RU2535336C1 - Red fluorescent biosensor for detection of hydrogen peroxide in living cells - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: group of inventions relates to the field of biotechnology and is directed to nucleic acid molecules which encode a protein exhibiting properties of a biosensor for detection of hydrogen peroxide in living cells having fluorescence in the red region of the spectrum. The nucleic acid molecules are derived by method of genetic engineering. Protein for detection of hydrogen peroxide has the amino acid sequence shown in SEQ ID NO:2. The host cell and the expression cassette are also provided, comprising the said nucleic acid molecules.

EFFECT: use of the inventions enables to carry out microscopy in thick layers of tissues, and provides the possibility of joint microscopy of several fluorescent structures.

4 cl 7 dwg

Description

1. Field of invention

This invention relates mainly to the field of biology and chemistry. In particular, the invention is directed to biosensors for the detection of hydrogen peroxide, constructed on the basis of fluorescent proteins.

2. The level of technology

[01] Fluorescent proteins of the GFP family (Green Fluorescent Protein, GFP), including the actual GFP from the jellyfish Aequorea victoria (avGFP), its mutants and homologs, are widely known today due to their intensive use as in vivo fluorescent markers in biomedical research, which is described in detail reviewed by Lippincott-Schwartz and Patterson in Science (2003) 300 (5616): 87-91 and Chudakov et al. (Physiol Rev. 2010 Jul; 90 (3): 1103-63).

[02] Fluorescent proteins are capable of fluorescence when irradiated with light of a suitable wavelength. The fluorescence properties of these proteins are due to the interaction of two or more amino acid residues that form the chromophore, and not the fluorescence of any one amino acid residue.

[03] GFP hydromedusa Aequorea aequorea (synonym for A. victoria) has been described by Johnson et al. at J Cell Comp Physiol. (1962), 60: 85-104, as part of a bioluminescent jellyfish system, where GFP plays the role of a secondary emitter that converts blue light from an equorin photo protein to green light. cDNA encoding A. victoria GFP was cloned by Prasher et al. (Gene (1992), 111 (2): 229-33). It turned out that this gene can be heterologically expressed in virtually any organism due to the unique ability of GFP to autocatalytically form a chromophore (Chalfie et al., Science 263 (1994), 802-805). This information has opened up broad prospects for the use of GFP in cell biology as a genetically encoded fluorescent label.

[04] GFP has been used in a wide range of applications, including the study of gene expression and protein localization (Chalfie et al., Science 263 (1994), 802-805, and Heim et al. In Proc. Nat. Acad. Sci. (1994) ), 91: 12501-12504), as a tool for visualizing the intracellular distribution of organelles (Rizzuto et al., Curr. Biology (1995), 5: 635-642), for visualizing the transport of proteins along the secretory pathway (Kaether and Gerdes, FEBS Letters (1995) 369: 267-271).

[05] Numerous studies have been carried out to improve the properties of avGFP (Aequorea victoria GFP) and to obtain GFP variants suitable and optimized for various research purposes. The avGFP (codon usage) genetic code was optimized to increase expression in mammalian cells (“humanized” GFP, Haas, et al., Current Biology (1996), 6: 315-324; Yang, et al., Nucleic Acids Research (1996), 24: 4592-4593). Various GFP mutants have been prepared, including the Enhanced Green Fluorescent Protein (EGFP) having two amino acid substitutions: F64L and S65T (Heim et al., Nature 373 (1995), 663-664). Other mutants are blue, blue, and yellow-green spectral variants of avGFP and contain substitutions of amino acid residues that form the chromophore and / or residues that form the environment of the chromophore.

[06] In 1999, GFP homologues were cloned from non-bioluminescent species of Anthozoa (Matz et al., Nature Biotechnol. (1999), 17: 969-973). This discovery has demonstrated that these proteins are not necessarily a component of the bioluminescent system. GFP-like proteins from Anthozoa had a large spectral diversity and included cyanic, green, yellow, red fluorescent proteins and violet-blue non-fluorescent chromoproteins (CPs) (Matz et al., Bioessays (2002), 24 (10): 953-959) . Subsequently, cDNAs of GFP-like proteins were cloned from a number of hydroid jellyfish and from copepods (Shagin et al., Mol Biol Evol. (2004), 21 (5): 841-850). Today, the family of GFP-like proteins includes hundreds of fluorescent and stained GFP homologs. The similarity of these proteins to GFP varies from 80-90% to less than 25% amino acid sequence identity.

[07] The crystal structure of wild-type avGFP, GFP S65T mutant, and a number of GFP homologues were obtained (Ormo et al. Science (1996) 273: 1392-1395; Wall et al. Nat Struct Biol (2000), 7: 1133-1138; Yarbrough et al. Proc Natl Acad Sci USA (2001) 98: 462-467; Prescott et al. Structure (Camb) (2003), 11: 275-284; Petersen et al. J Biol Chem (2003), 278: 44626 -44631; Wilmann et al. J Biol Chem (2005), 280: 2401-2404; Remington et al. Biochemistry (2005), 44, 202-212; Quillin et al. Biochemistry (2005), 44: 5774-5787) . It was postulated that all members of the family have a common 3D structure (GFP-like domain), which is the so-called “barrel” of 11 beta layers, forming a compact antiparallel structure, inside which there is an alpha helix containing a chromophore. A chromophore is formed within a GFP-like domain by oxidative cyclization of three conserved amino acid residues in the central region of the alpha helix (Cody et al., Biochemistry (1993) 32, 1212-1218). The positions of the amino acid residues forming the chromophore correspond to the Ser65-Tyr66-Gly67 region of avGFP. These amino acid residues can easily be identified in any GFP-like protein by aligning its sequence with the avGFP sequence.

[08] Fluorescent proteins are a unique family of structurally related proteins that are capable of forming a chromophore autocatalytically without the use of external substrates or cofactors. Under the influence of inducing light, the chromophore produces fluorescence that can be easily detected using modern laboratory equipment (spectrofluorimeter, fluorescence microscope, fluorescence-activated cell sorter, plate fluorimeter).

[09] The process of autocatalytic formation of a chromophore of proteins with different spectral properties is described in detail in a number of articles and includes several chemical reactions (Heim et al. Proc Natl Acad Sci USA. 1994; 91: 12501-12504; Ormo et al. Science. 1996; 273 : 1392-1395; Yang et al. Nat Biotechnol. 1996; 14: 1246-1251; Brejc et al. J. Proc Natl Acad Sci USA. 1997; 94: 2306-2311; Palm et al. Nat Struct Biol. 1997; 4: 361-365; Gurskaya et al., BMC Biochem. 2001; 2: 6; Gross et al. Proc Natl Acad Sci USA. 2000; 97: 11990-11995; Wall et al. Nat Struct Biol. 2000; 7: 1133-1138; Yarbrough et al., J. Proc Natl Acad Sci USA. 2001; 98: 462-467; Pakhomov, AA and Martynov, VI Chem. Biol. 2008, 15, 755-764; Quillinet al. (2005) Biochemistry 44, 5774-5787; Yampolsky et al. (2005) Biochemistry 44, 5788-5793; Shu et al. (2006) Biochemistry 45, 9639-9647; Kik uchi et al. (2008) Biochemistry 47, 11573-11580; Yampolsky et al., Biochemistry, 2009, 48 (33). pp 8077-8082).

[10] GFP-like proteins are widely used to create genetically encoded biosensors. The study of intracellular processes using such genetically encoded biosensors is becoming increasingly popular, since only such sensors can provide information about changes in the parameter under study directly in a living system. Such biosensors are in demand both in basic research of the body's signaling pathways, and in testing toxic and drug preparations on model cell lines or organisms. Compared with chemical and physical methods for recording biologically active substances with biosensors requiring exogenously added dyes, substrates, or cofactors, genetically encoded nanobiosensors belong to the class of non-reagent and reusable sensors. The use of fluorescent proteins as transmitters of information has become the most effective approach to the development of genetically encoded sensors.

[11] Fluorescence protein-based biosensors are chimeric proteins that include a sensory domain — a protein domain that is sensitive to changes in a specific cell parameter, such as changes in the concentration of an ion or molecule (calcium ions, hydrogen peroxide, hydrogen ions and etc.). As a signal part of the biosensor, GFP-like proteins or their variants subjected to circular permutation are used (Suslova and Chudakov, Trends Biotechnol. 23 (12) (2005), 605-13; Griesbeck, Curr Opin Neurobiol., 2004, v.14 ( 5), pp. 636-641; Bunt and Wouters, Int Rev Cytol., 2004, v. 237, pp. 205-277).

[12] The creation of permuted GFP-like proteins is necessary to increase the mobility of the chromophore environment and, therefore, for greater lability of the spectral properties of the protein. Circular permutation of fluorescent proteins is described by Topell S. and Glockshuber R. (Methods in Molecular Biology. 183, 31-48; 2002). For example, for circular permutation of avGFP, a gap is introduced into its primary structure between 144 and 149 amino acids, the native N and C ends are combined using a polypeptide linker. The new N- and C-ends are in close proximity to the chromophore and can affect its microenvironment. Circular permutation is performed at the nucleic acid level by operatively stitching the 3 ′ and 5 ′ ends of the nucleotide sequence encoding the fluorescent protein and introducing a gap in the sequence between codons encoding the new N and C-terminal amino acids. Methods for obtaining such structures are well known to specialists in this field.

[13] As a result of circular permutation, cpFB acquires the ability to respond to conformational rearrangements in the region of new N and C-ends by changing the fluorescence spectrum (sensors using conventional fluorescent proteins turned out to be insensitive).

[14] The efficacy of cpFB biosensors derived from avGFP has been demonstrated using a calcium ion sensor (Nagai et al, Prgos Natl Acad Sci USA. 98 (6) (2001), 197-202). cpFB was also used to fabricate the HyPer hydrogen peroxide sensor (Belousov et al., Nature Method. 4, 281-286; 2006).

[15] HyPer is a chimeric protein consisting of the regulatory domain of E. coli OxyR protein reacting with hydrogen peroxide and integrated into the OxyR peptide chain of the permuted yellow fluorescent protein cpYFP (Fig. 1). HyPer is a construct in which cpYFP is coupled to two fragments of the H ₂ O ₂ sensitive OxyR domain via short peptide linkers. Nucleotide sequences encoding fragments of the OxyR and cpYFP protein are quickly crosslinked using linker sequences. Linker sequences - nucleotide sequences encoding one or more amino acids are used to increase the mobility of parts of the chimeric protein relative to each other and contribute to its better folding (formation of the correct 3D structure) (Belousov et al. (2006), Nature Methods; 3 (4) : 281-286).

[16] Transcription factor Ε. coli OxyR activates the expression of a number of genes in response to an increase in H ₂ O ₂ concentration. OxyR consists of two domains that differ functionally. The N-terminal domain (the first 80 amino acids) forms a DNA-binding site, while the C-terminal domain (81-305 amino acids) has a regulatory function and promotes protein oligomerization (Choi et al., Cell. 105, 103-113; 2001). In the presence of H ₂ O ₂ , a disulfide bond forms between the cysteine residues of Cys-199 and Cys-208 OxyR protein, which leads to conformational changes in the entire regulatory domain of OxyR (Belousov et al., Nature Method.4, 281-286; 2006).

[17] HyPer allows you to analyze the change in the concentration of hydrogen peroxide inside living cells. Hydrogen peroxide (H ₂ O ₂ ) is an important signaling molecule responsible for the incorporation of a number of intracellular cascades (Yoshizumi et al, J Biol Chem. 2000, V.275 (16), pp. 11706-11712; Abe et al, J Biol Chem . 1996, V.271 (28), pp. 16586-16590; Schrecket al, EMBO J. 1991, V.10 (8), pp.2247-2258; Meyer et al, EMBO J. 1993, V.12 ( 5), pp. 2005-2015). Many external stimuli cause intracellular production of H ₂ O ₂ , which in turn leads to the activation of protein cascades. When hydrogen peroxide appears in the environment of HyPer, the OxyR domain is oxidized and its conformation changes, which in turn causes a change in the spectral characteristics of the fluorescent protein. These changes can be recorded using visual screening, spectrophotometry, spectrofluorimetry, fluorescence microscopy, using FACS or other generally accepted method for recording the spectral characteristics of fluorescence. After some time, the biosensor is restored due to the work of cell systems that restore disulfide bonds in the OxyR domain. In vitro reduction of OxyR with glutoredoxin-1 and thioredoxin has been shown by Aslund et al. (Biochemistry. 96, 6161-6165; 1999).

[18] Enhanced mutant HyPer variants have been developed with increased dynamic range (Markvicheva et al., Bioorg Med Chem. 2011; 19 (3): 1079-84) and modified half-life (Bilan et al., ACS Chem Biol. 2013, 15; 8 (3): 535-42).

[19] All developed HyPer variants are based on cpYFP and have the same excitation and fluorescence emission maxima. Most of the biosensors based on fluorescent proteins have similar spectral characteristics, which makes it impossible to use them simultaneously to monitor several biological processes inside the cell. Thus, the creation of biosensors having different spectral characteristics is extremely popular and will allow the simultaneous analysis of several cellular parameters directly in a living cell.

[20] Of greatest interest is the creation of biosensors with emission in the red region of the spectrum. Such biosensors can be used in in vivo systems, due to the better passage of red fluorescence through animal tissue. Absorption and autofluorescence of biomolecules such as melanins, keratin, hemoglobin in the 450–500 nm region impede the excitation and detection of fluorescence of biosensors based on yellow and green fluorescent proteins, YFP, and GFP. Red fluorescent proteins absorb light in the region of 550-580 nm and fluoresce in the wavelength range of 600-620 nm, so their spectral peaks are in the so-called “spectral window” of absorption of living samples, so the red proteins are excited by light of relatively low intensity, which penetrates well into thick layers of tissue, and allow detecting low-intensity fluorescence.

[21] While the need for sensors based on permuted red fluorescent proteins is great, such sensors appeared only recently. This is primarily due to difficulties in obtaining circular permutants of red fluorescent proteins. Until recently, there were few such proteins and the possibility of their use in sensors was discussed only theoretically. Moreover, the incorporation of red permuted proteins into other protein domains often did not result in a functional sensor (Li et al., Photochem Photobiol 2008, 84, 111-119; Akerboom et al., Front Mol Neurosci. 2013; 6: 2). Currently, the diversity of red biosensors based on permuted fluorescent proteins is limited to single sensor variants for monitoring changes in the concentration of calcium ions (Zhao et al, Science. 2011; 333 (6051): 1888-1891; Akerboom et al., Front Mol Neurosci. 2013; 6: 2) and it is impossible to predict whether the red fluorescent protein will retain the ability to fluorescence after incorporation into the OxyR sensory protein domain and whether conformational changes in the sensory domain will be effectively transmitted to the microenvironment of the chromophore of the red permuted protein, zyvaya change in the spectral properties of the biosensor.

[22] 3. Disclosure of the invention

[23] The present invention provides nucleic acid molecules encoding a fluorescent protein exhibiting biosensor properties for detecting hydrogen peroxide and having emission in the red spectral region (HyPer-Red).

[24] In some embodiments, the nucleic acid of the present invention encodes a fluorescent chimeric protein consisting of the regulatory domain of E. coli OxyR protein reacting with hydrogen peroxide and integrating the permuted red fluorescent mApple protein (cp-mApple) into the OxyR peptide chain. cp-mApple is operatively linked to two fragments of the OxyR domain sensitive to H ₂ O ₂ via short peptide linkers 1 and 2 (Fig. 1).

[25] In some embodiments, the nucleic acid of the present invention encodes a HyPer-Red fluorescent biosensor, the amino acid sequence of which is shown in SEQ ID NO: 2.

[26] In some embodiments, the nucleic acid of the present invention has the nucleotide sequence shown in SEQ ID NO: 01.

[27] In some embodiments, the nucleic acid of the present invention encodes a fluorescent biosensor protein for detecting hydrogen peroxide fused to an intracellular localization signal. Nucleic acid molecules that differ from the presented nucleotide sequences due to the degeneracy of the genetic code are also included in the scope of the present invention.

[28] In other embodiments, vectors comprising the nucleic acid of the present invention are also provided. In addition, the present invention provides expression cassettes comprising the nucleic acid of the present invention and the regulatory elements necessary for expression of the nucleic acid in a selected host cell. In addition, cells, stable cell lines, transgenic animals and transgenic plants including nucleic acids, vectors or expression cassettes of the present invention are also provided. In other embodiments, the functional fluorescent biosensors of the present invention are provided, which are encoded by the nucleic acids indicated above.

[29] Brief Description of the Drawings

[30] Figure 1 illustrates a schematic diagram of the structure of a biosensor for the detection of hydrogen peroxide.

[31] Figure 2 illustrates the dynamics of the signal of the HyPerRed biosensor after the addition of exogenous hydrogen peroxide in a bacterial suspension.

[32] Figure 3 shows graphs of the excitation and fluorescence emission spectra of HyPerRed.

[33] Figure 4 shows the signal level of HyPerRed in HeLa Kyoto cells by adding different concentrations of exogenous hydrogen peroxide.

[34] Figure 5 shows a graph of signal changes HyPerRed, HyPerRedC 199S and HyPer in HeLa Kyoto cells in response to the addition of EGF to the cell medium at a concentration of 100 ng / ml.

[35] Figure 6 shows the change in fluorescence of HyPerRed, Blue-GECO and the localization of EGFR-YFP in an individual HeLa Kyoto cell in response to the addition of EGF to the cell medium at a concentration of 100 ng / ml. The upper panel shows the redistribution of the EGFR-YFP fusion protein, the formation of endosomes on the plasma membrane of the cell (1-2 min) and their redistribution in the cytoplasm (8-15 min). The middle panel shows how short calcium emissions in the cytoplasm of cells cause sharp jumps in Blue-GECO fluorescence intensity (2, 15 min). The bottom panel shows a gradual increase in HyPerRed intensity in response to the production of hydrogen peroxide. The data processed in the Image J program are presented; the colors correspond to the level of cell fluorescence in accordance with the given scales.

[36] Figure 7 shows a graph of the change in fluorescence of HyPerRed, Blue-GECO and the localization of EGFR-YFP in an individual HeLa Kyoto cell in response to the addition of EGF to the cell medium at a concentration of 100 ng / ml. For EGFR-YFP, the ratio of the fluorescence intensity of YFP in the cytoplasm of the cell to the fluorescence intensity of YFP on the plasma membrane of the cell is shown; for HyPerRed, Blue-GECO is the change in the fluorescence intensity of these sensors in the cell cytoplasm.

[37] The implementation of the invention

[38] To more fully disclose the above characteristics of the present invention, a detailed description of the invention, summarized above, in the form of references to embodiments, some of which are illustrated by additional figures, is provided below. It should be noted that the accompanying figures illustrate only typical embodiments of the present invention and, therefore, should not be construed as limiting the scope of the invention, which may allow other equally effective embodiments.

[39] As indicated above, the present invention is directed to nucleic acid molecules that encode a fluorescence biosensor for the detection of hydrogen peroxide, which is a chimeric protein consisting of the regulatory domain of the E. coli OxyR protein reacting with hydrogen peroxide and integrated into the permuted OxyR peptide chain red fluorescent protein mApple (cp-mApple). cp-mApple is operably linked to two fragments of the H ₂ O ₂ sensitive OxyR domain via short peptide linkers 1 and 2.

[40] Nucleic acids of the present invention are obtained using recombinant technologies. In preferred embodiments, the nucleic acids of the present invention encode a protein having the amino acid sequence shown in SEQ ID NO: 2. In some embodiments, the nucleic acids encode a protein having the amino acid sequence shown in SEQ ID NO: 2 fused to a signal of a specific intracellular location. Also provided are vectors and expression cassettes comprising the nucleic acid of the present invention. In addition, provided are cells, stable cell lines, transgenic animals and transgenic plants, including nucleic acids, vectors or expression cassettes of the present invention.

[41] These nucleic acids are used in many different applications and methods to monitor changes in the concentration of hydrogen peroxide inside living cells and to identify cells in which the production of hydrogen peroxide has been activated. Finally, isolated proteins are provided that are biosensors encoded by the nucleic acids of the present invention.

[42] Definitions

[43] Various terms related to the biological molecules of the present invention are used above and also in the description and in the claims.

[44] As used here, the term "fluorescent protein" means a protein belonging to the family of GFP-like proteins, which has the ability to fluorescence; for example, it may exhibit low, medium, or intense fluorescence when irradiated with light at a suitable wavelength for excitation. The fluorescent property of a fluorescent protein is such a property, which is the result of the work of a chromophore formed by autocatalytic cyclization of three or more amino acid residues in a polypeptide chain. As such, the fluorescent proteins of the present invention do not include proteins that exhibit fluorescence due to individual fluorescent residues such as tryptophan, tyrosine and phenylalanine. The term “fluorescent protein” also refers to fluorescent proteins of the GFP family subjected to circular permutation.

[45] As used here, the term "avGFP" refers to a green fluorescent protein from Aequorea victoria jellyfish, including avGFP variants known in the art, designed to provide greater fluorescence or fluorescence intensity in other color areas. The wild-type avGFP sequence has been disclosed in Prasher et al. (1992, Gene 111: 229-33).

[46] As used here, the terms "fluorescent protein subjected to circular permutation", "permuted fluorescent protein" or "cpfb" refers to a protein derived from a fluorescent protein (eg, from avGFP) using genetic engineering modification of nucleic acid, as a result where the C- and N-ends of the original fluorescent protein are quickly fused, and the new C- and N-ends are formed near the chromophore. The mention of the protein permuted at positions N1-N2, where N1 and N2 are the numbers of amino acid residues in the polypeptide chain, means that N1 is the number of amino acid residue in the original protein, which formed the new C-terminus of cpFB, and N2 is the number of amino acid residue in the original the protein that formed the new N-terminus.

[47] Circular permutation, as a rule, does not affect the formation of a “barrel” of a GFP-like domain and the formation of an active (capable of fluorescence) chromophore. Circular permutation leads to an increase in the ability of cpFBs to change spectral characteristics upon conformational changes in protein domains or polypeptides operatively fused to its C- and N-ends.

[48] The term “humanized” refers to a change in the nucleotide sequence of a fluorescent protein made to optimize the genetic code of codons for expression in mammalian cells (Yang et al., 1996, Nucleic Acids Research 24: 4592-4593).

[49] As used here, the term "isolated" means a molecule or cell that is in an environment other than the environment in which the molecule or cell is in vivo.

[50] As used here, the term "mutant" or "derivative" refers to a protein disclosed in the present invention, in which one or more amino acids are added and / or substituted and / or removed (deleted) and / or inserted (inserted) into The N-terminus and / or C-terminus and / or within the native amino acid sequences of the proteins of the present invention. As used here, the term "mutant" refers to a nucleic acid molecule that encodes a mutant protein. In addition, the term “mutant” refers to any variant that is shorter or longer than a protein or nucleic acid.

[51] As used here, “homology” is a term used to describe the relationship of nucleotide or amino acid sequences to other nucleotide or amino acid sequences, which is determined by the degree of identity and / or similarity between said compared sequences.

[52] As used here, the amino acid or nucleotide sequences are "substantially similar" or "essentially the same as the reference sequence if the amino acid or nucleotide sequences have at least 85% identity with the specified sequence within the region selected for comparison. Thus essentially similar sequences include those that have, for example, at least 85% identity, at least 90% identity, at least 95% identity, or at least 96% , 97%, 98% or 99% identity Two sequences that are identical to one another are also essentially similar.

[53] The percentage of sequence identity is determined based on a reference sequence. Algorithms for sequence analysis are known in the art, such as BLAST described in Altschul et al., J. Mol. Biol., 215, pp. 403-10 (1990). For the purposes of the present invention, nucleotide and amino acid sequence comparisons performed using the Blast software package provided by the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/blast) using alignment breaks with standard parameters may be used to determine the level of identity and similarity between nucleotide sequences and amino acid sequences.

[54] As used here, the term "similar proteins" or "essentially similar proteins" refers to proteins that have amino acid sequences that are at least 85% identical, typically 90% or more identical, most often identical at least at least 95% or more (for example, 96% or more, 97% or more, 98%) and more, 99% or more, 100%). In this case, the length of homologous amino acid sequences of “similar proteins” can be at least 100 amino acid residues, more often at least 200 amino acid residues, or 300 amino acid residues.

[55] In some embodiments, the term “similar proteins” or “substantially similar proteins” refers to proteins that have the amino acid sequences of a whole protein that are at least 85% identical, typically 90% or more identical, most often identical at least 95% or more (for example, 96% or more, 97% or more, 98% or more, 99% or more, 100%).

[56] As used here, the term "functional" means that the nucleotide or amino acid sequence can function for the specified test or task. The term "functional" used to describe the biosensor for detecting hydrogen peroxide of the present invention means that it changes spectral characteristics with increasing concentration of hydrogen peroxide in the medium.

[57] As used here, the term "medium" in relation to a biosensor for the detection of hydrogen peroxide means any medium in which this biosensor can function. For an isolated protein, this can be any buffer solution in which this sensor retains functionality. For a protein expressed in a cell, it is the cytoplasm or intracellular compartment in which the biosensor is localized.

[58] As used herein, “biochemical properties” refer to protein folding (folding) and maturation rate, half-life of oxidation, half-life of recovery after reaction with hydrogen peroxide, half-life, ability to aggregate, ability to oligomerize, pH and temperature stability, and other similar properties.

[59] As used here, “fluorescence properties” or “spectral properties” refer to the molar extinction coefficient at a suitable wavelength, the quantum yield of fluorescence, the shape of the fluorescence excitation spectrum or emission spectrum, the wavelength corresponding to the maximum fluorescence excitation, and wavelength corresponding to the maximum emission, the ratio of the amplitude of the excitation of fluorescence at two different wavelengths, the ratio of the amplitude of the emission at two different wavelengths, the lifetime of the excited state scattering, and optical anisotropy. The measured difference can be defined as the amount of any quantitative fluorescence property, for example, the fluorescence intensity at a certain wavelength, or the integral fluorescence over the entire emission spectrum.

[60] Reference to the nucleotide sequence "encoding" a polypeptide means that the polypeptide is produced from the nucleotide sequence during translation and transcription of mRNA. In this case, both a coding strand identical to mRNA and usually used in the list of sequences can be indicated, as well as a complementary strand that is used as a template for transcription. As is obvious to any person skilled in the art, the term also includes any degenerate nucleotide sequences encoding the same amino acid sequence. Nucleotide sequences encoding a polypeptide include sequences containing introns.

[61] The term “operatively linked”, “operatively fused” or the like when describing the structure of a biosensor or chimeric proteins based on it refers to polypeptide sequences that are in physical and functional connection with each other. In the most preferred embodiments, the basic functions of the polypeptide components of the chimeric molecule are not changed compared with the functional properties of the isolated polypeptide components. For example, the OxyR domain, which is part of the biosensor of the present invention, retains the ability to bind hydrogen peroxide, and cpBP - the ability to fluorescence. In the case where the biosensor of the present invention is crosslinked with an intracellular localization signal of interest, the chimeric protein retains the ability to respond to the appearance of hydrogen peroxide, but is localized in a particular cellular compartment. As is obvious to any person skilled in the art, nucleotide sequences encoding a chimeric protein including “operably linked” components (proteins, polypeptides, linker sequences, protein domains, etc.) consist of fragments encoding these components, where these the fragments are covalently linked in such a way that a full-sized chimeric protein is produced during translation and transcription of the nucleotide sequence. In other words, the fragments are connected in such a way that there are no “failures” in the reading frame and stop codons at their junctions.

[62] As used here, the terms "hydrogen peroxide" and "hydrogen peroxide" refer to a molecule having the formula H ₂ About ₂ .

[63] As used here, the term "biosensor signal" means a detectable change in the spectral characteristics of the biosensor in response to hydrogen peroxide.

[64] As used here, the term “ligand biosensor sensitivity” means a range of ligand concentrations that trigger a detectable biosensor response.

[65] As used here, the term "dynamic range" means the ratio of the maximum signal level of the biosensor (in a fully oxidized state) to the level of the original signal (in a fully restored state).

[66] Nucleic acid molecules

[67] The present invention provides nucleic acid molecules encoding a fluorescent biosensor for detecting hydrogen peroxide having the amino acid sequence of SEQ ID NO: 2.

[68] As used here, a nucleic acid molecule is a DNA molecule, such as a genomic DNA or cDNA molecule, or an RNA molecule, such as an mRNA molecule. As used here, the term "cDNA" refers to nucleic acids that possess a sequence of sequence elements found in native mature types of mRNA, where sequence elements are exons and 5 'and 3' non-coding regions.

[69] The biosensor structure is shown schematically in Figure 1. Methods for producing such fusion proteins are well known in the art. For example, parts of a nucleic acid encoding various elements can be embedded in the polylinker of the vector so that these parts are quickly linked and there will be no stop codons in the reading frame and there will be no reading frame failures. Alternatively, the desired nucleotide sequence can be assembled from the fragments using DNA ligase or PCR with primers containing parts complementary to the terminal sequences of the fragments to be joined.

[70] A nucleic acid molecule encoding a fluorescent biosensor can be synthesized from suitable nucleoside triphosphates. For example, the availability of amino acid sequence information or nucleotide sequence information enables the production of isolated nucleic acid molecules of the present invention using oligonucleotide synthesis. In the case of amino acid sequence information, several nucleic acids can be synthesized that differ from each other due to the degeneracy of the genetic code. Methods for selecting codon variants for a desired host are well known in the art. Synthetic oligonucleotides can be prepared using the phosphoramidite method, and the resulting constructs can be purified using methods well known in the art, such as high performance liquid chromatography (HPLC) or other methods, as described, for example, in Sambrook et al., Molecular Cloning : A Laboratory Manual, 2nd Ed., (1989) Cold Spring Harbor Press, Cold Spring Harbor, NY, and following the instructions described in, for example, United States Dept. of HHS, National Institute of Health (NIH) Guidelines for Recombinant DNA Research. The long double-stranded DNA molecules of the present invention can be synthesized in the following stages: several smaller fragments with the necessary complementarity, which contain suitable ends capable of cohesion with the neighboring fragment, can be. Neighboring fragments can be crosslinked using a DNA ligase or PCR based method. A nucleic acid encoding a biosensor or fragment thereof can be isolated by any of many known methods.

[71] In addition, degenerate nucleic acid variants that encode the biosensor of the present invention are also provided. Degenerate nucleic acid variants include substitutions of nucleic acid codons with other codons encoding the same amino acids. In particular, degenerate nucleic acid variants are created to increase expression in the host cell. In this embodiment, nucleic acid codons that are not preferred or less preferred in the genes of the host cell are replaced by codons that are abundantly represented in the coding sequences of the genes in the host cell, where said substituted codons encode the same amino acid.

[72] Of particular interest are humanized versions of the nucleic acids of the present invention. As used herein, the term “humanized” refers to substitutions made in a nucleic acid sequence to optimize codons for protein expression in mammalian (human) cells (Yang et al., Nucleic Acids Research (1996) 24: 4592-4593). See also US Patent No. 5,795,737, which describes the humanization of proteins, the disclosure of which is incorporated herein by reference.

[73] In some embodiments, the nucleic acid molecule of the present invention is a DNA (or cDNA) molecule containing an open reading frame that encodes a biosensor of the present invention and is capable of being used under suitable conditions (eg, physiological intracellular conditions) for expression of a protein in a cell the owner. The present invention also encompasses nucleic acids that are homologous, substantially similar, identical or derived from nucleic acids encoding the proteins of the present invention. These nucleic acids are in a different environment from the environment in which they are found under natural conditions, for example, they are isolated, present in an increased amount, are or are expressed in vitro systems or in cells or organisms other than those in which they are located in vivo.

[74] The claimed nucleic acids can be isolated and obtained essentially in purified form. Essentially purified form means that the nucleic acids are at least 50% pure, usually at least 90% pure and usually are "recombinant", that is, flanked by one or more nucleotides - with which it usually not bound on the chromosome found naturally in its natural host organism.

[75] Changes or differences in the nucleotide sequence between highly similar nucleotide sequences can represent nucleotide substitutions in the sequence that occur during normal replication or duplication. Other substitutions can be specifically calculated and inserted into the sequence for specific purposes, such as changing the codons of certain amino acids or the nucleotide sequence of a regulatory region. Such special substitutions can be made in vitro using various mutagenesis technologies or obtained in host organisms under specific breeding conditions that induce or select these changes. Such specially prepared sequence variants may be called “mutants” or “derivatives” of the original sequence.

[76] Mutant or derivative nucleic acids can be obtained on a matrix nucleic acid selected from the above nucleic acids, by modifying, deleting or adding one or more nucleotides in the matrix sequence or a combination thereof, to obtain a variant of the matrix nucleic acid. Modifications, additions or deletions can be performed by any method known in the art (see, for example, Gustin et al., Biotechniques (1993) 14: 22; Barany, Gene (1985) 37: 111-123; and Colicelli et al., Mol. Gen. Genet. (1985) 199: 537-539, Sambrook et al., Molecular Cloning: A Laboratory Manual, (1989), CSH Press, pp. 15.3-15.108), including error prone PCR ), shuffling, oligonucleotide-directed mutagenesis, assembly PCR, pairwise PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive multiple mutagenesis, exponential multiple mutagenesis, site-specific mutagenesis, random mutagenesis, gene re assembly (gene reassembly), gene site-saturating mutagenesis (GSSM), artificial ligation rearrangement (SLR), or combinations thereof. Modifications, additions or deletions can also be carried out by a method including recombination, recursive recombination of sequences, phosphothioate-modified DNA mutagenesis, mutation on a uracil-containing matrix, double-pass mutagenesis, point-mutation reductive mutagenesis, strain mutagenesis, recovery-deficient mutagenesis, radioactive mutagenesis, deletion mutagenesis, restriction-selective mutagenesis, restriction mutagenesis with purification, synthesis of artificial enes, multiple mutagenesis, creation of multiple chimeric nucleic acids, and combinations thereof.

[77] Also provided are nucleic acids encoding chimeric proteins consisting of a biosensor of the present invention and a signal of a specific intracellular localization. Such chimeric proteins can be obtained by operatively combining a nucleic acid of the present invention encoding a biosensor and a nucleic acid encoding an intracellular localization signal. Methods for producing such nucleic acids are well known to those skilled in the art.

[78] A vector and other nucleic acid constructs containing the claimed nucleic acids are also provided. Suitable vectors include viral and non-viral vectors, plasmids, cosmids, phages, etc., preferably plasmids, and are used for cloning, amplification, expression, transfer, etc., of the nucleic acid sequence of the present invention in a suitable host. The selection of a suitable vector is readily apparent to those skilled in the art, and many such commercially available vectors are known. To prepare the construct, a full-length nucleic acid or part thereof is usually inserted into the vector by attaching a DNA ligase to a restriction enzyme cleaved site in a vector. Alternatively, the desired nucleotide sequence can be inserted by in vivo homologous recombination, usually by attaching homologous regions to a vector on the flanks of the desired nucleotide sequence. Homologous regions are added by ligation of oligonucleotides or by polymerase chain reaction using primers including, for example, both homologous regions and part of the desired nucleotide sequence.

[79] Also provided are expression cassettes or systems used inter alia to obtain the claimed biosensors or chimeric proteins based thereon or to replicate the claimed nucleic acid molecules. The expression cassette may exist as an extrachromosomal element or may be incorporated into the genome of a cell as a result of introducing said expression cassette into the cell.

[80] In the expression vector or expression cassette, said nucleic acid is operably linked to a regulatory sequence, which may include promoters, enhancers, terminators, operators, repressors and inducers, and initiates the reading of RNA (transcription) in the host cell. In the expression vector or expression cassette, the nucleic acid of the present invention may also be associated with transcription termination signals that are functional in the host cell. Methods for making expression cassettes or systems for expressing a desired product are known to those skilled in the art.

[81] The above expression systems may be used in prokaryotic or eukaryotic hosts. Cell lines that stably express the proteins of the present invention can be selected by methods known in the art (e.g., cotransfection with a selectable marker such as dhfr, gpt, neomycin, hygromycin, which makes it possible to identify and isolate transfected cells that contain the gene, included in the genome).

[82] The protein product encoded by the nucleic acid of the invention can be obtained by expression in any convenient expression system, including, for example, bacterial systems, yeast systems, insect cells, amphibians, or mammalian cells. For example, host cells such as E. coli, B. subtilis, S. cerevisiae, insect cells in combination with baculovirus vectors, or cells of a higher organism such as vertebrates such as COS 7 cells, HEK 293 can be used to produce protein , CHO, Xenopus oocytes, etc.

[83] If any of the aforementioned host cells or other suitable host cells or organisms are used to replicate and / or express the nucleic acids of the invention, the resulting replicated nucleic acid, expressed protein or polypeptide are within the scope of the invention as a product of the host cell or organism . The product can be isolated by a suitable method known in the art.

[84] Squirrels

[85] The nucleic acids of the present invention encode a fluorescent biosensor for detecting hydrogen peroxide. The amino acid sequence of the biosensor of the present invention is shown in SEQ ID NO: 2.

[86] The claimed biosensor has the ability to detect fluorescence, which can be detected using visual screening, spectrophotometry, spectrofluorimetry, fluorescence microscopy, using FACS or other generally accepted method for recording fluorescence.

[87] The claimed biosensor has a maximum (peak) emission in the range from 550 nm to 700 nm, usually in the range from 580 nm to 650 nm, for example at 605 nm.

[88] The claimed biosensor has a peak fluorescence excitation in the range from 450 nm to 600 nm, usually in the range from 500 nm to 580 nm, for example at 575 nm.

[89] When hydrogen peroxide appears, the claimed biosensor goes into an oxidized state. During biosensor oxidation, the intensity of fluorescence emission changes.

[90] The claimed biosensor has a wide dynamic range. The claimed biosensor has a dynamic range of 1.3 to 5, often from 1.5 to 3, for example, the claimed biosensor has a dynamic range of 1.8 in bacterial cells when oxidized with hydrogen peroxide at a concentration of 200-290 μm.

[91] Functional biosensors are also provided that are substantially similar to the above biosensor, where "substantially similar" means that these proteins have an amino acid sequence identical to the sequence of SEQ ID NO: 02, at least 85% identity, usually at least 90% and more often at least 95% (e.g. 95% and higher; 96% and higher, 97% and higher; 98% and higher: 99% and higher or 100% sequence identity).

[92] For example, biosensor mutants can be obtained using standard molecular biology methods, as described in detail in the section "nucleic acid molecules" above. The examples provide general techniques and the use of standard methods, so that those skilled in the art can easily obtain a number of additional mutants and check whether the biological (e.g., biochemical, spectral, etc.) property has been changed. For example, fluorescence intensity can be measured using a spectrofluorimeter at various excitation wavelengths.

[93] The biosensors of the present invention are present in an environment different from their natural environment; for example, they are recombinant. The proteins of the present invention can be in an isolated state, which means that the proteins are substantially free of other proteins and other biological molecules present in the natural environment, such as oligosaccharides, nucleic acids and fragments thereof and the like, where the term "essentially free "in this case means that less than 70%, usually less than 60% and more often less than 50% of the composition containing the isolated protein, are some other biological molecules than those found in nature. In some embodiments, the proteins are present in a substantially purified form, where “substantially purified form” means at least 95% purified, usually at least 97%, and more often at least 99%.

[94] The claimed biosensors can be obtained, for example, by expression of a recombinant nucleic acid encoding a sequence of a protein of interest in an appropriate host, as described above. For protein purification, any conventional methodology may be employed where suitable protein purification methods are described in Guide to Protein Purification (Deuthser ed., Academic Press, 1990). For example, a lysate can be prepared from a source and purified using HPLC, size exclusion chromatography, gel electrophoresis, affinity chromatography, and the like.

[95] Fusion proteins are also provided, including the biosensor of the present invention, fused to an intracellular localization sequence (for example, a localization signal in the nucleus, peroxisomes, Golgi apparatus, mitochondria, etc.). A polypeptide providing a certain intracellular localization can be operatively attached to the N-terminus and / or C-terminus of the biosensor. Signals of intracellular localization are well known to specialists in this field and are described, for example, by Nakai K. (Advances in Protein Chemistry, Vol. 54, 2000, p. 277-344).

[96] Transformants

[97] The nucleic acids of the present invention are used to produce transformants, including transgenic organisms or site-specific gene changes in cell lines. The transgenic cells of the invention comprise one or more nucleic acids of the invention as a transgene. For the purposes of the invention, any suitable host cell can be used, including prokaryotic (e.g., Escherichia coli, Streptomyces sp., Bacillus subtilis, Lactobacillus acidophilus, etc.) or eukaryotic host cells. A transgenic organism of the invention may be a prokaryotic or eukaryotic organism, including bacteria, cyanobacteria, fungi, plants, and animals in which one or more cells of the body contain a heterogeneous nucleic acid of the invention, introduced by human intervention, by methods such as technologies transgenosis, which are known in the art.

[98] The isolated nucleic acid of the present invention can be introduced into the host by methods known in the art, for example, infection, transfection, transformation or trans-conjugation. Methods for transferring a nucleic acid molecule (i.e., DNA) to such organisms are widely known and provided in references such as Sambrook et al. (Molecular Cloning: A Laboratory Manual, 3 ^nd Ed., (2001) Cold Spring Harbor Press, Cold Spring Harbor, NY).

[99] In one embodiment, the transgenic organism may be a prokaryotic organism. Methods for transforming prokaryotic hosts are well described in the art (e.g., see Sambrook et al. Molecular Cloning: A Laboratory Manual, 2 ^nd edition (1989) Cold Spring Harbor Laboratory Press and Ausubel et al., Current Protocols in Molecular Biology (1995) John Wiley & Sons, Inc).

[100] In another embodiment, the transgenic organisms may be fungi, for example, yeast. Yeast is widely used as a carrier for heterogeneous gene expression (e.g. see Goodey et al. Yeast biotechnology, DR Berry et al., Eds, (1987) Allen and Unwin, London, pp. 401-429) and King et al Molecular and Cell Biology of Yeasts, Ε F Walton and G Τ Yarronton, eds, Blackie, Glasgow (1989) pp. 107-133). Several types of yeast vectors are available, including integrative vectors that require recombination with the host genome to support them, and autonomously replicating plasmid vectors.

[101] In another embodiment, the transgenic organisms may be animals. Transgenic animals can be obtained by transgenic methods known in the art and provided in references such as Pinkert, Transgenic Animal Technology: a Laboratory Handbook, 2nd edition (2203) San Diego: Academic Press; Gersenstein and Vintersten, Manipulating the Mouse Embryo: A Laboratory Manual, 3rd ed, (2002) Nagy A. (Ed), Cold Spring Harbor Laboratory; Blau et al., Laboratory Animal Medicine, 2 ^nd Ed., (2002) Fox JG, Anderson LC, Loew FM, Quimby FW (Eds), American Medical Association, American Psychological Association; Gene Targeting: A Practical Approach by Alexandra L. Joyner (Ed.) Oxford University Press; 2nd edition (2000). For example, transgenic animals can be obtained by homologous recombination, where the endogenous locus changes. Alternatively, the nucleic acid construct is randomly incorporated into the genome. Vectors for sustainable incorporation include plasmids, retroviruses and other animal viruses, YAC, and the like. Nucleic acids can be introduced into a cell directly or indirectly, by introducing into a cell precursor, by intentional genetic manipulation, such as microinjection or infection with a recombinant virus or recombinant viral vector and the like. The term “genetic manipulation” does not include classical cross-fertilization or in vitro fertilization, but is preferably directed to the introduction of recombinant nucleic acid molecules. These nucleic acid molecules can be included in the chromosome or they can be extrachromosomal replicating DNA. DNA constructs for homologous recombination will contain at least a portion of the nucleic acid of the present invention, where the gene has the desired genetic modification (s) and includes homology regions with a target locus. DNA constructs for arbitrary incorporation do not necessarily contain a region of homology with a recombination mediator. Markers for positive and negative selection can easily be included. Methods for producing cells having targeted gene modifications through a homologous combination are known in the art. For various methods for transfecting mammalian cells, see Keown et al., Meth. Enzymol. (1990) 185: 527-537.

[102] Transgenic animals can be any non-human animal, including a non-human mammal (eg, mouse, rat), bird or amphibian, etc., and are used in functional research, drug screening, etc. .P.

[103] Transgenic plants can also be obtained. Methods for producing transgenic plant cells and plants are described in US patent No. 5767367; 5,750,870; 5,739,409; 5,689,049; 5,689,045; 5,674,731; 5656466; 5,633,155; 5,629,470; 5,595,896; 5,576,198; 5,538,879; 5,484,956; disclosures of which are incorporated herein by reference. Methods for producing transgenic plants are also discussed in Plant Biochemistry and Molecular Biology (eds. Lea and Leegood, John Wiley & Sons) (1993) pp. 275-295 and Plant Biotechnology and Transgenic Plants (eds. Oksman-Caldentey and Barz), ( 2002) 719 p. For example, embryogenic explants containing somatic cells can be used to produce a transgenic host. After collecting cells or tissues, exogenous DNA of interest is introduced into the plant cells, and a number of different methods are known for such introduction. In the presence of isolated protoplasts, it is possible to introduce DNA via indirect gene transfer protocols, including incubating protoplasts with purified DNA, such as a plasmid containing the desired exogenous sequence, in the presence of polyvalent cations (eg, PEG or PLO); or electroporation of protoplasts in the presence of isolated DNA, including the target exogenous sequence. Protoplasts that successfully incorporate exogenous DNA are then selected, grown in callus, and ultimately in a transgenic plant in contact with suitable amounts of stimulatory factors such as auxins and cytokines. Other suitable plant production methods that are available to those skilled in the art may be used, such as the use of a "gene gun" or Agrobacterium-mediated transformation.

[104] Uses

[105] The biosensors of the present invention are genetically encoded fluorescent proteins that change spectral properties in the presence of hydrogen peroxide. They can be used to monitor the production of hydrogen peroxide inside living cells in various biological processes. For monitoring, a nucleic acid encoding a biosensor must be obtained. Obtaining such designs is obvious to any person skilled in the art. The resulting construct must be integrated into the expression cassette (or vector), providing temporary or permanent expression of this nucleic acid in the host cells. The expression vector or cassette may contain elements that provide targeted delivery of the construct to cells of interest, or is part of particles that provide targeted delivery. After transfection of cells with a vector and after the time required for the expression product to develop in the cells, the production of hydrogen peroxide in these cells can be monitored.

[106] The biosensors of the present invention find use in the screening of drugs that induce the activation of certain protein cascades, for example, the activation of tyrosine kinases in cell lines. An example of the use of biosensors can serve as automated screening of multiple cells, described, for example, in US patent No. 5989835.

[107] The claimed biosensors also find use in sorting fluorescence activated cells (FACS). In such applications, the inventive biosensor is used to label a cell population stimulated by any factor to produce hydrogen peroxide, and the resulting labeled cell population is then sorted in a fluorescence activated cell sorting device, as is known in the art. FACS methods are described in US patent No. 5968738 and 5804387.

[108]

[109] Due to the emission of fluorescence in the red spectral region, the claimed biosensors also find application as labels for the production of hydrogen peroxide in vivo for transgenic animals. For example, expression of the claimed protein may be accompanied by tissue-specific promoters, where such methods are used in studies for gene therapy, such as testing the effectiveness of drugs in animal models. A typical application of fluorescent biosensors in transgenic animals, which explains such applications, is described in W0 00/02997.

[110] The following examples are provided as illustrative but not limiting.

[111] Examples

[112] Example 1

[113] Obtaining a HyPerRed biosensor for the detection of hydrogen peroxide.

[114] To obtain nucleic acids encoding chimeric proteins and circular permutants of the required composition, the "overlap extention" method was used for PCR products (Horton et al., 1989, Gene. 77, 61-8). For amplification, Tersus polymerase (Eurogen) and gene specific primers were used, in the structure of which overlapping regions, necessary linker sequences and / or restriction endonuclease sites were introduced. The following PCR parameters were used: denaturation 95 ° С -12 sec, annealing of primers 63 ° С - 30 sec, synthesis 72 ° С - 40-90 sec. The number of PCR cycles is 23-25.

[115] After amplification, the obtained fragments were separated from the matrix and primers by electrophoresis in 1% agarose gel in TAE buffer with ethidium bromide, and then they were extracted from the gel using commercially available DNA extraction kits from gel and reaction mixtures (Quigen, Germany). The isolated DNA fragments were combined into a full-size construct using the overlap extention method of PCR in the presence of Tersus polymerase. In this method, fragments with mutually complementary regions that are present in the reaction mixture are used as templates and primers. The following PCR parameters were used: denaturation 95 ° С - 12 sec, annealing of primers 60 ° С - 2 min, synthesis 72 ° С - 40 sec. The number of PCR cycles is 10-12.

[116] Amplification of the full-sized construct obtained in the previous step was carried out by PCR with the addition of gene-specific terminal primers. The following PCR parameters were used: denaturation 95 ° С - 12 sec, annealing of primers 60 ° С - 30 sec, synthesis 72 ° С - 1.5 min. The number of PCR cycles is 15.

[117] Nucleic acid encoding HyPer was obtained from the commercially available pHyPer-cyto vector (Eurogen, Russia). OxyR fragments flanking cpYFP were amplified using gene-specific primers.

[118] The cpTagFP635 protein subjected to circular permutation was obtained based on the TagFP635 sequence (also known as mKate) from the pTagFP635-C vector (Eurogen, Russia). For this, TagFP635 fragments were amplified using gene-specific primers that had a complementary region encoding a linker between the original C- and N-ends of the permutant. Several cp-TagFP635 variants were constructed with permutation at positions 15-14, 40-39, 42-41, 91-90, 105-104, 120-119, 176-175, 141-140, 144-143, 150-149 , 198-197, 201-200, and 200-199.

[119] The obtained nucleic acids were operatively fused to nucleic acids encoding OxyR fragments using randomized (random) linkers between the fluorescent portion of cpTagFP635 and the sensory portion of OxyR-RD with a length of 3 amino acid residues. Examples of the nucleotide and amino acid sequences of the variant OxyR-cpTagFP635 protein (permutation position cpTagFP635 141-140) are shown in SEQ ID No: 3 and 4, respectively. Next, the obtained constructs were cloned into the pQE30 bacterial expression vector (Qiagen) at the BamHI and HindIII sites and used to transform E. coli and prepare expression bacterial libraries.

[120] Ligation of the obtained fragments into a vector was carried out using T4 phage ligase (Sibenzyme). The reaction was carried out in a volume of 15 μl according to the manufacturer's protocol. Incubation was carried out for 16-18 hours at 14 ° C.

[121] Electroporation was performed to transform cells with a ligate, previously reprecipitated with ethanol and dissolved in sterile deionized water. Competent cells for electroporation, packaged in 40 μl in salt-free glycerol solution, were stored at -70 °. Before transformation, they were thawed for 10 minutes on ice, 50-100 ng of DNA was added and placed in pre-chilled electroporation cuvettes (BioRad). Electroporation was performed on a MicroPulser device (BioRad), Eel program. Then a suspension of competent cells was mixed with 1000 μl of SOB medium, incubated for 40 minutes with stirring at 37 ° C and dispersed on solid medium LB with antibiotic.

[122] Most of the libraries were not fluorescent. In the case of embedding positions 150-149 and 201-200, most of the library contained red fluorescent sensor variants, but they did not mature at 37 ° C (matured only at 25 ° C, which impedes their use in animal cells) and, most importantly, does not reacted to the addition of hydrogen peroxide.

[123] Random mutagenesis over the entire amino acid sequence of the obtained fluorescent variants of the chimeric protein OxyR-TagFP635 did not lead to the production of a functional protein exhibiting hydrogen peroxide biosensor properties.

[124] Changing linker sequences 1 and 2 to random sequences of 2 and 2 amino acids or 3 and 2 amino acids (as in HyPer) led to an increase in fluorescence brightness and an increase in maturation rate, but did not affect the ability of the protein to change spectral characteristics in the presence of hydrogen peroxide .

[125] Thus, it was not possible to obtain a fluorescent chimeric protein exhibiting biosensor properties on hydrogen peroxide based on cp-TagFP635.

[126] The circular candidate for permutation of the mApple protein was selected as the next candidate for the creation of a red fluorescent biosensor for the detection of hydrogen peroxide. The mApple protein belongs to the line of red fluorescent proteins mFruits, derivatives of the first red tetrameric protein DsRed, differing from DsRed in a reduced tendency to oligomerization (mainly monomeric proteins) and a higher fluorescence intensity. The maximum fluorescence excitation of the mApple protein is 575 nm, and the maximum fluorescence emission is 605 nm. The circularly permuted mApple protein (cpRFP) was previously used to produce a red fluorescent biosensor of intracellular calcium ion concentrations R-GECOl (Zhao et al., Science. 2011; 333 (6051): 1888-1891).

[127] Nucleic acid encoding cpRFP was amplified using gene-specific primers from plasmid No. 32444, CMV-R-GEC01, obtained through Addgene service (USA) and fused with nucleic acids encoding OxyR fragments using identical linkers those used to create the HyPer biosensor.

[128] The HyPerRed-SAG-GT protein (SEQ ID NO: 5, 6) obtained after expression in E. coli had weak fluorescence and did not respond to changes in the concentration of hydrogen peroxide in the medium. Thus, the direct replacement of the cpYFP circular permutant sequence in the HyPer sensor with the cpRFP sequence with preservation of the HyPer sensor linker sequences did not lead to the production of a hydrogen peroxide sensitive version of the sensor.

[129] The assumption that the fluorescence core of the sensor and its linker sites is a functionally efficient unit of transport was also not confirmed: HyPer sensor variants containing linker sites and the fluorescent core of the R-GEC01 sensor were also not sensitive to hydrogen peroxide and were extremely characterized low fluorescence intensity.

[130] Subsequently, we varied the length of the randomized linker sites in combinations of 2 and 2, 3, and 2, 3 and 3 amino acid residues on the first and second linker, respectively.

[131] To obtain a functional biosensor for hydrogen peroxide, a library of HyPer-cpRFP variants was prepared in which the link between the OxyR and cpRFP fragments was carried out using randomized (random) linker sequences with a length of 2 or 3 amino acid residues.

[132] The resulting libraries were screened to detect hydrogen peroxide sensitive versions of the protein. For this, Petri dishes with bacterial colonies at room temperature were photographed under a fluorescence binocular microscope (Olympus US SZX12), then they were sprayed from a finely divided atomizer with an aqueous solution of 200 μM hydrogen peroxide, kept for 1-1.5 min until the drops were absorbed, and photographed again. Both photographs were processed in the EMBL Image J program: the background signal value was subtracted and the fluorescence intensities of individual colonies were compared before and after spraying. If the intensity ratio exceeded 1.1, such colonies were subcultured onto new Petri dishes with LB agar and ampicillin and grown at 37 ° C for 10-12 hours, and then analyzed using a Varían Sagu Eclipse fluorescence spectrofluorimeter: cells that are descendants of one clone, in a small amount was selected and suspended in 1.5-2 ml of PBS buffer in a spectrofluorimeter cuvette. The excitation or emission spectra of the corresponding proteins were detected in the presence and absence of a saturating concentration of hydrogen peroxide.

[133] As a result, a variant called HyPerRed was selected that responds to the addition of hydrogen peroxide with an 80% increase in fluorescence intensity. Testing of a bacterial suspension in the presence of various concentrations of hydrogen peroxide showed that the minimum HyPerRed response in bacterial cells was observed with 30 μM hydrogen peroxide added, and the dynamic range of the sensor was a 1.8-fold increase in fluorescence (Figure 2).

[134] An expression vector based on pQE-30 encoding HyPerRed was purified from a bacterial culture using MiniPrep plasmid DNA purification columns (Eurogen) and sequenced. The nucleotide and amino acid sequences of HyPerRed are shown in SEQ ID NO: 1 and 2. It was shown that HyPerRed has linkers 1 and 2 with a length of 3 amino acid residues with the composition Pro, Arg, Thr and Ala, Gly, Val, respectively.

[135] Thus, it was shown that the length and composition of linkers are individual for each biosensor and can fundamentally affect its functionality.

[136] Example 2

[137] Investigation of the properties of the isolated HyperRed protein.

[138] An expression vector based on pQE-30 encoding HyPerRed was obtained as described in Example 1 and used for chemical transformation of E. coli cells of strain XL 1 Blue. Cells were seeded on Petri dishes with solid nutrient LB medium and ampicillin (100 μg / ml) and grown for 14 h at 37 ° C. Then the colonies were seeded in 200 ml of LB medium and grown at 25 ° С on a thermostatic shaker with constant stirring 190 rpm for 36-38 hours. Then the cells were pelleted by centrifugation with cooling in a Beckman J21 centrifuge at 4000g for 30 minutes. The cell pellet was resuspended in 7 ml of 40 mM Tris-HCl containing 150 mM KCl, 10 mM MgSO ₄ and 2 mM mercaptoethanol (buffer A) and lysed using a Sonic Vibra cell 5 ultrasonic homogenizer in an ice bath for 30 minutes. The resulting lysate was centrifuged in a Beckman J21 centrifuge at 18000g at 4 ° C for 30 minutes. Talon Metal Affinity Resin (Clontech, CIIIA) was used to purify the protein from the supernatant according to the manufacturer's protocol.

[139] The obtained protein was used to study its spectral properties on a Varían Caru Eclipse spectrophotometer. An aliquot of the protein was dissolved in 1.5-2 ml of PBS buffer in a fluorimeter cuvette so that the maximum fluorescence intensity was 100-150 units. The excitation and emission spectra of the protein, the reaction to mixing, and the change in the composition of the buffer were detected.

[140] Plots of the excitation and fluorescence emission spectra of HyPerRed are shown in Fig. 3.

[141] The sensitivity of the isolated protein to hydrogen peroxide was comparable to that for the original HyPer sensor. The lower limit of sensitivity was 50 nM, the upper - 290 nM, the dynamic range of the protein decreased from 80% to 66% increase in fluorescence intensity. A decrease in the dynamic range of the response to the hydrogen peroxide of the isolated protein relative to the response in the E. coli cell suspension may be due to the partial oxidation of the target protein, since in rare cases redox-active proteins can be isolated in the native and fully restored state (Kim et al., 2002, Cell. 109, 383-96; Brejc et al., 1997, Proceedings of the National Academy of Sciences of the United States of America. 94, 2306-11).

[142] The isolated protein did not respond to the addition of purified water, PBS, the incubation time in the buffer, and stirring the solution.

[143] Comparison of the brightness parameters of the purified protein relative to the proteins of the isolated mKate2 (Eurogen) and HyPer proteins obtained by the same protocol. The concentrations of the isolated proteins were determined by spectrophotometric and Bradford methods. Aliquots of the proteins were then diluted to the same molar concentration of the protein and the absorption and fluorescence emission of these solutions were measured.

[144] The quantum yield of fluorescence, QY (Quantum Yield) was determined by the formula:

,[145]

,

[146] Where Abs1 is the absorption of the studied protein at the absorption maximum, Abs2 is the absorption of the standard protein at its absorption maximum, Em1 is the fluorescence emission of the standard protein at the maximum of fluorescence emission, Em2 is the emission of the studied protein at the maximum of its fluorescence emission, QY1 is the quantum yield of the protein -standard, QY2 - quantum yield of the studied protein.

[147] The quantum yields of the studied proteins were 0.22 for HyPer, and 0.29 for HyPerRed. The absorption at the maximum of fluorescence excitation was 22,400 for HyPer and 39,000 for HyPerRed. Thus, the brightness of the recombinant HyPerRed fluorescence protein in vitro, defined as the product of absorption and quantum yield, was several times higher than that of HyPer, which makes HyPerRed the preferred sensor for microscopy in thick tissue layers.

[148] Example 3

[149] Investigation of the properties of HyperRed expressed in eukaryotic cells

[150] An expression vector based on pQE-30 encoding HyPerRed was obtained? as described in example 1.

[151] To clone the HyPerRed nucleic acid from the pQE30 vector to the pCleGFP vector (Clontech, USA), it was amplified using gene-specific terminal primers containing restriction sites for cloning and Tersus polymerase (Eurogen). Conducted 22 cycles of PCR in the mode: denaturation 95 ° C - 12 sec; annealing 60 ° С - 20 sec; elongation 72 ° C - 1 min. The resulting fragments were purified using phenol-chloroform extraction and ligated into pCleGFP, previously subjected to restriction at similar sites, to the EGFP site.

[152] As a control, the HyPer encoding pHyPer-cyto expression vector (Eurogen) was used.

[153] HeLa Kyoto cells were seeded a day before transfection into plates in DMEM containing 10% inactivated fetal serum (PAA Laboratories) at a rate of 20-50 thousand cells per cup.

[154] X-tremegene 9 reagent and a modified manufacturer's protocol were used for transfection. For 100 μl of Opti Mem medium, 3 μl of X-tremegene 9 was added, and after intensive mixing they were incubated for 6 minutes. Plasmid DNA (1 μg) in 50 μl of Opti Mem medium was added to the solution of liposomes formed, mixed and incubated for 30 minutes. Then, the transfection mixture was added in 2 ml of DMEM medium containing 10% inactivated fetal serum and the cells were incubated overnight.

[155] The next day, the medium was changed to 2 ml of fresh DMEM medium containing 10% inactivated fetal serum. 10 minutes before microscopy, the medium in all dishes was replaced with Hanks solution with heather without sodium bicarbonate.

[156] Measurement of sensor activity was performed on the first and second day after transfection.

[157] A Leica DMI 6000 fluorescence microscope was used to visualize the fluorescence of transfected cells the day after transfection. Cell microscopy was carried out at 37 ° C, fluorescence was detected in channels corresponding to the values of excitation and fluorescence emission of the fluorescent protein used in the work: HyPerRed - TX2, EGFR-YFP - channel GFP, HyPer - channels FRET (CFP-YFP) and GFP.

[158] The following lenses were used: НСХ Р2 ApoLambda blue 63 * 1.4 Oil and HCX PL АРО CS 40.0x1.25 OIL UV. The exposure was 100-300 ms at intervals of 5 seconds. 30 seconds after the start of the experiment, hydrogen peroxide was added to the cells to a final concentration of 180 μM. The resulting image series were analyzed using the Leica Application Suite Advanced Fluorescense (LAS AF) software. The final processing and calculation of images was carried out using the EMBL Image J program and Excel.

[159] Without an additional localization signal, HyPerRed is evenly distributed in the cell, fills the cytoplasm and nucleoplasm of cells. The reaction range of the sensor was similar to its reaction in bacterial cells and amounted to 80% - an increase in fluorescence in response to the addition of hydrogen peroxide. After oxidation, the sensor signal returned to its original value due to the restoration of the biosensor due to the work of cellular enzymatic systems. The range of recorded HyPerRed concentrations of hydrogen peroxide is shown in Fig. 4.

[160] The minimal detectable response causes hydrogen peroxide to be added to the cell medium to a final concentration of 6 μM, which is similar to the sensitivity of the HyPer sensor to hydrogen peroxide. according to available literature data, when hydrogen peroxide is added to eukaryotic cells, the cytoplasmic concentration of hydrogen peroxide reaches values 200-500 times lower. Thus, a signal change in response to the addition of 6 μM hydrogen peroxide to the cell medium is equivalent to a change in response to 12-24 nM hydrogen peroxide in the cell cytoplasm.

[161] HyPerRed and HyPer were tested for the ability to detect low physiological concentrations of ligands in living cells. Physiological stimulation of Hela Kyoto cells by epidermal growth factor (EGF) was used as a model system.

[162] Binding of EGF to a receptor (EGFR) on the cell surface causes activation of the tyrosine kinase domain of the receptor, which phosphorylates phosphatidylinositol-3-kinase (PI3K), activating it. PI3K converts fosfatidilinozitoldifosfat (PIP2) into fosfatidilinozitoltrisfosfat (PIP _3). PIP ₃ interacts with Racl, stimulating the exchange of GDP for GTP, which leads to activation of Racl. Activation of Racl leads to the activation of the NADPH oxidase complex and the production of hydrogen peroxide, which is capable of causing activation of the studied sensors. The concentration of hydrogen peroxide increases locally to 50-200 nM, but even a sensor evenly redistributed in the cytoplasm is able to detect these low concentrations of peroxide (Mishina et al., 2011, Antioxidants & redox signaling. 14, 1-7.), Because the half-period of its oxidation is several times less than the half-period of its reduction.

[163] During physiological stimulation, HeLa Kyoto cells on the first and second day after transfection were washed with 1 ml of Hanks solution with 20 mM HePesNa, the DMEM medium was replaced with 2 ml of Image Media (DMEM medium without phenol red and sodium bicarbonate without inactivated bovine serum and vitamins, added 20 mm HePesNa and 20 mm glutamine, low glucose concentration (1 g / l)). Cells were transferred to a temperature-controlled chamber at a temperature of 37 ° C for 2 hours.

[164] After this period, 300 μl of medium was taken from a cell plate into a separate tube, maintaining its temperature at 37 ° C.

[165] We started shooting with the following parameters: NSX PL ARC CS 40.0 × 1.25 OIL UV lens, exposure time was 100-150 ms at 20 second intervals. 2 minutes after the start of the experiment, a previously selected aliquot of the medium (300 μl) with added EGF was added to the cells to a final concentration of 100 ng / ml in the cell medium in the plate and shooting was continued for up to 20 min. The resulting image series were analyzed using the Leica Application Suite Advanced Fluorescense (LAS AF) software. The final processing and calculation of images was carried out using the EMBL Image J program and Excel.

[166] A mutant version of HyPerRed with the mutation C199S, a key amino acid OxyR protein, important for disulfide bond formation in response to the appearance of hydrogen peroxide, was used as a control. HyPerRed-C199S nucleic acid was obtained by site directed mutagenesis of HyPerRed nucleic acid. In experiments in bacterial suspension and expression in a HeLa Kyoto eukaryotic culture similar to those described in Examples 1 and 3 above, HyPerRed-C199S was not sensitive to the addition of exogenous hydrogen peroxide.

[167] During physiological stimulation of EGF, the signal of the HyPer and HyPerRed biosensors changed proportionally. The time and nature of the response differed in different cells in one experiment, because the response to stimulation is individual. Figure 5 shows a graph of a typical HeLa Kyoto cell response in response to EGF stimulation. The reaction is two-phase, the first phase is fast, the largest amount of H ₂ O ₂ is produced in it, the second phase is characterized by a less intense but longer production of hydrogen peroxide. HyPerRed-C199S did not change spectral properties in response to stimulation.

[168] Example 4

[169] Using HyperRed for multiparametric microscopy.

[170] When cotransfected with several vectors, the target plasmids were mixed in 50 μl of Opti Mem medium in equal amounts so that the final amount of DNA was 2 μg. Per 100 μl of Opti Mem medium, 6 μl of X-tremegene 9 was added; after vigorous stirring, they were incubated for 6 minutes. Prepared plasmid DNA was added to the solution of liposomes formed, mixed and incubated for 30 min at room temperature. Then, the transfection mixture was added in 2 ml of DMEM medium containing 10% inactivated fetal serum and the cells were incubated overnight. Subsequent actions were carried out similarly to the transfection protocol of one design described in Example 3.

[171] A model of HeLa Kyoto cell stimulation by epidermal growth factor was used for multiparametric microscopy.

[172] Cells were transfected with three constructs encoding a fusion epidermal growth factor receptor protein with a yellow fluorescent protein (EGFR-YFP), a blue fluorescent B-GECO bisensor protein to monitor changes in the concentration of calcium ions and HyPerRed. Expression vectors for the expression of EGFR-YFP and B-GECO were obtained through the Addgene service.

[173] HyPerRed and B-GECO did not carry additional localization signals and were evenly distributed in the cytoplasm and nucleoplasm of transfected cells, EGFR-YFP protein in the absence of growth factors was localized on the plasma membrane of cells, according to the normal localization of EGFR.

[174] As described above in Example 3, upon binding of EGFR to its ligand, the receptor tyrosine kinase domain is activated to form two secondary messengers - PIP3 and IP3 due to the action of PI3K and PLC, respectively, which, in turn, cause the release of calcium from the endoplasmic reticulum (action of IP3) and activation of NADPH oxidase (action of PIP3) followed by the production of hydrogen peroxide. These stages of the cell signaling system were recorded using multiparameter microscopy (Fig. 6).

[175] The formation of endocytic vesicles with EGFR-YFP is observed within a few seconds after the addition of EGF to the cell medium. Figure 7 shows the change in the fluorescence intensity of EGFR-YFP, endosomes with a receptor are formed on the cell membrane and transported to the cytoplasmic compartments of the cell. Then a short-term increase in the concentration of calcium in the cytoplasm of some cells occurs, and after 5-6 minutes the level of hydrogen peroxide in the cytoplasm of the cells increases, the reaction is similar to that observed on HeLa Kyoto cells transfected only with the HyPerRed construct.

[176] Thus, three stages of the activation of the tyrosine kinase signaling cascade, triggered by EGFR stimulation, namely, endocytosis of the receptor, release of calcium from the endoplasmic reticulum, and generation of hydrogen peroxide, in one living cell, are visualized. The possibility of joint microscopy of several fluorescent constructs opens up new possibilities for studying the relationship of signaling processes in vivo.

[177] All publications and patent applications cited in the present description are introduced into the present description by reference, as if each individual publication or patent application was specifically and separately introduced by reference. The citation of any publication is in accordance with the context and interpretation of the present invention and should not be construed as recognition of any such publication as a prototype of the present invention.

Claims (4)

1. The selected nucleic acid encoding a fluorescent protein exhibiting the properties of a biosensor for the detection of hydrogen peroxide, the amino acid sequence of which is shown in SEQ ID NO: 2. 2. The expression cassette, which, when integrated into the genome of a cell or when introduced into the cell as an extrachromosomal element, provides the expression of a fluorescent biosensor for the detection of hydrogen peroxide, and contains the nucleic acid according to claim 1 under the control of the regulatory elements necessary for the expression of nucleic acid in the cell - the owner. 3. A cell producing a fluorescent protein exhibiting biosensor properties encoded by the nucleic acid according to claim 1, comprising an expression cassette according to claim 2 in the form of an extrachromosomal element or an element integrated into the cell genome. 4. The selected fluorescent protein, the amino acid sequence of which is shown in SEQ ID NO: 2, showing the properties of a biosensor for the detection of hydrogen peroxide, encoded by the nucleic acid according to claim 1.

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