Self-organizing molecular photonic structures based on chromophore- and fluorophore-containing polynucleotides and methods of their use

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DESCRIPTION

1. Technical Field

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 biosensors, and DNA diagnostic assay systems.

2. Background of the Invention

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.

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).

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 Biology, 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.

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).

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.

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).

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 involves fluorophore-labelled oligonucleotides which carry out an efficient Forster non-radiative energy transfer process. 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); Heller et al., European Patent Application #0 229 943, 1987.

Forster non-radiative energy transfer is the 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 which emits it at a second wavelength. Lakowicz et al, in "Principles of Fluorescent Spectroscopy", Plenum Press, New York, N.Y., Chap. 10, pp. 305-337, (1983). In some cases a chemiluminescent group is used as the donor. In the above work, the two fluorophore labelled oligonucleotides are designed to bind (hybridize) to adjacent positions of a complementary target nucleic acid strand and then produce efficient fluorescent 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); Heller et al., European Patent Application #0 229 943, 1987. The binding or hybridization to the complementary sequence proximates the fluorescent donor group and fluorescent acceptor group at optimal distance for efficient Forster non-radiative energy transfer. The optimum distances between donor and acceptor were shown to be in the order of two to seven nucleotides. Heller et al., European Patent Application No. EPO 0229943, 1987. These initial demonstrations of simple energy transfer in nucleic acids were later corroborated by other workers. Cardullo et al., Proc. Natl. Acad. Sci. USA, 85:8790-8794, (1988); Morrision et al., Anal. Biochem., 183:231-244, (1989). Fluorescent energy transfer has also been utilized in other areas. Garner et al., Anal. Chem., 62:2193-2198, (1990); Morrison et al., Anal. Biochem., 174:101-120, (1988).

However, fluorescent energy transfer has never been demonstrated between donor chromophores.

SUMMARY OF THE INVENTION

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 extended non-radiative (Forster) energy transfer.

It has now been discovered that multiple chromophore donor groups can be arranged to absorb and transfer photonic energy to an acceptor, 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 the Forster 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 with appropriate acceptor fluorophores, leads to an efficient extended energy transfer process with unique properties. Since the relative positions of the functional molecular components (chromophores) can be programmed, via their nucleotide sequence, 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 structures. The combination of properties ultimately leads to the creation of photonic devices, photovoltaic devices, biosensors, and homogeneous and heterogeneous DNA diagnostic assays.

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. When positioned at an acceptor-donor transfer distance to an acceptor (fluorescent) chromophore, the multiple donors collect excitation light and transfer it to the acceptor which then re-emits the collected light.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings forming a portion of this disclosure:

FIG. 1 illustrates how two chromophore-labelled oligonucleotides (a donor oligomer (SEQID NO 1) and an acceptor (SEQID NO 2) oligomer) are designed to bind or hybridize to adjacent positions on a complementary target nucleic acid strand (SEQID NO 3)(target sequence). FIG. 1B shows the binding or hybridization to the target sequence proximates the fluorescent donor group and fluorescent acceptor group at a preselected donor-acceptor transfer distance so that when the system is irradiated by photonic energy at hv.sub.1- the donor group absorbs the energy and transfers it by non-radiative energy transfer (.fwdarw.) to the acceptor group which re-emits it at hv.sub.2. Irradiating and emitting photons are indicated by the wavy-lined arrows. The exact nucleotide sequence and position of donor and acceptor groups is shown for the un-hybridized (or disassociated system) in the upper portion of the figure. The hybridized figure (or associated system) is represented schematically for purposes of simplicity in the lower portion of the figure.

FIG. 2A illustrates a schematic representation of multiple donors groups (D) and a single acceptor group (A) incorporated into a single DNA polynucleotide strand hybridized or associated to a template DNA oligomer. FIG. 2B illustrates a multiple donor DNA polymer and an acceptor DNA polymer assembled into an organized structure on a template DNA polymer.

FIG. 3A, illustrates schematically the exemplary 14 nanometers (nm) photonic antenna structure described in Example 1 that is assembled and organized from four oligonucleotides: the 16-mer acceptor unit (AU), the 30-mer intermediate donor 1 unit (ID1), the 29-mer intermediate donor 2 unit (ID2), and the terminal donor unit (TD). FIG. 3B illustrates extended energy transfer when the assembled structure is illuminated wit light at 495 nm. The wavy lines indicate irradiating or emitting photons and the dashed arrow (.fwdarw.) shows the direction of the extended energy transfer process.

FIGS. 4A and 4B illustrate a homogeneous DNA hybridization assay method based on extended energy transfer as described in Example 3. The polynucleotides shown include the multiple donor-containing oligomer (MDO), the acceptor oligomer (AO), the quencher oligomer (QO) and a target DNA. FIG. 4A shows the homogeneous system before the target DNA is denatured. Note that the acceptor group is proximal to the quencher group, and therefor emission from the acceptor is quenched. FIG. 4B shows the homogeneous system after the target DNA is denatured whereupon the multiple donor and acceptor oligomers have hybridized to the target DNA at specific, programmed, complementary sites to produce a structure capable of extended energy transfer.

DETAILED DESCRIPTION OF THE INVENTION

A. Chromophore-Containing Polynucleotides

This invention relates to the design and synthesis of modified synthetic nucleic acid polymers/oligomers into which functional electronic/photonic properties are directly incorporated. Synthetic nucleic acids having inherent recognition properties are ideal materials for constructing molecular components which can self-organize into electronic and photonic structures and devices.

In one embodiment, the invention contemplates a polynucleotide having at least two donor chromophores, preferably multiple chromophores operatively linked to the polynucleotide by linker arms, such that the chromophores are positioned along the length of the polynucleotide at a donor-donor transfer distance effective for energy transfer as described by the present discoveries.

The polynucleotide has a predetermined sequence selected to be complementary to a target nucleic acid sequence, so that the donor chromophore-containing polynucleotide can be programmed to assemble by hybridization onto a preselected target nucleic acid.

In one embodiment, the polynucleotide further contains at least one fluorescing acceptor chromophore operatively linked to the polynucleotide by a linker arm, such that the fluorescing acceptor chromophore is positioned by linkage at a donor-acceptor transfer distance from at least one of the donor chromophores. This configuration provides the structure capable of non-radiative energy transfer described by the present invention.

For purposes of this invention and unless otherwise stated, the terms "oligonucleotide or oligomer" and "polynucleotide" will refer generally to nucleic acids in the form of single-stranded nucleic acid polymers, comprised of DNA, RNA, or modified sequences produced by totally synthetic procedures. The shorter sequences "oligonucleotides or oligomers" are usually 2 to 50 nucleotides in length, and the longer sequences "polynucleotides" are usually about 15 to 200 nucleotides in length, and typically are more than 50 nucleotides in length. However, it is to be understood that the terms are somewhat interchangeable insofar as they both denote nucleic acid polymers.

Important advantages of synthetic DNA as the support structure for providing the array to orient multiple donors and acceptor in a transfer structure are: (1) rapid synthesis with automated instruments, in lengths from 2 to 150 nucleotide units (0.8 nanometers (nm) to 50 nanometers (nm)); (2) programmable recognition with high specificity, via their nucleotide sequence; (3) easily modified with fluorophores, chromophores, affinity labels, metal chelates, and enzymes; (4) modifiable at any position in their sequence, and at several places within the base unit; (5) modifiable backbone structure to produce different properties (example; normally negatively charged DNA can be made in a neutral form); (6) linkable both covalently and noncovalently to solid surfaces: glass, metals, silicon, organic polymers, and bio-polymers; (7) reversible organizational properties; (8) ability to form three dimensional and branched structures; and (9) well understood and easily modelled structural and organizational properties.

1. Extended Energy Transfer

The particular functional electronic/photonic property which concerns this invention, is an extended non-radiative (Forster) energy transfer process. The basic Forster energy transfer process involves the ability of a donor group to absorb photonic energy at one wavelength (hv.sub.1) and transfer it, via a non-radiative dipole coupling process, to an acceptor group which re-emits the photonic energy at a longer wavelength (hv.sub.2). Energy transfer efficiency is dependent upon the parameters which are given in the equations below: ##EQU1## where E=the transfer efficiency, r=the distance between the donor and acceptor, k is a dipole orientation factor, n is the refractive index of the medium, O.sub.d is the quantum yield of the donor, and J is the overlap integral which express the degree of overlap between the donor emission and the acceptor absorption. All other parameters being optimal, the 1/r.sup.6 distance dependency requires a spacing of 2 nanometers (20 angstroms) or less between the donor and acceptor groups for efficient energy transfer to occur.

FIG. 1 shows how two fluorophore-labelled oligonucleotides (a donor and an acceptor) are designed to bind or hybridize to adjacent positions of a complementary target nucleic acid strand and then produce efficient fluorescent energy transfer. Relative efficiencies for the energy transfer process can be expressed in two simplistic ways. The first is in terms of the ratio of transferred energy to the energy absorbed by the donor; this is determined by measuring the relative amount of donor fluorescence quenching that occurs in the presence of the acceptor. The second way expresses relative efficiency in terms of the ratio of energy re-emitted by the acceptor to the energy absorbed by the donor; this is determined by measuring the relative increase in acceptor fluorescence due to donor group. While both methods are considered relative measures of energy transfer efficiency, the efficient transfer of energy from the donor to the acceptor (seen as donor quenching), does not necessarily lead to the same efficiency for re-emission by the acceptor. This occurs when secondary processes (acceptor quenching) cause the acceptor to dissipate its energy other than by re-emission.

Extended energy transfer is the process by which multiple donor groups absorb photonic energy at one wavelength (hv.sub.1), and directionally transfer it to an acceptor group, where it is then re-emitted as photonic energy at wavelength (hv.sub.2). Under conditions where hv.sub.1 is non-saturating, photonic energy can be collected by arrays of donor groups and directionally transferred to an appropriate acceptor, greatly enhancing its fluorescent emission at hv.sub.2. This can be considered a molecular antenna or amplifier mechanism. Alternatively, photonic energy (hv.sub.1) can be collected at one end of a structure by a donor group and be transferred by a linear array of donors, to an acceptor group at the other end of the structure where it is re-emitted as hv.sub.2. This type of molecular photonic transfer mechanism can be considered the equivalent of a photonic wire or connector. These mechanisms can also be used to interconnect different molecular structures, to connect molecular structures to surfaces, and to make molecular connections between surfaces (monolayers).

2. Chromophores And Fluorophores

A novel part of this invention relates to the selection and positioning of special chromophore and fluorophore groups to form appropriate donor and acceptor pairs which are capable of energy transfer by dipole coupling.

A chromophore refers to those groups which have favorable absorption characteristics, i.e, are capable of excitation upon irradiation by any of a variety of photonic sources. Chromophores can be fluorescing or non-fluorescing. Non-fluorescing chromophores typically do not emit energy in the form of photonic energy (hv.sub.2). Thus they can be characterized as having a low quantum yield, which is the ratio of emitted photonic energy to adsorbed photonic energy, typically less than 0.01. A fluorescing chromophore is referred to as a fluorophore, and typically emits photonic energy at medium to high quantum yields of 0.01 to 1.

Of particular importance to the present invention is the demonstration that non-fluorescent chromophores, such as 4-Dimethylaminophenyl-azophenyl-4'-isothiocyanate (or DABITC), can function as effective energy transfer donor groups. When these chromophore donor groups are closely proximated (5 to 40 Angstroms, preferably 10 to 20 angstroms or 1 to 2 nanometers) to a suitable acceptor group they produce a significant fluorescent re-emission by the acceptor. Chromophores capable of energy transfer to a suitable acceptor chromophore are referred to herein as donor chromophores or donors.

An acceptor chromophore for the purposes of the present invention is a fluorophore, that is capable of accepting energy transfer from a donor chromophore. Because energy transfer by dipole coupling can typically occur when there is an overlap in the emission spectrum of the donor and the excitation spectrum of the acceptor, a "suitable" acceptor typically has an excitation spectrum in the longer wavelengths than its corresponding suitable donor. In this regard, donors and acceptors can be paired for capacity to transfer energy on the basis of overlapping donor emission and acceptor excitation spectra. Therefore, potentially any chromophore can be paired with another chromophore to form an acceptor-donor pair, so long as the two chromophores have different emission spectrums, and have sufficiently overlapping donor emission and acceptor excitation spectra to effect energy transfer.

A non-fluorescent donor producing fluorescent re-emission in the acceptor group is an extremely valuable property. The non-fluorescing donor in a composition of the present invention provides the particular advantage of a low or absent level of emission by the donor, thereby not contributing to the detectable emitted light in a donor-acceptor system. Thus, non-fluorescent donors allow for very low background and are particularly preferred.

A multiple donor system comprised of such non-fluorescent chromophores would have very little inherent fluorescent background. This property overcomes a major limitation that has severely limited practical uses of fluorescent energy transfer in DNA diagnostic assay applications. It also opens opportunity to create more useful photonic mechanisms and applications.

With regard to unique properties in acceptors, most preferred are acceptors with the highest quantum yields, or with other properties that increase the signal-to-noise ratio between specific acceptor emissions and the background (non-specific) emissions attributable to the donor. Examples of approaches to reduce the signal-to-noise ratio include using donors having lower emission, preferably non-fluorescing donors, selection of acceptor-donor pairs in which the spectral distance between the emission spectrum of the donor and acceptor is maximized, and preferably selected as to be non-overlapping, and the like approaches described further herein.

Table 1 lists some of the potential chromophores and fluorophores which can be used as donors, acceptors, and quenches for the novel extended energy transfer mechanisms and applications disclosed in this invention. The list is not meant to be exclusive in that it identifies some specific types or classes of donors, acceptors, and quenchers which can produce these unique and desirable properties.

TABLE 1 ______________________________________ CHROMOPHORE DERIVATIVES USEFUL AS DONORS, ACCEPTORS, OR QUENCHERS FOR THE EXTENDED ENERGY TRANSFER PROCESS AND RELATED PHOTONIC MECHANISMS DERIVATIVE.sup.1 (EX).sup.2 (EM).sup.3 (QY).sup.4 ______________________________________ 4,4'-Diisothiocyanatodihydro- 286 none.sup.5 <0.01 stilbene-2,2'-disulfonic acid 4-acetamido-4'-isothiocyanato- 336 438 M stilbene-2,2'-disulfonic acid 4,4'-Diisothiocyanatostilbene 342 419 M 2,2'-disulfonic acid Succinimidyl pyrene butyrate 340 375,395 0.6 Acridine isothiocyanate 393 419 M 4-Dimethylaminophenylazophenyl 430 none.sup.5 <0.01 4'-isothiocyanate (DABITC) Lucifer Yellow vinyl sulfone 438 540 0.2 Fluorescein isothiocyanate 494 520 0.5 Reactive Red 4 (Cibacron 535 none.sup.5 <0.01 Brilliant Red 3B-A) Rhodamine X isothiocyanate 578 604 M-H Texas Red (Sulforhodamine 101, 596 615 H sulfonyl chloride) Malachite Green isothiocyanate 629 none.sup.5 <0.01 IR144.sup.6 745 825 M ______________________________________ .sup.1 The fluorophores and chromophores listed above are shown in derivatized forms suitable for direct coupling to the primary amino group incorporated into the DNA polymer. In many cases other types of derivatives (succinimidyl esters and haloacetyl) are available for coupling to amines. Also, derivatives specific for coupling to sulfhydryl and aldehyde functional groups are available. .sup.2 EX is the absorption maximum in nanometers (nm). .sup.3 EM is the emission maximum in nanometers (nm). .sup.4 For quantum yields (QY) the approximate ranges are: "Low", 0.01-0.1; "Medium", 0.1-0.3: and "High", 0.3-1.0. .sup.5 These are essentially nonfluorescent (QY <0.01) organic compounds, with medium to high molar absorptivity. They are more appropriately calle chromophores. .sup.6 IR144 (Kodak Laser Dye) is underivatized, and requires modificatio before it can be coupled to a DNA polymer.

3. Donor and Acceptor Pair Configurations

From the chromophores and fluorophores listed in Table 1 a number of donor/acceptor configurations or arrangements can be made that will produce efficient extended energy transfer processes and novel photonic mechanisms. These arrangements which are shown in Table 2 include:

(1) Arrangements of multiple donors groups (fluorescent and non-fluorescent) transferring energy to a single or smaller number of acceptor groups. Generally, multiple donors transfer to a single acceptor group, but under some conditions and for certain photonic mechanisms more than one acceptor group may be used. The preferred arrangements are those involving the non-fluorescent donors, which provide the important advantage of a low background extended energy transfer process. Other preferred arrangements involves multiple fluorescent donors, excited in the visible region, which transfer to an acceptor(s) which re-emits in the infra-red region. This is a useful mechanism because the infra-red emission can be detected by optoelectronic devices which are much less sensitive to background fluorescence produced in the visible region.

(2) Arrangements in which multiple donor groups (fluorescent and non-fluorescent) absorb light at hv.sub.1, and transfer to an intermediate donor-acceptor, which then transfers to a final acceptor group, which re-emits at hv.sub.2. These arrangements have the advantage of producing a large Stokes shift between the excitation wavelength (hv.sub.1) and the emission wavelength (hv.sub.2) of the system. This is important because the larger the separation between excitation and emission, the lower the background fluorescence for the system. Exemplary configurations are shown in Table 2, where three chromophores are shown in series. The preferred arrangements are those which transfer from non-fluorescent or fluorescent donors to an acceptor(s) which re-emit in the infra-red region. A preferred embodiment contemplates the use of IR144 (a Kodak Laser Dye), a chromophore that accepts excitation energy from donors that are excited in the visible region and then re-emits in the infra-red region.

(3) There are special arrangements in which certain chromophore groups with strong quenching properties are used to prevent fluorescent emission by the acceptor group. In this embodiment, the present invention contemplates the use of a quencher chromophore (or quencher), that has the capacity to accept, like an acceptor, the transfer of energy by dipole coupling, but does not have significant emission. Although similar in properties to a non-fluorescing donor, the term quencher refers to a non-fluorescing chromophore that is configured to draw the energy potential away from an excited acceptor so that the acceptor does not emit, i.e., the acceptor is quenched. An exemplary configuration utilizing a quencher chromophore in combination with a multiple donor oligonucleotide of the present invention is described in Example 3 and FIG. 4.

The mechanism for energy transfer to a quenching chromophore is the same as for donor-donor or donor-acceptor transfer, namely dipole coupling, and therefor is subject to the same requirements as described herein relating to transfer distances and optimum pairing configurations. Exemplary non-fluorescent chromophores suited for quenching are Reactive Red 4 or Malachite Green because they have no detectable emission and they are located at the "red" end of the spectrum, and therefore can be selected relative to a variety of acceptor chromophore to accept (quench) energy from the acceptor before it emits. The preferred arrangements are for the non-fluorescent chromophores Reactive Red 4 or Malachite to quench fluorescence in the Texas Red acceptor group.

TABLE 2 ______________________________________ MULTIPLE DONOR/ACCEPTOR, MULTIPLE DONOR 1/ACCEPTOR DONOR 2/ACCEPTOR, AND SPECIAL QUENCHING ARRANGEMENTS (* PREFERRED *) ______________________________________ DABITC ---> Fluorescein * DABITC ---> Texas Red * * DABITC ---> Texas Red ---> IR 144 * Lucifer Yellow ---> Texas Red Lucifer Yellow ---> Fluorescein ---> Texas Red * Lucifer Yellow ---> Texas Red ---> IR 144 * Fluorescein ---> Texas Red Fluorescein ---> IR 144 * Fluorescein ---> Texas Red ---> IR 144 * * Texas Red ---> IR 144 * * Malachite Green ::::> Texas Red * * Reactive Red 4 ::::> Texas Red * ______________________________________ The ---> indicates an energy transfer effect which leads to significant reemission by the acceptor group. The ::::> indicates an energy transfer effect that significantly quenches the fluorescence of the acceptor group

It is important to point out that the various arrangements and configurations of donor, acceptor, and quencher groups described above can be achieved by either incorporating them within a single DNA polymer; or by using a DNA template to assemble various combinations of multiple donor DNA polymers, acceptor DNA polymers, and quencher DNA polymers. Both types of arrangements are shown schematically in FIG. 2.

With regard to the proper positioning or spacing of "donor to acceptor" pairs and "donor to donor" pairs in multiple donor arrangements, the basic 1/r.sup.6 distance dependency for Forster transfer requires a spacing of about 5 to 40 Angstroms (.ANG.), and preferably a spacing of 2.0 nanometers (20 .ANG.) or less between the groups for reasonably efficient (.sup..about. 80-90%) energy transfer to occur. In terms of nucleotide spacing in double stranded DNA polymers, this optimum transfer distance is roughly equivalent to 3 to 7 bases, and preferably is about 4 to 6 bases. At shorter separation distances efficiency can theoretically approach 100%. However, in DNA polymers the incorporation of multiple donors at such close spacing might interfere with the ability of the DNA to hybridize with high specificity. Also, close spacing of donor-donor or donor-acceptor pairs can sometime introduce secondary quenching mechanisms or excitation traps which can greatly reduce energy transfer efficiency. At a distance of 4.0 nanometers (40 .ANG.) or 12 base pairs, energy transfer efficiency is at only 20%. The presently available chemistries for modifying synthetic DNA at internal and at terminal positions, allows for spacings of 4 to 6 base pairs between the two labelled bases to be achieved over reasonably long distances. This would mean about 10 donors could be incorporated in single oligonucleotide sequence of 50 nucleotides Spacing at further intervals from 7 to 15 base pairs can be carried out, but energy transfer efficiencies drop off rapidly as the separation distance increases.

For more critical connections, like the final donor to acceptor pair of an extended donor system, spacing can be compressed to 2 to 3 base pairs. Close spacing (0, 1 or 2 base pairs) can be carried out, but may require development of special linker arm chemistries which orient groups for optimal energy transfer and eliminate any secondary quenching mechanisms or excitation traps. In those case where quenching is a desired property, there can be 0 to 6 base pair spacing between the quencher group(s) and the acceptor group. It should be kept in mind that donor-donor, donor-acceptor, and quencher-acceptor pairs may not only be formed on the same DNA strand, but may also be formed between groups which are on opposite sides of double stranded DNA structures.

4. Synthesis and Labelling of Oligonucleotides and Polynucleotides

Synthesis of oligonucleotide and polynucleotide sequences can be carried out using any of the variety of methods including de novo chemical synthesis of polynucleotides such as by presently available automated DNA synthesizers and standard phosphoramidite chemistry, or by derivation of nucleic acid fragments from native nucleic acid sequences existing as genes, or parts of genes, in a genome, plasmid, or other vector, such as by restriction endonuclease digest of larger double-stranded nucleic acids and strand separation or by enzymatic synthesis using a nucleic acid template.

De novo chemical synthesis of a polynucleotide can be conducted using any suitable method, such as, for example, the phosphotriester or phosphodiester methods. See Narang et al, Meth. Enzymol., 68:90, (1979); U.S. Pat. No. 4,356,270; Itakura et al, Ann. Rev. Biochem., 53:323-56 (1989); and Brown et al, Meth. Enzymol., 68:109, (1979).

Derivation of a polynucleotide from nucleic acids involves the cloning of a nucleic acid into an appropriate host by means of a cloning vector, replication of the vector and therefore multiplication of the amount of the cloned nucleic acid, and then the isolation of subfragments of the cloned nucleic acids. For a description of subcloning nucleic acid fragments, see Maniatis et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, pp 390-401 (1982); and see U.S. Pat. Nos. 4,416,988 and 4,403,036.

In preferred embodiments, automated syntheses using an Applied Biosystems Model #381 DNA synthesizer and commercially available (Applied Biosystems) 5'-dimethoxytrityl nucleoside b-cyanoethyl phosphoramidite reagents and controlled pore glass synthesis columns were conducted for the work described in this patent application. In addition to the "standard phosphoramidite chemistry" o