RU2603060C2 - Modified biosensor for detecting intracellular ph - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: group of inventions relates to biotechnology. Group of inventions includes a nucleic acid, which encodes a fluorescent biosensor for detecting change in pH, amino acid sequence of which is shown in SEQ ID No: 4, as well as an expression cassette and eucariotic cell producing a biosensor.

EFFECT: group of inventions is aimed at biosensors for detecting change in pH in living cells, having high fluorescence brightness.

4 cl, 6 dwg, 4 ex

Description

1. Field of invention

[01] This invention relates generally to the field of biology and chemistry. In particular, the invention is directed to biosensors for pH detection, designed on the basis of fluorescent proteins.

2. The level of technology

[02] The study of the processes occurring in the brain tissues during normal electrical activity and pathology is of great interest both for identifying the fundamental mechanisms of neuronal activity and for biomedical applications.

[03] The main function of nerve cells, the generation and conduct of electrical impulses, is based on the movement of ions across the cell membrane. Within milliseconds, the concentration of Na ⁺ , K ⁺ , Ca ²⁺ and Cl ^{- ions} inside the cell can vary significantly, and maintaining homeostasis requires a complex regulatory system and high plasticity of metabolism.

[04] Intense neuronal activity is known to cause shifts in intracellular pH. Some studies have shown that long-term membrane depolarization and calcium entry into the cell are accompanied by significant acidification of the cytoplasm. The mechanism of this process has not been fully understood. One reason may be acid release during ATP hydrolysis by calcium pumps or proton entry through Ca / H and Na / H antiport (Wu, M.L., et al., 1999, The American journal of physiology. 277, C 717- 27). Another hypothesis suggests that acidification of the cytoplasm is caused mainly by the accumulation of lactate due to the acceleration of glycolysis processes (Zhan, R.Z., et al., 1998, Brain research. 780, 86-94).

[05] Changes in intracellular pH affect neuronal activity. It is known that the activity of voltage-gated calcium channels, NMDA and GABA receptors, etc., varies depending on pH. For example, the spontaneous incidental electrical activity of neurons in a medium with a low concentration of Mg2 + ions is suppressed by a slight acidification of the perfusion solution (Velisek, L., et al., 1994, Experimental brain research Experimentelle Hirnforschung Experimentation cerebrale. 101, 44-52). Transport of neurotransmitters also depends on the proton gradient across the membrane of synaptic vesicles (Blakely, R. D. & Edwards, R. N., 2012, Cold Spring Harbor perspectives in biology, 4). Therefore, pH shifts can play a significant role as a regulator of neuronal activity.

[06] Nerve cells have a complex morphological and functional structure, and measuring intracellular pH in such a system is not an easy task. Chemical indicators, such as SNARF and BCECF, do not allow comparison of pH values, for example, between the cytoplasm of dendrite and dendritic spines, which play a critical role in the processing of an incoming electrical signal (Lee, K.F. et al., 2012, Neural plasticity. 2012, 704103). It is possible, however, that the pH value varies non-uniformly in the neuron body and synaptic structures, taking into account some isolation of the latter from the main volume of the cell cytoplasm.

[07] The use of genetically encoded indicators circumvents this limitation. However, despite the existing variety of indicators, no designs have been created to visualize subtle changes in pH that occur during nervous activity, in particular in the presynaptic space.

[08] A significant part of the genetically encoded indicators developed to date contains fluorescent proteins of the GFP family, which change their spectral characteristics in response to changes in intracellular parameters.

[09] avGFP 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 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.

[10] GFP has been used in a wide range of applications, including studies 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).

[11] 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 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 were obtained, including a ′ ′ 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.

[12] In 1999, GFP homologues were cloned from non-bioluminescent Anthozoa species (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.

[13] GFP, its mutants and homologs are members of the protein family of the same name and are widely known for their intensive use as fluorescent markers in vivo in biomedical research, as discussed 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). Representatives of the GFP family 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.

[14] 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).

[15] 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.

[16] A GFP-like domain that can be easily identified by comparing the amino acid sequences of fluorescent proteins with avGFP (SEQ ID NO: 9) using software packages designed for domain structure analysis (for example, using the Conserved Domain Database (CDD) program available at http: // www. ′ ′ ncbi.nlm.nih.gov/Structure/cdd/ ′ ′ or using the Simple Modular Architecture Research Tool (SMART, available at http: // smart. ′ ′ ′ (Embl-heidelberg.de/). The GFP-like domain of avGFP begins with the 6th amino acid and ends with 229 amino acid.

[17] 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: 462-167; 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, 9639-9 647; Kikuchi et al. (2008) Biochemistry 47, 11573-11580; Yampolsky et al., Biochemistry, 2009, 48 (33), pp 8077-8082).

[18] 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 by 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 information transmitters has become the most effective approach to the development of genetically encoded sensors .

[19] 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.). 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).

[20] 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 may 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 a 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.

[21] 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 were found to be insensitive).

[22] The efficacy of cpFB biosensors derived from avGFP has been demonstrated using a calcium ion sensor (Nagai et al, Proc Natl Acad Sci USA. 98 (6) (2001), 197-202). cpFB was also used to fabricate the HyPer OxyR 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 cpYFP protein into the OxyR peptide chain. HyPer is a construct in which a circularly permutated cpYFP fluorescent protein is coupled to two fragments of the H2O2-sensitive OxyR domain via short peptide linkers. Nucleotide sequences encoding fragments of the OxyR protein and cpYFP are quickly crosslinked using linker sequences.

[23] An intracellular pH indicator called SypHer was also obtained from HyPer. The introduction of the C199S substitution into the HyPer sequence led to the loss of the ability to recognize hydrogen peroxide, and the appearance of a pronounced change in the fluorescent properties of the indicator in response to a change in pH.

[24] The obtained SypHer indicator has a ratiometric signal, which makes it independent of changes in cytoplasmic volume or protein concentration. The successful use of SypHer has been demonstrated in some in vitro systems (Poburko, D. et al., 2011, The Journal of biological chemistry. 286, 11672-84). Potentially, SypHer can be used to record pH changes in neurons.

[25] Nevertheless, the fluorescence brightness of the SypHer indicator is relatively low, which complicates its use in studies on tissue cultures and large living organisms. Thus, the aim of this study was to create a brighter version of the SypHer indicator and constructions based on this modified indicator, SypHer2, for recording pH changes in certain neuron compartments.

3. Disclosure of invention

[26] The present invention provides isolated nucleic acid molecules encoding a modified fluorescent biosensor for detecting pH within cells, different from SypHer (SEQ ID NO: 1 and 2) with increased brightness. The specified biosensor has the amino acid sequence shown in SEQ ID NO: 4. The specified biosensor finds its application for measuring pH inside living cells and in cell compartments, in particular, in live sections of the brain, and studying the dynamics of pH during electrical activity in various cell compartments on a culture of neurons and on short-lived sections of the brain.

[27] In some embodiments, the nucleic acid of the present invention encodes a fluorescent biosensor for detecting intracellular pH, fused to synapthysin protein, which provides localization of the sensor in the presynaptic zone of neurons. The amino acid sequence of this biosensor is shown in SEQ ID NO: 6.

[28] In some embodiments, the nucleic acid of the present invention encodes a fluorescent biosensor for detecting intracellular pH, fused to the Homer1 protein, which provides preferential localization of the sensor in the postsynaptic zone of neurons. The amino acid sequence of this biosensor is shown in SEQ ID NO: 8.

[29] In some embodiments, the nucleic acid of the present invention has a nucleotide sequence selected from SEQ ID NO: 03, SEQ ID NO: 05 and SEQ ID NO: 07.

[30] 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.

[31] 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 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.

4. Brief description of drawings

[01] Figure 1 illustrates the pH dependence of the ratiometric signal of SypHer and SypHer2 in a HeLa Kyoto culture in buffers containing 5 μM monensin and 5 μm nigericin. The number of cells is at least 30 at each point.

[02] Figure 2 illustrates a comparison of the mean fluorescence brightness of the SypHer and SypHer2 biosensors in mammalian cells. The average fluorescence brightness of the indicator is shown upon excitation with light of 488 nm for 15 cells in each line. The brightness is indicated in units relative to the average fluorescence brightness of SypHer2 in a given cell line.

[03] Figure 3A illustrates the dynamics of pH and Ca ⁺² in an electrically active nerve cell. The pH was measured using a SypHer2 biosensor, and Ca ²⁺ was measured using an RGeco biosensor. The spontaneous activity of neurons causes the entry of calcium into the cells, which can be observed by increasing fluorescence of RGeco. Calcium entry into cells is accompanied by a rapid decrease in SypHer2 fluorescence, which corresponds to cytoplasmic acidification. Fluorescence of SypHer2 was excited with 488 nm light.

[04] Figure 3B illustrates the dynamics of pH and Ca ²⁺ in a nerve cell in a medium containing an intracellular Ca ²⁺ chelator. SypHer fluorescence was excited with 488 nm light.

[05] Figure 4 shows cytoplasmic pH fluctuations caused by spontaneous electrical activity in sections of the brain in ACSF medium. SypHer fluorescence was excited with 488 nm light.

[06] Figure 5 shows cytoplasmic pH fluctuations caused by spontaneous electrical activity in sections of the brain in ACSF medium after adding 30 mM KCl. SypHer2 fluorescence was excited with 488 nm light.

4. The implementation of the invention

[07] To more fully disclose the above characteristics of the present invention, a detailed description of the invention, summarized above, is provided below in the form of links to embodiments, some of which are illustrated by additional figures. 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.

[08] As indicated above, the present invention is directed to nucleic acid molecules that encode a variant of the biosensor for measuring the intracellular pH of SypHer-SypHer2, which has increased fluorescence brightness and designs based on it, providing localization of the biosensor in certain compartments of the neuron.

[09] the 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.

[10] In some embodiments, the nucleic acids encode a chimeric protein comprising a SypHer2 biosensor operatively fused to a synaptophysin protein, the amino acid sequence of the chimeric protein is shown in SEQ ID NO: 6.

[11] In some embodiments, the nucleic acids encode a chimeric protein comprising a SypHer2 biosensor operatively fused to a Homerl protein, the amino acid sequence of the chimeric protein is shown in SEQ ID NO: 8

[12] Also provided are expression vectors and 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.

[13] These nucleic acids are used in many different applications and methods, in particular, for measuring pH in living sections of the brain and studying the dynamics of pH during spontaneous electrical activity in various cell compartments on neuron culture and on short-lived sections of the brain

[14] Definitions

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

[16] 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.

[17] As used here, the term “″ avGFP ″ refers to the green fluorescent protein from the jellyfish Aequorea victoria, 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).

[18] As used here, the term ″ fluorescent protein subjected to circular permutation ″ or ″ cfBP ″ refers to a protein derived from a fluorescent protein (eg, avGFP) using a genetic engineering modification of nucleic acid, as a result of which - and the N- ends of the original fluorescent protein are 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 that are operatively fused to its C- and N-ends.

[19] 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).

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

[21] 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 (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” here refers to any variant that is shorter or longer than a protein or nucleic acid.

[22] 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 these compared sequences.

[23] 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 specified 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% b 97% b 98 % or 99% identity. Two sequences that are identical to one another are also essentially similar.

[24] 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.

[25] As used here, 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, more often all identical at least 95% 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.

[26] In some embodiments, the term “’ like proteins ’’ or ’’ essentially similar proteins ’’ refers to proteins that have the whole protein amino acid sequence 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%).

[27] 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 the pH of the present invention means that it changes the spectral characteristics when the acidity in the medium changes.

[28] As used here, the term "environment" in relation to a biosensor means any environment 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 cell compartment in which the biosensor is localized.

[29] As used here, “biochemical properties” refer to protein folding (folding) and maturation rate, recovery rate after reaction with hydrogen peroxide, half-life, aggregation ability, oligomerization ability, pH and temperature stability, and others similar properties.

[30] As used here, the “fluorescence properties” or the “spectral properties” refers 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, lifetime the expected state, and anisotropy of the optical properties. 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.

[31] As used here, the term "biosensor signal" means a detectable change in the spectral characteristics of the biosensor in response to a change in pH of the medium.

[32] As used here, the “fluorescence brightness” is determined by the quantum yield of the biosensor fluorescence, multiplied by the maximum extinction coefficient and divided by 1000.

[33] A 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.

[34] The term “operatively linked” 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, when a biosensor of the present invention is crosslinked with an intracellular localization signal of interest or a protein, the chimeric protein retains the ability to respond to pH, but is localized in a particular cellular compartment. As is obvious to any person skilled in the art, the nucleotide sequences encoding a chimeric protein including the “operatively linked” components (proteins, polypeptides, linker sequences, protein domains, etc.) consist of fragments encoding these components, where these 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” of the reading frame and stop codons at the places of their connection.

[35] As used here, the term “pH” means “hydrogen index” - a measure that quantifies the acidity of a solution, which is calculated as the negative (taken with the opposite sign) decimal logarithm of the activity of hydrogen ions, expressed in moles per liter. Several standard techniques are widely used to determine the pH of solutions. The hydrogen index can be approximately estimated using indicators, accurately measured with a pH meter or determined analytically by acid-base titration.

[36] As used here, the term "physiological pH values" or the like means a range of cytoplasmic pH values observed under physiological conditions, which approximately corresponds to a pH of 6.8 to 7.8.

[37] Nucleic acid molecules

[38] The present invention provides nucleic acid molecules encoding a SypHer2 fluorescence biosensor for pH detection having the amino acid sequence of SEQ ID NO: 4. This biosensor has an increased fluorescence brightness compared to the SypHer biosensor and contains the A406V substitution (the amino acid numbering is in accordance with the amino acid sequence SypHer protein shown in SEQ ID NO: 2).

[39] Nucleic acid molecules are also provided that encode chimeric proteins containing the SypHer2 fluorescence biosensor for detecting pH operatively fused with proteins that localize the biosensor in specific neuron compartments.

[40] Methods for producing such fusion proteins are well known in the art. For example, parts of a nucleic acid encoding different elements can be inserted into the polylinker of the vector so that there are no stop codons between the different parts in the reading frame and there are no reading frame failures. Alternatively, the desired nucleotide sequence can be assembled from fragments using DNA ligase or PCR with primers containing parts complementary to the terminal sequences of the fragments to be joined.

[41] 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 the arrangement of sequence elements found in native mature types of mRNA, where sequence elements are exons and 5 ′ and 3 ′ non-coding regions.

[42] A nucleic acid molecule encoding a fluorescent biosensor can be synthesized from suitable nucleoside triphosphates or obtained by nucleic acid mutagenesis encoding a HyPer or SypHer biosensor or a homolog thereof. 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 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 no 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.

[43] 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.

[44] 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.

[45] 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.

[46] The claimed nucleic acids can be isolated and obtained essentially in purified form. Essentially, a purified form means that the nucleic acids are at least about 50% pure, usually at least about 90% pure, and are usually “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.

[47] 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 derived variants of a sequence can be called “mutants” or derivatives of the original sequence.

[48] Mutant or derivative nucleic acids can be obtained on a matrix nucleic acid selected from the above nucleic acids by modifying, deletion or addition of 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 p assembling (gene reassembly), gene site saturation mutagenesis (GSSM), artificial rearrangement ligation (SLR) or a combination thereof. Modifications, additions or deletions can also be performed by a method including recombination, recursive recombination of sequences, phosphotioate-modified DNA mutagenesis, mutagenesis on a uracil-containing matrix, double-pass mutagenesis, mismatch spot recovery mutagenesis, strain mutagenesis, recovery-deficient mutagenesis, radioactive mutagenesis, deletion mutagenesis, restriction-selective mutagenesis, restriction mutagenesis with purification, synthesis of artificial g new, multiple mutagenesis, creation of multiple chimeric nucleic acids, and combinations thereof.

[49] 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.

[50] 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 by introducing said expression cassette into the cell.

[51] 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 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.

[52] 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., co-transfection 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).

[53] 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.

[54] 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.

[55] Squirrels

[56] The nucleic acids of the present invention encode a fluorescent biosensor for detecting pH and chimeric proteins based thereon. In some embodiments, chimeric proteins include SypHer2 operatively fused to synapthysin protein (major synaptic vesicle protein p38). In some embodiments, chimeric proteins include SypHer2 operatively fused to the Homerl protein (PSD-Zip45).

[57] Synaptophysin (Synapthysin, major synaptic vesicle protein p38) is a membrane glycoprotein localized in synaptic vesicles. It is characterized by a high level of expression in neurons of all types and neuroendocrine cells, and can be a marker for the quantitative determination of synaptic contacts. Impaired synaptophysin expression causes minor impaired behavior in mice (McMahon HT et. Al., 1996, Proceedings of the National Academy of Sciences of the United States of America, 93, 4760-4764). The chimeric synaptiphysin-cypher2 protein is localized on the surface of synaptic vesicles at the presynaptic ends of neurons.

[58] Homerl (PSD-Zip45) is a cytoplasmic protein widely distributed in the cells of the central nervous system, as well as in the kidneys, heart, tissues of striated muscles, etc. Homer1 is one of the main components of the postsynaptic density of neurons, where it is involved in organizing complexes of channels, cell receptors, and cytoplasmic regulatory proteins (Hayashi MK et al, 2009, Cell 137, 159-171, Meyer D, Bonhoeffer T, Scheuss V, 2014, Neuron 82, 430-443). The combination of SypHer2-Homer into a single construct leads to the predominant localization of the pH sensor in the cytoplasm of postsynaptic endings in dendrites and on the bodies of neurons.

[59] The claimed biosensor and chimeric proteins based on it have 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.

[60] At physiological pH values, the 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). As the pH changes to higher values, the fluorescence intensity of the sensor decreases upon excitation in the range of 400–450 nm and the fluorescence intensity increases upon excitation in the range 470–510 nm.

[61] Thus, the ratio of the fluorescence intensity upon excitation in the range 470-510 nm to the fluorescence intensity upon excitation in the range 400-450 nm can be taken as a sensor signal. This parameter does not depend on the concentration of the biosensor, the level of its expression in the cell, the volume of the cell or the cell compartment, i.e. the sensor provides a ratiometric measurement of pH.

[62] In some embodiments, the sensor signal increases by more than 1.5 times when the pH changes from 7.0 to 7.5, more often more than 2 times, for example, 2.1 or 2.2 times.

[63] In some embodiments, the claimed biosensors are bright, where bright means that fluorescence of biosensors can be detected by conventional methods (eg, visual screening, spectrophotometry, spectrofluorimetry, fluorescence microscopy, FACS devices, etc.) In some embodiments, the claimed proteins have a fluorescence brightness in the range from about 1.8 to 4.5, usually from about 2.0 to 3.8, and most often from about 2.3 to 2.7 at a pH of 7.0.

[64] Specific biosensors of interest include a biosensor having the amino acid sequence of SEQ ID No: 4 and its functional chimeric proteins having the amino acid sequences of SEQ ID No: 6 and SEQ ID No: 8.

[65] Functional biosensors are also provided that are substantially similar to the biosensor above, 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 typically 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 )

[66] 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 purified at least 95%, usually at least 97%, and more often at least 99%.

[67] 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.

[68] Transformants

[69] The nucleic acids of the present invention can be used to obtain transformers, 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 may 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.

[70] 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) 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).

[71] 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, 2nd edition (1989) Cold Spring Harbor Laboratory Press and Ausubel et al., Current Protocols in Molecular Biology (1995) John Wiley & Sons, Inc).

[72] 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 EF Walton and GT 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.

[73] In another embodiment, animals may be transgenic organisms. 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, 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.

[74] 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.

[75] 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. When isolated protoplasts are present, it is possible to introduce through DNA-mediated gene transfer protocols, including incubating protoplasts with purified DNA, such as a plasmid containing the desired exogenous sequence of interest in the presence of polyvalent cations (e.g., 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 and ratios 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 an Agrobacterium-mediated transformation.

[76] Uses

[77] The biosensors of the present invention are genetically encoded fluorescent proteins that change spectral properties when the pH of the medium changes. They can be used to monitor changes in pH inside living cells during various biological processes. Due to the high brightness of fluorescence, the biosensors of the present invention can be used in live sections of the brain and studies of pH dynamics during spontaneous electrical activity in certain cell compartments in neuron culture and in short-lived sections of the brain.

[78] 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 vector may contain elements that provide targeted delivery of the structure to the cells of interest, or is part of the particles that provide targeted delivery. After transfection of the cells with the vector and after the time required for the expression product to develop in the cells, the pH changes in these cells can be monitored.

[79] The biosensors of the present invention can also be used in the screening of drugs that trigger the activation of certain protein cascades, such as 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.

[80] The claimed biosensors also find use as in vivo pH change labels for transgenic animals. For example, expression of a protein of interest may be accompanied by tissue-specific promoters, where such methods are used in studies for gene therapy, such as testing the effectiveness of transgenic expression. A typical application of fluorescent proteins in transgenic animals, which explains such applications, is described in W000 / 02997.

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

5. Examples

[82] Example 1

[83] Obtaining the SypHer2 biosensor.

[84] To obtain the nucleic acid encoding the SypHer protein, the nucleic acid sequence encoding HyPer from the commercially available pHyPer-cyto vector (Eurogen, Russia), the C199S was replaced by directional mutagenesis using “overlap extention” PCR products as described by Wurch et al., Methods in Molecular Biology. 12 (9), 653-657 (2004). For PCR, the Tersus PCR kit (Eurogen) was used according to the manufacturer's instructions. The resulting nucleotide sequence was cloned into a pHyPer-C vector using restriction enzymes NheI and HindIII instead of the previously contained nucleic acid encoding HyPer. The correctness of the obtained sequence (SEQ ID No: 1) was confirmed by sequencing.

[85] The obtained nucleic acid was subjected to targeted mutagenesis as described above, introducing point substitutions that could potentially affect the fluorescence brightness. The following options were checked:

[86] 1) A combination of amino acid substitutions 64F + 65G + 222E and 64F + 65S + 222Q, leading to an increase in the brightness and photostability of the yellow fluorescent protein YFP (amino acid numbering according to the sequence wtGFP, SEQ ID NO: 9);

[87] 2) Single mutations that increase the fluorescence brightness of a number of fluorescent proteins, including avGFP, selected from F64L, E222Q, and S65T (amino acid numbering according to the wtGFP sequence);

[88] 3) Single mutations in the OxyR domain sequence that increase the dynamic range of the HyPer biosensor selected from A406V (Markvicheva et al., 2011, Bioorganic & medicinal chemistry 19: 3, 1079-84) and H36Y (Bilan at al., ACS Chem. Biol., 2013, 8 (3), pp 535-542) and their combination (A406V + H36Y) (amino acid numbering according to the sequence SypHer, SEQ ID NO: 2).

[89] Substitutions were made using the “overlap extention” of PCR products as described by Wurch et al., Methods in Molecular Biology. 12 (9), 653-657 (2004). For PCR, the Tersus PCR kit (Eurogen) was used according to the manufacturer's instructions. The resulting nucleotide sequence was cloned into a pHyPer-C vector using the restriction enzymes Nhel and HindIII instead of the previously contained nucleic acid encoding HyPer. The correctness of the obtained sequences was confirmed by sequencing.

[90] Example 2

[91] Measurement of the dynamic range of the obtained biosensor options

[92] Expression vectors containing the coding sequences of the biosensor variants were obtained as described in Example 1. They were used to transfect cells of two cell lines HeLa Kyoto and NIH-3T3.

[93] Cells were cultured in DMEM medium supplemented with 10% FBS at 37 ° C in 5% CO ₂ incubator. Cells were plated on 35 mm plates and after 24 hours they were transfected with a mixture of DNA and x-tremeGENE 9 (Roche, Switzerland) according to the manufacturer's instructions.

[94] A study of the change in fluorescence as a function of pH was carried out 24 hours after transfection. Fluorescence changes were recorded on a Leica6000 epifluorescence microscope equipped with a 20x air lens and an NLC PL APO lbd. BL 63 × 1.4NA oil lens. Fluorescence was excited using sequentially bandpass filters 427/10 and 504/12; indicator emission was detected every 3 seconds using a 525/50 bandpass filter.

[95] To analyze the pH sensitivity, buffer solutions containing ionophores nigericin and monensin were used with a pH range of 6.0–9.5.

[96] Of all the tested variants, only the variant containing the single replacement A406V (SypHer2, SEQ ID NO: 4) had an increased fluorescence brightness. The influence of the remaining mutations was either insignificant or led to a decrease in the dynamic range of the sensor.

[97] SypHer2 showed a similar pH sensitivity to SypHer (Fig. 1), but has a 2-3 times greater brightness of the fluorescence of the cell culture (Fig. 2). The SypHer2 signal increases approximately 3 times with an increase in pH from 6.0 to 9.0, has a linear portion in the pH range of 7.5–8.5, and a pK value of approximately 8.1.

[98] Example 3

[99] Investigation of spontaneous pH oscillations in a primary culture of neurons

[100] To record pH changes during spontaneous activity, the primary neuron culture was transfected with a mixture of Rgeco and SypHer2 plasmids. Spontaneous activity was stimulated in a 15-20-day culture by the addition of 10 μM bicucullin, an inhibitor of GABA neuron receptors.

[101] A plasmid for the expression of SypHer2 was obtained as described in Example 1. A plasmid for expression of the Rgeco calcium sensor was purchased from Addgene (USA). To obtain a mixed primary culture of hippocampal neurons, a 1/6 C57B mouse was used on the 17th day of pregnancy. All procedures for the isolation of embryonic tissues were performed on ice. Embryos were anesthetized in ice-cold HBSS with a low Mg ²⁺ and Ca ²⁺ content, the brain was extracted, the hemispheres were separated, separated from the meninges and the hippocampi were collected in HBSS. The hippocampi were then washed in HBSS and incubated in 0.5% trypsin-EDTA for 15 minutes at 37 ° C. At the end of the incubation, the hippocampi were washed three times in warm DMEM supplemented with 10% FBS and 4 mM glutamine and carefully broken into cells by collecting and dropping into a 1 ml tip of an automatic pipette 10 times. The cells were placed in the center coated with poly-D-lysine 35 mm glass bottom plates density of 15 × 10 ⁴ cells in 100 ul of medium. 1-2 hours later, medium was replaced with 2 ml of Neurobasal medium supplemented with B27, 10% inactivated serum and 2 mM GlutaMax. Every 2-3 days, 1/3 of the volume of the medium was replaced with fresh neurobasal medium.

[102] On the 5th day in culture, cells were transfected with a Ca-phosphate transfection kit. 1 hour before transfection, the culture medium was taken into a 50 ml tube (conditioned medium), and 1.5 ml of pure DMEM was added to the cell culture. For each 35 mm dish, 100 μl of a DNA-CaCl ₂ mixture was added dropwise to 100 μl of 2xHBSS while mixing the tube with HBSS at low speed. After a 25 minute incubation, 200 μl of the solution was added to the plates, which were incubated for 15 minutes in a 5% CO ₂ incubator. Upon completion of the transfection, the neurons were washed twice with DMEM, then 2 ml of conditioned neurobasal medium was added.

[103] For fluorescence analysis, transfected cells of the primary hippocampal culture were transferred to Tyrode medium supplemented with 20 mM D-glucose and 20 mM Hepes and taken at 37 ° C using a Leica6000 epifluorescence microscope equipped with a 20x air lens and an NLC PL APO lbd. BL 63 × 1.4NA oil lens. SypHer fluorescence was excited using sequential bandpass filters 427/10 and 504/12; Indicator fluorescence was detected every 3 seconds using a 525/50 band pass filter. To excite fluorescence, RGeco and BCECF used a 561/10 band excitation filter and a 610/50 emission filter.

[104] The obtained images were analyzed in the Image J. software. To calculate the dynamics of the pH value, the images obtained by excitation of SypHer fluorescence with 420 nm and 500 nm light, after subtraction of the background, were converted to 32 bit format. For images corresponding to irradiation of 420 nm, a noise filtering threshold was set, the background signal was equal to the value of NaN. At each point, the average signal level in the selected area inside each individual cell was measured, then for each frame the values obtained by irradiation with light of 500 nm were divided into corresponding 420 nm images.

[105] The calibration of the pH sensor inside the cells was performed as indicated by Poburko et al. (Poburko et al., The Journal of biological chemistry. 2011, V. 286, p. 11672-84). The high K ⁺ buffer contained 5 μM nigericin, 5 μM monensin, 130 mm potassium gluconate, 20 mm sodium gluconate, 0.5 mm MgCl ₂ , 0.2 mm EDTA and 30 mm sodium borate (pH 9.5), Tris (pH 8.2-9.0) , HEPES (pH 7.0, 7.5), or MES (pH 6.0, 6.5).

[106] Spontaneous pH fluctuations in the culture were recorded (Fig. 3A). Parallel measurements of pH (SypHer2) and Ca ²⁺ (RGeco) showed that calcium entry into the cell is accompanied by rapid acidification of the cytoplasm (from about 7.4 to 7.35 pH units), after which a slow restoration of the initial pH value occurs. This allows us to conclude that pH fluctuations are caused by spontaneous electrical activity in a culture of neurons.

[107] In order to find out whether the pH fluctuations are directly related to calcium entry into the cell, we used media prepared with the addition of 2 mM CaCl ₂ or with the addition of 0.5 mM intracellular Ca ²⁺ BAPTA-AM chelator. It was shown that in the medium into which BAPTA-AM was added, there is no calcium entry into the cell, and pH fluctuations also disappear (Fig. 3B). The addition of a calcium transport inhibitor, 100 μM lanthanum chloride, to the cell medium caused a slight drop in the amplitude of the pH oscillations. All this allows us to talk about the participation of Ca ²⁺ transport systems in the acidification of the cytoplasm during spontaneous activity.

[108] Since the main events of signal transmission between neurons occur in synapses, it was decided to study the dynamics of pH values in these structures. For this, constructs were created in which the coding sequence of SypHer2 was quickly fused to the coding sequence of the postsynaptic density protein Homer1 (SEQ ID NOs: 7, 8) and the coding sequence of the synaptic vesicle protein Synapthysin (SEQ ID NOs: 5, 6), providing presynaptic localization . Expression of the obtained constructs on the culture of neurons showed that during the spontaneous activity of neurons, the pH in the postsynaptic end changes by 0.1 pH unit (7.4 to 7.3). In this case, the pH in the postsynaptic ending changes by 0.05 units, as in the cytoplasm.

[109] Example 4

[110] Study of the dynamics of pH values in the culture of sections

[111] To ensure that the Sypher2 pH indicator can be used in tissue culture, a cytoplasmic version of the sensor was expressed in brain sections of 10-day-old mice.

[112] Brain sections of a 10-day-old mouse CD57B 1/6 300 μm thick were obtained on a Leica VT1200S vibrotome in ice-cold ACSF (KC1, 2.5 mM; MgSO ₄ , 1.0 mM; NaH ₂ PO ₄ , 1.25 mM; NaHCO ₃ , 26 mM; D-glucose, 20 mM; CaCl ₂ , 2.0 mM and NaCl, 125 mM, pH 7.4). Sections were placed in a warm ACSF medium at 37 ° C, aerated with a mixture of 95% O ₂ + 5% CO ₂ . After 5-10 minutes, a ballistic transformation was performed using the Helios Gene Gun; the sections were incubated overnight (10-12 hours). The transformation and preparation of gold particles coated with plasmid pCl-SyPher2, was performed according to the manufacturer's protocol.

[113] Visualization, section cultures were performed on a Zeiss LSM 5 LIVE microscope equipped with a Zeiss ACHROPLAN 40x / 0.80w lens and a 488 nm laser, in a perfusion chamber in an aerated ACSF medium at 37 ° C.

[114] The indicator signal in individual neurons was detected already 10 hours after transformation (Fig. 4). To stimulate spontaneous neuronal activity, 30 mM KCl was added to the perfusion medium. This led to spontaneous pH fluctuations, similar to those observed in the primary culture of neurons (Fig. 5).

[115] The experiments performed show that the brightness and maturation rate of SypHer2 is sufficient to study the dynamics of pH changes in cultures of sections of mammalian brain sections.

Claims (3)

1. Nucleic acid encoding a fluorescent biosensor for detecting pH changes, the amino acid sequence of which is shown in SEQ ID No: 4. 2. The expression cassette, integrated into the genome of the cell or when introduced into the cell as an extrachromosomal element, is capable of ensuring the expression of the fluorescent biosensor according to claim 1 under the control of regulatory elements necessary for expression of the nucleic acid in the host cell. 3. A eukaryotic cell producing a biosensor encoded by a nucleic acid according to claim 1, containing an expression cassette according to claim 2 in the form of an extrachromosomal element or an element integrated into the genome of this cell.

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