PH probe based on quantum entanglement mechanism

Abstract

The invention provides a pH probe based on a quantum entanglement mechanism, and particularly provides a pH sensitive sensor, the sensor is a non-natural protein, and the sensor mutates at core amino acid sites, which correspond to SEQ ID NO: 1, are selected from the following groups and are related to pH sensitive characteristics, of wild type mTurqueise2 protein: D148, T203, W66, S65 and C48. The pH sensitive sensor disclosed by the invention has a relatively strong pH sensitive characteristic.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and specifically, to a pH probe based on the mechanism of quantum entanglement. Background Art

[0002] Precisely regulating the intracellular pH level is crucial for cell metabolism and overall function. Deviations from the normal pH level may have important implications for the occurrence and progression of various diseases. Specifically, abnormal pH changes in organelles (such as mitochondria) are closely related to the development of severe diseases, such as cancer and neurodegenerative diseases. Similarly, alterations in the pH of the Golgi apparatus may lead to abnormal glycosylation, thus causing the formation of cancer and cutis laxa. Dysregulation of the pH within lysosomes may result in lysosomal dysfunction, which may give rise to the occurrence of various diseases, including neurodegenerative diseases, inflammation and autoimmune diseases, lipid and glucose metabolism disorders, etc. Therefore, by precisely monitoring the pH of organelles, a better understanding of complex disease mechanisms can be achieved, providing valuable clues for the diagnosis and treatment of related diseases.

[0003] Currently, various strategies have been adopted to detect the pH of cytoplasmic organelles, including the use of pH microelectrodes, capillary electrophoresis, nuclear magnetic resonance (NMR), targeted avidin and fluorescein-biotin, fluorescent dyes, and fluorescent proteins, etc. Different fluorescent dyes and proteins have been used to monitor the pH levels of different cytoplasmic organelles. However, these methods for measuring the pH of cytoplasmic organelles have inconsistencies and many limitations. For example, pH microelectrodes require cell manipulation, which may cause damage to cells and are unable to simultaneously monitor multiple cytoplasmic organelles in real time. NMR measurements require expensive equipment and professional technical knowledge, so they are not applicable to in vivo experiments. Given these limitations, a single pH-sensitive fluorescent protein may provide a more consistent and desirable solution. pH-sensitive fluorescent proteins have the advantages of minimal cell damage, higher sensitivity characteristics, and stronger specificity when fused and expressed with organelle targeting sequences.

[0004] Currently, a variety of pH-sensitive fluorescent protein probes have been developed, including pHluorin, T-Sapphire, deGFP4, mNectarine, pHRed, SypHer, pHTomato, pHuji, pHoran, pH-Lemon, pHLARE, pHmScarlet, mOrange2, etc. However, these probes still have some limitations. For example, some probes have a low quantum yield, thus reducing their fluorescence signal intensity. Some other probes have a narrow pH response range, limiting their practicality for measuring the pH of different organelles. In addition, some of these proteins have single excitation and single emission characteristics, which may not be applicable to measuring the pH of all types of organelles.

[0005] Therefore, there is still a need to develop new pH probes suitable for measuring the pH of all types of organelles. Summary of the Invention

[0006] The main object of the present invention is to provide new pH probes suitable for measuring the pH of all types of organelles.

[0007] In a first aspect of the present invention, there is provided a pH-sensitive sensor, which is a non-natural protein, and the sensor has mutations at one or more core amino acid sites corresponding to SEQ ID NO: 1 of the wild-type mTurquoise2 protein and related to pH-sensitive characteristics, selected from the following group:

[0008] D148, T203, W66, S65, C48.

[0009] In another preferred embodiment, the Asp (D) at position 148 is mutated to any amino acid, preferably to the amino acid types shown in Table A, Table B, and Table C, preferably to one or more amino acids selected from the following group: Gly (G), Lys (K), Ser (S), Glu (E), Gln (Q), Ile (I), His (H), Trp (W), Pro (P), Tyr (Y), Arg (R), Asn (N), Met (M), Thr (T), Ala (A), Phe (F), Cys (C), Leu (L), Val (V), preferably mutated to Gly (G).

[0010] In another preferred embodiment, the Thr (T) at position 203 is mutated to Cys (C).

[0011] In another preferred embodiment, the Trp (W) at position 66 is mutated to Tyr (Y).

[0012] In another preferred embodiment, the Ser (S) at position 65 is mutated to Thr (T).

[0013] In another preferred embodiment, the Cys (C) at position 48 is mutated to Ser (S).

[0014] In another preferred embodiment, the mutations are selected from the following group: D148K, D148S, D148E, D148Q, D148I, D148H, D148W, D148P, D148Y, D148S, D148R, D148N, D148M, D148T, D148G, D148A, D148F, D148C, D148L, D148V, T203C, W66Y, S65T, C48S, or a combination thereof.

[0015] In another preferred embodiment, the mutations are selected from the following combinations:

[0016] (A) W66Y + D148C;

[0017] (B) S65T + W66Y + D148C;

[0018] (C) W66Y + D148C + T203C; or

[0019] (D) C48S + S65T + W66Y + T203C + X, where X is selected from the group: D148G, D148A, D148F, D148C, D148L or D148V.

[0020] In another preferred example, the mutations are as follows:

[0021] C48S + S65T + W66Y + T203C + D148G.

[0022] In another preferred example, except for the mutations (such as at positions 48, 203, 66, 65, and / or 48), the remaining amino acid sequence of the pH-sensitive sensor is the same as or substantially the same as the sequence of the wild-type mTurquoise2 protein.

[0023] In another preferred example, the substantially the same means that there are at most 50 (preferably 1 - 20, more preferably 1 - 10, even more preferably 1 - 5) amino acids that are different, where the differences include amino acid substitutions, deletions or additions, and the pH-sensitive sensor has strong pH-sensitive characteristics.

[0024] In another preferred example, the pH-sensitive sensor has at least 80% homology with the wild-type mTurquoise2 protein, preferably at least 85% or 90%, more preferably at least 95%, and most preferably at least 98% or 99%.

[0025] In another preferred example, the pH-sensitive sensor is formed by mutating the wild-type mTurquoise2 protein.

[0026] In another preferred example, the pH-sensitive sensor is selected from the group:

[0027] (A) A polypeptide having the amino acid sequence shown in SEQ ID NO:2;

[0028] (B) A polypeptide having at least 80% homology (preferably, at least 90% homology; equally preferably at least 95% homology; most preferably, at least 97% homology, such as above 98%, above 99%) with the amino acid sequence shown in SEQ ID NO:2, and the polypeptide has strong pH-sensitive characteristics;

[0029] (C) A derivative polypeptide formed by substituting, deleting or adding 1 to 15 amino acid residues in the amino acid sequence shown in SEQ ID NO: 2, and having strong pH-sensitive characteristics.

[0030] In another preferred embodiment, the amino acid sequence of the pH-sensitive sensor is as shown in SEQ ID NO: 2.

[0031] In another preferred embodiment, the pH-sensitive sensor has strong pH-sensitive characteristics.

[0032] In another preferred embodiment, the pH-sensitive sensor has one or more characteristics selected from the following groups:

[0033] (a) Having one main excitation peak and two emission peaks, and capable of performing ratio method for quantitative determination of the pH of organelles;

[0034] (b) Having a higher quantum yield (QY), thereby obtaining a stronger fluorescence signal;

[0035] (c) Having a wider pH response range, covering the range from pH 3.5 to 9.0, and being suitable for measuring the pH of most organelles;

[0036] (d) Having strong pH-sensitive characteristics;

[0037] (e) Localizing to the luminal surface of various organelles and their sub-organelles.

[0038] The second aspect of the present invention provides an isolated polynucleotide encoding the pH-sensitive sensor described in the first aspect of the present invention.

[0039] In another preferred embodiment, the polynucleotide is selected from the following groups: DNA sequence, RNA sequence.

[0040] In another preferred embodiment, the DNA sequence is selected from the following groups: genomic sequence, cDNA sequence.

[0041] In another preferred embodiment, the polynucleotide is selected from the following groups:

[0042] (a) A polynucleotide encoding the polypeptide shown in SEQ ID NO.: 2;

[0043] (b) A polynucleotide having a sequence as shown in SEQ ID NO.: 3;

[0044] (c) A polynucleotide having a nucleotide sequence with a homology of ≥ 95% (preferably ≥ 98%) to the sequence shown in SEQ ID NO.: 3 and encoding the polypeptide shown in SEQ ID NO.: 2;

[0045] (d) A polynucleotide complementary to any one of the polynucleotides of (a)-(c).

[0046] In another preferred embodiment, the polynucleotide further contains an auxiliary element selected from the group consisting of a signal peptide, a secretion peptide, a tag sequence (such as 6His), or a combination thereof, flanking the ORF of the mutant protein.

[0047] The third aspect of the present invention provides a vector that contains the polynucleotide described in the second aspect of the present invention.

[0048] The fourth aspect of the present invention provides a host cell that contains the vector described in the third aspect of the present invention or the polynucleotide described in the second aspect of the present invention is integrated into the genome.

[0049] The fifth aspect of the present invention provides a method for producing the pH-sensitive sensor described in the first aspect of the present invention, comprising the steps of:

[0050] Culturing the host cell described in the fourth aspect of the present invention under suitable expression conditions to express the pH-sensitive sensor described in the first aspect of the present invention; and

[0051] Optionally, isolating the pH-sensitive sensor.

[0052] The sixth aspect of the present invention provides a pharmaceutical composition, comprising:

[0053] The pH-sensitive sensor described in the first aspect of the present invention; and a pharmaceutically acceptable carrier.

[0054] The seventh aspect of the present invention provides a reagent for bioimaging detection, pH value detection, or cell microenvironment detection, comprising:

[0055] The pH-sensitive sensor described in the first aspect of the present invention.

[0056] The eighth aspect of the present invention provides a protein preparation that contains the pH-sensitive sensor described in the first aspect of the present invention.

[0057] In another preferred embodiment, the protein preparation includes an enzyme preparation.

[0058] In another preferred embodiment, the protein preparation includes an injection and / or a freeze-dried preparation.

[0059] The ninth aspect of the present invention provides a use of the pH-sensitive sensor described in the first aspect of the present invention for preparing a drug, a preparation, or a reagent for bioimaging detection, pH value detection, or cell microenvironment detection.

[0060] In another preferred embodiment, the drug, the preparation, or the reagent is further used for one or more uses selected from the group consisting of:

[0061] (i) Measuring the pH of organelles;

[0062] (ii) In vivo imaging and quantitative detection of pH changes in tissues and whole organisms.

[0063] In another preferred example, the organelles include mitochondrial sub-organelle regions, Golgi sub-organelle structures, endoplasmic reticulum, lysosomes, peroxisomes, and endosomes.

[0064] In another preferred example, the tissue includes: mouse brain tissue.

[0065] In another preferred example, the cells include COS-7, HCMEC / D3, and A172.

[0066] The tenth aspect of the present invention provides a method for bioimaging detection, pH value detection, or cell microenvironment detection, including the step of contacting the pH-sensitive sensor described in the first aspect of the present invention, the pharmaceutical composition described in the sixth aspect of the present invention, or the reagent described in the seventh aspect of the present invention with cells or tissues.

[0067] In another preferred example, the cells include: COS-7, HCMEC / D3, A172, U251, and U87.

[0068] In another preferred example, the tissue includes: mouse brain tissue.

[0069] In another preferred example, the cells or tissues are cells or tissues cultured in vitro.

[0070] In another preferred example, the method is an in vitro method.

[0071] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be repeated one by one here. Description of the Drawings

[0072] Figure 1: Spectral properties of mTurquoise2, mTurquoise2 S65T, and W66Y mutants. (A) Normalized fluorescence intensity of mTurquoise2 at an emission wavelength of 550 nm, and (B) emission spectra in buffers with pH ranging from 3.5 to pH 9.0 at an excitation wavelength of 450 nm. (C) Normalized fluorescence intensity of the mTurquoise2 S65T mutant at an emission wavelength of 550 nm, and (D) emission spectra at an excitation wavelength of 395 nm in different pH buffers. (E) Normalized fluorescence intensity of the mTurquoise2 W66Y mutant at an emission wavelength of 550 nm, and (F) emission spectra at an excitation wavelength of 365 nm in the same buffers. (G) Summarized the maximum excitation and emission peaks, full width at half maximum (FWHM), and pH of maximum emission for mTurquoise2, S65T, and W66Y. (H) The pH-sensitive curves were normalized with the emission spectrum peak as the standard, which were 475 nm (mTurquoise2), 475 nm (mTurquoise2 S65T), and 510 nm (mTurquoise2 W66Y), respectively.

[0073] Figure 2: Key residue D148 of mTurquoise2 and its saturation mutations. (A) Cartoon structures of mTurquoise2 S65T and W66Y mutants show that D148 is located in the loop of mTurquoise2 W66Y but in the β-sheet of mTurquoise2 S65T. (B) Aligned structures of mTurquoise2 S65T and W66Y show that the fluorophore (S65-Y66-G67) of mTurquoise2 W66Y forms hydrogen bonds with aspartic acid 148 (D148) and N121, while the fluorophore (T65-W66-G67) of mTurquoise2 S65T forms a hydrogen bond with S205. (C) and (D) Normalized excitation (dashed lines) and emission (solid lines) spectra of double mutant S65T / W66Y (C) and triple mutant S65T / W66Y / D148C (D), measured in buffers at pH 3.5 to 9.0 with excitation at a wavelength of 400 nm or emission at 550 nm. (E) and (F) Normalized pH-sensitive curves of quintuple mutants of mTurquoise2 containing C48S / S65T / W66Y / T203C / D148K (E) or C48S / S65T / W66Y / T203C / D148S (F), measured simultaneously at an excitation wavelength of 400 nm (dashed lines) or an emission wavelength of 550 nm (solid lines). (G) and (H) Representative fluorescence intensities of additional saturation mutations of D148 in 20 mTurquoise2 mutants C48S / S65T / W66Y / T203C, measured at an emission wavelength of 550 nm (G) or an excitation wavelength of 400 nm (H) in a buffer at pH 6.0. The preferred mutant is D148G, designated SITE-pHorin, which has the highest fluorescence intensity in both the excitation spectrum (G) and emission spectrum (H), marked with a red line.

[0074] Figure 3: pH-Sensitivity of SITE-pHorin. (A) and (B) Fluorescence intensities of SITE-pHorin (A) and deGFP4 (B) were measured in pH buffers from 3.5 to 9.0 at an excitation wavelength of 400 nm. (C) and (D) Excitation spectra of SITE-pHorin (C) and deGFP4 (D) were measured at an emission wavelength of 515 nm in buffers with different pH values. (E) Comparison of the logarithm of the ratio of emission peaks between 515 nm and 465 nm measured at an excitation wavelength of 400 nm for SITE-pHorin (blue) and deGFP4 (red) in buffers with different pH values. (F) Spectral properties of SITE-pHorin and deGFP4 were summarized (λex: main excitation peak; ε: extinction coefficient determined by absorption spectrum at pH 7.2; λemL: lower emission peak; λemH: higher emission peak; ΦL: quantum yield of λemL measured at pH 5.0; ΦH: quantum yield of λemH measured at pH 8.0). (G) SITE-pHorin protein was expressed and purified using Escherichia coli BL21(DE3) strain, and then its fluorescence intensity was detected in different buffers with pH ranging from 3.5 to 9.0. For clarity, the ratio of two emission peaks (green <480 nm and red >500 nm) in the image was represented by pseudocolor. Note that the leftmost scale of the pseudocolor is different from the other two scales.

[0075] Figure 4 : pH-Sensitivity and Quantum Entanglement Mechanism of SITE-pHorin. (A-D) Schematic diagrams of hydrogen bond network (A and C) and charge electrostatic surface potential (B and D) show the environment around the fluorescent group of SITE-pHorin at different pH values. Charge electrostatic surface potential (B and D) shows the phenomenon of quantum entanglement, that is, the simultaneous reprotonation and deprotonation between the fluorescent group of SITE-pHorin and the Y182 residue located outside the β-barrel structure (green sheet). The blue intensity on the surface represents the intensity of positive charge, and the red intensity represents the intensity of negative charge. Schematic diagrams of hydrogen bond network and charge electrostatic potential around the fluorescent group of SITE-pHorin and Y182 residue at pH 5.5 (A, B) or pH 7.0 (C, D). (E-G) The single mutant (C203E) of SITE-pHorin lacks quantum entanglement of the fluorescent group (opposite reprotonation and deprotonation of the phenol hydroxyl part at different pH values, as shown in A-D). (H) The single mutant (C203E) of SITE-pHorin loses the high sensitivity to pH changes.

[0076] Figure 5: SITE-pHorin targeted to organelles and their substructures. (A) SITE-pHorin was overexpressed in the COS-7 cell line, and fluorescence signals in two emission channels of 500 - 570 nm and 420 - 480 nm were captured in different pH buffers from pH 3.5 to 9.0 using a 405 nm excitation laser. For clarity, the pseudo-color image combining the two channels is shown on the far left. (B) Schematic diagram of specific organelle proteins fused with SITE-pHorin: H2B, nuclear marker; SITE-pHorin-SKL, peroxisome marker; Dnase2B, lysosome marker; Calreticulin, endoplasmic reticulum marker; TFR1, endosome marker; g subunit, mitochondrial cristae marker; 4Cox8, mitochondrial matrix marker; Fech, mitochondrial intermembrane space marker; ST6GAL1, trans-Golgi marker; MGAT2, medial-Golgi marker; GP73, cis-Golgi marker). (C) SITE-pHorin with specific organelle localization was overexpressed in the COS-7 cell line and imaged under the same conditions as in A. Note that COS-7 cells transfected with the lysosome-localized SITE-pHorin construct showed only green dots (signals in the 420 - 480 nm emission channel), while those expressing SITE-pHorin with TFR1 showed some green dots, red dots (in the 500 - 570 nm emission channel), and yellow dots after color merging. Scale bar, 3 μm.

[0077] Figure 6 : pH values and distributions of each organelle. (A) pH measurement data collected from approximately 100 COS-7 cells transfected with SITE-pHorin targeting each organelle. Gaussian fitting was performed on the pH measurements of various organelles, and the peak and standard deviation were calculated. Representative images of the cytoplasm, nucleus, and membrane-bound organelles including the endoplasmic reticulum (ER) and Golgi subunits are shown. The lower part is the original image after merging the two channels, and the upper part is the image shown in pseudo-color. (B) Representative images of peroxisomes, lysosomes, and endosomes containing TFR1, and the peak and standard deviation of pH measurements. Note that TFR1-positive endosomes can be divided into at least three populations with different pH values (4.0 - 9.0). (C) Representative images of mitochondrial cristae, matrix, and intermembrane space (IMS), and the peak and standard deviation of pH measurements. Note that the double Gaussian fitting of the mitochondrial matrix pH measurement shows two populations with pH values comparable to that of the cytoplasm. Scale bar, 3 μm.

[0078] Figure 7: Cartoon of the pH distribution in organelles. Summarizes the pH distribution map of organelles measured by using a single pH-sensitive probe SITE-pHorin with enhanced ultra-high pH sensitivity characteristics due to quantum entanglement effects. As shown, the pH measurement peaks and standard deviations of the nucleus, endoplasmic reticulum, Golgi substructures, peroxisomes, lysosomes, endosomes, and mitochondrial substructures are matched with corresponding pseudo-colors to their intracellular pH gradients.

[0079] Figure 8 . Spectral properties of mTurquoise2 mutants with single mutations C48S or T203C. (A) Normalized emission spectrum of mTurquoise2 mutant C48S at an excitation wavelength of 540 nm. (B) Excitation spectra detected at an emission wavelength of 395 nm in different pH buffers from pH 3.5 to 9.0 for single mutant C48S. (C) Normalized emission spectrum of single mutant T203C. (D) Normalized excitation spectrum of single mutant T203C. Fluorescence data were collected under the same shooting conditions as in A or B.

[0080] Figure 9 . Channels of mTurquoise2 S65T and W66Y. The channels of mTurquoise2 S65T and mTurquoise2 W66Y were analyzed using MOLE2.5 software. The structures of mTurquoise2 (A), mTurquoise2 S65T (B), and mTurquoise2 W66Y (C) were visualized using PyMOL 2.5.4. Green represents the cartoon of the structure, and the surface is shown with 60% transparency. The channels of mTurquoise2 S65T and mTurquoise2 W66Y are represented by gray channels. The structure of mTurquoise2 (A) lacks a channel or pore.

[0081] Figure 10 . Schematic diagrams of the hydrogen bond network and electrostatic surface formed by the mTurquiose2 and mTurquiose2 S65T fluorophores. (A) Network diagram of hydrogen bonds formed by the mTurquiose2 fluorophore, represented by dashed lines. (B) Charge surface potential near the mTurquiose2 fluorophore. (C and D) Schematic diagrams of the hydrogen bond network (C) and charge surface potential (D) formed by the mTurquiose2 S65T fluorophore. Dashed lines represent hydrogen bonds.

[0082] Figure 11 . Schematic diagrams of the hydrogen bond network and electrostatic surface formed by the mTurquiose2 W66Y fluorophore. (A) Shows the hydrogen bond network formed by the mTurquiose2 W66Y fluorophore and (B) the charge surface potential. Dashed lines represent hydrogen bonds.

[0083] Figure 12 . Spectral properties of mTurquiose2 double mutants, triple mutants, and quadruple mutants. (A-D) Normalized spectra of mTurquiose2 double mutants are plotted, including S65T / D148C (A), S65T / T203C (B), W66Y / D148C (C), and W66Y / T203C (D). The dashed line represents the excitation spectrum detected at an emission wavelength of 550 nm, and the solid line represents the emission spectrum excited at a wavelength of 400 nm, with the pH buffer ranging from 3.5 to 9.0. (E-G) Normalized excitation spectra (dashed lines) and emission spectra (solid lines) of mTurquiose2 triple mutants, including S65T / W66Y / T203C (E), S65T / D148C / T203C (F), and W66Y / D148C / T203C (G). (H) Normalized spectrum of the mTurquiose2 quadruple mutant containing C48S / S65T / W66Y / T203C.

[0084] Figure 13 . SITE-pHorin 148 mutants with dual excitation (main peak at 400 nm) and dual emission properties. (A-G) SITE-pHorin 148 mutants, including D148E (A), D148Q (B), D148I (C), D148H (D), D148W (E), D148P (F), and D148Y (G). The dashed line represents the excitation spectrum detected at an emission wavelength of 550 nm in a pH buffer ranging from 3.5 to 9.0, and the solid line represents the emission spectrum excited at a wavelength of 400 nm.

[0085] Figure 14 . SITE-pHorin 148 mutants with dual excitation (main peak at approximately 500 nm) and dual emission properties. (A-D) SITE-pHorin 148 mutants, including 148R (A), 148N (B), 148M (C), and 148T (D). The dashed line represents the excitation spectrum detected at an emission wavelength of 550 nm in a pH buffer ranging from 3.5 to 9.0, and the solid line represents the emission spectrum excited at a wavelength of 400 nm under the same pH conditions.

[0086] Figure 15. SITE-pHorin with one main excitation peak and dual emission and its 148 mutants. (A-F) SITE-pHorin 148 mutants, including 148G (SITE-pHorin) (A), 148A (B), 148F (C), 148C (D), 148L (E), 148V (F). The dotted line represents the excitation spectrum detected at an emission wavelength of 550 nm between pH buffers from 3.5 to 9.0, and the solid line represents the emission spectrum excited at a wavelength of 400 nm under the same pH conditions.

[0087] Figure 16 . Schematic diagram of the hydrogen bond network and electrostatic surface potential formed by the deGFP1 fluorophore and Y182. (A and B) Show the predicted hydrogen bond network (A) and charge surface potential (B) formed by deGFP1 under the crystallization buffer conditions at pH 5.5. (C and D) The hydrogen bond network (C) and charge surface potential (D) under the crystallization buffer conditions at pH 9.0. The long dotted line represents the distance between the phenol hydroxyl group of Y182 and the fluorophore.

[0088] Figure 17 . Schematic diagram of the hydrogen bond network and electrostatic surface potential formed by the GFP S65T fluorophore. (A and B) Show the predicted hydrogen bond network (A) and charge surface potential (B) formed by GFP S65T under the crystallization buffer conditions at pH 4.6. (C and D) The hydrogen bond network (C) and charge surface potential (D) formed under the crystallization buffer conditions at pH 8.0. The long dotted line represents the distance between the phenol hydroxyl group of Y182 and the fluorophore.

[0089] Figure 18 . pKa and pH-sensitive characteristics of SITE-pHorin. (A) Boltzmann fitting of pKa for the logarithmic data of the fluorescence intensity ratio in different pH buffers from pH 3.5 to 9.0. (B) pH-sensitive characteristic plotting and fitting. The data were normalized using the values around the pKa of SITE-pHorin. Fb represents the fluorescence intensity with an emission wavelength of 500 - 570 nm; Fa represents the fluorescence intensity with an emission wavelength of 420 - 480 nm measured by single excitation at a wavelength of 405 nm.

[0090] Figure 19 . Schematic diagram of the hydrogen bond network formed by the fluorophore of the SITE-pHorin mutant (C203E). (A and B) Show the predicted hydrogen bond network around the fluorophore of the SITE-pHorin mutant (C203E) at pH 5.0 (A) and pH 8.0 (B) in its crystallization buffer solution.

[0091] Figure 20.Analysis of the fluorophore and the benzene ring of Y182 in SITE-pHorin and GFP S65T. (A) Sum of the internal angles of the fluorophore and the benzene ring of Y182 in the SITE-pHorin crystal at pH 5.5. (B) Sum of the internal angles of the fluorophore and the benzene ring of Y182 in GFP S65T at pH 4.6. (C) Distribution of the sum of the internal angles of the benzene rings of phenylalanine and tyrosine in SITE-pHorin was calculated. The results showed that the sum of these internal angles followed a Gaussian distribution. The mean ± standard deviation of the fit of these internal angle sums was 719.98 ± 0.076 degrees. (D) Summary of the sum of the internal angles of the fluorophore and the benzene ring of Y182 in the SITE-pHorin crystal structure (pH 5.5, PDB: 8IZ3) and GFP S65T (pH 4.6, PDB: 1C4F). Interestingly, it was shown that the fluorophore of SITE-pHorin at pH 5.5 showed a significant distortion deviating from 720.0° to 719.7° (p = 0.000115, calculated using the fitting parameters in panel C), resulting in a discrete curvature of -0.3° for the fluorophore of SITE-pHorin at pH 5.5. Detailed Description of the Invention

[0092] Through extensive and in-depth research and a large number of screenings, the inventors unexpectedly discovered for the first time a new pH-sensitive sensor with strong pH-sensitive characteristics. The pH-sensitive sensor of the present invention also has the following characteristics: (a) having a main excitation peak and two emission peaks, and can perform ratio method to quantitatively measure the pH of organelles; and / or (b) having a higher quantum yield (QY), thus obtaining a stronger fluorescence signal; and / or (c) having a wider pH response range, covering the range from pH 3.5 to 9.0, suitable for measuring the pH of most organelles; and / or (d) localizing to the luminal surfaces of various organelles and their sub-organelles. On this basis, the inventors completed the present invention.

[0093] Terms

[0094] The following examples are only used to describe the present invention and do not limit the present invention. Unless otherwise specified, the experiments and methods described in the examples are basically carried out according to the conventional methods well-known in the art and described in various reference documents.

[0095] In addition, those not specifying specific conditions in the examples are carried out according to conventional conditions or conditions recommended by the manufacturer. Those reagents or instruments not indicating the manufacturer can be obtained as conventional products through commercial purchase. Those skilled in the art know that the examples describe the present invention by way of example and are not intended to limit the scope claimed by the present invention. All the published cases and other reference materials mentioned herein are incorporated herein by reference in their entirety.

[0096] To facilitate a better understanding of the present disclosure, certain terms are first defined. As used in this application, unless otherwise expressly specified herein, each of the following terms shall have the meanings given below. Other definitions are set forth throughout the application.

[0097] The term "about" can refer to a value or a component within an acceptable error range of a specific value or component determined by a person of ordinary skill in the art, which will depend in part on how the value or component is measured or determined. For example, as used herein, the expression "about 100" includes all values between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0098] As used herein, the term "comprising" or "including" can be open-ended, semi-closed, and closed. In other words, the term also includes "consisting essentially of" or "consisting of".

[0099] Sequence identity (or homology) is determined by comparing two aligned sequences along a predetermined comparison window, which can be 50%, 60%, 70%, 80%, 90%, 95%, or 100% of the length of a reference nucleotide sequence or protein, and determining the number of positions at which identical residues occur. Typically, this is expressed as a percentage. Methods for measuring sequence identity of nucleotide sequences are well known to those skilled in the art.

[0100] The term

[0101] As used herein, the term "AxxB" means that the amino acid A at position xx is changed to amino acid B. For example, "D148G" means that the amino acid D at position 148 is mutated to G, and so on.

[0102] mTurquoise2 gene and protein

[0103] In the present invention, mTurquoise2 can be from bacteria, jellyfish, or other animals, including non-human mammals (such as primates or rodents) or humans. The amino acid sequence of the mTurquoise2 protein encoded by a representative mTurquoise2 gene is shown in SEQ ID NO.1.

[0104] SEQ ID NO:1:

[0105] MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFIC

[0106] TTGKLPVPWPTLVTTLSWGVQCFARYPDHMKQHDFFKSAMPEGYVQE

[0107] RTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYN

[0108] YFSDNVYITADKQKNGIKANFKIRHNIEDGGVQLADHYQQNTPIGDGPV

[0109] LLPDNHYLSTQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYK

[0110] mTurquoise2 is a cyan fluorescent protein published in 2012 and is derived from the jellyfish Aequorea victoria (Goedhart et al., 2012).

[0111] Taking the mTurquoise2 gene as an example, it encodes a protein with 239 amino acids, and this protein has the characteristic of a fluorescence quantum yield as high as 93%.

[0112] The pH-sensitive sensor of the present invention and its encoding nucleic acid

[0113] As used herein, the terms "mutant protein", "mutant protein of the present invention", "pH-sensitive sensor", and "pH-sensitive sensor of the present invention" are used interchangeably and all refer to a non-naturally occurring mutant pH-sensitive sensor, and the pH-sensitive sensor is a protein artificially modified based on the protein shown in SEQ ID NO.: 1. Among them, the pH-sensitive sensor contains core amino acids related to pH-sensitive characteristics, and at least one of the core amino acids is artificially modified; and the pH-sensitive sensor of the present invention has strong pH-sensitive characteristics.

[0114] The term "core amino acid" refers to the corresponding site in a sequence that is at least 80% homologous to SEQ ID NO.: 1, such as 84%, 85%, 90%, 92%, 95%, 98% homology to SEQ ID NO.: 1, and is the specific amino acid described herein. For example, based on the sequence shown in SEQ ID NO.: 1, the core amino acids are:

[0115] D148, T203, W66, S65, C48.

[0116] Preferably, in the present invention, the core amino acids of the present invention are mutated in the following manner:

[0117] The Asp (D) at the 148th position is mutated to any amino acid, preferably to the amino acid types shown in Table A, Table B, and Table C, and preferably to one or more amino acids selected from the group consisting of: Gly (G), Lys (K), Ser (S), Glu (E), Gln (Q), Ile (I), His (H), Trp (W), Pro (P), Tyr (Y), Arg (R), Asn (N), Met (M), Thr (T), Ala (A), Phe (F), Cys (C), Leu (L), Val (V), and preferably mutated to Gly (G);

[0118] The Thr (T) at the 203rd position is mutated to Cys (C);

[0119] The Trp (W) at the 66th position is mutated to Tyr (Y);

[0120] The Ser (S) at the 65th position is mutated to Thr (T);

[0121] The Cys (C) at the 48th position is mutated to Ser (S).

[0122] It should be understood that the amino acid numbering in the pH-sensitive sensor of the present invention is based on the wild-type mTurquoise2 protein. When the homology of a specific pH-sensitive sensor with the sequence of the wild-type mTurquoise2 protein reaches 80% or more, the amino acid numbering of the pH-sensitive sensor may be misaligned relative to the amino acid numbering of the wild-type mTurquoise2 protein, such as being misaligned 1-100 positions towards the N-terminus or C-terminus of the amino acid. Using conventional sequence alignment techniques in the art, those skilled in the art can generally understand that such misalignment is within a reasonable range, and a pH-sensitive sensor with a homology of 80% (such as 90%, 95%, 98%) to the wild-type mTurquoise2 protein (SEQ ID NO.1) and having the same or similar strong pH-sensitive characteristics should not be excluded from the scope of the pH-sensitive sensor of the present invention due to the misalignment of amino acid numbering.

[0123] The pH-sensitive sensor of the present invention is a synthetic protein or a recombinant protein, that is, it can be a chemically synthesized product or produced using recombinant techniques from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, plants). Depending on the host used in the recombinant production protocol, the pH-sensitive sensor of the present invention can be glycosylated or can be non-glycosylated. The pH-sensitive sensor of the present invention may also include or not include the starting methionine residue.

[0124] The present invention also includes fragments, derivatives, and analogs of the pH-sensitive sensor. As used herein, the terms "fragment", "derivative", and "analog" refer to proteins that substantially retain the same biological function or activity of the pH-sensitive sensor.

[0125] The pH-sensitive sensor fragment, derivative, or analog of the present invention can be (i) a pH-sensitive sensor in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) a pH-sensitive sensor having a substituent group in one or more amino acid residues, or (iii) a pH-sensitive sensor formed by fusing a mature mutant protein with another compound (such as a compound that prolongs the half-life of the mutant protein, for example, polyethylene glycol), or (iv) a pH-sensitive sensor formed by fusing an additional amino acid sequence to this pH-sensitive sensor sequence (such as a leader sequence or a secretion sequence or a sequence used to purify this pH-sensitive sensor or a proprotein sequence, or a fusion protein formed with an antigen IgG fragment). According to the teachings herein, these fragments, derivatives, and analogs are within the scope well-known to those skilled in the art.

[0126] In certain embodiments, other selected groups of amino acids that are considered to be conservative substitutions of each other.

[0127] Table A

[0128]

[0129]

[0130] In certain embodiments, other selected groups of amino acids that are considered to be conservative substitutions of each other (see, for example, Creighton, Proteins (1984)):

[0131] Table B

[0132] Group 1 Ala(A), Gly(G) Group 2 Asp(D), Glu(E) Group 3 Asn(N), Gln(Q) Group 4 Arg(R), Lys(K) Group 5 Ile(I), Leu(L), Met(M), Val(V) Group 6 Phe(F), Tyr(Y), Trp(W) Group 7 Ser(S), Thr(T) Group 8 Cys(C), Met(M)

[0133] In certain embodiments, other selected groups of amino acids that are considered to be conservative substitutions of each other:

[0134] Table C

[0135] Group 1 Ala(A), Ser(S), Thr(T) Group 2 Asp(D), Glu(E) Group 3 Asn(N), Gln(Q) Group 4 Arg(R), Lys(K) Group 5 Ile(I), Leu(L), Met(M) Group 6 Phe(F), Tyr(Y), Trp(W)

[0136] The active pH-sensitive sensor of the present invention has stronger pH-sensitive characteristics compared to the wild-type mTurquoise2 protein (SEQ ID NO.1).

[0137] In addition, the pH-sensitive sensor of the present invention can also be modified. The modified (usually without changing the primary structure) forms include: chemically derived forms of the pH-sensitive sensor in vivo or in vitro, such as acetylation or carboxylation. Modification also includes glycosylation, such as those pH-sensitive sensors produced by glycosylation modification during the synthesis and processing of the pH-sensitive sensor or in further processing steps. Such modification can be accomplished by exposing the pH-sensitive sensor to enzymes that carry out glycosylation, such as mammalian glycosylating enzymes or deglycosylating enzymes. The modified forms also include sequences having phosphorylated amino acid residues (such as phosphotyrosine, phosphoserine, phosphothreonine). Also included are pH-sensitive sensors that are modified to improve their proteolytic resistance or optimize their solubility properties.

[0138] The term "polynucleotide encoding a mutant protein" can be a polynucleotide including the polynucleotide encoding the pH-sensitive sensor of the present invention, or can also be a polynucleotide further including additional coding and / or non-coding sequences.

[0139] The present invention also relates to variants of the above polynucleotides, which encode polypeptides having the same amino acid sequence as the present invention or fragments, analogs and derivatives of the pH-sensitive sensor. These nucleotide variants include substitution variants, deletion variants and insertion variants. As is known in the art, allelic variants are alternative forms of a polynucleotide, which may be substitutions, deletions or insertions of one or more nucleotides, but do not substantially change the function of the pH-sensitive sensor encoded thereby.

[0140] The present invention also relates to polynucleotides that hybridize to the above sequences and have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that can hybridize to the polynucleotides described in the present invention under stringent conditions (or stringent conditions). In the present invention, "stringent conditions" refer to: (1) hybridization and washing at lower ionic strength and higher temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) adding a denaturant during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization occurs only when the identity between the two sequences is at least 90% or more, preferably 95% or more.

[0141] The pH-sensitive sensor and polynucleotide of the present invention are preferably provided in isolated form, and more preferably, are purified to homogeneity.

[0142] The full-length sequence of the polynucleotide of the present invention can generally be obtained by PCR amplification, recombination or artificial synthesis. For PCR amplification, primers can be designed according to the nucleotide sequences disclosed in the present invention, especially the open reading frame sequences, and a commercially available cDNA library or a cDNA library prepared by conventional methods known to those skilled in the art can be used as a template for amplification to obtain the relevant sequences. When the sequence is relatively long, it is often necessary to perform PCR amplification twice or more times, and then splice the fragments amplified each time together in the correct order.

[0143] Once the relevant sequences are obtained, the relevant sequences can be obtained in large quantities by recombination. This is usually to clone it into a vector, then transfer it into cells, and then isolate the relevant sequences from the proliferated host cells by conventional methods.

[0144] In addition, the relevant sequences can also be synthesized by artificial synthesis, especially when the fragment length is relatively short. Usually, a very long fragment can be obtained by first synthesizing multiple small fragments and then ligating them.

[0145] At present, it is already possible to completely obtain the DNA sequence encoding the protein (or its fragment, or its derivative) of the present invention by chemical synthesis. Then this DNA sequence can be introduced into various existing DNA molecules (or such as vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequence of the present invention by chemical synthesis.

[0146] The method of applying PCR technology to amplify DNA / RNA is preferably used to obtain the polynucleotide of the present invention. Especially when it is difficult to obtain full-length cDNA from the library, the RACE method (rapid amplification of cDNA ends) can be preferably used. The primers for PCR can be appropriately selected according to the sequence information of the present invention disclosed herein and can be synthesized by conventional methods. The amplified DNA / RNA fragments can be separated and purified by conventional methods such as gel electrophoresis.

[0147] Expression vector

[0148] The present invention also relates to a vector containing the polynucleotide of the present invention, and a host cell genetically engineered with the vector of the present invention or the mutant protein coding sequence of the present invention, and a method for producing the polypeptide of the present invention by recombinant technology.

[0149] By conventional recombinant DNA technology, the polynucleotide sequence of the present invention can be used to express or produce a recombinant pH-sensitive sensor. Generally, there are the following steps:

[0150] (1). Transform or transduce a suitable host cell with the polynucleotide (or variant) encoding the pH-sensitive sensor of the present invention of the present invention, or with a recombinant expression vector containing the polynucleotide.

[0151] (2). A host cell cultured in a suitable medium;

[0152] (3). Isolate and purify the protein from the medium or cells.

[0153] In the present invention, the polynucleotide sequence encoding the pH-sensitive sensor can be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses or other vectors well-known in the art. Any plasmid and vector can be used as long as it can replicate and be stable in the host. An important feature of an expression vector is that it usually contains an origin of replication, a promoter, a marker gene and translation control elements.

[0154] Methods well-known to those skilled in the art can be used to construct an expression vector containing the DNA sequence encoding the pH-sensitive sensor of the present invention and appropriate transcriptional / translational control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, etc. The said DNA sequence can be effectively ligated to an appropriate promoter in the expression vector to direct mRNA synthesis. Representative examples of these promoters are: the lac or trp promoter of Escherichia coli; the λ phage PL promoter; eukaryotic promoters including the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the LTRs of retroviruses and some other known promoters that can control gene expression in prokaryotic or eukaryotic cells or their viruses. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

[0155] In addition, the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selecting transformed host cells, such as dihydrofolate reductase, neomycin resistance and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for Escherichia coli.

[0156] A vector containing the above-mentioned appropriate DNA sequence and an appropriate promoter or control sequence can be used to transform an appropriate host cell so that it can express the protein.

[0157] The host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples are: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; human cells such as HEK293t cells.

[0158] When the polynucleotide of the present invention is expressed in higher eukaryotic cells, the transcription will be enhanced if an enhancer sequence is inserted into the vector. Enhancers are cis-acting factors of DNA, usually about 10 to 300 base pairs in length, which act on promoters to enhance gene transcription. Examples include the 100 to 270 base pair SV40 enhancer on the late side of the replication origin, the polyoma enhancer on the late side of the replication origin, and the adenovirus enhancer, etc.

[0159] Those of ordinary skill in the art are well aware of how to select appropriate vectors, promoters, enhancers, and host cells.

[0160] Transformation of host cells with recombinant DNA can be carried out by conventional techniques well-known to those skilled in the art. When the host is a prokaryote such as Escherichia coli, competent cells capable of taking up DNA can be harvested after the exponential growth phase, treated with the CaCl2 method, and the steps used are well-known in the art. Another method is to use MgCl2. If necessary, transformation can also be carried out by electroporation. When the host is a eukaryote, the following DNA transfection methods can be selected: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0161] The obtained transformants can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention. Depending on the host cell used, the culture medium used in the culture can be selected from various conventional media. The culture is carried out under conditions suitable for the growth of the host cell. When the host cell grows to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature shift or chemical induction), and the cells are cultured for a further period of time.

[0162] The recombinant polypeptide in the above method can be expressed intracellularly, or on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be separated and purified by various separation methods using its physical, chemical, and other properties. These methods are well-known to those skilled in the art. Examples of these methods include but are not limited to: conventional renaturation treatment, treatment with protein precipitants (salting-out method), centrifugation, osmotic lysis, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC), and various other liquid chromatography techniques and combinations of these methods.

[0163] The main advantages of the present invention include:

[0164] (1) The present invention has for the first time discovered a new pH-sensitive sensor with strong pH-sensitive characteristics. The pH-sensitive sensor of the present invention also has the following characteristics: (a) having a main excitation peak and two emission peaks, and can perform ratio method to quantitatively measure the pH of organelles; and / or (b) having a higher quantum yield (QY), thereby obtaining a stronger fluorescence signal; and / or (c) having a wider pH response range, covering the range from pH 3.5 to 9.0, suitable for measuring the pH of most organelles; and / or (d) being localized to the luminal surfaces of various organelles and their sub-organelles.

[0165] (2) The present invention has constructed a fluorescent protein with quantum entanglement characteristics and sensitive to pH, called SITE-pHorin (single excitation and dual emission pH sensor protein). This protein has a main excitation wavelength and two different emission wavelengths, and can accurately measure the pH value of cytoplasmic organelles by using the ratio method. SITE-pHorin has a higher quantum yield (QY), thereby obtaining a stronger fluorescence signal. In addition, it also exhibits a wider pH response range, covering the range from pH 3.5 to 9.0. The pH value of the organelle is determined by calculating the fluorescence intensity ratio between the two emission wavelengths, thereby providing accurate and quantitative pH measurement results.

[0166] (3) The present invention has demonstrated that SITE-pHorin is a ratio-type pH-sensitive sensor. SITE-pHorin has several advantages over other fluorescent proteins, including a wide pH response range, which can cover from pH 3.5 to 9.0, enabling it to measure the pH of most organelles. Its main advantage is having a main excitation peak and two emission peaks, and can perform ratio method to quantitatively measure the pH of organelles. We have successfully measured the pH of various organelles and their sub-organelle structures, including mitochondrial sub-organelle regions, Golgi sub-organelle structures, endoplasmic reticulum, lysosomes, peroxisomes, and endosomes, by using SITE-pHorin combined with organelle-targeting labels.

[0167] (4) The research of the present invention has revealed that the lysosomal localization distribution of TFR1-SITE-pHorin is in at least three regions with different pH values.

[0168] (5) The present invention has for the first time discovered that this tightly coupled mechanism achieved through quantum entanglement enables the fluorophore of SITE-pHorin to sense the pH change of its surrounding environment through pH-sensitive residues (external Y182 residue), thereby resulting in the pH-sensitive characteristics of SITE-pHorin.

[0169] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are generally carried out under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are by weight percentage and weight parts.

[0170] Unless otherwise specified, the reagents and materials in the embodiments of the present invention are all commercially available products.

[0171] Materials and Methods

[0172] Molecular Cloning and Mutagenesis

[0173] The starting template was the pmTurquoise2-Mito plasmid (Addgene, #36208) provided by the Dorus Gadella laboratory. PCR was performed using KOD-Plus polymerase (TOYOBO, KOD-201) to clone mTurquoise2 into the pET28a vector (Novagen, #69337-3) for protein expression. Using II Site-Directed Mutagenesis Kit (Stratagene, 200523) for site-directed mutagenesis to introduce mutations at the C48, S65, W66, T203, and D148 sites to obtain single mutants, double mutants, triple mutants, and penta mutants. Then, these mutants were cloned into the pET28a vector for protein expression. The mutation (S65T / C48S / H148C / T203C) was introduced into EGFP to generate deGFP4, which was then cloned into the pET28a vector.

[0174] The construction of SITE-pHorin targeting organelles involves several steps as follows: The SITE-pHorin construct was initially cloned into the EGFP-N1 vector (Addgene, #6085-1) using the Age I and BsrG I restriction enzyme sites to construct the SITE-pHorin-N1 plasmid. The cDNAs of human H2B, human 4Cox8, and rat TFR1 were from the former Jiansheng-Kang laboratory. The cDNAs of human ATP synthase g subunit, human ST6GAL1, MGAT2, and GP73 were synthesized by Genewiz. The cDNAs of mouse heme synthase (Fech), mouse Dnase2B, and mouse Calreticulin were from mouse brain tissues (Shanghai Model Organisms Center, Inc.). The SITE-pHorin-SKL construct was created by replacing the last two amino acids YK with SKL. hH2B, hComplex V g subunit, cytochrome c oxidase subunit 8 (4Cox8), mFech, hST6GAL1, hMGAT2 (1-89), hGP73 (1-34), mCalreticulin, mDnase2B, and SITE-pHorin-SKL were cloned into the SITE-pHorin-N1 plasmid. The detailed information on the genes targeting organelles is summarized in Table 1. All plasmids were sequenced by GeneWiz before further analysis. Q5 polymerase, restriction enzymes, and T4 DNA ligase were purchased from New England Biolabs.

[0175] Table 1

[0176]

[0177] Protein expression, purification, and crystallization

[0178] Spectral measurement section: The mTurquoise2 mutants mTurquoise2 S65T and mTurquoise2 W66Y proteins were expressed in Escherichia coli BL21(DE3) strain (TransGen Biothech, Cat#CD601-02) using the pET28a expression vector with an N-terminal 6×His tag. The bacteria were cultured overnight at 18 °C with 0.3 mM IPTG. After centrifugation, the bacterial pellet was resuspended in 20 mM Tris-HCl, pH 7.5, 300 mM NaCl, 10 mM β-ME, 0.1 mM PMSF. Cell lysate was obtained by sonication for 10 minutes (200 w, working time 1 s, interval 2 s). Then, after centrifugation, Ni-agarose resin was added to the supernatant and rotated at 4 °C for 1 hour. The Ni-agarose-bound proteins were washed three times with 20 mM Tris-HCl pH 7.5, 300 mM NaCl, 20 mM Imidazole, 10 mM β-mercaptoethanol (β-ME). The proteins were eluted using three column volumes of 20 mM Tris-HCl pH 7.5, 300 mM NaCl, 200 mM Imidazole, 10 mM β-ME. The eluted proteins were concentrated to 2 ml using an ultrafiltration tube (Millipore, UFC201024). The buffer was exchanged to PBS using a PD10 column (GE, 17-0851-01). The protein concentration was determined using a BCA kit and adjusted to 5 mg / mL.

[0179] Crystalline fraction: mTurquoise2 S65T, W66Y, SITE-pHorin and SITE-pHorin_C203E were purified by affinity chromatography using a HisTrap HP (5 ml) column and then by gel filtration using a HiLoad 10 / 300 Superdex column. Affinity purification involved the following buffers: Buffer A: 50 mM Tris-HCl pH 8.0, 300 mM NaCl; Buffer B: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 20 mM Imidazole; Buffer C: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 250 mM Imidazole. The gel filtration buffer contained 10 mM Tris-HCl pH 8.0, 100 mM NaCl, 1 mM DTT. mTurquoise2 S65T was concentrated to 13 mg / ml and crystallized at 20% PEG 8000, 100 mM MgCl2, 100 mM HEPES pH 6.5. mTurquoise2 W66Y was concentrated to 15 mg / ml and crystallized at 10% PEG 8000, 100 mM MgCl2, 100 mM HEPES pH 6.5. SITE-pHorin was concentrated to 10 mg / ml and crystallized at 14% PEG 8000, 100 mM MgCl2, 100 mM HEPES pH 7.0 and 0.1 M Bis-tris pH 5.5, 25% PEG 3350. SITE-pHorin_C203E was also concentrated to 10 mg / ml and crystallized at 20% PEG 6000, 100 mM MgCl2, 100 mM citrate, pH 5.0 and 20% PEG 8000, 100 mM MgCl2, 100 mM Tris-HCl, pH 8.0. All crystals were grown using the hanging-drop vapor diffusion method at 289K.

[0180] Data collection and structure determination

[0181] Diffraction data were collected at the BL19U1 beamline of the Shanghai Synchrotron Radiation Facility (SSRF). The collected data were processed with HKL3000. The crystal structures of mTurquoise2 S65T, W66Y, SITE-pHorin (at pH 7.0 and pH 5.5) and SITE-pHorin_C203E (at pH 5.0 and pH 8.0) were solved by molecular replacement using the structure of mTurquoise2 resolved as the search model. Structure refinement and model building were performed using

[0182] Performed with Refmac, PHENIX, and Coot. All models were validated by MolProbity. Details of data processing and statistics are summarized in Table 2. All structural diagrams were generated using PyMOL software ( https: / / www.pymol.org ).

[0183] Table 2

[0184]

[0185]

[0186] Spectral measurements

[0187] Purified mTurquoise2, mTurquoise2 mutants, SITE-pHorin, and deGFP4 for testing absorption and emission spectra were measured using a UV-visible spectrometer (Agilent, Cary 100). Excitation and emission spectra were measured using a 96-well microplate fluorometer (Thermo, Varioskan Flash). For pH titration experiments, a series of buffers with a pH range from 3.5 to 9.0 were prepared, consisting of: 125 mM potassium gluconate, 20 mM sodium gluconate, 0.5 mM CaCl2, 0.5 mM MgCl2, and 25 mM of one of the following components: citric acid (pH 3.5, 4.0), acetic acid (pH 4.5, 5.0), MES (pH 5.5, 6.0), PIPES (pH 6.5), HEPES (pH 7.0), Tricine (pH 7.5), Tris (pH 8.0, 8.5), CHES (pH 9.0). The quantum yield (Φ) of the fluorescent proteins was calculated relative to deGFP4 with equal optical density and known (ΦL = 0.08, ΦH = 0.15).

[0188] Cell culture, transfection, and imaging

[0189] Cos-7 cells (ATCC, Cat# CRL-1651) were cultured in DMEM medium (GIBCO) containing 10% FBS in an incubator at 37 °C and 5% CO2. Cos-7 cells were seeded on 12 mm coverslips coated with Matrigel (Coring, 356234) and cultured in 35 mm culture dishes at a density of 3.0 - 5.0×10 5 cells / dish. Twenty-four hours after seeding, SITE-pHorin-N1 and SITE-pHorin plasmids targeting organelles were transfected into COS7 cells using the calcium phosphate transfection method according to the instructions.

[0190] The method for establishing the in vitro and in vivo pH-sensitive SITE-pHorin standard curve is as follows: For in vitro measurement, the purified SITE-pHorin protein was concentrated to 1 mg / mL and imaged using a confocal microscope (ZEISS, LSM980) with a single excitation wavelength of 405 nm and two emission wavelength channels: 420 - 480 nm and 500 - 570 nm. The fluorescence intensity ratio (log(Fb / Fa)) was calculated in the pH range from 3.5 to 9.0 to create a standard curve. The standard curve for in vivo measurement was obtained from Cos7 cells transfected with the SITE-pHorin-N1 plasmid. Initially, the transfected Cos7 cells were treated with Nigericin / Valinomycin at the same concentration as the intracellular pH calibration buffer (ThermoFisher, P35379). Subsequently, imaging was performed using the same excitation and emission wavelengths as above in buffers with pH from 3.5 to 9.0 using a confocal microscope. The in vivo standard curve was made by the fluorescence intensity ratio (log(Fb / Fa)) of Cos7 cells measured in the pH range from 3.5 to 9.0. Then, the pH value of the organelle-targeted SITE-pHorin can be calculated using the in vivo standard curve (Fb is the fluorescence intensity at the emission wavelength of 500 - 570 nm measured at the excitation wavelength of 405 nm; Fa is the fluorescence intensity at the emission wavelength of 420 - 480 nm measured at the excitation wavelength of 405 nm).

[0191] The quantum entanglement mechanism of SITE-pHorin

[0192] For a single acid-base balance,

[0193]

[0194] where K is the value of the acid dissociation constant; A represents the protonated form; B is the conjugate base of A.

[0195] According to the Henderson-Hasselbalch equation (I), we have:

[0196] pH = pK a + log 10 ([B] / [A]) (II)

[0197] where pKa is the value of the acid dissociation constant; [B] and [A] represent the concentrations of the base and acid, respectively.

[0198] The slope 1 / n of equation (II) represents the number of acids, i.e., the multi-proton model. For pH-sensitive fluorescent proteins, we assume that the protein needs to bind n hydrogen ions simultaneously to achieve pH sensitivity and the system is in equilibrium, and the ratio between the deprotonated and protonated forms can be expressed as:

[0199]

[0200] Then, we obtained the fluorescence intensity ratio between the base and acid forms under the multi-proton hypothesis:

[0201]

[0202] where Fa and Φa are the fluorescence and fluorescence quantum yield of the acid form, respectively; Fb and Φb are the fluorescence and fluorescence quantum yield of the base form, respectively; and g is a constant for the imaging acquisition settings.

[0203] Taking the negative logarithm of both sides of Equation (IV) and rearranging, the multi-proton hypothesis gives the following expression:

[0204]

[0205] When the pH is equal to pKa, if the fluorescence intensity ratio is normalized to Equation (V) reduces to:

[0206]

[0207] Obviously, the slope 1 / n of Equation (VI) reflects the sensitive characteristics of the protein to pH changes, which is exactly the hypothesis that the protein requires n simultaneous protonations to achieve its pH-sensitive characteristics.

[0208] Interestingly, the actual situation is very astonishing because there are simultaneous and opposite protonations and deprotonations between the fluorophore and the Y182 residue. The Y182 residue is located outside the β-barrel structure ( Figure 4 ). The results show that the protonation of the phenolic hydroxyl group in the Y182 residue is tightly coupled with the deprotonation of the phenolic hydroxyl group in the SITE-pHorin fluorophore, and vice versa, that is, the deprotonation of the phenolic hydroxyl group in the Y182 residue is tightly coupled with the protonation of the phenolic hydroxyl group in the SITE-pHorin fluorophore. Therefore, this coupling mechanism enables the SITE-pHorin fluorophore to connect the external environment with the inside of the β-barrel structure through the Y182 residue, thereby sensing pH changes.

[0209]

[0210] Therefore, the slope can be interpreted as a quantum effect (n = 1 / 2) of its pH-sensitive characteristics, which can be enhanced by the quantum entanglement (simultaneous and opposite protonations and deprotonations) between the SITE-pHorin fluorophore and Y182 ( Figure 4 ). The quantum effect and quantum entanglement in the pH-sensitive characteristics of SITE-pHorin may exactly reflect the resonance structure of the benzene ring ( Figure 4) The characteristics of the integrity of electron rotation and non-trivial topology in (Puentes, 2017).

[0211] Therefore, the interpretation of quantum entanglement can be well adapted to structural phenomena ( Figure 4 ) and fluorescence data within the physiological pH range (4.0 - 8.0) ( Figure 18 ), and well explains the mechanism and pH-sensitive characteristics of SITE-pHorin. On the other hand, there are some data points with less good fitting, such as at high pH conditions of pH 9.0 or very low pH conditions of pH 3.5. The situation of pH 3.5 may be partly due to the stability of the protein at such a low pH, although the fluorescence signal of SITE-pHorin is stable and good in lysosomes ( Figure 5 ). The high pH of pH 9.0 may interfere with the quantum entanglement between the fluorophore and Y182, because quantum entanglement seems to be stronger at lower pH (the distance between phenolic hydroxyl groups is shorter) ( Figure 4 ), which may also partly affect the stability of SITE-pHorin in acidic environments.

[0212] pH measurement with background noise and autofluorescence correction

[0213] Fluorescence images usually contain background noise or autofluorescence signals, which inevitably affect the accuracy of fluorescence intensity-based measurements. Therefore, it is necessary to understand how noise or autofluorescence will affect the results and how to correct these potential distortions.

[0214] The fluorescence intensity (F) contains the actual signal (r) and noise / autofluorescence interference (n), so the signal-to-noise ratio (snr) is r / n.

[0215] F = r + n, and snr = r / n (VIII)

[0216] Then substituting equation (VIII) into the logarithm of the fluorescence signal ratio, we get:

[0217]

[0218] Rearranging the equation, we get:

[0219]

[0220] Obviously, to obtain the logarithm of the true fluorescence signal ratio, we only need to calculate and adjust the measurement results according to the second term in Equation (IX), which involves the signal-to-noise ratio of the imaging channels. The second logarithmic term in Equation (IX) is linearly separated from the logarithm of the true signal ratio, making the correction simple and efficient. In addition, Equation (IX) also shows that if the imaging channels have comparable signal-to-noise ratios, the pH measurement will not be affected by background noise / spontaneous fluorescence, which is an advantageous advantage and reliability of ratio measurement. In our experiments, in most cases, the adjustment range of pH measurement is between 0.003 and 0.03.

[0221] If the signal-to-noise ratios of the two imaging channels are very good, the Taylor expansion of the second term in Equation (IX) is approximately

[0222]

[0223] Using Equations (VII), (IX) and (X), we get:

[0224]

[0225] Therefore, Equation (XI) tells us that the noise signal-to-noise ratio of the alkaline channel (peak at 515 nm) will cause the pH value to be overestimated, while the noise signal-to-noise ratio of the acidic channel (peak at 465 nm) will cause the pH measurement to be underestimated. In a worse case, the superposition and deviation effects of multiple noisy channels may occur.

[0226] Equation (XI) demonstrates how noise or spontaneous fluorescence affects the results. However, for better correction, it is recommended to use Equation (IX), especially for the correction of spontaneous fluorescence. When the signal is mixed with strong spontaneous fluorescence, the situation will be more serious, such as mitochondrial subcellular structures. The troublesome thing is that the adjustment of spontaneous fluorescence and the correction of background noise usually cannot be completed in the same experiment.

[0227] Due to the logarithmic analysis, the second logarithmic signal-to-noise ratio term for calibration in Equation (IX) can fortunately be linearly separated logarithmically from the true signal ratio. To minimize the error in pH measurement, it is necessary and feasible to conduct independent control experiments to measure and evaluate the parameters of noise and autofluorescence under the same conditions in order to accurately estimate the signal-to-noise ratio. In particular, the intensities of FAD autofluorescence and NADH autofluorescence are large, so the ratio of FAD and NADH autofluorescence is often used as a measurement of mitochondrial redox potential. Therefore, our previous study used this ratio to represent the dynamic changes in mitochondrial metabolism (Xie et al., 2017). Thus, minimizing the influence of autofluorescence is particularly important for the pH accuracy of mitochondrial substructural regions. In our experiment, the pH correction range in mitochondrial substructural regions was between 0.18 - 0.25, which is one order of magnitude higher than the adjustment range of other organelles due to the autofluorescence interference of mitochondrial metabolites NADH and FAD.

[0228] Example 1 Spectral Characteristics of mTurquoise2, mTurquoise2 S65T, and W66Y Mutants

[0229] In the research of the present invention, we successfully constructed a fluorescent protein with quantum entanglement properties and sensitive to pH, called SITE-pHorin (single excitation and dual emission pH sensor protein). This protein has a main excitation wavelength and two different emission wavelengths, and can accurately measure the pH value of cytoplasmic organelles using the ratio method. SITE-pHorin has a higher quantum yield (QY), thus obtaining a stronger fluorescent signal. In addition, it also exhibits a wider pH response range, covering the range from pH 3.5 to 9.0. The pH value of the organelle is determined by calculating the fluorescence intensity ratio between the two emission wavelengths, thus providing accurate and quantitative pH measurement results.

[0230] Due to the relatively fast maturation rate, high photostability, long single-exponential lifetime of mTurquoise2 (a monomeric blue-green fluorescent protein), and the highest quantum yield of up to 93%, it was selected as a template for engineering a brighter and pH-sensitive fluorescent protein. However, its extremely low pKa value of 3.1 limits its application in detecting the pH of cytoplasmic organelles. After extensive mutagenesis exploration, we found that the S65 and Y66 residues of mTurquoise2 may affect its pH-sensitive properties. Indeed, compared with mTurquoise2, the mutants of mTurquoise2 (S65T and W66Y) exhibit enhanced pH-sensitive properties ( Figure 1 ).

[0231] To verify their pH-sensitive and spectral properties, we expressed and purified mTurquoise2, mTurquoise2 S65T, and mTurquoise2 W66Y proteins using Escherichia coli BL21(DE3) strain. We recorded the excitation and emission spectra in the pH range from 3.5 to 9.0 using a 96-well microplate fluorometer. As expected, the S65T mutant did not alter the spectral properties of mTurquoise2. Whether it was the mTurquoise2 wild type ( Figure 1 A, 1B) or S65T ( Figure 1 C, 1D), the maximum excitation wavelength was at 440 nm, and the maximum emission wavelengths were at 475 nm, respectively. S65T did enhance the pH responsiveness. In contrast, W66Y restored the fluorophore of mTurquoise2 to that of GFP (S65-Y66-G67), and thus its spectral properties were similar to those of the wild-type green fluorescent protein GFP found in Aequorea victoria. It showed a maximum excitation wavelength of 395 nm and a maximum emission wavelength of 510 nm ( Figure 1 E, 1F). Compared with the mTurquoise2 wild type, the W66Y mutation also increased the pH-sensitive property of mTurquoise2 in the pH range from 3.5 to 9.0. By comparing the fluorescence intensities of the normalized emission peaks at 475 nm (mTurquoise2), 475 nm (mTurquoise2 S65T), and 510 nm (mTurquoise2 W66Y), we confirmed that the two mutants of mTurquoise2, S65T and W66Y, exhibited higher pH-sensitive properties compared with mTurquoise2 ( Figure 1 H). In addition, the full width at half maximum (FWHM) of excitation and emission, as well as the pH value of the maximum emission ( Figure 1 G), were plotted to further supplement the characterization of mTurquoise2, mTurquoise2 S65T, and mTurquoise2 W66Y.

[0232] Example 2 Key residue D148 and saturation mutagenesis

[0233] Although the pH-sensitive properties of mTurquoise2 S65T and W66Y are higher than those of mTurquoise2, they are still not ideal pH probes. An ideal probe should have a major excitation peak and two emission peaks, and use an accurate ratio method for pH quantitative analysis. The ratio method (Hanson et al., 2002) has obvious advantages in measurement, which can reduce or eliminate experimental data errors caused by factors such as probe abundance, illumination stability, and imaging settings. To further improve the spectral properties of mTurquoise2, we aimed to clarify its pH-sensitive mechanism by studying the crystal structures of mTurquoise2 S65T and W66Y at pH 6.5 ( Figure 2 A), and the specific method is described in the Methods section. Their structures were resolved by the molecular replacement method, with resolutions of and (Table 2). In mTurquoise2 W66Y, D148 adopts a loop conformation, while in mTurquoise2 S65T, it forms a β-sheet ( Figure 2 A). By comparing the crystal structures of mTurquoise2 S65T and W66Y, we found that the fluorophore (S65-Y66-G67, white) of mTurquoise2 W66Y forms hydrogen bonds with D148 and N121, while the fluorophore (T65-W66-G67, cyan) of mTurquoise2 S65T forms hydrogen bonds with S205 ( Figure 2 B). Subsequently, we used MOLE 2.5 software to analyze the channels of mTurquoise2, mTurquoise2 S65T, and mTurquoise2 W66Y. Interestingly, mTurquoise2 has no channel ( Figure 9 A), while mTurquoise2 S65T ( Figure 9 B) and mTurquoise2 W66Y ( Figure 9 C) exhibit channels that can easily pass water molecules. This indicates that mTurquoise2 S65T and W66Y may be more sensitive to changes in hydrogen ion concentration than mTurquoise2. According to the speculated hydrogen bond networks of mTurquoise2 and mTurquoise2 S65T, the fluorophores of these two proteins can form hydrogen bonds with the V61, Q94, R96, and S205 residues ( Figure 10 A, 10C). In contrast, mTurquoise2 W66Y also forms additional hydrogen bonds with the E222 residue ( Figure 11 A). Charge electrostatic surface analysis shows that the carbonyl group of glycine in the mTurquoise2 fluorophore (SWG) is converted to a hydroxyl group, deprotonated, and negatively charged (Figure 10 B). However, the carbonyl group of glycine in the mTurquoise2 S65T fluorophore (TWG) is uncharged ( Figure 10 D). In contrast, the mTurquoise2 W66Y fluorophore (SYG) not only contains the hydroxyl group of glycine, similar to the mTurquoise2 fluorophore which can be deprotonated, but also has the phenolic hydroxyl group of tyrosine as a deprotonation site ( Figure 11 B). Therefore, mTurquoise2 W66Y has two sites that can bind and dissociate hydrogen ions. In summary, biochemical analysis and crystal structure analysis confirmed that mTurquoise2 W66Y and mTurquoise2 S65T are more sensitive to pH changes than mTurquoise2 within the physiological pH range.

[0234] We also investigated whether the C48 and T203 residues of mTurquoise2 would affect the pH-sensitive properties of the protein. To study this, we mutated the C48 and T203 residues and examined the excitation ( Figure 8 A, 8C) and emission spectra ( Figure 8 B, 8D) of the single mutants C48S and T203C and found that these two mutations had no effect on the pH-sensitive properties and fluorescence spectra of mTurquoise2 ( Figure 1 A, 1B). Subsequently, we analyzed the excitation and emission spectra of the double mutant S65T / W66Y ( Figure 2 C), which showed the characteristics of a double-excitation and single-emission mode. In addition, we also detected various double mutants involving combinations of the S65T, W66Y, D148C, and T203C mutants, such as S65T / D148C ( Figure 12 A), S65T / T203C ( Figure 12 B) and triple mutants such as S65T / W66Y / T203C ( Figure 12 E), S65T / D148C / T203C ( Figure 12 F). We found that the double mutants (W66Y / D148C) ( Figure 12 C), the triple mutants (S65T / W66Y / D148C) ( Figure 12 D) and (W66Y / D148C / T203C) ( Figure 12 G) all had a single main excitation peak and two emission peaks.

[0235] Considering the above results and the imperfect spatial structure of D148 in mTurquoise2 W66Y ( Figure 2A), we speculated that D148 might be the key residue for pH sensitivity and spectra. Therefore, we performed saturation mutagenesis of D148 based on the mTurquoise2 quadruple mutant (C48S / S65T / W66Y / T203C)( Figure 12 H). Among these mutants with five mutations of mTurquoise2, D148K( Figure 2 E) and D148S( Figure 2 F) showed the characteristics of dual excitation peaks and dual emission peaks. The D148K mutant showed a major excitation peak at 400 nm in a pH 6.0 buffer solution, while D148E( Figure 13 A), D148Q( Figure 13 B), D148I( Figure 13 C), D148H( Figure 13 D), D148W( Figure 13 E), D148P( Figure 13 F) and D148Y( Figure 13 G) mutants had similar spectra. On the other hand, D148S had a major excitation peak around 500 nm, and D148R( Figure 14 A), D148N( Figure 14 B), D148M( Figure 14 C) and D148T( Figure 14 D) mutants were similar. Ideally, we found some mutants, namely D148G( Figure 15 A), D148A( Figure 15 B), D148F( Figure 15 C), D148C( Figure 15 D), D148L( Figure 15 E) and D148V( Figure 15 F), which had the characteristics of one major excitation peak and two emission peaks. Among these mutants, we observed that the D148G mutant showed the highest excitation efficiency( Figure 2 G) and emission intensity( Figure 2 H) in a pH 6.0 buffer solution. Therefore, we named this C48S / S65T / W66Y / T203C / D148G quintuple mutant SITE-pHorin. SITE-pHorin has the characteristics of one major excitation peak and two emission peaks, making it a promising probe for quantitative analysis. As implied by the pun of SITE, our goal is to obtain the pH map of cytoplasmic organelles using SITE-pHorin as a pH probe, so it is necessary to further study its pH-sensitive characteristics.

[0236] Example 3 pH-Sensitive Characteristics of SITE-pHorin

[0237] Currently, the pH-sensitive fluorescent protein deGFP4 is the only dual-emission probe with single-excitation and dual-emission characteristics that can be detected using the ratio method. Therefore, we analyzed and compared the emissions ( Figure 3 A, 3C) and excitations ( Figure 3 B, 3D) of SITE-pHorin ( Figure 3 A, 3B) and deGFP4 ( Figure 3 C, 3D) in different buffer solutions at pH 3.5 to 9.0. Notably, the main emission peak of SITE-pHorin showed stronger fluorescence intensity ( Figure 3 A) and higher excitation efficiency ( Figure 3 C), especially under alkaline conditions. Similarly, both SITE-pHorin and deGFP4 showed a weak emission peak around 465 nm, which increased under acidic conditions ( Figure 3 A, 3B). In addition, we plotted the logarithm of the fluorescence intensity ratio of the emission peaks of SITE-pHorin and deGFP4 in the pH range of 3.5 - 9.0 and showed that the pH-sensitive response range of SITE-pHorin was wider and more linear than that of deGFP4 ( Figure 3 E). In summary, the optical properties of SITE-pHorin and deGFP4 are listed in Figure 3 F. SITE-pHorin showed a higher fluorescence quantum yield (QY) of 0.56 at pH 8.0, while the QY of deGFP4 was 0.15. At the same time, both SITE-pHorin and deGFP4 had lower quantum yields at pH 5.0. To further verify the pH-sensitive characteristics of SITE-pHorin at the protein level, we expressed it in E. coli BL21(DE3) and measured its fluorescence intensity in different buffer solutions at pH 3.5 to 9.0 ( Figure 3 G). The fluorescence ratios obtained in the emission channels (alkaline-sensitive channel >500 nm and acidic-sensitive channel <480 nm) at pH 3.5 - 9.0 are represented by pseudocolors, and their scales are shown on the left side of the three panels in Figure 3 G. As shown, the wide scale range indicates that SITE-pHorin has strong pH-sensitive characteristics.

[0238] Example 4 Quantum Entanglement Mechanism of the pH-Sensitive Characteristics of SITE-pHorin

[0239] To study the pH-sensitive mechanism of SITE-pHorin, we obtained crystal structures under high pH 7.0 and low pH 5.5 crystallization conditions. The crystal structures were solved by the molecular replacement method, and the resolutions reached and (see Table 2). Analysis of the hydrogen-bonding network and charge distribution formed by the SITE-pHorin fluorophore revealed an interesting finding. Notably, the phenolic hydroxyl group of the Y182 residue located outside the β-barrel structure is deprotonated at pH 5.5, which cannot be explained by classical chemical theory because the pKa of phenol is approximately 10. Meanwhile, at low and high pH conditions, the phenolic hydroxyl groups of the Y182 residue and the fluorophore show opposite protonation or deprotonation states ( Figure 4 A-D). Under different pH conditions, this phenomenon does not exist in the published structures of deGFP1 ( Figure 16 A-D) and GFP S65T ( Figure 17 A-D). Interestingly, we also observed that the phenolic hydroxyl group of the Y182 residue in GFP S65T is also deprotonated at low pH ( Figure 17 A,17C), but the phenolic hydroxyl group of the fluorophore remains deprotonated at low and high pH conditions ( Figure 17 A,17C). Meanwhile, in GFP S65T, the distance between the phenolic hydroxyl groups of Y182 and the fluorophore remains unchanged at different pHs. In contrast, in SITE-pHorin, these distances were measured to be at pH 7.0 measured as These phenomena led us to speculate that the opposite protonation and deprotonation of the fluorophore and Y182 in SITE-pHorin can only be explained by quantum entanglement (for more discussion and theoretical modeling, see the Methods section), which may lead to quantum effects (n = 1 / 2), thus resulting in the pH-sensitive properties of SITE-pHorin ( Figure 18 A,18B).

[0240] To further confirm whether the potential quantum mechanism plays a role in the pH-sensitive properties of SITE-pHorin, we introduced a charged C203E mutant, which is a residue that forms a hydrogen bond with the key residue G148 ( Figure 4 A and 19A,19B), to interfere with the possible quantum effects in SITE-pHorin. Subsequently, we analyzed the crystal structures of SITE-pHorin C203E at pH 5.0 and pH 8.0, with resolutions reaching and (Table 2). Contrary to the hydrogen-bonding network and charge distribution of SITE-pHorin, we observed that the phenolic hydroxyl group of the fluorophore in SITE-pHorin_C203E carries two negative charges at pH 5.5 and pH7.0 ( Figure 4E-G, 19A, 19B). Thus, the C203E mutant eliminates the quantum entanglement between the phenolic hydroxyl group of residue Y182 and the fluorescent group in SITE-pHorin. In addition, we used real-time imaging of the fluorescence kinetics of SITE-pHorin and its C203E mutant by changing the buffer at pH 7.0 or pH 5.0 to observe its pH-sensitive properties. In Figure 4 H, the results showed that SITE-pHorin_C203E (red curve and red vertical axis) lost the pH-sensitive property of SITE-pHorin (blue curve). Thus, the structural and real-time imaging experiments of the single mutant (C203E) demonstrated that the quantum entanglement mechanism is specific for the pH-sensitive property of SITE-pHorin.

[0241] Example 5 SITE-pHorin Localized to Organelles and Their Sub-organelles

[0242] To use SITE-pHorin as a single pH probe to achieve the intracellular pH profile of organelles, we first evaluated the performance of SITE-pHorin in live cell imaging in COS-7 cells and demonstrated that the fluorescence in the emission channel of SITE-pHorin in COS-7 cells showed a wide pH response range and a good signal-to-noise ratio under different pH buffers from pH 3.5 to 9.0 ( Figure 5 A). Next, to localize SITE-pHorin to the luminal surface of various organelles and their sub-organelles, we determined appropriate markers through extensive screening ( Figure 5 B). Specifically, we used histone H2B as a classical nuclear marker, the g subunit of ATP synthase as a mitochondrial cristae marker (identified in the cryo-EM structure of mammalian ATP synthase), 4Cox8 as a mitochondrial matrix marker (composed of four repeated cytochrome c oxidase subunit 8), ferrochelatase (Fech) as a mitochondrial intermembrane space marker, Dnase2B as a lysosome marker, Calreticulin as an endoplasmic reticulum marker, ST6GAL1 as a trans-Golgi marker, MGAT2 as an intermediate Golgi marker, GP73 as a cis-Golgi marker, and SITE-pHorin-SKL as a peroxisome marker (where the last two amino acids YK of SITE-pHorin were mutated to SKL). TFR1 was selected as an endosome marker ( Figure 5 B). Details of these organelle localization sequences are provided in Table 1. The targeting specificity of these SITE-pHorin targeted to specific organelles was verified by confocal imaging ( Figure 5C). Interestingly, the fluorescence distribution of TFR1-tagged SITE-pHorin indicated that the vesicles localized with TFR1 could be divided into at least two populations with different pH values ( Figure 5 C).

[0243] Example 6 Organelle pH Distribution Map

[0244] Finally, to measure the pH of organelles, we analyzed COS-7 cells transfected with approximately 100 SITE-pHorin fusion plasmids targeting organelles. The measured ratios were converted to pH using Equation (VII) (see Materials and Methods). To accurately determine the pH value, background noise and autofluorescence were corrected using Equation (IX) (see Materials and Methods) to obtain accurate pH measurements, especially for organelles with strong autofluorescence, such as mitochondrial substructural regions. In addition, to obtain precise pH information of organelles and their substructural regions, the pH measurements unaffected by noise and autofluorescence were further fitted with single or multiple Gaussian distributions ( Figure 6 ). The pH of the cytoplasm was determined to be 7.41 ± 0.17, while the pH of the nucleus was approximately 7.43 ± 0.15. The pH range of the endoplasmic reticulum was approximately 7.20 ± 0.14 ( Figure 6 A). As expected, the pH within the Golgi apparatus showed a gradient distribution among its three sub-unit stacks, with a pH of 6.22 ± 0.20 in the trans-Golgi, 6.58 ± 0.22 in the medial-Golgi, and 6.64 ± 0.14 in the cis-Golgi ( Figure 6 A). In addition, the pH of peroxisomes was found to be 8.20 ± 0.21. In contrast, lysosomes maintained an acidic pH of 4.79 ± 0.17. Interestingly, our observations revealed three different types of endosomes labeled by TFR1, with pH values of 5.33 ± 0.50 (46%), 6.89 ± 0.70 (31%), and 7.82 ± 0.34 (23%), respectively, with acidic endosomes being the majority ( Figure 6 B).

[0245] Importantly, for mitochondrial sub-organelle structures, we found that there were two mitochondrial sub-populations with matrix pH values of 7.50 ± 0.16 (58%) and 7.20 ± 0.27 (42%); the pH distribution in the mitochondrial cristae space was 6.60 ± 0.40; and the pH value of the mitochondrial intermembrane space was measured to be 6.95 ± 0.30 ( Figure 6C). These measurements revealed a pH gradient of approximately 0.6 - 0.9 pH units between the mitochondrial cristae space and the mitochondrial matrix. This finding has significant implications for cellular energetics. Previously, this pH difference had been underestimated for a long time in the field of life sciences, especially in the context of mitochondria. Additionally, the pH value in the mitochondrial intermembrane space of approximately 6.95 ± 0.30 also corrected a long-standing misconception about the permeability of the mitochondrial outer membrane, showing a natural gradient between the mitochondrial intermembrane space and the mitochondrial cristae space. Finally, we created a cartoon summarizing the pH map of all organelles and their substructural regions measured by the pH-sensitive pH probe SITE-pHorin with quantum entanglement properties ( Figure 7 ).

[0246] Discussion:

[0247] In this study, we demonstrated that SITE-pHorin is a ratiometric pH-sensitive sensor. SITE-pHorin has several advantages over other fluorescent proteins, including a broad pH response range that can cover from pH 3.5 to 9.0, enabling it to measure the pH of most organelles. Its main advantage is that it has a major excitation peak and two emission peaks, allowing for ratiometric methods to quantitatively determine the pH of organelles. We successfully measured the pH of various organelles and their sub-organellar structures, including mitochondrial sub-organellar regions, Golgi sub-organellar structures, endoplasmic reticulum, lysosomes, peroxisomes, and endosomes, using SITE-pHorin conjugated with organelle-targeting tags.

[0248] Importantly, based on the recently published electron microscopy structural information of ATP synthase (Gu et al., 2019), we made a small but very crucial progress by fusing the g subunit of ATP synthase with SITE-pHorin to measure the pH value and gradient of the mitochondrial cristae. Previous studies found that the pH difference (ΔpH) across the mitochondrial inner membrane was 0.4 - 0.6 pH units (Kamo et al., 1979). The ΔpH we measured was 0.6 - 0.9 pH units, indicating that the previous understanding underestimated the ΔpH, probably due to the effects of uncorrected noise and autofluorescence. Since ΔpH is an important factor driving the formation of the proton motive force, the underestimated ΔpH led to an underestimation of the mitochondrial proton motive force. Another of our studies showed that the underestimated mitochondrial membrane potential might be an inducer of aging and late-onset diseases, as reducing the mitochondrial membrane potential by a few millivolts using a newly developed mitochondrial optogenetic tool could reduce DNA double-strand breaks in COS-7 cells.

[0249] Furthermore, our study interestingly revealed that the lysosomal localization distribution of TFR1-SITE-pHorin is in at least three regions with different pH values (Figure 6 B). pH measurements indicate the presence of diverse TFR1 endosomes, including acidic endosomes (early and late endosomes), as well as lysosomes; on the other hand, based on the fitting results and pH maps ( Figure 7 ), we speculate that the basic endosomes labeled with TFR1 may interact with peroxisomes and mitochondria. In the near future, SITE-pHorin may enable in vivo imaging and quantitative detection of pH changes in tissues and whole organisms under various physiological and pathological conditions, thereby providing in-depth understanding of the role of pH regulation in biological processes and revealing key factors in disease onset and progression. However, further research is needed to verify and clarify these speculations.

[0250] The most unexpected phenomenon is quantum entanglement, which appears in the structural evolution and optimization of fluorescent proteins. Figure 4 It is shown that the protonation / deprotonation of the phenolic hydroxyl group of Y182 is tightly and conversely coupled with the state of the phenolic hydroxyl group of the SITE-pHorin fluorophore, and vice versa. Thus, this tight coupling mechanism achieved through quantum entanglement enables the fluorophore of SITE-pHorin to sense pH changes in its surrounding environment through pH-sensitive residues (the external Y182 residue), resulting in the pH-sensitive property of SITE-pHorin.

[0251] The quantum entanglement mechanism of the pH-sensitive property of SITE-pHorin is not yet clear. We carried out computational analysis based on density functional theory (DFT) within the B3LYP / 6-31G** computational framework, which is a widely recognized method in quantum chemistry. This method enables us to calculate the interaction energy between the Y182 residue and the fluorophore under different pH conditions. Specifically, we explored the cases under low and high pH conditions to comprehensively evaluate the impact of protonation / deprotonation changes on the interaction energy (see Table 3). As a control, we studied GFP-S65T, which also shows deprotonation of its Y182 residue ( Figure 10 ). Surprisingly, under low pH conditions, deprotonation of the Y182 residue in both SITE-pHorin and GFP-S65T is sufficient to induce interaction, while under high pH conditions, the interaction between the protonated Y182 residue and the fluorophore is very small (Table 3). Apparently, just having deprotonation of Y182 is not sufficient to trigger quantum entanglement.

[0252] Table 3

[0253]

[0254] To ensure the accuracy of the calculations, we recalculated the interaction energy of the fluorophore in SITE-pHorin under low pH conditions using a larger basis set (Aug-cc-pVDZ), obtaining a value of -0.3653 Hartree (approx. -9.94 eV). This is close to the energy of a distorted benzene ring (Casanova and Alemany, 2010), so we measured the internal angles of the benzene ring of Y182 in SITE-pHorin ( Figure 20 A) and GFP-S65T ( Figure 20 B). We found that at pH 5.5, the benzene ring angle in the SITE-pHorin fluorophore was 719.7°, differing from the standard internal angle sum of the benzene ring of 720.0° by -0.3°. Considering the rigidity of the benzene ring structure and the relatively large mean square error of the bond angles (approx. 0.966 - 1.938°) (see the bond angles of each crystal structure in Table 2), we comprehensively analyzed the internal angle sum and distribution of the benzene ring structures in the phenylalanine and tyrosine residues of SITE-pHorin under different pH conditions. The sum of the internal angles of these benzene ring structures follows a Gaussian distribution, with a fitting peak position of 719.98° and a relatively low standard deviation of 0.076° ( Figure 20 C), which is consistent with the rigidity of the benzene ring structure. Therefore, the distribution of the internal angle sum of the benzene ring structure indicates that the benzene ring angle in the SITE-pHorin fluorophore at pH 5.5 is 719.7°, significantly deviating from the normal value of the standard 720.0° (p = 0.000115), indicating the presence of discrete curvature (Gu et al., 2013), which is -0.3° ( Figure 20 D). The difference in the interaction energy (-0.0161 Hartree / approx. -0.438 eV) between SITE-pHorin and GFP-S65T in Table 3 may be related to the curvature, which may be necessary for quantum entanglement because simply deprotonating Y182 is not sufficient. In the field of physics, gravity and Yang-Mills field strength are intertwined with the form of curvature (Baez and Muniain, 1994). Quantum gravity was once considered weak and may now be closely related to the quantum entanglement mechanism of SITE-pHorin. This potential connection comes from the square relationship between gravity and gauge field strength (Borsten, 2020). Although little is known about quantum entanglement under high pH conditions, it may have a similar mechanism to that in a low pH environment. Quantum entanglement in SITE-pHorin is most likely triggered during the structural maturation process after its expression. Therefore, the pH-regulated quantum properties exhibited by SITE-pHorin are not only attractive to biologists but also have potential implications for chemists and physicists exploring quantum chemistry, quantum mechanics, and quantum gravity.

[0255] Sequence information:

[0256] SEQ ID NO:1:

[0257] MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLSWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYFSDNVYITADKQKNGIKANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYK

[0262] SEQ ID NO:2:

[0263] MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFISTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYFSGNVYITADKQKNGIKANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSCQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYK

[0264] SEQ ID NO.:3:

[0265] ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCAGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCGCCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCA AGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACTTTAGCGGAAACGTCTATATCACCGCCGACAAGCAGAAGAACGGCATCAAGGCCAACTTCAAGATCCGCCACAACATCGAGGACGGCGGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCTGCCAGTCCAAGCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA

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[0283] All documents mentioned in this invention are cited herein as references, as if each document was cited individually as a reference. In addition, it should be understood that after reading the above teachings of this invention, those skilled in the art can make various changes or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

1. A pH-sensitive sensor, characterized in that, The sensor is a non-natural protein, and the sensor has mutations at one or more core amino acid sites related to pH-sensitive characteristics corresponding to SEQ ID NO: 1 of the wild-type mTurquoise2 protein, selected from the group consisting of: D148, T203, W66, S65, C48.

2. An isolated polynucleotide, characterized in that, The polynucleotide encodes the pH-sensitive sensor according to claim 1.

3. A carrier, characterized in that, It contains the polynucleotide according to claim 2.

4. A host cell, characterized in that, It contains the vector according to claim 3 or the polynucleotide according to claim 2 is integrated into the genome.

5. A method for producing the pH-sensitive sensor according to claim 1, characterized in that, Comprising the steps of: Culturing the host cell according to claim 4 under conditions suitable for expression, so as to express the pH-sensitive sensor according to claim 1; and Optionally, isolating the pH-sensitive sensor.

6. A pharmaceutical composition, characterized in that, Comprising: The pH-sensitive sensor according to claim 1; and a pharmaceutically acceptable carrier.

7. A reagent for biological imaging detection, pH detection or cell microenvironment detection, characterized in that, Comprising: The pH-sensitive sensor according to claim 1.

8. A protein preparation, characterized in that, The protein preparation contains the pH-sensitive sensor according to claim 1.

9. Use of the pH-sensitive sensor according to claim 1, characterized in that, For the preparation of a drug or preparation or reagent for biological imaging detection, pH value detection or cell microenvironment detection.

10. A method for biological imaging detection, pH value detection or cell microenvironment detection, characterized in that, Comprising the step of: contacting the pH-sensitive sensor according to claim 1 or the pharmaceutical composition according to claim 6 or the reagent according to claim 7 with cells or tissues.

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