Preparation and application of fluorescent probe applicable to single-molecule FRET (Fluorescence Resonance Energy Transfer) technology

Abstract

The invention relates to a p-toluenesulfonyl functionalized fluorescent probe capable of being specifically combined with a histidine label and application of the p-toluenesulfonyl functionalized fluorescent probe in single-molecule FRET (Fluorescence Resonance Energy Transfer) research. The probe is based on a Cyanine 5 dye, and can realize covalent binding with a protein with a His-tag by performing chemical modification on toluenesulfonyl. Compared with a traditional Ni < 2 + >-NTA non-covalent fluorescent probe, the probe provides higher binding stability and site specificity, and is suitable for high-resolution single-molecule fluorescence imaging of protein. The invention further establishes a protein fluorescence labeling strategy which is suitable for smFRET analysis and is universal, simple and convenient to operate and stable in labeling, and a novel technical means is provided for exploring protein conformation change and a dynamic regulation and control mechanism.

Description

TECHNICAL FIELD

[0001] The present application relates to a p-toluenesulfonyl functionalized fluorescent probe capable of specifically binding to a protein methionine tag and its application in single-molecule FRET research. BACKGROUND

[0002] Single-molecule fluorescence resonance energy transfer (smFRET) technology has been widely used to study the interactions and conformational changes of biological macromolecules at the single-molecule level in real time. A typical smFRET experiment requires labeling the target biomolecule with a pair of fluorescent donor and acceptor molecules. In contrast, the specific labeling of nucleic acid molecules has become a routine operation and can even be obtained directly through commercial channels; however, there is still a lack of a universal strategy for protein-specific site labeling for smFRET analysis.

[0003] The commonly used protein fluorescence labeling strategies include genetically encoding fluorescent proteins and small organic molecule fluorescent probes. Fluorescent proteins have the advantages of transient transfection and direct integration into proteins, without the need for chemical synthesis, but their relatively large structure may interfere with the positioning and function of proteins. In contrast, small molecule organic fluorescent dyes have less interference with protein function and are therefore more promising. Organic small molecule fluorescent probes include reaction of maleimide functionalized dyes with cysteine residues in proteins, or reaction of N-hydroxysuccinimide ester (NHS ester) with lysine residues on the surface of proteins. In these methods, the number of cysteine residues is usually limited and requires the introduction of specific site mutations; while the number of lysine residues is large and widely distributed, resulting in lack of site specificity of labeling, making the number and position of fluorescent dyes on each protein molecule uncertain.

[0004] To achieve high specificity labeling required for single-molecule research, small-tag labeling strategies have emerged in recent years, i.e., through genetic engineering means to fuse target proteins with specific recognition short peptides or chemical groups, thereby achieving site-specific binding and labeling. Such strategies include the introduction of unnatural amino acids, short peptide tags, chemical tags, and enzyme-catalyzed tags, etc. Among them, the use of two different self-labeling tags (such as SNAP and CLIP) to orthogonally label polyproline peptides of different lengths has been successfully applied to smFRET labeling. However, these tags have relatively large volumes and may not be suitable for proteins sensitive to the flexibility of the structure between the labeling site and the fluorophore.

[0005] His n His-tag is considered an attractive alternative labeling strategy in recent years due to its small volume, simple structure and wide use in protein purification. With His6-Ni2+ -NTA (nickel-trifluoroacetate) and fluorophore conjugated (His6-Ni 2+ -NTA-Fluorophore) are widely used for protein labeling. This His6-Ni 2+ -NTA based non-covalent labeling method has the advantages of simple operation and no need for chemical modification of proteins, but it still has many limitations in application. First, this labeling method relies on the reversible coordination of metal chelation, which is easy to dissociate under low concentration or washing conditions, making it difficult to meet the high requirements of single molecule fluorescence imaging for labeling stability. Second, the Ni 2+ -NTA system is sensitive to environmental conditions and is easily deactivated by factors such as pH, chelating agents or reducing agents. In addition, this method has a certain risk of non-specific background binding, which may interact with the natural histidine residues in the protein, affecting the signal-to-noise ratio of the experiment. Because the labeling is not covalently fixed, it also limits its use in dynamic imaging, single molecule surface modification, and long-term tracking, which require high stability. Therefore, it is urgent to develop a covalently labeled fluorescent probe with higher specificity and stability to expand the application potential of His-tag in single molecule fluorescence analysis. SUMMARY

[0006] The technical problem to be solved by the present application is to overcome the shortcomings of the prior art and provide a novel p-toluenesulfonyl functionalized Cyanine 5 fluorescent probe (fluorescent probe I) which can specifically conjugate and label proteins with a specific His-tag (His-tag) by covalent p-toluenesulfonyl method. 2+ The applicability of the traditional Ni

[0007] Invention idea

[0008] The present application provides a specific binding protein histidine tag p-toluenesulfonyl functionalized fluorescent probe, and a synthesis method thereof and its application in single molecule FRET research.

[0009] To solve the above technical problems, the technical scheme adopted by the present application is as follows: a p-toluenesulfonyl functionalized fluorescent probe which can specifically bind to a protein histidine tag, the compound structure of the fluorescent probe is as follows:

[0010]

[0011] The fluorescent probe exhibits a coexistence of the two compounds described above in a system with a pH of 6.0.

[0012] The method for preparing the p-toluenesulfonyl-functionalized fluorescent probe includes the following steps:

[0013] The compound 2,5-dioxopyrrolidone-1-yl 4-(3-p-toluenesulfonyl-2-(p-toluenesulfonylmethyl)propionyl)benzoate was dissolved in an organic solvent and ultrasonically dispersed. Then, the compounds Cy5 amine and triethylamine were added, mixed thoroughly, and stirred until the reaction was complete. The mixture was then purified by preparative chromatography to obtain the fluorescent probe.

[0014] The organic solvent is dimethylformamide.

[0015] The molar ratio of 2,5-dioxopyrrolidone-1-yl 4-(3-p-toluenesulfonyl-2-(p-toluenesulfonylmethyl)propionyl)benzoate to Cy5 amine is 1:3.

[0016] The reaction temperature is room temperature, and the reaction time is more than 1 hour.

[0017] The application of the fluorescent probe in single-molecule FRET fluorescence imaging.

[0018] Application of the fluorescent probe in fluorescence imaging of His-tag OmpC protein expressed in situ in live bacteria.

[0019] Beneficial effects:

[0020] (1) This invention, based on Cyanine 5 dye, employs toluenesulfonyl chemical modification to achieve covalent binding to His-tag proteins, effectively overcoming the limitations of traditional Ni dyes. 2+ The defect of easy dissociation of the -NTA system at low concentrations or under rinsing conditions improves the stability of the label in the smFRET experiment.

[0021] (2) The probe and His-tag have good binding specificity, which reduces non-specific binding to natural histidine residues in the protein, thereby significantly reducing background signal and improving signal-to-noise ratio.

[0022] (3) Compared with fluorescent proteins or macromolecular chemical tags, this probe is smaller in size and has less interference with protein conformation and function, making it more suitable for conformation-sensitive single-molecule studies.

[0023] (4) Compared with fluorescent proteins or macromolecular chemical tags, this probe is smaller in size and has less interference with protein conformation and function, making it more suitable for conformation-sensitive single-molecule studies.

[0024] (5) This probe can be applied to various single-molecule level experiments such as smFRET imaging, protein immobilization and long-term kinetic tracking, providing a universal tool for studying protein functional dynamics. Attached Figure Description

[0025] Figure 1 This is a schematic diagram of the structure of fluorescent probe I prepared in Example 1.

[0026] Figure 2 This is the preparative HPLC chromatogram of fluorescent probe I prepared in Example 1.

[0027] Figure 3 This is a schematic diagram of the reaction between fluorescent probe I and His-tag.

[0028] Figure 4 This is a high-performance liquid chromatography (HPLC) chromatogram of the reaction products of fluorescent probe I with different histidine peptides in Example 2.

[0029] Figure 5 This is a graph showing the MST binding analysis of fluorescent probe I with His-tag-containing SBD protein, His-tag-removed SBD protein, and His-tag-free MBP protein in Example 3.

[0030] Figure 6 This is a comparison of fluorescence imaging of OmpC-His-tag expression and non-expression states in E. coli live cells labeled with fluorescent probe I in Example 4.

[0031] Figure 7 This is a two-dimensional distribution of efficiency-diffusion parameter (E–S) for single-molecule FRET of a protein labeled with fluorescent probe I. Detailed Implementation

[0032] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; the reagents and materials described are commercially available unless otherwise specified.

[0033] This invention discloses a p-toluenesulfonyl-functionalized fluorescent probe (I), the compound structural formulas of which are shown in the figure below for two forms.

[0034] The structural formula of the compound represented by Formula I is as follows:

[0035]

[0036] Furthermore, the present invention provides a method for preparing the above-mentioned fluorescent probe, comprising the following steps:

[0037] Compound 2,5-dioxopyrrolidone-1-yl 4-(3-p-toluenesulfonyl-2-(p-toluenesulfonylmethyl)propionyl)benzoate (1.0 mg, 0.00167 mmol, Bio-Pharmaceutical) was dissolved in dimethylformamide (0.1 mL) and ultrasonically dispersed. Compound Cy5amine (3.7 mg, 0.005 mmol, Lumiprobe) was dissolved in 1.0 mL of dimethylformamide, and triethylamine (3.5 μL, 0.0025 μmol) was added and ultrasonically dispersed. The prepared solutions were then mixed to obtain a reaction solution, which was stirred at room temperature for more than 1 h. Subsequently, preparative reversed-phase high-performance liquid chromatography (prep-RP-HPLC) was performed using a C18 column with a gradient solution of acetonitrile-water containing 0.1% (v / v) formic acid to obtain fluorescent probe I.

[0038] Fluorescent probe I exhibits two coexisting forms, compounds 3 and 4, in a system at approximately pH 6.0. This system allows for rapid, reliable, and site-specific covalent modification of the His-tag via a two-step Michael addition reaction, where the two tosylate functional groups react with the two histidine residues in the His-tag to generate stable heterocyclic structures. Notably, at pH 6.0, the lysine residues lose their nucleophilic activity due to protonation, while the imidazole ring nitrogen of histidine retains good nucleophilicity, thus endowing the reaction with excellent selectivity.

[0039] In some embodiments, the organic solvent is dimethylformamide, which is used to dissolve or disperse the raw material evenly.

[0040] In some embodiments, in step (1), the molar ratio of the ester compound to the amine compound is 3:1.

[0041] In some embodiments, in step (1), the reaction time is more than 1 hour.

[0042] In some embodiments, the mobile phase used in step (1) is an acetonitrile-water gradient elution solution containing 0.1% (volume ratio) formic acid, used to prepare and purify the target compound.

[0043] The application of the aforementioned fluorescent probes in single-molecule FRET fluorescence is also within the scope of protection of this invention.

[0044] The application of the aforementioned fluorescent probe in fluorescence imaging of His-tag OmpC protein expressed in situ in live bacteria is also within the scope of protection of this invention.

[0045] Example 1: Preparation of Fluorescent Probe I

[0046] Compound 2,5-dioxopyrrolidone-1-yl 4-(3-p-toluenesulfonyl-2-(p-toluenesulfonylmethyl)propionyl)benzoate (1.0 mg, 0.00167 mmol) was dissolved in dimethylformamide (0.1 mL) and ultrasonically dispersed. Compound Cy5amine (3.7 mg, 0.005 mmol) was dissolved in 1.0 mL LDM, and triethylamine (3.5 μL) was added and ultrasonically dispersed. The prepared solutions were then mixed to obtain a reaction solution, which was stirred at room temperature for at least 1 h. The solution was then separated and purified by preparative liquid chromatography for subsequent use.

[0047] Example 2: HPLC detection of fluorescent probe I with different histidine peptides

[0048] In this embodiment, 12 peptides containing different numbers and positions of histidine residues were used for verification. Each of the 12 different peptides (5 mg) was dissolved in an appropriate volume of PBS buffer (pH 6.0) to prepare a 10 mM peptide solution. 1 μL of this 10 mM peptide solution was taken, followed by the addition of 1 μL of fluorescent probe I solution (10 mM) and 18 μL of methanol solution. After shaking and mixing, the mixture was reacted at room temperature for at least 3 hours. The reaction was then analyzed by high-performance liquid chromatography (HPLC). The same procedure was performed on the remaining 11 peptides.

[0049] The reactions of fluorescent probe I with different histidine peptides were characterized by high-performance liquid chromatography (HPLC). Specific HPLC chromatograms are shown below. Figure 4 .like Figure 4 As shown, the binding ability of the peptide to fluorescent probe I increases with the increase of histidine content. The binding was most significant when the histidine content was 100%. Furthermore, under the same histidine content, the binding effect was better when histidine and glycine were arranged alternately than when histidine was arranged continuously. The corresponding new peak signal in the HPLC chromatogram was more obvious, indicating that this arrangement helps to improve the binding efficiency of the peptide and fluorescent probe I.

[0050] Example 3: Detection of MST with / without His-tag protein using fluorescent probe I

[0051] Three protein stock solutions were prepared at a concentration of 5 μM: SBD (the first substrate-binding structural domain in the GlnPQ transporter, PDB ID: 4LA9), His-tag-removed SBD, and His-tag-free MBP (maltose-binding protein of the MalEFGK2ABC transporter system, PDB ID: 1OMP). SBD without His-tag and MBP served as negative controls, while SBD was the experimental group, for a total of three groups. Sixteen 0.2 mL PCR tubes were prepared for each group, numbered 1 to 16, for serial dilution. 10 μL of PBST buffer was added to tubes 2 through 16, and 20 μL of the protein stock solution was added to tube 1. Then, 10 μL of the solution from tube 1 was transferred to tube 2, mixed, and then another 10 μL was transferred to tube 3, and so on, until tube 16. After mixing in tube #16, discard the excess 10 μL of solution to complete the serial dilution of the protein. Then, add 10 μL of fluorescent probe I solution (50 nM) to each tube and mix thoroughly to complete sample preparation. Insert a capillary tube into each PCR tube to fully aspirate the mixture and fill the lumen. Finally, use a microscale thermophoresis (MST) system to detect and analyze the samples. Each experiment was repeated three times. The binding curves of fluorescent probe I with the three proteins are shown in [Figure number missing]. Figure 5 .like Figure 5 As shown, with increasing concentrations of His-tag-containing SBD protein, the MST signal exhibited significant changes, forming a typical S-shaped binding curve, indicating a specific interaction between this protein and fluorescent probe I. This result was consistent in repeated experiments, demonstrating good reproducibility. In contrast, when His-tag-removed SBD protein and His-tag-free MBP protein were subjected to MST detection under the same conditions, no significant signal changes were observed; their binding curves were approximately linear, indicating no significant specific interaction between them and fluorescent probe I. These results further validate that fluorescent probe I can specifically recognize and bind to the His-tag in proteins.

[0052] Example 4: Fluorescent probe I and E. coli live-cell fluorescence imaging

[0053] Experimental Procedure: 1) Induction of Membrane Protein Expression: First, the constructed pET-21a(+) recombinant plasmid carrying the membrane protein OmpC gene or OmpC-His fusion gene, and the empty pET-21a(+) plasmid, were transformed into BL21(DE3)pLysS supercompetent *E. coli*. After transformation, single colonies were picked from LB solid medium containing resistance selection agents and inoculated into LB liquid medium containing ampicillin and chloramphenicol, and cultured overnight at 37°C on a shaker. The next day, the overnight culture was diluted 1:100 into fresh LB liquid medium and cultured until the optical density (OD) of the bacterial culture was reached. 600 The concentration was approximately 0.6. Subsequently, isopropyl-β-D-thiogalactoside (IPTG) at a final concentration of 20 μM and rhamnose at a final concentration of 0.1% were added to the bacterial culture, and expression was induced at 30°C for 18 hours.

[0054] 2) Incubation of bacterial culture with fluorescent probe I: After culture, collect the bacterial cells by centrifugation at 2500×g for 5 min, discard the supernatant, and then resuspend the cells in PBS (pH 7.4) buffer and wash them three times to remove residual culture medium. Resuspend the washed bacterial cells in PBS buffer (pH 6.0) and adjust the bacterial culture to OD200. 600 The value is approximately 0.3. Take 300 μL of the resuspended bacterial culture and place it in a centrifuge tube. Add 2 μL of fluorescent probe I solution and incubate at room temperature for 1-2 hours. After incubation, wash three times with PBS buffer (pH 7.4) (3000×g, 5 min) to remove excess dye. Finally, resuspend the bacterial culture in PBS buffer (pH 7.4) for later use.

[0055] 3) Sample Preparation: First, a standard glass coverslip with dimensions of 22×22mm and a thickness of 0.17mm was selected as the sample slide. 35mg of agarose powder was weighed and dissolved in 35mL of ultrapure water. The solution was heated in a microwave oven until it became transparent, obtaining an agarose solution. Then, 500μL of the agarose solution was added dropwise to the surface of the glass slide, and another glass slide was used to flatten it evenly. The slide was allowed to stand for 20 minutes until the agarose solidified naturally. The top glass slide used for flattening was then removed, resulting in a smooth agarose gel layer. Next, 1.5μL of the treated bacterial suspension was added dropwise to the pre-prepared agarose gel slide. The slide was allowed to stand for 5 minutes until the bacterial suspension air-dried, and then covered with a coverslip for subsequent observation.

[0056] 4) Confocal Microscopy Imaging Test: Bacterial imaging was performed using a Zeiss LSM 900 system, which includes: an inverted confocal microscope, a regulated power supply, a laser cabinet, a confocal scanning detector, a stage control lever, a 63× oil immersion microscope, image acquisition and control software (ZEN), a manual focusing system, a stage, and a vibration-damping platform. The appropriate Cy5 dye channel and ESID brightfield channel were selected within the software. Professional immersion oil was added to the objective lens, and the prepared sample was placed on the stage. The coarse adjustment knob was slowly rotated to raise the objective lens until it was in close contact with the bottom of the slide. The formation of a complete, transparent oil film without air bubbles was observed. The fine adjustment knob was then used to find the focal plane and adjust until the image was clear.

[0057] The imaging parameters were set as follows: In ZEN software, Laser Power was adjusted to approximately 0.6%, and Master Gain was set to 650V. The image storage format was set to TIFF, the resolution to 1024×1024 pixels, and the data save path was specified. This data will be used for subsequent bacterial imaging detection research. See [link to data]. Figure 6 .Depend on Figure 6 Strong fluorescence signals were observed in the expression group, localized to the cell membrane, indicating that the His-tag transmembrane protein was correctly expressed and recognized by the fluorescent probe. Cells without the transporter protein showed no background fluorescence, indicating that the probe had high specificity for the target protein. Bright-field images showed that the two groups of cells were similar in number and morphology, ruling out the possibility that expression affected cell viability.

Claims

1. A p-tosyl functionalized fluorescent probe that can specifically bind to a proteome isoleucine tag, characterized in that, The compound structure of the fluorescent probe is as follows: The fluorescent probe presents the above two compounds coexisting form in a system with pH of 6.

0.

2. A method of preparing the p-toluenesulfonyl functionalized fluorescent probe of claim 1, characterized in that, The method comprises the following steps: The compound 2,5-dioxopyrrolidin-1-yl 4-(3-p-tolylsulfonyl-2-(p-tolylsulfonylmethyl) propanoyl) benzoate is dissolved in an organic solvent and ultrasonically dispersed, then the compound Cy5 amine and triethylamine are added, the mixture is uniformly mixed, then the reaction is stirred for sufficient time, and the fluorescent probe is finally obtained by purification through a preparative chromatograph.

3. The method of claim 2, wherein, The organic solvent is dimethylformamide.

4. The method of claim 2, wherein, The molar ratio of the compound 2,5-dioxopyrrolidin-1-yl 4-(3-p-tolylsulfonyl-2-(p-tolylsulfonylmethyl) propanoyl) benzoate to the Cy5 amine is 1:

3.

5. The method of claim 2, wherein, The reaction temperature is room temperature, and the reaction time is more than 1 hour.

6. The fluorescent probe of claim 1 is applied to single-molecule FRET fluorescence imaging.

7. The fluorescent probe of claim 1 is applied to fluorescence imaging of His-tagged OmpC protein expressed in situ by living bacteria.

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