the loop hybridizes to this sequence. This process is energetically favored as the intermolecular duplex formed is longer, and therefore more stable, than the intramolecular duplex formed in the stem region. As this intermolecular double helix forms, torsional forces are generated that cause the stem region to unwind. As a result, the fluorescent group and the quenching group become spatially separated such that the quenching group is no longer able to efficiently absorb light emitted from the fluorescent group. Thus, binding of the molecular beacon to its target nucleic acid sequence is accompanied by an increase in fluorescence emission from the fluorescent group.
It is possible to simultaneously use two or more molecular beacons with different sequence specificities in the same assay. In order to do this, each molecular beacon is labeled with at least a different fluorescent group. The assay is then monitored for the spectral changes characteristic for the binding of each particular molecular beacon to its complementary sequence. In this way, molecular beacons have been used to determine whether an individual is homozygous wild-type, homozygous mutant or heterozygous for a particular mutation. For example, using one quenched-fluorescein molecular beacon that recognizes the wild-type sequence and another rhodamine-quenched molecular beacon that recognizes a mutant allele, it is possible to genotype individuals for the &bgr;-chemokine receptor (Kostrikis et al. (1998) Science 279:1228-1229). The presence of only a fluorescein signal indicates that the individual is wild-type, and the presence of rhodamine signal only indicates that the individual is a homozygous mutant. The presence of both rhodamine and fluorescein signal is diagnostic of a heterozygote. Tyagi et al. ((1998) Nature Biotechnology 16: 49-53) have even described the simultaneous use of four differently labeled molecular beacons for allele discrimination.
Although useful for the detection of nucleic acid targets, molecular beacons have not been used for detecting other types of molecules. Indeed, there has been no suggestion made in the extensive art that molecular beacons can be used for anything other than detecting specific nucleic acids in mixtures containing a plurality of nucleic acids. Detection of nucleic acids is undeniably important, but in many applications—especially medical diagnostic scenarios—detection of non-nucleic acid molecules, such as proteins, sugars, and small metabolites, is required.
In general, the detection of non-nucleic acid target molecules is a more complicated matter than the detection of nucleic acids, and no single method is universally applicable. Specific proteins may be detected through the use of antibody-based assays, such as an enzyme linked immunoassay (ELISA). In one form of ELISA, a primary antibody binds to the protein of interest, and signal amplification is achieved by using a labeled secondary antibody that can bind to multiple sites on the primary antibody. This technique can only be used to detect molecules for which specific antibodies exist. The generation of new antibodies is a time consuming and very expensive procedure, and many proteins are not sufficiently immunogenic to generate antibodies in host animals. Furthermore, it is often necessary to measure and detect small molecules, such as hormones and sugars, that are generally not amenable to antibody recognition. In these cases, enzymatic assays for the specific molecule are often required.
The dogma for many years was that nucleic acids had primarily an informational role. Through a method known as Systematic Evolution of Ligands by EXponential enrichment, termed the SELEX™ process, it has become clear that nucleic acids have three dimensional structural diversity similar to or even more than proteins. The SELEX™ process is a method for the in vitro evolution of nucleic acid molecules with highly specific binding to target molecules and is described in U.S. patent application Ser. No. 07/536,428, filed Jun. 11, 1990, entitled “Systematic Evolution of Ligands by Exponential Enrichment,” now abandoned; U.S. patent application Ser. No. 07/714,131, filed Jun. 10, 1991, entitled “Nucleic Acid Ligands,” now U.S. Pat. No. 5,475,096; U.S. patent application Ser. No. 07/93 1,473, filed Aug. 17, 1992, entitled “Methods for Identifying Nucleic Acid Ligands,” now U.S. Pat. No. 5,270,163 (see also, WO 91/19813), each of which is specifically incorporated by reference herein. Each of these applications, collectively referred to herein as the SELEX™ Patent Applications, describes a fundamentally novel method for making a nucleic acid ligand to any desired target molecule. The SELEX™ process provides a class of products which are referred to as nucleic acid ligands, each ligand having a unique sequence, and which has the property of binding specifically to a desired target compound or molecule. Each SELEX™ process-identified nucleic acid ligand is a specific ligand of a given target compound or molecule. The SELEX™ process is based on the unique insight that inucleic acids have sufficient capacity for forming a variety of two- and three-dimensional structures and sufficient chemical versatility available within their monomers to act as ligands (form specific binding pairs) with virtually any chemical compound, whether monomeric or polymeric. Molecules of any size or composition can serve as targets.
The SELEX™ method involves selection from a mixture of candidate oligonucleotides and step-wise iterations of binding, partitioning and amplification, using the same general selection scheme, to achieve virtually any desired criterion of binding affinity and selectivity. Starting from a mixture of nucleic acids, preferably comprising a segment of randomized sequence, the SELEX™ method includes steps of contacting the mixture with the target under conditions favorable for binding, partitioning unbound nucleic acids from those nucleic acids which have bound specifically to target molecules, dissociating the nucleic acid-target complexes, amplif
Gold Larry
Jayasena Sumedha
Horlick Kenneth R.
NeXstar Pharmaceuticals, Inc.
Swanson & Bratschun LLC
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