Luciferase sequences that utilize infrared-emitting substrates to produce enhanced luminescence

Abstract

To provide a near-infrared bioluminescence system with a longer-wavelength and lower-energy emission for small animal optical imaging applications, which makes it possible to detect signals from deep tissue, where standard near-infrared bioluminescence systems (450-620 nm) tend to be strongly absorbed.SOLUTION: Provided herein are isolated polynucleotide encoding modified click beetle luciferase polypeptides that have enhanced luminescence and longer wavelength near-infrared signals. The disclosure also relates to near-infrared bioluminescence systems that include the modified click beetle luciferase polypeptides and novel luciferin derivatives, as well as methods of using the modified click beetle luciferase polypeptides and bioluminescence systems.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 049,150, filed September 11, 2014, which is incorporated herein by reference in its entirety.

[0002] The present invention relates to isolated polynucleotides encoding click beetle luciferase mutant polypeptides that have enhanced luminescence and produce longer, near-infrared wavelength signals. The present invention also relates to near-infrared bioluminescence systems comprising the click beetle luciferase mutant polypeptides and infrared-emitting substrates, as well as methods of using the click beetle luciferase mutant polypeptides and near-infrared bioluminescence systems. [Background technology]

[0003] Long-wavelength, low-energy-emitting bioluminescence has attracted considerable attention for both multiplexing applications using multiple emission colors and deep-tissue imaging, where short wavelengths are strongly absorbed. Many standard optical imaging systems have limited utility in whole-animal imaging due to reduced light transmission through biological samples. Optical penetration is limited by the absorption coefficients of certain components in blood. Strong absorption by hemoglobin (Hb) and oxygenated hemoglobin (HbO2) reduces light transmission and penetration depth through blood and animal tissue. Luminescent systems emitting light in the far-red and near-infrared range (680–900 nm) enable optimal imaging because the absorption spectra of Hb and HbO2 are minimal. This region of maximum optical penetration is known as the "optical window" for whole-animal imaging. Bioluminescent reporter systems are widely used in research animals but still struggle with the limitations of reduced tissue penetration. Typical bioluminescent emission wavelengths (460–620 nm) occur in a region with limited penetration depth. For an ideal bioluminescent reporter system in whole-body animals, bright emission in the 680–900 nm region would be highly beneficial. While many bioluminescent systems have been engineered to red-shift their visible light emission, none have achieved strong red emission that significantly overlaps the critical “optical window” of blood penetration.

[0004] Previous approaches for in vivo molecular imaging include quantum dot conjugates that emit light via bioluminescence resonance energy transfer (BRET) in the absence of external excitation. These conjugates, prepared by coupling carboxylate-displaying quantum dots to mutants of the Renilla reniformis luciferase bioluminescent protein, emit long-wavelength (red to near-infrared) bioluminescent light in cells and animals, as well as in deep tissues. However, this approach is limited by the signal strength associated with Renilla luciferase and the relatively low aqueous solubility of the coelenterazine substrate. Another approach, e.g., Akarumine (Wako), utilizes a luciferin derivative that is dim red-purple (675 nm) (see Figures 19A and 19B).

[0005] Therefore, near-infrared bioluminescence systems emitting longer wavelengths and lower energy, which would enable detection of signals from deep tissues where conventional near-infrared bioluminescence systems (450–620 nm) tend to be strongly absorbed, are needed for small animal optical imaging applications. Summary of the Invention

[0006] The present invention relates to oligonucleotides having at least 80% amino acid sequence identity to SEQ ID NO: 1 and containing at least one amino acid substitution at positions 4, 16, 34, 47, 51, 52, 55, 72, 73, 74, 79, 82, 83, 87, 89, 104, 109, 113, 117, 119, 124, 130, 131, 133, 136, 144, 146, 156, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 170, 179, 186, 200, 211, 218, 224, 225, 226, 228, 229, 234, 247, 251, 252, 253, 255, 280, 281, 285, 308, 309, 310, 319, 329, 334, 335, 337, 346, 348, 349, 350, 352, 354, 355, 358, 363, 370, 377, 39 539, 444, 445, 453, 455, 467, 471, 473, 479, 484, 489, 496, 501, 503, 508, 516, 528, 531, 535, 537, 539, or a combination thereof, wherein the mutant CBR polypeptide has at least one of enhanced light emission, a shift in emitted light wavelength (emission wavelength), a change in substrate specificity, or a combination thereof, compared to the CBR polypeptide of SEQ ID NO: 1. The CBR mutant polypeptide may further comprise at least one amino acid substitution at a position corresponding to position 351, 389, 457, or a combination thereof, of SEQ ID NO: 1.CBR mutant polypeptides include R4H, H16Q, H34Y, D47E, S51N, Y52C, F55L / V, K72E, I79V, M73K / T, N74S, E82G, N83H, F87S, I89V, V104D, I109N / V, L113Q, M117T, I119F / T, I124V, N130K, I131N / T, N133D, K136N, F144L, and R4H of SEQ ID NO: 1. K146E, N156D, N156K, G159D, Y170C, K179S, V186A, G200G, N211N, H218L / Y, G225S, T226C / G / H / N / Q / Y, L 228P, I229V, V234A, G251S, G251I, Y252C, V255D / F, E253K, R280S, S281N / Q, V285A, I309T, E319G, N329 D, R334E / Q / H / S / N / K, C335S, K337E, I346N, Q348H / E, L350P, G351K / R, D352N, R355G, S358P, T363A / S, I 370T, I389F / G / S / V, I390I, M393K / L, V394M, N400D, N401S, I409T, D412G, F420F, Y422C, V431A, E437G, The CBR mutant polypeptide may include substitutions corresponding to at least one of I439V, S444C / R / T, Q445H, E453K, V455D, K457N, D471V, E473A, S479T, K484E / M / R, E489V, Y496H, E501G, V503M, Y508C, V516A, T528A, E531G, Q535H, L537W, K539R, or combinations thereof. The CBR mutant polypeptide may include amino acid substitutions at positions corresponding to positions 389, 444, and 251 of SEQ ID NO:1. The amino acid substitutions may include I389F, S444R, and G251S. The CBR mutant polypeptide may include amino acid substitutions at positions corresponding to positions 334 and 351 of SEQ ID NO:1. The amino acid substitutions may include R334S and G351R. The CBR mutant polypeptide may further include amino acid substitutions at positions corresponding to positions 51 and 444 of SEQ ID NO: 1. The amino acid substitutions may include S51N and S444R. The CBR mutant polypeptide may include the amino acid polypeptide of SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4.The CBR mutant polypeptide may have enhanced luminescence compared to the CBR polypeptide of SEQ ID NO: 1. The CBR mutant polypeptide may have enhanced luminescence when luciferin is utilized by the CBR mutant polypeptide to generate luminescence. The CBR mutant polypeptide may have enhanced luminescence when a luciferin derivative is utilized by the CBR mutant polypeptide to generate luminescence. The luciferin derivative is TIFF0007780560000001.tif39162. The luminescence of the CBR mutant polypeptide may be increased by at least two-fold compared to the CBR polypeptide of SEQ ID NO: 1. The luminescence of the CBR mutant polypeptide may be increased by at least four-fold compared to the CBR polypeptide of SEQ ID NO: 1. The CBR mutant polypeptide may have an altered emission light spectrum compared to the CBR polypeptide of SEQ ID NO: 1. The CBR mutant polypeptide may be capable of emitting light at a longer wavelength when luciferin is utilized by the CBR mutant polypeptide to generate luminescence. The CBR mutant polypeptide may be capable of emitting light at a longer wavelength when a luciferin derivative is utilized by the CBR mutant polypeptide to generate luminescence. The luciferin derivative may be TIFF0007780560000002.tif39162. The luciferin derivative may be JPEG0007780560000003.jpg3380, the CBR mutant polypeptide may emit light having a spectral maximum shifted by at least about 1 nm to at least about 100 nm relative to the light produced by the CBR polypeptide of SEQ ID NO: 1. The CBR mutant polypeptide may emit light having a spectral maximum between about 650 nm and about 800 nm. The CBR mutant polypeptide may emit light having a spectral maximum between about 725 nm and about 775 nm. The CBR mutant polypeptide may emit light having a spectral maximum at about 750 nm. When a luciferin derivative is used, JPEG0007780560000004.jpg3377, the CBR mutant polypeptide may emit light having a shift in spectral maximum of at least about 1 nm to at least about 100 nm relative to the light produced by the CBR polypeptide of SEQ ID NO: 1. The CBR mutant polypeptide may emit light having a shift in spectral maximum of at least about 75 nm relative to the light produced by the CBR polypeptide of SEQ ID NO: 1. The CBR mutant polypeptide may emit light having a spectral maximum between about 650 nm and about 800 nm. The CBR mutant polypeptide may emit light having a spectral maximum between about 700 nm and about 775 nm. The CBR mutant polypeptide may emit light having a spectral maximum at about 725 nm. The CBR mutant polypeptide may have altered substrate specificity compared to the CBR polypeptide of SEQ ID NO: 1. The CBR mutant polypeptide may have altered relative specificity for the CBR mutant polypeptide in the presence of luciferin compared to a luciferin derivative. A CBR mutant polypeptide can have a change in relative specificity for the CBR mutant polypeptide in the presence of a luciferin derivative compared to a different luciferin derivative. TIFF0007780560000005.tif39162. The mutant CBR polypeptide may have luciferase activity. The mutant CBR polypeptide may have a Km for PBI-4813 of at least about 0.01 μM to at least about 5.00 μM. The mutant CBR polypeptide may have a Km for PBI-4813 of at least about 0.50 μM to at least about 3.00 μM. The mutant CBR polypeptide may have a Km for PBI-4813 of at least about 0.82 μM or 2.41 μM. The mutant CBR polypeptide may have a Km for PBI-4739 of at least about 0.01 μM to at least about 5.00 μM. The mutant CBR polypeptide may have a Km for PBI-4739 of at least about 1.50 μM to at least about 4.50 μM. The mutant CBR polypeptide may have a Km for PBI-4739 of at least about 2.33 μM or 3.95 μM. The mutant CBR polypeptide may have a relative Vmax using PBI-4813 as a substrate that is at least two-fold higher than the relative Vmax of the CBR polypeptide of SEQ ID NO: 1. The mutant CBR polypeptide may have a relative Vmax using PBI-4739 as a substrate that is at least two-fold higher than the relative Vmax of the CBR polypeptide of SEQ ID NO: 1. The sequence may be codon-optimized. The sequence may comprise a polynucleotide of SEQ ID NO: 6-9. The polynucleotide may further encode a polypeptide of interest linked to the CBR mutant polypeptide, and the polypeptide of interest and the CBR mutant polypeptide may be expressed as a fusion protein. The polypeptide of interest may comprise HALOTAG®.

[0007] The present invention relates to a vector comprising the above-mentioned polynucleotide, or a fragment thereof. The polynucleotide may be operably linked to a promoter.

[0008] The present invention relates to a cell comprising the above-mentioned polynucleotide or the above-mentioned vector.

[0009] The present invention relates to a non-human transgenic animal comprising the above-described cells.

[0010] The present invention relates to a non-human transgenic animal comprising the above-mentioned polynucleotide or the above-mentioned vector.

[0011] The present invention relates to CBR mutant polypeptides encoded by the above-described polynucleotides.

[0012] The present invention relates to a circularly permuted luciferase comprising a polypeptide encoded by the above-described polynucleotide or a fragment thereof.

[0013] The present invention relates to fusion proteins comprising the CBR mutant polypeptides encoded by the above-described polynucleotides.

[0014] The present invention relates to a near-infrared bioluminescence system comprising the above-mentioned polynucleotide and a luciferin derivative. It may contain TIFF0007780560000006.tif39162.

[0015] The present invention relates to a method for producing a CBR mutant polypeptide, comprising growing the above-described cells under conditions that allow expression of the CBR mutant polypeptide.

[0016] The present invention relates to a method for producing a CBR mutant polypeptide, comprising introducing the vector described above into a cell under conditions that allow expression of the CBR mutant polypeptide.

[0017] The present invention relates to a kit comprising the above-mentioned polynucleotide or the above-mentioned vector.

[0018] The present invention also relates to a kit comprising the above-described CBR mutant polypeptide. (a) TIFF0007780560000007.tif36164 and (b) at least one of the buffer reagents.

[0019] The present invention relates to a bioluminescence resonance energy transfer (BRET) system, comprising: a first fusion protein comprising a first target protein and a bioluminescent donor molecule, wherein the bioluminescent donor molecule is a CBR mutant encoded by the polynucleotide described above; a second fusion protein comprising a second target protein and a fluorescent acceptor molecule; and a CBR substrate. The CBR substrate may be luciferin or a luciferin derivative. The luciferin derivative may be: It may contain TIFF0007780560000008.tif39162.

[0020] The present invention relates to methods for measuring bioluminescence using at least one of the above polynucleotides, the above vectors, the above cells, the above animals, the above CBR mutant polypeptides, the above circularly permuted luciferases, the above fusion proteins, or the above near-infrared bioluminescence systems. Bioluminescence may be measured in a living, intact non-human animal.

[0021] The present invention relates to a method for measuring the enzymatic activity of a luminogenic protein, the method comprising contacting a luminogenic protein, a deprotecting enzyme, and a protected luminophore; and detecting light produced from the composition, wherein the luminogenic protein is a CBR mutant encoded by the polynucleotide described above, and the luminophore is TIFF0007780560000009.tif35156. Enzyme activity may be measured in living, intact non-human animals.

[0022] The present invention relates to a method for measuring the activity of a non-luminescent enzyme of interest, the method comprising: (a) providing a luminescent molecule, wherein the molecule is a substrate for the non-luminescent enzyme of interest and a pro-substrate for a CBR mutant encoded by the polynucleotide described above; (b) contacting the luminescent molecule with at least one non-luminescent enzyme of interest and at least one CBR mutant to produce a reaction mixture; and (c) determining the activity of the non-luminescent enzyme of interest by measuring the luminescence of the reaction mixture. The non-luminescent enzyme of interest may be a protease enzyme, a cytochrome P450 enzyme, a monoamine oxidase, or a glutathione S-transferase. The activity of the non-luminescent enzyme may be measured in a living, intact animal.

[0023] The present invention relates to a method for detecting the presence of at least two molecules in a sample or cell, the method comprising contacting the sample or cell with a first reporter molecule comprising a CBR variant encoded by the polynucleotide described above, wherein the first reporter molecule is operably linked to a first component of the sample or cell; contacting the sample with a second reporter molecule, wherein the second reporter molecule is operably linked to a second component of the sample or cell; and detecting the presence of the first and second reporter molecules to determine the presence and / or amount of the first and second components in the sample or cell.

[0024] The present invention relates to a method for detecting an interaction between a first protein and a second protein in a sample, the method comprising: The method includes contacting a sample with a first polynucleotide encoding a first fusion protein, wherein the first fusion protein comprises a first fragment of a luciferase and a first protein; and a second polynucleotide encoding a second fusion protein, wherein the second fusion protein comprises a second fragment of the luciferase and a second protein; and detecting luminescence in the sample. The detection of luminescence indicates an interaction between the first protein and a second protein, wherein the luciferase is encoded by the isolated polynucleotide described above. When the first protein and the second protein interact, the first fragment of the luciferase and the second fragment of the luciferase can reconstitute a full-length enzyme capable of stably binding to a cell-permeable substrate.

[0025] The present invention relates to a method for detecting an interaction between a first protein and a second protein in a sample, the method comprising: The sample TIFF0007780560000012.tif39157, wherein the sample comprises a first polynucleotide encoding a first fusion protein, wherein the first fusion protein comprises a luciferase and a first protein, the luciferase being encoded by the isolated polynucleotide of any one of claims 1 to 46; and a second polynucleotide encoding a second fusion protein, wherein the second fusion protein comprises a fluorescent acceptor molecule and a second protein; and (b) detecting bioluminescence resonance energy transfer (BRET) in the sample, which indicates an interaction or proximity of a bioluminescent donor and a fluorescent acceptor. [Brief explanation of the drawings]

[0026] [Figure 1] The chemical structures of PBI-4739 and PBI-4813 are shown. [Figure 2]2A and 2B show spectral scans of click beetle red luciferase (CBR) using PBI-4813 (FIG. 2A) and PBI-4739 (FIG. 2B) as substrates. [Figure 3] 3A and 3B show spectral scans of Ultra-Glo™ luciferase using PBI-4813 (FIG. 3A) and PBI-4739 (FIG. 3B) as substrates. [Figure 4] 4A and 4B show spectral scans of QuantiLum® recombinant luciferase (“firefly”) using PBI-4813 (FIG. 4A) and PBI-4739 (FIG. 4B) as substrates. [Figure 5] Normalized luminescence of CBR mutants and CBR ("wild type") using PBI-4813 (dark bars) and PBI-4739 (light bars) as substrates is shown. [Figure 6] Normalized luminescence of CBR mutants and CBR in HEK293 cells using PBI-4813 (dark bars) and PBI-4739 (light bars) as substrates is shown. [Figure 7] Normalized luminescence of CBR mutants and CBR in HeLa cells using PBI-4813 (dark bars) and PBI-4739 (light bars) as substrates is shown. [Figure 8] Normalized luminescence of CBR mutants and CBR ("wild type") using PBI-4813 (dark bars) and PBI-4739 (light bars) as substrates is shown. [Figure 9] Normalized luminescence of CBR mutants and CBR ("ATG685") using luciferin ("D-LH2"; dark bars), PBI-4813 (medium dark bars), and PBI-4739 (light bars) as substrates is shown. [Figure 10] Normalized luminescence of CBR mutants ATG1230 and ATG1250 and CBR using PBI-4813 (dark bars) and PBI-4739 (light bars) as substrates is shown. [Figure 11]11A and 11B show the spectral properties of CBR mutants ATG1230 and ATG1240 compared to CBR using PBI-4813 (FIG. 11A) and PBI-4739 (FIG. 11B) as substrates. [Figure 12] Figures 12A and 12B show Km titrations of CBR mutants ATG1230 and ATG1240 compared to CBR for PBI-4813 (Figure 12A) and PBI-4739 (Figure 12B) as substrates. [Figure 13] 1 shows the kinetic profiles of CBR and CBR mutants ATG1230 and ATG1240 in viable cells at 37° C. using PBI-4813 and PBI-4739 as substrates. [Figure 14] Luminescence between CBR mutants ATG1230 and ATG1240 is shown compared to CBR using PBI-4813 (medium dark bars) and PBI-4739 (light bars) as substrates and firefly luciferase ("Luc2") using luciferin ("D-LH2") as a substrate (dark bars). [Figure 15] Normalized luminescence of CBR mutants using luciferin ("D-LH2"; light bars), PBI-4813 (dark bars), and PBI-4739 (medium dark bars) as substrates is shown. [Figure 16] Figures 16A and 16B show viable cell substrate titrations of CBR mutant ATG1240 compared to CBR using PBI-4739 (Figure 16A) and PBI-4813 (Figure 16B) as substrates. [Figure 17] Luminescence measured using a long-pass filter to mimic tissue attenuation of the CBR mutant ATG1240 using luciferin ("D-LH2"; medium dark bar), PBI-4813 (medium light bar), and PBI-4739 (light bar) as substrates compared to firefly luciferase ("Luc2") using luciferin (dark bar). [Figure 18] The amino acid sequence of the CBR is shown with the target amino acid sites for insertional mutagenesis in bold and underlined. [Figure 19]Figures 19A and 19B show the luminescence of CBR using acarmine, PBI-4739, and PBI-4813 (Figure 19A) and the spectral scan of QuantiLum® recombinant luciferase using acarmine as a substrate (Figure 19B). [Figure 20] Figures 20A and 20B show the luminescence of codon-optimized CBR mutants expressed in CHO cells (Figure 20A) or 3T3 cells (Figure 20B) using Bright-Glo™ assay buffer. [Figure 21] Figures 21A and 21B show the Km titration of CBR with PBI-4813 and D-luciferin ("D-LH2") as substrates in live HeLa cells. [Figure 22] 1 shows the Km titration of CBR against PBI-4813, PBI-4739, and D-luciferin as substrates in live HEK293T cells. [Figure 23] Figures 23A and 23B show the Km titration of the CBR mutant ATG1230 compared to CBR with PBI-4813 (Figure 23A) and PBI-4739 (Figure 23B) as substrates in live HEK293T cells. DETAILED DESCRIPTION OF THE INVENTION

[0027] Disclosed herein are click beetle red luciferase (CBR) mutants with improved performance in the near-infrared (near-IR) range using luciferin and / or novel luciferin derivatives. The disclosed CBR mutants have improved performance in vitro and in living cells, e.g., increased brightness and longer wavelength. Also disclosed herein are near-IR bioluminescence systems comprising the CBR mutants and novel luciferin derivatives. The disclosed near-IR bioluminescence systems have improved performance in the near-IR range and address sensitivity limitations associated with other bioluminescence detection systems related to tissue absorption. Unlike other bioluminescence systems, such as VivoGlo™ (Promega Corp.), the disclosed near-IR bioluminescence systems provide near-IR luminescence signals that penetrate tissue well and improve detection of signals from deep animal tissues. The disclosure also encompasses kits and methods of use that include the CBR mutants and / or bioluminescence systems.

[0028] 1.Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and are not intended to be limiting.

[0029] As used herein, the terms "comprise(s)," "include(s)," "having," "has," "can," "containing," and variations thereof are intended to be open-ended conjunctions, terms, or words that do not exclude the possibility of additional acts or structures. The terms used herein are for the purpose of describing particular embodiments only and are not intended to be limiting. As used in the specification and the appended claims, the singular forms "a," "and," and "the" include plural references unless the context clearly dictates otherwise. The present disclosure contemplates other embodiments that "comprising," "consisting of," and "consisting essentially of" the embodiments or elements presented herein, whether or not expressly stated.

[0030] For the recitation of numerical ranges herein, each intervening value is expressly contemplated to the same degree of precision. For example, for the range of 6 to 9, the values ​​7 and 8 are contemplated in addition to 6 and 9, and for the range of 6.0 to 7.0, the values ​​6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are expressly contemplated.

[0031] As used herein, "bioluminescence" or "luminescence" is the light produced as a result of a reaction between an enzyme and a light-producing substrate.

[0032] Generally, "enhanced" means that a particular property (e.g., bioluminescence or luminescence) is increased relative to that of a reference luciferase+luciferin combination or luciferase under test, where the increase is at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 20%, at least 25%, at least 50%, at least 75%, at least 90%, at least 100%, at least 200%, at least 500%, or at least 1000% greater than the reference luciferase+luciferin combination or luciferase under test.

[0033] The term "luciferin substrate," as used herein, refers to a molecule (e.g., luciferin, a luciferin derivative, or a functional analog thereof) that can create light through a chemical or biochemical reaction. Suitable luciferin substrates for luciferase enzymes include luciferin, luciferin derivatives, and functional analogs of luciferin. In some embodiments, functional analogs of luciferin include modified luciferins, including derivatives of these compounds. Exemplary compounds include those disclosed in U.S. Patent Application No. 14 / 200,563.

[0034] The naturally occurring substrate for beetle luciferase is firefly luciferin, which is a polytherocyclic organic acid, D-(-)-2-(6'-hydroxy-2'-benzothiazolyl)-Δ 2 The most common luciferin is thiazoline-4-carboxylic acid ("D-luciferin," also known as "luciferin"). Luciferin may be isolated from natural sources (e.g., from fireflies) or synthesized. Synthetic luciferin can have the same structure as naturally occurring luciferin, or it can be derivatized so long as it functions similarly. Examples of luciferin derivatives include D-luciferin methyl ester and other esters of luciferase, as well as naphthyl- and quinolyl-luciferin, which are hydrolyzed or acted upon by esterases in the sample to produce luciferin (Branchini et al., 1989). There are numerous sources that commercially offer luciferin (e.g., Promega Corp., Madison, Wisconsin).

[0035] The term "luciferin derivative," as used herein, refers to a type of luminescent molecule or compound having the substantial structure of D-luciferin and is a luciferase substrate, e.g., aminoluciferin, or a luciferase substrate disclosed in U.S. Patent Publication No. 2007 / 0015790.

[0036] A "luciferase reaction mixture" contains a luciferase enzyme and materials that will enable the luciferase enzyme to generate a light signal. The materials needed to generate a luminescent signal, as well as the specific concentrations and / or amounts of the materials needed, will vary depending on the luciferase enzyme used and the type of luciferase-based assay being performed. Generally, for click beetle luciferase, including CBR mutants, these materials can include ATP, a magnesium (Mg2+) salt, e.g., magnesium sulfate, a click beetle luciferase enzyme, and a luciferin or novel luciferin derivative capable of generating light when the luciferin or novel luciferin derivative is used as a substrate for the click beetle luciferase. Often, other materials will be added to the solution, including a buffer to maintain the reaction at an appropriate pH, additives such as PRIONEX or bovine serum albumin (BSA) that help maintain luciferase activity, a reducing agent, a detergent, an esterase, salts, an amino acid, e.g., D-cysteine, etc. An exemplary luciferase reaction mixture would contain click beetle luciferase, MgSO4, ATP, Tergitol NP-9, and tricine.

[0037] A "luciferase detection mixture" contains materials that will enable detection of a luciferase enzyme. The materials needed to generate a luminescent signal, as well as the specific concentrations and / or amounts of the materials needed, will vary depending on the luciferase enzyme used and the type of luciferase-based assay being performed. Generally, for click beetle luciferase, including the CBR mutants disclosed herein, these materials can include ATP, a magnesium (Mg2+) salt, e.g., magnesium sulfate, and a luciferase substrate, e.g., luciferin, a luciferin derivative, a functional analog, or a novel luciferin derivative, that can generate light when a luciferase substrate, e.g., luciferin, a luciferin derivative, a functional analog, or a novel luciferin derivative, is used as a substrate for click beetle luciferase. Often, other materials will be added to the solution, including a buffer to maintain the reaction at the appropriate pH, additives such as PRIONEX or bovine serum albumin (BSA) to help maintain luciferase activity, reducing agents, detergents, esterases, salts, amino acids such as D-cysteine, etc. An exemplary luciferase detection mixture would contain luciferase substrate, MgSO, ATP, Tergitol NP-9, and tricine.

[0038] The term "luminescence" refers to the light output of a luciferase, e.g., a CBR mutant, under appropriate conditions, e.g., in the presence of an appropriate substrate, such as luciferin or a novel luciferin derivative. Light output may be measured as an instantaneous or near-instantaneous measurement of light output at the start of a luminescence reaction, which may be initiated upon addition of luciferin or a novel luciferin derivative substrate (sometimes referred to as "T=0" luminescence or "flash"). In various embodiments, the luminescence reaction is carried out in solution. In other embodiments, the luminescence reaction is carried out on a solid support. The solution may contain a lysate, e.g., obtained from cells in a prokaryotic or eukaryotic expression system. In other embodiments, expression is cell-free or the luciferase protein is secreted into the extracellular medium, eliminating the need to generate a lysate. In some embodiments, the reaction is initiated by injecting appropriate materials, e.g., luciferin substrate, buffer, etc., into a reaction chamber (e.g., a well of a multiwell plate, such as a 96-well plate) containing the luminescent protein. In yet other embodiments, the luciferase substrate is introduced into a host, potentially expressing a luciferase, e.g., a CBR mutant, and luminescence measurements are made in the host or a portion thereof, which may include the whole organism or cells, tissues, explants, or extracts thereof. The reaction chamber may be placed in a reader capable of measuring light output, e.g., using a luminometer or photomultiplier tube. Light output or luminescence may be measured over time, e.g., over a period of seconds, minutes, hours, etc., in the same reaction chamber. Light output or luminescence may be reported as an average measurement over time, a half-life of signal decay, a sum of the signal over a period of time, or a peak output. Luminescence may be measured in relative light units (RLU).

[0039] "Relative substrate specificity" is determined by dividing the luminescence of a luciferase in the presence of a test luciferin substrate by the luminescence of the luciferase in the presence of a reference luciferin substrate. For example, relative specificity can be determined by dividing the luminescence of a luciferase and a novel luciferin derivative by the luminescence of a luciferase and a different luciferin (e.g., D-luciferin or a novel luciferin derivative). The test luciferin substrate and the reference luciferin substrate being compared are considered to be a comparative substrate pair for determining relative substrate specificity.

[0040] "Change in relative substrate specificity" is measured by dividing the relative substrate specificity of the test luciferase using a comparative substrate pair by the relative substrate specificity of the reference luciferase using the same comparative substrate pair. For example, the change in relative specificity can be determined by dividing the relative substrate specificity of the test luciferase using a novel luciferin derivative compared to a different luciferin (e.g., D-luciferin or a novel luciferin derivative) by the relative substrate specificity of the reference luciferase using the same novel luciferin derivative compared to the same different luciferin used for the test luciferase.

[0041] The term "fusion polypeptide" or "fusion protein" refers to a chimeric protein containing a reference protein (e.g., a CBR variant) joined at its N- and / or C-terminus to one or more heterologous sequences (e.g., a non-CBR polypeptide).

[0042] The term "identity," in the context of two or more nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences that are identical, or that have a specified percentage of identical amino acid residues or nucleotides, when compared and aligned for maximum correspondence over a comparison window, or designated region, as measured using any number of sequence comparison algorithms or by manual alignment and visual inspection. Methods for aligning sequences for comparison are well known in the art. The optimal alignment of sequences for comparison can be achieved using the algorithm of Smith et al. (J. Mol. Biol. 147:195-197 (1981)), the homology alignment algorithm of Needleman and Wunsch (J. Mol. Biol., 48:443-453 (1970)), the similarity search method of Pearson and Lipman (Proc. Natl. Acad. Sci. USA, 85:2444-2448 (1988)), algorithms such as FASTA, SSEARCH, and GGSEARCH (available on the FASTA server at the University of Virginia by William R. Pearson http: / / fasta.bioch.virginia.edu / fasta_www2 / fasta_intro.shtml), and the Clustal series of programs (Chenna et al., Nucl. Acids. Res. 31(13):3497-3500 (2003); examples are available at http: / / www.ebi.ac.uk or http: / / www.ch.embnet.org), or by computer implementation of other sequence analysis software. It is well known in the art that creating maximum match alignments between polypeptide sequences with significant sequence changes (e.g., domain reordering, domain deletion / addition, domain repeats, domain shuffling, circular permutations) may involve the use of specialized methods such as the ABA method (Raphael et al., Genome Res. 14(11):2336-2346 (2004)), other suitable methods, or performing alignments using two linked identical copies of the polypeptide sequences.

[0043] Nucleic acid is known to contain different types of " mutation ", and mutation refers to the change in the sequence of nucleotide at specific base position compared with wild-type sequence.Mutation can also refer to the insertion or deletion of one or more bases, or the substitution of stop codon, which makes nucleic acid sequence different from reference sequence, for example, wild-type sequence." Substitution " refers to the change of amino acid at specific position in sequence.

[0044] The terms "nucleic acid molecule," "polynucleotide," or "nucleic acid sequence," as used herein, refer to a nucleic acid, including DNA or RNA, that contains coding sequences necessary for the production of a polypeptide or protein precursor. The encoded polypeptide can be a full-length polypeptide, a fragment (less than full-length) thereof, or a fusion of either the full-length polypeptide or a fragment thereof with another polypeptide, resulting in a fusion polypeptide.

[0045] A polynucleotide encoding a protein or polypeptide refers to a nucleic acid sequence comprising the coding region of a gene, or in other words, a nucleic acid sequence that encodes a gene product. The coding region may be present in the form of cDNA, genomic DNA, or RNA. If present in DNA form, the oligonucleotide may be single-stranded (e.g., the sense strand) or double-stranded. Suitable regulatory elements, such as enhancers / promoters, splice junctions, polyadenylation signals, etc., may be positioned adjacent to the coding region of the gene, as necessary to enable proper transcription initiation and / or accurate processing of the primary RNA transcript. Other regulatory or regulatory elements include, but are not limited to, transcription factor binding sites, splicing signals, polyadenylation signals, termination signals, and enhancer elements.

[0046] "Peptide," "protein," and "polypeptide" refer to amino acid chains of various lengths, regardless of post-translational modification (e.g., glycosylation or phosphorylation). The nucleic acid molecules of the invention encode artificial (i.e., synthetic) mutant protein variants or polypeptide fragments thereof having an amino acid sequence that is at least 60%, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identical to the amino acid sequence of the parent protein from which it is derived, where the parent protein can be a naturally occurring (native or wild-type) sequence or a variant sequence, which is subsequently further modified.

[0047] As used herein, "pure" or "purified" means that the target species is the predominant species present (i.e., more abundant, on a molar and / or mass basis, than any other individual species, excluding water, solvent, buffer, or other common components of the aqueous system in the composition); in some embodiments, a purified fraction is a composition in which the target species constitutes at least about 50% (on a molar basis) of all macromolecular species present. Generally, a "substantially pure" composition will contain greater than about 80% of all macromolecular species present in the composition, and in some embodiments, greater than about 85%, greater than about 90%, greater than about 95%, or greater than about 99%. In some embodiments, the target species is purified to essential homogeneity (contaminating species cannot be detected in the composition by conventional detection methods), where the composition consists essentially of a single macromolecular species.

[0048] The term "variant" refers to an altered version of a starting polypeptide or polynucleotide sequence. The term "parent" is a related term that refers to a starting sequence that is subsequently modified. The parent sequence is typically used as a reference for the protein encoded by the resulting modified sequence, for example, to compare activity levels or other properties of the proteins encoded by the parent and modified sequences. The starting sequence can be a naturally occurring (i.e., native or wild-type) sequence. The starting sequence can also be a variant sequence that is subsequently further modified. A polypeptide sequence is "modified" when one or more amino acids (whether naturally occurring or synthetic) are substituted, deleted, and / or added at the beginning, middle, i.e., internal positions, and / or end of the sequence. A polynucleotide sequence is "modified" when one or more nucleotides are substituted, deleted, and / or added at the beginning, middle, i.e., internal positions, and / or end of the sequence, although the amino acids encoded by the sequence may or may not be changed. In some embodiments, the modification produces a variant that is a functional fragment of CBR or a specific CBR variant. A functional fragment is a fragment of a full-length parent sequence that has the same functional activity as the full-length parent sequence. The functional activity is the ability to emit light. In some embodiments, the modification produces variants that are permuted sequences of the parent sequence, such as circularly permuted sequences and permuted sequences containing deletions and / or insertions.

[0049] Variant is also used herein to describe a protein having an amino acid sequence substantially identical to a reference protein that retains at least one biological activity. Conservative amino acid substitutions, i.e., replacing an amino acid with a different amino acid with similar properties (e.g., hydrophilicity, degree, and distribution of charged regions), are recognized in the art as typically involving minor changes. These minor changes can be identified, in part, by considering the hydropathic index of an amino acid, as understood in the art. Kyte et al., J. Mol. Biol. 157:105-132 (1982). The hydropathic index of an amino acid is based on consideration of its hydrophobicity and charge. It is known in the art that amino acids with similar hydropathic indices can be substituted while retaining protein function. In one embodiment, amino acids with hydropathic indices of ±2 are substituted. The hydrophilicity of an amino acid can also be used to identify substitutions that will result in a protein that retains biological function.

[0050] Substitution of amino acids with similar hydrophilicity values ​​can result in peptides that retain biological activity, as is understood in the art. Substitutions can be made with amino acids having hydrophilicity values ​​within ±2 of each other. Both the hydrophobicity index and hydrophilicity value of an amino acid are influenced by the specific side chain of that amino acid. Consistent with this observation, amino acid substitutions that are compatible with biological function are understood to depend on the relative similarity of amino acids, particularly their side chains, as manifested by hydrophobicity, hydrophilicity, charge, size, and other properties. "Variant" can also be used to describe a polypeptide or fragment thereof that is differentially processed, such as by proteolysis, phosphorylation, or other post-translational modification, while still retaining its biological activity.

[0051] The term "vector" refers to a nucleic acid molecule into which a fragment of DNA may be inserted or cloned, used to transfer the DNA segment into a cell, and which is replicable in the cell. Vectors may be derived from plasmids, bacteriophages, viruses, cosmids, etc.

[0052] The terms "wild-type" or "native," as used herein, refer to a gene or gene product that has the characteristics of that gene or gene product isolated from a naturally occurring source. A wild-type gene is that which is most frequently observed in a population and is therefore arbitrarily designated the "wild-type" form of the gene. In contrast, the term "mutant" refers to a gene or gene product that exhibits alterations in sequence and / or functional properties (i.e., altered characteristics) when compared to the wild-type gene or gene product. Note that it is possible to isolate naturally occurring mutants; these are identified by the fact that they have altered characteristics when compared to the wild-type gene or gene product.

[0053] 2. CBR mutant The present disclosure provides mutants of click beetle red luciferase (CBR) that utilize luciferin and luciferin derivatives to produce longer, near-IR (NIR) wavelength signals. Using various techniques described herein, CBR was evolved to produce brighter light output through extended conjugation of luciferin, and sites of amino acid substitution were identified to produce improved synthetic CBR polypeptides. Making one or more amino acid substitutions, alone or in various combinations, was found to produce synthetic CBR-type polypeptides with enhanced light emission (e.g., increased brightness) and longer wavelength emission. Also provided are polynucleotides encoding the CBR mutants or fusions thereof of the invention, isolated host cells harboring the polynucleotides or CBR mutants or fusions thereof, and methods of using the polynucleotides, CBR mutants or fusions thereof, or host cells of the invention.

[0054] CBR variants contain at least one amino acid substitution at positions 4, 16, 34, 47, 51, 52, 55, 72, 73, 74, 79, 82, 83, 87, 89, 104, 109, 113, 117, 119, 124, 130, 131, 133, 136, 144, 146, 156, 159, 170, 179, 186, 200, 211, 218, 224, 225, 226, 228, 229, 234, 247, 251, 252, 253, 255, 280, 281, 285, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 328, 329, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367 , 310, 319, 329, 334, 335, 337, 346, 348, 349, 350, 352, 354, 355, 358, 363, 370, 377, 390, 393, 394, 400, 401, 409, 412, 420, 422, 431, 437, 439, 444, 445, 453, 455, 467, 471, 473, 479, 484, 489, 496, 501, 503, 508, 516, 528, 531, 535, 537, 539, or a combination thereof. The CBR variant may further comprise at least one amino acid substitution at a position corresponding to position 351, 389, 457, or a combination thereof of SEQ ID NO:1.CBR mutants are R4H, H16Q, H34Y, D47E, S51N, Y52C, F55L / V, K72E, I79V, M73K / T, N74S, E82G, N83H, F87S, I89V, V104D, I109N / V, L113Q, M117T, I119F / T, I124V, N130K, I131N / T, N133D, K136N, F144L, K146E, N147L, and N148L of SEQ ID NO: 1. 156D, N156K, G159D, Y170C, K179S, V186A, G200G, N211N, H218L / Y, G225S, T226C / G / H / N / Q / Y, L228P, I22 9V, V234A, G251S, G251I, Y252C, V255D / F, E253K, R280S, S281N / Q, V285A, I309T, E319G, N329D, R334E / Q / H / S / N / K, C335S, K337E, I346N, Q348H / E, L350P, G351K / R, D352N, R355G, S358P, T363A / S, I370T, I389F / G / S / V, I390I, M393K / L, V394M, N400D, N401S, I409T, D412G, F420F, Y422C, V431A, E437G, I439V, S444C The CBR mutant polypeptide may comprise at least one amino acid substitution corresponding to at least one of the following, or a combination thereof: / R / T, Q445H, E453K, V455D, K457N, D471V, E473A, S479T, K484E / M / R, E489V, Y496H, E501G, V503M, Y508C, V516A, T528A, E531G, Q535H, L537W, K539R. The CBR mutant polypeptide may comprise amino acid substitutions at positions corresponding to positions 389, 444, and 251 of SEQ ID NO:1. The amino acid substitutions may include I389F, S444R, and G251S. The CBR mutant polypeptide may comprise amino acid substitutions at positions corresponding to positions 334 and 351 of SEQ ID NO:1. The amino acid substitutions may include R334S and G351R. The CBR variant may comprise the amino acid polypeptide of SEQ ID NO:2 or SEQ ID NO:3.

[0055] Some CBR mutants and parent CBRs disclosed herein have been assigned abbreviations to facilitate discussion. The term "ATG343" (also referred to as "343" and "pF4Ag-CBR") refers to the CBR polypeptide of SEQ ID NO: 1. The CBR polypeptide of SEQ ID NO: 1 is encoded by the polynucleotide sequence of SEQ ID NO: 5. The term "ATG685" (also referred to as "685" and "pF4Ag-HT7-CBR") refers to the CBR polypeptide sequence of SEQ ID NO: 1 fused with HALOTAG®. The term "ATG1230" (SEQ ID NO: 3) refers to a CBR mutant having the amino acid substitutions I389F, S444R, and G251S relative to SEQ ID NO: 1 (where the format "x#y" indicates that the parent amino acid "x" is at position '#' and has been changed to the mutant amino acid "y"). The term "ATG1240" (SEQ ID NO: 2) refers to a CBR mutant having the amino acid substitutions R334S and G351R relative to SEQ ID NO: 1. The term "ATG1240+S51N+S444R" (SEQ ID NO: 4) refers to a CBR mutant with amino acid substitutions of ATG1240, i.e., R334S and G351R of SEQ ID NO: 1, and amino acid substitutions S51N and S444R relative to SEQ ID NO: 1.

[0056] The sequence of a CBR variant is substantially identical to the amino acid sequence of the corresponding parent CBR, e.g., SEQ ID NO: 1. A polypeptide or peptide having substantially the same sequence means that the amino acid sequence is the same for most, but not all, of the sequences to which it is related and retains the functional activity of the sequence to which it is related. Generally, two amino acid sequences are substantially identical if they have at least 60%, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity, but less than 100%. In some embodiments, the CBR variant is encoded by a recombinant polynucleotide.

[0057] In some embodiments, the CBR variant has at least 60%, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, or 100% amino acid sequence identity to SEQ ID NO: 1, 2, 3, or 4. Typically, the variant fragment is at least about 50 amino acids in length, often at least about 60 amino acids in length, and more often at least about 70, 80, 90, 100, 150, 200, 300, 400, 500, or 550 amino acids in length or longer, and retains the ability to generate luminescence. Full-length luciferase, fragments thereof, or variants thereof may be fused to heterologous amino acid sequences and still be functional in the present invention.

[0058] These CBR mutants exhibit at least one of the following: enhanced luminescence (including increased brightness, improved signal stability, signal duration, and / or reduced sensitivity to substrate inhibition); altered light emission spectra; and altered substrate specificity (i.e., altered relative substrate specificity). In various embodiments, the invention encompasses novel luciferases that exist in solution, as soluble proteins chemically linked to other molecules (e.g., fusion proteins), or as soluble proteins attached to solid surfaces (e.g., particles, capillaries, or assay tubes or plates).

[0059] a. Enhanced luminescence The enhanced luminescence of CBR mutants can be attributed to one or more of the following characteristics: enhanced light output (i.e., brightness), improved substrate specificity, improved signal stability, improved signal duration, and / or reduced sensitivity to substrate inhibition. Improved signal stability includes, for example, an increase in the length of time that a signal from a luciferase remains luminescent, as measured by the half-life of the signal decay over time. The term "substrate inhibition," as used herein, refers to the inhibition of luciferase enzyme activity (e.g., inhibition of luminescence production and reduced RLU values) at high substrate concentrations. CBR mutants with reduced sensitivity to substrate inhibition do not exhibit a decrease in RLU values ​​with increasing amounts of substrate. For example, PBI-4813 at concentrations greater than 10-fold the Km inhibits the activity of CBR and ATG1230, thereby reducing the luminescence produced, i.e., the RLU values, whereas ATG1240 does not exhibit this inhibition with PBI-4813 and has reduced sensitivity to substrate inhibition.

[0060] Enhanced luminescence can be determined relative to comparable properties of a luciferase, such as a wild-type CBR, a CBR mutant protein, Renilla luciferase (e.g., hRluc), or firefly luciferase (e.g., Luc2; luciferase from Photinus pyralis), combined with a native, known, or novel substrate, as shown in the Examples below. For example, the luminescence of a given CBR mutant combined with a particular luciferin (including a native, known, or novel luciferin, or derivative thereof) can be compared to the properties of CBR combined with any of the native, known, or novel luciferins disclosed herein, or derivatives thereof, using one or more of the assays disclosed in the Examples below. Specifically, enhanced luminescence can be determined by measuring the luminescence signal (RLU) resulting from incubation of a bacterial or mammalian lysate containing the CBR mutant of interest with a luciferase substrate, e.g., a novel luciferin derivative such as PBI-4813 or PBI-4739. The luminescent signal can be compared to that of a reference point such as CBR and D-luciferin or a luciferin derivative such as PBI-4813 or PBI-4739, or Luc2 (firefly) luciferase and D-luciferin.

[0061] In certain embodiments, the CBR variants have increased luminescence emission, e.g., at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, or at least about 10-fold, in prokaryotic and / or eukaryotic cells using the same or a different substrate, such as D-luciferin, PBI-4813, or PBI-4739, relative to a corresponding reference luciferase, such as the CBR of SEQ ID NO: 1. In some embodiments, one or more properties of the CBR variants are compared to a comparable property of a luciferase from another species, e.g., firefly luciferase or Renilla luciferase.

[0062] b. Change in the emission spectrum The CBR mutant polypeptide may have an altered emission light spectrum compared to a parent CBR, e.g., SEQ ID NO: 1. The CBR mutant polypeptide may be capable of emitting light at a longer wavelength when luciferin is utilized by the CBR mutant polypeptide to generate luminescence. The CBR mutant polypeptide may be capable of emitting light at a longer wavelength when a luciferin derivative, such as PBI-4813 or PBI-4739, is utilized by the CBR mutant polypeptide to generate luminescence.

[0063] The CBR mutant polypeptides can use luciferin or novel luciferin derivatives, such as PBI-4813 or PBI-4739, to emit light with a shifted spectral maximum relative to the light produced by the CBR polypeptide of SEQ ID NO:1. In some embodiments, the CBR variant polypeptide has a wavelength of at least about 1 nm to at least about 200 nm, at least about 2 nm to at least about 200 nm, at least about 3 nm to at least about 200 nm, at least about 4 nm to at least about 200 nm, at least about 5 nm to at least about 200 nm, at least about 6 nm to at least about 200 nm, at least about 7 nm to at least about 200 nm, at least about 8 nm to at least about 200 nm, at least about 9 nm to at least about 200 nm, at least about 10 nm to at least about 200 nm, at least about 15 nm to at least about 200 nm, at least about 20 nm to at least about 200 nm, at least about 30 nm to at least about 200 nm, at least about 40 nm to at least about 200 nm, at least about 50 nm to at least about 200 nm, at least about 60 nm to at least about 200 nm, at least about 70 nm to at least about 200 nm 0 nm, at least about 80 nm to at least about 200 nm, at least about 90 nm to at least about 200 nm, at least about 100 nm to at least about 200 nm, at least about 150 nm to at least about 200 nm, at least about 1 nm to at least about 150 nm, at least about 2 nm to at least about 150 nm, at least about 3 nm to at least about 150 nm, at least about 4 nm to at least about 150 nm, at least about 5 nm to at least about 150 nm, at least about 6 nm to at least about 150 nm, at least about 7 nm to at least about 150 nm, at least about 8 nm to at least about 150 nm, at least about 9 nm to at least about 150 nm, at least about 10 nm to at least about 150 nm, at least about 15 nm to at least about 150 nm, at least about 20 nm to at least about 150 nm, at least about 30 nm to at least about 150 nm, at least about 40 nm to at least about 150 nm, at least about 50 nm to at least about 150 nm,at least about 60 nm to at least about 150 nm, at least about 70 nm to at least about 150 nm, at least about 80 nm to at least about 150 nm, at least about 90 nm to at least about 150 nm, at least about 100 nm to at least about 150 nm, at least about 1 nm to at least about 100 nm, at least about 2 nm to at least about 100 nm, at least about 3 nm to at least about 100 nm, at least about 4 nm to at least about 100 nm, at least about 5 nm to at least about 100 nm, at least about 6 nm to at least about 100 nm, At least about 7 nm to at least about 100 nm, at least about 8 nm to at least about 100 nm, at least about 9 nm to at least about 100 nm, at least about 10 nm to at least about 100 nm, at least about 15 nm to at least about 100 nm, at least about 20 nm to at least about 100 nm, at least about 30 nm to at least about 100 nm, at least about 40 nm to at least about 100 nm, at least about 50 nm to at least about 100 nm, at least about 60 nm to at least about 100 nm, at least about 70 nm to at least about 100 nm m, at least about 80 nm to at least about 100 nm, at least about 90 nm to at least about 100 nm, at least about 1 nm to at least about 70 nm, at least about 2 nm to at least about 70 nm, at least about 3 nm to at least about 70 nm, at least about 4 nm to at least about 70 nm, at least about 5 nm to at least about 70 nm, at least about 6 nm to at least about 70 nm, at least about 7 nm to at least about 70 nm, at least about 8 nm to at least about 70 nm, at least about 9 nm to at least about 70 nm, at least about 10 nm at least about 70 nm, at least about 15 nm to at least about 70 nm, at least about 20 nm to at least about 70 nm, at least about 30 nm to at least about 70 nm, at least about 40 nm to at least about 70 nm, at least about 50 nm to at least about 70 nm, at least about 60 nm to at least about 70 nm, at least about 1 nm to at least about 50 nm, at least about 2 nm to at least about 50 nm, at least about 3 nm to at least about 50 nm, at least about 4 nm to at least about 50 nm, at least about 5 nm to at least about 50 nm,The light may be emitted with an increase or decrease in the spectral maximum of at least about 6 nm to at least about 50 nm, at least about 7 nm to at least about 50 nm, at least about 8 nm to at least about 50 nm, at least about 9 nm to at least about 50 nm, at least about 10 nm to at least about 50 nm, at least about 15 nm to at least about 50 nm, at least about 20 nm to at least about 50 nm, at least about 30 nm to at least about 50 nm, at least about 40 nm to at least about 50 nm, at least about 1 nm to at least about 20 nm, at least about 2 nm to at least about 20 nm, at least about 3 nm to at least about 20 nm, at least about 4 nm to at least about 20 nm, at least about 5 nm to at least about 20 nm, at least about 6 nm to at least about 20 nm, at least about 7 nm to at least about 20 nm, at least about 8 nm to at least about 20 nm, at least about 9 nm to at least about 20 nm, at least about 10 nm to at least about 20 nm, or at least about 15 nm to at least about 20 nm. The CBR variant polypeptide can emit light that has an increase or decrease in spectral maximum shifted by at least about 1 nm, at least about 2 nm, at least about 3 nm, at least about 4 nm, at least about 5 nm, at least about 6 nm, at least about 7 nm, at least about 8 nm, at least about 9 nm, at least about 10 nm, at least about 15 nm, at least about 20 nm, at least about 30 nm, at least about 40 nm, at least about 50 nm, at least about 60 nm, at least about 70 nm, at least about 80 nm, at least about 90 nm, at least about 100 nm, at least about 150 nm, or at least about 200 nm relative to the light produced by the CBR polypeptide of SEQ ID NO: 1.

[0064] When luciferin or a novel luciferin derivative, for example, PBI-4813 or PBI-4739, is used as a substrate, the CBR mutant polypeptide has a wavelength of about 650 nm to about 1000 nm, about 700 nm to about 1000 nm, about 725 nm to about 1000 nm, about 750 nm to about 1000 nm, about 775 nm to about 1000 nm, about 800 nm to about 1000 nm, about 850 nm to about 1000 nm, about 900 nm to about 1000 nm, about 950 nm to about 1000 nm, about 650 nm to about 900 nm, about 700 nm to about 900 nm, about 725 nm to about 900 nm, about 750 nm to about 9 ... The compound may emit light having a spectral maximum between about 5 nm and about 900 nm, between about 800 nm and about 900 nm, between about 850 nm and about 900 nm, between about 650 nm and about 800 nm, between about 700 nm and about 800 nm, between about 725 nm and about 800 nm, between about 750 nm and about 800 nm, between about 775 nm and about 800 nm, between about 650 nm and about 775 nm, between about 700 nm and about 775 nm, between about 725 nm and about 775 nm, between about 750 nm and about 775 nm, between about 650 nm and about 750 nm, between about 700 nm and about 750 nm, between about 725 nm and about 750 nm, between about 650 nm and about 725 nm, between about 700 nm and about 725 nm, or between about 725 nm and about 750 nm. The CBR mutant polypeptide can emit light having a spectral maximum of at least about 600 nm, at least about 650 nm, at least about 700 nm, at least about 725 nm, at least about 750 nm, at least about 775 nm, at least about 800 nm, at least about 900 nm, or at least about 1000 nm when luciferin or a novel luciferin derivative, e.g., PBI-4813 or PBI-4739, is used as a substrate.

[0065] c. change in substrate specificity The CBR mutant polypeptide may have altered substrate specificity compared to the CBR polypeptide of SEQ ID NO: 1. The CBR mutant polypeptide may have increased or decreased substrate specificity for luciferin or a novel luciferin derivative, e.g., PBI-4739 or PBI-4813, compared to the CBR polypeptide of SEQ ID NO: 1. The CBR mutant polypeptide may have a change, such as an increase or decrease in relative specificity for the CBR mutant polypeptide in the presence of luciferin, compared to a luciferin derivative such as PBI-4739 or PBI-4813. The CBR mutant polypeptide may have a change, such as an increase or decrease in relative specificity for the CBR mutant polypeptide in the presence of one luciferin derivative, compared to a different luciferin derivative, such as PBI-4739 or PBI-4813.

[0066] The CBR variant polypeptides may have an altered Km or Vmax using D-luciferin and / or novel luciferin derivatives, compared to the CBR polypeptide of SEQ ID NO: 1. A CBR variant polypeptide with a lower Km for a particular substrate may be advantageous for in vivo imaging when it is difficult to "saturate" the substrate.

[0067] The CBR variants have an activity against PBI-4813 of at least about 0.10 μM, at least about 0.20 μM, at least about 0.30 μM, at least about 0.40 μM, at least about 0.50 μM, at least about 0.60 μM, at least about 0.70 μM, at least about 0.80 μM, at least about 0.81 μM, at least about 0.82 μM, at least about 0.83 μM, at least about 0.84 μM, at least about 0.85 μM, at least about 0.86 μM, at least about 0.87 μM, at least about 0.88 μM, at least about 0.89 μM, at least about 0.90 μM, at least about 0.91 μM, at least about 0.92 μM, at least about 0.93 μM, at least about 0.94 μM, at least about 0.95 μM, at least about 0.96 μM, at least about 0.97 μM, at least about 0.98 μM, at least about 0.99 μM, at least about 100 μM, at least about 101 μM, at least about 102 μM, at least about 103 μM, at least about 104 μM, at least about 105 μM, at least about 106 μM, at least about 107 μM, at least about 108 μM, at least about 109 μM, at least about 110 μM, at least about 111 μM, at least about 112 μM, at least about 113 μM, at least about 114 μM, at least about 115 μM, at least about 116 μM, at least about 117 μM, at least about 118 μM, at least about 119 or may have a Km of at least about 0.01 μM to at least about 5.00 μM, at least about 0.90 μM, at least about 1.00 μM, at least about 1.50 μM, at least about 2.00 μM, at least about 2.10 μM, at least about 2.20 μM, at least about 2.30 μM, at least about 2.40 μM, at least about 2.50 μM, at least about 2.60 μM, at least about 2.70 μM, at least about 2.80 μM, at least about 2.90 μM, at least about 3.00 μM, at least about 4.00 μM, or at least about 5.00 μM, or 0.01 μM to at least about 4.00 μM, at least about 0.01 μM to at least about 3.00 μM, at least about 0.01 μM to at least about 2.50 μM, at least about 0.01 μM to at least about 2.00 μM, at least about 0.01 μM to at least about 1.00 μM, at least about 0.01 μM to at least about 0.80 μM, at least about 0.01 μM to at least about 0.50 μM, at least about 0.05 μM to at least about 5.00 μM, at least about 0.05 μM to at least about 4.00 μM, at least about 0.05 μM to at least about 3.0 0 μM, at least about 0.05 μM to at least about 2.50 μM, at least about 0.05 μM to at least about 2.00 μM, at least about 0.05 μM to at least about 1.00 μM, at least about 0.05 μM to at least about 0.80 μM, at least about 0.08 μM to at least about 5.00 μM, at least about 0.08 μM to at least about 4.00 μM, at least about 0.08 μM to at least about 3.00 μM, at least about 0.08 μM to at least about 2.50 μM, at least about 0.08 μM to at least about 2.00 μM, or at least about 0.It has a K in the range of 0.8 μM to at least about 1.00 μM.

[0068] The CBR variants have an activity against PBI-4739 of at least about 0.10 μM, at least about 0.50 μM, at least about 1.00 μM, at least about 1.50 μM, at least about 2.00 μM, at least about 2.10 μM, at least about 2.20 μM, at least about 2.30 μM, at least about 2.40 μM, at least about 2.50 μM, at least about 2.60 μM, at least about 2.70 μM, at least about 2.80 μM, at least about 2.90 μM, at least about 3.00 μM, or may have a Km of at least about 3.10 μM, at least about 3.20 μM, at least about 3.30 μM, at least about 3.40 μM, at least about 3.50 μM, at least about 3.60 μM, at least about 3.70 μM, at least about 3.80 μM, at least about 3.90 μM, at least about 4.00 μM, or at least about 5.00 μM, or at least about 0.01 μM to at least about 5.00 μM, at least about 0.01 μM to at least about 4.00 μM, at least about 0.01 μM to at least about 3.00 μM, at least about 0.01 μM to at least about 2.50 μM, at least about 0.01 μM to at least about 2.00 μM, at least about 0.01 μM to at least about 1.00 μM, at least about 0.01 μM to at least about 0.80 μM, at least about 0.01 μM to at least about 0.50 μM, 1.00 μM to at least about 5.00 μM, at least about 1.00 μM to at least about 4.00 μM, at least about 1.00 μM to at least about having a Km in the range of 3.00 μM, at least about 1.00 μM to at least about 2.50 μM, at least about 1.00 μM to at least about 2.00 μM, 2.00 μM to at least about 5.00 μM, at least about 2.00 μM to at least about 4.00 μM, at least about 2.00 μM to at least about 3.00 μM, at least about 2.00 μM to at least about 2.50 μM, 3.00 μM to at least about 5.00 μM, or at least about 3.00 μM to at least about 4.00 μM.

[0069] The CBR variant can have a Km for PBI-4813 that is at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 30-fold, at least about 4-fold, at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold, at least about 90-fold, at least about 100-fold lower, at least about 150-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, or at least about 1000-fold lower than the Km of the CBR polypeptide of SEQ ID NO: 1 for PBI-4813.

[0070] The CBR variant can have a Km for PBI-4813 that is at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 30-fold, at least about 4-fold, at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold, at least about 90-fold, at least about 100-fold lower, at least about 150-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, or at least about 1000-fold lower than the Km of the CBR polypeptide of SEQ ID NO: 1 for D-luciferin.

[0071] The CBR variant can have a Km for PBI-4739 that is at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 30-fold, at least about 4-fold, at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold, at least about 90-fold, at least about 100-fold lower, at least about 150-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, or at least about 1000-fold lower than the Km of the CBR polypeptide of SEQ ID NO: 1 for PBI-4739.

[0072] The CBR variant can have a Km for PBI-4739 that is at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 30-fold, at least about 4-fold, at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold, at least about 90-fold, at least about 100-fold lower, at least about 150-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, or at least about 1000-fold lower than the Km of the CBR polypeptide of SEQ ID NO: 1 for D-luciferin.

[0073] The CBR variant may have a relative Vmax to PBI-4813 that is at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 30-fold, at least about 4-fold, at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold, at least about 90-fold, at least about 100-fold lower, at least about 150-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, or at least about 1000-fold higher than the relative Vmax of the CBR polypeptide of SEQ ID NO: 1 to PBI-4813.

[0074] The CBR variant may have a relative Vmax to PBI-4739 that is at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 30-fold, at least about 4-fold, at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold, at least about 90-fold, at least about 100-fold lower, at least about 150-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, or at least about 1000-fold higher than the relative Vmax of the CBR polypeptide of SEQ ID NO: 1 to PBI-4739.

[0075] d. fusion protein In some embodiments, the CBR variants of the invention have one or more heterologous amino acid sequences at the N-terminus, C-terminus, or both ends (e.g., fusion polypeptides with epitopes or fusion tags), which optionally interact directly or indirectly with a molecule of interest. In some embodiments, the presence of the heterologous sequence(s) does not substantially alter the luminescence of the CBR variant either before or after interaction with the molecule of interest. The heterologous amino acid sequence can be any protein of interest, e.g., RNasin or RNase, and / or a channel protein, receptor, membrane protein, cytosolic protein, nuclear protein, structural protein, phosphorylated protein, kinase, signaling protein, metabolic protein, mitochondrial protein, receptor-associated protein, fluorescent protein, enzyme substrate, transcription factor, transporter protein, and / or a targeting sequence, e.g., a myristilation sequence, a mitochondrial localization sequence, or a nuclear localization sequence, which directs the hydrolase fragment, e.g., the fusion protein, to a specific location.

[0076] In some embodiments, the heterologous amino acid sequence is an epitope tag. In some embodiments, the heterologous amino acid sequence undergoes a conformational change during or after interaction with a molecule of interest, thereby changing the activity of the CBR mutant; for example, a CBR mutant having such an amino acid sequence is useful for detecting allosteric interactions. The CBR mutant or a fusion with the CBR mutant or a fragment thereof can be employed as a reporter.

[0077] The CBR variants of the present invention can be coupled to any protein or molecule of interest. In some embodiments, the variants are fusion proteins; for example, some variants are coupled to a HALOTAG® polypeptide (also referred to as "HT7") attached at either the N- or C-terminus. In some embodiments, the fusion or chimeric protein contains a CBR variant joined at the N-terminus to a HALOTAG® fusion protein (Promega). In other embodiments, the fusion or chimeric protein contains a CBR variant joined at the C-terminus to a HALOTAG® fusion protein. A signal sequence, in combination with a membrane anchor sequence, may be used to position or display the CBR variant on the outer surface of the cell membrane. Other methods known in the art may also be used to position a CBR variant on the membrane or elsewhere within the cell.

[0078] e. Vectors and host cells encoding modified luciferases or fusions thereof Once a desired nucleic acid molecule encoding a CBR variant or a fragment thereof, such as one that has luminescence activity or can be complemented with another molecule to provide luminescence activity, or a fusion thereof that has luminescence activity, has been prepared, an expression cassette can be prepared that encodes the CBR variant or a fragment thereof, e.g., for complementation, or a fusion thereof that has luminescence activity. For example, a nucleic acid molecule comprising a nucleic acid sequence encoding a CBR variant is optionally operably linked to a transcription control sequence, such as one or more enhancers, promoters, transcription termination sequences, or a combination thereof, to form an expression cassette. The nucleic acid molecule or expression cassette can be introduced into a vector, e.g., a plasmid or viral vector, optionally containing a selectable marker gene, and the vector can be introduced into a cell of interest, e.g., prokaryotic cells such as E. coli, Streptomyces spp., Bacillus spp., Staphylococcus spp., as well as eukaryotic cells, lysates thereof, or in vitro transcription / translation mixtures, including plant (dicotyledonous or monocotyledonous), fungi (yeast, e.g., Pichia, Saccharomyces, or Schizosaccharomyces), or mammalian cells. Mammalian cells include, but are not limited to, bovine, caprine, ovine, canine, feline, non-human primate cells, e.g., monkey cells, and human cells. Mammalian cell lines include, but are not limited to, CHO, COS, HEK293, HeLa, CV-1, SH-SY5Y, and NIH3T3 cells; many other cell lines can also be used.

[0079] Expression of the encoded CBR variant can be controlled by any promoter capable of expression in prokaryotic or eukaryotic cells, including synthetic promoters. Prokaryotic promoters include, but are not limited to, SP6, T7, T5, tac, bla, trp, gal, lac, or maltose promoters, including any fragments having promoter activity. Eukaryotic promoters include, but are not limited to, constitutive promoters, e.g., viral promoters such as CMV, SV40, and RSV promoters, and regulatable promoters, e.g., inducible or repressible promoters, such as Tet promoters, hsp70 promoters, and synthetic promoters regulated by CRE, including any fragments having promoter activity. Expression of the encoded CBR variant can also be controlled by post-transcriptional processes, such as regulation of RNA processing or translation, e.g., by RNAi, miRNA, shRNA, siRNA, or by RNA or protein degradation. The nucleic acid molecules, expression cassettes, and / or vectors of the present invention can be introduced into cells by any method, including, but not limited to, calcium-mediated transformation, electroporation, microinjection, lipofection, etc.

[0080] f. Optimized sequence encoding the CBR variant Also provided are isolated nucleic acid molecules (polynucleotides) comprising a nucleic acid sequence encoding a CBR variant of the invention, a functional fragment thereof, or a fusion protein thereof. In some embodiments, the isolated nucleic acid molecule comprises a nucleic acid sequence optimized for expression in at least one selected host. Optimized sequences include codon-optimized sequences, i.e., codons more frequently used in one organism relative to another, e.g., a distantly related organism, as well as modifications to add or modify Kozak sequences and / or introns and / or remove undesirable sequences, e.g., potential transcription factor binding sites. Such optimized sequences can provide enhanced expression, e.g., increased protein expression levels, when introduced into a host cell. Examples of optimized sequences are disclosed in U.S. Pat. No. 7,728,118 and U.S. Patent Application Publication Nos. 2008 / 0070299 and 2008 / 0090291, each of which is incorporated herein by reference.

[0081] In some embodiments, a polynucleotide comprises a nucleic acid sequence encoding a CBR variant of the invention, wherein the nucleic acid sequence is optimized for expression in mammalian host cells in culture (e.g., CHO cells), in a live animal (e.g., a mouse), or in tissue-specific cells within a live animal. In some embodiments, the polynucleotide may comprise the codon-optimized sequence of any one of SEQ ID NOS: 2-4. In some embodiments, the polynucleotide may comprise the polynucleotide sequence of any one of SEQ ID NOS: 7-9. In some embodiments, the polynucleotide may comprise the polynucleotide sequence of SEQ ID NOS: 10. In some embodiments, the nucleic acid sequence is optimized for expression in bacterial cells or plants. In some embodiments, an optimized polynucleotide will not hybridize to a corresponding non-optimized sequence, e.g., under moderate or high stringency conditions. The term "stringent" refers to the conditions of temperature, ionic strength, and the presence of other compounds under which nucleic acid hybridization occurs. Under "high stringency" conditions, nucleic acid base pairing will occur only between nucleic acid fragments that frequently have complementary base sequences. Thus, "moderate" or "low" stringency conditions are often used when it is desired to hybridize or anneal nucleic acids that are not completely complementary to one another. It is well known in the art that numerous equivalent conditions can be employed to encompass moderately or lowly stringent conditions.

[0082] In some embodiments, the polynucleotides have less than 90%, e.g., less than 80%, nucleic acid sequence identity to the corresponding non-optimized sequence, and optionally encode a polypeptide having at least 60%, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the polypeptide encoded by the non-optimized sequence. Also provided are constructs, e.g., expression cassettes and vectors, comprising the isolated nucleic acid molecules, e.g., with optimized nucleic acid sequences, as well as kits comprising the isolated nucleic acid molecules, constructs, or vectors.

[0083] A nucleic acid molecule comprising a nucleic acid sequence encoding a CBR variant of the present invention, a fragment thereof, or a fusion thereof is optionally optimized for expression in a particular host cell and is optionally operably linked to a transcription control sequence, such as one or more enhancers, promoters, transcription termination sequences, or a combination thereof, to form an expression cassette.

[0084] In some embodiments, nucleic acid sequences encoding the CBR variants, fragments thereof, or fusions thereof of the present invention are optimized by codon substitution, e.g., by substituting at least 25% of the codons in the parent CBR sequence with codons preferentially used in a particular (selected) cell. Preferred codons preferably have a relatively high codon usage frequency in the selected cell, and their introduction results in relatively few transcription factor binding sites and other undesirable structural attributes for transcription factors present in the selected host cell. Examples of undesirable structural attributes include, but are not limited to, restriction enzyme sites, eukaryotic sequence elements, vertebrate promoter modules and transcription factor binding sites, response elements, E. coli sequence elements, and mRNA secondary structures. Thus, optimized nucleic acid products may have improved expression levels as a result of improved codon usage and a reduced risk of inappropriate transcription behavior due to a reduced number of undesirable transcriptional regulatory sequences.

[0085] An isolated, optimized nucleic acid molecule may have a codon composition that differs from that of the corresponding wild-type nucleic acid sequence by more than 30%, 35%, 40%, or 45% of the codons, e.g., 50%, 55%, 60%, or more. Exemplary codons for use in the present invention are those that are more frequently used than at least one other codon for the same amino acid in a particular organism, and in some embodiments are not codons that are under-used in that organism or in the organism used to clone or screen for expression of the nucleic acid molecule. Furthermore, codons for a particular amino acid (i.e., amino acids with three or more codons) may contain two or more codons that are more frequently used than other (non-preferred) codon(s). The presence in a nucleic acid molecule of codons that are more frequently used in one organism than in another organism results in the nucleic acid molecule, when introduced into cells of an organism that uses those codons more frequently, being expressed in those cells at levels greater than the expression of the wild-type or parent nucleic acid sequence in those cells.

[0086] In some embodiments of the present invention, the different codons are those more frequently used in mammals, while in still other embodiments, the different codons are those more frequently used in plants or bacteria. Preferred codons for different organisms are known in the art; see, for example, http: / / www.kazusa.or.jp. / codon / . A particular type of mammal, such as a human, may have a set of preferred codons that differs from another type of mammal. Similarly, a particular type of plant or bacterium may have a set of preferred codons that differs from another type of plant or bacterium. In some embodiments of the present invention, the majority of the different codons are those that are preferred codons in the desired host cell. Preferred codons for organisms, including mammals (e.g., humans) and plants, are known in the art (e.g., Wada et al., Nucl. Acids Res., 18:2367 (1990); Murray et al., Nucl. Acids Res., 17:477 (1989)).

[0087] 3. Near-infrared Bioluminescence System The present disclosure also provides near-IR bioluminescence systems comprising the above-described CBR mutants and novel luciferin derivatives. Certain combinations of CBR mutants and novel luciferin derivatives offer significant technical advantages for bioluminescence assays, particularly for deep-tissue imaging, because near-IR light emission is produced. The disclosed invention provides improved detection of signals from deep animal tissues, in part because the longer wavelengths are not absorbed by tissue, and also because light emission is improved over the parent CBR and known luciferin substrates. In addition, other reagents may be included in the bioluminescence system, including, but not limited to, those that inhibit or prevent inactivation of the CBR mutants or that otherwise prolong or enhance the luminescence signal.

[0088] a. Novel luciferin derivatives The near-IR bioluminescence system can include novel red-shifted luciferin derivatives, such as those disclosed in PCT Application No. PCT / US2014 / 021678, which is incorporated herein by reference in its entirety. For example, the luciferin derivative can include a compound of one of formulas (Ia), (Ib), (Ic), (II), (III), (IV), (V), (VI), or (VII), or a pro-substrate thereof, such as a reductase substrate, a glycosidase substrate, a protease and protease-dependent protein modification substrate, an oxidase substrate, a carboxyl-based pro-substrate, a glutathione transferase substrate, a beta-lactamase substrate, and other pro-substrates. In some embodiments, the novel luciferin derivative can be PBI-4739 or PBI-4813 (see FIG. 1; disclosed in PCT Application No. PCT / US2014 / 021678).

[0089] (1) A compound of formula(s) (Ia), (Ib), (Ic), (II), (III), (IV), (V), (VI), or (VII) Luciferin derivatives are represented by formulas (Ia), (Ib) and (Ic): TIFF0007780560000013.tif4360(Ia) TIFF0007780560000014.tif2669(Ib) TIFF0007780560000015.tif3959(Ic) and During the ceremony, X is CN or TIFF0007780560000016.tif2643; Each Y is independently halo, SO3H, C 1-4 Alkyl, substituted C 1-4 Alkyl, OR 1 or NR 1 R 2 and; Each R 1 is H, C 1-10 Alkyl or substituted C 1-10 is alkyl; Each R 2 is H, C 1-10 Alkyl or substituted C 1-10 is alkyl; or R 1 and R 2 together form a 4- to 8-membered ring; n is 1 to 6; two Y substituents may be joined together to form a ring containing 5 to 7 ring atoms; wherein at least one Y is OH or NR 1 R 2 Either:

[0090] Luciferin derivatives may include compounds such as Not limited to TIFF0007780560000017.tif30134.

[0091] In other embodiments, the luciferin derivative has the formula (II): TIFF0007780560000018.tif3968(II) and During the ceremony X is CN or TIFF0007780560000019.tif2643; Y is for OR 1 or NR 1 R 2 and; R 1 is H, C 1-10 Alkyl or substituted C 1-10 is alkyl; R 2 is H, C 1-10 Alkyl or substituted C 1-10 is alkyl; or R 1 and R 2 together form a ring.

[0092] In a further embodiment, the luciferin derivative has the formula (III): TIFF0007780560000020.tif5289(III) and During the ceremony X is CN or TIFF0007780560000021.tif2643; Y is for OR 1 or NR 1 R 2 and; R 1 is H, C 1-10 Alkyl or substituted C 1-10 is alkyl; R 2 is H, C 1-10 Alkyl or substituted C 1-10 is alkyl; or R 1 and R 2 together form a ring.

[0093] In still other embodiments, the luciferin derivative has the formula (IV): TIFF0007780560000022.tif3958(IV) and During the ceremony X is CN or TIFF0007780560000023.tif2643; Y is for OR 1 or NR 1 R 2 and; R 1 is H, C 1-10 Alkyl or substituted C 1-10 is alkyl; R 2 is H, C 1-10 Alkyl or substituted C 1-10 is alkyl; or R 1 and R 2 together form a ring.

[0094] In other embodiments, the luciferin derivative has the formula (V): TIFF0007780560000024.tif3968(V) and During the ceremony X is CN or TIFF0007780560000025.tif2643; Y is for OR 1 or NR 1 R 2 and; R 1 is H, C 1-10 Alkyl or substituted C 1-10 is alkyl; R 2 is H, C 1-10 Alkyl or substituted C 1-10 is alkyl; or R 1 and R 2 together form a ring.

[0095] In other embodiments, the luciferin derivative has the formula (VI): TIFF0007780560000026.tif3967(VI) and During the ceremony X is CN or TIFF0007780560000027.tif2643; Y is for OR 1 or NR 1 R 2 and; R 1 is H, C 1-10 Alkyl or substituted C 1-10 is alkyl; R 2 is H, C 1-10 Alkyl or substituted C 1-10 is alkyl; or R 1 and R 2 together form a ring.

[0096] In additional embodiments, the luciferin derivative has the formula (VII): TIFF0007780560000028.tif5359(VII) and During the ceremony X is CN or TIFF0007780560000029.tif2643; Each Y is independently OR 1 or NR 1 R 2 and; Each R 1 are independently H, C 1-10 Alkyl or substituted C 1-10 is alkyl; Each R 2 are independently H, C 1-10 Alkyl or substituted C 1-10 alkyl; or R 1 and R 2 together form a ring.

[0097] In certain embodiments, the emission maximum of the compound is at least about 650 nm, about 655 nm, about 680, or about 760 nm.

[0098] (2) Pro-substrate The present invention also provides compounds that are substrates for a variety of nonluciferase enzymes and pro-substrates for luciferase enzymes, including, but not limited to, reductases, glycosidases, proteases, peptidases, oxidases, esterases, cytochrome P450, beta-lactamases, glycosylases, and glutathione transferases.

[0099] In some embodiments, these pro-substrates have a substituent at one or more Yi positions that is a substrate for a non-luciferase site, which is cleaved to yield: TIFF0007780560000030.tif4360(VIII) Forming During the ceremony X is CN or TIFF0007780560000031.tif2643; each Y is OH or NH; n is 1 to 3.

[0100] (a) Reductase substrate In some embodiments, the compound is a reductase substrate. In some embodiments, the reductase substrate has the formula: TIFF0007780560000032.tif3968(IX) and During the ceremony X is CN or TIFF0007780560000033.tif2643; Y is OR; R is TIFF0007780560000034.tif3030; W, S, NR N , or O; Z, S, NR N , O or CH; R N is H, C 1-4 Alkyl or substituted C 1-4 It is alkyl.

[0101] In some embodiments, the reductase substrate has the formula: TIFF0007780560000035.tif4866(X) and During the ceremony X is CN or TIFF0007780560000036.tif2643; Y is for OR 1 or NR 1 R 2 is; and R 1 is H, C 1-10 Alkyl or substituted C 1-10 is alkyl; R 2 is H, C 1-10 Alkyl or substituted C 1-10 is alkyl; or R 1 and R 2 together form a 4- to 8-membered ring.

[0102] In some embodiments, the reductase substrate has the formula: TIFF0007780560000037.tif3968(XI) and During the ceremony X is CN or TIFF0007780560000038.tif2643; Y is OR; R is The file is TIFF0007780560000039.tif46116.

[0103] The reductase substrates are the following compounds: Including, but not limited to, TIFF0007780560000040.tif139164.

[0104] (b) Glycosidase substrate In some embodiments, the compound is a glycosidase substrate. In some embodiments, the glycosidase substrate has the formula: TIFF0007780560000041.tif3968(XII) is a compound of During the ceremony X is CN or TIFF0007780560000042.tif2643; Y is OR; R is TIFF0007780560000043.tif57164; A is OR or NHAc; Each R5 is independently H, a monosaccharide, or a polyethylene glycol moiety of up to 40 units.

[0105] Glycosidase substrates include the following compounds: Including but not limited to TIFF0007780560000044.tif63149.

[0106] (c) Proteases and protease-dependent protein-modified substrates In some embodiments, the compound is a protease or a protease-dependent protein-modifying substrate. In some embodiments, the substrate has the formula: TIFF0007780560000045.tif3968(XIII) is a compound of During the ceremony X is CN or TIFF0007780560000046.tif2643; Y is NHR; R is TIFF0007780560000047.tif3036; R7 is an amino acid side chain; R6 is H, a nitrogen protecting group, or a chain of up to 20 amino acids.

[0107] Suitable nitrogen protecting groups include, but are not limited to, those conventionally known to those skilled in the art, such as Boc, Cbz, Ac, and Fmoc.

[0108] These substrates include the following compounds: Including, but not limited to, TIFF0007780560000048.tif123164.

[0109] (d) Oxidase substrate In some embodiments, the compound is an oxidase substrate. In some embodiments, the oxidase substrate has the formula: TIFF0007780560000049.tif3968(XIV) is a compound of During the ceremony X is -CH(OR 10 )2; R 10 is C 1-4 Alkyl, substituted C 1-4 alkyl, benzyl, or substituted benzyl; Y is OR 1 or NR 1 R 2 is; and R 1 is H, C 1-10 Alkyl or substituted C 1-10 is alkyl; R 2 is H, C 1-10 Alkyl or substituted C 1-10 is alkyl; or R 1 and R 2 together form a 4- to 8-membered ring.

[0110] In some embodiments, the oxidase substrate has the formula: TIFF0007780560000050.tif3968(XV) is a compound of During the ceremony X is CN or TIFF0007780560000051.tif2643; Y is H or OR; R is C 1-10 Alkyl, substituted C 1-10It is alkyl, aryl, substituted aryl, aralkyl or substituted aralkyl.

[0111] In some embodiments, the oxidase substrate has the formula: TIFF0007780560000052.tif3994(XVI) is a compound of During the ceremony X is -CH(OR 10 )2; R 10 is C 1-4 Alkyl, substituted C 1-4 alkyl, benzyl or substituted benzyl; Y is for OR 1 or NR 1 R 2 is; and R 1 is H, C 1-10 Alkyl or substituted C 1-10 is alkyl; R 2 is H, C 1-10 Alkyl or substituted C 1-10 is alkyl; or R 1 and R 2 together form a 4- to 8-membered ring.

[0112] Oxidase substrates include the following compounds: Including, but not limited to, TIFF0007780560000053.tif62164.

[0113] (e) Carboxyl pro-substrate In some embodiments, the compound is a carboxylic pro-substrate. In some embodiments, the carboxylic pro-substrate has the formula: TIFF0007780560000054.tif4094(XVII) is a compound of During the ceremony R8 is CH2OH, C(O)R 10 or -C(O)ZR9; Z is O or NH; R9 is C 1-7 Alkyl or substituted C 1-7 is alkyl; R 10 is a peptide; Y is for OR 1 or NR 1 R 2 is; and R 1 is H, C 1-10 Alkyl or substituted C 1-10 is alkyl; R 2 is H, C 1-10 Alkyl or substituted C 1-10 is alkyl; or R 1 and R 2 together form a 4- to 8-membered ring.

[0114] Carboxyl pro-substrates include the following compounds: Including, but not limited to, TIFF0007780560000055.tif63149.

[0115] (f) glutathione transferase substrate In some embodiments, the compound can be a glutathione transferase substrate. In some embodiments, the glutathione transferase substrate has the formula: TIFF0007780560000056.tif3968(XVIII) is a compound of During the ceremony X is CN or TIFF0007780560000057.tif2643; Y is OR; R is TIFF0007780560000058.tif3750; Each R 11 are independently H, C 1-6 Alkyl, substituted C 1-6 Alkyl, CF3, halogen, NO2, CO2R 12 or R 11If at least one of the R is NO2, then any two adjacent R 11 can form a fused ring; R 12 is H, C 1-6 Alkyl or substituted C 1-6 It is alkyl.

[0116] Glutathione transferase substrates include the following compounds: Including, but not limited to, TIFF0007780560000059.tif36164.

[0117] (g) beta-lactamase substrates In some embodiments, the compound is a beta-lactamase substrate. In some embodiments, the beta-lactamase substrate has the formula: TIFF0007780560000060.tif60129(XIX) is a compound of During the ceremony X is CN or TIFF0007780560000061.tif2643; Y is O, NH, N(C 1-7 alkyl), or N(substituted C 1-7 alkyl); Z is absent or O; A is C 1-4 Alkylene or substituted C 1-4 is alkylene; R 14 is H, phenacetyl, or a cephalosporin side chain.

[0118] Suitable cephalosporin side chains include those known to those skilled in the art.

[0119] Beta-lactamase substrates include the following compounds: Including, but not limited to, TIFF0007780560000062.tif62164.

[0120] (h) Other pro-substrates The present invention also provides compounds that react with various biologically important small molecules, such as hydrogen peroxide, and are pro-substrates for the luciferase enzyme. In some embodiments, these compounds are reactive with hydrogen peroxide. In some embodiments, these compounds have the formula: TIFF0007780560000063.tif3968(XX) and During the ceremony X is CN or TIFF0007780560000064.tif2643; Y is LR; L is a linker; R is a boronic acid or boronic ester.

[0121] In some embodiments, R is -B(OR 15 )2; where each R 15 are independently H and C 1-4 In some embodiments, R is selected from: TIFF0007780560000065.tif3043; where each R 16 and R 17 are independently H, C 1-4 Alkyl, substituted C 1-4 alkyl, CF, phenyl or substituted phenyl; or R 16 and R 17 can be joined together to form an alkyl ring having 3 to 7 carbons or can be replaced by a fused 6-membered aromatic ring.

[0122] In some embodiments, the linker is a direct bond. In other embodiments, the linker is TIFF0007780560000066.tif2039; During the ceremony A is -C 6( R 20 )4-, -O-C6(R 20 )4- or -(CR 21 =CR 21 ) n-or-S-C6(R 20 )4- or -NR'-C6(R 20 ) 4 or direct bond; R' is H, C 1-4 Alkyl or substituted C 1-4 is alkyl; Each R 23 are independently halo, H, C 1-4 Alkyl, substituted C 1-4 Alkyl, C 1-4 Hydroxyalkyl, substituted C 1-4 Hydroxyalkyl, C 1-4 Alkyl carboxylic acid or substituted C 1-4 It is an alkyl carboxylic acid; Each R 20 are independently H, halo, CH3, OCH3, or NO2; Each R 21 are independently H or CH3; n is 1 or 2; X is -O-, Selected from TIFF0007780560000067.tif57149.

[0123] These compounds include the following compounds: Including, but not limited to, TIFF0007780560000068.tif41164.

[0124] 4. Methods Using CBR Mutants and / or Bioluminescence Systems CBR variants and near-IR bioluminescence systems can be used in any method in which luciferases and luciferase substrates, such as luciferin and luciferin derivatives, are used. CBR variants and / or near-IR bioluminescence systems can be used with samples (including cells, tissues, animals, etc.) or in vivo imaging, and detected using various microscopy and imaging techniques. CBR variants and / or near-IR bioluminescence systems can be used as transcriptional reporters or biosensors. CBR variants and / or near-IR bioluminescence systems can be used in two-color assays or multiplexing. CBR variants and / or near-IR bioluminescence systems can be used in assays to detect the presence or activity of enzymes, such as non-luminescent enzymes. For example, they can be used in bioluminescence source methods that employ luciferin analogs to detect one or more molecules in a sample, such as enzymes, cofactors for enzymatic reactions, enzyme substrates, enzyme inhibitors, enzyme activators, or OH radicals, or one or more conditions, such as redox conditions. In some embodiments, the CBR mutants and / or near-IR bioluminescence systems can be used as energy donors to another molecule (e.g., a fluorophore, chromophore, or nanoparticle). The CBR mutants and near-IR bioluminescence systems can be used in protein proximity assays or protein complementation assays. The CBR mutants and / or near-IR bioluminescence systems disclosed herein are also useful in in situ methods for analyzing cells. Methods for performing in situ analysis of cells using luciferase are known in the art. The CBR mutants and / or near-IR bioluminescence systems disclosed herein can be used to distinguish between substrates and inhibitors of an enzyme. Screening can be performed either in vitro or in vivo.

[0125] Samples may include animals (e.g., vertebrates), plants, fungi, physiological fluids (e.g., blood, plasma, urine, mucosal secretions, etc.), cells, cell lysates, cell supernatants, or purified fractions of cells (e.g., subcellular fractions). The presence, amount, spectral distribution, emission kinetics, or specific activity of such molecules may be detected or quantified. The molecules may be detected or quantified in solution, including multiphase solutions (e.g., emulsions or suspensions), or on solid supports (e.g., particles, capillaries, or assay vessels).

[0126] The luciferase reaction may be limited or confined by time, enzyme concentration, and / or substrate concentration. Reaction conditions may be adjusted to run under conditions that result in the luciferase reaction being about, at least about, or at most about 20, 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, or 100% complete, or any range derivable therebetween. For example, the reaction can be carried out at a temperature between about 20°C and about 45°C, between about 20°C and about 40°C, between about 20°C and about 35°C, between about 20°C and about 30°C, between about 20°C and about 25°C, between about 25°C and about 45°C, between about 25°C and about 40°C, between about 25°C and about 35°C, between about 25°C and about 30°C, between about 30°C and about 45°C, between about 30°C and about 40°C, between about 30°C and about 35°C, between about 35°C and about 45°C, or between about 35°C and about 40°C. The reaction may be carried out at 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, or 45°C. These temperature conditions and / or reactions may be maintained or measured for 1 minute, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 minutes, 65 minutes, 70 minutes, 75 minutes, 80 minutes, 85 minutes, 90 minutes, 100 minutes, 110 minutes, 120 minutes, 130 minutes, 140 minutes, 150 minutes, 160 minutes, 170 minutes, 180 minutes, 190 minutes, 200 minutes, 210 minutes, 220 minutes, 230 minutes, 240 minutes, 250 minutes, or more.

[0127] a. In vivo imaging CBR variants and near-IR (NIR) bioluminescence systems may be used for in vivo imaging and may improve the speed, detection limit, and deep penetration of bioluminescence imaging. CBR variants and near-IR bioluminescence systems provide a means for noninvasive animal imaging for a variety of applications, such as understanding tumor biology and evaluating compounds with therapeutic potential. For example, the methods described herein can be used for rapid and inexpensive assessment of tumor progression and response to anticancer therapeutic agents in small animals, using, for example, transgenic nonhuman animals, such as mice expressing a luciferase reporter gene linked to a promoter or gene expressed, e.g., selectively expressed, in cells desired to be imaged. Expression of a selected protein of interest can be imaged in real time in living cells or animals using cells or transgenic animals expressing a reporter construct comprising a nucleic acid encoding the selected protein of interest or a nucleic acid encoding a CBR variant linked in-frame to the promoter of the selected protein.

[0128] The method can be carried out in cells or animals (e.g., non-human mammals, e.g., rodents, e.g., experimental animals such as rats or mice) that express the CBR mutant reporter construct. Generating such cells or animals can be carried out using standard molecular biology techniques. A sufficient amount of the novel luciferin derivatives described herein can be added to cells or administered to animals, and images of NIR bioluminescence can be obtained using standard imaging methods. Promoter activity, protein expression, protein subcellular localization, protein translocation, and protein half-life can be assessed in real time in living cells and animals.

[0129] When using experimental animals, cells containing NIR bioluminescence can be identified and excised and further evaluated using, for example, assays for gene expression, protein expression, or other genetic or biochemical parameters. Bioluminescence systems can be designed using alternative luciferin / luciferase pairs (e.g., with different emission maxima) to allow simultaneous imaging of bioluminescence from two or more luciferases.

[0130] (1) Imaging method The methods described herein can be practiced with any imaging system capable of detecting near-infrared bioluminescence. Common imaging systems are available from Xenogen (e.g., IVIS), Hamamatsu, Roper, and Kodak.

[0131] (2) Viable cells In various embodiments, CBR mutants can be used to detect luminescence in living cells. In some embodiments, CBR mutants can be expressed in cells (as reporters or otherwise), and cells treated with a luciferase substrate, e.g., luciferin, a luciferin derivative, e.g., a novel luciferin derivative such as PBI-4739 or PBI-4813, or a functional analog that can permeate cells in culture, react with the CBR mutant and generate luminescence. PBI-4739 or PBI-4813 exhibits non-toxicity comparable to D-luciferin in cytotoxicity tests. In some embodiments, variants of PBI-4739 or PBI-4813 containing chemical modifications known to increase the stability of native luciferin derivatives in culture medium can be synthesized and used for more robust, live-cell CBR mutant-based reporter assays. In still other embodiments, samples (including cells, tissues, animals, etc.) containing the CBR variants and / or novel luciferin derivatives of the invention can be assayed using a variety of microscopy and imaging techniques. In still other embodiments, secretable CBR variants can be expressed in cells as part of a live cell reporter system.

[0132] b. Use as a transcriptional reporter The CBR mutant and near-IR bioluminescence system can be used as a gene transcription reporter system. The CBR mutant or a fragment thereof can be used to examine the transcriptional expression pattern of any promoter and / or gene, such as a gene involved in development. In some embodiments, the CBR mutant or a fragment thereof can be operably linked to a transcriptional control sequence, such as one or more enhancers, promoters, transcription termination sequences, or a combination thereof, to form an expression cassette. For example, the CBR mutant can be operably linked to a minimal promoter and a cAMP-response element (CRE).

[0133] In certain embodiments, a method for measuring the activity of a promoter in a sample is provided, wherein the promoter is operably linked to a gene encoding a CBR mutant enzyme. The method includes: (a) contacting the sample with luciferin or a novel luciferin derivative; and (b) determining the activity of the promoter by measuring the luminescence of the sample, wherein the sample contains the promoter. The promoter can be operably linked to the gene via a translational or transcriptional fusion. A biological pathway of interest can be examined, for example, by treating cells containing the promoter operably linked to a gene encoding a luminescent enzyme with an inducer of the pathway. The promoter activity can then be measured and observed to study any correlation between the promoter activity and the pathway of interest, as well as to obtain kinetic measurements related to gene expression (e.g., inducibility, repression, and activation).

[0134] c.Multiplexing The CBR mutant and bioluminescence system can be used in a multiplex reaction with another enzyme (e.g., luciferase) that emits light at a different wavelength. In some embodiments, the CBR mutant can be used as a transcriptional reporter and paired with a luciferase that emits light at a different wavelength contained in the assay reagent. In some embodiments, the CBR mutant can be used with one or more additional luciferases, where the luminescence of each luciferase can be measured separately through the use of selective enzyme inhibitors. For example, the luminescence of the CBR mutant can be measured upon addition of an appropriate substrate and buffer, followed by measurement of the second luciferase upon subsequent addition of an appropriate substrate and buffer and one or more inhibitors selective for the first luciferase.

[0135] In some embodiments, a CBR mutant of the invention may be used as a functional reporter for a particular gene, and a second enzyme in a multiplexed reaction may be used as a functional reporter for a second gene. In some embodiments, a CBR mutant may be used with one or more additional luciferases, where the luminescence of each luciferase can be easily distinguished using a luminometer equipped with wavelength discrimination filters that allow measurement of both signals from the same sample. For example, the luminescence generated from the CBR mutant enzyme (approximately between 725 and 750 nm) and the green CHROMA-LUC™ (approximately 537 nm) can be easily distinguished using a luminometer equipped with wavelength discrimination filters that allow measurement of both signals from the same sample.

[0136] In some embodiments, CBR mutants may be used as transcriptional reporters, and can be simultaneously coupled with either aequorin or a cAMP-cyclically permuted firefly luciferase biosensor, or both, to detect multiple pathways in a single sample. In such a system, for example, aequorin can be used to detect and / or measure calcium, the biosensor can be used to detect and / or measure cAMP, and the CBR mutant can be used to monitor downstream gene expression.

[0137] The CBR variants can be multiplexed with luciferases from beetles, such as fireflies (e.g., Pothinus pularis (e.g., Luc2; Promega Corp.) or Photuris pennsylvanica luciferase) or different click beetle luciferases (Pyrophorus plagiophthalamus or Pyrearinus termitilluminans), such as green click beetle luciferase (CHROMA-LUC™; Promega Corp.), bioluminescent decapods, such as Oplophorus luciferase, e.g., Oplophorus gracilirostris luciferase or a variant thereof, such as NanoLuc® luciferase (Promega Corp.). Corporation, which are described in U.S. Patent Nos. 8,557,970 and 8,669,103, and U.S. Patent Application Nos. 2014 / 0223590 and 2014 / 0227759, and which are directed to the production of luciferases from marine organisms, e.g., cnidarians (e.g., Renilla reniformis luciferase), copepod luciferases, e.g., Gaussia luciferases, e.g., Gaussia princeps luciferase, Metridia luciferases, e.g., Metridia longa and Metridia pacifica luciferase, Vargula luciferases, e.g., Vargula hilgendorfii luciferase, Pleuromamma cyphias luciferase, and the like. xiphias luciferase, glowworm luciferase (e.g., Phrixothrix hirtus), and variants, recombinants, and mutants thereof.CBR variants can be multiplexed with green fluorescent protein (GFP), luminescent proteins such as aequorin, obelin, iPhotina, and variants, recombinants, and mutants thereof. For example, when a CBR variant of the invention is used as a functional reporter, green firefly luciferase or green CHROMA-LUC™ luciferase can be used to control for nonspecific effects on genetic regulation or to normalize for transfection efficiency.

[0138] d. Detection of non-luminescent enzymes Bioluminescence systems containing the pro-substrates and CBR variants described herein can be used in luminescence-based assays to detect the activity of non-luminescent enzymes. The CBR variants and pro-substrates can be included in assay reagents for measuring specific aspects of cellular physiology, such as ATP to estimate cell viability, or caspase activity to estimate cell apoptosis. The CBR variants and pro-substrates can be added to a sample suspected of containing the non-luminescent enzyme of interest. Any non-luminescent enzyme present in the sample reacts with the pro-substrate, releasing the luciferin substrate for the CBR variant for use as a substrate, thereby generating and measuring luminescence. In some embodiments, non-luminescent enzymes of interest are reductases, glycosidases, proteases, serine proteases, threonine proteases, cysteine ​​proteases such as caspase-3 and caspase-8, aspartic acid proteases, glutamic acid proteases, and metalloproteases, peptidases, oxidases such as monoamine oxidases, esterases, cytochrome P450, beta-lactamases, glycosylases, and glutathione transferase protease enzymes such as glutathione S-transferases. In some embodiments, pro-substrates as described herein can be used to detect non-luminescent enzymes.

[0139] e. Bioluminescence Resonance Energy Transfer (BRET) The CBR mutants and bioluminescence systems can be used in any method for detecting ligand-protein and / or protein-protein interactions. In various embodiments, the CBR mutant enzymes can be used to transfer energy to an energy acceptor. One such method is bioluminescence resonance energy transfer (BRET). With BRET, energy transfer from a bioluminescent donor to a fluorescent acceptor results in a shift in the spectral distribution of light emission. This energy transfer can enable real-time monitoring of protein-protein or ligand-protein interactions in vitro or in vivo.

[0140] In some embodiments, the CBR mutant enzyme used in a BRET assay can be used to determine whether two molecules can bind to each other or coexist in a cell. For example, a CBR mutant enzyme can be used as a bioluminescent donor molecule that is combined with a molecule or protein of interest to create a first fusion protein. In various embodiments, the first fusion protein contains a CBR mutant enzyme and the protein of interest. In various embodiments, a first fusion protein containing a CBR mutant enzyme can be used in a BRET assay to detect protein / protein interactions in systems including, but not limited to, cell lysates, intact cells, and live animals. In various embodiments, HALOTAG® can be used as a fluorescent acceptor molecule. In some embodiments, HALOTAG® can be fused to a second protein of interest or to a CBR mutant enzyme. For example, a CBR mutant enzyme can be fused to HALOTAG®, expressed in a cell or animal, and labeled with a fluorescent HALOTAG® ligand, such as the HALOTAG® TMR ligand. The fusion can then be excited to fluoresce in the presence of a cell-permeable CBR mutant enzyme substrate. In some embodiments, BRET can be performed using a CBR mutant enzyme in combination with a fluorescent protein, including, but not limited to, green fluorescent protein (GFP), red fluorescent protein (RFP), far-red fluorescent protein, and near-infrared fluorescent protein, e.g., iRFP and IFP1.4, or a fluorescent label including fluorescein, rhodamine green, Oregon green, Alexa488, to name a few non-limiting examples. Fluorescent labels include luciferase (e.g., 650-750 nm), such as IRDye® 800CW, IRDye® 800RS, IRDye® 800 phosphoramidite, IRDye® 750, IRDye® 700DX, IRDye® 700 phosphoramidite, IRDye® 680LT, IRDye® 680RD, IRDye® IRDye® e650, 1,1',3,3,3',3'-Hexamethylindotricarbocyanine iodide, 1,1'-diethyl-2,2'-dicarbocyanine iodide, 1,1'-diethyl-4,4'-carbocyanine iodide, 1,4,8,11,15,18,22,25-octabutoxy-29H,31H-phthalocyanine, 2,11,20,29-tetra-tert-butyl-2,3-naphthalocyanine, 2,3,9,10,16,17,23,24-octakis(octyloxy)-29H,31H-phthalocyanine, 2,3-naphthalocyanine dyes containing Tetra-tert-butyl-29H,31H-phthalocyanine, 29H,31H-phthalocyanine β-form, 3,3'-diethylthiadicarbocyanine iodide, 3,3'-diethylthiatricarbocyanine iodide, 3,3'-diethylthiatricarbocyanine perchlorate, aluminum 1,8,15,22-tetrakis(phenylthio)-29H,31H-phthalocyanine chloride, aluminum 2,9,16,23-tetrakis(phenylthio)-29H,31H-phthalocyanine chloride, aluminum 2,9,16,23-tetrakis(phenylthio)-29H,31H-phthalocyanine chloride 29H,31H-phthalocyanine chloride, aluminum 2,9,16,23-tetraphenoxy-29H,31H-phthalocyanine hydroxide, aluminum phthalocyanine chloride, aluminum phthalocyanine hydroxide, cobalt(II) 1,2,3,4,8,9,10,11,15,16,17,18,22,23,24,25-hexadecafluoro-29H,31H-phthalocyanine, cobalt(II) 2,3-naphthalocyanine, cobalt(II) phthalocyanine β-form, copper phthalocyanine-3,4',4?,4?'-tetrasulfur phthalocyanine, copper(II) 1,2,3,4,8,9,10,11,15,16,17,18,22,23,24,25-hexadecafluoro-29H,31H-phthalocyanine, copper(II) 1,4,8,11,15,18,22,25-octabutoxy-29H,31H-phthalocyanine, copper(II) 2,3,9,10,16,17,23,24-octakis(octyloxy)-29H,31H-phthalocyanine, copper(II) 2,3-naphthalocyanine, copper(II) 2,9,16,23-tetra-tert-butyl-29H,31H-Phthalocyanine, Copper(II) Phthalocyanine-Tetrasulfonic Acid Tetrasodium Salt, Copper(II) Phthalocyanine, Copper(II) Phthalocyanine β-Form, Copper(II) Phthalocyanine Sublimation Purified Grade, Copper(II) Phthalocyanine, Phthalocyanine Dilithium, Phthalocyanine Disodium, Gallium(III) Phthalocyanine Chloride, IR-775 Chloride, IR-780 Iodide, IR-783, IR-792 Perchlorate, IR-797 Chloride, Iron(II) Phthalocyanine, Iron(III) Phthalocyanine Chloride, Iron(III) Phthalocyanine -4,4',4'',4''''-Tetrasulfonic acid, compounds with oxygen monosodium salt hydrate, lead(II) phthalocyanine, lead(II) tetrakis(4-cumylphenoxy)phthalocyanine, magnesium phthalocyanine, manganese(II) phthalocyanine, manganese(III) phthalocyanine chloride, methylsilicon(IV) phthalocyanine hydroxide, naphthol green B, nickel(II) 1,4,8,11,15,18,22,25-octabutoxy-29H,31H-phthalocyanine, nickel(II) phthalocyanine-tetrasulfone Acid tetrasodium salt, Nickel(II) phthalocyanine, Poly(copper phthalocyanine), Silicon 2,3-naphthalocyanine bis(trihexylsilyl oxide), Silicon 2,3-naphthalocyanine dichloride, Silicon 2,3-naphthalocyanine dihydroxide, Silicon 2,3-naphthalocyanine dioctyl oxide, Silicon 2,9,16,23-tetra-tert-butyl-29H,31H-phthalocyanine dihydroxide, Silicon phthalocyanine dichloride, Silicon phthalocyanine dihydroxide, Tin(IV) phthalocyanine oxide Cide, titanium (IV) phthalocyanine dichloride, titanyl phthalocyanine, titanyl phthalocyanine, vanadyl 2,3-naphthalocyanine, vanadyl 3,10,17,24-tetra-tert-butyl-1,8,15,22-tetrakis(dimethylamino)-29H,31H-phthalocyanine pigment containing 80%, zinc 1,2,3,4,8,9,10,11,15,16,17,18,22,23,24,25-hexadecafluoro-29H,31H-phthalocyanine, zinc 1,4,8,11,15,18,22,25-octabutoxy-29H,The fluorescent BRET acceptor may be a fluorescent BRET acceptor that absorbs emission from 31H-phthalocyanine, zinc 2,11,20,29-tetra-tert-butyl-2,3-naphthalocyanine, zinc 2,3,9,10,16,17,23,24-octakis(octyloxy)-29H,31H-phthalocyanine, zinc 2,9,16,23-tetra-tert-butyl-29H,31H-phthalocyanine, zinc phthalocyanine, or zinc(II) tetranitrophthalocyanine.

[0141] f. Protein proximity assays in live cells or soluble formats In some embodiments, CBR mutants can be used as circularly permuted (CP) or linearly cleaved (SS) luciferase fusion proteins to measure protein proximity. The CBR mutant enzyme is displaced or cleaved via insertion of a protease substrate amino acid sequence (e.g., TEV) to generate low bioluminescence. The CBR mutant luciferase is tethered to a monitor protein (e.g., via gene fusion). A potential interacting protein is tethered to a protease (e.g., TEV) (e.g., via gene fusion). When the two monitor proteins interact (e.g., via constitutive interaction, drug stimulation, or pathway response) or are in sufficient proximity, the CBR mutant enzyme is cleaved, generating increased bioluminescence activity. This example can be applied to measuring protein proximity in cells or biochemical assays.

[0142] g. Protein complementation assay In some embodiments, compounds of the present disclosure may be used in other methods for detecting ligand-protein interactions and protein-protein interactions or proximity, such as protein complementation assays (PCAs) or enzyme fragmentation assays. Protein complementation assays (PCAs) provide a means for detecting interactions between two biomolecules, e.g., polypeptides. PCAs utilize two fragments of the same protein, e.g., a CBR mutant, that can be reconstituted into a functional, active protein when brought into close proximity with each other. In some embodiments, PCAs utilizing a CBR mutant can be used to detect molecular proximity by reconstitution of the CBR mutant through binding interactions of enzyme components or subunits. A fragment of the CBR mutant is fused to a protein of interest. When the proteins of interest interact, the fragments of the CBR mutant interact to reconstitute the full-length CBR mutant enzyme.

[0143] For example, a CBR mutant enzyme can be separated into two fragments at a site(s) tolerant to separation, and each fragment of the separated CBR mutant enzyme can be fused to one of a peptide pair of interest (e.g., FKBP and FRB) that are thought to interact. If the two polypeptides of interest indeed interact, the CBR mutant enzyme fragments can be brought into close proximity with each other to reconstitute a functional, active CBR mutant enzyme. In some embodiments, the activity of the reconstituted CBR mutant enzyme can then be detected and measured using compounds of the present disclosure and cell-permeable substrates. In some embodiments, the cleaved CBR mutant enzyme can be used in a more general complementation system similar to lac-Z (Langley et al., PNAS, 72:1254-1257 (1975)) or ribonuclease S (Levitt and Berger, J. Biol. Chem., 251:1333-1339 (1976)). In some embodiments, a CBR mutant enzyme fragment ("A") known to complement another CBR mutant enzyme fragment (designated "B") can be fused to a target protein, and the resulting fusion can be monitored via luminescence in cells or cell lysates containing fragment B. In some embodiments, the source of fragment B can be the same cell (e.g., if the gene for fragment B is integrated into the cell's genome or contained on a separate plasmid within the cell), or it can be a lysate or purified protein from another cell. In some embodiments, this same fusion protein (fragment A) can be captured or immobilized using a fusion between fragment B and a polypeptide, such as HALOTAG®, that can be attached to a solid support. In some embodiments, luminescence can be used to demonstrate successful capture or to quantify the amount of captured material.

[0144] h. Biosensors CBR mutants may be used as biosensors, which have one or more altered activities in the presence of another molecule (e.g., one or more molecules of interest) or under certain conditions. Upon interacting with a molecule of interest or subjected to certain conditions, the biosensor undergoes a conformational change or is chemically altered to cause a change in enzymatic activity or luminescence, e.g., specific activity, spectral distribution, or luminescence kinetics. For example, CBR mutants of the invention, e.g., circularly permuted mutants, can contain an interaction domain for a molecule of interest. Alternatively, for example, a CBR mutant can contain an interaction domain that can couple to an energy acceptor, e.g., a fluorescent protein, and alter the efficiency of energy transfer from the enzyme to the energy acceptor. For example, biosensors can be generated by inserting a suitable sensor region into a CBR mutant sequence to detect binding proteins such as proteases, kinases, ligands, antibodies, cyclic nucleotides such as cAMP or cGMP, or metals such as calcium. One or more sensor regions can be inserted at the C-terminus, N-terminus, and / or one or more suitable positions in the polypeptide sequence, where the sensor region comprises one or more amino acids. In the case of circularly permuted CBR mutants, the sensor region can be inserted between the N-terminus and C-terminus of the parent CBR mutant. In addition, one or all of the inserted sensor regions can include linker amino acids to couple the sensor to the remainder of the CBR mutant polypeptide.

[0145] In some embodiments, full-length circularly permuted CBR mutant enzymes can be fused to their respective binding partners, e.g., FRB and FKBP, and used in protein complementation-type assays using luciferin or novel luciferin derivatives. The key difference between the methods disclosed herein and traditional protein complementation is not the absence of complementation, but rather the dimerization of two full-length enzymes, e.g., circularly permuted CBR mutant enzymes.

[0146] Briefly, circularly permuted reporter proteins similarly configured for low activity are fused to both fusion protein partners. For example, each fusion partner can be linked to a permuted reporter of the same configuration. The interaction of the fusion partners brings the permuted reporters into close proximity, thereby allowing the reconstitution of a hybrid reporter with higher activity.

[0147] 5. Sample The disclosed CBR variants and / or near-IR bioluminescence systems may be used with samples containing biological components. The samples may include cells and / or tissues. The samples may contain heterogeneous mixtures of components (intact cells, cell extracts, cell lysates, bacteria, viruses, organelles, and mixtures thereof) or homogeneous groups of single components or components (e.g., natural or synthetic amino acid, nucleic acid, or carbohydrate polymers, or lipid membrane complexes). The compounds are typically non-toxic to viable cells and other biological components within the concentrations used.

[0148] Samples may include animals (e.g., vertebrates), plants, fungi, physiological fluids (e.g., blood, plasma, urine, mucosal secretions, etc.), cells, cell lysates, cell supernatants, or purified fractions of cells (e.g., subcellular fractions). In certain embodiments, the sample may be a cell. In some embodiments, the sample may be a viable cell. The cell may be a eukaryotic cell, such as a yeast, avian, plant, insect, or mammalian cell, including but not limited to a human, simian, murine, canine, bovine, equine, feline, ovine, caprine, or porcine cell, or a prokaryotic cell, or a cell from two or more different organisms, or a cell lysate or supernatant thereof. The cells may not be genetically modified through recombinant techniques (non-recombinant cells), or they may be recombinant cells transiently transfected with recombinant DNA and / or genomes stably propagated with recombinant DNA, or their genomes may be modified to disrupt a gene, e.g., disrupt a promoter, intron, or open reading frame, or replace one DNA fragment with another. The recombinant DNA or replacement DNA fragment may encode a molecule to be detected by the methods of the invention, a moiety that alters the level or activity of the molecule to be detected, and / or a gene product unrelated to the molecule or moiety that alters the level or activity of the molecule. The cells may be genetically modified through recombinant techniques.

[0149] 6. Kit Kits for using the CBR variants and / or near-IR bioluminescence systems are provided herein. Such kits include an active CBR variant and a luciferin substrate. The kits may further include buffers and instructions. The kit components, compositions, and buffers may be modified by adding suitable components. Suitable kit components, compositions, and buffers that can be used in the methods described herein are also commercially available. Different components may include subsets of these parts and may be combined in any way that facilitates application of the invention or extends shelf life.

[0150] In some embodiments, the kit comprises a separate container containing a lyophilized luciferase, hi some embodiments, the container contains a lyophilized luciferase that further comprises a luciferase substrate, lyophilized luciferin or a derivative thereof.

[0151] One or more reagents may be supplied in a solid form or liquid buffer suitable for inventory storage and for later addition to the reaction medium when the method using the reagent is performed. Suitable containers are provided.

[0152] (1) Container / Container The reagents included in the kit can be supplied in any type of container so that the shelf life of the different components is preserved and not absorbed or altered by the container material. For example, a sealed glass ampoule can contain lyophilized luciferase or buffer solution packaged under a neutral, non-reactive gas such as nitrogen. The ampoule can be made of any suitable material, such as glass, organic polymers such as polycarbonate or polystyrene, ceramic, metal, or any other material typically used to hold reagents. Other examples of suitable containers include simple bottles, which can be manufactured from materials similar to ampoules, and envelopes with foil-lined interiors such as aluminum or alloys. Other containers include test tubes, vials, flasks, bottles, syringes, etc. The container may have a sterile access port, such as a bottle with a stopper that can be pierced by a hypodermic needle. Other containers may have two compartments separated by a readily removable membrane, which, upon removal, allows the components to mix. The removable membrane may be glass, plastic, rubber, etc.

[0153] (2) Explanation materials The kit may be supplied with further instructional materials. The instructions may be printed on paper or other substrate and / or supplied as an electronically readable medium, such as a floppy disk, CD-ROM, DVD-ROM, Zip disk, videotape, audiotape, etc. The detailed instructions need not physically accompany the kit; instead, the user may be directed to an internet website designated by the kit manufacturer or distributor, or may be supplied as an e-mail. [Example]

[0154] 7. Working Example The present invention can be utilized as illustrated in the following non-limiting examples.

[0155] Example 1 Characterization of Near-IR Substrates PBI-4739 and PBI-4813 with Ultra-Glo™ Luciferase, QuantiLum® Recombinant Luciferase, and Purified Click Beetle Red Luciferase (CBR) Materials: The following were used in the examples: Ultra-Glo™ Luciferase (Promega Catalog No. E140); QuantiLum® Recombinant Luciferase (Promega Catalog No. E1701); Click Beetle Red Luciferase (CBR; 0.5 mg / mL purified; Promega); Bright-Glo™ Assay Buffer (Promega Catalog No. E264A); PBI-4739 (Promega - see Figure 1); and PBI-4813 (Promega - see Figure 1).

[0156] Experimental Details: The substrates, PBI-4813 and PBI-4739, were diluted to 1 mM (final concentration) in Bright-Glo™ assay buffer + 1 mM ATP. 50 μL of substrate solution was added in triplicate to 50 μL of Click Beetle Red, UltraGlo®, or QuantiLum® purified enzyme (0.5 mg / mL diluted in DMEM + 0.1% Prionex), and the samples were then assayed in spectral scanning mode using a Tecan M-1000 plate reader.

[0157] Figure 2 provides spectral data for click beetle red luciferase using PBI-4813 (A) and PBI-4739 (B), where the spectral maxima were 655 nm for PBI-4813 and 760 nm for PBI-4739. Figure 3 provides spectral data for UltraGlo® luciferase using PBI-4813 (A) and PBI-4739 (B), where the spectral maxima were 670 nm for PBI-4813 and 660 nm for PBI-4739. Figure 4 provides spectral data for QuantiLum® luciferase (firefly) using PBI-4813 (A) and PBI-4739 (B), where the spectral maxima were 680 nm for PBI-4813 and >700 nm for PBI-4739.

[0158] Example 2 Library screening A library of click beetle red mutants was prepared using the Diversify™ PCR Random Mutagenesis Kit (Clontech) according to the manufacturer's instructions, using pF4Ag-HT7-CBR, i.e., the CBR-HALOTAG® fusion protein, as a template. The library of mutant DNA was cloned into pF4Ag-HT7 (Promega) and transformed into 50 μL KRX competent cells (Promega Corporation). Cells were grown overnight at 37° C. on LB-ampicillin plates.

[0159] Colonies were picked and grown overnight in wells of a 96-well plate in 200 μL of M9 minimal medium (1× M9 salts, 0.1 mM CaCl, 2 mM MgSO, 1 mM thiamine-HCl, 1% gelatin, 0.2% glycerol, and 100 μg / mL ampicillin) at 37° C. 10 μL of the overnight culture was diluted into 190 μL M9 minimal medium and grown overnight at 37° C. 10 μL of the second overnight culture was diluted into 190 μL M9 minimal induction medium (M9 minimal medium plus 0.05% glucose and 0.02% rhamnose) and grown overnight at 25° C.

[0160] Cells were assayed via robotics. Briefly, 25 μL of cell culture was added to 25 μL of lysis buffer (50 mM HEPES pH 7.5, 0.3× Passive Lysis Buffer (PLB; Promega Corporation), 0.006 U RQ1 DNase 1 (Promega Corporation)) and incubated for 3 minutes. 50 μL of Bright-Glo™ Assay Reagent (Promega Corporation) or PBI-4813 Assay Reagent (Bright-Glo™ Assay Buffer + ATP (up to 1 mM) and PBI-4813 (up to 5 μM)) was added to the lysed cells, and luminescence was detected on a Tecan Genios Pro (Table 1). Table 1 TIFF0007780560000069.tif228166

[0161] Example 3 Click Beetle Red Mutation Combination Some of the mutations identified in the library screen outlined in Example 2 were combined to identify beneficial combinations of mutations. Mutations were introduced into click beetle red (CBR) luciferase (SEQ ID NO: 1) using the QuikChange Multiple Site-Directed Mutagenesis Kit (Agilent). Mutants were cloned, expressed, and screened as described in Example 2. Table 2 and Figure 5 list the mutants and the fold change in luminescence relative to CBR luciferase with substrates PBI-4813 or PBI-4739. Table 2 TIFF0007780560000070.tif77156

[0162] Example 4 Mutants tested in mammalian cells Some of the mutants identified in Examples 2 and 3 were screened for their performance in mammalian cells. DNA was prepared by Plasmid.com. HEK293 and HeLa cells were cultured at 0.05 x 10 in wells of a 24-well plate. 6 Cells were seeded at 2000 cells / mL and grown overnight at 37°C and CO2. 2.2 μg mutant DNA was mixed with 80 μL OptiMEM and 6.6 μL FuGENE® HD Transfection Reagent (Promega Corporation) and incubated at room temperature for 5 minutes. 25 μL of the DNA mixture was added to each well.

[0163] After overnight incubation, the medium was removed from the cells and 1 mL DPBS (Life Technologies) was added. The cells were then freeze-thawed to generate cell lysates. 50 μL of the lysate was dispensed into wells of a 96-deep-well plate. 50 μL of assay reagent (Bright-Glo™ Assay Buffer containing 20 μM PBI-4813 or PBI-4739, containing 1 mM ATP) was added to the lysate, and luminescence was detected on an ImageQuant. The luminescence of the mutants was normalized to the average of the two CBR samples (Figures 6 and 7).

[0164] Example 5 Hydrogen acceptor mutagenesis design Amino acid differences between CBR and CBG99 contribute to the color change, including the major contributing amino acid changes Y224V, H247S, and Q348H, and the minor contributing amino acid changes I346N and T349S. At position 351, the amino acid is glycine in CBR and CBG99 and arginine in CBG68.

[0165] Efficient emission from some red-shifted luciferin derivatives may require the presence of an amino acid capable of acting as a hydrogen acceptor (H acceptor) at the appropriate position in the active site of the CBR mutant. For example, PBI-4739 may require an H acceptor near its hydroxyl group. The following computational experiments were performed using Discovery Studio software (Accelrys). A homology model of the CBR was generated based on the X-ray structure template of the related Luciola cruciata luciferase in complex with DLSA (Protein Data Bank accession 2D1S). The PBI-4739 ligand was manually docked into this model to match the position of the DLSA ligand in 2D1S, and the amino acid side chains within 5 Å of PBI-4739 were energy minimized. Molecular dynamics simulations were performed on the CBR amino acid side chains within 5 Å of PBI-4739. Representative output configurations were used to in silico examine which CBR mutations (amino acid substitutions) could provide the desired H acceptor.

[0166] The first group of mutations was identified based on reconstructing the network of interacting side chains. The target positions were near the PBI-4739 hydroxyl and at least partially solvent-exposed. Based on the alignment of representative beetle luciferases (firefly, click beetle, and glowworm), the side chains at these positions were determined to be conserved (E308), mostly conserved (R334 and T226), or variable (G351). Various combinations of side chains at these positions protected the ligand from solvent and allowed the formation of a stabilized H-bond network that bridged multiple secondary structure elements. The following combinations of mutations were intended to provide different H-bond networks while simultaneously allowing one of the side chains to act as an H-acceptor for interaction with the PBI-4739 hydroxyl:

[0167] G351R+R334(E, Q, D, N, H, S, T, C, Y)

[0168] G351K+R334(E, Q, D, N, H, S, T, C, Y)

[0169] G351K+T226N+R334(E, Q, D, N, H, S, T, C, Y)

[0170] G351E+E308R+R334(E, Q, D, N, H, S, T, C, Y)

[0171] E308R+R334(E, Q, D, N, H, S, T, C, Y)

[0172] T226N+R334(E, Q, D, N, H, S, T, C, Y)

[0173] A second group of mutations was developed based on targeting distinct positions that can accommodate various H receptors. Substitutions that would not be accessible by random mutagenesis techniques, i.e., single base substitutions, were prioritized:

[0174] T226(N, Q, E, D, H, C, Y)

[0175] C310(Q, E, N, D, H)

[0176] Q348(H, E)

[0177] S281(N, Q, H, Y)

[0178] Example 6 Hydrogen acceptor mutagenesis mutant screening A. Mutagenesis. Hydrogen acceptor mutagenesis was performed using the QuikChange Multiple Site-Directed Mutagenesis Kit (Agilent) as previously described, using condensation oligos specific for the following amino acids. The following mutants were cloned and expressed as described in Example 2:

[0179] T226(Q, N, E, D, H, C, Y)

[0180] C310(Q, E, N, D, H)

[0181] S281(Q, N, H, Y)

[0182] Q348((H, E)

[0183] G351R+R334(E, Q, D, N, H, S, T, C, Y)

[0184] G351K+R334(E, Q, D, N, H, S, T, C, Y)

[0185] G351+226N+R334(E, Q, D, N, H, S, T, C, Y)

[0186] G351E+E308R+R334(E, Q, D, N, H, S, T, C, Y)

[0187] E308R+R334(C, Y)

[0188] T226N+R334(E, D, S, T, C, Y)

[0189] B. Primary Screening. 1.1 plates of clones were picked for each of the 10 sets of amino acid combinations listed. These plates were then sequenced and assayed as described above.

[0190] 2. Cell lysates were prepared from cells containing the mutants listed in Table 3 by diluting 100 μL of induced culture with 100 μL of lysis buffer (0.3× PLB, 0.006 U RQ DNAse 1) using a Tecan liquid handling robot. 50 μL of each cell lysate was assayed with either PBI-4813 or PBI-4739 (20 μM in Bright-Glo™ assay buffer containing 1 mM ATP), and luminescence was detected on an ImageQuant CCD imager. Mutant luminescence was normalized to CBR luciferase (Figure 8).

[0191] C. Secondary Screening. 1. Hits from the primary screen were selected and then processed for secondary screening. Each sample was assayed in quadruplicate using the same assay method described above for the primary screen.

[0192] 2. Mutants were assayed as described above for the primary screen. Luminescence of mutants was normalized to CBR luciferase (Table 3). Table 3 TIFF0007780560000071.tif176162

[0193] Example 7 H receptor mutagenesis and library screening mutant combinations Combinations of hydrogen acceptor mutants identified in Example 6 were designed and prepared using the QuikChange Multiple Site-Directed Mutagenesis Kit (Agilent). Mutants were cloned and expressed as described in Example 2, except the template was pF4Ag-CBR. Mutants were screened as described in Example 6. Luminescence of the mutants using luciferin ("D-LH2"), PBI-4813, and PBI-4739 was normalized to CBR luciferase (Table 4 and Figure 9). Table 4 TIFF0007780560000072.tif161166

[0194] Example 8 Evaluation of clones 1230 and 1240 The mutant clones identified as ATG1230 (CBR + 389F + 444R + 251S) and ATG1240 (CBR + 334S + 351R) were further evaluated for their luminescence, spectral properties, Km, and viable cell kinetics with either PBI-4813 or PBI-4739. These mutants were cloned and expressed as described in Example 2, except that the template was pF4Ag-CBR, i.e., without HALOTAG®.

[0195] A. Luminescence with PBI-4813 and PBI-4739. Cell lysates of these mutant clones were prepared and assayed for luminescence as described in Example 6. Luminescence was detected on a GloMax® Discover equipped with a red-sensitive photomultiplier tube (PMT) and compared to CBR luciferase (Table 5 and Figure 10). Table 5 TIFF0007780560000073.tif25143

[0196] B. Spectral Measurements. Purified ATG1230 and ATG1240 enzymes were prepared. CBR was diluted 1:10 in 0.3x PLB + 0.1% Prionex. The enzymes were assayed in triplicate by adding 50 μL of enzyme to 50 μL of assay reagent (Bright-Glo™ assay buffer containing 20 μM PBI-4813 or PBI-4739, containing 1 mM ATP). Luminescence was detected using spectral scanning mode on a Tecan-M1000 (FIG. 11). Using PBI-4813 as the substrate, ATG-1240 had a spectral maximum at approximately 725 nm, while CBR and ATG-1230 had spectral maxima at approximately 650 nm. Using PBI-4739 as the substrate, ATG-1240 had a spectral maximum at approximately 750 nm, while CBR and ATG-1230 had a spectral maximum at approximately 760 nm.

[0197] C. Km Titration. Cell lysates of ATG1230 and ATG1240 expressed in HEK293 cells were prepared as described in Example 4. The cell lysates were then diluted 1:10 in 0.3x PLB + 0.1% Prionex. Dilutions (2-fold) of each substrate (PBI-4813 or PBI-4739) were prepared in Bright-Glo™ assay buffer containing 1 mM ATP. 50 μL of diluted cell lysate was assayed with 50 μL of each substrate assay solution. Luminescence was detected on a GloMax® Discover (FIG. 12). Figures 12A and 12B show the relative Vmax (RLU) and Km (μM) values ​​for ATG1230 and ATG1240 lysates using PBI-4813 and PBI-4739 as substrates. Because the enzyme concentration was not known for the lysates of the viable cell titrations, it was not possible to express Vmax as a velocity. Instead, relative Vmax was calculated, which is the maximum intensity for each lysate using different substrates. The Km for the lysate containing PBI-4813 and CBR was approximately 30-fold lower than that for CBR and D-luciferin. The Km for PBI-4813 and ATG1240 was approximately 5-fold lower than that for CBR and D-luciferin.

[0198] D. Viability Cell Kinetics. HEK293 cells were seeded into wells of a 96-well plate at 10,000 cells / well for kinetic readings and 5,000 cells / well for luminescence detection. Cells were transfected using 17 μg of each DNA (ATG1230 or ATG1240) in a total of 776 μL of OptiMEM (n=24 for each sample). 50 μL of FuGENE® HD (Promega Corporation) was then added, and the samples were incubated for 10 minutes at room temperature. 5 μL of DNA complex was added to each plate up to 12 wells / clone. The plates were then incubated overnight at 37°C with CO2. Cells were assayed by first removing the growth medium and replacing it with CO2-independent medium + 0.5% FBS containing diluted substrate (100 μM PBI-4813, 1 mM PBI-4739, or 3 mM D-luciferin). For kinetic readings, plates were incubated at room temperature for 5 minutes and kinetics was measured on a GloMax® Discover for 150 minutes at 37° C. (FIG. 13). For luminescence detection, plates were incubated in a CO2 incubator for 90 minutes and luminescence was detected on a GloMax® Discover (FIG. 14).

[0199] HeLa cells were seeded at 20,000 cells / well into wells of a 96-well white assay plate and grown overnight. A transfection mixture was made containing 13 μg of CBR DNA and 40 μL FuGENE® HD in a total volume of 620 μL OptiMEM. The mixture was incubated at room temperature for 10 minutes. Cells were then transfected in triplicate with 5 μL of the transfection complex and grown overnight.

[0200] The transfected cells were then assayed. 3-fold serial dilutions of D-luciferin, starting at 3 mM, and PBI-4813, starting at 0.3 mM, were prepared in CO2-independent medium + 0.5% FBS. Growth medium was removed from the transfected cells and replaced with medium containing one of the 3-fold serially diluted substrates. Cells were incubated for 60 minutes at 37°C. Luminescence was detected on a GloMax®-Multi+ luminometer (Figures 21A and 21B). For CBR in viable cells, the Km for PBI-4813 was approximately 20-fold lower than the Km for D-luciferin.

[0201] HEK293T cells were seeded at 15,000 cells / well into wells of a 96-well white assay plate and grown overnight. A transfection mixture was made containing 13 μg of CBR DNA and 40 μL FuGENE® HD in a total volume of 620 μL OptiMEM. The mixture was incubated at room temperature for 10 minutes. Cells were then transfected in triplicate with 5 μL of transfection complex and grown overnight.

[0202] The transfected cells were then assayed. Serial two-fold dilutions of D-luciferin, starting at 2 mM, and PBI-4813 and PBI-4739, starting at 1 mM, were prepared in CO2-independent medium + 0.5% FBS. Growth medium was removed from the transfected cells and replaced with medium containing one of the three-fold serially diluted substrates. Cells were incubated for 60 minutes at 37°C, and the cells were imaged on an ImageQuant CCD imager (Figure 22). For CBR, the Km for PBI-4739 was approximately 5-fold lower than D-luciferin, while the Km for PBI-4813 was approximately 23-fold lower than D-luciferin.

[0203] HEK293T cells were seeded at 15,000 cells / well into wells of a 96-well white assay plate and grown overnight. A transfection mixture was made containing 6.6 μg of CBR or ATG-1240 DNA and 20 μL FuGENE® HD in a total volume of 310 μL OptiMEM. The mixture was incubated at room temperature for 10 minutes. Cells were then transfected in triplicate with 5 μL of transfection complex and grown overnight.

[0204] The transfected cells were then assayed. Serial two-fold dilutions of PBI-4813 and PBI-4739, starting at 2 mM, were prepared in CO2-independent medium + 0.5% FBS. Growth medium was removed from the transfected cells and replaced with medium containing one of the serially diluted substrates. Cells were incubated for 60 minutes at 37°C, and luminescence was detected on a modified GloMax® Discover luminometer containing a red-shifted PMT (see Figures 23A and 23B). The Km of ATG1240 was approximately 5-fold higher than the Km of CBR with PBI-4813. The Km of ATG1240 was approximately 3-fold higher than the Km of CBR with PBI-4739.

[0205] Example 9 Secondary library screening using ATG1240 as a template A random clone library was prepared using ATG1240 as a template as described in Example 2. Mutants were then cloned into pF4Ag (without HT7). The library was screened as follows (50 plates total).

[0206] i. Primary Screening. Cells were grown as described in Example 2. Cell lysates were prepared by diluting 100 μL of induced culture with 100 μL of lysis buffer (0.3×PLB + 0.006U RQDNAse1) using a Tecan liquid handling robot. 50 μL of each cell lysate was then assayed with either PBI-4813 or PBI-4739 (20 μM substrate in Bright-Glo™ assay buffer containing 1 mM ATP). Luminescence was detected on a GloMax® Discover.

[0207] ii. Secondary Screening. Cells from each hit in the primary screen were streaked onto new plates and grown overnight. Colonies were picked in quadruplicate, and cultures were grown as described in Example 2. 50 μL of each cell lysate was assayed in 50 μL of BrightGlo™ assay buffer containing 1 mM ATP and 30 μM PBI-4813 or 50 μM PBI-4739. Luminescence was detected on a GloMax® Discover multimode detection system (Table 6). Mutants within this library were identified as having an average of between two and three mutations per gene. Table 6 TIFF0007780560000074.tif207166

[0208] Example 10 Insertional mutagenesis: engineering the click beetle red-luciferin binding pocket The goal was to significantly increase light emission from click beetle red (CBR) mutant(s) with red-shifted substrates, e.g., PBI-4739 (redder and less soluble) and PBI-4813 (brighter, potentially substrate-inhibiting). Random and site-directed mutagenesis, as previously described, yielded approximately a three-fold improvement in light emission.

[0209] Approach 1 was to perform cassette mutagenesis of a contiguous stretch of residues near the luciferin ligand binding site, and approach 2 was to attempt to expand the luciferin ligand binding pocket by inserting a single Ala residue at a specific site.

[0210] Suitable insertion sites were identified by visual inspection of a previously generated three-dimensional structural model of the CBR with PBI-4739, as described in Example 5. Further information on sites that tolerate insertions / deletions was obtained from alignments of multiple beetle luciferase protein sequences (e.g., firefly, click beetle, and glow worm) retrieved from the public GenBank and UniProt databases. Further information was obtained by superimposing the three-dimensional structures of a luciferase (e.g., firefly luciferase with a ligand, PDB accession 2D1S) and a related nonluciferase (e.g., CoA-ligase with a ligand, PDB accession 3N12) and examining how the insertions / deletions alter the sequence, such as allowing secondary structural elements to move to accommodate different ligands within the binding pocket.

[0211] Based on the combined analysis of the three-dimensional structure and protein sequence alignment, several sites in the CBR were identified as targets for insertional mutagenesis. Single Ala residues were inserted after these positions in the CBR (bold and underlined in Figure 18).

[0212] Mutant constructs generated by insertional mutagenesis were prepared by Gene Dynamics. Cells from each mutant clone were grown overnight on LB-ampicillin plates, and colonies were picked in quadruplicate and expanded in culture as described in Example 6. Cell lysates were prepared as previously described, and 50 μL of cell lysate was assayed with 50 μL of Bright-Glo™ assay buffer containing 1 mM ATP and 30 μM PBI-4813 or PBI-4739. Luminescence was detected on a GloMax® Discover and normalized to CBR (Table 7). Table 7 TIFF0007780560000075.tif93163

[0213] Example 11 Recombination of ATG1240 library mutants Combinations of the ATG1240 library mutants identified in Example 9 were designed and prepared using DNA shuffling (Stemmer (1994) PNAS USA 91:10747-10751). Briefly, a library of mutant genes was created via DNA shuffling. The mutant genes were then cloned into the pF4Ag vector and screened as described in Example 6. Luminescence was detected on a GloMax® Discover multimode detection system and normalized to ATG-1240 luciferase (Table 8 and Figure 15). Table 8 TIFF0007780560000076.tif118164

[0214] Example 12 Viable cell substrate titration HEK293 cells were seeded into wells of a 96-well plate at 5,000 cells / well and grown overnight. The cells were then transfected using 17 μg of ATG1240 DNA in a total of 776 μL of OptiMEM (n=24 for each sample). 50 μL of FuGENE® HD (Promega Corporation) was then added, and the samples were incubated for 10 minutes at room temperature. 5 μL of the DNA complex was added to each plate up to 18 wells / sample. The plates were then incubated overnight at 37°C with CO2.

[0215] Cells were assayed by first removing the growth medium and replacing it with CO2-independent medium + 0.5% FBS containing 2-fold diluted substrate (PBI-4813 or PBI-4739). 100 μL of each diluted substrate was added to transfected cells in triplicate and incubated for 60 minutes. Luminescence was detected on a GloMax® Discover (FIG. 16).

[0216] Example 13 Mimicking tissue attenuation using long-pass filters This example demonstrates how much light is produced in cells transfected with Luc2 (Potinus polaris luciferase) and assayed with D-luciferin compared to ATG1240 assayed with D-luciferin, PBI-4813, or PBI-4739, using various long-pass cutoff filters.

[0217] Transfection complexes were prepared by diluting ATG1240 DNA or Luc2 DNA (pF4Ag-Luc2) to 100 ng / μL, followed by two-fold serial dilutions in 50 ng / μL pGEM carrier DNA. 40 μL of diluted DNA was mixed with 160 μL phenol red-free OptiMEM and 16 μL FuGENE® HD and incubated for 20 minutes. 5 μL of each transfection complex was added to a well of a 96-well black assay plate, followed by 100 μL of diluted HEK293T cells (200,000 / mL in DMEM + 10% FBS). Cells were grown overnight at 37°C with CO2.

[0218] To assay cells, 4 mM D-luciferin, 2 mM PBI-4813, and 2 mM PBI-4739 substrate solutions were prepared in DMEM + 10% FBS. 100 μL of D-luciferin solution was added to cells expressing Luc2 and ATG1240, and 100 μL of PBI-4813 or PBI-4739 solution was added to cells expressing ATG1240. Samples were incubated for 10 minutes at 37°C, and luminescence was detected on a GloMax® Discover (heated to 37°C) using no filter, a 610 nm long-pass (LP) filter, a 665 nm LP filter, a 695 nm LP filter, a 720 nm LP filter, or a 760 nm LP filter.

[0219] These results in Figure 17 show that ATG1240 assayed with D-luciferin can be brighter than with PBI-4739, even when light is attenuated with a long-pass filter. PBI-4813 and D-luciferin are comparable in maximum light attenuation. Note that in living cells, the K for PBI-4813 is more than 10-fold less than that for D-luciferin. This may be advantageous for PBI-4813 in living animals.

[0220] Example 14 Codon optimization 600,000 cells (HEK293, 3T3, or CHO cells) in 3 mL were seeded into a 6-well plate. Briefly, for each cell type grown in a T-75 flask, the medium was removed from the T-75 flask and the cells were washed with DPBS. 3 mL of trypsin was added to the cells and the cells were incubated for 3 minutes. 10 mL of growth medium (DMEM + 10% FBS for HEK293T and 3T3, Ham's F12 + 10% FBS for CHO) was added. The cells were centrifuged at 500 rpm for 5 minutes. The cells were counted and diluted to 200,000 cells per mL. 3 mL of cells were added to each well of a 6-well plate (i.e., 600,000 cells).

[0221] Protocol for transfection of HEK293 cells grown in 3,000 μL of medium in 6-well plates using a FuGENE® HD:DNA ratio of 3.0:1. This protocol will prepare enough DNA / FuGENE® HD reagent to transfect 3 wells at 3,000 μL / well.

[0222] Cell seeding. HEK293 cells were seeded at 5 × 10 per well of a 6-well plate in 3 mL of complete growth medium (DMEM + 10% fetal bovine serum). 5 Cells were seeded the day before transfection at a density of 1000 x g.

[0223] Complex Preparation: 465 μL of a 0.020 μg / μL plasmid solution was prepared in OptiMEM, OptiPro, or sterile deionized water. Briefly, 9.9 μg of test DNA was added to a total volume of 465 μL. 10 ng of Nanoluc DNA ("ATG42") was added to each reaction for normalization. 30 μL of FuGENE® HD Reagent was added and carefully mixed by pipetting (15 times) or briefly vortexing. The mixture was incubated for 5-10 minutes at room temperature. 150 μL of complex was added to the cells per well and mixed thoroughly. The reagent:DNA ratio can range from 2.5:1.0 to 3.5:1.0. The recommended reagent:DNA ratio for HEK293 cells is 3.0:1, with 3.0 μg DNA per well.

[0224] Re-seeding cells: Growth medium was removed and cells were washed with 1 ml of DPBS. 3 ml of growth medium was added and cells were centrifuged at 500 rpm. For each sample, the pellet was resuspended in 1 ml of medium and cells were counted. HEK cells had a very small pellet and cell counts were less than 100,000 / ml. See Table 9 for dilutions of 3T3 and CHO cells. 100 μL of each sample (n=16) was seeded into a 96-well plate. One plate was used for each cell type. Table 9 TIFF0007780560000077.tif82162

[0225] Assay: Bright-Glo assay buffer was reconstituted with Bright-Glo assay substrate. 500 μL of furimazine was added to 25 ml of NanoGlo assay buffer. 100 μL of reconstituted Bright-Glo was added to each sample (n=8) and 100 μL of NanoGlo was added to the other samples (n=8) and read on a GloMax®-Multi+.

[0226] Legend / Background information for codon optimization experiments:

[0227] Mutant 343 = CBR sequence ("ATG343")

[0228] Mutant 1240 = CBR sequence + R334S / G351R ("ATG1240")

[0229] Mutant 1929 = CBR sequence + R334S / G351R (codons optimized for RNA structure and codon usage in rodents) ("ATG1929")

[0230] Mutant 1944 = CBR sequence + R334S / G351R (codons optimized for RNA structure and codon usage in CHO cells) ("ATG1944")

[0231] Mutant 1945 = CBR sequence + R334S / G351R (codons optimized for RNA structure and codon usage in mouse lung and liver cells) ("ATG1945")

[0232] While the present invention has been described in connection with what are presently considered to be the most practical and preferred embodiments, it is to be understood that the invention is not limited to the disclosed embodiments, but is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims. Modifications and variations in the present invention can be made without departing from the novel aspects of the invention as defined in the appended claims. The appended claims should be interpreted broadly and in a manner consistent with the spirit and scope of the invention herein.

[0233] For completeness, various aspects of the invention are set out in the following numbered clauses.

[0234] Clause 1. Having at least 80% amino acid sequence identity to SEQ ID NO:1 and comprising a sequence selected from the group consisting of a nucleotide sequence at positions 4, 16, 34, 47, 51, 52, 55, 72, 73, 74, 79, 82, 83, 87, 89, 104, 109, 113, 117, 119, 124, 130, 131, 133, 136, 144, 146, 156, 159, 170, 179, 186, 200, 210, 212, 214, 216, 218, 219, 220, 222, 224, 226, 228, 229, 230, 231, 233, 236, 237, 238, 240, 242, 244, 246, 256, 259, 260, 261, 262, 263, 264, 265, 266, 267, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 289, 290, 300, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 31 211, 218, 224, 225, 226, 228, 229, 234, 247, 251, 252, 253, 255, 280, 281, 285, 308, 309, 310, 319, 329, 334, 335, 337, 346, 348, 349, 350, 352, 354, 355, 358, 363, 370, 377, 390, 393, 394, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 452, 454, 455, 458, 460, 461, 462, 463, 464, 465, 466, 467, 468, 1. An isolated polynucleotide encoding a click beetle red luciferase (CBR) mutant polypeptide comprising at least one amino acid substitution at a position corresponding to SEQ ID NO: 01, 409, 412, 420, 422, 431, 437, 439, 444, 445, 453, 455, 467, 471, 473, 479, 484, 489, 496, 501, 503, 508, 516, 528, 531, 535, 537, 539, or a combination thereof, wherein the mutant CBR polypeptide has at least one of enhanced light emission, a shift in emitted light wavelength (emission wavelength), a change in substrate specificity, or a combination thereof, compared to the CBR polypeptide of SEQ ID NO: 1.

[0235] Clause 2. The isolated polynucleotide of Clause 1, wherein the CBR mutant polypeptide further comprises at least one amino acid substitution at a position corresponding to position 351, 389, 457, or a combination thereof, of SEQ ID NO:1.

[0236] Clause 3: The CBR mutant polypeptide is selected from the group consisting of R4H, H16Q, H34Y, D47E, S51N, Y52C, F55L / V, K72E, I79V, M73K / T, N74S, E82G, N83H, F87S, I89V, V104D, I109N / V, L113Q, M117T, I119F / T, I124V, N130K, I131N / T, N133D, K136N, F144L, K146L, and K146L of SEQ ID NO: 1. E, N156D, N156K, G159D, Y170C, K179S, V186A, G200G, N211N, H218L / Y, G225S, T226C / G / H / N / Q / Y, L228P, I2 29V, V234A, G251S, G251I, Y252C, V255D / F, E253K, R280S, S281N / Q, V285A, I309T, E319G, N329D, R334E / Q / H / S / N / K, C335S, K337E, I346N, Q348H / E, L350P, G351K / R, D352N, R355G, S358P, T363A / S, I370T, I389F / G / S / V, I390I, M393K / L, V394M, N400D, N401S, I409T, D412G, F420F, Y422C, V431A, E437G, I439V, S444C / R / T, 3. The isolated polynucleotide of clause 1 or 2, comprising a substitution corresponding to at least one of Q445H, E453K, V455D, K457N, D471V, E473A, S479T, K484E / M / R, E489V, Y496H, E501G, V503M, Y508C, V516A, T528A, E531G, Q535H, L537W, K539R, or a combination thereof.

[0237] Clause 4. The isolated polynucleotide of any one of Clauses 1-3, wherein said CBR mutant polypeptide comprises amino acid substitutions at positions corresponding to positions 389, 444, and 251 of SEQ ID NO:1.

[0238] Clause 5. The isolated polynucleotide of any one of clauses 1-4, wherein said amino acid substitutions comprise I389F, S444R, and G251S.

[0239] Clause 6. The isolated polynucleotide of any one of Clauses 1-5, wherein said CBR mutant polypeptide comprises amino acid substitutions at positions corresponding to positions 334 and 351 of SEQ ID NO:1.

[0240] Clause 7. The isolated polynucleotide of any one of Clauses 1 to 6, wherein said amino acid substitutions comprise R334S and G351R.

[0241] Clause 8. The isolated polynucleotide of any one of Clauses 1-7, wherein said CBR mutant polypeptide further comprises amino acid substitutions at positions corresponding to positions 51 and 444 of SEQ ID NO:1.

[0242] Clause 9. The isolated polynucleotide of any one of clauses 1 to 8, wherein said amino acid substitutions comprise S51N and S444R.

[0243] Clause 10. The isolated polynucleotide of Clause 1, wherein said CBR mutant polypeptide comprises the amino acid polypeptide of SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4.

[0244] Clause 11. The isolated polynucleotide of any one of the preceding clauses, wherein said CBR mutant polypeptide has enhanced luminescence compared to the CBR polypeptide of SEQ ID NO:1.

[0245] Clause 12. The isolated polynucleotide of Clause 11, wherein said CBR mutant polypeptide has enhanced luminescence when luciferin is utilized by said CBR mutant polypeptide to generate luminescence.

[0246] Clause 13. The isolated polynucleotide of Clause 11, wherein said CBR mutant polypeptide has enhanced luminescence when a luciferin derivative is utilized by said CBR mutant polypeptide to generate luminescence.

[0247] Clause 14: The luciferin derivative, 14. The isolated polynucleotide of clause 13, comprising TIFF0007780560000078.tif36164.

[0248] Clause 15. The isolated polynucleotide of Clause 14, wherein the luminescence of said CBR mutant polypeptide is increased by at least two-fold compared to the CBR polypeptide of SEQ ID NO:1.

[0249] Clause 16. The isolated polynucleotide of Clause 14, wherein the luminescence of said CBR mutant polypeptide is increased by at least four-fold compared to the CBR polypeptide of SEQ ID NO:1.

[0250] Clause 17. The isolated polynucleotide of any one of the preceding clauses, wherein said CBR mutant polypeptide has an altered emission spectrum compared to the CBR polypeptide of SEQ ID NO:1.

[0251] Clause 18. The isolated polynucleotide of Clause 17, wherein said CBR mutant polypeptide is capable of emitting light at a longer wavelength when luciferin is utilized by said CBR mutant polypeptide to generate luminescence.

[0252] Clause 19. The isolated polynucleotide of Clause 17, wherein the CBR mutant polypeptide is capable of emitting light at a longer wavelength when a luciferin derivative is utilized by the CBR mutant polypeptide to generate luminescence.

[0253] Clause 20: The luciferin derivative, 19. The isolated polynucleotide of clause 18, comprising TIFF0007780560000079.tif36164.

[0254] Clause 21: The luciferin derivative, JPEG0007780560000080.jpg3480, wherein the CBR mutant polypeptide emits light having a spectral maximum shift of at least about 1 nm to at least about 100 nm relative to the light produced by the CBR polypeptide of SEQ ID NO: 1.

[0255] Clause 22. The isolated polynucleotide of Clause 21, wherein said CBR mutant polypeptide emits light having a spectral maximum between about 650 nm and about 800 nm.

[0256] Clause 23. The isolated polynucleotide of Clause 21, wherein said CBR mutant polypeptide emits light having a spectral maximum between about 725 nm and about 775 nm.

[0257] Clause 24. The isolated polynucleotide of Clause 21, wherein said CBR mutant polypeptide emits light having a spectral maximum of about 750 nm.

[0258] Clause 25: The luciferin derivative JPEG0007780560000081.jpg3478, wherein the CBR mutant polypeptide emits light having a spectral maximum shift of at least about 1 nm to at least about 100 nm relative to the light produced by the CBR polypeptide of SEQ ID NO: 1.

[0259] Clause 26. The isolated polynucleotide of Clause 25, wherein said CBR mutant polypeptide emits light having a spectral maximum shift of at least about 75 nm relative to the light produced by said CBR polypeptide of SEQ ID NO:1.

[0260] Clause 27. The isolated polynucleotide of Clause 25, wherein said CBR mutant polypeptide emits light having a spectral maximum between about 650 nm and about 800 nm.

[0261] Clause 28. The isolated polynucleotide of Clause 25, wherein said CBR mutant polypeptide emits light having a spectral maximum between about 700 nm and about 775 nm.

[0262] Clause 29. The isolated polynucleotide of Clause 25, wherein said CBR mutant polypeptide emits light having a spectral maximum of about 725 nm.

[0263] Clause 30. The isolated polynucleotide of any one of the preceding clauses, wherein said CBR mutant polypeptide has altered substrate specificity compared to the CBR polypeptide of SEQ ID NO:1.

[0264] Clause 31. The isolated polynucleotide of Clause 30, wherein said CBR mutant polypeptide has a change in relative specificity for said CBR mutant polypeptide in the presence of luciferin compared to a luciferin derivative.

[0265] Clause 32. The isolated polynucleotide of Clause 30, wherein said CBR mutant polypeptide has a change in relative specificity for said CBR mutant polypeptide in the presence of a luciferin derivative compared to a different luciferin derivative.

[0266] Clause 33: The luciferin derivative, 33. The isolated polynucleotide of clause 31 or 32, comprising TIFF0007780560000082.tif39162.

[0267] Clause 34. The isolated polynucleotide of any one of the preceding clauses, wherein said mutant CBR polypeptide has luciferase activity.

[0268] Clause 35. The isolated polynucleotide of any one of the preceding clauses, wherein said mutant CBR polypeptide has a Km for PBI-4813 of at least about 0.01 μM to at least about 5.00 μM.

[0269] Clause 36. The isolated polynucleotide of Clause 35, wherein said mutant CBR polypeptide has a Km for PBI-4813 of at least about 0.50 μM to at least about 3.00 μM.

[0270] Article 37 36. The isolated polynucleotide of clause 35, wherein said mutant CBR polypeptide has a Km for PBI-4813 of at least about 0.82 μM or 2.41 μM.

[0271] Clause 38. The isolated polynucleotide of any one of the preceding clauses, wherein said mutant CBR polypeptide has a Km for PBI-4739 of at least about 0.01 μM to at least about 5.00 μM.

[0272] Clause 39. The isolated polynucleotide of Clause 38, wherein said mutant CBR polypeptide has a Km for PBI-4739 of at least about 1.50 μM to at least about 4.50 μM.

[0273] Clause 40. The isolated polynucleotide of Clause 38, wherein said mutant CBR polypeptide has a Km for PBI-4739 of at least about 2.33 μM or 3.95 μM.

[0274] Clause 41. The isolated polynucleotide of any one of the preceding clauses, wherein said mutant CBR polypeptide has a relative Vmax that is at least 2-fold higher than the relative Vmax of the CBR polypeptide of SEQ ID NO:1 using PBI-4813 as a substrate.

[0275] Clause 42. The isolated polynucleotide of any one of the preceding clauses, wherein said mutant CBR polypeptide has a relative Vmax that is at least 2-fold higher than the relative Vmax of the CBR polypeptide of SEQ ID NO:1 using PBI-4739 as a substrate.

[0276] Clause 43. The isolated polynucleotide of any one of the preceding clauses, wherein said sequence is codon optimized.

[0277] Clause 44. The isolated polynucleotide of any one of the preceding clauses, wherein said sequence comprises the polynucleotide of SEQ ID NOs: 6-9.

[0278] Clause 45. The isolated polynucleotide of any one of the preceding clauses, wherein said polynucleotide further encodes a polypeptide of interest linked to said CBR mutant polypeptide, wherein said polypeptide of interest and said CBR mutant polypeptide can be expressed as a fusion protein.

[0279] Clause 46. The isolated polynucleotide of Clause 45, wherein said polypeptide of interest comprises HALOTAG®.

[0280] Clause 47. A vector comprising a polynucleotide according to any one of the preceding clauses, or a fragment thereof.

[0281] Clause 48. The vector of Clause 47, wherein said polynucleotide is operably linked to a promoter.

[0282] Clause 49. A cell comprising the polynucleotide of any one of Clauses 1 to 46 or the vector of Clause 47 or 48.

[0283] Clause 50. A non-human transgenic animal comprising a cell according to Clause 49.

[0284] Clause 51. A non-human transgenic animal comprising a polynucleotide according to any one of clauses 1 to 46 or a vector according to clause 47 or 48.

[0285] Clause 52. A CBR mutant polypeptide encoded by a polynucleotide according to any one of clauses 1 to 46.

[0286] Clause 53. A circularly permuted luciferase comprising a polypeptide encoded by the polynucleotide or fragment thereof according to any one of Clauses 1 to 46.

[0287] Clause 54. A fusion protein comprising a CBR mutant polypeptide encoded by the polynucleotide of any one of Clauses 1-46.

[0288] Clause 55. A near-infrared bioluminescence system comprising the polynucleotide according to any one of Clauses 1 to 46 and a luciferin derivative.

[0289] Clause 56: The luciferin derivative, 56. The near-infrared bioluminescence system of clause 55, comprising TIFF0007780560000083.tif39162.

[0290] Clause 57. A method of producing a CBR mutant polypeptide, the method comprising growing the cell of Clause 47 under conditions that allow expression of said CBR mutant polypeptide.

[0291] Clause 58. A method of producing a CBR mutant polypeptide, the method comprising introducing the vector of Clause 47 or 48 into a cell under conditions that allow expression of said CBR mutant polypeptide.

[0292] Clause 59. A kit comprising a polynucleotide according to any one of Clauses 1 to 46 or a vector according to Clause 47 or 48.

[0293] Clause 60. A kit comprising a CBR mutant polypeptide according to Clause 46.

[0294] Article 61 et seq.: (a) At least one of TIFF0007780560000084.tif39162; and (b) Buffer Reagent 61. The kit of clause 59 or 60, further comprising:

[0295] Clause 62. A bioluminescence resonance energy transfer (BRET) system comprising a first fusion protein comprising a first target protein and a bioluminescent donor molecule, a second fusion protein comprising a second target protein and a fluorescent acceptor molecule, and a CBR substrate, wherein the bioluminescent donor molecule is a CBR variant encoded by the polynucleotide of any one of clauses 1 to 46.

[0296] Clause 63. The BRET system of Clause 62, wherein said CBR substrate is luciferin or a luciferin derivative.

[0297] Clause 64: The luciferin derivative, 63. The BRET system described in clause 63, including TIFF0007780560000085.tif39162.

[0298] Clause 65. A method for measuring bioluminescence using at least one of a polynucleotide described in any one of clauses 1 to 46; a vector described in any one of clauses 47 or 48; a cell described in clause 49; an animal described in any one of clauses 50 or 51; a CBR mutant polypeptide described in clause 52; a circularly permuted luciferase described in clause 53; a fusion protein described in clause 54; or a near-infrared bioluminescence system described in clause 55 or 56.

[0299] Clause 66. The method of Clause 65, wherein said bioluminescence is measured in a living, intact non-human animal.

[0300] Clause 67. A method for measuring the enzymatic activity of a luminogenic protein, said method comprising contacting a luminogenic protein, a deprotecting enzyme, and a protected luminophore; and detecting light produced from said composition, wherein said luminogenic protein is a CBR mutant encoded by the polynucleotide of any one of clauses 1-46, and said luminophore is The method according to claim 1, wherein the luciferin derivative is a luciferin derivative containing TIFF0007780560000086.tif39162.

[0301] Clause 68. The method of Clause 67, wherein said enzyme activity is measured in a living, intact non-human animal.

[0302] Article 69 47. A method for measuring the activity of a non-luminescent enzyme of interest, said method comprising: (a) providing a luminescent molecule; (b) contacting said luminescent molecule with at least one non-luminescent enzyme of interest and at least one CBR mutant to produce a reaction mixture; and (c) determining the activity of said non-luminescent enzyme of interest by measuring the luminescence of said reaction mixture, wherein said molecules are substrates for said non-luminescent enzyme of interest and a pro-substrate for a CBR mutant encoded by the polynucleotide of any one of clauses 1 to 46;

[0303] Clause 70: The luminescent molecule comprising: 69. The method of claim 69, which is a variant of TIFF0007780560000087.tif39162.

[0304] Clause 71. The method of any one of clauses 69 and 70, wherein said non-luminescent enzyme of interest is a protease enzyme, a cytochrome P450 enzyme, a monoamine oxidase, or a glutathione S-transferase.

[0305] Clause 72. The method of any one of clauses 69 to 71, wherein the activity of said non-luminescent enzyme is measured in a living, intact animal.

[0306] Clause 73. A method for detecting the presence of at least two molecules in a sample or cell, said method comprising contacting said sample or cell with a first reporter molecule comprising a CBR variant encoded by a polynucleotide of any one of Clauses 1 to 46, wherein said first reporter molecule is operably linked to a first component of said sample or cell; contacting said sample with a second reporter molecule, wherein said second reporter molecule is operably linked to a second component of said sample or cell; and detecting the presence of said first and second reporter molecules to determine the presence and / or amount of said first and second components in said sample or cell.

[0307] Clause 74. A method of detecting an interaction between a first protein and a second protein in a sample, said method comprising: (a) A sample is TIFF0007780560000088.tif39162, wherein said sample is (i) a first polynucleotide encoding a first fusion protein, wherein the first fusion protein comprises a first fragment of a luciferase and a first protein; and (ii) a second polynucleotide encoding a second fusion protein, wherein the second fusion protein comprises a second fragment of the luciferase and a second protein; and

[0308] (b) detecting luminescence in the sample; 47. The method of claim 1, wherein said detecting luminescence indicates an interaction between said first protein and said second protein, and wherein said luminescent enzyme is encoded by the isolated polynucleotide of any one of clauses 1 to 46.

[0309] Clause 75. The method of Clause 74, wherein when said first protein and second protein interact, said first fragment of said luciferase and said second fragment of said luciferase reconstitute a full-length enzyme capable of stably binding to a cell-permeable substrate.

[0310] Clause 76. A method of detecting an interaction between a first protein and a second protein in a sample, said method comprising: (a) A sample is TIFF0007780560000089.tif39162, wherein said sample is (i) a first polynucleotide encoding a first fusion protein, wherein said first fusion protein comprises a luciferase and a first protein, and said luciferase is encoded by the isolated polynucleotide of any one of clauses 1 to 46; and (ii) a second polynucleotide encoding a second fusion protein, wherein the second fusion protein comprises a fluorescent receptor molecule and a second protein; and (b) detecting bioluminescence resonance energy transfer (BRET) in the sample, which indicates an interaction or proximity of the bioluminescent donor and the fluorescent acceptor.

Claims

1. An isolated polynucleotide encoding a click beetle red luciferase (CBR) mutant polypeptide consisting of an amino acid sequence of SEQ ID NO: 1 with one amino acid substitution at position 348 of SEQ ID NO: 1, wherein the one amino acid substitution is Q348H or Q348E, and the CBR mutant polypeptide has enhanced luminescence compared to the CBR polypeptide of SEQ ID NO:

1.

2. The isolated polynucleotide of claim 1, which is codon-optimized.

3. 3. The isolated polynucleotide of claim 1 or 2, wherein the polynucleotide further encodes a polypeptide of interest linked to the CBR mutant polypeptide, and wherein the polypeptide of interest and the CBR mutant polypeptide can be expressed as a fusion protein.

4. A vector comprising the polynucleotide according to any one of claims 1 to 3.

5. The vector of claim 4 , wherein the polynucleotide is operably linked to a promoter.

6. A cell comprising the polynucleotide of any one of claims 1 to 3 or the vector of claim 4 or 5.

7. A CBR mutant polypeptide encoded by the polynucleotide of any one of claims 1 to 3.

8. A fusion protein comprising the CBR mutant polypeptide of claim 7.

9. A bioluminescence system comprising the polynucleotide according to any one of claims 1 to 3 and a luciferin derivative.

10. A kit comprising a polynucleotide according to any one of claims 1 to 3, a vector according to claim 4 or 5, or a CBR mutant polypeptide according to claim 7.

11. below: (a) , and (b) Buffer Reagent 11. The kit of claim 10, further comprising at least one of:

12. 10. A bioluminescence resonance energy transfer (BRET) system comprising: a first fusion protein comprising a first target protein and a bioluminescent donor molecule; a second fusion protein comprising a second target protein and a fluorescent acceptor molecule; and a CBR substrate, wherein the bioluminescent donor molecule is the CBR variant described in claim 7.

13. An in vitro method for measuring bioluminescence using at least one of the polynucleotide described in any one of claims 1 to 3; the vector described in claim 4 or 5; the cell described in claim 6; the CBR mutant polypeptide described in claim 7; the fusion protein described in claim 8; or the bioluminescence system described in claim 9.

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