Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-06-21
2001-10-23
Riley, Jezia (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S007100, C435S007200, C536S022100, C536S023100, C536S024300, C536S025330, C536S024330, C436S518000, C436S528000, C422S051000, C422S067000, C422S082050, C422S082090
Reexamination Certificate
active
06306598
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to methods and compositions for the direct detection of analytes using color changes that occur in biopolymeric material in response to selective binding of analytes.
BACKGROUND OF THE INVENTION
DNA synthesis via the automated solid-phase method, whereby the DNA fragment is built up by the sequential addition of activated nucleotides to a growing chain that is linked to an insoluble support, has provided for the synthesis of DNA chains of up to 100 nucleotides long at an approximate rate of 10 minutes per base. Such artificial DNA strands with known sequence, the single stranded probe DNA, have been used to find the complementary counterpart in DNA samples by hybridization. Above a certain temperature (T
m
) the DNA double helix “melts” to form two complementary single strands which recombine upon cooling. If a single strand from the sample has the complementary sequence to the probe DNA they can hybridize to form a double helix. Detection of the DNA hybridization process is important for the development of methods and compositions for DNA synthesis and detection of specific nucleic acid sequences (e.g., detection of mutations, pathogens, and particular alleles). One approach for detecting DNA hybridization utilizes a quartz crystal microbalance, which is a very sensitive device to measure mass changes in the nanogram regime (Okahata et al., J. Am. Chem. Soc. 114:8299 [1992]. Another method of detecting DNA hybridization at surfaces employed the electrogenerated chemiluminescence (ECL) by intercalating an ECL marker into the double helix of the sample-probe DNA tethered to a surface (Xu et al., J. Am. Chem. Soc. 117:2627 [1995]). However, both methods are rather sensitive to interferences, such as chemical, pH, temperature, etc., and require sophisticated equipment.
There has been an increasing interest in the field of DNA sensors due to the impact of such devices on diverse areas of medical, environmental, and biological applications. (See e.g., Fodor et al., Science 251:767-773 [1991]; Maeda et al., Anal. Sciences 8:83-34 [1992]; Sakurai et al., Anal. Chem. 64:1996-1997 [1992]; Okahata et al., J. Am. Chem. Soc. 114:8299-8300 [1992]; Xu et al., J. Am. Chem. Soc. 117:2627-2631 [1995]; and Wang et al., Anal. Chem. 68:2629-2634 [1996]). Besides pure sequencing applications, such DNA sensors could help detect infectious or inherited diseases, or as RNA sensors, aid in monitoring expression levels of specific metabolic pathways to determine environmental pollution. DNA hybridization between a synthetic oligodeoxynucleotide of known sequence and its complement in a given sample provides a powerful tool for the detection and sequencing of DNA and RNA. The hybridization event itself is usually monitored by introducing fluorescent markers and radioactive labels or by applying antibody assays and enzyme reactions to the specifically modified DNA (or RNA) pair, which generally requires labor intensive and time consuming multistep procedures.
Thus, there remains a need of analyte detectors that provide for DNA detection that can be visually monitored by the naked eye, thus, making any further detection procedures ancillary or unnecessary.
SUMMARY OF THE INVENTION
The present invention relates to methods and compositions for the direct detection of analytes using color changes that occur in biopolymeric material in response to selective binding of analytes. In one embodiment, the biopolymeric material comprises self-assembling monomers. In another embodiment, the self-assembling monomers are lipids.
The present invention contemplates biopolymeric materials comprising a plurality of polymerized self-assembling monomers and one or more nucleic acid ligands, wherein said biopolymeric materials change color in the presence of an analyte. In some embodiments, the nucleic acids have affinity for an analyte. In other embodiments, the nucleic acid ligands are single stranded nucleic acid sequences. In a further embodiment, the nucleic acid ligands are linked to said polymerized self-assembling monomers through one or more covalent bonds. In yet another embodiment, the covalent bonds are selected from the group consisting of amine bonds, thiol bonds, and aldehyde bonds.
In a preferred embodiment of the present invention, the biopolymeric materials contains nucleic acids as ligands that have affinity for an analyte. In one embodiment, the nucleic acid ligands have affinity for an analyte selected from the group of nucleic acid molecules, enzymes, pathogens, drugs, receptor ligands, antigens, ions, proteins, hormones, blood components, antibodies, and lectins. In further embodiments, the analytes are nucleic acid molecules are from any organism (including microorganisms, including, but not limited to bacteria, fungi, viruses, etc.), cell, plasmid, or expression vector. In another embodiment, the analytes which are nucleic acid molecules are selected from ribosomal RNA, transfer RNA, messenger RNA, intron RNA, double stranded RNA, single stranded RNA, single stranded DNA, double stranded DNA, DNA-RNA hybrid molecules, PNA, PNA-DNA or PNA-RNA hybrid molecules, nucleic acid sequences characteristic of human pathogens, nucleic acid sequences characteristic of non-human pathogens, and nucleic acid sequences characteristic of genetic abnormalities (e.g., cystic fibrosis, Tay-Sachs disease, cretinism, phenylketonuria (PKU), sickle-cell anemia, diabetes insipidus, retinoblastoma, hemophilia, Deuchenne-type muscular dystrophy, Klinefelter's syndrome, Turner's syndrome, and trisomy-21 (i.e., Down's syndrome)). In additional embodiments, the analytes are enzymes including, but not limited to, polymerases, nucleases, ligases, telomerases, and transcription factors.
The present invention also contemplates biopolymeric materials comprising nucleic acid ligands that have affinity for analytes that are pathogens. It is not intended that the present invention be limited to any particular pathogen analyte(s), as a variety of pathogen analytes are contemplated. In one embodiment, the pathogens are selected from viruses, bacteria, parasites, and fingi. In further embodiments, the pathogens are viruses selected from influenza, rubella, varicella-zoster, hepatitis A, hepatitis B, other hepatitis viruses, herpes simplex, polio, smallpox, human immunodeficiency virus, vaccinia, rabies, Epstein Barr, retroviruses, and rhinoviruses. In another embodiment, the pathogens are bacteria selected from
Escherichia coli, Mycobacterium tuberculosis
, Salmonella, Chiamydia and Streptococcus. In yet a further embodiment, the pathogens are parasites selected from Plasmodium, Trypanosoma,
Toxoplasma gondii
, and Onchocerca. However, it is not intended that the present invention be limited to the specific genera and/or species listed above.
In certain embodiments, the biopolymeric materials comprise biopolymeric films. In other embodiments, the biopolymeric materials comprise biopolymeric liposomes. In yet other embodiments, the biopolymeric materials are selected from the group consisting of tubules, braided assemblies, lamellar assemblies, helical assemblies, fiber-like assemblies, solvated rods, and solvated coils.
In some embodiments, the self-assembling monomers of the biopolymeric material of the present invention comprise diacetylene monomers. In certain embodiments, the diacetylene monomers are selected from the group consisting of 5,7-docosadiynoic acid, 5,7-pentacoadiynoic acid, 10,12-pentacosadiynoic acid, and combinations thereof, although all diacetylene monomers are contemplated by the present invention. In other embodiments, the self-assembling monomers are selected from the group consisting of acetylenes, alkenes, thiophenes, polythiophenes, imides, acrylamides, methacrylates, vinylether, malic anhydride, urethanes, allylamines, siloxanes anilines, pyrroles, vinylpyridinium, and combinations thereof. In certain embodiments, the self-assembling monomers contain head groups se
Charych Deborah H.
Jonas Ulrich
Medlen & Carroll LLP
Regents of the University of California
Riley Jezia
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