Nucleic acid-based detection

Details Nucleic acid-based detection Nucleic acid-based detection

2000-05-12

2004-01-20

Zitomer, Stephanie (Department: 1634)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C536S023100, C436S501000, C435S006120, C435S018000, C435S019000, C435S183000

Reexamination Certificate

active

06680377

ABSTRACT:

FIELD OF THE INVENTION
The invention relates to compositions, systems, and methods for detecting molecular species using nucleic acids.
BACKGROUND OF THE INVENTION
Various types of systems have been used to detect the presence of a particular chemical or molecule in a complex sample. For example, antibodies are used to detect the presence of a protein in a sample, and DNA microarray chips have been used to identify genes and study gene expression. Most existing molecular detection systems are designed to detect the presence of a single type or single category of target molecule. In the case of antibody detection, existing systems are typically limited to detecting only a subset of a type of molecule.
Recently, it has been shown that RNA and DNA aptamers can substitute for monoclonal antibodies in various applications (Jayasena, “Aptamers: an emerging class of molecules that rival antibodies in diagnostics.” Clin. Chem., 45(9):1628-50, 1999; Morris et al., “High affinity ligands from in vitro selection: complex targets.” Proc. Natl. Acad. Sci., USA, 95(6):2902-7, 1998). The relatively fast selection process of the specific aptamers and the inexpensive synthesis makes the aptamer useful alternatives for monoclonal antibodies. These nucleic acids can be easily synthesized, readily manipulated, and can be stored for over long time. These benefits make nucleic acids more attractive biotechnology tools than their counterpart of proteins, antibodies. Additionally these nucleic acid probes can also be labeled by radioisotope, biotin, or fluorescent tags and can be used to detect targets under various conditions. An increasing number of DNA and RNA aptamers that recognize their non-nucleic acid targets has been developed by SELEX and has been characterized (Gold et al., “Diversity of Oligonucleotide Functions,” Annu. Rev. Biochem., 64:763-97.1995; Bacher & Ellington, “Nucleic Acid Selection as a Tool for Drug Discovery,” Drug Discovery Today, 3(6):265-273,1998).
SUMMARY OF THE INVENTION
The invention relates to new compositions, systems, and methods for simultaneously detecting the presence and quantity of one or more different compounds in a sample using novel nucleic acid sensor molecules. Nucleic acids have previously been shown to be capable of specifically binding with high affinity to non-nucleotide target molecules, such as proteins, small organic molecules, or inorganic molecules. These nucleic acids are commonly referred to as aptamers. An aptamer can be either an RNA or a DNA composed of naturally occurring or modified nucleotides.
In the new compositions, standard aptamers are bioengineered such that binding of a bioengineered aptamer to a target molecule causes a change in the conformation of the bioengineered aptamer. Furthermore, one or more reporter moieties or groups are included in the bioengineered aptamers such that the change in bioengineered aptamer conformation results in a detectable change of a physical property of the reporter group (or the engineered aptamer itself). These bioengineered aptamers are referred to herein as aptamer beacons.
Aptamer beacons having binding regions configured to bind to different target molecules can be used in various detection methods and systems. For example the new aptamer beacons can be used in solution-based assays, or can be attached to a solid support, e.g., at different predetermined points in a one or two-dimensional array, for use in solid-based assays. The aptamer beacons or aptamer beacon arrays are then exposed to the sample, such that target molecules in the sample bind to their respective aptamer beacons. The presence of bound target molecules can be detected by measuring a change in a physical property of the aptamer beacon reporter group, e.g., by observing a change in fluorescence efficiency of the aptamer beacon.
To assist in analyzing the sample, the new detection systems can include pattern recognition software. The software compares the target molecule binding pattern corresponding to the unknown sample with binding patterns corresponding to known compounds. From these comparisons, the software can determine the composition of the sample, or deduce information about the source of the sample.
The systems can be used to detect the existence of characteristic compounds, or “molecular fingerprints,” associated with certain chemicals or conditions. For example, the systems can be used for human drug testing by detecting the presence of metabolites of particular drugs. The systems can also be used to infer the existence of a disease (e.g., cancer) by detecting the presence of compounds associated with the disease state, or for pollution monitoring by detecting compounds characteristic of the discharge of certain pollutants. Numerous other applications are also possible.
In general, the invention features an aptamer beacon that binds to a non-nucleic acid target molecule and that includes an oligonucleotide including a loop portion, a first segment, and a second segment complementary to the first segment, wherein the first and second segments form a stem portion when hybridized together; a binding region formed by the oligonucleotide and configured to bind to the non-nucleic acid target molecule; a first reporter moiety, e.g., a fluorophore, attached to the first segment; and a second reporter moiety, e.g., a chemical quencher, attached to the second segment, wherein the first and second reporter moieties interact to produce a detectable signal when the distance between them is changed; wherein binding of the target molecule to the binding region breaks base pair bonds in the stem portion, causing a change in conformation of the aptamer beacon that separates the first and second segments, thereby altering the distance between the first and second reporter moieties, and producing a detectable signal.
A conformational change in an aptamer beacon is an alteration in the secondary and/or tertiary structure of the oligonucleotide that makes up the aptamer beacon. A conformational change typically results in the addition and/or deletion of basepairing interactions in the between alternate forms of the aptamer beacon.
In these new aptamer beacons, when the first reporter moiety is a fluorophore and the second reporter moiety is a chemical quencher, the quencher quenches the fluorophore when the first and second segments are hybridized together to form the stem portion, and wherein binding of a target molecule to the binding region breaks base pair bonding in the stem portion, causing the first and second segments to separate and the fluorophore to separate from the chemical group, thereby ending the quenching and enabling the fluorophore to emit detectable fluorescence.
In these aptamer beacons, the binding region can be located entirely or partially within the loop portion, the stem portion, or at least partially in both. In addition, the first and second reporter moieties can be an enzyme and a corresponding ligand, and the first and second segments can include 4, 5, 6, or 7 nucleotides each.
The invention also features an aptamer beacon that binds to a non-nucleic acid target molecule and that includes an oligonucleotide including a first segment, a second segment, and a third segment located between the first and second segments, wherein the first and second segments form a complex, e.g., a hybrid duplex or other secondary or tertiary structure, when the aptamer beacon is not bound to the target molecule; a binding region formed by the aptamer beacon when contacting the target molecule; a first reporter moiety attached to the first segment; and a second reporter moiety attached to the second segment, wherein the first and second reporter moieties interact to produce a detectable signal when the distance between them is changed; wherein binding of the target molecule to the binding region breaks base pair bonds in the complex causing a change in conformation of the aptamer beacon that alters the distance between the first and second reporter moieties, and producing a detectable signal.
In these aptamer beacons, the firs

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