Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-10-15
2003-05-27
Zitomer, Stephanie W. (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C536S022100, C536S023100
Reexamination Certificate
active
06569630
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to the field of detection of biological agents using novel compositions, methods and apparatus comprising one or more nucleic acid ligands operably coupled to an organic semiconductor. More particularly, the present invention relates to the production and use of nucleic acid ligands against anthrax spores.
2. Description of Related Art
There is a great need for the development of methods, compositions and apparatus capable of detecting and identifying known or unknown chemical and biological agents (herein referred to as analytes), which include but are not limited to nucleic acids, proteins, illicit drugs, explosives, toxins, pharmaceuticals, carcinogens, poisons, allergens, contaminants, pathogens and infectious agents.
As one skilled in the art will readily appreciate, any method, technique or device capable of such detection and identification would have numerous medical, industrial forensic and military applications. For instance, such methods, techniques and devices could be employed in the diagnosis and treatment of disease, to develop new compounds for pharmaceutical, medical or industrial purposes, or to identify chemical and biological warfare agents.
Current methods, techniques and devices that have been applied to identification of chemical and biological analytes typically involve capturing the analyte through the use of a non-specific solid surface or through capture deoxyribonucleic acids (DNA) or antibodies. A number of known binding agents must then be applied, particularly in the case of biological analytes, until a binding agent with a high degree of affinity for the analyte is identified. A labeled antiligand (e.g., labeled DNA or labeled antibodies) must be applied, where the antiligand causes, for example, the color or fluorescence of the analyte to change if the binding agent exhibits affinity for the analyte (i.e., the binding agent binds with the analyte). The analyte may be identified by studying which of the various binding agents exhibited the greatest degree of affinity for the analyte.
There are a number of problems associated with current methods of chemical and biological agent identification. It takes a great deal of time and effort to repetitiously apply each of the known labeled antiligands, until an antiligand exhibiting a high degree of affinity is found. Accordingly, these techniques are not conducive to easy automation. Current methods are also not sufficiently robust to work in the heat, dust, humidity or other environmental conditions that might be encountered, for example, on a battlefield or in a food processing plant. Portability and ease of use are also problems seen with current methods for chemical and biological agent identification.
Within the field of biological warfare, there is a great need for a rapid, sensitive method to detect and identify pathogenic spores of Bacillus anthrax (hereafter “anthrax”). Anthrax is a highly pathogenic biological agent that is relatively simple to produce and distribute in the field. Present methods for detection of anthrax are not sufficiently rapid, sensitive, and robust to allow early detection of exposure to anthrax under field conditions, such as might be encountered on a battlefield. No good method presently exists for neutralization of anthrax under field conditions.
SUMMARY OF THE INVENTION
The present invention fulfills an unresolved need in the art, by providing methods, compositions and apparatus for the production of nucleic acid ligands capable of binding to, identifying and/or neutralizing anthrax. The methods and compositions disclosed herein provide substantial improvements over earlier methods for anthrax detection (e.g., Reif et al., 1994; Gatto-Menking et al., 1995; Bruno and Yu, 1996), by utilizing anthrax-binding nucleic acid ligands.
The compositions of the present invention comprise a recognition complex or a recognition complex system that are capable of detecting, identifying, characterizing or purifying a chemical or biological agent (hereafter, “analyte”), preparing or purifying high affinity nucleic acid ligands for selected known analytes, using high affinity nucleic acid ligands to measure the concentration of analyte in a sample or to neutralize an analyte, or to perform high through-put screening of libraries of compounds or native plant extracts for compounds that are structural analogs of known inhibitors, activators or binding agents of bioactive molecules. The recognition complex and recognition complex system and the corresponding techniques should be capable of full automation.
Each recognition complex is comprised of a nucleic acid ligand operably coupled to an organic semiconductor. In certain embodiments, the organic semiconductor is DALM (diazoluminomelanin), although the use of other organic semiconductors, such as polyphenylenes, is contemplated within the scope of the invention. In various embodiments, the organic semiconductor may be attached to the nucleic acid ligand by either covalent or non-covalent interaction.
In preferred embodiments, the nucleic acid ligand is DNA, although it is contemplated within the scope of the invention that other nucleic acids comprised of RNA or synthetic nucleotide analogs could be utilized as well. In certain embodiments, the nucleic acid ligand sequences are random, or may be generated from libraries of random DNA sequences. In other embodiments, the nucleic acid ligand sequences may not be random, but may rather be designed to react with specific target analytes. In a preferred embodiment, the nucleic acid ligand sequences are aptamers (Lorsch and Szostak, 1996; Jayasena, 1999; U.S. Pat. Nos. 5,270,163; 5,567,588; 5,650,275; 5,670,637; 5,683,867; 5,696,249; 5,789,157; 5,843,653; 5,864,026; 5,989,823 and PCT application WO 99/31275, each incorporated herein by reference).
In certain embodiments, the analyte to be identified may be added in the form of a complex mixture that may include, for example, aqueous or organic solvent, proteins, lipids, nucleic acids, detergents, particulates, intact cells, bacteria, viruses and spores, as well as other components. In other embodiments, the analyte may be partially or fully purified before exposure to the array. In particularly preferred embodiments, the analyte is anthrax spore.
In certain embodiments, a recognition complex system, comprising two or more recognition complexes, may be used in methods for identifying an analyte. After the analyte is contacted with the recognition complexes, certain recognition complexes will bind the analyte, while others will not. Binding of analyte to a recognition complex may be detected by changes in the electrochemical properties of the nucleic acid ligand/organic semiconductor couplet upon binding to the analyte. Nonlimiting examples of electrochemical signals include photochemical, fluorescent or luminescent signals, changes in color or changes in electrical conductivity. The degree to which the electrochemical properties change is a function of the degree to which the nucleic acid ligand binds the analyte. Accordingly, the electrochemical changes that occur across all of the recognition complexes, when taken as a whole, can be used as a unique signature to identify the analyte.
To facilitate detection of such electrochemical changes, the recognition complex system may be associated with a detection unit operably coupled to the recognition complexes. Non-limiting examples of detection units include a charge coupled device (CCD), a CCD camera, a photomultiplier tube, a spectrophotometer or a fluorometer. The recognition complex system may also be associated with system memory for storing electrochemical signals, as well as a data processing unit that may comprise a neural network or lookup tables. For embodiments where the binding of analyte is detected by changes in electrical conductivity of the recognition complex, the complexes may be positioned between a pair of electrodes attached to a conductivity meter.
In addition to analyte identification, re
Kiel Johnathan L.
Vivekananda Jeevalatha
Blakely & Sokoloff, Taylor & Zafman
Conceptual MindWorks, Inc.
Nakashima Richard A.
Zitomer Stephanie W.
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