Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-07-17
2003-03-11
Horlick, Kenneth R. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S007100, C436S094000
Reexamination Certificate
active
06531286
ABSTRACT:
FIELD OF THE INVENTION
This invention is directed to novel methods for the highly selective detection of specific target molecules. In one embodiment the methods described can be used to detect exceedingly low concentrations of said target molecules by virtue of a highly efficient signal amplification mechanism. In another embodiment 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
Techniques that allow specific detection of target molecules or analytes are important for many areas of research, as well as for clinical diagnostics. Central to most detection techniques are ligands that dictate specific and high affinity binding to a target molecule of interest. In immunodiagnostic assays antibodies mediate specific and high affinity binding, whereas in assays detecting nucleic acid target sequences, complementary oligonucleotide probes fulfill this role. To date, antibodies have been able to provide molecular recognition needs for a wide variety of target molecules and have been the popular choice of the class of ligands for developing diagnostic assays.
Recently, a novel class of oligonucleotide probes, referred to as molecular beacons, that facilitate homogeneous detection of specific nucleic acid target sequences has been described (Piatek et al. (1998) Nature Biotechnology 16:359-363; Tyagi and Kramer (1996) Nature Biotechnology 14:303-308). Molecular beacons are nucleic acid sequences that contain a fluorophore and a quencher (
FIG. 1
; star and filled circle, respectively). By design, molecular beacons are expected to fold into stem-loop structures in which the fluorophore is placed in close proximity to the quencher. When the molecular beacon is illuminated with light corresponding to the excitation wavelength of the fluorescent group, no fluorescence is observed, 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.
The loop region of molecular beacons is designed to contain a nucleotide sequence complementary to the target sequence of interest. 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. Molecular beacons undergo intermolecular hybridization upon interaction with the specific target sequence. Molecular beacons have been used for homogeneous detection of specific nucleic acid sequences, both DNA and RNA (Leone et al. (1998) Nucleic Acids Research 26:2150-2155; Piatek et al. (1998) Nature Biotechnol. 16:359-363; Tyagi and Kramer (1996) Nature Biotecnol. 14:303-308).
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 and coworkers have even described the simultaneous use of four differently labeled molecular beacons for allele discrimination. (Tyagi et al. (1998) Nature Biotechnology 16:49-53).
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 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 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 discovery of the SELEX™ (Systematic Evolution of Ligands by EXponential enrichment) process enables the identification of nucleic acid-based ligands, referred to as aptamers, that recognize molecules other than nucleic acids with high affinity and specificity (Ellington and Szostak (1990) Nature 346:818-822; Gold et al. (1995) Ann. Rev. Biochem. 64:763-797; Tuerk and Gold (1990) Science 249:505-510). Aptamers have been selected to recognize a broad range of targets, including small organic molecules as well as large proteins (Gold et al. (1995) Ann. Rev. Biochem. 64:763-797; Osborne and Ellington (1997) Chem. Rev. 97:349-370). In most cases, affinities and specificities of aptamers to these targets are comparable or better than those of antibodies. In contrast to antibodies whose identification and production completely rest on animals and/or cultured cells, both the identification and production of aptamers takes place in vitro without any requirement for animals or cells. Aptamers are produced by solid phase chemical synthesis, an accurate and reproducible process with consistency among production batches. Aptamers are stable to long-term storage at room temperature. Moreover, once denatured, aptamers can easily be renatured, a feature not shared by antibodies. These inherent characteristics of aptamers make them attractive for diagnostic applications.
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.
Gold Larry
Jayasena Sumedha
Gilead Sciences, Inc.
Horlick Kenneth R.
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