Branched tandem dye scaffolds

Abstract

Branched tandem dye scaffolds are provided. Embodiments of the invention include: a non-conjugated backbone comprising a branch comprising two or more donor fluorophores; and an acceptor fluorophore(s) linked to the non-conjugated backbone; wherein the acceptor fluorophore(s) and the donor fluorophores are in energy transfer relationship. Also provided are methods of making and using the branched tandem dye scaffolds, as well as kits that include the branched tandem dye scaffolds that find use in embodiments of the methods.

Description

[0001] BRANCHED TANDEM DYE SCAFFOLDS

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to the filing date of United States Provisional Patent Application Serial No. 63 / 611 ,646 filed on December 18, 2023; the disclosure of which application is incorporated herein by reference.

[0004] INTRODUCTION

[0005] Fluorescent dyes are compounds which, when irradiated with light of a wavelength which they absorb, emit light of a (usually) different wavelength. Fluorescent dyes find use in a variety of applications in biochemistry, biology and medicine, e.g., in diagnostic kits, in microscopy or in drug screening. Fluorescent dyes are characterized by a number of parameters allowing a user to select a suitable dye depending on the desired purpose. Parameters of interest include the excitation wavelength maximum, the emission wavelength maximum, the Stokes shift, the extinction coefficient, the fluorescence quantum yield and the fluorescence lifetime. Dyes may be selected according to the application of interest in order to, e.g., allow penetration of exciting radiation into biological samples, to minimize background fluorescence and / or to achieve a high signal- to-noise ratio.

[0006] Molecular recognition involves the specific binding of two molecules. Molecules which have binding specificity for a target analyte find use in a variety of research and diagnostic applications, such as the labelling and separation of analytes, flow cytometry, in situ hybridization, enzyme-linked immunosorbent assays (ELISAs), western blot analysis, magnetic cell separations and chromatography. Target analytes may be detected by labelling with a fluorescent dye.

[0007] Some applications, such as flow cytometry applications, employ combinations or panels of differentially fluorescently labeled specific binding members, such as antibodies, for the detection of multiple different targets, e.g., internal or cell surface markers. In such applications, multiple different fluorecent dyes are employed with the same sample, where the multiple different fluorescent dyes are distinguishable from each other in terms of excitation and / or emission maxima. One type of fluorescent dye that finds use in such applications is a tandem dye. Tandem dyes are compounds having two covalently linked different fluorophores, which fluorophores may be covalently Inked to each other directly or through a linking group. One of the fluorophores serves as donor fluorophore and the other fluorphore acts as acceptor fluorophore. The donor and acceptor fluorophores together form a fluorescence-resonance energy transfer (FRET) pair. Such FRET pairs behave as a unique dye that has the excitation properties of the donor fluorophore and the emission properties of the acceptor fluorophore.

[0008] SUMMARY

[0009] The inventors have realized that branched structures allow for the manufacture of more compact tandem dye systems that lead to enhanced light harvesting and energy transfer. By locating donor fluorophores around a smaller number of acceptor fluorophores, distances for energy transfer are reduced while increasing the number of donor fluorophores present. A branched approach brings the donor fluorophores and acceptor fluorophores together in a smaller area, with donor fluorophores configured close and with more uniform distances between donor fluorophores and acceptor fluorophores. These structures can help reduce unwanted donor fluorophore emission and increase the efficiency of energy transfer between donor fluorophores and acceptor fluorophores. Embodiments of the invention satisfy this need.

[0010] Branched tandem dye scaffolds are provided. Embodiments of the invention include: a non-conjugated backbone comprising a branch comprising two or more donor fluorophores; and an acceptor fluorophore(s) linked to the non-conjugated backbone; wherein the acceptor fluorophore(s) and the donor fluorophores are in energy transfer relationship. Also provided are methods of making and using the branched tandem dye scaffolds, as well as kits that include the branched tandem dye scaffolds and find use in embodiments of the methods.

[0011] BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The invention may be best understood from the following detailed description when read in conjunction with the accompanying drawings. Included in the drawings are the following figures:

[0013] FIG. 1 provides the synthesis scheme for BTDS 1 according to an embodiment of the invention. As illustrated in FIG. 1 , the backbone structure in BTDS 1 is synthesized by conjugating protected lysine and homopropargylglycine to a PEG chain. The donor is conjugated to the backbone after selective deprotection of amine on the lysine residue while the acceptor is attached using Cu(l)-catalyzed azide-alkyne cycloaddition (CuAAC) click chemistry to the homopropargylglycine residue. Finally, the terminal amine of the PEG chain is exposed and SMCC is conjugated to the structure for further antibody conjugation application.

[0014] FIG. 2 provides a schematic of a branched tandem dye according to an embodiment.

[0015] DEFINITIONS

[0016] The term “unit” refers to a structural subunit of a scaffold. The term unit is meant to include monomers, co-monomers, co-blocks, repeating units, and the like. It is understood that a variety of arrangements of units are possible and that in the depicted formulas of the non-conjugated backbones any convenient arrangements of various lengths can be included within the structure of the overall branched tandem dye scaffold. It is understood that the branched tandem dye scaffold may also be represented by a formula in terms of mol% values of each unit in the branched tandem dye scaffold and that such formula may represent a variety of arrangements. In certain instances, the unit possesses a portion of the non-conjugated backbone and a branch, or a portion of the nonconjugated backbone and a branch comprising two or more donor fluorophores, or a portion of the non-conjugated backbone and a branch comprising one or more acceptor fluorophore(s), or a terminal unit of the scaffold.

[0017] In certain instances, the scaffold is a polymer. A “repeating unit” or “repeat unit” is a subunit of a scaffold that is defined by the minimum number of distinct structural features that are required for the unit to be considered monomeric, such that when the unit is repeated n times, the resulting structure describes the polymer or a block thereof. In some cases, the polymer may include two or more different repeating units, e.g., when the polymer is a multiblock polymer, a random arrangement of units or a defined sequence, each block may define a distinct repeating unit. In some cases, a repeating unit of the polymer includes a single monomer group. In certain instances, a repeating unit of the polymer includes two or more monomer groups, i.e., co-monomer groups, such as two, three, four or more co-monomer groups. The term “co-monomer” or “co-monomer group” refers to a structural unit of a polymer that may itself be part of a repeating unit of the polymer. The non-conjugated backbone may have a random configuration of non-conjugated repeat units. The non-conjugated backbone may include a block or co-block configuration of non-conjugated repeat units. Alternatively, the non-conjugated backbone may include a particular defined sequence of non-conjugated repeat units, e.g., amino acid residues of a polypeptide sequence. These configurations can be characterized by polymeric segments of repeat units (e.g., as described herein), which segments can themselves be repeated throughout the modular scaffold.

[0018] The backbone of the branched tandem dye scaffold may have any convenient length. In some cases, the particular number of units of the branched tandem dye scaffold may fall within the range of 1 to 100, such as 2 to 100, such as 2 to 500, 2 to 100, 3 to 100, 4 to 100, 5 to 100, 6 to 100, 7 to 100, 8 to 100, 9 to 100, or 10 to 100 units. In some cases, the particular number of units of the branched tandem dye scaffold may fall within the range of 2 to 20, such as 2 to 15, 2 to 14, 2 to 13, 2 to 12, 2 to 1 1 , 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, or 2 to 3 units. In some cases, the particular number of units of the branched tandem dye scaffold may be 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 units. In some cases, the particular number of units of the branched tandem dye scaffold may be 2, 3, 4, or 5 units.

[0019] By “non-conjugated” is meant that pi conjugation or an extended delocalized electronic structure is precluded along the backbone from one unit to the next. It is understood that a unit may include within it one or more isolated unsaturated groups including an unsaturated bond (e.g., of an alkenylene group or an alkynylene group) and / or an aryl or heteroaryl group, which groups can be a part of the backbone.

[0020] As used herein, the term “specific binding member” refers to one member of a pair of molecules which have binding specificity for one another. One member of the pair of molecules may have an area on its surface, or a cavity, which specifically binds to an area on the surface of, or a cavity in, the other member of the pair of molecules. Thus, the members of the pair have the property of binding specifically to each other to produce a binding complex. In some embodiments, the affinity between specific binding members in a binding complex is characterized by a Kd (dissociation constant) of 10’6M or less, such as 10’7M or less, including 10-8M or less, e.g., 10-9M or less, 1 O-10M or less, 1 O’11M or less, 10’12M or less, 10’13M or less, 10’14M or less, including 10’15M or less. In some embodiments, the specific binding members specifically bind with high avidity. By high avidity is meant that the binding member specifically binds with an apparent affinity characterized by an apparent Kd of 10 x 10’9M or less, such as 1 x 10-9M or less, 3 x 10’10M or less, 1 x 10’10M or less, 3 x 10’11M or less, 1 x 10’11M or less, 3 x 10’12M or less or 1 x 10’12M or less.

[0021] In certain cases, the specific binding member is a biomolecule. The specific binding member can be proteinaceous. As used herein, the term “proteinaceous” refers to a moiety that is composed of amino acid residues. A proteinaceous moiety can be a polypeptide. In certain cases, the proteinaceous specific binding member is an antibody. In certain embodiments, the proteinaceous specific binding member is an antibody fragment. As used herein, the terms “antibody” and “antibody molecule” are used interchangeably and refer to a protein consisting of one or more polypeptides substantially encoded by all or part of the recognized immunoglobulin genes. The recognized immunoglobulin genes, for example in humans, include the kappa (k), lambda (I), and heavy chain genetic loci, which together comprise the myriad variable region genes, and the constant region genes mu (u), delta (d), gamma (g), sigma (e), and alpha (a) which encode the IgM, IgD, IgG, IgE, and IgA isotypes respectively. An immunoglobulin light or heavy chain variable region consists of a "framework” region (FR) interrupted by three hypervariable regions, also called “complementarity determining regions” or "GDRs”. The extent of the framework region and CDRs have been precisely defined (see, “Sequences of Proteins of Immunological Interest,” E. Kabat et aL, U.S. Department of Health and Human Services, (1991 )). The numbering of all antibody amino acid sequences discussed herein conforms to the Kabat system. The sequences of the framework regions of different light or heavy chains are relatively conserved within a species. The framework region of an antibody, that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs. The GDRs are primarily responsible for binding to an epitope of an antigen. The term antibody is meant to include full length antibodies and may refer to a natural antibody from any organism, an engineered antibody, or an antibody generated recombinantly for experimental, therapeutic, or other purposes as further defined below'.

[0022] Antibody fragments of interest include, but are not limited to, Fab, Fab’, F(ab5)2. Fv, scFv, or other antigen-binding subsequences of antibodies, either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA technologies. Antibodies may be monoclonal or polyclonal and may have other specific activities on cells (e.g., antagonists, agonists, neutralizing, inhibitory, or stimulatory antibodies). It is understood that the antibodies may have additional conservative amino acid substitutions which have substantially no effect on antigen binding or other antibody functions.

[0023] In certain embodiments, the specific binding member is a Fab fragment, a Ffab’k fragment, a scFv, a diabody or a triabody. In certain embodiments, the specific binding member is an antibody, hi some cases, the specific binding member is a murine antibody or binding fragment thereof, in certain instances, the specific binding member is a recombinant antibody or binding fragment thereof.

[0024] The term “linker” or “linkage” refers to a linking moiety that connects two groups and has a length of 1 to 600 atoms. A linker or linkage may be a covalent bond that connects two groups or a chain of between 1 and 100 atoms in length, for example a chain of 1 , 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20 or more carbon atoms in length, where the linker may be linear, branched, cyclic or a single atom.

[0025] In certain embodiments, the linker has between 10 and 300 atoms, or between 20 and 260 atoms, or between 230 and 250 atoms, or between 236 and 250 atoms, or between 30 and 40 atoms, or between 60 and 70 atoms, or between 70 and 80 atoms, or between 75 and 85 atoms, or between 95 and 105 atoms, or between 110 and 120 atoms, or between 125 and 135 atoms. In certain embodiments, the linker has between between 1 and 30 atoms, or between 1 and 5 atoms, or between 2 and 5 atoms, or between 7 and 10 atoms, or between 18 and 22 atoms. In certain embodiments, the linker has between 160 and 200 atoms, between 165 and 195 atoms, between 170 and 190 atoms, between 175 and 185 atoms, between 178 and 182 atoms, and 180 atoms. In certain embodiments, the linker has between 350 and 400 atoms, between 355 and 395 atoms, between 360 and 390 atoms, between 365 and 385 atoms, between 370 and 380 atoms, between 373 and 377 atoms, and 375 atoms.

[0026] In some cases, the linker is a branching linker that refers to a linking moiety that connects three or more groups. In certain cases, one, two, three, four or five or more carbon atoms of a linker backbone may be optionally substituted with a sulfur, nitrogen or oxygen heteroatom. In some cases, the linker backbone includes a linking functional group, such as an ether, thioether, amino, amide, sulfonamide, carbamate, thiocarbamate, urea, thiourea, ester, thioester or imine. The bonds between backbone atoms may be saturated or unsaturated, and in some cases not more than one, two, or three unsaturated bonds are present in a linker backbone. The linker may include one or more substituent groups, for example with an alkyl, aryl or alkenyl group. A linker may include, without limitations, polyethylene glycol; ethers, thioethers, tertiary amines, alkyls, which may be straight or branched, e.g., methyl, ethyl, n-propyl, 1 -methylethyl (isopropyl), n-butyl, n-pentyl, 1 ,1 -dimethylethyl (t-butyl), and the like. The linker backbone may include a cyclic group, for example, an aryl, a heterocycle or a cycloalkyl group, where 2 or more atoms, e.g., 2, 3 or 4 atoms, of the cyclic group are included in the backbone. A linker may be cleavable or non-cleavable.

[0027] As used herein, the terms “chemoselective functional group” and “chemoselective tag” are used interchangeably and refer to a functional group that can selectively react with another compatible functional group to form a covalent bond, in some cases, after optional activation of one of the functional groups. Chemoselective functional groups of interest include, but are not limited to, thiols and maleimide or iodoacetamide, amines and carboxylic acids or active esters thereof, as well as groups that can react with one another via Click chemistry, e.g., azide and alkyne groups (e.g., cyclooctyne groups), tetrazine, transcyclooctene, dienes and dienophiles, and azide, sulfur(VI) fluoride exchange chemistry (SuFEX), sulfonyl fluoride, as well as hydroxyl, hydrazido, hydrazino, aldehyde, ketone, azido, alkyne, phosphine, epoxide, and the like.

[0028] As used herein, the term “sample” relates to a material or mixture of materials, in some cases in liquid form, containing one or more analytes of interest. In some embodiments, the term as used in its broadest sense, refers to any plant, animal or bacterial material containing cells or producing cellular metabolites, such as, for example, tissue or fluid isolated from an individual (including without limitation plasma, serum, cerebrospinal fluid, lymph, tears, saliva and tissue sections) or from in vitro cell culture constituents, as well as samples from the environment. The term “sample” may also refer to a “biological sample”. As used herein, the term “a biological sample” refers to a whole organism or a subset of its tissues, cells or component parts (e.g., body fluids, including, but not limited to, blood, mucus, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluid and semen). A “biological sample” can also refer to a homogenate, lysate or extract prepared from a whole organism or a subset of its tissues, cells or component parts, or a fraction or portion thereof, including but not limited to, plasma, serum, spinal fluid, lymph fluid, the external sections of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, milk, blood cells, tumors and organs. In certain embodiments, the sample has been removed from an animal or plant. Biological samples may include cells. The term “cells” is used in its conventional sense to refer to the basic structural unit of living organisms, both eukaryotic and prokaryotic, having at least a nucleus and a cell membrane. In certain embodiments, cells include prokaryotic cells, such as from bacteria. In other embodiments, cells include eukaryotic cells, such as cells obtained from biological samples from animals, plants or fungi.

[0029] The terms “support bound” and “linked to a support” are used interchangeably and refer to a moiety (e.g., a specific binding member) that is linked covalently or non-covalently to a support of interest. Covalent linking may involve the chemical reaction of two compatible functional groups (e.g., two chemoselective functional groups, an electrophile and a nucleophile, etc.) to form a covalent bond between the two moieties of interest (e.g., a support and a specific binding member). In some cases, non-covalent linking may involve specific binding between two moieties of interest (e.g., two affinity moieties such as a hapten and an antibody or a biotin moiety and a streptavidin, etc.). In certain cases, non-covalent linking may involve absorption to a substrate.

[0030] The term “polypeptide” refers to a polymeric form of amino acids of any length, including peptides that range from 2-50 amino acids in length and polypeptides that are greater than 50 amino acids in length. The terms “polypeptide” and “protein” are used interchangeably herein. The term “polypeptide” includes polymers of coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones in which the conventional backbone has been replaced with non-naturally occurring or synthetic backbones. A polypeptide may be of any convenient length, e.g., 2 or more amino acids, such as 4 or more amino acids, 10 or more amino acids, 20 or more amino acids, 50 or more amino acids, 100 or more amino acids, 300 or more amino acids, such as up to 500 or 1000 or more amino acids. “Peptides” may be 2 or more amino acids, such as 4 or more amino acids, 10 or more amino acids, 20 or more amino acids, such as up to 50 amino acids. In some embodiments, peptides are between 5 and 30 amino acids in length.

[0031] As used herein the term “isolated,” refers to a moiety of interest that is at least 60% free, at least 75% free, at least 90% free, at least 95% free, at least 98% free, and even at least 99% free from other components with which the moiety is associated with prior to purification.

[0032] A “plurality” contains at least 2 members. In certain cases, a plurality may have 5 or more, such as 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, 100 or more, 300 or more, 1000 or more, 3000 or more, 10,000 or more, 100,000 or more members. Numeric ranges are inclusive of the numbers defining the range.

[0033] The methods described herein may include multiple steps. Each step may be performed after a predetermined amount of time has elapsed between steps, as desired. As such, the time between performing each step may be 1 second or more, 10 seconds or more, 30 seconds or more, 60 seconds or more, 5 minutes or more, 10 minutes or more, 60 minutes or more and including 5 hours or more. In certain embodiments, each subsequent step is performed immediately after completion of the previous step. In other embodiments, a step may be performed after an incubation or waiting time after completion of the previous step, e.g., a few minutes to an overnight waiting time.

[0034] As used herein, the terms “evaluating”, “determining,” “measuring,” and “assessing," and “assaying” are used interchangeably and include both quantitative and qualitative determinations. The term “separating”, as used herein, refers to physical separation of two elements (e.g., by size or affinity, etc.) as well as degradation of one element, leaving the other intact.

[0035] As used herein, the terms “water solubilizing group”, “water soluble group” and WSG are used interchangeably and refer to a group or substituent that is well solvated in aqueous environments e.g., under physiological conditions, and which imparts improved water solubility upon the molecule to which it is attached. A WSG can increase the solubility of a tandem dye or component thereof, e.g., donor or acceptor fluorophore, in a predominantly aqueous solution, as compared to a control tandem dye or component thereof which lacks the WSG. The water solubilizing groups may be any convenient hydrophilic group that is well solvated in aqueous environments.

[0036] A variety of water soluble polymer groups can be adapted for use in the WSG of the subject dyes. Any convenient water solubilizing groups (WSGs) may be included in the dyes described herein to provide for increased water-solubility. While the increase in solubility may vary, in some instances the increase (as compared to the compound without the WSG(s)) is 2 fold or more, e.g., 5 fold, 10 fold, 25 fold, 50 fold, 100 fold or more. In some cases, the hydrophilic water solubilizing group is charged, e.g., positively or negatively charged. In certain cases, the hydrophilic water solubilizing group is a neutral hydrophilic group. In some embodiments, the WSG is branched (e.g., as described herein). In certain instances, the WSG is linear. In some embodiments, the WSG is a hydrophilic polymer, e.g., a polyethylene glycol, a modified PEG, a peptide sequence, a peptoid, a carbohydrate, an oxazoline, a polyol, a dendron, a dendritic polyglycerol, a cellulose, a chitosan, or a derivative thereof. Water solubilizing groups of interest include, but are not limited to, carboxylate, phosphonate, phosphate, sulfonate, sulfate, sulfinate, sulfonium, ester, polyethylene glycols (PEG) and modified PEGs, hydroxyl, amine, amino acid, ammonium, guanidinium, pyridinium, polyamine and sulfonium, polyalcohols, straight chain or cyclic saccharides, primary, secondary, tertiary, or quaternary amines and polyamines, phosphonate groups, phosphinate groups, ascorbate groups, glycols, including, polyethers, -COOM', -SO3M', -PO3M', -NRs+, Y', (CH2CH2O)PR and mixtures thereof, where Y' can be any halogen, sulfate, sulfonate, or oxygen containing anion, p can be 1 to 500, each R can be independently H or an alkyl (such as methyl) and M' can be a cationic counterion or hydrogen, -(CH2CH2O)yyCH2CH2XRyy, -(CH2CH2O)yyCH2CH2X-, - X(CH2CH2O)yyCH2CH2--, glycol, and polyethylene glycol, wherein yy is selected from 1 to 1000, X is selected from O, S, and NRZZ, and Rzzand RYYare independently selected from H and C1-3 alkyl. In some cases, a WSG is (CH2)x(OCH2CH2)yOCH3where each x is independently an integer from 0-20, each y is independently an integer from 0 to 50. In some cases, the water solubilizing group includes a non-ionic polymer (e.g., a PEG polymer) substituted at the terminal with an ionic group (e.g., a sulfonate).

[0037] In some embodiments of the formulae, the pendant group of interest includes a substituent selected from (CH2)x(OCH2CH2)yOCH3 where each x is independently an integer from 0-20, each y is independently an integer from 0 to 50; and a benzyl optionally substituted with one or more halogen, hydroxyl, C1- C12 alkoxy, or (OCH2CH2)zOCH3 where each z is independently an integer from 0 to 50. In some instances, the substituent is (CH2)3(OCH2CH2)nOCH3. In some embodiments, one or more of the substituents is a benzyl substituted with at least one WSG groups (e.g., one or two WSG groups) selected from (CH2)x(OCH2CH2)yOCH3 where each x is independently an integer from 0-20 and each y is independently an integer from 0 to 50.

[0038] Multiple WSGs may be included at a single location in the subject dyes via a branching linker. In certain embodiments, the branching linker is an aralkyl substituent, further di-substituted with water solubilizing groups. As such, in some cases, the branching linker group is a substituent of the dye that connects the dye to two or more water solubilizing groups. In certain embodiments, the branching linker is an amino acid, e.g., a lysine amino acid that is connected to three groups via the amino and carboxylic acid groups. In some cases, the incorporation of multiple WSGs via branching linkers imparts a desirable solubility on the dye. In some instances, the WSG is a non-ionic sidechain group capable of imparting solubility in water in excess of 50 mg / mL. In some instances, the WSG is a non-ionic sidechain group capable of imparting solubility in water in excess of 100 mg / mL. In some embodiments, the dye includes substituent(s) selected from the group consisting of, an alkyl, an aralkyl and a heterocyclic group, each group further substituted with a include water solubilizing groups hydrophilic polymer group, such as a polyethylene glycol (PEG) (e.g., a PEG group of 6-24 units).

[0039] Water soluble polymers of interest that can be utilized in the WSG include polyethylene glycol (PEG) groups or modified PEG groups. Water-soluble polymers of interest include, but are not limited to, polyalkylene oxide based polymers, such as polyethylene glycol “PEG” (See. e.g., “Polyethylene glycol) Chemistry: Biotechnical and Biomedical Applications”, J. M. Harris, Ed., Plenum Press, New York, N.Y. (1992); and “Poly(ethylene glycol) Chemistry and Biological Applications”, J. M. Harris and S. Zalipsky, Eds., ACS (1997); and International Patent Applications: WO 90 / 13540, WO 92 / 00748, WO 92 / 16555, WO 94 / 04193, WO 94 / 14758, WO 94 / 17039, WO 94 / 18247, WO 94 / 28937, WO 95 / 11924, WO 96 / 00080, WO 96 / 23794, WO 98 / 07713, WO 98 / 41562, WO

[0040] 98 / 48837, WO 99 / 30727, WO 99 / 32134, WO 99 / 33483, WO 99 / 53951 , WO

[0041] 01 / 26692, WO 95 / 13312, WO 96 / 21469, WO 97 / 03106, WO 99 / 45964, and U.S.

[0042] Pat. Nos. 4,179,337; 5,075,046; 5,089,261 ; 5,100,992; 5,134,192; 5,166,309; 5,171 ,264; 5,213,891 ; 5,219,564; 5,275,838; 5,281 ,698; 5,298,643; 5,312,808; 5,321 ,095; 5,324,844; 5,349,001 ; 5,352,756; 5,405,877; 5,455,027; 5,446,090; 5,470,829; 5,478,805; 5,567,422; 5,605,976; 5,612,460; 5,614,549; 5,618,528; 5,672,662; 5,637,749; 5,643,575; 5,650,388; 5,681 ,567; 5,686,1 10; 5,730,990; 5,739,208; 5,756,593; 5,808,096; 5,824,778; 5,824,784; 5,840,900; 5,874,500; 5,880,131 ; 5,900,461 ; 5,902,588; 5,919,442; 5,919,455; 5,932,462; 5,965,119; 5,965,566; 5,985,263; 5,990,237; 6,011 ,042; 6,013,283; 6,077,939; 6,113,906; 6,127,355; 6,177,087; 6,180,095; 6,194,580; 6,214,966).

[0043] Examples of water soluble polymers of interest include, but are not limited to, those containing a polyalkylene oxide, polyamide alkylene oxide, or derivatives thereof, including polyalkylene oxide and polyamide alkylene oxide comprising an ethylene oxide repeat unit of the formula -(CH2-CH2-O)-. Further examples of polymers of interest include a polyamide having a molecular weight greater than 1 ,000 Daltons of the formula -[C(O)-X-C(O)-NH-Y-NH]n- or -[NH-Y- NH-C(O)-X-C(O)]n-, where X and Y are divalent radicals that may be the same or different and may be branched or linear, and n is a discrete integer from 2-100, such as from 2 to 50, and where either or both of X and Y comprises a biocompatible, substantially non-antigenic water-soluble repeat unit that may be linear or branched. Further examples of water-soluble repeat units comprise an ethylene oxide of the formula -(CH2-CH2-O)- or -(O-CH2-CH2)- . The number of such water-soluble repeat units can vary significantly, with the number of such units being from 2 to 500, 2 to 400, 2 to 300, 2 to 200, 2 to 100, 6-100, for example from 2 to 50 or 6 to 50. An example of an embodiment is one in which one or both of X and Y is selected from: -((CH2)ni-(CH2-CH2-O)n2-(CH2)- or - ((CH2)ni-(O-CH2-CH2)n2-(CH2)ni-), where n1 is 1 to 6, 1 to 5, 1 to 4, or 1 to 3, and where n2 is 2 to 50, 2 to 25, 2 to 15, 2 to 10, 2 to 8, or 2 to 5. A further example of an embodiment is one in which X is -(CH2-CH2)-, and where Y is -(CH2-(CH2- CH2-O)3-CH2-CH2-CH2)- or -(CH2-CH2-CH2-(O-CH2-CH2)3-CH2)-.

[0044] The term modified polymer, such as a modified PEG, refers to water soluble polymers that have been modified or derivatized at either or both terminals, e.g., to include a terminal substituent (e.g., a terminal alkyl, substituted alkyl, alkoxy or substituted alkoxy, etc.) and / or a terminal linking functional group (e.g., an amino or carboxylic acid group suitable for attachment via amide bond formation) suitable for attachment of the polymer to a molecule of interest. These water soluble polymers can be adapted to include any convenient linking groups. It is understood that in some cases, the water soluble polymer can include some dispersity with respect to polymer length, depending on the method of preparation and / or purification of the polymeric starting materials. In some instances, the water soluble polymers are monodisperse.

[0045] The water soluble polymer can include one or more spacers or linkers. Examples of spacers or linkers include linear or branched moieties comprising one or more repeat units employed in a water-soluble polymer, diamino and or diacid units, natural or unnatural amino acids or derivatives thereof, as well as aliphatic moieties, including alkyl, aryl, heteroalkyl, heteroaryl, alkoxy, and the like, which can contain, for example, up to 18 carbon atoms or even an additional polymer chain.

[0046] The water soluble polymer moiety, or one or more of the spacers or linkers of the polymer moiety when present, may include polymer chains or units that are biostable or biodegradable. For example, polymers with repeat linkages have varying degrees of stability under physiological conditions depending on bond lability. Polymers with such bonds can be categorized by their relative rates of hydrolysis under physiological conditions based on known hydrolysis rates of low molecular weight analogs, e.g., from less stable to more stable, e.g., polyurethanes (-NH-C(O)-O-) > polyorthoesters (-O-C((OR)(R’))-O-) > polyamides (-C(O)-NH-). Similarly, the linkage systems attaching a water-soluble polymer to a molecule may be biostable or biodegradable, e.g., from less stable to more stable: carbonate (-O-C(O)-O-) > ester (-C(O)-O-) > urethane (-NH-C(O)- O-) > orthoester (-O-C((OR)(R’))-O-) > amide (-C(O)-NH-). In general, it may be desirable to avoid use of a sulfated polysaccharide, depending on the lability of the sulfate group. In addition, it may be less desirable to use polycarbonates and polyesters. These bonds are provided by way of example, and are not intended to limit the types of bonds employable in the polymer chains or linkage systems of the water-soluble polymers useful in the WSGs disclosed herein.

[0047] The water soluble group (WSG) can be capable of imparting solubility in water in excess of 10 mg / mL to the donor fluorophore or acceptor fluorophore, such as in excess of 20 mg / mL, in excess of 30 mg / mL, in excess of 40 mg / mL, in excess of 50 mg / mL, in excess of 60 mg / mL, in excess of 70 mg / mL, in excess of 80 mg / mL, in excess of 90 mg / mL or in excess of 100 mg / mL. In certain cases, the branched non-ionic water soluble group (WSG) is capable of imparting solubility in water (e.g., an aqueous buffer) of 20 mg / mL or more to the donor fluorophore or acceptor fluorophore, such as 30 mg / mL or more, 40 mg / mL or more, 50 mg / mL or more, 60 mg / mL or more, 70 mg / mL or more, 80 mg / mL or more, 90 mg / mL or more, 100 mg / mL or more, or even more. The terms “polyethylene oxide”, “PEO”, "polyethylene glycol” and “PEG” are used interchangeably and refer to a polymeric group including a chain described by the formula -(CH2-O--)n- or a derivative thereof. In some embodiments, "n" is 5000 or less, such as 1000 or less, 500 or less, 200 or less, 100 or less, 50 or less, 40 or less, 30 or less, 20 or less, 15 or less, such as 3 to 15, or 10 to 15. It is understood that the PEG polymeric group may be of any convenient length and may include a variety of terminal groups and / or further substituent groups, including but not limited to, alkyl, aryl, hydroxyl, amino, acyl, acyloxy, and amido terminal and / or substituent groups. PEG groups that may be adapted for use in the branched tandem dye scaffolds include those PEGs described by S. Zalipsky in “Functionalized polyethylene glycol) for preparation of biologically relevant conjugates”, Bioconjugate Chemistry 1995, 6 (2), 150- 165; and by Zhu et al in “Water-Soluble Conjugated Polymers for Imaging, Diagnosis, and Therapy”, Chem. Rev., 2012, 112 (8), pp 4687-4735 .

[0048] The term “alkyl” by itself or as part of another substituent refers to a saturated branched or straight-chain monovalent hydrocarbon radical derived by the removal of one hydrogen atom from a single carbon atom of a parent alkane. Alkyl groups of interest include, but are not limited to, methyl; ethyl, propyls such as propan-1 -yl or propan-2-yl; and butyls such as butan-1 -yl, butan-2-yl, 2-methyl-propan-1 -yl or 2-methyl-propan-2-yl. In some embodiments, an alkyl group includes from 1 to 20 carbon atoms. In some embodiments, an alkyl group includes from 1 to 10 carbon atoms. In certain embodiments, a lower alkyl group includes from 1 to 6 carbon atoms, such as from 1 to 4 carbon atoms. This term includes, by way of example, linear and branched hydrocarbyl groups such as methyl (CH3-), ethyl (CH3CH2-), n-propyl (CH3CH2CH2-), isopropyl ((CH3)2CH- ), n-butyl (CH3CH2CH2CH2-), isobutyl ((CH3)2CHCH2-), sec-butyl ((CH3)(CH3CH2)CH-), t-butyl ((CH3)3C-), n-pentyl (CH3CH2CH2CH2CH2-), and neopentyl ((CH3)3CCH2-).

[0049] The term “substituted alkyl” refers to an alkyl group as defined herein substituted with from 1 to 5 substituents selected from the group consisting of alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO- aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-aryl, -S02-heteroaryl, and -NRaRb, wherein R’ and R” may be the same or different and are chosen from hydrogen, optionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl and heterocyclic.

[0050] “Alkoxy” refers to the group -O-alkyl, wherein alkyl is as defined herein. Alkoxy includes, by way of example, methoxy, ethoxy, n-propoxy, isopropoxy, n- butoxy, t-butoxy, sec-butoxy, n-pentoxy, and the like. The term “alkoxy” also refers to the groups alkenyl-O-, cycloalkyl-O-, cycloalkenyl-O-, and alkynyl-O-, where alkenyl, cycloalkyl, cycloalkenyl, and alkynyl are as defined herein.

[0051] The term “substituted alkoxy” refers to the groups substituted alkyl-O-, substituted alkenyl-O-, substituted cycloalkyl-O-, substituted cycloalkenyl-O-, and substituted alkynyl-O- where substituted alkyl, substituted alkenyl, substituted cycloalkyl, substituted cycloalkenyl and substituted alkynyl are as defined herein.

[0052] “Alkenyl" refers to a monoradical, branched or linear, cyclic or non-cyclic hydrocarbonyl group that comprises a carbon-carbon double bond. Exemplary alkenyl groups include ethenyl, n-propenyl, isopropenyl, n-butenyl, isobutenyl, octenyl, decenyl, tetradecenyl, hexadecenyl, eicosenyl, and tetracosenyl. In some cases the alkenyl group comprises 1 to 24 carbon atoms, such as 1 to 18 carbon atoms or 1 to 12 carbon atoms. The term "lower alkenyl" refers to an alkyl groups with 1 to 6 carbon atoms.

[0053] “Alkynyl” or “alkyne” refers to straight or branched monovalent hydrocarbyl groups having from 2 to 6 carbon atoms and preferably 2 to 3 carbon atoms and having at least 1 and preferably from 1 to 2 sites of triple bond unsaturation. Examples of such alkynyl groups include acetylenyl (-C=CH), and propargyl (-CH2C=CH). The term “substituted alkynyl” or “substituted alkyne” refers to an alkynyl group as defined herein having from 1 to 5 substituents, or from 1 to 3 substituents, selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, - SO-heteroaryl, -SO2-alkyl, -SC -substituted alkyl, -SO2-aryl, and -SC -heteroaryL

[0054] “Heterocyclyl” refers to a monoradical, cyclic group that contains a heteroatom (e.g. O, S, N) in as a ring atom and that is not aromatic (i.e. distinguishing heterocyclyl groups from heteroaryl groups). Exemplary heterocyclyl groups include piperidinyl, tetrahydrofuranyl, dihydrofuranyl, and thiocanyl.

[0055] “Amino” refers to the group -NH2. The term “substituted amino” refers to the group -NRR where each R is independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, cycloalkenyl, substituted cycloalkenyl, alkynyl, substituted alkynyl, aryl, heteroaryl, and heterocyclyl provided that at least one R is not hydrogen.

[0056] "Aryl" by itself or as part of another substituent refers to a monovalent aromatic hydrocarbon radical derived by the removal of one hydrogen atom from a single carbon atom of an aromatic ring system. Aryl groups of interest include, but are not limited to, groups derived from aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene, coronene, fluoranthene, fluorene, hexacene, hexaphene, hexalene, as-indacene, s- indacene, indane, indene, naphthalene, octacene, octaphene, octalene, ovalene, penta-2,4-diene, pentacene, pentalene, pentaphene, perylene, phenalene, phenanthrene, picene, pleiadene, pyrene, pyranthrene, rubicene, triphenylene, trinaphthalene and the like. In certain embodiments, an aryl group includes from 6 to 20 carbon atoms. In certain embodiments, an aryl group includes from 6 to 12 carbon atoms. Examples of an aryl group are phenyl and naphthyl.

[0057] “Substituted aryl”, unless otherwise constrained by the definition for the aryl substituent, refers to an aryl group substituted with from 1 to 5 substituents, or from 1 to 3 substituents, selected from acyloxy, hydroxy, thiol, acyl, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl, substituted alkoxy, substituted alkenyl, substituted alkynyl, substituted cycloalkyl, substituted cycloalkenyl, amino, substituted amino, aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl, carboxylalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, -SO-alkyl, -SO- substituted alkyl, -SO-aryl, -SO-heteroaryl, -SC -alkyl, -SC -substituted alkyl, - SO2-aryl, -SO2-heteroaryl and trihalomethyl.

[0058] "Heteroaryl" by itself or as part of another substituent, refers to a monovalent heteroaromatic radical derived by the removal of one hydrogen atom from a single atom of a heteroaromatic ring system. Heteroaryl groups of interest include, but are not limited to, groups derived from acridine, arsindole, carbazole, [3-carboline, chromane, chromene, cinnoline, furan, imidazole, indazole, indole, indoline, indolizine, isobenzofuran, isochromene, isoindole, isoindoline, isoquinoline, isothiazole, isoxazole, naphthyridine, oxadiazole, oxazole, perimidine, phenanthridine, phenanthroline, phenazine, phthalazine, pteridine, purine, pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pyrrole, pyrrolizine, quinazoline, quinoline, quinolizine, quinoxaline, tetrazole, thiadiazole, thiazole, triazole, benzotriazole, thiophene, triazole, xanthene, benzodioxole and the like. In certain embodiments, the heteroaryl group is from 5-20 membered heteroaryl. In certain embodiments, the heteroaryl group is from 5-10 membered heteroaryl. In certain embodiments, heteroaryl groups are those derived from thiophene, pyrrole, benzothiophene, benzofuran, indole, pyridine, quinoline, imidazole, oxazole and pyrazine. “Heterocycle,” “heterocyclic,” “heterocycloalkyl,” and “heterocyclyl” refer to a saturated or unsaturated group having a single ring or multiple condensed rings, including fused bridged and spiro ring systems, and having from 3 to 20 ring atoms, including 1 to 10 hetero atoms. These ring atoms are selected from the group consisting of nitrogen, sulfur, or oxygen, wherein, in fused ring systems, one or more of the rings can be cycloalkyl, aryl, or heteroaryl, provided that the point of attachment is through the non-aromatic ring. In certain embodiments, the nitrogen and / or sulfur atom(s) of the heterocyclic group are optionally oxidized to provide for the N-oxide, -S(O)-, or -SO2- moieties.

[0059] Examples of heterocycles and heteroaryls include, but are not limited to, azetidine, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, dihydroindole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine, imidazolidine, imidazoline, piperidine, piperazine, indoline, phthalimide, 1 , 2,3,4- tetrahydroisoquinoline, 4,5,6,7-tetrahydrobenzo[b]thiophene, thiazole, thiazolidine, thiophene, benzo[b]thiophene, morpholinyl, thiomorpholinyl (also referred to as thiamorpholinyl), 1 ,1 -dioxothiomorpholinyl, piperidinyl, pyrrolidine, tetrahydrofuranyl, and the like.

[0060] “Substituted heteroaryl”, unless otherwise constrained by the definition for the substituent, refers to an heteroaryl group substituted with from 1 to 5 substituents, or from 1 to 3 substituents, selected from acyloxy, hydroxy, thiol, acyl, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl, substituted alkoxy, substituted alkenyl, substituted alkynyl, substituted cycloalkyl, substituted cycloalkenyl, amino, substituted amino, aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl, carboxylalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, - SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2- substituted alkyl, -SO2-aryl, -SO2-heteroaryl and trihalomethyl. The term “alkaryl” or “aralkyl” refers to the groups -alkylene-aryl and substituted alkylene-aryl where alkylene, substituted alkylene and aryl are defined herein.

[0061] “Alkylene” refers to divalent aliphatic hydrocarbyl groups preferably having from 1 to 6 and more preferably 1 to 3 carbon atoms that are either straight- chained or branched, and which are optionally interrupted with one or more groups selected from -O-, -NR10-, -NR10C(O)-, -C(O)NR10- and the like. This term includes, by way of example, methylene (-CH2-), ethylene (-CH2CH2-), n- propylene (-CH2CH2CH2-), iso-propylene (-CH2CH(CH3)-), (-C(CH3)2CH2CH2-), (-C(CH3)2CH2C(O)-), (-C(CH3)2CH2C(O)NH-), (-CH(CH3)CH2-), and the like. “Substituted alkylene” refers to an alkylene group having from 1 to 3 hydrogens replaced with substituents as described for carbons in the definition of “substituted” below.

[0062] “Substituted” refers to a group in which one or more hydrogen atoms are independently replaced with the same or different substituent(s). Substituents of interest include, but are not limited to, alkylenedioxy (such as methylenedioxy), halogen, -R60, -O’, -OH, =0, -OR60, -SR60, -S , -SH, =S, -NR60R61, =NR60, -CF3, -NR62C(NR63)NR60R61, and -C(NR62)NR60R61, wherein R60, R61, R62, and R63are independently hydrogen, alkyl, substituted alkyl, alkoxy, substitued alkoxy, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, aryl, substituted aryl, heteroaryl or substituted heteroaryl, or optionally R60and R61together with the nitrogen atom to which they are bonded form a cycloheteroalkyl or substituted cycloheteroalkyl ring; and R64and R65are independently hydrogen, alkyl, substituted alkyl, aryl, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, aryl, substituted aryl, heteroaryl or substituted heteroaryl, or optionally R64and R65together with the nitrogen atom to which they are bonded form a cycloheteroalkyl or substituted cycloheteroalkyl ring. In certain embodiments, substituents include halogen, - R60, =0, -OR60, -SR60, =S, -NR60R61, =NR60, -CF3, -CN, -OCN, -SON, -NO, -NO2, =N2, -N3, -S(O)2R60, -0S(0)20-, -OS(O)2OH, -OS(O)2R60, -P(O)(Oj2, - P(O)(OR60)(O ), -OP(O)(Oj2, -OP(O)(OR60)(OH), -OP(O)(OR60)(OR61), -C(O)R60, -C(S)R60, -C(0)0-, -C(O)OH, -C(O)OR60, -C(O)NR60R61, -NR62C(O)NR60R61. In certain embodiments, substituents include halogen, -R60, =0, -OR60, -SR60, - NR60R61, -CF3, -CN, -N02, -S(O)2R60, -P(O)(OR60)(Oj, -OP(O)(OR60)(OR61), - C(O)R60, -C(O)OR60, and -C(0)0_. In certain embodiments, substituents include halogen, -R60, =0, -OR60, -SR60, -NR60R61, -CF3, -CN, -NO2, -S(O)2R60, - OP(O)(OR60)(OR61), -C(O)R60, -C(O)OR60, and -C(O)Q-. For example, a substituted group may bear a methylenedioxy substituent or one, two, or three substituents selected from a halogen atom, a (Ci-4)alkyl group and a (Ci-4)alkoxy group. When the group being substituted is an aryl or heteroaryl group, the substituent(s) (e.g., as described herein) may be referred to as “aryl substituent(s)”.

[0063] “Ester” refers to a group of formula -C(O)(OH).

[0064] “Substituted ester” refers to a group of formula -C(O)(O)(R’), wherein R’ is selected from the group consisting of alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocylyl, substituted heterocyclyl, cycloalkyl, and substituted cycloalkyl.

[0065] It is understood that in all substituted groups defined above, polymers arrived at by defining substituents with further substituents to themselves (e.g., substituted aryl having a substituted aryl group as a substituent which is itself substituted with a substituted aryl group, which is further substituted by a substituted aryl group, etc.) are not intended for inclusion herein. In such cases, the maximum number of such substitutions is three. For example, serial substitutions of substituted aryl groups specifically contemplated herein are limited to substituted aryl-(substituted aryl)-substituted aryl. “Acyl” refers to a group of formula -C(O)R wherein R is alkyl, alkenyl, or alkynyl. For example, the acetyl group has formula -C(0)CH3.

[0066] “Halo” and “halogen” refer to the chloro, bromo, fluoro, and iodo groups.

[0067] “Carboxyl”, “carboxy”, and “carboxylate” refer to the -CO2H group and salts thereof.

[0068] “Imine” refers to a group of =NRX, wherein Rxis any substituent, e.g., alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, substituted heterocyclyl.

[0069] “Sulfonyl” refers to the group -SO2R, wherein R is alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, and substituted versions thereof. Exemplary sulfonyl groups includes -SO2CH3 and -SC^CeHs).

[0070] Unless otherwise specified, reference to an atom is meant to include all isotopes of that atom. For example, reference to H is meant to include1H,2H (i.e., D) and3H (i.e., T), and reference to C is meant to include12C and all isotopes of carbon (such as13C). In addition, any groups described include all stereoisomers of that group.

[0071] Unless indicated otherwise, the nomenclature of substituents that are not explicitly defined herein are arrived at by naming the terminal portion of the functionality followed by the adjacent functionality toward the point of attachment. For example, the substituent “arylalkyloxycarbonyl” refers to the group (aryl)- (alkyl)-O-C(O)-.

[0072] As to any of the groups disclosed herein which contain one or more substituents, it is understood, of course, that such groups do not contain any substitution or substitution patterns which are sterically impractical and / or synthetically non-feasible. In addition, the subject compounds include all stereochemical isomers arising from the substitution of these compounds. DETAILED DESCRIPTION

[0073] Branched tandem dye scaffolds are provided. Embodiments of the invention include: a non-conjugated backbone comprising a branch comprising two or more donor fluorophores; and an acceptor fluorophore(s) linked to the non-conjugated backbone; wherein the acceptor fluorophore(s) and the donor fluorophores are an energy transfer relationship. Also provided are methods of making and using the branched tandem dye scaffolds, as well as kits that include the branched tandem dye scaffolds and find use in embodiments of the methods.

[0074] Before describing exemplary embodiments in greater detail, the following definitions are set forth to illustrate and define the meaning and scope of the terms used in the description.

[0075] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Still, certain terms are defined below for the sake of clarity and ease of reference. Further, although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, representative illustrative methods and materials are described herein.

[0076] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention. Certain ranges are presented herein with numerical values being preceded by the term "about." The term "about" is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number.

[0077] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

[0078] It is noted that, as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.

[0079] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible. While the apparatus and method has or will be described for the sake of grammatical fluidity with functional explanations, it is to be expressly understood that the claims, unless expressly formulated under 35 U.S.C. §1 12, are not to be construed as necessarily limited in any way by the construction of "means" or "steps" limitations, but are to be accorded the full scope of the meaning and equivalents of the definition provided by the claims under the judicial doctrine of equivalents, and in the case where the claims are expressly formulated under 35 U.S.C. §1 12 are to be accorded full statutory equivalents under 35 U.S.C. §112.

[0080] In further describing various embodiments of the invention, branched tandem dye scaffolds are reviewed first in greater detail, followed by a review of methods of using and making the scaffolds, as well as a review of kits and systems that include the scaffolds.

[0081] Compositions

[0082] Branched tandem dye scaffolds

[0083] As summarized above, branched tandem dye scaffolds of embodiments of the invention include: a non-conjugated backbone comprising a branch comprising two or more donor fluorophores; and an acceptor fluorophore(s) linked to the non-conjugated backbone; wherein the acceptor fluorophore(s) and the donor fluorophores are in energy transfer relationship. Each of these components is now reviewed in greater detail.

[0084] In certain instances, the non-conjugated backbone comprises a branch comprising two or more donor fluorophores. In certain instances, the nonconjugated backbone comprises a branch comprising two, three, or four donor fluorophores. In certain instances, the non-conjugated backbone comprises a branch comprising five, six, or seven donor fluorophores. In certain instances, the non-conjugated backbone comprises a branch comprising eight, nine, or ten donor fluorophores. In some instances, the non-conjugated backbone is made up of non-conjugated repeat units wherein at least one comprises a branch comprising two or more donor fluorophores. The configuration of donor fluorophores and / or acceptor fluorophore(s) can be installed during or after synthesis of the non-conjugated backbone. The incorporation of donor fluorophores and / or acceptor fluorophore(s) can be achieved with a random configuration, a block configuration, or in a sequence-specific manner via stepwise synthesis, depending on the particular method of synthesis utilized.

[0085] The backbone of the branched tandem dye scaffold may have any convenient length. In some cases, the particular number of units of the branched tandem dye scaffold may fall within the range of 1 to 100, such as 2 to 100, such as 2 to 500, 2 to 100, 3 to 100, 4 to 100, 5 to 100, 6 to 100, 7 to 100, 8 to 100, 9 to 100, or 10 to 100 units. In some cases, the particular number of units of the branched tandem dye scaffold may fall within the range of 2 to 20, such as 2 to

[0086] 15, 2 to 14, 2 to 13, 2 to 12, 2 to 1 1 , 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, or 2 to 3 units. In some cases, the particular number of units of the branched tandem dye scaffold may be 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15,

[0087] 16, 17, 18, 19, or 20 units. In some cases, the particular number of units of the branched tandem dye scaffold may be 2, 3, 4, or 5 units.

[0088] Where desired, the backbones present in tandem dye scaffolds of embodiments of the invention may be substituted with one or more water solubilizing groups (WSG), e.g., as defined above.

[0089] In certain instances, all of the donor fluorophores in the branched tandem dye scaffold are in an energy transfer relationship with the acceptor fluorophore(s) in the branched tandem dye scaffold. In certain instances, 30% or more of the energy harvested by each of the donor fluorophores is transferred to the acceptor fluorophore(s). In certain instances, between 30% and 100% of the energy harvested by each of the donor fluorophores in the branched tandem dye scaffold is transferred to the acceptor fluorophore(s) in the branched tandem dye scaffold.

[0090] In certain instances, the ratio of donor fluorophore to acceptor fluorophore is between 3:1 and 100:1 in the branched tandem dye scaffold. In certain instances, the ratio of donor fluorophore to acceptor fluorophore is between 4:1 and 16:1 in the branched tandem dye scaffold. In certain instances, the ratio of donor fluorophore to acceptor fluorophore is 12:1 in the branched tandem dye scaffold.

[0091] In certain instances, the donor fluorophores are arranged in a three- dimensional configuration relative to the acceptor fluorophore(s) in the branched tandem dye scaffold in an aqueous solution. In certain instances, the three- dimensional configuration in the branched tandem dye scaffold is selected from spherical, ellipsoidal, tetrahedral, pyramidal, cuboidal, cylindrical, tetrapodal, and stellate, or combinations thereof. In certain instances, the non-conjugated backbone comprises two or more branches comprising two or more donor fluorophores, and the branches are arranged in a three-dimensional configuration relative to the acceptor fluorophore in an aqueous solution. In certain instances, the two or more branches are arranged equidistantly from each other. In certain instances, the two or more branches are arranged symmetrically in the three- dimensional configuration relative to the acceptor fluorophore(s) in the branched tandem dye scaffold in an aqueous solution.

[0092] In certain instances, the donor fluorophores are between 3 Angstroms and 60 Angstroms from the acceptor fluorophore(s) in the branched tandem dye scaffold in an aqueous solution. In certain instances, the donor fluorophores are between 3 Angstroms and 30 Angstroms from the acceptor fluorophore(s) in the branched tandem dye scaffold in an aqueous solution.

[0093] In certain instances, the non-conjugated backbone is peptidic. In certain instances, the non-conjugated backbone comprises one or more amino acids. In certain instances, the non-conjugated backbone comprises an amino acid which comprises the branch comprising two or more donor fluorophores. In certain instances, the non-conjugated backbone comprises an amino acid through which the acceptor fluorophore is linked.

[0094] In certain instances, the non-conjugated backbone comprises an amidoamine which comprises the branch comprising two or more donor fluorophores. In certain instances, the non-conjugated backbone comprises a glycerol which comprises the branch comprising two or more donor fluorophores. In certain instances, the non-conjugated backbone comprises an ethylene glycol which comprises the branch comprising two or more donor fluorophores. In certain instances, the non-conjugated backbone comprises a polyethylene glycol).

[0095] The present disclosure provides branched tandem dye scaffolds that include donor fluorophores and acceptor fluorophore(s).

[0096] Tandem dyes are compounds having two different, covalently linked fluorophores, which fluorophores may be covalently linked to each other directly or through a linking group, e.g., a non-conjugated backbone. In the tandem dye, two or more of the fluorophores serve as donor fluorophores and at least one other fluorophore acts as acceptor fluorophore. The donor and acceptor fluorophores together form a fluorescence-resonance energy transfer (FRET) pair. Such FRET pairs behave as a unique dye that has the excitation properties of the donor fluorophore and the emission properties of the acceptor fluorophore.

[0097] Excitation of the donor can lead to energy transfer to, and emission from, the covalently attached acceptor fluorophore. Mechanisms for energy transfer between the donor fluorophores to a linked acceptor fluorophore include, for example, resonant energy transfer (e.g., Forster (or fluorescence) resonance energy transfer, FRET), quantum charge exchange (Dexter energy transfer) and the like. These energy transfer mechanisms can be relatively short range; that is, close proximity of donor fluorophores to each other and / or to an acceptor fluorophore provides for efficient energy transfer. Under conditions for efficient energy transfer, amplification of the emission from the acceptor fluorophore can occur where the emission from the acceptor fluorophore is more intense when the incident light (the "pump light") is at a wavelength which is absorbed by, and transferred from, the donor fluorophores than when the acceptor fluorophore is directly excited by the pump light. By “efficient” energy transfer is meant 10% or more, such as 20% or more or 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, of the energy harvested by the donor fluorophores is transferred to the acceptor. By “amplification” is meant that the signal from the acceptor fluorophore is 1 .5x or greater when excited by energy transfer from the donor fluorophores as compared to direct excitation of the acceptor fluorophore with incident light of an equivalent intensity. The signal may be measured using any convenient method. In some cases, the 1 .5x or greater signal refers to an intensity of emitted light. In certain cases, the 1 .5x or greater signal refers to an increased signal to noise ratio. In certain embodiments, the acceptor fluorophore emission is 1 .5 fold greater or more when excited by the donor fluorophores as compared to direct excitation of the acceptor fluorophore with incident light, such as 2-fold or greater, 3-fold or greater, 4-fold or greater, 5-fold or greater, 6-fold or greater, 8-fold or greater, 10- fold or greater, 20-fold or greater, 50-fold or greater, 100-fold or greater, or even greater as compared to direct excitation of the acceptor fluorophore with incident light.

[0098] In some instances, the branched tandem dye scaffold exhibits an effective Stokes shift ranging from 25 nm to 300 nm, such as from 50 nm to 250 nm or from 75 nm to 200 nm. In some cases the effective Stokes shift is 25 nm or more, such as 50 nm or more, 75 nm or more, 100 nm or more, such as 110 nm or more, 120 nm or more, 130 nm or more, 140 nm or more, 150 nm or more, 160 nm or more, 170 nm or more, 180 nm or more, 190 nm or more, 200 nm or more, 250 nm or more when the branched tandem dye scaffold is directly excited with incident light.

[0099] The emission of the branched tandem dye scaffold can have a quantum yield of 0.03 or more, such as a quantum yield of 0.04 or more, 0.05 or more, 0.06 or more, 0.07 or more, 0.08 or more, 0.09 or more, 0.1 or more, 0.15 or more, 0.2 or more, 0.3 or more or even more. In some instances, the branched tandem dye scaffold has an extinction coefficient of 5 x 105cnr1M_1or more, such as 6 x 105cnr1M-1or more, 7 x 105cnr1M-1or more, 8 x 105cnr1M-1or more, 9 x 105cnr1M-1or more, such as 1 x 106cm_1M'1or more, 1.5 x 106cm-1M1or more, 2 x 106cm1M1or more, 2.5 x 106cm1M1or more, 3 x 106cm1M1or more, 4 x 106cm-1M-1or more, 5 x 106cm-1M-1or more, 6 x 106cm-1M-1or more, 7 x 106cm-1l\ / l“1or more, or 8 x 106cm-1M-1or more. In some embodiments, the branched tandem dye scaffold has a molar extinction coefficient of 5 x 105h / Hcirr1or more. In certain embodiments, the tandem dye has a molar extinction coefficient of 1 x 106M‘1cm1or more.

[0100] In some embodiments, the donor fluorophores can be independently selected from the group consisting of cyanine dyes, rhodamine dyes, xanthene dyes, coumarin dyes, polymethines, pyrenes, dipyrromethene borondifluorides, napthalimides, thiazine dyes, and acridine dyes. In some cases, the acceptor fluorophore(s) can be selected from the group consisting of cyanine dyes, rhodamine dyes, xanthene dyes, coumarin dyes, polymethines, pyrenes, dipyrromethene borondifluorides, napthalimides, thiazine dyes, and acridine dyes.

[0101] Molecules that may be employed as donor fluorophores or acceptor fluorphore(s) include, but are not limited to, fluorescein, 6-FAM, rhodamine, Texas Red, tetramethylrhodamine, carboxyrhodamine, carboxyrhodamine 6G, carboxyrhodol, carboxyrhodamine 1 10, Cascade Blue, Cascade Yellow, coumarin, Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy-Chrome, phycoerythrin, PerCP (peridinin chlorophyll-a Protein), PerCP-Cy5.5, JOE (6-carboxy-4',5'-dichloro- 2',7'-dimethoxyfluorescein), NED, ROX (5-(and-6)-carboxy-X-rhodamine), HEX, Lucifer Yellow, Marina Blue, Oregon Green 488, Oregon Green 500, Oregon Green 514, Alexa Fluor 350, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, 7-amino-4- methylcoumarin-3-acetic acid, BODIPY FL, BODIPY FL-Br.sub.2, BODIPY 530 / 550, BODIPY 558 / 568, BODIPY 564 / 570, BODIPY 576 / 589, BODIPY 581 / 591 , BODIPY 630 / 650, BODIPY 650 / 665, BODIPY R6G, BODIPY TMR, BODIPY TR, Dyonomics dyes (e.g. DY 431 , DY 485XL, DY 500XL, DY 610, DY 640, DY 654, DY 682, DY 700, DY 701 , DY 704, DY 730, DY 731 , DY 732, DY 734, DY 752, DY 778, DY 782, DY 800, DY 831 ), dipyrromethene borondifluoride (BODIPY), Biotium CF 555, diethylamino coumarin, and derivatives thereof. In certain embodiments, the branched tandem dye scaffold comprises formula (la): wherein * and ** each optionally represent a point of attachment with the branched tandem dye scaffold; wherein at least one of * and ** is present; each M1and M2is an independently selected non-conjugated backbone portion of the unit; each S1and S2is an independently selected optional spacer nonconjugated backbone portion of the unit; each X1is a linker; f is an integer between 1 and 16; g is 0 or 1 ; each D is an independently selected donor fluorophore linked to X1; each X2is a linker; each A is an independently selected acceptor fluorophore linked to X2; x is an integer between 1 to 20; y is an integer between 1 to 5; the ratio of x:y is between 1 :1 to 20:1 ; and wherein at least one X1-(D)t is the branch comprising two or more fluorophores.

[0102] In certain embodiments, the branched tandem dye scaffold comprises formula (lb): wherein G1and G2are each independently selected from a terminal unit or a linker; each M1and M2is an independently selected non-conjugated backbone portion of the unit; each S1and S2is an independently selected optional spacer non-conjugated backbone portion of the unit; each X1is a linker; f is an integer between 1 and 16; g is 0 or 1 ; each D is an independently selected donor fluorophore linked to X1; each X2is a linker; each A is an independently selected acceptor fluorophore linked to X2; x is an integer between 1 to 20; y is an integer between 1 to 5; the ratio of x:y is between 1 :1 to 20:1 ; and wherein at least one X1-(D)t is the branch comprising two or more fluorophores.

[0103] In certain embodiments, the branched tandem dye scaffold comprises formula (Ila): wherein * and ** each optionally represent a point of attachment with the branched tandem dye scaffold; wherein at least one of * and ** is present; each M1and M2is an independently selected non-conjugated backbone portion of the unit; each S1and S2is an independently selected optional spacer nonconjugated backbone portion of the unit; each X1is a linker; f is an integer between 1 and 16; each D is an independently selected donor fluorophore linked to X1; each X2is a linker; each A is an independently selected acceptor fluorophore linked to X2; x is an integer between 1 to 20; y is an integer between 1 to 5; the ratio of x:y is between 1 :1 to 20:1 ; and wherein at least one X1-(D)t is the branch comprising two or more fluorophores.

[0104] In certain embodiments, the branched tandem dye scaffold comprises formula (lib): wherein G1and G2are each independently selected from a terminal group or a linker; each M1and M2is an independently selected non-conjugated backbone portion of the unit; each S1and S2is an independently selected optional spacer non-conjugated backbone portion of the unit; each X1is a linker; f is an integer between 1 and 16; each D is an independently selected donor fluorophore linked to X1; each X2is a linker; each A is an independently selected acceptor fluorophore linked to X2; x is an integer between 1 to 20; y is an integer between 1 to 5; the ratio of x:y is between 1 :1 to 20:1 ; and wherein at least one X1-(D)t is the branch comprising two or more fluorophores.

[0105] In certain instances, the non-conjugated backbone comprises formula (Illa): wherein * and ** each optionally represent a point of attachment with the branched tandem dye scaffold; wherein at least one of * and ** is present; each M1and M2is an independently selected non-conjugated backbone portion of the unit; each X1is a linker; f is an integer between 1 and 16; each D is an independently selected donor fluorophore linked to X1; each X2is a linker; each A is an independently selected acceptor fluorophore linked to X2; x is an integer between 1 to 20; y is an integer between 1 to 5; the ratio of x:y is between 1 :1 to 20:1 ; and wherein at least one X1-(D)t is the branch comprising two or more fluorophores.

[0106] In certain embodiments, the branched tandem dye scaffold comprises formula (lllb) : wherein G1and G2are each independently selected from a terminal group or a linker; each M1and M2is an independently selected non-conjugated backbone portion of the unit; each X1is a linker; f is an integer between 1 and 16; each D is an independently selected donor fluorophore linked to X1; each X2is a linker; each A is an independently selected acceptor fluorophore linked to X2; x is an integer between 1 to 20; y is an integer between 1 to 5; the ratio of x:y is between 1 :1 to 20:1 ; and wherein at least one X1-(D)t is the branch comprising two or more fluorophores. o H II H *— N — C— C— In certain embodiments, M1is selected from # ** each optionally represent a point of attachment with the branched tandem dye scaffold, wherein at least one of * and *“ is present, and # represents a point of attachment with X1. In certain embodiments, M2is selected from , wherein ** and **** each optionally represent a point of attachment with the branched tandem dye scaffold, wherein at least one of ** and **** is present, and ## represents a point of attachment with X2.

[0107] "4o-c4'

[0108] In certain embodiments, S1 or 'zv , wherein1and each represent a point of attachment with the branched tandem dye scaffold, each v is an integer independently selected from between 1 and 20. In certain embodiments, S22is , wherein ‘ and " each represent a point of attachment with the branched tandem dye scaffold, each v is an integer independently selected from between 1 and 20.

[0109] In the following paragraph,Arepresents a point of attachment with M1,AAArepresents a point of attachment with D,AAAArepresents a point of attachment with A. In certain embodiments, wherein n and o are each independently selected from an integer between 1 and 10, or n is 4 or o is 4. In certain embodiments, X1is , wherein n, o, p, and q are each independently selected from an integer between 1 and 10, or n is 4 or o is 4 or p is 4 or q is 4. In certain embodiments, X1is wherein a, b, c, d, and e are each independently selected from an integer between 1 and 10, or wherein e is 1 or 2, or a is 4, or b is 4, or c is 4, or d is 4. In certain embodiments, X1is wherein n, o, p, and q are each an integer independently selected from between 1 and 10, or n is 3, or o is 3, or p is 3, or q is 3. In certain embodiments, X1is wherein n, a, b, c, d, e, f, g, and h are each an integer independently selected from between 1 and 5, or a is 3, or b is 3, or c is 3, or d is 3, or e is 3, or f is 3, or g is 3, or h is 3. In certain

[0110] embodiments, wherein j, k, I, m, n, o, p, and q are each an integer independently selected from between 1 and 5, , wherein n, a, b, c, d, e, and f are each an integer independently selected from between 1 and 5, or n is 1 or 2, or a is 2, or b is 2, or c is 2, or d is 2, or e is 2, or f is 2. In certain embodiments, X1is wherein n, a, b, and c are each an integer independently selected from between 1 and 5, or n is 1 or 2, or a is 2, or b is 2, or c is 2. In certain embodiments, X1is wherein I, m, n, o, p, q, r, s, t, u, y, z, and a1 are each an integer independently selected from between

[0111] 1 and 5, or I is 2, or m is 2, or n is 2, or o is 2, or p is 2, or q is 2, or r is 2, or s is

[0112] 2, or t is 2, or u is 2, or y is 2, or z is 2, or a1 is 2. In certain embodiments, X1is wherein a, b, c, d, e, f, g, h, j, k, I, m, n, o, p, q, r, s, t, u, v, w, x, y, z, a1 , b1 , c1 , d1 , and e1 are each an integer independently selected from between 1 and 5, or a is 2, or b is 2, or c is 2, or d is 2, or e is 2, or f is 2, or g is 2, or h is 2, or j is 2, or k is 2, or I is 2, or m is 2, or n is 2, or o is 2, or p is 2, or q is 2, or r is 2, or s is 2, or t is 2, or u is 2, or v is 2, or w is 2, or x is 2, or y is 2, or z is 2, or a1 is 2, or b1 is 2, or c1 is 2, or d1 is 2, or e1 is 2. In certain embodiments, X1is wherein a, b, c, d, and e are each an integer independently selected from between 1 and 5, or a is 1 or 2, or b is 4, or c is 4, or d is 4, or e is 4. In certain embodiments, X1is a linker with between 10 and 300 atoms, or between 20 and 260 atoms, or between 230 and 250 atoms, or between 236 and 250 atoms, or between 30 and 40 atoms, or between 60 and 70 atoms, or between 70 and 80 atoms, or between 75 and 85 atoms, or between 95 and 105 atoms, or between 110 and 120 atoms, or between 125 and 135 atoms.

[0113] In certain embodiments, X2is selected from whereinAArepresents a point of attachment with M2, andAAAArepresents a point of attachment with A, and n is an integer between 1 and 5, or n is 1 or n is 2. In certain embodiments, X2is a linker with between 1 and 30 atoms, or between 1 and 5 atoms, or between 2 and 5 atoms, or between 7 and 10 atoms, or between 18 and 22 atoms.

[0114] In certain embodiments, f is selected from 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 ,

[0115] 12, 13, 14, 15, and 16. In certain embodiments, f is selected from 2, 4, 6, 8, 10, 12, 14, and 16. In certain embodiments, f is selected from 2, 4, or 8. In certain embodiments, g is 0. In certain embodiments, g is 1.

[0116] In certain embodiments, D is a donor fluorophore described herein. In certain embodiments, D comprises BODIPY. In certain embodiments, A is an acceptor fluorophore described herein. In certain embodiments, A comprises indoline.

[0117] In certain embodiments, x is an integer between 1 to 20, y is an integer between 1 to 5; and the ratio of x:y is between 1 :1 to 20:1 . In certain embodiments, the ratio of x:y is 1 :1 , 2:1 , 3:1 , 4:1 , or 5:1 . In certain embodiments, the ratio of x:y is 6:1 , 7:1 , 8:1 , 9:1 , or 10:1 .

[0118] Any convenient end unit (e.g., G1and G2) may be utilized at the terminals of the branched tandem dye scaffolds. As used herein, the terms “end unit” and “terminal unit” are used interchangeably to refer to the groups located at the terminals of the scaffold, e.g., as described herein. G1and G2groups of interest include, but are not limited to H, a linker, a donor fluorophore, an acceptor fluorophore, an amino acid, a glycerol, an amidoamide, a peptoid, an amino acid linked to one or more donor fluorophores, an amino acid linked to one or more acceptor fluorophores, a glycerol linked to one or more donor fluorophores, a glycerol linked to one or more acceptor fluorophores, an amidoamine linked to one or more donor fluorophores, an amidoamine linked to one or more acceptor fluorophores, a peptoid linked to one or more donor fluorophores, a peptoid linked to one or more acceptor fluorophores. In certain instances, G1and G2groups of interest include, but are not limited to an amino acid linked to one or more donor fluorophores, a glycerol linked to one or more donor fluorophores, an amidoamine linked to one or more donor fluorophores, and a peptoid linked to one or more donor fluorophores.

[0119] In this paragraph, ***** is a point of attachment with the branched tandem dye scaffold andAAAis a point of attachment with a donor fluorophore. In certain o embodiments, G1or G2is H or ”"*-CH2-LNH2|ncertain embodiments, G1or G2

[0120] , wherein r, s, t, and u are each an integer independently selected from between 1 and 10, or r is 3, or s is 3, or t is 3, or u is 3.

[0121] In certain embodiments, G1or G2is , wherein a, b, c, d, e, f, g, and h are each an integer independently selected from between 1 and 10, or a is 3, or b is 3, or c is 3, or d is 3, or e is 3, or f is 3, or g is 3, or h is 3.

[0122] In certain embodiments, G1or G2is a linker. In certain embodiments, G1or G2is a linker that comprises polyethylene glycol, which can be represented as X3or (X3)zwherein z is the number of polyethylene glycol units. In certain embodiments, the polyethylene glycol or (X3)zhas between 1 and 100 units, or between 2 and 90 units, or between 5 and 60 units, or between 10 and 30 units, or between 15 and 27 units, or between 20 and 27 units, or between 23 and 25 units, or between 40 and 60 units, or between 42 and 55 units, or between 45 and 50 units, or 48 units, or 24 units.

[0123] FIG. 2 provides an illustration of branched tandem dye scaffold according an embodiment. As illustrated in FIG. 2, branched tandem dye scaffold 200 includes a non-conjugated backbone 210 having 8 different donor branches, each of which is bound to 8 donor molecules 220. Also shown is acceptor chromomphor 230 linked to the non-conjugated backbone 210. A functional group 240 for linking to a biosensor is linked to one end of the non-conjugated backbone 210 by a linker 250.

[0124] Labeled Specific Binding Member

[0125] Aspects of the present disclosure include labeled specific binding members. In certain instances, the labeled specific binding member comprises the branched tandem dye scaffold (e.g., as described herein) and a specific binding member linked to the branched tandem dye scaffold. Any of the branched tandem dye scaffolds described herein may be conjugated to a specific binding member. In certain instances, the specific binding member and the branched tandem dye scaffold can be conjugated (e.g., covalently linked) to each other at any convenient locations of the two molecules, via an optional linker. In certain embodiments, the specific binding member is a Fab fragment, a F(ab')2 fragment, a scFv, a diabody or a triabody. In certain embodiments, the specific binding member is an antibody. In certain embodiments, the specific binding member is an antibody fragment or binding derivative thereof. In certain embodiments, the antibody fragment or binding derivative thereof is selected from a Fab fragment, a F(ab')2 fragment, a scFv, a diabody and a triabody. In some cases, the specific binding member is a murine antibody or binding fragment thereof. In certain instances, the specific binding member is a recombinant antibody or binding fragment thereof.

[0126] In certain embodiments of the formulae described herein, G1and / or G2is a linker, such as a linker including a functional group suitable for conjugation to a specific binding member, or SBM. It is understood that linkers located at the G1and / or G2positions may be selected so as to be orthogonal to any other linkers including chemoselective tags (e.g., as described herein) that may be present.

[0127] In certain instances, SBM is a biomolecule. Biomolecules of interest include, but are not limited to, polypeptides, polynucleotides, carbohydrates, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs thereof and combinations thereof. In certain instances, SBM is an antibody. In some instances, SBM is an antibody fragment or binding derivative thereof. In some cases, the antibody fragment or binding derivative thereof is selected from the group consisting of a Fab fragment, a F(ab')2 fragment, a scFv, a diabody and a triabody.

[0128] In certain embodiments, the labeled specific binding member comprises formula wherein SBM is a specfic binding member, and the other variables are as described herein. In certain embodiments, the labeled specific binding member comprises formula binding member, and the other variables are as described herein. In certain embodiments, the labeled specific binding member comprises formula (He): wherein SBM is a specfic binding member, and the other variables are as described herein. In certain embodiments, the labeled specific binding member comprises formula (I Id): (lid), wherein SBM is a specfic binding member, and the other variables are as described herein. In certain embodiments, the labeled specific binding member comprises formula (I He): wherein SBM is a specfic binding member, and the other variables are as described herein. In certain embodiments, the labeled specific binding member comprises formula (Hid): wherein SBM is a specfic binding member, and the other variables are as described herein.

[0129] In certain embodiments, SBM-G1or SBM-G2has a structure which is , wherein z is as described herein, z1 is an integer selected from 1 and 10, or z1 is 2, ***** is a point of attachment with the branched tandem dye scaffold, and $ is a point of attachment with the specific binding member. In certain embodiments, SBM-G1or SBM-G2has a structure which i wherein z is as described herein, z1 is an integer selected from 1 and 10, or z1 is 2, ***** is a point of attachment with the branched tandem dye scaffold, and $ is a point of attachment with the specific binding member. In certain instances, SBM- G1or SBM-G2has a structure which is and ***** are as defined herein.

[0130] Methods

[0131] Methods of Making Branched Tandem Dye Scaffolds

[0132] Also provided are methods of producing branched tandem dye scaffolds, e.g., as described herein. In some embodiments, the method includes: producing a precursor peptidic scaffold and conjugating donor fluorophores and acceptor fluorophores the precursor peptidic scaffold. Precursor peptidic scaffolds may be synthesized using any convenient protocol, e.g., using conventional peptide synthesis protocols (see e.g., Chanduru et al., "Chemical Methods for Peptide and Protein Production," Molecules (2013)18(4): 4373-4388). Where desired, orthogonally reactive side chains, e.g., as defined above, may be employed. In some instances, orthogonally reactive side chains include orthogonally reactive conjugation tags that may be used to covalently link the donor and acceptor dyes and the sensor to the peptidic scaffold. The term “conjugation tag” refers to a group that includes a chemo-selective functional group (e.g., as described herein) that can covalently link with a compatible functional group of a specific binding member, after optional activation and / or deprotection. Any convenient conjugation tags may be utilized in the subject in order to conjugate the fluors and sensor to the peptidic backbone. In some embodiments, the conjugation tag includes a terminal functional group selected from an amino, a carboxylic acid or a derivative thereof, a thiol, a hydroxyl, a hydrazine, a hydrazide, an azide, an alkyne and a protein reactive group (e.g., amino-reactive, thiol-reactive, hydroxyl-reactive, imidazolyl-reactive or guanidinyl-reactive). Any convenient methods and reagents may be adapted for use in the subject methods in order to covalently link the conjugation tag to the specific binding member. Methods of interest for labeling a target, include but are not limited to, those methods and reagents described by Hermanson, Bioconjugate Techniques, Third edition, Academic Press, 2013. The contacting step may be performed in an aqueous solution. In some instances, the conjugation tag includes an amino functional group and the target molecule includes an activated ester functional group, such as a NHS ester or sulfo-NHS ester, or vice versa. In certain instances, the conjugation tag includes a maleimide functional group and the target molecule includes a thiol functional group, or vice versa. In certain instances, the conjugation tag includes an alkyne (e.g., a cyclooctyne group) functional group and the target molecule includes an azide functional group, or vice versa, which can be conjugated via Click chemistry. In some cases, the method includes a separating step where the product labeled specific binding member is separated from the reaction mixture, e.g., excess reagents or unlabeled specific binding member. A variety of methods may be utilized to separate a target from a sample, e.g., via immobilization on a support, precipitation, chromatography, and the like. The order in which the donor fluorophore, acceptor fluorophore and sensor are conjugated with their respective side chains to produce the desired tandem dye with internally positioned sensor may vary as desired. For example, the donors may be conjugated first, followed by the acceptor and then the sensor. Alternatively, the acceptor may be conjugated first, following by the donor and sensor. In yet other embodiments, the senor may be conjugated first, following which the donors and acceptor(s) may be conjugated, in any desired order. In those instances where the donor and acceptors are conjugated to the peptidic scaffold prior to conjugation of the sensor, the construct prior to conjugation of the sensor may be referred to as a fluorescently labeled peptidic scaffold. In such instances, the fluorescently labeled peptidic scaffold may include: one or more donorfluor amino acid residues each conjugated to a pendant donor fluorophore; one or more acceptor-fluor amino acid residues each conjugated to a pendant acceptor fluorophore; and an internal amino acid residue conjugated to a chemoselective group or tage; wherein donor and acceptor fluorophores of the fluorescently labeled molecular entity are in energy transfer relationship. An example of synthesis of a tandem dye in accordance with an embodiment of the invention is provided in FIG. 1 . In some instances, the method further includes detecting and / or analyzing the product tandem dye. In some instances, the method further includes fluorescently detecting the labeled specific binding member. Any convenient methods may be utilized to detect and / or analyze the specific binding member in conjunction with the subject methods and compositions. Methods of analyzing a target of interest that find use in the subject methods, include but are not limited to, flow cytometry, fluorescence microscopy, in-situ hybridization, enzyme-linked immunosorbent assays (ELISAs), western blot analysis, magnetic cell separation assays and fluorochrome purification chromatography. Detection methods of interest include but are not limited to fluorescence spectroscopy, fluorescence microscopy, nucleic acid sequencing, fluorescence in-situ hybridization (FISH), protein mass spectroscopy, flow cytometry, and the like.

[0133] Detection may be achieved directly via the polymeric tandem dye, or indirectly by a secondary detection system. The latter may be based on any one or a combination of several different principles including, but not limited to, antibody labelled anti-species antibody and other forms of immunological or non-immunological bridging and signal amplification systems (e.g., biotin-streptavidin technology, protein-A and protein-G mediated technology, or nucleic acid probe / anti-nucleic acid probes, and the like). Suitable reporter molecules may be those known in the field of immunocytochemistry, molecular biology, light, fluorescence, and electron microscopy, cell immunophenotyping, cell sorting, flow cytometry, cell visualization, detection, enumeration, and / or signal output quantification. More than one antibody of specific and / or non-specific nature might be labelled and used simultaneously or sequentially to enhance target detection, identification, and / or analysis.

[0134] Methods of Making Labeled Specific Binding Member

[0135] Also provided are methods of making the labeled specific binding member, e.g., as described herein. In some embodiments, the methods include: contacting a specific binding member with a branched tandem dye scaffold (e.g., as described herein) to produce a labeled specific binding member, wherein the labeled specific binding member includes a conjugation tag that covalently links the branched tandem dye scaffold to the specific binding member. The term “conjugation tag” refers to a group that includes a chemo-selective functional group (e.g., as described herein) that can covalently link with a compatible functional group of a specific binding member, after optional activation and / or deprotection. Any convenient conjugation tags may be utilized in the branched tandem dye scaffold in order to conjugate the branched tandem dye scaffold to a specific binding member, e.g., antibody or fragment thereof, of interest. In some embodiments, the conjugation tag includes a terminal functional group selected from an amino, a carboxylic acid or a derivative thereof, a thiol, a hydroxyl, a hydrazine, a hydrazide, an azide, an alkyne and a protein reactive group (e.g., amino-reactive, thiol-reactive, hydroxyl-reactive, imidazolyl-reactive or guanidinyl- reactive). Any convenient methods and reagents may be adapted for use in the subject methods in order to covalently link the conjugation tag to the specific binding member. Methods of interest for labeling a target, include but are not limited to, those methods and reagents described by Hermanson, Bioconjugate Techniques, Third edition, Academic Press, 2013. The contacting step may be performed in an aqueous solution. In some instances, the conjugation tag includes an amino functional group and the target molecule includes an activated ester functional group, such as a NHS ester or sulfo-NHS ester, or vice versa. In certain instances, the conjugation tag includes a maleimide functional group and the target molecule includes a thiol functional group, or vice versa. In certain instances, the conjugation tag includes an alkyne (e.g., a cyclooctyne group) functional group and the target molecule includes an azide functional group, or vice versa, which can be conjugated via Click chemistry. In some cases, the method includes a separating step where the labeled specific binding membertarget analyte binding complex is separated from the reaction mixture, e.g., excess reagents or unlabeled specific binding member. A variety of methods may be utilized to separate a labeled specific binding member-target analyte binding complex from a sample, e.g., via immobilization on a support, precipitation, chromatography, and the like. Methods of Using

[0136] Aspects of the invention include methods of evaluating a sample for the presence of a target analyte. Aspects of the methods include contacting a sample with a labeled specific binding member that specifically binds the target analyte to produce a labeled composition contacted sample. In certain aspects, the labelled composition contacted sample is assayed for the presence of a labeled specific binding member-target analyte binding complex to evaluate whether the target analyte is present in the sample.

[0137] The labeled specific binding member employed in embodiments of methods of the invention includes a specific binding member conjugated to a branched tandem dye scaffold, e.g., as described above. In the following section, the target analyte may be a target molecule of interest or reagent, e.g., primary antibody, bound to the target molecule, depending on whether the labeled specific binding member is employed as a primary or secondary label. Any convenient method may be used to contact the sample with a labeled specific binding member that specifically binds to the target analyte to produce the assay composition. In some instances, the sample is contacted with the labeled specific binding member under conditions in which the labeled specific binding member specifically binds to the target analyte, if present. For specific binding of the labeled specific binding member with the target analyte, an appropriate medium may be used that maintains the biological activity of the components of the sample and the singal domain antibody. The medium may be a balanced salt solution, e.g., normal saline, PBS, Hank’s balanced salt solution, etc., conveniently supplemented with fetal calf serum, human platelet lysate or other factors, in conjunction with an acceptable buffer at low concentration, such as from 5-25 mM. Convenient buffers include HEPES, phosphate buffers, lactate buffers, etc. Various media are commercially available and may be used according to the nature of the target analyte, including dMEM, HBSS, dPBS, RPMI, Iscove’s medium, etc., in some cases supplemented with fetal calf serum or human platelet lysate. The final components of the medium, which may be a solution, may be selected depending on the components of the sample which are included. The temperature at which specific binding of the labeled specific binding member to the target analyte takes place may vary, and in some instances may range from 5°C to 50°C, such as from 10°C to 40°C, 15°C to 40°C, 20°C to 40°C, e.g., 20°C, 25°C, 30°C, 35°C or 37°C (e.g., as described above). In some instances, the temperature at which specific binding takes place is selected to be compatible with the biological activity of the specific binding member and / or the target analyte. In certain instances, the temperature is 25°C, 30°C, 35°C or 37°C. In certain cases, the temperature at which specific binding takes place is room temperature (e.g., 25°C), 30°C, 35°C or 37°C. Any convenient incubation time for specific binding may be selected to allow for the formation of a desirable amount of binding complex, and in some instances, may be 1 minute (min) or more, such as 2 min or more, 10 min or more, 30 min or more, 1 hour or more, 2 hours or more, or even 6 hours or more.

[0138] Any convenient specific binding members may be utilized in the labeled specific binding members employed in methods of the invention. Specific binding members of interest include, but are not limited to, those specific binding members that specifically bind cell surface proteins of a variety of cell types, including but not limited to, stem cells, e.g., pluripotent stem cells, hematopoietic stem cells, T cells, T regulator cells, dendritic cells, B Cells, e.g., memory B cells, antigen specific B cells, granulocytes, leukemia cells, lymphoma cells, virus cells (e.g., HIV cells) NK cells, macrophages, monocytes, fibroblasts, epithelial cells, endothelial cells, and erythroid cells. Target cells of interest include cells that have a convenient cell surface marker or antigen that may be captured by a convenient specific binding member conjugate. In some embodiments, the target cell is selected from HIV containing cell, a Treg cell, an antigen-specific T -cell populations, tumor cells or hematopoetic progenitor cells (CD34+) from whole blood, bone marrow or cord blood. Any convenient cell surface proteins or cell markers may be targeted for specific binding to the conjugates employed in the subject methods. In some embodiments, the target cell includes a cell surface marker selected from a cell receptor and a cell surface antigen. In some cases, the target cell may include a cell surface antigen such as CD1 1 b, CD123, CD14, CD15, CD16, CD19, CD193, CD2, CD25, CD27, CD3, CD335, CD36, CD4, CD43, CD45RO, CD56, CD61 , CD7, CD8, CD34, CD1c, CD23, CD304, CD235a, T cell receptor alpha / beta, T cell receptor gamma / delta, CD253, CD95, CD20, CD105, CD117, CD120b, Notch4, Lgr5 (N-Terminal), SSEA-3, TRA-1 -60 Antigen, Disialoganglioside GD2 and CD71.

[0139] Any convenient targets may be selected for evaluation utilizing the subject methods. Targets of interest include, but are not limited to, a nucleic acid, such as an RNA, DNA, PNA, CNA, HNA, LNA or ANA molecule, a protein, such as a fusion protein, a modified protein, such as a phosphorylated, glycosylated, ubiquitinated, SUMOylated, or acetylated protein, or an antibody, a peptide, an aggregated biomolecule, a cell, a small molecule, a vitamin and a drug molecule. As used herein, the term “a target protein” refers to all members of the target family, and fragments thereof. The target protein may be any protein of interest, such as a therapeutic or diagnostic target, including but not limited to: hormones, growth factors, transcription factor, receptors, enzymes, cytokines, osteoinductive factors, colony stimulating factors and immunoglobulins. The term “target protein” is intended to include recombinant and synthetic molecules, which can be prepared using any convenient recombinant expression methods or using any convenient synthetic methods, or purchased commercially. Any convenient target analyte that specifically binds an antibody or antibody fragment of interest may be targeted in the subject methods.

[0140] In some embodiments, the target analyte is associated with a cell. In certain instances, the target analyte is a cell surface marker of the cell. In certain cases, the cell surface marker is selected from the group consisting of a cell receptor and a cell surface antigen. In some instances, the target analyte is an intracellular target, and the method further includes treating the cell so as to provide access of the labeled specific binding memberto the intracellular target, e.g., by permeabilizing or lysing the cell. As such, a labeled specific binding member employed in methods of the invention may target a cell surface or intracellular antigen. Alternatively, a labeled specific binding member employed in methods of the invention may target a primary antibody that in turn specifically binds to a target cell surface or intracellular antigen.

[0141] In some embodiments, the sample may include a heterogeneous cell population from which target cells are isolated. In some instances, the sample includes peripheral whole blood, peripheral whole blood in which erythrocytes have been lysed prior to cell isolation, cord blood, bone marrow, density gradient- purified peripheral blood mononuclear cells or homogenized tissue. In some cases, the sample includes hematopoetic progenitor cells (e.g., CD34+ cells) in whole blood, bone marrow or cord blood. In certain embodiments, the sample includes tumor cells in peripheral blood. In certain instances, the sample is a sample including (or suspected of including) viral cells (e.g., HIV).

[0142] The labeled specific binding members find use in the subject methods, e.g., for labeling a target cell, particle, target or analyte with a branched tandem dye scaffold. For example, labeled specific binding members find use in labeling cells to be processed (e.g., detected, analyzed, and / or sorted) in a flow cytometer. The labeled specific binding members may include specific binding members, e.g., antibodies or binding fragments thereof, that specifically bind to, e.g., cell surface proteins of a variety of cell types (e.g., as described herein). The labeled specific binding members may be used to investigate a variety of biological (e.g., cellular) properties or processes such as cell cycle, cell proliferation, cell differentiation, DNA repair, T cell signaling, apoptosis, cell surface protein expression and / or presentation, and so forth. Labeled specific binding members may be used in any application that includes (or may include) antibody-mediated labeling of a cell, particle or analyte.

[0143] Aspects of the methods include assaying the assay composition, i.e., labeled specific binding member contacted sample, for the presence of a labeled specific binding member-target analyte binding complex to evaluate whether the target analyte is present in the sample. Once the sample has been contacted with the labeled specific binding member, any convenient method may be utilized in assaying the assay composition that is produced for the presence of a labeled specific binding member-target analyte binding complex. The labeled specific binding member-target analyte binding complex is the binding complex that is produced upon specific binding of the labeled specific binding member to the target analyte (or primary binding member, e.g., primeary antibody, to the target antigent depending on the embodiment), if present. Assaying the assay composition may include detecting a fluorescent signal from the binding complex, if present.

[0144] In some cases, the assaying includes a separating step where the target analyte, if present, is separated from the sample. A variety of methods can be utilized to separate a target analyte from a sample, e.g., via immobilization on a support. Assay methods of interest include, but are not limited to, any convenient methods and assay formats where pairs of specific binding members such as avidin- biotin or hapten-anti-hapten antibodies find use, are of interest. Methods and assay formats of interest that may be adapted for use with the subject compositions include, but are not limited to, flow cytometry methods, in- situ hybridization methods, enzyme-linked immunosorbent assays (ELISAs), western blot analysis, magnetic cell separation assays and fluorochrome purification chromatography.

[0145] In certain embodiments, the method further includes contacting the sample with a second specific binding member that specifically binds the target analyte. In certain instances, the second specific binding member is support bound. Any convenient supports may be utilized to immobilize a component of the subject methods (e.g., a second specific binding member). In certain instances, the support is a particle, such as a magnetic particle. In some instances, the second specific binding member and the labeled specific binding member-target analyte binding complex produce a sandwich complex that may be isolated and detected, if present, using any convenient methods. In some embodiments, the method further includes flow cytometrically analyzing the labeled specific binding member-target analyte binding complex, i.e., a fluorescently labeled target analyte. Assaying for the presence of a labeled specific binding member-target analyte binding complex may provide assay results (e.g., qualitative or quantitative assay data) which can be used to evaluate whether the target analyte is present in the sample.

[0146] Any convenient supports may be utilized in the subject methods to immobilize any convenient component of the methods, e.g., labeled specific binding member, target analyte, secondary specific binding member, etc. Supports of interest include, but are not limited to: solid substrates, where the substrate can have a variety of configurations, e.g., a sheet, bead, or other structure, such as a plate with wells; beads, polymers, particle, a fibrous mesh, hydrogels, porous matrix, a pin, a microarray surface, a chromatography support, and the like. In some instances, the support is selected from the group consisting of a particle, a planar solid substrate, a fibrous mesh, a hydrogel, a porous matrix, a pin, a microarray surface and a chromatography support. The support may be incorporated into a system that it provides for cell isolation assisted by any convenient methods, such as a manually-operated syringe, a centrifuge or an automated liquid handling system. In some cases, the support finds use in an automated liquid handling system for the high throughput isolation of cells, such as a flow cytometer.

[0147] In some embodiments of the method, the separating step includes applying an external magnetic field to immobilize a magnetic particle. Any convenient magnet may be used as a source of the external magnetic field (e.g., magnetic field gradient). In some cases, the external magnetic field is generated by a magnetic source, e.g. by a permanent magnet or electromagnet. In some cases, immobilizing the magnetic particles means the magnetic particles accumulate near the surface closest to the magnetic field gradient source, i.e. the magnet.

[0148] The separating may further include one or more optional washing steps to remove unbound material of the sample from the support. Any convenient washing methods may be used, e.g., washing the immobilized support with a biocompatible buffer which preserves the specific binding interaction of the labeled specific binding member and the target analyte. Separation and optional washing of unbound material of the sample from the support provides for an enriched population of target cells where undesired cells and material may be removed.

[0149] In certain embodiments, the method includes detecting the labeled specific binding member-target analyte binding complex. Detecting the labeled specific binding member-target analyte binding complex may include exciting the branched tandem dye scaffold with one or more lasers and subsequently detecting fluorescence emission from the branched tandem dye scaffold using one or more optical detectors. Detection of the labeled specific binding membertarget analyte binding complex can be performed using any convenient instruments and methods, including but not limited to, flow cytometry, FACS systems, fluorescence microscopy; fluorescence, luminescence, ultraviolet, and / or visible light detection using a plate reader; high performance liquid chromatography (HPLC); and mass spectrometry. When using fluorescently labeled components in the methods and compositions of the present disclosure, it is recognized that different types of fluorescence detection systems can be used to practice the subject methods. In some cases, high throughput screening can be performed, e.g., systems that use 96 well or greater microtiter plates. A variety of methods of performing assays on fluorescent materials can be utilized, such as those methods described in, e.g., Lakowicz, J. R., Principles of Fluorescence Spectroscopy, New York: Plenum Press (1983); Herman, B., Resonance energy transfer microscopy, in: Fluorescence Microscopy of Living Cells in Culture, Part B, Methods in Cell Biology, vol. 30, ed. Taylor, D. L. & Wang, Y.-L., San Diego: Academic Press (1989), pp. 219-243; Turro, N.J., Modern Molecular Photochemistry, Menlo Park: Benjamin / Cummings Publishing Col, Inc. (1978), pp. 296-361.

[0150] Fluorescence in a sample can be measured using a fluorimeter. In some cases, excitation radiation, from an excitation source having a first wavelength, passes through excitation optics. The excitation optics cause the excitation radiation to excite the sample. In response, fluorescently labeled specific binding member-target analyte binding complexes in the sample emit radiation which has a wavelength that is different from the excitation wavelength. Collection optics then collect the emission from the sample. The device can include a temperature controller to maintain the sample at a specific temperature while it is being scanned. In certain instances, a multi-axis translation stage moves a microtiter plate holding a plurality of samples in order to position different wells to be exposed. The multi-axis translation stage, temperature controller, auto-focusing feature, and electronics associated with imaging and data collection can be managed by an appropriately programmed digital computer. The computer also can transform the data collected during the assay into another format for presentation.

[0151] In some embodiments, the method of evaluating a sample for the presence of a target analyte further includes detecting fluorescence in a flow cytometer. In some embodiments, the method of evaluating a sample for the presence of a target analyte further includes imaging the labelling composition contacted sample using fluorescence microscopy. Fluorescence microscopy imaging can be used to identify a labeled specific binding member-target analyte binding complex in the contacted sample to evaluate whether the target analyte is present. Microscopy methods of interest that find use in the subject methods include laser scanning confocal microscopy.

[0152] In some instances, the method further includes detecting and / or analyzing the labeled specific binding member-target analyte binding complex. In some instances, the method further includes fluorescently detecting the labeled specific binding member-target analyte binding complex. Any convenient methods may be utilized to detect and / or analyze the labeled specific binding member-target analyte binding complex in conjunction with the subject methods and compositions. Methods of analyzing a target of interest that find use in the subject methods, include but are not limited to, flow cytometry, fluorescence microscopy, in-situ hybridization, enzyme-linked immunosorbent assays (ELISAs), western blot analysis, magnetic cell separation assays and fluorochrome purification chromatography. Detection methods of interest include but are not limited to fluorescence spectroscopy, fluorescence microscopy, nucleic acid sequencing, fluorescence in-situ hybridization (FISH), protein mass spectroscopy, flow cytometry, and the like.

[0153] Detection may be achieved directly via the branched tandem dye scaffold, or indirectly by a secondary detection system. The latter may be based on any one or a combination of several different principles including, but not limited to, antibody labeled anti-species antibody and other forms of immunological or non- immunological bridging and signal amplification systems (e.g., biotin-streptavidin technology, protein-A and protein-G mediated technology, or nucleic acid probe / anti-nucleic acid probes, and the like). Suitable reporter molecules may be those known in the field of immunocytochemistry, molecular biology, light, fluorescence, and electron microscopy, cell immunophenotyping, cell sorting, flow cytometry, cell visualization, detection, enumeration, and / or signal output quantification. More than one antibody of specific and / or non-specific nature might be labeled and used simultaneously or sequentially to enhance target detection, identification, and / or analysis.

[0154] Kits

[0155] Aspects of the invention further include kits for use in practicing the subject methods and compositions. The compositions of the invention can be included as reagents in kits either as starting materials or provided for use in, for example, the methodologies described above. A kit can include a labeled specific binding member, e.g., as described above, and a container. Any convenient containers can be utilized, such as tubes, bottles, or wells in a multi-well strip or plate, a box, a bag, an insulated container, and the like. The subject kits can further include one or more components selected from a labeled specific binding member for a given target analyte, a support bound specific binding member, a cell, a support, a biocompatible aqueous elution buffer, a control (postive and / or negative), etc., and instructions for use, as desired. A given kit may include reagents suitable for a detection of a single target analyte, or multiple reagents suitable for detection of two or more different target analytes, e.g., where a given kit is configured for multiplex detection applications.

[0156] In certain embodiments, the kit finds use in evaluating a sample for the presence of a target analyte, such as an intracellular target. As such, in some instances, the kit includes one or more components suitable for permeabilizing or lysing cells. The one or more additional components of the kit may be provided in separate containers (e.g., separate tubes, bottles, or wells in a multi-well strip or plate).

[0157] In certain aspects, the kit further includes reagents for performing a flow cytometric assay. Reagents of interest include, but are not limited to, buffers for reconstitution and dilution, buffers for contacting a cell sample with the branched tandem dye scaffold, wash buffers, control cells, control beads, fluorescent beads for flow cytometer calibration and combinations thereof. The kit may also include one or more cell fixing reagents such as paraformaldehyde, glutaraldehyde, methanol, acetone, formalin, or any combinations or buffers thereof. Further, the kit may include a cell permeabilizing reagent, such as methanol, acetone or a detergent (e.g., triton, NP-40, saponin, tween 20, digitonin, leucoperm, or any combinations or buffers thereof. Other protein transport inhibitors, cell fixing reagents and cell permeabilizing reagents familiar to the skilled artisan are within the scope of the subject kits.

[0158] The compositions of the kit may be provided in a liquid composition, such as any suitable buffer. Alternatively, the compositions of the kit may be provided in a dry composition (e.g., may be lyophilized), and the kit may optionally include one or more buffers for reconstituting the dry composition. In certain aspects, the kit may include aliquots of the compositions provided in separate containers (e.g., separate tubes, bottles, or wells in a multi-well strip or plate).

[0159] In addition, one or more components may be combined into a single container, e.g., a glass or plastic vial, tube or bottle. In certain instances, the kit may further include a container (e.g., such as a box, a bag, an insulated container, a bottle, tube, etc.) in which all of the components (and their separate containers) are present. The kit may further include packaging that is separate from or attached to the kit container and upon which is printed information about the kit, the components of the and / or instructions for use of the kit.

[0160] In addition to the above components, the subject kits may further include instructions for practicing the subject methods. These instructions may be present in the subject kits in a variety of forms, one or more of which may be present in the kit. One form in which these instructions may be present is as printed information on a suitable medium or substrate, e.g., a piece or pieces of paper on which the information is printed, in the packaging of the kit, in a package insert, etc. Yet another means would be a computer readable medium, e.g., diskette, CD, DVD, portable flash drive, etc., on which the information has been recorded. Yet another means that may be present is a website address which may be used via the Internet to access the information at a removed site. Any convenient means may be present in the kits.

[0161] Systems

[0162] Aspects of the invention further include systems for use in practicing the subject methods and compositions. A sample analysis system can include sample field of view or a flow channel loaded with a sample and labeled specific binding member of the invention, e.g., as described above. In some embodiments, the system is a flow cytometric system including: a flow cytometer including a flow path; a composition in the flow path, wherein the composition includes: a sample and a labeled specific binding member (e.g., as described herein). In some embodiments, the system for analyzing a sample is a fluorescence microscopy system, including: a fluorescence microscope comprising a sample field of view; and a composition disposed in the sample field of view, wherein the composition comprises a sample; and a labeled specific binding member (e.g., as described herein).

[0163] In certain embodiments of the systems, the composition further includes a second specific binding member that is support bound and specifically binds the target analyte. In some cases, the support includes a magnetic particle. As such, in certain instances, the system may also include a controllable external paramagnetic field configured for application to an assay region of the flow channel.

[0164] The sample may include a cell. In some instances, the sample is a cellcontaining biological sample. In some instances, the sample includes a labeled specific binding member specifically bound to a target cell. In certain instances, the target analyte that is specifically bound by the specific binding member is a cell surface marker of the cell. In certain cases, the cell surface marker is selected from a cell receptor and a cell surface antigen.

[0165] In certain aspects, the system may also include a light source configured to direct light to an assay region of the flow channel or sample field of view. The system may include a detector configured to receive a signal from an assay region of the flow channel or a sample field of view, wherein the signal is provided by the fluorescent composition. Optionally further, the sample analysis system may include one or more additional detectors and / or light sources for the detection of one or more additional signals.

[0166] In certain aspects, the system may further include computer-based systems configured to detect the presence of the fluorescent signal. A “computer- based system" refers to the hardware means, software means, and data storage means used to analyze the information of the present invention. The minimum hardware of the computer-based systems of the present invention includes a central processing unit (CPU), input means, output means, and data storage means. A skilled artisan can readily appreciate that any one of the currently available computer-based system are suitable for use in the subject systems. The data storage means may include any manufacture including a recording of the present information as described above, or a memory access means that can access such a manufacture.

[0167] To "record" data, programming or other information on a computer readable medium refers to a process for storing information, using any such methods as known in the art. Any convenient data storage structure may be chosen, based on the means used to access the stored information. A variety of data processor programs and formats can be used for storage, e.g., word processing text file, database format, etc.

[0168] A “processor” references any hardware and / or software combination that will perform the functions required of it. For example, any processor herein may be a programmable digital microprocessor such as available in the form of an electronic controller, mainframe, server or personal computer (desktop or portable). Where the processor is programmable, suitable programming can be communicated from a remote location to the processor, or previously saved in a computer program product (such as a portable or fixed computer readable storage medium, whether magnetic, optical or solid state device based). For example, a magnetic medium or optical disk may carry the programming, and can be read by a suitable reader communicating with each processor at its corresponding station.

[0169] In addition to the sensor device and signal processing module, e.g., as described above, systems of the invention may include a number of additional components, such as data output devices, e.g., monitors and / or speakers, data input devices, e.g., interface ports, keyboards, etc., fluid handling components, power sources, etc.

[0170] In certain aspects, the system includes a flow cytometer. Suitable flow cytometry systems may include, but are not limited to, those described in Ormerod (ed.), Flow Cytometry: A Practical Approach, Oxford Univ. Press (1997); Jaroszeski et al. (eds.), Flow Cytometry Protocols, Methods in Molecular Biology No. 91 , Humana Press (1997); Practical Flow Cytometry, 3rd ed., Wiley- Liss (1995); Virgo, et al. (2012) Ann Clin Biochem. Jan;49(pt 1 ):17-28; Linden, et. al., Semin Throm Hemost. 2004 Oct;30(5):502-11 ; Alison, et al. J Pathol, 2010 Dec; 222(4):335-344; and Herbig, et al. (2007) Crit Rev Ther Drug Carrier Syst. 24(3):203-255; the disclosures of which are incorporated herein by reference. In certain instances, flow cytometry systems of interest include BD Biosciences FACSCanto™ flow cytometer, BD Biosciences FACSCanto™ II flow cytometer, BD Accuri™ flow cytometer, BD Accuri™ C6 Plus flow cytometer, BD Biosciences FACSCelesta™ flow cytometer, BD Biosciences FACSLyric™ flow cytometer, BD Biosciences FACSVerse™ flow cytometer, BD Biosciences FACSymphony™ flow cytometer, BD Biosciences LSRFortessa™ flow cytometer, BD Biosciences LSRFortessa™ X-20 flow cytometer, BD Biosciences FACSPresto™ flow cytometer, BD Biosciences FACSVia™ flow cytometer and BD Biosciences FACSCalibur™ cell sorter, a BD Biosciences FACSCount™ cell sorter, BD Biosciences FACSLyric™ cell sorter, BD Biosciences Via™ cell sorter, BD Biosciences Influx™ cell sorter, BD Biosciences Jazz™ cell sorter, BD Biosciences Aria™ cell sorter, BD Biosciences FACSAria™ II cell sorter, BD Biosciences FACSAria™ III cell sorter, BD Biosciences FACSAria™ Fusion cell sorter and BD Biosciences FACSMelody™ cell sorter, BD Biosciences FACSymphony™ S6 cell sorter or the like.

[0171] In some embodiments, the subject systems are flow cytometric systems, such those described in U.S. Patent Nos. 10,663,476; 10,620,111 ; 10,613,017; 10,605,713; 10,585,031 ; 10,578,542; 10,578,469; 10,481 ,074; 10,302,545; 10,145,793; 10,113,967; 10,006,852; 9,952,076; 9,933,341 ; 9,726,527; 9,453,789; 9,200,334; 9,097,640; 9,095,494; 9,092,034; 8,975,595; 8,753,573; 8,233,146; 8,140,300; 7,544,326; 7,201 ,875; 7,129,505; 6,821 ,740; 6,813,017; 6,809,804; 6,372,506; 5,700,692; 5,643,796; 5,627,040; 5,620,842; 5,602,039; 4,987,086; 4,498,766; the disclosures of which are herein incorporated by reference in their entirety.

[0172] In certain instances, flow cytometry systems of the invention are configured for imaging particles in a flow stream by fluorescence imaging using radiofrequency tagged emission (FIRE), such as those described in Diebold, et al. Nature Photonics Vol. 7(10); 806-810 (2013) as well as described in U.S. Patent Nos. 9,423,353; 9,784,661 ; 9,983,132; 10,006,852; 10,078,045; 10,036,699; 10,222,316; 10,288,546; 10,324,019; 10,408,758; 10,451 ,538; 10,620,111 ; and U.S. Patent Publication Nos. 2017 / 0133857; 2017 / 0328826; 2017 / 0350803; 2018 / 0275042; 2019 / 0376895 and 2019 / 0376894 the disclosures of which are herein incorporated by reference.

[0173] Other systems may find use in practicing the subject methods. In certain aspects, the system may be a fluorimeter or microscope loaded with a sample having a fluorescent composition of any of the embodiments discussed herein. The fluorimeter or microscope may include a light source configured to direct light to the assay region of the flow channel or sample field of view. The fluorimeter or microscope may also include a detector configured to receive a signal from an assay region of the flow channel or field of view, wherein the signal is provided by the fluorescent composition.

[0174] Utility

[0175] The branched tandem dye scaffolds and labeled specific binding members comprising the same, compositions, methods and systems as described herein may find use in a variety of applications, including diagnostic and research applications, in which the labelling, detection and / or analysis of a target of interest is desirable. Such applications include methodologies such as cytometry, microscopy, immunoassays (e.g. competitive or non-competitive), assessment of a free analyte, assessment of receptor bound ligand, and so forth. The compositions, system and methods described herein may be useful in analysis of any of a number of samples, including but not limited to, biological fluids, cell culture samples, and tissue samples. In certain aspects, the compositions, system and methods described herein may find use in methods where analytes are detected in a sample, if present, using fluorescent labels, such as in fluorescent activated cell sorting or analysis, immunoassays, immunostaining, and the like. In certain instances, the compositions and methods find use in applications where the evaluation of a sample for the presence of a target analyte is of interest.

[0176] In some cases, the methods and compositions find use in any assay format where the detection and / or analysis of a target from a sample is of interest, including but not limited to, flow cytometry, fluorescence microscopy, in- situ hybridization, enzyme-linked immunosorbent assays (ELISAs), western blot analysis, magnetic cell separation assays and fluorochrome purification chromatography. In certain instances, the methods and compositions find use in any application where the fluorescent labelling of a target molecule is of interest.

[0177] The subject compositions may be adapted for use in any convenient applications where pairs of specific binding members find use, such as biotin-streptavidin and hapten-anti-hapten antibody. The following examples are offered by way of illustration and not by way of limitation.

[0178] EXAMPLES

[0179] Example 1 : Branched tandem dye scaffolds

[0180] Embodiment 1 : BTDS 1 Embodiment 2: BTDS 2 Embodiment 4: BTDS 4 Embodiment 6: BTDS 6

[0181] Embodiment 7: BTDS 7

[0182] Embodiment 8: BTDS 8

[0183] Embodiment 9: BTDS 9 Embodiment 10: BTDS10

[0184] Embodiment 11 : BTDS 1 1

[0185] Embodiment 12: BTDS 12

[0186] The above specific tandem dyes may be synthesized using any convenient protocol, such as the protocols described herein and illustrated in FIG. 1 for BTDS 1 . As illustrated in FIG. 1 , the backbone structure of BTDS 1 is synthesized by conjugating protected lysine and homopropargylglycine to a PEG chain. The donor is conjugated to the backbone after selective deprotection of amine on the lysine residue while the acceptor is attached using Cu(l)-catalyzed azide-alkyne cycloaddition (CuAAC) click chemistry to the homopropargylglycine residue. Finally, the terminal amine of the PEG chain is exposed and SMOG is conjugated to the structure for further antibody conjugation application. Notwithstanding the appended claims, the disclosure is also defined by the following clauses:

[0187] 1 . A branched tandem dye scaffold comprising: a non-conjugated backbone comprising a branch comprising two or more donor fluorophores; and an acceptor fluorophore(s) linked to the non-conjugated backbone; wherein the acceptor fluorophore(s) and the donor fluorophores are in energy transfer relationship.

[0188] 2. The branched tandem dye scaffold according to clause 1 , wherein a majority of the donor fluorophores are in an energy transfer relationship with the acceptor fluorophore(s).

[0189] 3. The branched tandem dye scaffold according to any one of clauses 1 -2, wherein 30% or more of the energy harvested by the donor fluorophores is transferred to the acceptor fluorophore(s).

[0190] 4. The branched tandem dye scaffold according to any one of clauses 1 -3, wherein from 30% to 100% of the energy harvested by each of the donor fluorophores is transferred to the acceptor fluorophore(s).

[0191] 5. The branched tandem dye scaffold according to any one of clauses 1 -4, wherein the ratio of donor fluorophores to acceptor fluorophore(s) ranges from 3:1 to 100:1.

[0192] 6. The branched tandem dye scaffold according to any one of clauses 1 -5, wherein the ratio of donor fluorophores to acceptor fluorophore(s) ranges from 4:1 to 16:1.

[0193] 7. The branched tandem dye scaffold according to any one of clauses 1 -6, wherein the ratio of donor fluorophores to acceptor fluorophore(s) is 12:1 . 8. The branched tandem dye scaffold according to any one of clauses 1 -7, wherein the donor fluorophores are arranged in a three-dimensional configuration relative to the acceptor fluorophore(s) in an aqueous solution.

[0194] 9. The branched tandem dye scaffold according to clause 8, wherein the three-dimensional configuration is selected from spherical, ellipsoidal, tetrahedral, pyramidal, cuboidal, cylindrical, tetrapodal, and stellate, or combinations thereof.

[0195] 10. The branched tandem dye scaffold according to any one of clauses 1 -9, wherein the non-conjugated backbone comprises two or more branches comprising two or more donor fluorophores, and the branches are arranged in a three-dimensional configuration relative to the acceptor fluorophore(s) in an aqueous solution.

[0196] 1 1 . The branched tandem dye scaffold according to clause 10, wherein the two or more branches are arranged equidistantly from each other.

[0197] 12. The branched tandem dye scaffold according to clause 10, wherein the two or more branches are arranged symmetrically in the three-dimensional configuration relative to the acceptor fluorophore(s) in an aqueous solution.

[0198] 13. The branched tandem dye scaffold according to any one of clauses 1 -12, wherein the donor fluorophores are from 3 Angstroms to 60 Angstroms from the acceptor fluorophore(s) in an aqueous solution.

[0199] 14. The branched tandem dye scaffold according to clause 13, wherein the donor fluorophores are from 3 Angstroms to 30 Angstroms from the acceptor fluorophore(s) in an aqueous solution.

[0200] 15. The branched tandem dye scaffold according to any one of clauses 1 -14, wherein the non-conjugated backbone is peptidic.

[0201] 16. The branched tandem dye scaffold according to any one of clauses 1 -14, wherein the non-conjugated backbone comprises one or more amino acids. 17. The branched tandem dye scaffold according to any one of clauses 1 -14 and 16, wherein the non-conjugated backbone comprises an amino acid which comprises the branch comprising two or more donor fluorophores.

[0202] 18. The branched tandem dye scaffold according to any one of clauses 1 -14 and 16-17, wherein the non-conjugated backbone comprises an amino acid through which the acceptor fluorophore is linked.

[0203] 19. The branched tandem dye scaffold according to any one of clauses 1 -14 and 16-18, wherein the non-conjugated backbone comprises an amidoamine which comprises the branch comprising two or more donor fluorophores.

[0204] 20. The branched tandem dye scaffold according to any one of clauses 1 -14 and 16-19, wherein the non-conjugated backbone comprises a glycerol which comprises the branch comprising two or more donor fluorophores.

[0205] 21 . The branched tandem dye scaffold according to any one of clauses 1 -14 and 16-20, wherein the non-conjugated backbone comprises an ethylene glycol which comprises the branch comprising two or more donor fluorophores.

[0206] 22. The branched tandem dye scaffold according to any one of clauses 1 -14 and 16-21 , wherein the non-conjugated backbone comprises a polyethylene glycol).

[0207] 23. The branched tandem dye scaffold according to any one of clauses 1 -14, comprising formula wherein * and ** each optionally represent a point of attachment with the branched tandem dye scaffold; wherein at least one of * and ** is present; each M1and M2is an independently selected non-conjugated backbone portion of the unit; each S1and S2is an independently selected optional spacer nonconjugated backbone portion of the unit; each X1is a linker; f is an integer between 1 and 16; g is 0 or 1 ; each D is an independently selected donor fluorophore linked to X1; each X2is a linker; each A is an independently selected acceptor fluorophore linked to X2; x is an integer between 1 to 20; y is an integer between 1 to 5; and the ratio of x:y is between 1 :1 to 20:1 wherein at least one X1-(D)t is the branch comprising two or more fluorophores.

[0208] 24. The branched tandem dye scaffold according to any one of clauses 1 -14, comprising formula wherein G1and G2are each independently selected from a terminal group or a linker; each M1and M2is an independently selected non-conjugated backbone portion of the unit; each S1and S2is an independently selected optional spacer non-conjugated backbone portion of the unit; each X1is a linker; f is an integer between 1 and 16; g is 0 or 1 ; each D is an independently selected donor fluorophore linked to X1; each X2is a linker; each A is an independently selected acceptor fluorophore linked to X2; x is an integer between 1 to 20; y is an integer between 1 to 5; and the ratio of x:y is between 1 :1 to 20:1 wherein at least one X1-(D)t is the branch comprising two or more fluorophores.

[0209] 25. The branched tandem dye scaffold according to clause 23 or 24, wherein the M1-X1is selected from an amino acid, a glycerol, an amidoamine, and a peptoid.

[0210] 26. The branched tandem dye scaffold according to clause 24 or 25, wherein the M1-X1is an amino acid, and the amino acid is lysine. 27. The branched tandem dye scaffold according to clause 23, wherein g is 0, and the M1-X1-(D)t is an amino acid having a structure which is: wherein *** and **** represent points of attachment with the branched tandem dye scaffold, wherein at least one of *** and **** is present; n and o are each independently selected from an integer between 1 and 10, p and q, when present, are each independently selected from an integer between 1 and 10, each L is independently selected from a covalent bond and a linker, and each D is one of the donor fluorophores. 28. The branched tandem dye scaffold according to clause 26, wherein n is 4, o is 4, p, when present, is 4, and q, when present, is 4.

[0211] 29. The branched tandem dye scaffold according to clause 23 or 24, wherein g is 1 , and the has a structure which is: wherein *** and **** represent points of attachment with the branched tandem dye scaffold, wherein at least one of *** and **** is present; a, b, c, d, and e are each independently selected from an integer between 1 and 10, each L is independently selected from a covalent bond and a linker, and each D is one of the donor fluorophores.

[0212] 30. The branched tandem dye scaffold according to clause 29, wherein e is 1 , and a, b, c, and d are each 4.

[0213] 31 . The branched tandem dye scaffold according to clause 24, wherein the amino acid further comprises branched polyethylene glycol).

[0214] 32. The branched tandem dye scaffold according to clause 31 , wherein the amino acid is asparagine substituted with branched polyethylene glycol) or glutamine substituted with branched polyethylene glycol).

[0215] 33. The branched tandem dye scaffold according to clause 24, wherein g is 0, and the M1-X1-(D)t is an amino acid having a structure which is: wherein *** and **** represent points of attachment with the branched tandem dye scaffold, wherein at least one of *** and **** is present; n, a, b, c, d, e, and f are each an integer independently selected from between 1 and 5; each L is independently selected from a covalent bond and a linker; and each D is one of the donor fluorophores. 34. The branched tandem dye scaffold according to clause 33, wherein n is 1 or 2, and a, b, c, d, e, and f are each 2.

[0216] 35. The branched tandem dye scaffold according to clause 23, wherein g is 0, and the M1-X1-(D)t is an amino acid having a structure which is: wherein *** and **** represent points of attachment with the branched tandem dye scaffold, wherein at least one of *** and **** is present; n is an integer between 1 and 5; o, p, and q are each an integer independently selected from between 1 and 10; each L is independently selected from a covalent bond and a linker; and each D is one of the donor fluorophores.

[0217] 36. The branched tandem dye scaffold according to clause 35, wherein n is 1 or 2, q is 1 , and o and p are each 2.

[0218] 37. The branched tandem dye scaffold according to clause 24, wherein g is 0 and the M1-X1-(D)t comprises a glycerol.

[0219] 38. The branched tandem dye scaffold according to clause 37, wherein M1-X1- (D)t has a structure which is: and wherein G1is wherein *** represents a point of attachment with the branched tandem dye scaffold; ***** represents a point of attachment with the branched tandem dye scaffold; n, o, p, q, r, s, t, and u are each an integer independently selected from between 1 and 10, each L is independently selected from a covalent bond and a linker; and each D is one of the donor fluorophores.

[0220] 39. The branched tandem dye scaffold according to clause 38, wherein n, o, p, q, r, s, t, and u are each 3. 40. The branched tandem dye scaffold according to clause 37, wherein M1-X1- (D)t has a structure which is: and wherein G1is wherein *** represents a point of attachment with the branched tandem dye scaffold; ***** represents a point of attachment with the branched tandem dye scaffold; a, b, c, d, e, f, g, h, j, k, I, m, n, o, p, and q are each an integer independently selected from between 1 and 5, each L is independently selected from a covalent bond and a linker; and each D is one of the donor fluorophores.

[0221] 41 . The branched tandem dye scaffold according to clause 40, wherein a, b, c, d, e, f, g, h, j, k, I, m, n, o, p, and q are each 3. 42. The branched tandem dye scaffold according to clause 23 or 24, wherein g is 0 and the M1-X1-(D)t comprises an amidoamine.

[0222] 43. The branched tandem dye scaffold according to clause 42, wherein the M1-X1-(D)r has a structure which is: and wherein G1is wherein *** represents a point of attachment with the branched tandem dye scaffold; ***** represents a point of attachment with the branched tandem dye scaffold; a, b, c, d, e, f, g, h, j, k, I, m, n, o, p, q, r, s, t, u, v, w, x, y, z, a1 , b1 , c1 , and d1 are each an integer independently selected from between 1 and 5, each L is independently selected from a covalent bond and a linker; and each D is one of the donor fluorophores. 44. The branched tandem dye scaffold according to clause 43, wherein a, b, c, d, e, f, g, h, j, k, I, m, n, o, p, q, r, s, t, u, v, w, x, y, z, a1 , b1 , c1 , and d1 are each 2.

[0223] 45. The branched tandem dye scaffold according to clause 42, wherein the M1-X1-(D)f has a structure which is: wherein *** and **** represent points of attachment with the branched tandem dye scaffold, wherein at least one of *** and **** is present; a, b, c, d, e, f, g, h, j, k, I, m, n, o, p, q, r, s, t, u, v, w, x, y, z, a1 , b1 , c1 , d1 , and e1 are each an integer independently selected from between 1 and 5, each L is independently selected from a covalent bond and a linker; and each D is one of the donor fluorophores.

[0224] 46. The branched tandem dye scaffold according to clause 45, wherein a, b, c, d, e, f, g, h, j, k, I, m, n, o, p, q, r, s, t, u, v, w, x, y, z, a1 , b1 , c1 , d1 and e1 are each 2. 47. The branched tandem dye scaffold according to clause 23 or 24, wherein g is 0 and the M1-X1-(D)t comprises a peptoid.

[0225] 48. The branched tandem dye scaffold according to clause 47, wherein the

[0226] M1-X1-(D)r has a structure which is: wherein *** and **** represent points of attachment with the branched tandem dye scaffold, wherein at least one of *** and **** is present; ***** represents a point of attachment with the branched tandem dye scaffold; a, b, c, d, and e are each an integer independently selected from between 1 and 5, each L is independently selected from a covalent bond and a linker; and each D is one of the donor fluorophores.

[0227] 49. The branched tandem dye scaffold according to clause 48, wherein a, b, c, d, and e are each 4.

[0228] 50. The branched tandem dye scaffold according to any one of clauses 23-49, wherein each D comprises BODIPY.

[0229] 51 . The branched tandem dye scaffold according to any one of clauses 23-50, wherein the M2-X2is selected from an amino acid, a glycerol, an amidoamine, and a peptoid.

[0230] 52. The branched tandem dye scaffold according to clause 51 , wherein the M2-X2comprises an amino acid. 53. The branched tandem dye scaffold according to clause 23, wherein the

[0231] M2-X2-A has a structure according to: wherein *** and **** optionally represent points of attachment with the formula (la) or the branched tandem dye scaffold, wherein at least one of *** and **** is present; n is an integer between 1 and 5; L is a covalent bond or a linker; and A is the acceptor fluorophore.

[0232] 54. The branched tandem dye scaffold according to clause 53, wherein the amino acid has a structure according to: 55. The branched tandem dye scaffold according to clause 53, wherein the amino acid has a structure according to: 56. The branched tandem dye scaffold according to any one of clauses 23-55, wherein each A comprises indoline.

[0233] 57. The branched tandem dye scaffold according to any one of clauses 23-56, wherein the ratio of x:y is 1 :1 , 2:1 , 3:1 , 4:1 , or 5:1 .

[0234] 58. A labeled specific binding member, comprising: the branched tandem dye scaffold according to any one of clauses 1 -57; and a specific binding member linked to the branched tandem dye scaffold.

[0235] 59. The labeled specific binding member according to clause 58, wherein the specific binding member is an antibody.

[0236] 60. The labeled specific binding member according to clause 58, wherein the specific binding member is an antibody fragment or binding derivative thereof.

[0237] 61 . The labeled specific binding member according to clause 60, wherein the antibody fragment or binding derivative thereof is selected from a Fab fragment, a F(ab')2 fragment, a scFv, a diabody and a triabody.

[0238] 62. The labeled specific binding member according to any one of clauses 58- 61 , wherein the specific binding member is linked to the branched tandem dye scaffold through the non-conjugated backbone.

[0239] 63. The labeled specific binding member according to any one of clauses 58- 61 , wherein the specific binding member is linked to the branched tandem dye scaffold through the M1, a linker attached to the M1, the M2, or a linker attached to the M2.

[0240] 64. A method of evaluating a sample for the presence of a target analyte, the method comprising: (a) contacting the sample with the labeled specific binding member according to any one of clauses 58-63, that specifically binds the target analyte to produce a labeled composition contacted sample; and (b) assaying the labeled composition contacted sample for the presence of a labeled specific binding member-target analyte binding complex to evaluate whether the target analyte is present in the sample.

[0241] 65. The method according to clause 64, further comprising contacting the sample with a second specific binding member that is support bound and specifically binds the target analyte.

[0242] 66. The method according to clause 65, wherein the support comprises a magnetic particle.

[0243] 67. The method according to any one of clauses 64-66, wherein the target analyte is associated with a cell.

[0244] 68. The method according to clause 67, wherein the target analyte is a cell surface marker of the cell.

[0245] 69. The method according to clause 68, wherein the cell surface marker is selected from a cell receptor and a cell surface antigen.

[0246] 70. The method according to clause 64, wherein the target analyte is an intracellular target, and the method further comprises lysing the cell.

[0247] 71 . The method according to any one of clauses 64-70, wherein the method further comprises flow cytometrically analyzing the fluorescently labeled target analyte.

[0248] 72. A method of labelling a target molecule, the method comprising: contacting the target molecule with a branched tandem dye scaffold to produce a labeled target molecule, wherein: the branched tandem dye scaffold according to any one of clauses 1 -57 and comprises a conjugation tag that covalently links to the target molecule.

[0249] 73. A kit comprising: the branched tandem dye scaffold according to any one of clauses 1 -57; instructions for use of the kit. In at least some of the previously described embodiments, one or more elements used in an embodiment can interchangeably be used in another embodiment unless such a replacement is not technically feasible. It will be appreciated by those skilled in the art that various other omissions, additions and modifications may be made to the methods and structures described above without departing from the scope of the claimed subject matter. All such modifications and changes are intended to fall within the scope of the subject matter, as defined by the appended claims.

[0250] It will be understood by those within the art that, in general, terms used herein, and especially in the appended claims (e.g., bodies of the appended claims) are generally intended as “open” terms (e.g., the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” etc.). It will be further understood by those within the art that if a specific number of an introduced claim recitation is intended, such an intent will be explicitly recited in the claim, and in the absence of such recitation no such intent is present. For example, as an aid to understanding, the following appended claims may contain usage of the introductory phrases “at least one” and “one or more” to introduce claim recitations. However, the use of such phrases should not be construed to imply that the introduction of a claim recitation by the indefinite articles “a” or “an” limits any particular claim containing such introduced claim recitation to embodiments containing only one such recitation, even when the same claim includes the introductory phrases “one or more” or “at least one” and indefinite articles such as “a” or “an” (e.g., “a” and / or “an” should be interpreted to mean “at least one” or “one or more”); the same holds true for the use of definite articles used to introduce claim recitations. In addition, even if a specific number of an introduced claim recitation is explicitly recited, those skilled in the art will recognize that such recitation should be interpreted to mean at least the recited number (e.g., the bare recitation of “two recitations,” without other modifiers, means at least two recitations, or two or more recitations). Furthermore, in those instances where a convention analogous to “at least one of A, B, and C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “ a system having at least one of A, B, and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). In those instances where a convention analogous to “at least one of A, B, or C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “ a system having at least one of A, B, or C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or “B” or “A and B.”

[0251] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0252] As will be understood by one skilled in the art, for any and all purposes, such as in terms of providing a written description, all ranges disclosed herein also encompass any and all possible sub-ranges and combinations of subranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like include the number recited and refer to ranges which can be subsequently broken down into sub-ranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1 -3 articles refers to groups having 1 , 2, or 3 articles. Similarly, a group having 1 -5 articles refers to groups having 1 , 2, 3, 4, or 5 articles, and so forth.

[0253] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it is readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

[0254] Accordingly, the preceding merely illustrates the principles of the invention. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. Moreover, nothing disclosed herein is intended to be dedicated to the public regardless of whether such disclosure is explicitly recited in the claims.

[0255] The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of present invention is embodied by the appended claims. In the claims, 35 U.S.C. §1 12(f) or 35 ll.S.C. §112(6) is expressly defined as being invoked for a limitation in the claim only when the exact phrase "means for" or the exact phrase "step for" is recited at the beginning of such limitation in the claim; if such exact phrase is not used in a limitation in the claim, then 35 U.S.C.

[0256] § 1 12 (f) or 35 U.S.C. §1 12(6) is not invoked.

Claims

What is claimed is:1 . A branched tandem dye scaffold comprising: a non-conjugated backbone comprising a branch comprising two or more donor fluorophores; and an acceptor fluorophore(s) linked to the non-conjugated backbone; wherein the acceptor fluorophore(s) and the donor fluorophores are in energy transfer relationship.

2. The branched tandem dye scaffold according to claim 1 , wherein the donor fluorophores are arranged in a three-dimensional configuration relative to the acceptor fluorophore(s) in an aqueous solution.

3. The branched tandem dye scaffold according to claim 2, wherein the three-dimensional configuration is selected from spherical, ellipsoidal, tetrahedral, pyramidal, cuboidal, cylindrical, tetrapodal, and stellate, or combinations thereof.

4. The branched tandem dye scaffold according to any one of claims 1 -3, wherein the non-conjugated backbone comprises two or more branches comprising two or more donor fluorophores, and the branches are arranged in a three-dimensional configuration relative to the acceptor fluorophore(s) in an aqueous solution.

5. The branched tandem dye scaffold according to claim 4, wherein the two or more branches are arranged equidistantly from each other.

6. The branched tandem dye scaffold according to claim 5, wherein the two or more branches are arranged symmetrically in the three-dimensional configuration relative to the acceptor fluorophore(s) in an aqueous solution.

7. The branched tandem dye scaffold according to any one of claims 1 -6, wherein the non-conjugated backbone is peptidic.

8. The branched tandem dye scaffold according to any one of claims 1 -7, wherein the non-conjugated backbone comprises an amino acid which comprises the branch comprising two or more donor fluorophores.

9. The branched tandem dye scaffold according to any one of claims 1 -8, wherein the non-conjugated backbone comprises an amino acid through which the acceptor fluorophore is linked.

10. The branched tandem dye scaffold according to any one of claims 1 -9, comprising formula (la):wherein* and ** each optionally represent a point of attachment with the branched tandem dye scaffold; wherein at least one of * and ** is present; each M1and M2is an independently selected non-conjugated backbone portion of the unit; each S1and S2is an independently selected optional spacer non-conjugated backbone portion of the unit; each X1is a linker; f is an integer between 1 and 16; g is 0 or 1 ; each D is an independently selected donor fluorophore linked to X1; each X2is a linker; each A is an independently selected acceptor fluorophore linked to X2; x is an integer between 1 to 20; y is an integer between 1 to 5; and the ratio of x:y is between 1 :1 to 20:1wherein at least one X1-(D)t is the branch comprising two or more fluorophores.1 1 . The branched tandem dye scaffold according to any one of claims 1 -9, comprising formula (lb):whereinG1and G2are each independently selected from a terminal group or a linker; each M1and M2is an independently selected non-conjugated backbone portion of the unit; each S1and S2is an independently selected optional spacer non-conjugated backbone portion of the unit; each X1is a linker; f is an integer between 1 and 16; g is 0 or 1 ; each D is an independently selected donor fluorophore linked to X1; each X2is a linker; each A is an independently selected acceptor fluorophore linked to X2; x is an integer between 1 to 20; y is an integer between 1 to 5; and the ratio of x:y is between 1 :1 to 20:1 wherein at least one X1-(D)t is the branch comprising two or more fluorophores.

12. A labeled specific binding member, comprising: the branched tandem dye scaffold according to any one of claims 1 -1 1 ; and a specific binding member linked to the branched tandem dye scaffold.

13. A method of evaluating a sample for the presence of a target analyte, the method comprising:(a) contacting the sample with the labeled specific binding member according to claim 12, that specifically binds the target analyte to produce a labeled composition contacted sample; and(b) assaying the labeled composition contacted sample for the presence of a labeled specific binding member-target analyte binding complex to evaluate whether the target analyte is present in the sample.

14. A method of labelling a target molecule, the method comprising: contacting the target molecule with a branched tandem dye scaffold to produce a labeled target molecule, wherein: the branched tandem dye scaffold according to any one of claims 1 -1 1 and comprises a conjugation tag that covalently links to the target molecule.

15. A kit comprising: the branched tandem dye scaffold according to any one of claims 1 -1 1 ; instructions for use of the kit.

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