Fluorescence-based detection methods, compound panels, and arrays including the same for fluorescence detection

Abstract

A panel of oligonucleotide-conjugated fluorescent donor dyes, wherein the oligonucleotide is complementary to a first sequence of a target oligonucleotide; a plurality of oligonucleotide-conjugated acceptor dyes, wherein the oligonucleotide is complementary to a second sequence of a target oligonucleotide; the first sequence and second sequence are spaced apart to allow fluorescent resonance energy transfer between one donor dye and one acceptor dye when each is bonded to the target oligonucleotide, which creates a spectral signal distinct from the spectra of the donor dye, the acceptor dye, or the sum thereof. An n-plex array of oligonucleotide-conjugated fluorescent donor dye and acceptor dye pairs, a method of detecting the n-plex array of target oligonucleotide, a method for detecting a target nucleic acid with a padlock probe and the n-plex array of target oligonucleotides, and a method of detecting an array of antigens with the n-plex array of labelled antibodies are described.

Description

FLUORESCENCE-BASED DETECTION METHODS, COMPOUND PANELS, ANDARRAYS INCLUDING THE SAME FOR FLUORESCENCE DETECTIONCROSS-REFERENCE TO RELATED APPLICATIONSThis application claims priority to U.S. Provisional Application Ser. No. 63 / 650,683 filed May 22, 2024, which is incorporated by reference herein in its entirety.FEDERAL RESEARCH STATEMENT

[0001] This invention was made with government support under DPI DA051608 awarded by the National Institutes of Health / National Institute on Drug Abuse. The government has certain rights in the invention.FIELD OF THE DISCLOSURE

[0002] This disclosure relates to fluorescence-based detection methods, compound panels, and arrays for use of the methods. In particular, the invention relates to methods for providing increased spectrally distinct fluorescent colors for detection, including compound panels and arrays allowing such increased fluorescent color detection.BACKGROUND

[0003] Fluorescence-based detection methods, including microscopy and flow cytometry, are used extensively in every branch of the biomedical sciences. These methods rely on using spectrally distinct fluorescent dyes to label different biological molecules. Traditional fluorescence measurements are generally limited to six to seven colors, meaning that only 6 to seven distinct molecules of interest can be labeled. However, thousands of genes and proteins exist, spurring great interest in increasing the number of colors available for detection. There accordingly remains a need in the art for methods, compound panels, and arrays that allow analysis and detection of a large number of fluorescent colors.SUMMARY

[0004] This disclosure includes a panel of fluorescent dye probes, the panel comprising: a plurality of oligonucleotide-conjugated fluorescent donor dyes, wherein the oligonucleotide is complementary to a first sequence of a target oligonucleotide; a plurality of oligonucleotide-conjugated acceptor dyes, wherein the oligonucleotide is complementary to a second sequence of a target oligonucleotide; andwherein the first sequence and second sequence are spaced apart a distance effective to allow fluorescent resonance energy transfer between one donor dye and one acceptor dye when each is bonded to the target oligonucleotide, to create a unique spectral signal with the excitation spectrum of the donor and the emission spectrum of the acceptor, wherein the spectral signal is distinct from a spectra of the donor dye, a spectra of the acceptor dye, or an additive overlay spectra of the donor dye and acceptor dye; and wherein the panel comprises at least 2, for example from 12 to 128 oligonucleotide- conjugated fluorescent donor dye pairs, wherein each dye pair creates a spectral signal different from each other dye pair and from each individual donor and acceptor dye.

[0005] The panel of fluorescent dye probes can have 2 to 512, preferably 10 to 512, preferably 10 to 256, more preferably 10 to 128 oligonucleotide-conjugated fluorescent donor / acceptor dye pairs.

[0006] The panel of fluorescent dye probes can be a panel in which the first sequence and the second sequence comprise from 5 to 25 nucleotides.

[0007] The first sequence and the second sequence can be sequences separated by 3, 4, or 5 nucleotides.

[0008] In the panel, the donor and acceptor dyes can be Alexa Fluor 350, Cascade Blue, Alexa Fluor 405, Alexa Fluor 430, nitrobenzoxadiazole (NBD), fluorescein, Alexa Fluor 488, Oregon Green 488, BODIPY 493 / 503, Rhodamine Green, BODIPY FL, Oregon Green 514, Alexa Fluor 514, Eosin, Rhodamine 6G, BODIPY R6G, Alexa Fluor 532, BODIPY 530 / 550, BODIPY TMR, Alexa Fluor 555, tetramethyl rhodamine (TMR), Alexa Fluor 546, BODIPY 558 / 568, QSY 7, QSY 9, BODIPY 564 / 570, Lissamine rhodamine B, Rhodamine Red, BODIPY 576 / 589, Alexa Fluor 568, X-rhodamine, Body BODIPY 581 / 591, BODIPY TR, Alexa Fluor 594, Texas Red, Alexa Fluor 610-X, BODIPY 630 / 650, Alexa Fluor 633, Alexa Fluor 635, BODIPY 650 / 665, Alexa Fluor 647, QSY 21, Alexa Fluor 660, Alex Sa Fluor (Alexa Fluor) 680, Alexa Fluor (Alexa Fluor) 700, Alexa Fluor 750, Alexa Fluor 790, ATTO, AF568, AF594, AF610, AF633, AF647, AF680, AF700, Alexa Fluor 405, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 633, ATTO 488, ATTO 532, ATTO425, ATTO465, ATTO488, ATTO520, ATTO532, ATTO542, ATTO550, ATTO565, Cy5.5, iFluor488, or a combination thereof.

[0009] The disclosure includes a method of making of an n-plex array of oligonucleotide-conjugated fluorescent donor dye and acceptor dye pairs as described above for detection of an n-plex array of target oligonucleotides, wherein n is a number of at least two, for example from 10 to 128, the method comprising selecting a first oligonucleotide-conjugatedfluorescent donor dye and acceptor dye pair from the panel of donor / acceptor dye pairs, wherein the oligonucleotide dye sequences are complementary to a first target oligonucleotide; selecting a second oligonucleotide-conjugated fluorescent donor dye and acceptor dye pair from the panel of donor / acceptor dye pairs as described above and herein, wherein the oligonucleotide dye sequences are complementary to a second target oligonucleotide; and repeating the selecting up to n times for each oligonucleotide in the n-plex array to provide n pairs of oligonucleotide-conjugated fluorescent donor dye and acceptor dye pairs.

[0010] The disclosure includes an n-plex array of oligonucleotide-conjugated fluorescent donor dye and acceptor dye pairs for detection of an n-plex array of target oligonucleotides as described above and herein, the array comprising: a first oligonucleotide-conjugated fluorescent donor dye and acceptor dye pair from the panel, wherein the oligonucleotide dye sequences are complementary to a first target oligonucleotide; a second oligonucleotide-conjugated fluorescent donor dye and acceptor dye pair from the panel, wherein the oligonucleotide dye sequences are complementary to a second target oligonucleotide; and an additional number of oligonucleotide-conjugated fluorescent donor dye and acceptor dye pairs from the panel, wherein the oligonucleotide dye sequences are complementary to each of an additional target oligonucleotide, wherein the additional number is n-2.

[0011] The disclosure includes a method of detecting an n-plex array of target oligonucleotides wherein n is a number of at least 2, for example from 10-128, comprising contacting n pairs of the oligonucleotide-conjugated fluorescent donor dyes and acceptor dyes of the n-plex array of oligonucleotide-conjugated fluorescent donor dye and acceptor dye pairs with the n-plex array of target oligonucleotides to provide a fluorescent energy transfer spectral signal; and analyzing the energy transfer signal to determine the pairs of oligonucleotide-conjugated fluorescent donor dyes and acceptor dyes contributing to the signal.

[0012] The n-plex array can be an array in which the oligonucleotide sequence of each of the array of n pairs of the oligonucleotide-conjugated fluorescent donor dyes and acceptor dyes is part of a padlock probe. The oligonucleotide sequences can be part of a bridge probe. The bridge probe contains the same barcode sequence as the padlock probe and thus hybridizes to the RCP. The other part of the bridge probe contains reverse complement of the dye-labeled oligo sequences.

[0013] The disclosure includes a method for detecting a target nucleic acid, comprisingcontacting a padlock probe with the target nucleic acid; circularly linking the padlock probe using a ligase; amplifying the target nucleic acid by binding of a polymerase; contacting the amplified nucleic acid with a bridge oligonucleotide; contacting the bridge oligonucleotide with the n-plex array of target oligonucleotides as described above and herein to provide a fluorescent energy transfer spectral signal; and analyzing the energy transfer signal to determine the pairs of oligonucleotide-conjugated fluorescent donor dyes and acceptor dyes contributing to the signal.

[0014] The disclosure includes a method for detecting a target nucleic acid, comprising contacting a padlock probe with the target nucleic acid; circularly linking the padlock probe using a ligase; amplifying the padlock probe sequence by binding of a polymerase; contacting the amplified padlock probe sequence with a bridge oligonucleotide; contacting the bridge oligonucleotide with the n-plex array of target oligonucleotides as described above and hereon to provide a fluorescent energy transfer spectral signal; and analyzing the energy transfer signal to determine the pairs of oligonucleotide-conjugated fluorescent donor dyes and acceptor dyes contributing to the signal.

[0015] This disclosure includes a method of labeling an antibody, comprising linking a target oligonucleotide to the antibody; contacting the target oligonucleotide with an oligonucleotide-conjugated fluorescent donor dye wherein the donor dye oligonucleotide is complementary to a first sequence of the target oligonucleotide together with an oligonucleotide-conjugated acceptor dye wherein the acceptor dye oligonucleotide is complementary to a second sequence of a target oligonucleotide; wherein the first sequence and second sequence are spaced apart a distance effective to allow fluorescent resonance energy transfer between one donor dye and one acceptor dye, in other words, a fluorescent resonance energy transfer signal between the donor dye and the acceptor dye when each is bonded to the target nucleotide, to create a novel spectral signal with the excitation spectrum of the donor and the emission spectrum of the acceptor that is distinct from a spectra of the donor dye, a spectra of the acceptor dye, or an additive overlay spectra of the donor dye and acceptor dye.

[0016] This disclosure includes a method of labeling an n-plex array of target antibodies, wherein n is a number of at least 2, the method comprising linking each of the target antibodies to a unique target oligonucleotide;selecting a first oligonucleotide-conjugated fluorescent donor dye and acceptor dye pair from the panel as described above and herein, wherein the oligonucleotide dye sequences are complementary to a first target oligonucleotide; selecting a second oligonucleotide-conjugated fluorescent donor dye and acceptor dye pair from the panel of claim 1, wherein the oligonucleotide dye sequences are complementary to a second target oligonucleotide; and repeating the selecting up to n times for each oligonucleotide of each antibody in the n- plex array to provide an n-plex array of the antibodies labelled with n pairs of oligonucleotide- conjugated fluorescent donor dye and acceptor dye pairs.

[0017] This disclosure includes an-plex array of target antibodies, wherein n is a number at least two, for example, from 10 to 128, the array comprising a first antibody comprising a first target oligonucleotide hybridized with a first nucleotide-conjugated fluorescent donor dye and acceptor dye pair of the panel as described above and herein, wherein the oligonucleotide dye sequences are complementary to the first target oligonucleotide; a second antibody comprising a second oligonucleotide hybridized to a second conjugated fluorescent donor dye and acceptor dye pair of the panel, wherein the oligonucleotide dye sequences are complementary to the second target oligonucleotide; and an additional number of n antibodies hybridized to an additional number of oligonucleotide-dye pairs, wherein each of the additional oligonucleotide dye sequences are complementary to each of an additional target oligonucleotide, wherein the additional number is n-2.

[0018] This disclosure includes a method of detecting an array of antigens, the method comprising contacting the n-plex array of labelled antibodies as described above and herein with a sample comprising an antigen that binds to at least one of the labelled antibodies, to provide a fluorescent energy transfer spectral signal; and analyzing the energy transfer signal to determine the pairs of oligonucleotide-conjugated fluorescent donor dyes and acceptor dyes contributing to the signal.

[0019] The embodiments disclosed herein are given by way of example only and are not intended to be limiting on the scope of this disclosure and appended claims. Additional objects and advantages associated with the compositions, methods, and processes will be appreciated by one of ordinary skill in the art in light of the instant claims, description, and examples. Forexample, the various aspects and embodiments can be used in numerous combinations, all of which are expressly contemplated by this disclosure.BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The accompanying drawings, which are incorporated into and form a part of the specification, illustrate embodiments of the disclosure and, together with the description, serve to explain the principles of the disclosure. The drawings are only for the purpose of illustrating embodiments of the disclosure and are not to be construed as limiting the disclosure. Further objects, features, and advantages of the disclosure will become apparent from the following detailed description taken in conjunction with the accompanying figures showing illustrative embodiments of the disclosure.

[0021] FIG. 1A shows that in traditional epifluorescence microscopy, a small part of the full spectrum of a fluorophore is sampled with low resolution (FIG. 1A left), whereas spectral unmixing instruments sample the spectrum at higher density (FIG. 1A, right). Fluorescence intensity is shown in grayscale, with white indicating strong signal and black indicating background or no fluorescence.

[0022] FIG. IB is a schematic illustration showing that fluorophores have a characteristic spectrum consisting of their fluorescence at every combination of excitation and emission wavelengths (three shown), and that if these spectra are sampled adequately, spatially overlapping fluorophores can be unmixed. Fluorescence intensity is shown in grayscale, with white indicating strong signal and black indicating background or no fluorescence.

[0023] FIG. 1C illustrates that in theory, spectral unmixing can resolve hundreds of colors, but in practice this is limited by the fact that spectra of typical fluorescent dyes (ATTO dyes shown) are crowded near the diagonal (short Stokes shifts), leaving most of the spectral space unused. Fluorescence intensity is shown in grayscale, with white indicating strong signal and black indicating background or no fluorescence.

[0024] FIG. ID illustrates that long Stokes shift dyes (mFluor-green shown) access the off-diagonal part of the spectrum, but they typically have broad spectra unfavorable for spectral unmixing, inconvenient chemistry for conjugation to oligonucleotides, and rapid photobleaching. Fluorescence intensity is shown in grayscale, with white indicating strong signal and black indicating background or no fluorescence.

[0025] FIG. IE illustrates that the dye-conjugated oligonucleotides as described herein are placed in close apposition, where fluorescent resonance energy transfer (FRET) interactions between them create a novel spectral peak with the excitation spectrum of the donor and theemission spectrum of the acceptor. Unlike long Stokes shift dyes, these peaks are narrow and favorable for spectral unmixing. Fluorescence intensity is shown in grayscale, with white indicating strong signal and black indicating background or no fluorescence.

[0026] FIG. IF illustrates that by combinatorially pairing short Stokes shift dyes (the same 6 dyes shown in FIG. 1C), one can tile the entire spectral space densely with unique narrow peaks representing novel colors that can be unmixed. Fluorescence intensity is shown in grayscale, with white indicating strong signal and black indicating background or no fluorescence.

[0027] FIG. 2A shows the first steps in the direct RNA- hybridization-based in situ sequencing fluorescent in situ hybridization (dRNA-HyblSS FISH) method as reported by Lee el al. (Scientific Reports, (2022) 12, article number: 7976), which is hybridizing a padlock probe to mRNA, followed by ligation and rolling circle amplification. The resulting rolling circle product (RCP) is a DNA “spot” in the range of 500 nm, containing many copies of a genespecific barcode.

[0028] FIG. 2B (left) is a schematic diagram showing that in the reported dRNA-HyblSS method, the barcode identity is read out by hybridizing a sequence of bridge probes and fluorescent dye probes over multiple rounds of imaging.

[0029] FIG. 2B (right) is a schematic diagram showing an exemplary embodiment of the PUFRFISH method disclosed herein, in which each RCP is labeled with a pair of two fluorescent probes (a donor dye-oligonucleotide and acceptor-dye-oligonucleotide) that are FRET partners.

[0030] FIG. 2C is a graph that shows that 4 base pair (bp) spacing between the two fluorescent probes yields optimal FRET efficiency.

[0031] FIG. 2D shows photographs showing that GAPDH mRNA is visualized in cultured A549 cells. The strong FRET signal (as indicated by the bright, punctate, or diffuse fluorescence in the bottom-right image) and near-complete quenching of the donor (in the topright image) indicate high FRET efficiency. Fluorescence intensity is shown in grayscale, with white indicating strong signal and black indicating background or no fluorescence.

[0032] FIG. 3A is a reference spectra measured on a Zeiss LSM880 spectral microscope using an embodiment of the PUFRFISH method with purified rolling circle products (RCPs). Four donor / acceptor pairs were used to create a 10-plex FRET panel.

[0033] FIG. 3B shows the result of images of in vitro RCPs for each of the 10 FRET pairs being added together. Spectral unmixing was able to recover the original ground truth images accurately (only 2 out of 10 RCPs in the mixed image are shown). Fluorescence intensityis shown in grayscale, with white indicating strong signal and black indicating background or no fluorescence.

[0034] FIG. 3C is an image showing spot-based 39-plex PUFRFISH data in mouse brain sections. A preliminary analysis with blind unmixing uncovered robust labeling with multiple FRET pairs (three shown), and manual inspection of the spot spectra revealed that most FRET pairs in the panel were visible, though some were dim. Fluorescence intensity is shown in grayscale, with white indicating strong signal and black indicating background or no fluorescence.

[0035] FIG. 3D illustrates expansion of an embodiment of the PUFRFISH method to 128 colors using a panel of 14 donors and 14 acceptors, which can be imaged with a hyperspectral microscope having a much higher spectral resolution than the Zeiss confocal (FIG. 1A).

[0036] FIG. 4 A shows an exemplary method of using a panel of the oligonucleotide- conjugated donor and acceptor dyes to detect antibodies. As shown, a single bridge oligonucleotide (402) synthesized with a 5’ amine group and conjugated to an antibody in a panel of antibodies. A pair of oligonucleotide-conjugated donor and acceptor dyes is selected for the antibody and hybridized to the bridge oligonucleotide at a distance effective to provide a signal. The process can be carried out with multiple antibodies, where each antibody is labelled with a different pair of donor / acceptor dyes.

[0037] FIG. 4B illustrates another embodiment, in which the oligonucleotide contains a click chemistry group, enabling labelling of the oligonucleotides with, for example, azide-linked dyes.DETAILED DESCRIPTION

[0038] Disclosed herein is a method for rapidly creating virtual dyes with customized spectral properties that are optimal for spectral unmixing. This fulfills a large unmet need for massively multiplexed fluorescence applications such as spatial transcriptomics with RNA fluorescence in situ hybridization (FISH) or immunofluorescence with large antibody panels. This method has numerous applications in both research and clinical diagnostics. In particular, using relatively small panels of different donor and acceptor dyes, a multiplicity of pairwise combinations (i.e., a panel, also known as a library) can be generated, where each donor and acceptor dye pair generate a unique, spectrally-unmixable pseudocolor.

[0039] In particular, combinatorially pairing dye-conjugated oligonucleotides provides a large panel of virtual tandem dyes. FRET interactions between the donor and acceptor havenarrow spectral peaks that can be placed strategically at any location in spectral space, providing a unique spectral signature for each pair.

[0040] In some embodiments, the panel of fluorescent dye probes comprises at least 2 oligonucleo tide-conjugated fluorescent donor / acceptor dye pairs, and each dye pair creates a novel, i.e., unique and distinct, spectral signal that is different from each other dye individually or a sum of the spectral signals of the donor and acceptor. The unique spectral signal also differs from each other dye pair. In some embodiments, the panel of fluorescent dye probes comprises at least 2, at least 4, at least 6, at least 8, at least 10, at least 12, at least 50, at least 100, at least 128, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, or at least 500 fluorescent donor / acceptor dye pairs, where each dye pair creates a spectral signal different from each other dye individually, a sum of the spectra of the donor / acceptor pair, and from each other dye pair. The number of dye pairs can be theoretically higher, depending on the sensitivity of the instrumentation used. For example, the number n being from 10 to 128 represents one range of n-plex sizes but n can be any number from 2 to any number of target antibodies available, e.g. up to 512, 1000, 1024, 2000, 10,000, or 100,000.

[0041] However, in some embodiments, from a practical perspective, the panel of fluorescent dye probes comprises less than or equal to 500, less than or equal to 450, less than or equal to 350, less than or equal to 250, less than or equal to 150, less than or equal to 128, less than or equal to 100, less than or equal to 50, or less than or equal to 25, or less than or equal to 12, or less than or equal to 10, or less than or equal to oligonucleotide-conjugated fluorescent donor / acceptor dye pairs, wherein each dye pair creates a spectral signal different from each other dye individually, a sum of the spectra of the donor / acceptor pair, and from each other dye pair.

[0042] In some embodiments, the panel of fluorescent dye probes comprises 2 to 512, preferably 10 to 512, preferably 10 to 256, more preferably 10 to 128 oligonucleotide- conjugated fluorescent donor / acceptor dye pairs, for example 2 to 500, 6 to 400, 8 to 10 to 300, 12 to 200, 12 to 150, 10 to 128, or 12 to 128 oligonucleotide-conjugated fluorescent donor / acceptor dye pairs, wherein each dye pair creates a spectral signal different from each other dye individually, a sum of the spectra of the donor / acceptor pair, and from each other dye pair.

[0043] Tandem dyes, i.e., two dyes that are covalently combined into a single molecule, are known. The covalent linkage between the dyes places the dyes in close proximity, forming a FRET -based dye with a novel spectrum. One of the dyes acts as a donor and the other as an acceptor; when the donor is excited by an incident photon at its excitation wavelength, theenergy is transferred to the acceptor, which then emits a photon at its emission wavelength. The FRET interaction thus creates a new long-Stokes-shift spectra signature with the excitation spectrum of the donor and the emission spectrum of the acceptor. However, each tandem dye must be chemically synthesized and purified individually, which is labor-intensive and costly, such that a limited number of few tandem dyes exist. Instead, the methods described herein enable the user to select any two dyes from donor and acceptor panels and combine them to make a virtual tandem dye. The speed and low cost of the method enables large panels of virtual tandem dyes that would not be feasible with synthetic chemistry approaches. In addition, not all dyes are chemically compatible for use as tandem dyes. With this method, any two dyes that can be conjugated to an oligonucleotide can be linked, enabling the selection of desired spectral properties.

[0044] Still further, many tandem dyes have poor properties for practical use, e.g., low water solubility, low stability, high batch-to-batch variability in labelling efficiency, and synthetic drawbacks. Most tandem dyes use a protein-based donor that cannot be readily conjugated to oligonucleotides.

[0045] Spectral unmixing allows imaging of many colors with overlapping spectra, then “unmixing” them to isolate each of the colors cleanly. In principle, spectral unmixing could be used to unmix over 100 colors, but in practice it cannot achieve these numbers because existing fluorescent dyes have suboptimal spectral properties for this purpose. Unmixing of a large number of colors is potentially enabled by the method described herein, the method also being referred to herein as FRAINBOW (fluorescence resonance-assisted identification based on wavelength). The method further greatly decreases costs of detection.

[0046] The underlying concept of spectral unmixing is that a given fluorescent dye has a full excitation-emission spectrum, but traditional fluorescence microscopy only samples it at low density (FIG. 1 A, left). Spectral unmixing instruments sample the spectrum at higher density (FIG. 1 A, right), enabling them to resolve dyes whose spectral peaks are similar or even overlapping. This high-density spectral sampling enables linear unmixing algorithms to determine how much of each dye is present at each peak. Spectral unmixing confocal microscopy accordingly samples more of the spectrum with higher resolution, enabling it to unmix spectrally overlapping fluorophores. Commercially available spectral confocal microscopes use either emission-only unmixing (Zeiss, few excitation channels) or excitation- only unmixing (Leica, few emission channels). Unmixing the most colors requires a hyperspectral fluorescence microscope, which uses combined excitation-emission unmixing.

[0047] As illustrated in FIG. IB, fluorophores have a characteristic spectrum consisting of their fluorescence at every combination of excitation and emission wavelengths (three fluorophores shown). Emission wavelengths are always longer than excitation wavelengths, so the spectrum is left of the black diagonal line. Spectral unmixing can distinguish spectrally distinct fluorophores even if their spectra overlap partially. Reference spectra (right) must first be collected for each fluorophore individually. The microscope then images the tissue section, collecting a mixed signal (left) at each point in the sample. An unmixing algorithm then determines how much of each reference fluorophore is present at that location in the sample.

[0048] FIG. 1C illustrates that even though in theory, spectral unmixing can resolve hundreds of colors, in practice, resolution is limited by the fact that spectra of typical fluorescent dyes (ATTO dyes shown) are crowded near the diagonal (short Stokes shifts), leaving most of the spectral space unused. FIG. ID shows that long Stokes shift (LSS) dyes (mFluor-green shown) access the off-diagonal part of the spectrum. These dyes are used extensively in spectral flow cytometry, enabling cells to be routinely labeled with up to 40 colors. However, the spectra of LSS dyes are broad, thus using the spectral space inefficiently, and many cannot be conjugated to oligonucleotides easily.

[0049] In contrast, as shown in FIG. IE, when the dye-conjugated oligonucleotide pairs as described herein are placed in close apposition, fluorescent resonance energy transfer (FRET) interactions between them create a novel spectral peak with the excitation spectrum of the donor and the emission spectrum of the acceptor. By combinatorially pairing short Stokes shift dyes (e.g., the same 6 dyes shown as in FIG. 1C), all or substantially all of the spectral space can be densely filled with unique narrow peaks representing novel pseudocolors with ideal properties for spectral unmixing, as shown in FIG. IF (wherein only a small portion of the visible spectrum is shown). Thus, the combinatorial labeling scheme using FRET between a relatively small number of donor and acceptor oligonucleotides can be used to generate a large panel of spectrally-unmixable pseudocolors.

[0050] The number of pseudocolors can be as described above in connection with the dye pairs, e.g., up to 500 or even higher. For example, 2-512, 10-512, 10-256, or 10-128, or 1- 140 pseudocolors, or 2-140, 10-140, 20-140, 30-140, 40-140, or 50-140 pseudocolors can be unmixed. In other embodiments, 1-60, 2-60, 10-60, 20-70, or 30-60 pseudocolors can be unmixed. In another embodiment, 35-45, or 35-55, or 35-65 pseudocolors can be unmixed. In still other embodiments, 30-130, or 39-128 pseudocolors can be unmixed.

[0051] Accordingly, a panel of virtual or actual oligonucleotide-conjugated donor dye and acceptor dye pairs can be generated to provide a selected set of signals, wherein the numberof unique dye pairs can be as described above, e.g., up to 500 or above. For example, the panel can include 2-512, 10-512, 10-256, or 10-128 virtual or actual oligonucleotide-conjugated fluorescent donor / acceptor dye pairs 1-140, or 2-140 oligonucleotide-conjugated donor dye and acceptor dye pairs, or, for example 10-140, 20-140, 30-140, 40-140, or 50-140 virtual or actual oligonucleotide-conjugated donor dye and acceptor dye pairs. In another embodiment, the panel can include 1-60, 2-60, 10-60, 20-70, or 130-60 virtual or actual oligonucleotide-conjugated donor dye and acceptor dye pairs. In another embodiment, the panel can include 35-45, or 35- 55, or 35-65 virtual or actual dye-conjugated oligonucleotide pairs. In still other embodiments, the panel can include 30-130, or 39-128 virtual or actual oligonucleotide-conjugated donor dye and acceptor dye pairs.

[0052] The donor and acceptor dyes can include a moiety such as a small molecule dye group, a fluorescent protein group, a quantum dot, or a combination thereof. In some embodiments, the small molecule dye group is a xanthene, a rhodamine, a fluorescein, a coumarin, a pyrene, a phenanthridine, a boron difluoride dipyrromethene (BODIPY), a cyanine, a phthalocyanine, an oxazine, an acridine, a fluorone, a combination thereof, or derivatives thereof. In some embodiments, the acceptor includes a fluorescent protein group, for example green fluorescent protein (GFP), enhanced GFP (EGFP), superfolder GFP (sfGFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP), blue fluorescent proteins (BFP), cyan fluorescent proteins, mCherry, mRFPl, tdTomato, TagRFP, mKate2, mNeptune, mCardinal, or iRFP713.

[0053] Exemplary donor and acceptor dyes can be Alexa Fluor 350, Cascade Blue, Alexa Fluor 405, Alexa Fluor 430, nitrobenzoxadiazole (NBD), fluorescein, Alexa Fluor 488, Oregon Green 488, BODIPY 493 / 503, Rhodamine Green, BODIPY FL, Oregon Green 514, Alexa Fluor 514, Eosin, Rhodamine 6G, BODIPY R6G, Alexa Fluor 532, BODIPY 530 / 550, BODIPY TMR, Alexa Fluor 555, tetramethyl rhodamine (TMR), Alexa Fluor 546, BODIPY 558 / 568, QSY 7, QSY 9, BODIPY 564 / 570, Lissamine rhodamine B, Rhodamine Red, BODIPY 576 / 589, Alexa Fluor 568, X-rhodamine, Body BODIPY 581 / 591, BODIPY TR, Alexa Fluor 594, Texas Red, Alexa Fluor 610-X, BODIPY 630 / 650, Alexa Fluor 633, Alexa Fluor 635, BODIPY 650 / 665, Alexa Fluor 647, QSY 21, Alexa Fluor 660, Alex Sa Fluor (Alexa Fluor) 680, Alexa Fluor 700, Alexa Fluor 750, Alexa Fluor 790, ATTO, AF568, AF594, AF610, AF633, AF647, AF680, AF700, Alexa Fluor 405, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 633, ATTO 488, ATTO 532, ATTO425, ATTO465, ATTO488, ATTO520, ATTO532, ATTO542, ATTO550, ATTO565, Cy5.5, iFluor488, or a combination thereof.

[0054] In some embodiments, the donor and acceptor dyes can be AF568, AF594, AF610, AF633, AF647, AF680, AF700, Alexa Fluor 405, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 633, ATTO 488, ATTO 532, ATTO425, ATTO465, ATTO488, ATTO520, ATTO532, ATTO542, ATTO550, ATTO565, Cy5.5, iFluor488, or a combination thereof.

[0055] Methods detection using the FRET-based panel is useful in a variety of applications requiring detection of multiple targets, and can be applied to most biomedical applications relying on fluorescence.

[0056] For example, the method can be used with multiplexed RNA FISH methods, which have increased in popularity because single-cell RNA-seq has revealed a very large diversity of cell type-specific transcriptomic states. Multiplexed RNA FISH further has important applications beyond basic research, including the analysis of surgical pathology specimens in cancer diagnosis. An exemplary embodiment of a method of fluorescence detection using a panel of actual oligonucleotide-conjugated donor dye and acceptor dye pairs is illustrated in FIG. 2A and FIG. 2B. As shown in FIG. 2A, a padlock probe is hybridized to mRNA, followed by ligation and rolling circle amplification. The resulting rolling circle product (RCP) is a DNA “spot” of 500 nm, containing many copies of a gene-specific barcode. In the reported dRNA-HyblSS methods of detection, the barcode identity is read out by hybridizing a sequence of bridge probes and fluorescent dye probes over multiple rounds of imaging (FIG. 2B, left). FIG. 2B at right shows an exemplary embodiment of the method described herein, also known as the PUFRFISH (probes unmixed in first round RNA FISH, pronounced “pufferfish”) method. In the PUFRFISH method, each RCP is labeled with a pair of two fluorescent probes (a donor dye-conjugated oligonucleotide and acceptor dye conjugated oligonucleotide) that are FRET partners, thereby generating a unique signal that can be unmixed.

[0057] FIG. 2C is a graph that shows that 4 base pair (bp) spacing between the two fluorescent probes yields optimal FRET efficiency. Without being bound by theory, it is believed that the decreased FRET signal with 2-3 bp spacing is likely due to contact quenching, an unrelated phenomenon, and which is consistent with theoretical calculations.

[0058] Two commercial RNA FISH technologies that can analyze panels of hundreds of mRNAs, including 10X Genomic’s Xenium platform and Vizgen’s MERSCOPE platform, have low throughput and have 4 colors or at most 6-7 colors. The PUFRFISH method, in contrast, can use a larger number of colors using 39-128 colors, which provides many advantages. It greatly increases throughput and solves the “spot crowding” problem that limits panel size.

[0059] FIG. 2D shows PUFRFISH data showing GAPDH mRNA visualized in cultured A549 cells (a human lung carcinoma cell line). The strong FRET signal and near-complete quenching of the donor signal indicate high FRET efficiency.

[0060] In another embodiment, the PUFRFISH method using the FRET-based labeling panel is effective in vitro, as illustrated in FIGS. 3A and 3B. In particular, it has been tested with a 39-plex oligonucleotide-conjugated donor dye and acceptor dye pair PUFRFISH panel that labeled 39 mRNAs with 39 different FRET pairs (FIG. 3C). This panel can be expanded up to 128 mRNAs with 128 different FRET pairs (FIG. 3D). In principle the panel can be expanded beyond 128 pairs, for example the panel can include up to 150, 160, 170, 180, 190, 200, 210, 220, 230, 250, 250, 256, 300 or more mRNA and an equivalent number of different FRET pairs.

[0061] In still another embodiment, the panels and the FRET-based methods can be used to label antibodies, for example antibodies used in immunohistochemistry (IHC) and flow cytometry, enabling larger immunofluorescence panels with less complexity and lower cost. Exemplary antibodies for IHC include those targeting CD3 (T cells), CD4 (helper T cells), CD8 (cytotoxic T cells), CD20 (B cells), CD68 (macrophages), CD31 (endothelial cells), Ki-67 (a proliferation marker), and pan-cytokeratin (epithelial cell marker). In flow cytometry, exemplary antibodies include those targeting CD45 (leukocytes), CD 14 (monocytes), CD 16 (natural killer cells), CD 19 (B cells), CD25 (activated T cells), CD34 (hematopoietic stem cells), CD44 and CD90 (mesenchymal stem cells), and intracellular markers like FoxP3 (regulatory T cells) or granzyme B (cytotoxic granules).

[0062] Other applications include proteomics, epigenomics, and the like. Indeed, with the increasing prevalence of “omics” approaches, there is an increasing demand for massively multiplexed fluorescence-based assays.

[0063] For example, in an embodiment, the method includes conjugating a bridge oligonucleotide to a selected antibody. Methods and commercial arrays are known and available for such labeling, allowing labeling of all or substantially all of the selected antibody in the sample. A panel of oligonucleotide-conjugated donor dye and acceptor dye pairs is provided, wherein each of the oligonucleotides of the oligonucleotide-conjugated acceptor dyes is complementary to a first sequence of the bridge oligonucleotide, and is linked to a different acceptor dye; and each of the oligonucleotides of the oligonucleotide-conjugated donor dyes is complementary to a second sequence of the bridge oligonucleotide, and is linked to a different donor dye. The first and second sequences of the bridge oligonucleotide is separated by the appropriate number of base pairs (e.g., 3, 4, or 5). Depending on the desired color signal, anappropriate pair of oligonucleotide-conjugated donor dye and acceptor dyes is selected and hybridized to the antibody.

[0064] FIG. 4 shows an exemplary embodiment of this step of the method, wherein an antibody is labeled with pairs of dyes that interact via FRET to generate novel spectral signatures. FIG. 4A shows a single bridge oligonucleotide (402) synthesized with a 5’ amine group and conjugated to every antibody in the panel. The desired donor and acceptor FRET probes for each antibody are then selected from a panel as described above, and hybridized to the bridge oligonucleotide. The hybridization occurs under low-salt conditions, but the antibody is used at physiological salt concentrations (154 mM Na+, oligonucleotide Tm > 50°C), preventing the FRET probes from detaching.

[0065] Different oligonucleotide-conjugated donor and acceptor dye pairs can be selected and used for each antibody of interest, such that in a panel of antibodies, each antibody can be labeled with a unique spectral signature that can be detected and quantified. The abovedescribed panels and methods provide major advantages over current protocols. First, the end user can select donor and acceptor probes from a panel and choose which combination to label each antibody immediately prior to using the antibody. This would ensure that each antibody in the panel had a unique spectral signature. Furthermore, the methods increase the number of antibodies that could be used in parallel.

[0066] Arrays for fluorescent analysis using the above-described methods can include antibodies pre-complexed with a panel of the oligonucleotide-conjugated donor dye and acceptor dye pairs. The arrays can be used in diagnostics, immunohistochemistry, flow cytometry, or multiplexed immunoassays.

[0067] Still another embodiment for labeling an antibody is shown in FIG. 4B. In this embodiment, the antibody is linked to an oligonucleotide sequence as described above. The oligonucleotide sequence is labeled with a click chemistry group, one specific for an acceptor dye, and one specific for a donor dye (for example, an azide-linked dye). Each antibody can then be readily labeled with a selected donor and acceptor dye pair.DEFINITIONS

[0068] The terms “and / or” mean “either . . . or . . ., or both . . . and ...” when referring to two elements, and mean “either . . ., . . ., or . . ., or any combination or all thereof’ when referring to three or more elements. As an example, the phrase “A and / or B” means “either A or B, or both A and B”, and the phrase “A, B and / or C” means “either A, B or C, or any combination or all thereof’.

[0069] As used in the specification and the claims, all transitional terms such as “comprising”, “containing”, “having”, “including”, “composed of’, and the like are open-ended and inclusive, that is, mean including but not limited to and do not exclude additional, unrecited element(s) or method step(s). Only the transitional term “consisting of’ is closed, that is, excludes any additional, unrecited element or method step, and the transitional term “consisting essentially of’ is semi-closed, that is, only allows inclusion of additional, unrecited element(s) or method step(s) that do not materially affect the basic and novel characteristic(s) of that particular embodiment.

[0070] The term “exemplary” as used herein means “serving as an example, instance or illustration”. Any embodiment or feature characterized herein as “exemplary” is not necessarily to be construed as preferred or advantageous over other embodiments or features.

[0071] Whenever the term “at least” or “greater than” precedes the first numerical value in a series of two or more numerical values, the term “at least” or “greater than” applies to each one of the numerical values in that series of numerical values.

[0072] Whenever the term “no more than” or “less than” precedes the first numerical value in a series of two or more numerical values, the term “no more than” or “less than” applies to each one of the numerical values in that series of numerical values.

[0073] The term “oligonucleotide” refers to a nucleic acid molecule typically comprising a sequence of nucleotide residues (e.g., DNA or RNA) ranging from 5 to 100 nucleotides in length, although longer or shorter sequences may also be included depending on the context. Oligonucleotides may be single-stranded or double-stranded and can include naturally occurring or modified bases, sugars, or intemucleotide linkages, such as phosphorothioates, 2’-O-methyl modifications, locked nucleic acids (LNAs), peptide nucleic acids (PNAs), or other synthetic analogs. The oligonucleotide may be linear or circular and may be chemically synthesized or recombinantly produced.

[0074] The term “polynucleotide” refers to a polymer composed of nucleotide units. Polynucleotides can contain naturally occurring nucleic acids (e.g., deoxyribonucleic acid (“DNA”) and ribonucleic acid (“RNA”)), or / and nucleic acid analogs. Polynucleotides containing one or more nucleic acid or nucleotide analogs are sometimes called “aptamers”. Nucleic acid or nucleotide analogs include without limitation those which have a non-naturally occurring base / nucleobase, have a sugar or non-sugar moiety other than 2’ -deoxyribose or ribose, or engage in linkages with other nucleotides other than the naturally occurring phosphodiester bond, or a combination or all thereof. Non-limiting examples of nucleic acid or nucleotide analogs include xeno(biotic) nucleic acids (XNAs) having a backbone other than thenaturally occurring sugar-phosphate backbone present in DNA or RNA (e.g., 2’-O-substituted ribonucleotides (e.g., 2’-0-methyl ribonucleotides and 2’-O-(2-methoxyethyl) ribonucleotides), cyclohexene nucleic acids (CeNAs)), 2’-deoxy-2’-fluoroarabino nucleic acids (FANAs)), glycol nucleic acids (GNAs)), 1,5-anhydrohexitol nucleic acids (HNAs)), locked nucleic acids (LNAs) (also called bridged nucleic acids (BNAs)), morpholino nucleic acids (MNAs), peptide nucleic acids (PNAs), and threose nucleic acids (TNAs)), 5-methylcytosine, 5 -methyluracil, phosphorothioates / thiophosphates, phosphorodithioates, phosphoramidates, phosphorodiamidates, boranophosphates, phosphorotriesters, methylphosphonates, chiral-methyl phosphonates, and the like. DNA and RNA polynucleotides can be synthesized using a DNA or RNA polymerase or an automated DNA or RNA synthesizer. Polynucleotides containing nucleic acid analogs can be synthesized using, e.g., an engineered DNA or RNA polymerase that recognizes the nucleic acid analogs, a phosphoramidite strategy, or an automated peptide synthesizer in the case of PNAs. The term “nucleic acid molecule” typically refers to a larger polynucleotide. The term “oligonucleotide” typically refers to a shorter polynucleotide. In certain embodiments, an oligonucleotide contains no more than 100 nucleotides. In some embodiments, when a polynucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes the corresponding, or the complementary, RNA sequence (i.e., A, U, G, C) in which “U” replaces “T”.

[0075] The term “complementary” refers to the topological compatibility or matching together of interacting surfaces of two polynucleotides. Thus, the two molecules can be described as complementary, and furthermore the contact surface characteristics are complementary to each other. A first polynucleotide is complementary to a second polynucleotide if the nucleotide sequence of the first polynucleotide is identical to the nucleotide sequence of the polynucleotide binding partner of the second polynucleotide. Complementarity of polynucleotides typically refers to C / G and A / T (or U in the case of RNA) base pairings between antiparallel DNA / DNA, DNA / RNA or RNA / RNA sequences. Thus, the polynucleotide whose sequence is 5 ’-TAT AC-3’ is complementary to a polynucleotide whose sequence is 5’- GTATA-3’. A nucleotide sequence is “substantially complementary” to a reference nucleotide sequence if the sequence complementary to the subject nucleotide sequence is substantially identical to the reference nucleotide sequence.

[0076] The term “padlock probe” refers to an oligonucleotide capable of circularization upon hybridization to a target nucleic acid sequence. A padlock probe typically comprises two target-complementary regions (also referred to as “arms”) located at the 5' and 3' ends of the probe, separated by a non-complementary linker region. Upon specific hybridization of botharms to adjacent sequences on the target nucleic acid, the ends of the probe are brought into juxtaposition and can be covalently joined — typically by a ligase enzyme — resulting in the formation of a closed circular DNA molecule. This circularized padlock probe can then serve as a template for amplification, such as by rolling circle amplification (RCA), to detect the presence of the target sequence with high specificity and sensitivity.

[0077] The term “donor dye” is used interchangeable with “donor” to refer to a group of chemical that is capable of absorbing light energy and then transferring at least some of such light energy to an acceptor. The donor might be a fluorescent resonance energy transfer (FRET) donor.

[0078] The term “acceptor dyes” is used interchangeable with “acceptor” to refer to a group that is capable of receiving or absorbing energy transferred from a donor group and converting at least some of the transferred energy to emitted light.

[0079] The term “excitation spectrum” refers to the ability of a group to absorb and be excited by electromagnetic radiation as a function of the wavelength of the electromagnetic radiation.

[0080] The term “emission spectrum” is used to refer to the relative emission of photons, as a function of wavelength, of an excited group. The compositions, methods, and articles can alternatively comprise, consist of, or consist essentially of, any appropriate materials, steps, or components herein disclosed. The compositions, methods, and articles can additionally, or alternatively, be formulated so as to be devoid, or substantially free, of any materials (or species), steps, or components that are otherwise not necessary to the achievement of the function or objectives of the compositions, methods, and articles.

[0081] The terms “a” and “an” and “the” do not denote a limitation of quantity, but rather denote the presence of at least one of the referenced items. The term “or” means “and / or” unless clearly indicated otherwise by context. Reference throughout the specification to “an embodiment”, means that a particular element (e.g., feature, structure, step, or characteristic) described in connection with the embodiment is included in at least one embodiment described herein, and may or may not be present in other embodiments. In addition, it is to be understood that the described elements may be combined in any suitable manner in the various embodiments.

[0082] The endpoints of all ranges directed to the same component or property are inclusive of the endpoints, are independently combinable, and include all intermediate points and ranges. For example, ranges of “up to 25 wt%, or 5 to 20 wt%” is inclusive of the endpoints and all intermediate values of the ranges of “5 to 25 wt%,” such as 10 to 23 wt%, etc.

[0083] The term “combination” is inclusive of blends, mixtures, alloys, reaction products, and the like. Also, “at least one of” means that the list is inclusive of each element individually, as well as combinations of two or more elements of the list, and combinations of at least one element of the list with like elements not named.

[0084] Unless defined otherwise, technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this invention belongs.

[0085] All cited patents, patent applications, and other references are incorporated herein by reference in their entirety. However, if a term in the present application contradicts or conflicts with a term in the incorporated reference, the term from the present application takes precedence over the conflicting term from the incorporated reference.

[0086] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims. It is understood that the detailed examples and embodiments described herein are given by way of example for illustrative purposes only, and are in no way considered to be limiting to the invention. Various modifications or changes in light thereof will be suggested to persons skilled in the art and are included within the spirit and purview of this application and are considered within the scope of the appended claims. For example, the relative quantities of the components can be varied to optimize the desired effects, additional components can be added, and / or similar components can be substituted for one or more of the components described.

[0087] Additional advantageous features and functionalities associated with the systems, methods, and processes of the present invention will be apparent from the appended claims.

Claims

CLAIMSWhat is claimed is:

1. A panel of fluorescent dye probes, the panel comprising a plurality of oligonucleotide-conjugated fluorescent donor dyes, wherein the oligonucleotide is complementary to a first sequence of a target oligonucleotide; a plurality of oligonucleotide-conjugated acceptor dyes, wherein the oligonucleotide is complementary to a second sequence of a target oligonucleotide; wherein the first sequence and second sequence are spaced apart a distance effective to allow fluorescent resonance energy transfer between one donor dye and one acceptor dye when each is bonded to the target oligonucleotide, to create a spectral signal with the excitation spectrum of the donor and the emission spectrum of the acceptor that is distinct from a spectra of the donor dye, a spectra of the acceptor dye, or an additive overlay spectra of the donor dye and acceptor dyethe panel comprises from at least 2 oligonucleotide-conjugated fluorescent donor dye pairs, wherein each dye pair creates a spectral signal different from each other dye pair.

2. The panel of claim 1, wherein the panel comprises 2 to 512, preferably 10 to 512, preferably 10 to 256, more preferably 10 to 128 oligonucleotide-conjugated fluorescent donor / acceptor dye pairs.

3. The panel of claim 1, wherein the first sequence and the second sequence comprise from 5 to 25 nucleotides.

4. The panel of claim 1 or claim 3, wherein the first sequence and the second sequence are separated by 3, 4, or 5 nucleotides.

5. The panel of any one of claims 1 to 4, wherein the donor and acceptor dyes are Alexa Fluor 350, Cascade Blue, Alexa Fluor 405, Alexa Fluor 430, nitrobenzoxadiazole (NBD), fluorescein, Alexa Fluor 488, Oregon Green 488, BODIPY 493 / 503, Rhodamine Green, BODIPY FL, Oregon Green 514, Alexa Fluor 514, Eosin, Rhodamine 6G, BODIPY R6G, Alexa Fluor 532, BODIPY 530 / 550, BODIPY TMR, Alexa Fluor 555, tetramethyl rhodamine (TMR), Alexa Fluor 546, BODIPY 558 / 568, QSY 7, QSY 9, BODIPY 564 / 570, Lissamine rhodamine B, Rhodamine Red, BODIPY 576 / 589, Alexa Fluor 568, X-rhodamine, Body BODIPY 581 / 591, BODIPY TR, Alexa Fluor 594, Texas Red, Alexa Fluor 610-X, BODIPY 630 / 650, Alexa Fluor633, Alexa Fluor 635, BODIPY 650 / 665, Alexa Fluor 647, QSY 21, Alexa Fluor 660, Alex Sa Fluor (Alexa Fluor) 680, Alexa Fluor 700, Alexa Fluor 750, Alexa Fluor 790, ATTO, AF568, AF594, AF610, AF633, AF647, AF680, AF700, Alexa Fluor 405, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 633, ATTO 488, ATTO 532, ATTO425, ATTO465, ATTO488, ATTO520, ATTO532, ATTO542, ATTO550, ATTO565, Cy5.5, iFluor488, or a combination thereof.

6. A method of making of an n-plex array of oligonucleotide-conjugated fluorescent donor dye and acceptor dye pairs of any one of claims 1 to 5 for detection of an n-plex array of target oligonucleotides, wherein n is a number of at least two, the method comprising selecting a first oligonucleotide-conjugated fluorescent donor dye and acceptor dye pair from the panel of any one of claims 1 to 5, wherein the oligonucleotide dye sequences are complementary to a first target oligonucleotide; selecting a second oligonucleotide-conjugated fluorescent donor dye and acceptor dye pair from the panel of claim 1, wherein the oligonucleotide dye sequences are complementary to a second target oligonucleotide; and repeating the selecting up to n-2 times for each oligonucleotide in the n-plex array to provide n pairs of oligonucleotide-conjugated fluorescent donor dye and acceptor dye pairs.

7. An n-plex array of oligonucleotide-conjugated fluorescent donor dye and acceptor dye pairs of any one of claims 1-5 for detection of an n-plex array of target oligonucleotides, the array comprising a first oligonucleotide-conjugated fluorescent donor dye and acceptor dye pair from the panel, wherein the oligonucleotide dye sequences are complementary to a first target oligonucleotide; a second oligonucleotide-conjugated fluorescent donor dye and acceptor dye pair from the panel, wherein the oligonucleotide dye sequences are complementary to a second target oligonucleotide; andan additional number of oligonucleotide-conjugated fluorescent donor dye and acceptor dye pairs from the panel, wherein the oligonucleotide dye sequences are complementary to each of an additional target oligonucleotide, wherein the additional number is n-2.

8. A method of detecting an n-plex array of target oligonucleotides wherein n is a number of at least two, the method comprising contacting n pairs of the oligonucleotide-conjugated fluorescent donor dyes and acceptor dyes of the n-plex array of oligonucleotide-conjugated fluorescent donor dye and acceptor dye pairs of claim 7 with the n-plex array of target oligonucleotides to provide a fluorescent energy transfer spectral signal; and analyzing the energy transfer signal to determine the pairs of oligonucleotide-conjugated fluorescent donor dyes and acceptor dyes contributing to the signal.

9. The array of claim 7, wherein the oligonucleotide sequence of each of the array of n pairs of the oligonucleotide-conjugated fluorescent donor dyes and acceptor dyes is part of a padlock probe.

10. A method for detecting a target nucleic acid, comprising contacting a padlock probe with the target nucleic acid; circularly linking the padlock probe using a ligase; amplifying the target nucleic acid by binding of a polymerase; contacting the amplified nucleic acid with a bridge oligonucleotide; contacting the bridge oligonucleotide with the n-plex array of target oligonucleotides of claim 9 to provide a fluorescent energy transfer spectral signal; and analyzing the energy transfer signal to determine the pairs of oligonucleotide-conjugated fluorescent donor dyes and acceptor dyes contributing to the signal.

11. A method of labeling an antibody, comprising linking a target oligonucleotide to the antibody; contacting the target oligonucleotide with an oligonucleotide-conjugated fluorescent donor dye wherein the donor dye oligonucleotide is complementary to a first sequence of the target oligonucleotide together with an oligonucleotide-conjugated acceptor dye wherein the acceptor dye oligonucleotide is complementary to a second sequence of a target oligonucleotide;wherein the first sequence and second sequence are spaced apart a distance effective to allow fluorescent resonance energy transfer between the donor dye and the acceptor dye when each is bonded to the target nucleotide, to create a spectral signal with the excitation spectrum of the donor and the emission spectrum of the acceptor, and wherein the spectral signal is distinct from a spectra of the donor dye, a spectra of the acceptor dye, or an additive overlay spectra of the donor dye and acceptor dye.

12. A method of labeling an n-plex array of target antibodies, wherein n is a number of at least two, the method comprising linking each of the target antibodies to a unique target oligonucleotide ; selecting a first oligonucleotide-conjugated fluorescent donor dye and acceptor dye pair from the panel of any one of claims 1-5, wherein the oligonucleotide dye sequences are complementary to a first target oligonucleotide; selecting a second oligonucleotide-conjugated fluorescent donor dye and acceptor dye pair from the panel of claim 1, wherein the oligonucleotide dye sequences are complementary to a second target oligonucleotide; and repeating the selecting up to n-2 times for each oligonucleotide of each antibody in the n- plex array to provide an n-plex array of the antibodies labelled with n pairs of oligonucleotide- conjugated fluorescent donor dye and acceptor dye pairs.

13. An n-plex array of target antibodies, wherein n is a number of at least two, the array comprising a first antibody comprising a first target oligonucleotide hybridized with a first nucleotide-conjugated fluorescent donor dye and acceptor dye pair of the panel of any one of claims 1-5, wherein the oligonucleotide dye sequences are complementary to the first target oligonucleotide; a second antibody comprising a second oligonucleotide hybridized to a second conjugated fluorescent donor dye and acceptor dye pair of the panel of any one of claims 1-5, wherein the oligonucleotide dye sequences are complementary to the second target oligonucleotide; and an additional number of n antibodies hybridized to an additional number of oligonucleo tide-dye pairs from the panel of claims 1-5, wherein each of the additional oligonucleotide dye sequences are complementary to each of an additional target oligonucleotide, wherein the additional number is n-2.

14. A method of detecting an array of antigens, the method comprising contacting the n-plex array of labelled antibodies of claim 13 with a sample comprising an antigen that binds to at least one of the labelled antibodies, to provide a fluorescent energy transfer spectral signal; and analyzing the energy transfer signal to determine the pairs of oligonucleo tide-conjugated fluorescent donor dyes and acceptor dyes contributing to the signal.

Images