| Hybridization of polynucleotides conjugated with chromophores and fluorophores to generate donor-to-donor energy transfer system -> Monitor Keywords |
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Hybridization of polynucleotides conjugated with chromophores and fluorophores to generate donor-to-donor energy transfer systemRelated Patent Categories: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip, Involving Nucleic AcidHybridization of polynucleotides conjugated with chromophores and fluorophores to generate donor-to-donor energy transfer system description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20070042386, Hybridization of polynucleotides conjugated with chromophores and fluorophores to generate donor-to-donor energy transfer system. Brief Patent Description - Full Patent Description - Patent Application Claims
[0001] This is a continuation of U.S. application Ser. No. 09/724,753, filed on Nov. 28, 2000, which in turn is a continuation of U.S. application Ser. No. 09/123,638, filed on Jul. 28, 1998, now U.S. Pat. No. 6,162,603, which in turn is a continuation of U.S. application Ser. No. 08/232,233, filed on May 5, 1994, now U.S. Pat. No. 5,565,322, which was filed under 35 U.S.C. .sctn.371 based on International Application PCT/US92/09827, filed Nov. 6, 1992, which in turn is a continuation-in-part of U.S. application Ser. No. 08/250,951, filed May 27, 1994, now U.S. Pat. No. 5,532,129, which in turn is a continuation of U.S. application Ser. No. 07/790,262, filed Nov. 7, 1991, now abandoned. Each of the above-identified patents and applications is expressly incorporated herein by reference. TECHNICAL FIELD [0002] This invention relates to design and synthesis of modified synthetic nucleic acid polymers/oligomers with directly incorporated electronic/photonic transfer properties. In particular, it relates to the property of extended directional non-radiative energy transfer. These unique components can be programmed to self-assemble and organize into larger more complex structures. The directly incorporated electronic/photonic functional properties allow connections and novel mechanisms to be formed within the organized structures. The combination of the properties allows ultimately for the creation of useful photonic and photovoltaic devices, DNA bio-sensors, and DNA diagnostic assay systems. BACKGROUND OF THE INVENTION [0003] The fields of molecular electronics/photonics and nanotechnology offer immense technological promise for the future. Nanotechnology is defined as a projected technology based on a generalized ability to build objects to complex atomic specifications. Drexler, Proc. Natl. Acad. Sci USA, 78:5275-5278, (1981). Nanotechnology means an atom-by-atom or molecule-by-molecule control for organizing and building complex structures all the way to the macroscopic level. Nanotechnology is a bottom-up approach, in contrast to a top-down strategy like present lithographic techniques used in the semiconductor and integrated circuit industries. The success of nanotechnology will be based on the development of programmable self-assembling molecular units and molecular level machine tools, so-called assemblers, which will enable the construction of a wide range of molecular structures and devices. Drexler, in "Engines of Creation," Doubleday Publishing Co., New York, N.Y. (1986). Thus, one of the first and most important goals in nanotechnology is the development of programmable self-assembling molecular construction units. [0004] Present molecular electronic/photonic technology includes numerous efforts from diverse fields of scientists and engineers. Carter, ed. in "Molecular Electronic Devices II," Marcel Dekker, Inc, New York, N.Y. (1987). Those fields include organic polymer based rectifiers, Metzger et al., in "Molecular Electronic Devices II," Carter, ed., Marcel Dekker, New York, N.Y., pp. 5-25 (1987), conducting conjugated polymers, MacDiarmid et al., Synthetic Metals. 18:285 (1987), electronic properties of organic thin films or Langmuir-Blogett films, Watanabe et al., Synthetic Metals, 28:C473 (1989), molecular shift registers based on electron transfer, Hopfield et al., Science, 241:817 (1988), and a self-assembly system based on synthetically modified lipids which form a variety of different "tubular" microstructures. Singh et al., in "Applied Bioactive Polymeric Materials," Plenum Press, New York, N.Y., pp. 239-249 (1988). Molecular optical or photonic devices based on conjugated organic polymers, Baker et al., Synthetic Metals, 28:D639 (1989), and nonlinear organic materials have also been described. Potember et al., Proc. Annual Conf. IEEE in Medicine and Biology, Part 4/6:1302-1303 (1989). [0005] However, none of the cited references describe a sophisticated or programmable level of self-organization or self-assembly. Typically the actual molecular component which carries out the electronic and/or photonic mechanism is a natural biological protein or other molecule. Akaike et al., Proc. Annual Conf. IEEE in Medicine and Biolog Part 4/6:1337-1338 (1989). There are presently no examples of a totally synthetic programmable self-assembling molecule which produces an efficient electronic or photonic structure, mechanism or device. [0006] Progress in understanding self-assembly in biological systems is relevant to nanotechnology. Drexler, Proc. Natl. Acad. Sci USA, 78:5275-5278 (1981). Drexler, in "Engines of Creation," Doubleday Publishing Co., New York, N.Y. (1986). Areas of significant progress include the organization of the light harvesting photosynthetic systems, the energy transducing electron transport systems, the visual process, nerve conduction and the structure and function of the protein components which make up these systems. The so called bio-chips described the use of synthetically or biologically modified proteins to construct molecular electronic devices. Haddon et al., Proc. Natl. Acad. Sci. USA, 82:1874-1878 (1985). (McAlear et al., in "Molecular Electronic Devices II," Carter ed., Marcel Dekker, Inc., New York N.Y., pp. 623-633 (1987). Some work on synthetic proteins (polypeptides) has been carried out with the objective of developing conducting networks. McAlear et al., in "Molecular Electronic Devices," Carter ed., Marcel Dekker, New York, N.Y., pp. 175-180 (1982). Other workers have speculated that nucleic acid based bio-chips may be more promising. Robinson et al., "The Design of a Biochip: a Self-Assembling Molecular-Scale Memory Device," Protein Engineering. 1:295-300 (1987). [0007] Great strides have also been made in our understanding of the structure and function of the nucleic acids, deoxyribonucleic acid or DNA, Watson, et al., in "Molecular Biology of the Gene," Vol. 1, Benjamin Publishing Co., Menlo Park, Calif. (1987), which is the carrier of genetic information in all living organisms. In DNA, information is encoded in the linear sequence of nucleotides by their base units adenine, guanine, cytosine, and thymidine (A, G, C, and T). Single strands of DNA (or polynucleotides) have the unique property of recognizing and binding, by hybridization, to their complementary sequence to form a double stranded nucleic acid duplex structure. This is possible because of the inherent base-pairing properties of the nucleic acids; A recognizes T, and G recognizes C. This property leads to a very high degree of specificity since any given polynucleotide sequence will hybridize only to its exact complementary sequence. [0008] In addition to the molecular biology of nucleic acids, great progress has also been made in the area of the chemical synthesis of nucleic acids (16). This technology has developed so automated instruments can now efficiently synthesize sequences over 100 nucleotides in length, at synthesis rates of 15 nucleotides per hour. Also, many techniques have been developed for the modification of nucleic acids with functional groups, including: fluorophores, chromophores, affinity labels, metal chelates, chemically reactive groups and enzymes. Smith et al., Nature, 321:674-679 (1986); Agarawal et al., Nucleic Acids Research, 14:6227-6245 (1986); Chu et al., Nucleic Acids Research, 16:3671-3691 (1988). [0009] An impetus for developing both the synthesis and modification of nucleic acids has been the potential for their use in clinical diagnostic assays, an area also referred to as DNA probe diagnostics. Simple photonic mechanisms have been incorporated into modified oligonucleotides in an effort to impart sensitive fluorescent detection properties into the DNA probe diagnostic assay systems. This approach involved fluorophore and chemiluminescent-labeled oligonucleotides which carry out Forster nonradiative energy transfer. Heller et al., in "Rapid Detection and Identification of Infectious Agents," Kingsbury et al., eds., Academic Press, New York, N.Y. pp. 345-356 (1985). Forster nonradiative energy transfer is a process by which a fluorescent donor (D) group excited at one wavelength transfers its absorbed energy by a resonant dipole coupling process to a suitable fluorescent acceptor (A) group. The efficiency of energy transfer between a suitable donor and acceptor group has a 1/r.sup.6 distance dependency (see Lakowicz et al., in "Principles of Fluorescent Spectroscopy," Plenum Press, New York, N.Y., Chap. 10, pp. 305-337 (1983)). [0010] In the work of Heller et al., supra, two fluorophore labeled oligonucleotides are designed to bind or hybridize to adjacent positions of a complementary target nucleic acid strand and then produce efficient fluorescent energy transfer in terms of re-emission by the acceptor. The first oligonucleotide is labeled in the 3' terminal position with a suitable donor group, and the second is labeled in the 5' terminal position with a suitable acceptor group. The binding or hybridization to the complementary sequence positions the fluorescent donor group and fluorescent acceptor groups so they are at optimal distance (theoretically) for most efficient Forster nonradiative energy transfer. However, the observed energy transfer efficiency in terms of re-emission by the acceptor was relatively low (.about.20%) for this particular arrangement. [0011] In later work (Heller et al., European Patent Application No. EPO 0229943, 1987; and Heller et al., U.S. Pat. No. 4,996,143, Feb. 26, 1991), the advances in synthetic chemistry provided methods for the attachment of fluorophores at any position within an oligonucleotide sequence using a linker arm modified nucleotide. Also, with this synthetic linkage technique it was possible to incorporate both a donor and an acceptor fluorophore within the same oligonucleotide. Using the particular linker arm, it was found that the most efficient energy transfer (in terms of re-emission by the acceptor) occurred when the donor and acceptor were spaced by 5 intervening nucleotide units, or approximately 1.7 nm apart. Heller et al., U.S. Pat. No. 4,996,143 also showed that as the nucleotide spacing decreases from 4 to 0 units (1.4 nm to 0 nm), the energy transfer efficiency also decreases; which is not in accordance with Forster theory. As the nucleotide spacing was increased from 6 to 12 units (2 nm to 4.1 nm), the energy transfer efficiency was also found to decrease; which is in accordance with Forster theory. At the time, it was not explained nor understood why the more closely spaced donor and acceptor arrangements had reduced energy transfer efficiency and were not in agreement with Forster theory. In particular, the teachings of Heller et al. did not address multiple donor resonance and extended energy transfer from donors beyond Forster distances of >5 nm. [0012] Fluorescent energy transfer has been utilized in other areas which include immunodiagnostics and liquid chromatography analysis. Morrison et al., Anal. Biochem. 174:101-120 (1988); and Garner et al., Anal. Chem. 62:2193-2198 (1990). Also, some of the initial demonstrations of simple fluorescent donor/acceptor energy transfer in nucleic acids were later corroborated by other workers. Cardullo et al., Proc. Natl. Acad. Sci. USA, 85:8790-8794 (1988); and Morrision et al., Anal. Biochem., 183:231-244 (1989). In the Cardullo et al. work, an arrangement is studied where two short (12-mer) oligonucleotide sequences, each terminally labeled with rhodamine acceptors and hybridized to a complementary 29-mer sequence, are associated with several intercalating donors (acridine orange). The arrangements described by Cardullo show some added energy transfer due to the additional donors. However, this increase in energy transfer efficiency is entirely consistent with direct donor to acceptor transfer, as none of the donors were described as functioning beyond the Forster distance necessary for efficient transfer. To date, there has been no descriptions of an organized structure capable of extended energy transfer from multiple donors and to an acceptor beyond normal Forster distances. SUMMARY OF THE INVENTION [0013] This invention relates to the design and synthesis of modified synthetic nucleic acid polymers/oligomers into which functional electronic/photonic properties are directly incorporated. In particular, it concerns incorporating the property of an extended nonradiative energy transfer process into arrangements of synthetic nucleic acids. [0014] It has now been discovered that multiple chromophore donor groups which are located beyond the normal Forster distance (>5 nm) can be arranged to absorb and transfer photonic energy to a terminal acceptor group, thereby acting as a light antenna or photonic conductor. This property involves the ability of an array of donor groups to absorb photonic energy at one wavelength (hv.sub.1), and directionally transfer it, via a coupled resonance process, to an acceptor group, where it is then re-emitted as photonic energy at a longer wavelength (hv.sub.2). The selection and relative positioning of special donor chromophore groups, which include nonfluorescent chromophores, with appropriate acceptor fluorophores, leads to an efficient extended energy transfer process with unique properties. Additionally, appropriate designs for oligonucleotides and polynucleotides have found which allow a primary donor group to be placed in close proximity with an acceptor group. [0015] Since the relative positions of the functional molecular components (chromophores) can be programmed, via their placement upon nucleotide sequences, nucleic acid containing the chromophores can be designed to self-assemble and organize into larger and more complex defined structures. The programmability and functional electronic/photonic properties of the molecular components enable connections, amplification mechanisms, and antenna arrays to be made within the nucleic acid structures. The combination of properties ultimately leads to the creation of photonic devices, photovoltaic devices, biosensors, and homogeneous and heterogeneous DNA diagnostic assay. [0016] The present invention therefore describes a polynucleotide having at least two (multiple) donor chromophores operatively linked to the polynucleotide by linker arms, such that the chromophores are positioned by the linkage along the length of the polynucleotide at a donor-donor transfer distance. Typically the donor chromophores are nonfluorescing chromophores. [0017] The polynucleotide can further contain a fluorescing acceptor chromophore operatively linked to the polynucleotide by a linker arm, wherein the fluorescing acceptor chromophore is positioned by the linkage at a donor-acceptor transfer distance from the donor chromophores such that the multiple donors can collect excitation light and transfer it to the acceptor which then re-emits the collected light. [0018] In another embodiment the donor chromophores and acceptor chromophores can be displayed upon more than one polynucleotide such that upon their hybridization, the acceptor fluorescing chromophore is brought into donor-acceptor transfer distance to at least one of the donor chromophores. Thus, combinations of polynucleotides are contemplated containing preselected sequences and the requisite donor and acceptor chromophores that can be adapted for a variety of uses as described herein. [0019] For example, a diagnostic assay system is described that contains a polynucleotide capable of donor-donor transfer as described above. The system can utilize an acceptor chromophore that is present on a separate polynucleotide, or the acceptor chromophore can be present on the same polynucleotide as the donor chromophores. [0020] The sequences of the polynucleotides can be selected for purposes of complementary hybridization to facilitate assembly of larger structures capable of donor-donor transfer and ultimate donor-acceptor transfer. Alternatively, the sequences of the polynucleotides can be selected to be complementary to target nucleic acid sequences such that the polynucleotides are used diagnostically to detect the target sequences in samples. [0021] In another embodiment, the invention describes structures in the form of a nucleic acid duplex that are comprised of at least two polynucleotides hybridized together by conventional complementary nucleotide base hybridization. Multiple polynucleotides can be hybridized to form the duplex as is represented in FIG. 3. The polynucleotides contain operatively linked donor and acceptor chromophores to provide a larger structure upon which the disclosed donor-donor and donor-acceptor energy transfers can occur. The chromophores can be arranged along a single strand of the duplex structure, but are preferably positioned such that the energy transfer alternates between the strands of the duplex. Continue reading about Hybridization of polynucleotides conjugated with chromophores and fluorophores to generate donor-to-donor energy transfer system... 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