RU2493260C2 - Isolated nucleic acid coding fluorescent biosensor for detection of hydrogen peroxide, expression cassette, cell that produces biosensor, isolated fluorescent biosensor for detection of hydrogen peroxide, isolated nucleic acid that codes fluorescent biosensor, efficiently fused with nucleic acid that codes signal of intracellular localisation - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: isolated nucleic acid is proposed, which codes a fluorescent biosensor for detection of hydrogen peroxide, and also an appropriate expression cassette, a biosensor producent cell and an isolated fluorescent biosensor.

EFFECT: group of inventions may be used to detect hydrogen peroxide in live cells having increased recovery half-life.

5 cl, 1 tbl, 3 dwg, 4 ex

Description

The present 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.

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 was examined in detail by Lippincott Schwartz and Patterson in Science (2003) 300 (5616): 87-91 and Chudakov et al. (Physiol Rev 2010 Jul; 90 (3): 1103-63).

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.

GFP hydromedusa Aequorea aequorea (synonym for A. victoria), has been described by Johnson et al. in 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, and

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 almost 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.

GFP has been used in a wide range of applications, including gene expression studies 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 protein transport along the secretory pathway (Kaether and Gerdes, FEBS Letters (1995) 369: 267-271).

Numerous studies have been conducted to improve the properties of avGFP (Aequorea victoria GFP) and to obtain GFP variants suitable and optimized for various research purposes. Genetic code avGFP (codon usage) 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.

In 1999, GFP homologues were cloned from non-bioluminescent species of Anthozoa (Matz et al., Nature Biotechnol. (1999), 17: 969-973). This discovery demonstrated that these proteins are not necessarily a component of the bioluminescent system. Anthozoa CPP-like proteins had a great spectral diversity and included cyanic, green, yellow, red fluorescent proteins and purple-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 homologues. The similarity of these proteins to GFP varies from 80-90% to less than 25% amino acid sequence identity.

The crystal structures 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 Stmct Biol (2000), 7: 1133-1138; Yarbrough et al . Proc Nati 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 position of the amino acid residues forming the chromophore corresponds 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.

Fluorescent proteins are a unique family of structurally related proteins that are able to form the 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, fluorescent-activated cell sorter, plate fluorimeter).

The process of autocatalytic formation of a chromophore of proteins with different spectral properties is described in detail in several articles and includes several chemical reactions (Heim et al. Proc Nati 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 Nati 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 Nati Acad Sci USA. 2000; 97: 11990-11995; Wall et al. Nat Struct Biol. 2000; 7: 1133-1138 ; Yarbrough et al., J. Proc Nati Acad Sci USA. 2001; 98: 462-467; Pakhomov, AA and Marynov, 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; Kiku chi et al. (2008) Biochemistry 47, 11573-11580; Yampolsky et al., Biochemistry, 2009, 48 (33), pp 8077-8082).

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 studied parameter 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 ;, reagentless 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.

Biosensors based on fluorescent proteins are chimeric proteins, which include a sensory domain - a protein domain that is sensitive to a change in a specific cell parameter, for example, a change in the concentration of any ion or molecule (calcium ions, hydrogen peroxide, hydrogen ions, etc. .). GFP-like proteins or their variants subjected to circular permutation are used as a signal part of the biosensor (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).

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 the codons encoding the new N and C terminal amino acids. Methods for obtaining such structures are well known to specialists in this field.

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

HyPer is a chimeric protein consisting of the regulatory domain of E. coli OxyR protein reacting with hydrogen peroxide and integrating the permuted yellow fluorescent protein cpYFP into the OxyR peptide chain (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 protein and cpYFP 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).

The transcription factor E. coli OxyR activates the expression of a number of genes in response to an increase in the concentration of H ₂ O ₂ . 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 HgO2, 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) .

HyPer is a unique tool for analyzing changes 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; Schreck et 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 detected by visual screening, spectrophotometry, spectrofluorimetry, fluorescence microscopy, using FACS or other conventional 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).

A HyPer variant has been developed called HyPer2 with an increased dynamic range (Markvicheva et al., Bioorg Med Chem. 2011; 19 (3): 1079-84). HyPer2 was obtained by incorporation; A406V mutations in the HyPer sequence. HyPer2 has an approximately 2-fold improved dynamic range than HyPer.

HyPer and HyPer2 allow real-time monitoring of dynamic changes in H3O2 concentration, however, due to the relatively high recovery rates of the OxyR domain, it is not possible to detect cells that have responded to stimulation by the production of hydrogen peroxide for a long time. In other words, the HyPer and HyPer2 biosensors work as “dynamic” sensors, but cannot be used as “storage” ones. This limits the scope of HyPer and HyPer2 for monitoring changes in the concentration of НзОз, for example, in in vivo systems, or when sorting cells by activated fluorescence (FACS). That is, when it is necessary that the biosensor signal is maintained for the time necessary for processing biological samples (for example, when it is necessary to fix tissues and make tissue sections for further study or when time is needed for applying samples to a FACS machine).

The present invention provides isolated nucleic acid molecules encoding a fluorescent biosensor for detecting hydrogen peroxide, having a reduced recovery rate after reaction with hydrogen peroxide compared to HyPer and HyPer2. In some embodiments, the nucleic acid of the present invention encodes a fluorescent biosensor for detecting hydrogen peroxide, the amino acid sequence of which differs from the HyPer2 sequence (SEQ ID NO: 1) by at least one amino acid substitution. In some embodiments, this is a replacement in the OxyR regulatory domain. In some embodiments, this is a substitution at position 34 in the amino acid sequence shown in (SEQ ID NO: 1). In some embodiments, this is a replacement for H34Y.

In some embodiments, the nucleic acid of the present invention encodes a fluorescent biosensor for detecting hydrogen peroxide having the amino acid sequence shown in SEQ ID NO: 4. In some embodiments, the nucleic acid of the present invention has the nucleotide sequence shown in SEQ ID NO: 03. In some embodiments, the nucleic acid of the present invention encodes a fluorescent biosensor for detecting hydrogen peroxide fused to an intracellular localization signal.

In some embodiments, the nucleic acid of the present invention encodes a fluorescent biosensor for detecting hydrogen peroxide, which has a lower recovery rate after oxidation during the interaction of the sensor with hydrogen peroxide than with HyPer and HyPer2.

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. 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. In addition, a kit is provided comprising nucleic acids and / or vectors and / or expression cassettes comprising said nucleic acids of the present invention.

Brief Description of Drawings

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

Figure 2 illustrates the dynamics of the signal variation of the HyPer biosensor over time after addition of hydrogen peroxide.

Figure 3 shows a comparison of the average value for the half-period of oxidation and reduction of variants of the HyPer biosensor (n = 20, P <0.05).

The implementation of the invention

To more fully disclose the above characteristics of the present invention, a detailed description of the invention, summarized above, in the form of links 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.

As indicated above, the present invention is directed to nucleic acid molecules that encode a mutant version of the HyPer biosensor, having a reduced recovery rate after reaction with hydrogen peroxide. Nucleic acids of the present invention 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: 4. In some embodiments, the nucleic acids encode a protein having the amino acid sequence shown in SEQ ID NO: 4 fused to a specific intracellular localization signal. 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.

These nucleic acids are used in many different applications and methods, in particular, 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, kits are provided for their use in such methods and applications.

Definitions

Various terms relating to the biological molecules of the present invention are used above and also in the description and in the claims.

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.

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).

As used herein, the term “circularly permuted fluorescent protein” or “cfpb” refers to a protein derived from a fluorescent protein (eg, avGFP) using genetic engineering modification of a nucleic acid, resulting in the C- and N- ends of the original fluorescent the protein is quickly fused, and new C- and N-ends are formed near the chromophore. Circular permutation 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 the fact that cpFB acquires the ability to change spectral characteristics upon conformational changes in protein domains or polypeptides operatively fused to its C- and N-ends.

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).

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.

As used herein, 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 deleted (delegated) and / or inserted (inserted) at 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.

As used herein, “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.

As used herein, an amino acid or nucleotide sequence is “substantially similar” or “substantially the same as a reference sequence if the amino acid or nucleotide sequence has at least 85% identity with the indicated sequence within the region selected for comparison. Thus, substantially similar sequences include those that have, for example, at least 85% identity, at least 90% identity, at least 95% identity, or at least 96% and 97% b 98% or 99% identity Two sequences that are identical to one another are also essentially similar.

The percentage of sequence identity is determined based on the 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 can be used to determine the level of identity and similarity between nucleotide sequences and amino acid sequences.

As used herein, the term “similar proteins” or “substantially similar proteins” refers to proteins that have amino acid sequences that are at least 85% identical, typically 90% or more identical, most often at least 95 identical % or more (for example, 96% or more, 97% or more, 98% or 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.

In some embodiments, the term “similar proteins” or “substantially similar proteins” refers to proteins that have the amino acid sequences of a whole protein 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%).

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.

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.

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 .

As used herein, “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 the 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 effects, and anisotropy of optical properties. The measured difference can be defined as the amount of any quantitative fluorescence property, for example, the fluorescence intensity at a specific wavelength, or the integral fluorescence over the entire emission spectrum.

As used herein, “aggregation” refers to the tendency or ability of an expressed protein to form an insoluble precipitate (aggregates). "Aggregation" should be distinguishable from "oligomerization". In particular, mutants with reduced ability to aggregate, for example, with increased solubility, do not necessarily have a reduced ability to oligomerization.

As used herein, “oligomerization” refers to the tendency or ability of an expressed protein to form complexes (oligomers) as a result of the specific interaction of two or more polypeptides. The specified specific interaction is observed under special conditions, for example, in physiological conditions, and is relatively stable under these conditions. A reference to the “ability” of proteins to oligomerize means that proteins can form dimers, trimmers, tetramers, or similar complexes under special conditions. As a rule, fluorescent proteins are capable of oligomerization under physiological conditions. Fluorescent proteins can also oligomerize under conditions other than, for example, pH than pH under physiological conditions. The conditions under which fluorescent proteins form oligomers or show a tendency to oligomerization can be determined using well-known methods such as gel filtration or otherwise known in the art.

Reference to the nucleotide sequence of the "coding" polypeptide means that the polypeptide is produced from the nucleotide sequence during translation and transcription of mRNA. In this case, both a coding chain identical to mRNA and usually used in the list of sequences can be indicated, as well as a complementary chain 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.

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 specific 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.

As used here, the terms "hydrogen peroxide" and "hydrogen peroxide" refer to a molecule having the formula H ₂ O ₂ .

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

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).

As used here, the term "response rate" or "oxidation rate" in relation to the biosensor of the present invention refers to the rate at which the biosensor molecule reacts to an increase in the concentration or appearance of hydrogen peroxide in the medium. For the needs of the present invention, the response rate can be characterized by the “oxidation half-period” (T1 / 20X) of the biosensor — half the time during which the biosensor reaches its maximum signal value, counting from the moment hydrogen peroxide is added to the medium.

As used here, the term "recovery rate" in relation to the biosensor of the present invention, refers to the speed at which the molecule of the oxidized biosensor restores the ability to respond to the appearance of hydrogen peroxide in the environment through interaction with cellular systems that restore disulfide bonds. For the needs of the present invention, the recovery rate can be characterized by a “half recovery period” (T 1/2 RED) - half the time during which the biosensor reaches the minimum (initial, characteristic for the restored form) signal value, counting from the moment the signal reaches its maximum at oxidation.

Nucleic acid molecules

The present invention provides nucleic acid molecules encoding a fluorescent biosensor for detecting hydrogen peroxide having the amino acid sequence of SEQ ID NO: 4.

As used herein, a nucleic acid molecule is a DIC molecule, such as a genomic DIC or cDNA molecule, or an RNA molecule, such as an mRNA molecule. As used here, the term "cDNA" refers to nucleic acids that possess the arrangement of sequence elements found in native mature types of mRNA, where sequence elements are exons and 5 'and 3' non-coding regions. 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 may be assembled from fragments using DNA ligase or PCR with primers containing parts complementary to the terminal sequences of the fragments to be joined.

A nucleic acid molecule encoding a fluorescent biosensor can be synthesized from suitable nucleoside triphosphates or isolated from biological sources. Both methods are based on protocols well known in the art. For example, the availability of amino acid sequence information or nucleotide sequence information enables the production of the isolated nucleic acid molecules of the present invention using oligonucleotide synthesis. In the case of amino acid sequence information, several nucleic acids that differ from each other due to the degeneracy of the genetic code can be synthesized. 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, 2 ^nd Ed., (1989) Cold Spring Harbor Press, Cold Spring Harbor, NY, and according to 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.

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 replaced codons encode the same amino acid.

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. 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 (e.g., physiological intracellular conditions) for expression of the protein in the host cell. 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 located in a medium different from the environment in which they are found in vivo, 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.

The claimed nucleic acids can be isolated and obtained essentially in purified form. A substantially 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 is usually not linked on the chromosome found naturally in its natural host organism.

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.

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 a 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, paired PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive multiple mutagenesis, exponential multiple mutagenesis, site-specific mutagenesis, random mutagenesis, gene p disassemble (gene reassembly), gene site-saturation mutagenesis (GSSM), artificial restructuring with ligirova-Niemi (SLR), or a combination thereof. Modifications, additions or deletions can also be carried out by a method including recombination, recursive recombination of sequences, phosphothioate-modified DNA mutagenesis, mutagenesis on a uracil-containing matrix, double-pass mutagenesis, mismatched reductive mutagenesis, strain mutagenesis, recovery-deficient mutagenesis, radioactive mutagenesis, deletion mutagenesis, restriction-selective mutagenesis, restriction mutagenesis with purification, synthesis of artificial genes, multiple mutagenesis, the creation of chimeric multiple nucleic acids and combinations thereof.

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 a signal of intracellular localization. Methods for producing such nucleic acids are well known to those skilled in the art.

Also provided are a vector and other nucleic acid constructs containing the claimed nucleic acids. 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.

Also provided are expression cassettes or systems used inter alia to obtain the claimed biosensors or chimeric proteins based on them 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 by introducing said expression cassette into the cell.

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 provides the initiation of RNA (transcription) reading 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 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.

The above expression systems can 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).

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.

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 is within the scope of the invention as a product of the host cell or organism. The product may be isolated by a suitable method known in the art.

Proteins 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 differs from the HyPer amino acid sequence (SEQ ID NO: 2) by the presence of amino acid substitutions A406V and H34Y. The amino acid sequence of the biosensor of the present invention differs from the amino acid sequence of HyPer2 (SEQ ID NO: 1) by the presence of the amino acid substitution H34Y. Specific biosensors of interest include a biosensor having the amino acid sequence of SEQ ID No: 04.

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. In the absence of hydrogen peroxide, the claimed biosensor has a maximum (peak) emission in the range from 500 nm to 550 nm, for example, 520 nm, and two peaks of fluorescence excitation; the first in the range of 400-450 nm (e.g., 420 nm) and the second in the range of 470-510 nm (e.g., 500 nm).

When hydrogen peroxide appears, the claimed biosensor goes into an oxidized state. During biosensor oxidation, the fluorescence intensity changes upon excitation by light with a wavelength of 500 nm (F500), to the fluorescence intensity upon excitation by light with a wavelength of 420 nm (F420). For the needs of the present invention, the “F500 / F420” ratio is a biosensor signal. The claimed biosensor has a wide dynamic range. The claimed biosensor has a dynamic range from 3 to 10, more often from 5 to 8.

The claimed biosensor has a half-cycle of oxidation (T1 / 20X) ranging from 15 to 80 seconds, more often from 40 to 75 seconds, usually from 50 to 70 seconds.

The claimed biosensor has a recovery rate that is lower than the recovery rate of the HyPer and HyPer2 biosensors. This means that the biosensor indicated exhibits a longer half-recovery period (T1 / 2RED) than HyPer and HyPer2. The half-recovery period of the biosensor of the present invention is more than 11 minutes, more often more than 13 minutes and is in the range from 11 to 25 minutes, more often from 13 to 21 minutes.

To determine T1 / 20X and T1 / 2RED for hydrogen peroxide biosensors, a biosensor expressed in a heterologous expression system can be used. For example, HeLa cells can be used.

Kyoto. To determine T1 / 20X and T1 / 2RED after transfection of cells with a construct for expression of the biosensor in host cells (vector, expression cassette, etc.) and cultivation under suitable conditions for the time required for cell growth and production of the biosensor in them cultivation medium add hydrogen peroxide in a certain concentration and record the dependence of the fluorescence intensity upon excitation with light with a wavelength of 420 nm and 500 nm from time to time. Based on the data obtained, T1 / 20X and T1 / 2RED are determined. 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: 04, at least 85% identity, usually at least 90% and more often, at least 95%, (for example 95% and more; 96% and more, 97% and more; 98% and more: 99% and more, or 100% sequence identity).

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. 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 essentially 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 "substantially 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%.

The claimed biosensors can be obtained artificially, 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.

Also provided are fusion proteins comprising the biosensor of the present invention, fused to a sequence of intracellular localization (for example, with a localization signal in the nucleus, in 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 those skilled in the art and are described, for example, by K. Nakai (Advances in Protein Chemistry, Vol. 54,2000, p. 277-344).

Transformants

The nucleic acids of the present invention are used to obtain 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 pro-karyotic (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.

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. Replacement sheet

Methods for transferring a nucleic acid molecule (i.e., DNA) into such organisms are widely known and provided in references such as Sambrook et al. (Molecular Cloning: A Laboratory Manual, 3nd Ed., (2001) Cold Spring Harbor Press, Cold Spring Harbor, NY).

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

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) Alien and Unwin, London, pp 401-429) and King et al Molecular and Cell Biology of Yeasts, EF Walton and G.T. 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.

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, 2nd Ed., (2002) Fox J.G., Anderson L.C., Loew F.M., Quimby F.W. (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, retro viruses 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.

Transgenic animals can be any non-human animals, including a non-human mammal (e.g., mouse, rat), bird or amphibian, etc., and are used in functional research, drug screening, and the like. "

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 obtain 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. With isolated protoplasts, it is possible to introduce DNA through 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 (for example, 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.

Methods of use 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. Due to the extended recovery period, the biosensors of the present invention are stored in an oxidized form for a long time, which makes it possible to identify and track cells that have been stimulated by the production of hydrogen peroxide. 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 should 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.

The biosensors of the present invention find use in the screening of drugs that cause 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.

The claimed biosensors also find application in the sorting of fluorescence activated cells (FACS). In such applications, the inventive biosensor is used to label a population of cells stimulated by any factor to produce hydrogen peroxide, and the resulting labeled population of cells 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.

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 WO 00/02997.

Sets

Kits are also provided in accordance with the present invention for use in carrying out one or more of the above applications. In preferred embodiments, the kits typically comprise a nucleic acid encoding a biosensor, preferably with elements for expressing the biosensor in the host cell. For example, the kits may include a vector comprising a nucleic acid encoding the claimed protein. The components of the kits are usually present in an appropriate storage medium, such as an aqueous or buffer solution, usually in an appropriate container. In separate embodiments, the kit includes many different vectors, each of which encodes the claimed protein, where the vectors are designed for expression in different environments and / or under different conditions. For example, for constitutive expression, the kit includes a vector having a strong promoter for expression in mammalian cells, and a vector with a weakened promoter. Multiple cloned site vectors for selectively inserted promoter and / or vectors for expression in various organisms may also be included.

In addition to the components described above, the claimed kits will further include instructions for implementing the claimed methods. These instructions may be present in the claimed sets in various forms (for example, in print or on electronic media in the form of a text and / or graphic file) in an amount of one or more.

The following examples are provided as illustrative but not limiting.

Examples

Example 1

Obtaining mutant variants of Well Per biosensor for the detection of hydrogen peroxide.

For targeted mutagenesis, nucleic acid encoding HyPer was used from the commercially available pHyPer-cyto vector (Eurogen, Russia). To obtain constructs containing the desired mutations, site-directed mutagenesis was performed using "overlap extention" of PCR products as described by Wurch et all, Methods in Molecular Biology. 12 (9), 653 - 657 (2004). For PCR, primers were used, the structures of which are shown in SEQ ID NOs: 5-10, and Tersus polymerase (Eurogen). The following amplification mode was used in 22 PCR cycles: denaturation 95 ° С - 12 sec; annealing 55 ° С - 20 sec; elongation 72 ° C - 1 min. Amplification was performed on a RTS-100 Thermal Cycler (MJ Reserch). Each reaction sample also contained primers (0.5 μM), an equimolar mixture of dNTP (0.5 μM), template DNA (10-100 ng). To bring the mixture to the desired volume, milliQ purification water was used.

After amplification, the obtained fragments were purified by electrophoresis in 1% agarose gel and further extraction. DNA fragments containing the desired mutation isolated from the gel were combined into a complete construct containing a point-wise substitution by overlap PCR method (denaturation 95 ° С - 12 sec; annealing 55 ° С - 2 min; elongation 72 ° С - 1 min, 8 PCR cycles). This method implies that, as a matrix and seed, the fragments present in the reaction mixture with complementary regions are used.

Further, the amplification of the entire construct obtained in the previous step was performed by standard PCR with the addition of end primers, the structures of which are shown in SEQ ID NOs: 11, 12. The following amplification mode was used: 14 PCR cycles; denaturation 95 ° С - 12 sec; annealing 55 ° С - 20 sec; elongation 72 ° C - 1 min. The obtained nucleic acids were cloned into the pQE30 vector according to the standard protocol; the correspondence of the insert structure was checked by sequencing using standard plasmid primers. The following new HyPer variants were obtained containing the following mutations: H34Y and A406V (HyPer-H34Y-A406V), H34Y (HyPer-H34Y), I32D (HyPer-I32D), F392A (HyPer-F392A) and L46D (HyPer-L46D). All of these mutations are in the region of the regulatory domain of OxyR, responsible for dimerization.

Example 2

Measurement of the dynamic range and half-periods of oxidation and reduction of the obtained mutant HyPer variants.

Vectors based on pQE-30 for expression of mutant HyPer variants in E. coli were obtained as described in Example 1. To carry out the re-cloning of the obtained constructs from the pQE30 vector into the pCleGFP vector (Clontech, USA), nucleic acid fragments containing the biosensor coding region were amplified using primers (SEQ ID NO: 13, 14) specific to the ends of the fragments and containing restriction sites for cloning, and Tersus polymerase (Eurogen). Conducted 22 cycles of PCR in the mode: denaturation 95 ° C - 12 sec; annealing 55 ° С - 20 sec; elongation 72 ° C - 1 min. The obtained fragments were purified by phenol-chloroform extraction and ligated into pCleGFP, which was previously subjected to restriction at similar sites to the EGFP site.

HeLa Kyoto cells were plated on slides (p-Slide VI, Ibidi) in DMEM containing 10% inactivated fetal serum (PAA Laboratories) at the rate of 5-7 thousand cells per lane, transfected with the corresponding vector and cultured according to the standard protocol.

Before microscopy, the medium in all slides was replaced with a Hanks solution without sodium bicarbonate. To visualize the fluorescence of transfected cells, a Leica DMI 6000 V fluorescence microscope was used. Cell microscopy was performed at 37 ° C, fluorescence was detected in two channels corresponding to two cpYFP chromophore excitation peaks (420 nm and 500 nm), emission 510 nm. For the channel corresponding to the protonated form of the cpYFP chromophore (420 nm), filters EFW Excitation: 427/10 (CFP), Emission: BP 542/27 (YFP) were used. For the deprotonated form of cpYFP chromophore (500 nm) - Excitation filters: BP 504/12 (YFP); Emission: BP 542/27 (YFP). Used the lens NX2 ApoLambda blue 63 * 1.4 Oil. The shooting intervals were 5 seconds. Hydrogen peroxide was added to the cells to a final concentration of 100 μM. The biosensor signal was recorded and half-periods of oxidation and reduction were calculated.

The following results were obtained: sensors with mutations 13 2D, L46D, F392A showed a low dynamic range compared to the original HyPer. The response amplitudes of HyPer-H34Y-A406V and HyPer-H34Y to the addition of H ₂ O ₂ significantly exceeded that of HyPer, not inferior to HyPer-2.

HyPer-H34Y showed higher oxidation and reduction rates compared to HyPer and HyPer-2, while HyPer-H34Y-A406V had longer oxidation and reduction half-periods than HyPer and HyPer-2.

The experimental results are summarized in Table 1. Graphs showing the dynamics of the signal for the biosensor options are shown in Fig. 2. A comparison of the average half-periods of oxidation and reduction of biosensor variants is shown in Fig. 3.

Variant HyPer-H34Y-A406V (SEQ ID NOs: 3, 4) was selected for further studies as having the longest half-recovery period and wide dynamic range.

Table 1.

The main characteristics of the biosensor options.

Biosensor Range T1 / 20X T1 / 2RED HyPer 2-2.5 17 ± 2 sec 3.2 ± 1 min HyPer2 (HyPer-A406V) 5-8 39 ± 2 sec 5 ± 0.8 min HyPer-H34Y 5-8 21 ± 2 sec 1.8 ± 1 min HyPer-H34Y-A406V 5-8 60 ± 10 sec 17 ± 4 min Hyper-I32D No more than 1.4 - - HyPer-F392A No more than 1,2 - - HyPer-L46D No more than 1,2 - -

Example 3

Testing the HyPer-H34Y-A406V biosensor variant in stably transfected cell lines

The HeLa Kyoto cell line stably expressing HyPer-H34Y-A406V protein was obtained by lentiviral transduction. For this, the open reading frame HyPer-H34Y-A406V obtained as described in Example 1 was cloned into the lentiviral vector pRRLSIN.EFl.WPRE. Transduction was performed according to standard protocols. The population of cells with the highest fluorescence intensity was selected using a flow-through fluorescence cell sorter MoFlo (Dako). The resulting line stably expressed HyPer-H34Y-A406V. The addition of H ₂ O ₂ in various concentrations to the medium caused the expected change in the spectral characteristics of the biosensor, confirming its functionality. Testing was carried out as described in Example 2. The oxidized form of the biosensor was detected in the cells 20-30 minutes after the addition of peroxide, while in the cell line expressing HyPer and prepared according to the same protocol, the oxidized form of the biosensor could not be detected later than after 5 min after addition of hydrogen peroxide.

Example 4

Obtaining and testing of the HyPer-H34Y-A406V biosensor with different location in mammalian cells.

The nucleic acid encoding HyPer-H34Y-A406V, obtained as described in example 1, was quickly inserted into the following commercially available vectors in place of the fluorescent markers TagRFP and mKate2:

1. pTagRFP-Golgi (Eurogen) containing a sequence of beta-1,4-galactosyltransferase of a person, providing localization of the protein in the Golgi apparatus;

2. pTagRFP-mito (Eurogen) containing a sequence of the VIII subunit of cytochrome C oxidase (Rizzuto et al., J Biol Chem. 1989; 264 (18): 10595-600 .; Rizzuto et al., Curr Biol. 1995 ; 5 (6): 635-42), providing localization of the protein in the mitochondria;

3. pmKate2-peroxi (Eurogen) containing SKL tripeptide, which provides protein localization in peroxisomes. The obtained vectors were used to transfect 293 T human renal epithelial cells. Transfection led to the appearance of a fluorescent signal in the expected cell compartments. Testing of the expressed fusion proteins for their ability to respond to the addition of hydrogen peroxide was confirmed as described in Example 2.

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 (5)

1. The selected nucleic acid encoding a fluorescent biosensor for the detection of hydrogen peroxide, the amino acid sequence of which is shown in SEQ ID NO: 4. 2. The expression cassette, which, being integrated into the genome of the cell or when introduced into the cell as part of an extrachromosomal element, is capable of expressing a fluorescent biosensor for detecting hydrogen peroxide and contains the nucleic acid according to claim 1 under the control of regulatory elements necessary for expression of nucleic acid in host cell. 3. A cell producing a biosensor encoded by a nucleic acid according to claim 1, containing an expression cassette according to claim 2 as part of an extrachromosomal element or an element integrated into the genome of this cell. 4. The selected fluorescent biosensor for the detection of hydrogen peroxide encoded by the nucleic acid according to claim 1. 5. An isolated nucleic acid encoding a fluorescent biosensor, the amino acid sequence of which is shown in SEQ ID NO: 4, operatively fused to a nucleic acid encoding an intracellular localization signal, which allows the biosensor to be localized in a particular compartment of the cell, where the specified nucleic acid is capable of expressing the fluorescent biosensor hydrogen peroxide detection in this cell compartment.

Images