Mutant of fluorescent protein and application thereof

Abstract

The application discloses a mutant of fluorescent protein and application thereof, and belongs to the field of cell biology, chemical biology and molecular biology. The application provides a mutant of fluorescent protein, wherein compared with a wild-type mEYFP fluorescent protein sequence, the mutant comprises a mutation at at least one position corresponding to positions 47, 154, 164 and 176; and the mutant is sensitive to fluorescence lifetime of LLPS or liquid-solid phase transition, or even to both processes, by modifying the wild-type mEYFP.

Description

TECHNICAL FIELD

[0001] The present application relates to a mutant of fluorescent protein and application thereof, in particular to a fluorescent protein for detecting phase separation or phase change and application thereof, and belongs to the field of cell biology, chemical biology and molecular biology. BACKGROUND

[0002] Liquid-liquid phase separation (LLPS) of biomolecules is a fundamental cellular process that is essential for maintaining homeostasis and facilitating biochemical activities. On the other hand, abnormal phase separation can change the fluidity of condensates and cause a transition from liquid-like condensates to solid-like condensates, which can lead to the formation of pathological aggregates commonly seen in neurodegenerative diseases such as Alzheimer's disease and amyotrophic lateral sclerosis. Therefore, being able to quickly and real-time detect such changes in phase separation is of great significance for studying the impact of phase separation on biological functions, especially for exploring the mechanisms of neurodegenerative diseases and clinical detection. The fluidity of condensates is usually evaluated by fluorescence recovery after photobleaching (FRAP), but this method requires high-intensity laser photobleaching, which has a greater impact on the organism. At the same time, due to the need for photobleaching and post-bleaching signal collection, it is difficult to perform real-time dynamic detection of multiple targets. Fluorescence lifetime is defined as the average residence time of a fluorophore in an excited state, usually in the nanosecond range. Fluorescence lifetime is sensitive to the local environment of the fluorophore, but not to the concentration of the fluorophore and the intensity of illumination. Fluorescence lifetime imaging microscopy (FLIM) using organic fluorophores has been used to probe the microenvironment within protein condensates, including polarity and viscosity, ionic effects within condensates, and aggregation caused by protein misfolding. Forster resonance energy transfer fluorescence lifetime microscopy (FRET-FLIM) has also been used to visualize the binding of regulatory proteins in microtubule-associated protein tau (MAPT, simply Tau) condensates. Although the fluorescent protein CFP has been used in FRET-FLIM experiments to reveal the molecular packing density of Tau in cells, it is still unknown whether the fluorescence lifetime of genetically encoded fluorescent proteins can be used to detect LLPS and liquid-solid phase transitions. SUMMARY

[0003] The invention addresses the problem

[0004] Due to technical limitations, the traditional coacervate FRAP detection method needs to use high-intensity laser for photobleaching, which has a greater impact on the organism, the fluorescence lifetime is not sensitive to the concentration of the fluorophore and the illumination intensity, and it is still not possible to quickly and real-time detect the phase separation change, and it is still unknown whether the fluorescence lifetime of the genetically encoded fluorescent protein can be used to detect LLPS and liquid-solid phase transition.

[0005] In view of this, the present application develops a new fluorescent protein with sensitive fluorescence lifetime to phase separation by mutating the key amino acids of wild-type mEYFP.

[0006] The solution to the problem

[0007] [1]. A fluorescent protein mutant, wherein the fluorescent protein mutant is selected from any one of the group consisting of (i)-(v):

[0008] (i) the mutant comprises a mutation at at least one position corresponding to positions 47, 154, 164, 176 of the sequence set forth in SEQ ID NO: 1, as compared to the sequence set forth in SEQ ID NO: 1, and the change in fluorescence signal of the mutant is more sensitive to liquid-liquid phase separation and / or liquid-solid phase transition of biological molecules, as compared to the fluorescent protein set forth in SEQ ID NO: 1;

[0009] (ii) a mutant having at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, most preferably at least 99% sequence identity to the amino acid sequence set forth in (i), and excluding the mutation of the sequence set forth in SEQ ID NO: 1;

[0010] (iii) a mutant encoded by a polynucleotide that hybridizes under very high stringency conditions to a polynucleotide set forth in (a) or (b):

[0011] (a) a polynucleotide encoding a mutant of the amino acid sequence set forth in (i);

[0012] (b) a polynucleotide complementary in whole length to (a);

[0013] (iv) a fragment of the mutant set forth in any one of (i), (ii) or (iii), and the change in fluorescence signal of the fragment still has improved sensitivity to liquid-liquid phase separation and / or liquid-solid phase transition of biological molecules;

[0014] (v) a polypeptide in which one or more amino acids are added to or deleted from at least one of the N-terminus and the C-terminus of the polypeptide of the amino acid sequence set forth in (i), (ii), (iii) or (iv);

[0015] Preferably, the fluorescent protein mutant has a mutation of amino acid at at least one of the following positions:

[0016] F47L, M154P, V164G, S176G.

[0017] [2]. The fluorescent protein mutant according to [1], wherein the fluorescent protein mutant corresponds to the sequence set forth in SEQ ID NO: 1, has a mutation as set forth in (i)-(v):

[0018] (i) F47L, M154P, V164G, S176G;

[0019] (ii) F47L;

[0020] (iii) M154P;

[0021] (iv) V164G;

[0022] (v) S176G.

[0023] [3]. The fluorescent protein mutant according to [1] or [2], wherein the fluorescent protein mutant comprises a polypeptide as set forth in any one of SEQ ID NOs: 2-6, or a polypeptide having at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, most preferably at least 99% sequence identity to any one of SEQ ID NOs: 2-6.

[0024] [4]. A fusion protein, wherein the fusion protein comprises the fluorescent protein mutant according to any one of [1]-[3], and a biological molecule to be detected, preferably the biological molecule to be detected comprises a protein.

[0025] [5]. An isolated polynucleotide, wherein the polynucleotide comprises a sequence encoding the fluorescent protein mutant according to any one of [1]-[3] or a sequence encoding the fusion protein according to [4].

[0026] [6]. An expression vector, wherein the expression vector comprises the isolated polynucleotide according to [5].

[0027] [7]. A recombinant host cell, wherein the recombinant host cell comprises the fluorescent protein mutant according to any one of [1]-[3], the fusion protein according to [4], the isolated polynucleotide according to [5], or the expression vector according to [6].

[0028] [8]. A method for detecting liquid-liquid phase separation and / or liquid-solid phase transition of a biomolecule, wherein the method uses the fluorescent protein mutant according to any one of [1]-[3], the fusion protein according to [4], the isolated polynucleotide according to [5], the expression vector according to [6], or the recombinant host cell according to [7].

[0029] [9]. The method according to [8], wherein the method detects liquid-liquid phase separation and / or liquid-solid phase transition of a biomolecule using a change in fluorescent signal of the fluorescent protein mutant, wherein the change in fluorescent signal comprises at least one of a change in fluorescence intensity, a change in fluorescence lifetime, a change in fluorescence polarization, a change in fluorescence spectrum.

[0030] Preferably, the change in fluorescent signal comprises a change in fluorescence lifetime.

[0031]

[10] . Use of the fluorescent protein mutant according to any one of [1]-[3], the fusion protein according to [4], the isolated polynucleotide according to [5], the expression vector according to [6], or the recombinant host cell according to [7] in any one of the following:

[0032] (1) use in preparing a reagent or a kit for detecting liquid-liquid phase separation and / or liquid-solid phase transition of a biomolecule;

[0033] (2) use for detecting liquid-liquid phase separation and / or liquid-solid phase transition of a biomolecule.

[0034] Effects of the invention

[0035] The present application studies the sensitivity of the fluorescence lifetime of fluorescent proteins to LLPS and liquid-solid phase transition, identifies several key residues that can be used to adjust the sensitivity of the fluorescence lifetime to phase separation, and modifies the wild-type mEYFP which is insensitive to the fluorescence lifetime of phase separation by using these identified residues, and invents a new mEFYP fluorescent protein mutant which is sensitive to the fluorescence lifetime of phase separation. BRIEF DESCRIPTION OF DRAWINGS

[0036] Figure 1 A in FIG. 1 is that the fluorescence lifetime of wild-type mEYFP is not sensitive to phase transition;

[0037] Figure 1 A in FIG. 1 is that the fluorescence lifetime of wild-type mEYFP is not sensitive to phase transition; Figure 1 B in FIG. 1 is a starburst formed by G156E (electron microscopy picture), wherein, in Figure 1 In B in FIG. 1, the left blue box is the center part, and the red box is the fiber part. In Figure 1B in FIG. 1A, the two right-hand side figures show the enlarged center portion and the fiber structure.

[0038] Figure 2 The sensitivity of LLPS and liquid-solid phase transition detection for different mEYFP mutants. Among them:

[0039] Figure 2 A in FIG. 1A is a mutant schematic diagram of mEYFP; Figure 2 B in FIG. 1A is the sensitivity of fluorescence lifetime of different mEYFP mutants to LLPS and liquid-solid phase transition detection. DETAILED DESCRIPTION

[0040] Various illustrative embodiments, features and aspects of the present application are described in detail below. The word "exemplary" is used herein to mean "serving as an example, instance, or illustration." Any implementation described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other implementations.

[0041] In addition, in order to better illustrate the present application, numerous specific details are given in the following detailed description. Those skilled in the art will understand that the present application can be practiced without certain specific details. In some instances, well-known methods, means, instruments and steps have not been described in detail in order to highlight the principles of the present application.

[0042] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The meaning of "a", "an", and "the" includes plural references unless otherwise indicated. The meaning of "in" includes "in" and "on" unless otherwise indicated. The meaning of "including" and "comprising" includes "open" terms such as "comprising" and "including" but also "closed" terms such as "consisting of" and "consisting essentially of".

[0043] In this specification, the meaning expressed by "may" includes both the meaning of performing a certain process and the meaning of not performing a certain process.

[0044] In this specification, "some specific / preferred embodiments / implementations", "other specific / preferred embodiments / implementations", "embodiments", "implementations" and the like refer to the specific elements (e.g. features, structures, properties and / or characteristics) described in relation to the embodiments / implementations, which are included in at least one embodiment / implementation described herein, and can or can not be present in other embodiments / implementations. In addition, it should be understood that the elements can be combined in various embodiments / implementations in any suitable manner.

[0045] In this specification, the numerical range expressed by "numerical value A to numerical value B" refers to a range including the end point values A and B.

[0046] In the present specification, the terms "polypeptide", "peptide", and "protein" are used interchangeably herein and refer to a polymer of amino acids of any length. The polymer can be linear or branched, it can comprise modified amino acids, and it can be interrupted by non-amino acids. The term also encompasses an amino acid polymer that has been modified, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component.

[0047] In the present specification, the term "wild type" refers to a naturally occurring amino acid or nucleotide sequence that can be found directly in nature and has not been modified by man. The term "mutant" refers to an amino acid or nucleotide sequence in which there is one or more substitutions, deletions or insertions compared to the naturally occurring amino acid or nucleotide sequence. As used herein, "naturally occurring" and "wild type" are synonymous.

[0048] In the present specification, the term "mutant" refers to a polynucleotide or polypeptide that comprises an alteration (i.e., a substitution, insertion, and / or deletion) at one or more (e.g., several) positions relative to a "wild type" or "compared" polynucleotide or polypeptide, wherein a substitution refers to the replacement of a nucleotide or amino acid occupying a position with a different nucleotide or amino acid. A deletion refers to the removal of a nucleotide or amino acid occupying a position. An insertion refers to the addition of a nucleotide or amino acid after the nucleotide or amino acid occupying a position, adjacent and immediately following.

[0049] In the present specification, the term "amino acid mutation" or "nucleotide mutation" includes "a substitution, duplication, deletion, or addition of one or more amino acids or nucleotides". In the present invention, the term "mutation" refers to an alteration of a nucleotide sequence or an amino acid sequence.

[0050] In some embodiments, the "mutation" of the present invention can be selected from "conservative mutations". In the present invention, the term "conservative mutation" refers to a mutation that can normally maintain the function of a protein. A representative example of a conservative mutation is a conservative substitution.

[0051] In the present specification, the term "conservative substitution" relates to the replacement of an amino acid residue by an amino acid residue with similar side chain. Families of amino acid residues with similar side chains have been defined in the art and include those with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan), beta-branched side chains (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine).

[0052] In the present specification, the term "sequence identity" or "percent identity" in the comparison of two nucleic acids or polypeptides means that they are identical or have a certain percentage of identical sequence when compared and aligned for maximum correspondence, using a nucleotide or amino acid residue sequence comparison algorithm or by visual inspection. That is, the identity of a nucleotide or amino acid sequence can be defined using the proportion of nucleotides or amino acids that are identical over the length of two or more nucleotide or amino acid sequences aligned for maximum correspondence, and gaps introduced to achieve maximum correspondence, as necessary. The proportion is the fraction of nucleotides or amino acids that are identical over the total number of nucleotides or amino acids in the aligned portion.

[0053] According to the present application, "moderate to very high stringency conditions" include "moderate stringency", "moderate-high stringency", "high stringency" or "very high stringency", which describe conditions for nucleic acid hybridization and washing. Guidance in the choice of hybridization and wash conditions can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6, which is incorporated herein by reference. Both aqueous and nonaqueous methods are described in this document, and either can be used. For example, specific hybridization conditions are as follows: (1) low stringency hybridization conditions in 6x sodium chloride / sodium citrate (SSC) at about 45°C, followed by at least one wash in 0.2x SSC, 0.1% SDS at at least 50°C (for low stringency conditions, the wash temperature can be increased to 55°C); (2) moderate stringency hybridization conditions in 6x SSC at about 45°C, followed by one or more washes in 0.2x SSC, 0.1% SDS at 60°C; (3) high stringency hybridization conditions in 6x SSC at about 45°C, followed by one or more washes in 0.2x SSC, 0.1% SDS at 65°C and preferably; (4) very high stringency hybridization conditions are 0.5M sodium phosphate, 7% SDS at 65°C, followed by one or more washes in 0.2x SSC, 1% SDS at 65°C.

[0054] In the present specification, the term "expression" includes any step involved in the production of a polypeptide including, but not limited to: transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0055] In the present specification, the term "expression vector" refers to a DNA construct containing a DNA sequence operably linked to suitable control sequences, so as to direct the expression of a gene of interest in a suitable host. A "recombinant expression vector" refers to a DNA construct used to express, for example, a polynucleotide encoding a desired exogenous polypeptide. A recombinant expression vector can include, for example, a transcriptional unit comprising i) a collection of genetic elements having a regulatory role in gene expression, such as promoters and enhancers; ii) a structural or coding sequence that is transcribed into mRNA and translated into a protein; and iii) appropriate transcriptional and translational initiation and termination sequences. The recombinant expression vector is constructed in any suitable manner. The nature of the vector is not critical and any vector, including plasmid, virus, bacteriophage, and transposon, can be used. Possible vectors for use in the present application include, but are not limited to, chromosomal, non-chromosomal, and synthetic DNA sequences, such as bacterial plasmids, bacteriophage DNA, yeast plasmids, and vectors derived from combinations of plasmids and bacteriophage DNA, DNA from viruses such as vaccinia, adenovirus, fowlpox, baculovirus, SV40, and pseudorabies.

[0056] In the present specification, the terms "isolated" and "purified" are used to refer to a molecule (e.g., an isolated nucleic acid, polypeptide, etc.) or other component that is removed from at least one other component with which it is naturally associated. The term "purified" does not require absolute purity; rather, it is intended as a relative term.

[0057] In the present specification, the term "liquid-liquid phase separation" or "LLPS" refers to the intracellular or in vitro biological macromolecules (such as proteins, RNAs) through interactions, forming phase-separated droplets with different components and properties.

[0058] In the present specification, the term "liquid-solid phase transition" refers to the intracellular or in vitro biological macromolecules (such as proteins, RNAs) through interactions, forming phase-separated droplets with different components and properties and coagulating into solid matter.

[0059] Unless otherwise defined or indicated by context, all technical and scientific terms used in the present application have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0060] The technical solutions of the present invention are described in detail as follows:

[0061] <First aspect>

[0062] In the first aspect of the present application, a fluorescent protein mutant sensitive to at least one of phase separation (e.g., liquid-liquid phase separation) and phase change (e.g., liquid-solid phase transition) of biological molecules is provided.

[0063] In some embodiments, the mutant has a higher sensitivity of change in fluorescence signal to liquid-liquid phase separation and / or liquid-solid phase transition of biomolecules compared to the wild-type fluorescent protein mEYFP as shown in SEQ ID NO: 1.

[0064] In some embodiments, the fluorescent protein mutant is selected from any one of the group consisting of (i)-(v):

[0065] (i) the mutant comprises a mutation at at least one position corresponding to positions 47, 154, 164, 176 of the sequence as shown in SEQ ID NO: 1 compared to the sequence as shown in SEQ ID NO: 1, and the mutant has a higher sensitivity of change in fluorescence signal to liquid-liquid phase separation and / or liquid-solid phase transition of biomolecules compared to the fluorescent protein as shown in SEQ ID NO: 1.

[0066] (ii) a mutant having at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, most preferably at least 99% sequence identity with the amino acid sequence as shown in (i) and excluding the sequence as shown in SEQ ID NO: 1;

[0067] (iii) a mutant encoded by a polynucleotide which hybridizes under very high stringency conditions with a polynucleotide as shown in (a) or (b):

[0068] (a) a polynucleotide encoding a mutant as shown in (i);

[0069] (b) a polynucleotide which is the full-length complement of (a);

[0070] (iv) a fragment of a mutant as shown in any one of (i), (ii) or (iii), and the fragment still has an improved sensitivity of change in fluorescence signal to liquid-liquid phase separation and / or liquid-solid phase transition of biomolecules;

[0071] (v) a polypeptide having one or more amino acids added to or deleted from at least one of the N- and C-termini of a polypeptide as shown in (i), (ii), (iii) or (iv);

[0072] In some preferred embodiments, the fluorescent protein mutant corresponds to the sequence as shown in SEQ ID NO: 1, having a mutation at at least one of the following positions: F47L, M154P, V164G, S176G.

[0073] In some specific embodiments, the fluorescent protein mutant corresponds to the sequence as shown in SEQ ID NO: 1, having a mutation as shown in (i)-(v):

[0074] (i) F47L, M154P, V164G, S176G (in the embodiments of the present application, it is FLmEYFP, and its amino acid sequence is shown as SEQ ID No. 2);

[0075] (ii) F47L (in the embodiments of the present application, it is mEYFP F47L or FLSmEYFP, and its amino acid sequence is shown as SEQ ID No. 3);

[0076] (iii) M154P (in the embodiments of the present application, it is mEYFP M154P , and its amino acid sequence is shown as SEQ ID No. 4);

[0077] (iv) V164G (in the embodiments of the present application, it is mEYFP V164G , and its amino acid sequence is shown as SEQ ID No. 5);

[0078] (v) S176G (in the embodiments of the present application, it is mEYFP S176G , and its amino acid sequence is shown as SEQ ID No. 6).

[0079] In some embodiments, the change in the fluorescence signal of the fluorescent protein mutant comprises at least one of a change in fluorescence intensity, a change in fluorescence lifetime, a change in fluorescence polarization, and a change in fluorescence spectrum.

[0080] In some specific embodiments, the change in the fluorescence signal is a change in fluorescence lifetime.

[0081] In some preferred embodiments, the fluorescent protein mutant corresponds to the sequence shown in SEQ ID NO: 1, and has the mutations shown in (i) and / or (ii). Specifically, FLSmEYFP is more sensitive to the liquid-solid phase transition (i.e., liquid- solid phase transition) of biomolecules than wild-type mEYFP in terms of fluorescence lifetime; and FLmEYFP is more sensitive to the LLPS and / or liquid-solid phase transition of biomolecules than wild-type mEYFP in terms of fluorescence lifetime.

[0082] The above mutation sites enable the fluorescent protein of the present application to have good sensitive fluorescence lifetime, and can quickly detect phase separation and phase transition.

[0083] <Second aspect>

[0084] In the second aspect of the present application, a fusion protein is provided, which comprises the fluorescent protein mutant of the first aspect of the present application, and a biomolecule to be detected.

[0085] In some preferred embodiments, the biomolecule to be detected comprises a protein.

[0086] In some exemplary embodiments, the biomolecule to be detected can be a biomolecule that can undergo LLPS and / or liquid-solid phase transition.

[0087] In some exemplary embodiments, the biomolecule to be detected can be a biomolecule (marker) that can undergo LLPS and / or liquid-solid phase transition in a neurodegenerative disease.

[0088] In some embodiments, the neurodegenerative disease comprises an acute neurodegenerative disease or a chronic neurodegenerative disease.

[0089] In some specific embodiments, the neurodegenerative disease comprises amyotrophic lateral sclerosis (ALS) or frontotemporal lobar degeneration (FTLD) disease.

[0090] In some specific embodiments, the biomolecule marker is Fused in sarcoma (FUS) or a mutant thereof associated with amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD).

[0091] In some embodiments, the fusion protein further comprises a tag.

[0092] In the present specification, the term "tag" refers to a short peptide that is fused or linked to a protein of interest (e.g., a fluorescent protein mutant of the present application or a fusion protein comprising the fluorescent protein mutant) and thereby facilitates the soluble expression, detection and / or purification of the recombinant protein. The tag can be fused or linked to the N-terminus and / or C-terminus of the protein of interest (optionally through a linker or a protease cleavage site), and in some embodiments, the tag can also be fused or linked to the interior of the protein of interest.

[0093] In some exemplary embodiments, the tag comprises a maltose binding protein (MBP) tag, and in some specific embodiments, the tag sequence is set forth in SEQ ID No. 9.

[0094] In some exemplary embodiments, the tag comprises a 6xHis tag.

[0095] According to the present application, the term "protease cleavage site" refers to a site that can be specifically recognized and cleaved by a protease. Various specific proteases and their recognition sites are well known to those skilled in the art and are found in many prior art documents. Those skilled in the art can use a suitable protease cleavage site in the fusion protein and cleave it with the corresponding protease according to the actual situation. The use of a protease cleavage site can be advantageous, for example, it can be used to remove the signal peptide and / or the tag from the fusion protein, thereby obtaining the mature protein with the desired activity.

[0096] In some specific embodiments, the protease cleavage site sequence is as shown in SEQ ID No. 10.

[0097] In some embodiments, the various parts in the fusion protein (such as the fluorescent protein mutant, the biological molecule to be detected, the tag, and the protease cleavage site, etc.) can be connected by an optional linker.

[0098] According to the present application, the term "linker" or "Linker" refers to a connection between two molecules or parts, for example, two domains of a fusion protein. Typically, the linker is located between or flanked by two groups, molecules or other parts, and is connected to each by a covalent bond, thereby connecting the two. In some embodiments, the linker is an organic molecule, group, polymer or chemical moiety. In some embodiments, the linker can be a polynucleotide. In some embodiments, the linker can be a DNA molecule. In some embodiments, the linker can be an RNA molecule. In some embodiments, the linker can include an aptamer capable of binding to a ligand. In some embodiments, the linker is an amino acid or a plurality of amino acids (such as a peptide or a protein).

[0099] <Third aspect>

[0100] In a third aspect of the present application, an isolated polynucleotide is provided, which encodes the fluorescent protein mutant of the first aspect of the present application or the fusion protein of the second aspect of the present application.

[0101] The polynucleotide of the present application can be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The DNA can be a coding strand or a non-coding strand.

[0102] The polynucleotide encoding the mutant or fusion protein of the present application includes: only the coding sequence of the mutant or fusion protein; the coding sequence of the mutant or fusion protein and various additional coding sequences; the coding sequence of the mutant or fusion protein (and optional additional coding sequences) and non-coding sequences.

[0103] <Fourth aspect>

[0104] In a fourth aspect of the present application, an expression vector is provided, which comprises the polynucleotide of the third aspect of the present application.

[0105] In some embodiments, the polynucleotide of the third aspect is operably linked to one or more heterologous regulatory sequences that control gene expression to produce a polynucleotide capable of expressing a polypeptide.

[0106] In some exemplary embodiments, the expression vector can be constructed by genetically engineering the expression vector to be linked to the biomolecule to be detected and / or the fluorescent protein mutant.

[0107] In some specific embodiments, the recombinant expression vector comprises a coding sequence of the fluorescent protein mutant, a coding sequence of the biomolecule to be detected (e.g., a protein to be detected), and a coding sequence of the tag.

[0108] <5th Aspect>

[0109] In a fifth aspect of the present application, a recombinant host cell is provided, wherein the recombinant host cell comprises the fluorescent protein mutant of the first aspect of the present application, the fusion protein of the second aspect of the present application, the isolated polynucleotide of the third aspect of the present application, or the expression vector of the fourth aspect of the present application.

[0110] In some alternative embodiments, an expression vector containing a polynucleotide encoding the fluorescent protein mutant or the fusion protein is introduced into a suitable host cell to express the corresponding fluorescent protein mutant or fusion protein.

[0111] <6th Aspect>

[0112] In a sixth aspect of the present application, a method for detecting liquid-liquid phase separation and / or liquid-solid phase transition of a biomolecule is provided, wherein the method uses the fluorescent protein mutant of the first aspect of the present application, the fusion protein of the second aspect of the present application, the polynucleotide of the third aspect of the present application, the expression vector of the fourth aspect of the present application, or the recombinant host cell of the fifth aspect of the present application.

[0113] In some embodiments, the method for detecting comprises labeling the biomolecule to be detected with the fluorescent protein mutant to form a fusion protein (as described in the second aspect above).

[0114] In some embodiments, the method for detecting utilizes a change in the fluorescent signal of the fluorescent protein mutant to detect the liquid-liquid phase separation and / or liquid-solid phase transition of the biomolecule, wherein the change in the fluorescent signal comprises at least one of a change in the fluorescence intensity, a change in the fluorescence lifetime, a change in the fluorescence polarization, and a change in the fluorescence spectrum.

[0115] In some preferred embodiments, the change of the fluorescent signal comprises a change of the fluorescent lifetime.

[0116] In some specific embodiments, the fluorescent protein mutant labels the biomolecule to be detected, and when the phase separation or phase transition occurs in the labeled biomolecule, the fluorescent lifetime of the fluorescent protein mutant changes, which can quickly and real-time detect the change of the state of the biomolecule to be detected.

[0117] In some exemplary embodiments, the fluorescent protein mutant labels the FUS mutant G156E protein, and when the FUS protein undergoes liquid-liquid phase separation and / or liquid-solid phase transition, the fluorescent lifetime of the fluorescent protein mutant changes, and by observing the fluorescent lifetime, further phase separation and / or phase transition information is obtained by mathematical fitting.

[0118] In some specific embodiments, the fluorescent lifetime can be observed by a confocal microscope and measured by a fluorescent lifetime imaging system and / or a bioluminescence resonance energy transfer system.

[0119] In some embodiments, the fluorescent lifetime imaging comprises using a fluorescent lifetime imaging microscope, such as a single photon counting confocal microscope, a single molecule time-resolved confocal fluorescence microscopy system, a FLIM (fluorescence lifetime imaging microscopy) system, etc.

[0120] In some embodiments, the bioluminescence resonance energy transfer system comprises a BRET (Bioluminescence Resonance Energy Transfer) system and a FRET (Fluorescence Resonance Energy Transfer) system.

[0121] <Seventh aspect>

[0122] In the seventh aspect of the present application, any one of the following uses of the fluorescent protein mutant of the first aspect of the present application, the fusion protein of the second aspect of the present application, the polynucleotide of the third aspect of the present application, the expression vector of the fourth aspect of the present application, the recombinant host cell of the fifth aspect of the present application, or the method of the sixth aspect of the present application is provided:

[0123] (1) a use for detecting liquid-liquid phase separation and / or liquid-solid phase transition of a biomolecule;

[0124] (2) a use for preparing a reagent or a kit for detecting liquid-liquid phase separation and / or liquid-solid phase transition of a biomolecule.

[0125] In some specific embodiments, FLSmEYFP can be used in the preparation of reagents and / or kits for the detection of biomolecules through liquid-liquid phase separation; and / or in the preparation of reagents and / or kits for the detection of biomolecules through liquid-solid phase transitions.

[0126] Example

[0127] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0128] Example 1

[0129] This invention investigated the sensitivity of the fluorescence lifetime of fluorescent proteins to LLPS and liquid-solid phase transitions, finding that the fluorescence lifetime of wild-type mEYFP is not sensitive to changes in phase separation (e.g., Figure 1 (As shown in Figure A). This invention uses a fusion protein (Fused in sarcoma, FUS) associated with amyotrophic lateral sclerosis (ALS) and frontotemporal degeneration (FTLD) as a model. The patient-derived FUS mutant G156E (abbreviated as FUS(G156E)) has been reported to have a stronger tendency to aggregate and accelerate the liquid-to-solid phase transition. Therefore, this invention uses the fluorescent protein fusion FUS(G156E) to test whether the lifetime of the fluorescent protein can reflect changes in at least one state of phase separation and phase transition when liquid-like condensates and solid-like condensates are fully formed. The specific experimental steps are as follows.

[0130] I. Experimental Materials

[0131] Tris-HCl (Sigma-Aldrich), KCl (Sigma-Aldrich), NaCl (Sigma-Aldrich), MgCl2 (Sigma-Aldrich), PMSF (Amresco), imidazole (Sigma-Aldrich), EGTA (Biodee), TCEP (Goldbio), glycerol (GPC biotech), BL21 (DE3) competent cells (Full Gold), TEV protease (laboratory purification).

[0132] II. Experimental Procedure

[0133] 1. Protein preparation steps

[0134] The specific acquisition process of the mEYFP and mutant protein labeled FUS (G156E) protein is as follows: taking the mEYFP labeled FUS (G156E) protein as an example, using the initial expression plasmid MBP-GFP-FUS (G156E) (provided by the laboratory of Professor Li Pulong of Tsinghua University), the mEYFP labeled FUS (G156E) protein expression plasmid MBP-mEYFP-FUS (G156E) is constructed by polymerase chain reaction (PCR) and homologous recombination: the amino acid sequence of the protein expressed is shown as SEQ ID No. 8, and after the sequence of the constructed plasmid is confirmed to be correct (Table 1), subsequent protein purification is carried out. The plasmid used to express the mEYFP labeled FUS (G156E) protein introduces a maltose binding protein (MBP) tag at the N terminus of the fluorescent protein, and introduces an amino acid sequence that can be recognized and cut by TEV protease between the fluorescent protein and the MBP tag, so as to realize the removal of the MBP tag in the subsequent experiment; the C terminus of the fluorescent protein is connected with the FUS (G156E) protein, and there is a 6×His tag between them for affinity purification with Ni-NTA agarose purification resin.

[0135] Table 1 Protein amino acid sequence

[0136]

[0137]

[0138]

[0139] (Note: the mutation site is represented by italic and underlined in the table)

[0140] (1) Preparation of solution: the buffer used in the protein purification process is as follows (Table 2), which is prepared in advance, filtered with a 0.22 μm filter membrane, and stored at 4°C. TCEP needs to be prepared and added to the buffer every time the protein is purified, to prevent oxidation and loss of the ability to reduce disulfide bonds in the protein due to long-term placement in the air.

[0141] Table 2 Buffer composition in protein purification process

[0142]

[0143] (2) Transformation induction: Transform the plasmid expressing the protein into BL21(DE3) competent cells, and use a coating rod to coat the LB solid medium plate with 100 μg / mL ampicillin, and place it in a 37°C incubator overnight. The next day, 5-10 single colonies are picked from the plate and placed in 5 mL of LB liquid medium containing 100 μg / mL ampicillin, and cultured in a 37°C shaker at 220 rpm for 5 h until the bacterial solution is turbid. Then, the whole bacterial solution is transferred to 1 L of LB liquid medium containing 100 μg / mL ampicillin, and cultured in a 37°C shaker at 220 rpm until the optical density (OD600) of the bacterial solution at 600 nm is in the range of 0.6-0.8. Then, the shaker and the bacterial solution are cooled to 16°C, and IPTG (Goldbio) is added to the bacterial solution to a final concentration of 0.5 mM. After 16-18 h of protein expression induction, the bacterial cells are collected by high-speed centrifugation, frozen in liquid nitrogen, and stored in a -80°C refrigerator for subsequent purification.

[0144] (3) Protein purification: The frozen bacterial cells are thawed on ice, resuspended with 40 mL of lysis buffer, and dissolved until no visible bacterial clumps are present. Then, the solution is transferred to a 50 mL beaker, and the bacterial solution is broken by an ultrasonic disrupter at 350 W power, working for 2 s and then pausing for 5 s, for a total of 30 min. The broken bacterial solution is transferred to a high-speed centrifuge tube, and centrifuged at 12000 rpm for 1 h at 4°C. After centrifugation, the supernatant is collected in a 50 mL centrifuge tube, and 1-2 mL of Ni-NTA agarose purification resin (Cytiva) washed with lysis buffer is added. The mixture is incubated at 4°C for 1.5-2 h, and then centrifuged at 5000 rpm for 10 min at 4°C. The Ni-NTA agarose purification resin combined with the target protein is separated and transferred to an empty gravity column, and the target protein is eluted with washing buffer and elution buffer in sequence. The eluted protein is collected. Then, the protein is purified by a chromatography column Superdex TM 200Increase 10 / 300 column (GE healthcare), and the chromatography column is eluted with 1 column volume (25 mL) of molecular sieve elution buffer. During the elution process, the protein flowing out is collected in different tubes according to the change in absorbance at 280 nm, and each tube of protein is identified by SDS-PAGE to determine the purity and size of the protein. The final selected protein is stored in the corresponding tube, which is identified according to the volume position of the protein eluted after the chromatography column is loaded. The protein is combined, concentrated to a final concentration of 50 μM by a 30K ultrafiltration centrifuge tube (Amicon-Ultra), and aliquoted at 5 μL per tube. The protein is frozen in liquid nitrogen and stored in a -80°C refrigerator, thereby obtaining the mEYFP-labeled FUS(G156E) protein.

[0145] 2. Liquid-liquid phase separation and liquid-solid phase transition steps

[0146] To initiate phase separation of mEYFP-tagged FUS(G156E) protein obtained in Step 1, take the aliquoted mEYFP-tagged FUS(G156E) protein, configure the phase separation reaction system (Table 3), mix uniformly, and add to a glass-bottom 384-well plate (Cellvis). At this time, the protein state is referred to as "before phase separation" or "before liquid-liquid phase separation". After the mEYFP-tagged FUS(G156E) protein before phase separation is left to stand at room temperature for 2 h, the mEYFP-tagged FUS(G156E) protein forms droplets (greater than 200 nm) that can be observed under an optical microscope. For experimental observation, the protein state at this time is referred to as "2-hour condensate". Subsequently, the glass-bottom 384-well plate containing the droplets of mEYFP-tagged FUS(G156E) protein that have undergone liquid-liquid phase separation (2-hour condensate) is fixed on a multi-purpose decolorization shaker, the rotation speed is adjusted to about 200 rpm / min, and the sample is subjected to 24 h horizontal shaking to obtain the condensate of solid mEYFP-tagged FUS(G156E) protein after liquid-solid phase transition, including 24-hour fiber-free condensate or 24-hour fibrous condensate (both are products obtained after liquid-solid phase transition, only different in morphology), which is used for subsequent experiments.

[0147] Table 3 FUS(G156E) liquid-liquid phase separation reaction system

[0148]

[0149]

[0150] 3. Data acquisition and analysis step

[0151] The FLIM data acquisition and analysis system (Picoquant, Symphotime) used in this experiment is used in conjunction with the FV1200 confocal microscope (Olympus), both of which use independent light paths and software. In the experiment, a 100x oil objective was selected on the FV1200 for imaging first, then switched to the FLIM data acquisition system for related data collection, and finally analyzed the data on the software. The software used for FLIM data acquisition and analysis is SymPhoTime 64, and the specific process is as follows: find the area and appropriate focal plane for FLIM observation on the FV1200, enlarge the target field of view for FLIM experiment to the appropriate size, take a picture of the target field of view with 512x512 pixels, and save the image. Then adjust the light path on the FV1200 software to the light path of the FLIM system, so that the emitted light does not pass through the confocal detector but directly enters the fluorescence lifetime detector. Then set the FLIM data acquisition parameters, change the imaging conditions on the FLIM software to match the image resolution on the FV1200, then optimize the FLIM acquisition conditions, and adjust the intensity of the laser by changing the decay of the laser to test the real-time TSCPC curve under the condition. Confirm that the laser intensity is appropriate and that the parameters are set correctly, turn on the laser key on the laser driver, click the control button on the FLIM software, select the target field of view for XY repeated scanning on the FV1200 software, start collecting fluorescence lifetime images, record enough photon counts, stop data collection, and perform subsequent data analysis on the obtained FLIM images.

[0152] 4. Agglomerate internal structure observation step

[0153] In order to confirm the internal structure of the liquid-solid phase transition of the mEYFP-labeled FUS(G156E) protein (i.e., 24-hour fibrous aggregates), a transmission electron microscope (Hitachi HT7800) was used for observation. The specific steps are as follows: The mEYFP-labeled FUS(G156E) protein liquid-liquid phase separation reaction system was prepared (the specific method is the same as that in step 2 above), and the liquid-solid phase transition experiment was performed by adding drops in the center of a single grid confocal culture dish (the specific method is the same as that in step 2 above). After imaging was performed using a laser confocal microscope FV1200, the corresponding area was marked on the dish bottom with a marker pen after the formation of fibrous aggregates. Then, an equal volume of 2.5(v / v) % glutaraldehyde fixing solution (source leaf biological) was added, and the sample was fixed at room temperature for 20 min. Subsequently, 1 mL of 2.5(v / v) % glutaraldehyde was added to fill the center area of the culture dish, and the sample was fixed at 4°C overnight for subsequent electron microscope sample preparation. The sample was embedded with resin, polymerized, and the target area was sectioned. The sample was picked up with a support net for staining, then washed with distilled water, and placed at room temperature for 5 h after excess water was absorbed. The sample was dried and ready for electron microscope observation. When observing the sample using a transmission electron microscope, the target structure to be photographed was first selected, and then the objective lens focal length and the electron beam spot size and brightness were adjusted for imaging and photographing at a voltage of 80 kV.

[0154] 5. Image processing steps

[0155] In the FLIM software, the image file to be processed was selected and opened using the image fitting program provided by the software. To improve the fitting quality, the image was merged by 2 times the number of pixels, and the image size became 256x256 pixels. The target area for which the fluorescence lifetime was to be analyzed was circled on the image, and fitting was performed. The parameters were set in the fitting area in the software, the fitting model was selected as "n-Exponential Reconvolution", the fitting object was selected as the circled target area, and the n value in the model was set to 1 for fitting. In the software, the TCSPC curve was displayed, and if the coincidence degree of the curve obtained by fitting and the TCSPC curve was not good, or the reference parameter χ 2 for fitting goodness was obviously not suitable (the closer to 1, the better the fitting quality), the n value was increased and the same fitting process was performed again until a better fitting result was obtained. In this application, when the n value was 2, a satisfactory fitting result was obtained, and the fluorescence lifetime τ AvInt (Intensity weighted average lifetime, hereinafter referred to as fluorescence lifetime or intensity weighted average lifetime) (Formula 1-1).

[0156]

[0157] Where τ AvInt τ represents the fluorescence lifetime based on the average fluorescence intensity, where I represents the fluorescence intensity per photon and τ represents the fluorescence lifetime. sum The total fluorescence intensity is represented by k, a specific photon is represented by n, and the total number of photons is represented by n.

[0158] Furthermore, the fluorescence lifetime of mEYFP or its mutants was calculated to be sensitive to liquid-liquid phase separation and to liquid-solid phase transition by measuring the ratio of fluorescence lifetimes of products with different flow rates. Specifically, the sensitivity of mEYFP or its mutants to liquid-liquid phase separation was calculated as the ratio of the 2-hour fluorescence lifetime of the mEYFP or its mutant-labeled FUS(G156E) protein aggregate to the fluorescence lifetime of the mEYFP or its mutant-labeled FUS(G156E) protein before phase separation. The sensitivity of mEYFP or its mutants to liquid-solid phase transition was calculated as the ratio of the fluorescence lifetime of the central portion of the 24-hour fibrillary aggregate of the mEYFP or its mutant-labeled FUS(G156E) protein aggregate to the 2-hour fluorescence lifetime of the mEYFP or its mutant-labeled FUS(G156E) protein aggregate. A sensitivity value closer to 1 indicates lower sensitivity.

[0159] III. Experimental Results

[0160] like Figure 1 As shown, the unmodified mEYFP-labeled FUS(G156E) protein under free diffusion before phase separation, 2 hours before phase separation, 24 hours before phase separation without fibrous aggregates, and 24 hours before phase separation with fibrous aggregates (i.e., starbursts with fibers formed by FUS(G156E), see [reference]). Figure 1 The intensity-weighted average lifetime (mean ± standard error, n>9, from three independent experiments) of B (which includes the central portion, referred to as the "24-hour center," and the surrounding fibers, referred to as the "24-hour fibers") was calculated. Statistical significance between the 24-hour center and 24-hour fibers was assessed using a paired two-tailed t-test, while other significance was assessed using an unpaired two-tailed t-test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. The fluorescence lifetime ratio of the 2-hour aggregate to the 24-hour center showed that the fluorescence lifetime change was smaller in the 2-hour aggregate compared to the 24-hour center. Figure 1 As can be seen, the fluorescence lifetime of the unmodified mEYFP is less sensitive to the change in fluorescence lifetime from liquid to solid state.

[0161] Example 2

[0162] This invention discovers a novel fluorescent protein whose fluorescence lifetime is sensitive to at least one of phase separation and phase transition by mutating key amino acids of wild-type mEYFP, using the modified mEYFP mutant to label the FUS(G156E) protein, and detecting fluorescence lifetime during phase separation and phase transition.

[0163] I. Experimental Procedures and Reagents

[0164] The specific experimental steps and reagents are similar to steps one and two in Example 1. The difference is that in the construction of the mEYFP-labeled FUS(G156E) protein expression plasmid MBP-mEYFP-FUS(G156E), mEYFP is replaced with the corresponding mEYFP mutant.

[0165] II. Experimental Results

[0166] This invention identified four residues affecting fluorescence lifetime sensitivity through analysis, corresponding to positions 47, 154, 164, and 176 in mEYFP (Table 1). To further verify whether these sites indeed affect lifetime sensitivity, this invention mutated four residues in mEYFP (…). Figure 2 A). Unlike the unmodified mEYFP-tagged FUS(G156E) protein (mEYFP-WT), mEYFP F47L / M154P / V164G / S176G The mutant (hereinafter referred to as FLmEYFP)-tagged FUS(G156E) protein is much more sensitive to LLPS and liquid-solid phase transitions. Figure 2 In the B group, the fluorescence lifetime decreased from 2.75 ± 0.01 ns in solution (before phase separation, i.e., before liquid-liquid phase separation) to 2.535 ± 0.003 ns in the liquid condensate (2-hour condensate), and further to 2.281 ± 0.005 ns in the center of the 24-hour fibrous condensate. When these residues were mutated one by one, mEYFP F47L The mutant (hereinafter referred to as FLSmEYFP)-tagged FUS(G156E) protein exhibited the highest sensitivity to liquid-solid phase transitions, while mEYFP... M154P mEYFP V164G and mEYFP S176G The lifespan of the mutant-labeled FUS(G156E) protein is primarily affected by LLPS and is little or no sensitive to liquid-solid phase transitions. Figure 2 (See B in Table 4).

[0167] Table 4. Total fluorescence lifetime of the mEYFP mutant in the liquid-solid phase transition process of the mEYFP mutant-labeled FUS(G156E) protein.

[0168]

[0169]

[0170] In summary, the transition from liquid condensates to solid condensates plays a key role in neurodegenerative diseases. Fluorescence recovery after photobleaching (FRAP) is a widely used tool to observe changes in fluidity within condensates. However, due to technical limitations, FRAP is mainly used to examine one or a few micrometer-sized condensates at a time. It is not feasible in FRAP and other commonly used methods to probe the fluidity changes of multiple condensates in parallel in real time without relying on fluorescence intensity and non-destructively. Therefore, the present invention systematically investigates the fluorescence lifetime of fluorescent proteins for real-time probing of liquid-liquid phase separation (LLPS) and liquid-solid phase transition, and enhances their sensitivity to LLPS or liquid-solid phase transition, even to both processes, by engineering wild-type mEYFP.

[0171] It should be noted that although the technical solutions of the present application are described with specific examples, those skilled in the art can understand that the present application should not be limited thereto.

[0172] The above has described various embodiments of the present application, and the above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and changes are obvious to those skilled in the art without departing from the scope and spirit of the described embodiments. The choice of terms used herein is intended to best explain the principles of the embodiments, practical application, or technical improvement in the market, or to enable other ordinary skilled persons in the art to understand the embodiments disclosed herein.

Claims

1. A fluorescent protein mutant, characterized in that, The amino acid sequence of the fluorescent protein mutant is shown in SEQ ID NO:2 or 3.

2. A fusion protein, characterized in that, The fusion protein comprises the fluorescent protein mutant of claim 1, the biomolecule to be detected, and the purification tag, wherein the sequence of the biomolecule to be detected is shown in SEQ ID No.

7.

3. An isolated polynucleotide, characterized in that, The polynucleotide encodes the fluorescent protein mutant of claim 1 or the fusion protein of claim 2.

4. An expression carrier, characterized in that, The expression vector comprises the isolated polynucleotides according to claim 3.

5. A recombinant host cell, characterized in that, The recombinant host cell comprises the fluorescent protein mutant according to claim 1, the fusion protein according to claim 2, the isolated polynucleotide according to claim 3, or the expression vector according to claim 4.

6. A method for detecting biomolecules by liquid-liquid phase separation and / or liquid-solid phase transition for non-diagnostic and non-therapeutic purposes, characterized in that, The method uses the fluorescent protein mutant according to claim 1, the fusion protein according to claim 2, the isolated polynucleotide according to claim 3, the expression vector according to claim 4, and the recombinant host cell according to claim 5.

7. The method according to claim 6, characterized in that, The method utilizes changes in the fluorescence signal of fluorescent protein mutants to detect liquid-liquid phase separation and / or liquid-solid phase transition of biomolecules, wherein the changes in the fluorescence signal include at least one of changes in fluorescence intensity, changes in fluorescence lifetime, changes in fluorescence polarization, and changes in fluorescence spectrum.

8. The method according to claim 6 or 7, characterized in that, The changes in the fluorescence signal include changes in fluorescence lifetime.

9. Use of the fluorescent protein mutant according to claim 1, the fusion protein according to claim 2, the isolated polynucleotide according to claim 3, the expression vector according to claim 4, or the recombinant host cell according to claim 5 in the preparation of reagents or kits for the detection of biomolecules in liquid-liquid phase separation and / or liquid-solid phase transition.

Images