Homo-doubly labeled compositions for the detection of enzyme activity in biological samples   

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a Continuation of U.S. Ser. No. 11/015,864, filed on Dec. 15, 2004, which is a Divisional of U.S. Ser. No. 09/747,287, filed on Dec. 22, 2000, which is a Continuation-in-Part of U.S. Ser. No. 09/394,019, filed on Sep. 10, 1999, which is a continuation-in-part of U.S. Ser. No. 08/802,981, filed on Feb. 20, 1997. This is also a continuation in part of PCT/US00/24882, filed on Sep. 11, 2000 designating the United States, which is a continuation-in-part of U.S. Ser. No. 09/394,019, filed on Sep. 10, 1999, which is a continuation-in-part of U.S. Ser. No. 08/802,981, filed on Feb. 20, 1997. All of these documents are incorporated herein by reference in their entirety for all purposes.

FIELD OF THE INVENTION

This invention pertains to a class of novel fluorogenic compositions whose fluorescence level increases in the presence active proteases. These fluorogenic protease indicators typically fluoresce at visible wavelengths and are thus highly useful for the detection and localization of protease activity in biological samples.

BACKGROUND OF THE INVENTION

Proteases represent a number of families of hydrolytic enzymes that catalytically hydrolyze peptide bonds. Principal groups of proteases include metalloproteases, serine proteases, cysteine proteases and aspartic proteases. Proteases, in particular serine proteases, are involved in a number of physiological processes such as blood coagulation, fertilization, inflammation, hormone production, the immune response and fibrinolysis.

Numerous disease states are caused by and can be characterized by alterations in the activity of specific proteases and their inhibitors. For example emphysema, arthritis, thrombosis, cancer metastasis and some forms of hemophilia result from the lack of regulation of serine protease activities (see, for example, Textbook of Biochemistry with Clinical Correlations, John Wiley and Sons, Inc. N.Y. (1993)). In case of viral infection, the presence of viral proteases have been identified in infected cells. Such viral proteases include, for example, HIV protease associated with AIDS and NS3 protease associated with Hepatitis C. These viral proteases play a critical role in the virus life cycle.

Proteases have also been implicated in cancer metastasis. Increased synthesis of the protease urokinase has been correlated with an increased ability to metastasize in many cancers. Urokinase activates plasmin from plasminogen which is ubiquitously located in the extracellular space and its activation can cause the degradation of the proteins in the extracellular matrix through which the metastasizing tumor cells invade. Plasmin can also activate the collagenases thus promoting the degradation of the collagen in the basement membrane surrounding the capillaries and lymph system thereby allowing tumor cells to invade into the target tissues (Dano, et al. (1985) Adv. Cancer. Res., 44: 139.

Clearly, measurement of changes in the activity of specific proteases is clinically significant in the treatment and management of the underlying disease states. Proteases, however, are not easy to assay. Typical approaches include ELISA using antibodies that bind the protease or RIA using various labeled substrates. With their natural substrates assays are difficult to perform and expensive. With currently available synthetic substrates the assays are expensive, insensitive and nonselective. In addition, many “indicator” substrates require high quantities of protease which results, in part, in the self destruction of the protease.

Recent approaches to protease detection rely on a cleavage-induced spectroscopic change in a departing chromogen or fluorogen located in the P1′ position (the amino acid position on the carboxyl side of the cleavable peptide bond) (see, for example U.S. Pat. Nos. 4,557,862 and 4,648,893). However, many proteases require two or four amino acid residues on either side of the scissile bond for recognition of the protease (a specific protease may require up to 6 amino acid residues) and thus, these approaches lack protease specificity.

Recently however, fluorogenic indicator compositions have been developed in which a “donor” fluorophore is joined to an “acceptor” chromophore by a short bridge containing a (7 amino acid) peptide that is the binding site for an HIV protease and linkers joining the fluorophore and chromophore to the peptide (Wang et al. (1990) Tetra. Letts. 45: 6493-6496). The signal of the donor fluorophore was quenched by the acceptor chromophore through a process believed to involve resonance energy transfer (RET). Cleavage of the peptide resulted in separation of the chromophore and fluorophore, removal of the quench and a subsequent signal was measured from the donor fluorophore.

The design of the bridge between the donor and the acceptor led to relatively inefficient quenching limiting the sensitivity of the assay. In addition, the chromophore and/or fluorophore absorbed light in the ultraviolet range reducing the sensitivity for detection in biological samples which typically contain molecules that absorb strongly in the ultraviolet. Broad utility of these substrates was also limited by the modifications to existing equipment required for optimal measurements.

Clearly fluorogenic protease indicators that show a high signal level when cleaved, and a very low signal level when intact, that show a high degree of protease specificity, and that operate exclusively in the visible range thereby rendering them suitable for use in biological samples are desirable. The compositions of the present invention provide these and other benefits.

SUMMARY

The present invention provides for novel reagents whose fluorescence increases in the presence of particular proteases. These fluorogenic protease indicators provide a high intensity fluorescent signal at a visible wavelength when they are digested by a protease. Because of their high fluorescence signal in the visible wavelengths, these protease indicators are particularly well suited for detection of protease activity in biological samples, in particular, in frozen tissue section and cultured or freshly isolated cells. The measurement can be carried out, e.g., using a fluorescence microscope for histological samples, cells, and the like and using a flow cytometer or microscope for cell suspensions and adherent cell cultures. Hence, the fluorogenic compositions of this invention allow detection of intracellular protease activity.

The fluorogenic protease indicators of the present invention are compositions suitable for detection of the activity of a protease. These compositions have the general formula:

in which P is a peptide comprising a protease binding site for said protease consisting of 2 to about 15, preferably 2 to about 12, preferably 2 to about 10, preferably 2 to about 8, 2 to about 6, or 2 to about 4 amino acids; F1 and F2 are fluorophores; S1 and S2 are peptide spacers ranging in length from 1 to about 50 amino acids; i and r are independently 0 or 1; and C1 and C2 are conformation determining regions comprising peptides ranging in length from 1 to about 8, amino acids, more preferably from 1 to about 6 amino acids. The conformation determining regions each introduce a bend into the composition or otherwise restrict the degrees of freedom of the peptide backbone, thereby juxtaposing the fluorophores with a separation of less than about 100 Å. When either of the spacers (S1 and S2) are present they are linked to the protease binding site by a peptide bond to the alpha carbon of the terminal amino acid. Thus, when i is 1, S1 is joined to C1 by a peptide bond through a terminal α-amino group of C1, and when r is 1, S2 is joined to C2 by a peptide bond through a terminal alpha carboxyl group of C2.

The amino acid residues comprising a protease binding site are, by convention, numbered relative to the peptide bond hydrolyzed by a particular protease. Thus the first amino acid residue on the amino side of the cleaved peptide bond is designated P1 while the first amino acid residue on the carboxyl side of the cleaved peptide bond is designated P1′. The numbering of the residues increases with distance away from the hydrolyzed peptide bond. Thus a four amino acid protease binding region would contain amino acids designated:

P2—P1—P1′—P2′

and the protease would cleave the binding region between P1 and P1′.

In particularly preferred embodiments, the fluorogenic compositions of this invention are compositions of Formula II and Formula V as described herein. Preferred fluorogenic indicators according to this invention have conformation determining regions and, optionally, spacers as described herein. In a most preferred embodiment, the compositions bear a single species of fluorophore. Fluorophores suitable for these “homolabeled” compositions include fluorophores that form H-type dimers. It was a surprising discovery of this invention that a single species of fluorophore is capable of “self-quenching” when it participates in the formation of an H-type dimer. Such self-quenching dimer formation is not limited to a particular backbone, but may be accomplished in a wide variety of configurations and thus the principle can be applied in many contexts. Thus, in one embodiment, this invention provides a fluorogenic composition comprising a polypeptide backbone or a nucleic acid backbone joining two fluorophores of the same species where the fluorophores form an H-dimer resulting in quenching of fluorescence of the fluorophores. Preferred polypeptide backbones range comprise a protease binding site ranging in length from about 2 to about 8, more preferably from about 2 to about 15 amino acids and certain polypeptide backbones range in length from about 4 to about 31 amino acids.

Similarly, preferred nucleic acid backbones range in length from about 10 to about 100 nucleotides, more preferably from about 15 to about 50 nucleotides. Certain preferred nucleic acid backbones comprise a restriction site.

In certain embodiments, the fluorogenic compositions are attached to a solid support, while in other embodiments, the fluorogenic compositions are inside a cell (e.g. a mammalian cell, an insect cell, a yeast cell, etc.). The fluorogenic compositions can also bear one or more hydrophobic groups (e.g. Fmoc, 9-fluoreneacetyl group, 1-fluorenecarboxylic group, 9-florenecarboxylic group, and 9-fluorenone-1-carboxylic group, benzyloxycarbonyl, Xanthyl (Xan), Trityl (Trt), 4-methyltrityl (Mtt), 4-methoxytrityl (Mmt), 4-methoxy-2,3,6-trimethyl-benzenesulphonyl (Mtr), Mesitylene-2-sulphonyl (Mts), 4,4′-dimethoxybenzhydryl (Mbh), Tosyl (Tos), 2,2,5,7,8-pentamethyl chroman-6-sulphonyl (Pmc), 4-methylbenzyl (MeBzl), 4-methoxybenzyl (MeOBzl), Benzyloxy (BzlO), Benzyl (Bzl), Benzoyl (Bz), 3-nitro-2-pyridinesulphenyl (Npys), 1-(4,4-dimentyl-2,6-diaxocyclohexylidene)ethyl (Dde), 2,6-dichlorobenzyl (2,6-DiCl-Bzl), 2-chlorobenzyloxycarbonyl (2-Cl-Z), 2-bromobenzyloxycarbonyl (2-Br-Z), Benzyloxymethyl (Bom), t-butoxycarbonyl (Boc), cyclohexyloxy (cHxO), t-butoxymethyl (Bum), t-butoxy (tBuO), t-Butyl (tBu), Acetyl (Ac), and Trifluoroacetyl (TFA), etc.). In certain particularly preferred embodiments, the hydrophobic group is attached to a terminus of the backbone (e.g. the carboxyl or amino terminus of a polypeptide backbone).

Particularly preferred fluorophores used in the compositions of this invention have an excitation wavelength between about 300 and 800 nm, more preferably between about 310 nm and about 750 nm, most preferably between about 315 nm and about 700 nm. In certain preferred embodiments, the fluorophores include, but are not limited to, carboxytetramethylrhodamine, carboxyrhodamine-X, carboxyrhodamine 110, diethylaminocoumarin, and carbocyanine dyes.

In still another embodiment, this invention provides a cell (e.g. mammalian cell, insect cell, yeast cell, etc.) comprising one or more of the fluorogenic indicators of this invention (e.g. as described above).

This invention also provides a method of detecting the activity of a protease. The method typically involves contacting the protease with a fluorogenic composition comprising a polypeptide backbone joining two fluorophores of the same species whereby the fluorophores form an H-dimer resulting in quenching of the fluorescence of said fluorophores (e.g. a peptide-backbone fluorogenic composition as described above); and detecting a change in fluorescence or absorbance of said fluorogenic composition where an increase in fluorescence or a change in absorbance indicates that the protease cleaves the polypeptide backbone. In certain preferred embodiments, the fluorogenic composition is attached to a solid support and/or is inside a cell (e.g. a mammalian cell). In certain embodiments, the contacting is in a histological section, a cell culture, a seeded or cultured adherent cell, or a cell suspension derived from a biological sample (e.g tissue, blood, urine, saliva, lymph, biopsy). Detection is by any of a number of methods known to those of skill in the art. Such methods include, but are not limited to fluorescence microscopy, confocal microscopy, fluorescence microplate reader, flow cytometry, fluorometry, and absorption spectroscopy.

In still another embodiment, this invention provides a method of detecting the activity of a nuclease or the presence of a nucleic acid. The method involves contacting the nuclease or the nucleic acid with a fluorogenic composition comprising a nucleic acid backbone joining two fluorophores of the same species whereby said fluorophores form an H-dimer resulting in quenching of the fluorescence of said fluorophores (e.g. a nucleic acid-backbone fluorogenic composition as described above); and detecting a change in fluorescence or absorbance of the fluorogenic composition where an increase in fluorescence or a change in absorbance indicates that the nuclease cleaves said nucleic acid backbone or that the nucleic acid hybridizes to the backbone. In certain preferred embodiments, the fluorogenic composition is attached to a solid support and/or is inside a cell (e.g. a mammalian cell). In certain embodiments, the contacting is in a histological section, a cell culture, a seeded or cultured adherent cell, or a cell suspension derived from a biological sample (e.g., tissue, blood, urine, saliva, lymph, biopsy). Detection is by any of a number of methods known to those of skill in the art. Such methods include, but are not limited to fluorescence microscopy, confocal microscopy, fluorescence microplate reader, flow cytometry, fluorometry, and absorption spectroscopy.

In yet another embodiment, this invention provides a method of detecting the interaction of a first and a second molecule. The method involves providing a first molecule having a first fluorophore attached thereto; providing a second molecule having a second fluorophore attached thereto wherein the first and second fluorophore are the same species of fluorophore and, when juxtaposed, form an H-dimer thereby quenching fluorescence produced by the fluorophores; and iii) detecting a change in fluorescence or absorbance produced by the fluorophores where a decrease in fluorescence or a change in absorbance indicates that the first molecule and the second molecule are interacting. Preferred first and second molecules include, but are not limited to a receptor and a receptor ligand, an antibody and an antigen, a lectin and a carbohydrate, a first protein and a second protein, and a nucleic acid and a nucleic acid binding protein. In particularly preferred embodiments, the fluorophore is linked to the first molecule by a linker. Preferred fluorophores include, but are not limited to, those described above.

This invention also provides a method of detecting a change in conformation or cleavage of a macromolecule. The method involves providing a macromolecule having attached thereto two fluorophores of the same species where the fluorophores form an H-dimer resulting in quenching of fluorescence of the fluorophores; and detecting a change in fluorescence or absorbance of the fluorophores wherein a change in fluorescence or fluorescence indicates a change in conformation or cleavage of the macromolecule. Preferred macromolecules, include, but are not limited to a polypeptide, a nucleic acid, a lipid, a polysaccharide, or an oligosaccharide. In various embodiments, the macromolecule is attached to a solid support or is inside a cell (e.g. a mammalian cell, an insect cell, a yeast cell, etc.). The macromolecule can, optionally, bear one or more hydrophobic groups e.g. a described above. Preferred fluorophores include, but are not limited to those described above. In certain embodiments, the contacting is in a histological section, a cell culture, a seeded or cultured adherent cell, or a cell suspension derived from a biological sample (e.g., tissue, blood, urine, saliva, lymph, biopsy). Detection is by any of a number of methods known to those of skill in the art. Such methods include, but are not limited to fluorescence microscopy, confocal microscopy, fluorescence microplate reader, flow cytometry, fluorometry, and absorption spectroscopy.

In still another embodiment, this invention provides a method of screening a test agent for the ability to modulate a protease (or a nuclease, lipase, etc.). The method involves contacting a protease or a cell comprising a protease with the test agent; contacting the protease with a fluorogenic indicator composition as described herein; and detecting a signal or lack of signal produced by the fluorogenic composition where a difference in the signal produced by the protease or cell contacted with the test agent compared to a control (e.g. a negative control) in which the protease or cell is contacted by said test agent at a lower concentration indicates that the test agent modulates activity of the protease. In preferred embodiments, the control comprises the absence of the test agent. Typically, an increase in signal produced by the protease or cell contacted with the test agent as compared to the control indicates that the test agent increases the activity of said protease, while a decrease in signal (e.g. fluorescence) produced by the protease or cell contacted with the test agent as compared to the control indicates that the test agent decreases the activity of said protease. The protease is contacted with the fluorogenic composition in the presence of the test agent in certain embodiments. In certain other embodiments, the protease is contacted with the fluorogenic composition after removal of the test agent. The method can further entail entering test agents that modulate activity of said protease into a database comprising a list of test agents modulating said protease. In various embodiments, the detecting comprises detecting an intracellular signal (e.g., via microscopy, flow cytometry, etc.). In certain particularly preferred embodiments, the detecting comprises high-throughput screening of whole cells.

The term “protease binding site” is used herein to refer to an amino acid sequence that is characteristically recognized and cleaved by a protease. The protease binding site contains a peptide bond that is hydrolyzed by the protease and the amino acid residues joined by this peptide bond are said to form the cleavage site. These amino acids are designated P1 and P1′ for the residues on the amino and carboxyl sides of the hydrolyzed bond respectively.

A fluorophore is a molecule that absorbs light at a characteristic wavelength and then re-emits the light most typically at a characteristic different wavelength. Fluorophores are well known to those of skill in the art and include, but are not limited to rhodamine and rhodamine derivatives, fluorescein and fluorescein derivatives, coumarins and chelators with the lanthanide ion series. A fluorophore is distinguished from a chromophore which absorbs, but does not characteristically re-emit light.

The terms “polypeptide”, “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. The term also includes variants on the traditional peptide linkage joining the amino acids making up the polypeptide. Preferred “peptides, eptides”, and “proteins” are chains of amino acids whose α carbons are linked through peptide bonds. The terminal amino acid at one end of the chain (amino terminal) therefore has a free amino group, while the terminal amino acid at the other end of the chain (carboxy terminal) has a free carboxyl group. As used herein, the term “amino terminus” (abbreviated N-terminus) refers to the free α-amino group on an amino acid at the amino terminal of a peptide or to the α-amino group (imino group when participating in a peptide bond) of an amino acid at any other location within the peptide. Similarly, the term “carboxy terminus” refers to the free carboxyl group on the carboxy terminus of a peptide or the carboxyl group of an amino acid at any other location within the peptide. Peptides also include essentially any polyamino acid including, but not limited to peptide mimetics such as amino acids joined by an ether as opposed to an amide bond.

The polypeptides described herein are preferably written with the amino terminus at the left and the carboxyl terminus at the right. The amino acids comprising the peptide components of this invention are numbered with respect to the protease cleavage site, with numbers increasing consecutively with distance in both the carboxyl and amino direction from the cleavage site. Residues on the carboxyl site are either notated with a “′” as in P1′, or with a letter and superscript indicating the region in which they are located. The “′” indicates that residues are located on the carboxyl side of the cleavage site.

The term “residue” or “amino acid” as used herein refers to an amino acid that is incorporated into a peptide. The amino acid may be a naturally occurring amino acid and, unless otherwise limited, may encompass known analogs of natural amino acids that can function in a similar manner as naturally occurring amino acids.

The term “domain” or “region” refers to a characteristic region of a polypeptide. The domain may be characterized by a particular structural feature such as a β turn, an alpha helix, or a β pleated sheet, by characteristic constituent amino acids (e.g. predominantly hydrophobic or hydrophilic amino acids, or repeating amino acid sequences), or by its localization in a particular region of the folded three dimensional polypeptide. As used herein, a region or domain is composed of a series of contiguous amino acids.

The terms “protease activity” or “activity of a protease” refer to the cleavage of a peptide by a protease. Protease activity comprises the “digestion” of one or more peptides into a larger number of smaller peptide fragments. Protease activity of particular proteases may result in hydrolysis at particular peptide binding sites characteristically recognized by a particular protease. The particular protease may be characterized by the production of peptide fragments bearing particular terminal amino acid residues.

The terms “nucleic acid” or “oligonucleotide” refer to at least two nucleotides covalently linked together. A nucleic acid of the present invention is preferably single-stranded or double stranded and will generally contain phosphodiester bonds, although in some cases, as outlined below, nucleic acid analogs are included that may have alternate backbones, comprising, for example, phosphoramide (Beaucage et al. (1993) Tetrahedron 49(10):1925) and references therein; Letsinger (1970) J. Org. Chem. 35:3800; Sprinzl et al. (1977) Eur. J. Biochem. 81: 579; Letsinger et al. (1986) Nucl. Acids Res. 14: 3487; Sawai et al. (1984) Chem. Lett. 805, Letsinger et al. (1988) J. Am. Chem. Soc. 110: 4470; and Pauwels et al. (1986) Chemica Scripta 26: 1419), phosphorothioate (Mag et al. (1991) Nucleic Acids Res. 19:1437; and U.S. Pat. No. 5,644,048), phosphorodithioate (Briu et al. (1989) J. Am. Chem. Soc. 111:2321, O-methylphosphoroamidite linkages (see Eckstein, Oligonucleotides and Analogues: A Practical Approach, Oxford University Press), and peptide nucleic acid backbones and linkages (see Egholm (1992) J. Am. Chem. Soc. 114:1895; Meier et al. (1992) Chem. Int. Ed. Engl. 31: 1008; Nielsen (1993) Nature, 365: 566; Carlsson et al. (1996) Nature 380: 207). Other analog nucleic acids include those with positive backbones (Denpcy et al. (1995) Proc. Natl. Acad. Sci. USA 92: 6097; non-ionic backbones (U.S. Pat. Nos. 5,386,023, 5,637,684, 5,602,240, 5,216,141 and 4,469,863; Angew. (1991) Chem. Intl. Ed. English 30: 423; Letsinger et al. (1988) J. Am. Chem. Soc. 110:4470; Letsinger et al. (1994) Nucleoside & Nucleotide 13:1597; Chapters 2 and 3, ASC Symposium Series 580, “Carbohydrate Modifications in Antisense Research”, Ed. Y. S. Sanghui and P. Dan Cook; Mesmaeker et al. (1994), Bioorganic & Medicinal Chem. Lett. 4: 395; Jeffs et al. (1994) J. Biomolecular NMR 34:17; Tetrahedron Lett. 37:743 (1996)) and non-ribose backbones, including those described in U.S. Pat. Nos. 5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580, Carbohydrate Modifications in Antisense Research, Ed. Y.S. Sanghui and P. Dan Cook. Nucleic acids containing one or more carbocyclic sugars are also included within the definition of nucleic acids (see Jenkins et al. (1995), Chem. Soc. Rev. pp 169-176). Several nucleic acid analogs are described in Rawls, C & E News Jun. 2, 1997 page 35. These modifications of the ribose-phosphate backbone may be done to facilitate the addition of additional moieties such as labels, or to increase the stability and half-life of such molecules in physiological environments. Preferred nucleic acid backbones used in this invention range from about 5 nucleotides to about 500 nucleotides, preferably from about 10 nucleotides to about 100 nucleotides, more preferably from about 10 nucleotides to about 50 nucleotides, and most preferably from about 12 or 15 nucleotides to about 30, 40, or 50 nucleotides in length.

The term “test agent” refers to an agent that is to be screened in one or more of the assays described herein. The agent can be virtually any chemical compound. It can exist as a single isolated compound or can be a member of a chemical (e.g. combinatorial) library. In a particularly preferred embodiment, the test agent will be a small organic molecule.

The term “small organic molecule” refers to a molecule of a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macromolecules (e.g., proteins, nucleic acids, etc.). Preferred small organic molecules range in size up to about 3000 Da, more preferably up to 2000 Da, and most preferably up to about 1000 Da.

The term macromolecule refers to a “large” molecule. Biopolymers (e.g. proteins, glycoproteins, carbohydrates, lipids, polysaccharides, and the like) are typical macromolecules. Typical macromolecules have a molecular weight greater than about 1000 Da, preferably greater than about 2000 Da, more preferably greater than about 3000 Da, and most preferably greater than about 4,000 or 5,000 Da.

The term database refers to a means for recording and retrieving information. In preferred embodiments the database also provides means for sorting and/or searching the stored information. The database can comprise any convenient media including, but not limited to, paper systems, card systems, mechanical systems, electronic systems, optical systems, magnetic systems or combinations thereof. Preferred databases include electronic (e.g. computer-based) databases. Computer systems for use in storage and manipulation of databases are well known to those of skill in the art and include, but are not limited to “personal computer systems”, mainframe systems, distributed nodes on an inter- or intra-net, data or databases stored in specialized hardware (e.g. in microchips), and the like.

The term “biological sample”, as used herein, refers to a sample obtained from an organism, from components (e.g., cells or tissues) of an organism, and/or from in vitro cell or tissue cultures. The sample may be of any biological tissue or fluid (e.g. blood, serum, lymph, cerebrospinal fluid, urine, sputum, etc.). Biological samples can also include whole organisms, organs or sections of tissues such as frozen sections taken for histological purposes.

The term “specifically binds”, when referring to the interaction of a nucleic acid binding protein and a nucleic acid binding site or two proteins or other binding pairs refers to a binding reaction which is determinative of the presence of the one or other member of the binding pair in the presence of a heterogeneous population of molecules (e.g., proteins and other biologics). Thus, for example, in the case of a receptor/ligand binding pair the ligand would specifically and/or preferentially select its receptor from a complex mixture of molecules, or vice versa. An enzyme would specifically bind to its substrate, etc. The binding may be by one or more of a variety of mechanisms including, but not limited to ionic interactions, covalent interactions, hydrophobic interactions, van der Waals interactions, etc.

The terms “binding partner”, or a member of a “binding pair”, or “cognate ligand” refers to molecules that specifically bind other molecules to form a binding complex such as antibody/antigen, lectin/carbohydrate, nucleic acid/nucleic acid, receptor/receptor ligand (e.g. IL-4 receptor and IL-4), avidin/biotin, etc.

The term ligand is used to refer to a molecule that specifically binds to another molecule. Commonly a ligand is a soluble molecule, e.g. a hormone or cytokine, that binds to a receptor. The decision as to which member of a binding pair is the ligand and which the “receptor” is often a little arbitrary when the broader sense of receptor is used (e.g., where there is no implication of transduction of signal). In these cases, typically the smaller of the two members of the binding pair is called the ligand. Thus, in a lectin-sugar interaction, the sugar would be the ligand (even if it is attached to a much larger molecule, recognition is of the saccharide).

The terms “hybridizing specifically to” and “specific hybridization” and “selectively hybridize to,” as used herein refer to the binding, duplexing, or hybridizing of a nucleic acid molecule preferentially to a particular nucleotide sequence under stringent conditions. The term “stringent conditions” refers to conditions under which a probe will hybridize preferentially to its target subsequence, and to a lesser extent to, or not at all to, other sequences. Stringent hybridization and stringent hybridization wash conditions in the context of nucleic acid hybridization are sequence dependent, and are different under different environmental parameters. An extensive guide to the hybridization of nucleic acids is found in, e.g., Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes part 1, chapt 2, Overview of principles of hybridization and the strategy of nucleic acid probe assays, Elsevier, N.Y. (Tijssen). Generally, highly stringent hybridization and wash conditions are selected to be about 5° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Very stringent conditions are selected to be equal to the Tm for a particular probe. An example of stringent hybridization conditions for hybridization of complementary nucleic acids which have more than 100 complementary residues on an array or on a filter in a Southern or northern blot is 42° C. using standard hybridization solutions (see, e.g., Sambrook (1989) Molecular Cloning: A Laboratory Manual (2nd ed.) Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor Press, NY, and detailed discussion, below), with the hybridization being carried out overnight. An example of highly stringent wash conditions is 0.15 M NaCl at 72° C. for about 15 minutes. An example of stringent wash conditions is a 0.2×SSC wash at 65° C. for 15 minutes (see, e.g., Sambrook supra.) for a description of SSC buffer). Often, a high stringency wash is preceded by a low stringency wash to remove background probe signal. An example medium stringency wash for a duplex of, e.g., more than 100 nucleotides, is 1×SSC at 45° C. for 15 minutes. An example of a low stringency wash for a duplex of, e.g., more than 100 nucleotides, is 4× to 6×SSC at 40° C. for 15 minutes.

The term “nucleic acid array” refers to a collection of nucleic acids comprising a multiplicity of different nucleic acids (nucleic acid species). The nucleic acids are typically attached to a solid support. The support can be contiguous and of virtually any convenient geometry (e.g. a glass or quartz slide). In other embodiments, the support is not contiguous, e.g., where the array nucleic acids are disposed on a collection of particles, e.g. beads. The nucleic acids comprising the array can be chemically synthesized nucleic acids, naturally occurring nucleic acids, cloned nucleic acids, or any combination thereof. Preferred nucleic acid arrays are “high density arrays” or “microarrays”. Typically such microarrays have a density of greater than about 100, preferably greater than about 1000, more preferably greater than about 10,000, and most preferably greater than about 100,000 array elements per square centimeter.

The term “array element” refers to a domain of an array comprising substantially one species of nucleic acid.

Two fluorophores are said to quench each other in an H-dimer when their aggregate fluorescence in an H-dimer formation is detectably less than the aggregate fluorescence of the fluorophores when they are separated (e.g. in solution at approximately 10 μM or less). In preferred embodiments the fluorophores quench by at least 50%, preferably by at least 70%, more preferably by at least 80%, and most preferably by at least 90%, 95%, or even at least 99%.

Certain amino acids referred to herein are described by shorthand designations as shown in Table 1.

TABLE 1 Amino acid nomenclature. Abbreviation Name 3 Letter 1 Letter Alanine Ala A βAlanine (NH2—CH2—CH2—COOH) βAla Arginine Arg R Asparagine Asn N Aspartic Acid Asp D Cysteine Cys C Glutamic Acid Glu E Glutamine Gln Q Glycine Gly G Histidine His H Homoserine Hse — Isoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met M Methionine sulfoxide Met (O) — Methionine methylsulfonium Met (S-Me) — Norleucine Nle — Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V episilon-aminocaproic acid Ahx J (NH2—(CH2)5—COOH) 4-aminobutanoic acid (NH2—(CH2)3—COOH) gAbu — tetrahydroisoquinoline-3-carboxylic acid — O 8-aminocaprylic acid —

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