CN111337666B

CN111337666B - I-motif recombination mediated FRET probe and application thereof in-situ imaging cancer cell surface protein homodimerization

Info

Publication number: CN111337666B
Application number: CN202010096115.0A
Authority: CN
Inventors: 姜玮; 阚爱玲; 张楠
Original assignee: Shandong University
Current assignee: Shandong University
Priority date: 2020-02-12
Filing date: 2020-02-17
Publication date: 2021-04-02
Anticipated expiration: 2040-02-17
Also published as: CN111337666A

Abstract

The present invention relates to an i-motif recombination-mediated FRET probe and its application for in situ imaging of the homodimerization of surface proteins of cancer cells. In this strategy, the aptamer of the target protein, the half-i of the i-motif forming sequence and the FRET donor-acceptor pair are integrated as probes. The probe is bound to the target protein through aptamer binding. The homodimerized protein brings the two probes closer to each other, allowing two identical Half-i sequences to recombine into i-motifs in the acidic tumor extracellular environment, triggering the FRET effect. The FRET occurrence pattern in this design matches the protein homodimerization pattern more closely. This strategy enables accurate and dynamic in situ imaging of mesenchymal epithelial transition (Met) receptor homodimerization on the surface of live tumor cells, with great potential in biomedical research.

Description

I-motif recombination mediated FRET probe and application thereof in-situ imaging cancer cell surface protein homodimerization

Technical Field

The invention belongs to the field of visual monitoring of protein homodimerization, and particularly relates to an i-motif recombination mediated Fluorescence Resonance Energy Transfer (FRET) strategy for in-situ imaging of homodimerization of live cancer cell surface proteins.

Background

The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.

Membrane proteins perform most of the essential biological functions through protein-protein interactions. In particular, it has been found that homodimerization of membrane proteins is an initial step in many signaling pathways, and is critical in both physiological regulation and cancer progression. For example, the mesenchymal epithelial transition (Met) receptor, a widely expressed transmembrane protein, plays an important role in homeostasis and cancer metastasis. Met-mediated activation of the signal transduction pathway is a typical homodimerization model. Upon stimulation by its ligand (hepatocyte growth factor, HGF), inactive Met monomers are converted to active homodimers, which in turn cause subsequent Met receptor phosphorylation and downstream signaling cascades. Revealing information such as expression level, spatial distribution and dynamic response of the protein monomers and homodimers can greatly contribute to the research of signal transduction networks.

Because of the ability to respond sensitively and reversibly to distance changes, FRET techniques are widely used to develop visualization strategies for protein homodimerization. The general principle of these strategies is to bind the FRET fluorescence pair to the target protein via a specific recognition element, thereby converting the invisible protein behavior into a visible fluorescence change. These methods do help people to understand the behavior characteristics of protein homodimerization more intuitively, and promote the research on the action mechanism of the protein homodimerization. However, the inventors found that: to accurately monitor subtle changes in cell membrane protein homodimerization, current FRET methods are not accurate enough. Specifically, protein homodimers are formed from two molecules of the same protein, so the donor and acceptor will bind randomly to the target protein monomer carrying the same recognition element. This random binding results in three combinations of donor and acceptor: one-half of the "donor-acceptor", one-quarter of the "donor-donor" and one-quarter of the "acceptor-acceptor" combinations. Of these three combinations, only the "donor-acceptor" combination is able to generate the expected FRET signal to indicate homodimerization of the protein. The other two combinations, while occupying homodimerized proteins, do not generate FRET signals indicative of protein dimers. This mismatch between FRET signal and protein homodimerization results in only half of the homodimerized protein being detectable by these methods, with a severe loss of signal.

Disclosure of Invention

To overcome the above problems, the present invention provides an i-motif recombination mediated FRET strategy for in situ imaging of the homodimerization of live cancer cell surface proteins. The FRET generation pattern in this design matches the protein homo-dimerization pattern more closely. The strategy realizes accurate and dynamic in-situ imaging of mesenchymal epithelial transition (Met) receptor homodimerization on the surface of a living cancer cell, and has great potential in biomedical research.

In order to achieve the technical purpose, the technical scheme adopted by the invention is as follows:

in a first aspect of the present invention, there is provided an i-motif recombination mediated FRET probe comprising: executing a chain Half-i @ Apt and a prebraked chain packer;

the executive chain Half-i @ Apt comprises a Half-i sequence, a Linker sequence, an aptamer sequence of a target protein, a Linker complementary to the Linker and a partial sequence of the Half-i, and Cy5 and Cy3 are respectively marked at the 5' ends of the Half-i and the Linker to be used as an acceptor and a donor for FRET;

the whole Linker, part of Half-i and part of the protein aptamer sequence are blocked by a Blocker chain in advance;

the Half-i is a fully symmetric two part resulting from the cleavage of the forming sequence of i-motif.

The probe is based on a FRET strategy mediated by i-motif recombination, and integrates an aptamer of a target protein, Half of an i-motif forming sequence (Half-i) and a FRET donor-acceptor into a probe. The probe is bound to the target protein by aptamer binding. The homodimerization of the proteins leads the two probes to be close to each other, so that two identical Half-interface sequences can be recombined into i-motif in an acidic cancer extracellular environment, and further the FRET effect is triggered.

It is to be noted that the present invention is directed to the homodimerization of proteins on cancer cell membranes, and that the realization of this technique must also rely on the acidic microenvironment characteristic of outside cancer cells.

In a second aspect of the present invention, there is provided a method for preparing a nucleic acid probe based on an i-motif recombination mediated FRET strategy, comprising:

mixing concentrated solutions of a Half-i @ Apt chain and a Blocker chain, heating to 90-95 ℃, maintaining for 4-5 minutes, slowly cooling to room temperature, and then placing in a dark place at 3-5 ℃ for more than 8 hours to ensure that the two chains are fully hybridized.

The probe shows satisfactory stability, specificity and real-time imaging capability, and can be used for in situ imaging of homodimerization of live cancer cell surface proteins.

In a third aspect of the invention, there is provided the use of any of the above probes for in situ imaging of homodimerization of a surface protein of a living cancer cell.

This strategy enables accurate, dynamic in situ imaging of Met receptor homodimerization on the surface of living cancer cells. Since aptamers to various membrane proteins can be obtained by in vitro screening, this strategy can be extended to monitoring of other proteins of interest for homodimerization by simply replacing the recognition sequence.

The invention has the beneficial effects that:

(1) the present invention proposes an i-motif recombination mediated FRET strategy for in situ imaging of the homodimerization of live cancer cell surface proteins. The probe exhibits satisfactory stability, specificity and real-time imaging capabilities. Importantly, the FRET signal used in this strategy to indicate protein homodimers is induced by recombination of two identical Half-i into i-motif in close proximity, which is highly matched to the mode of homodimerization, thereby making the strategy more fidelity in imaging homodimerization events. Using this strategy, we achieved accurate, dynamic in situ imaging of Met receptor homodimerization on the surface of living cancer cells. Since aptamers to various membrane proteins can be obtained by in vitro screening, this strategy can be extended to monitoring of other proteins of interest for homodimerization by simply replacing the recognition sequence. Given that membrane protein homodimerization is involved in multiple signaling pathways and plays an important role in the development and progression of cancer, this strategy will provide a powerful tool for relevant signaling networks and pathological studies.

(2) The operation method is simple and quick, has high accuracy, universality and strong practicability.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.

FIG. 1 is a schematic representation of the principle of the i-motif recombination mediated FRET strategy used to image in situ the homo-dimerization of live cancer cell surface proteins in example 1.

FIG. 2 is a non-denaturing polyacrylamide gel electrophoresis of a Half-i-linker and an exact-i-linker in a buffer solution of pH 7.4 and pH6.5 (Lane M: 20bp DNAmarker; Lane 1: Half-i-linker; Lane 2: exact-i-linker) in example 1 (A). (B) Circular dichroism spectra of Half-i in buffer pH 7.4 and pH 6.5. (C) UV absorption spectra of Half-i in buffers pH 7.4 and pH 6.5.

FIG. 3 is the ellipticity of Half-i at 287nm as a function of time in PBS pH6.5 from example 1 (with the ellipticity of Half-i in PBS pH 7.4 being taken as zero).

FIG. 4 is the ellipticity of Half-i at 287nm as a function of temperature in the buffer pH6.5 of example 1.

FIG. 5 is the cell viability of HepG2 and LNCaP cells treated with different concentrations of probe in example 1 (A). (B) Western blotting analysis of Met and p-Met expression in probe-treated HepG2 cells.

FIG. 6 is a time-dependent change (25 μm scale) of the CLSM image of HepG2 cells treated with Q-probe in example 1 (A). (B) Mean fluorescence intensity of CLSM images.

FIG. 7 is a western blotting analysis of Met receptors in (A) LNCaP and HepG2 cells in example 1. (B) Probes were used to image CLSM on LNCaP and HepG2 cells (scale bar 25 μm).

Fig. 8 is a CLSM image (scale bar 25 μm) of Met monomers and dimers in HepG2 cells treated with different concentrations of HGF in example 1 (a). (B) Western blotting analysis of Met (monomeric Met) and p-Met (dimeric Met) in HepG2 cells treated with different concentrations of HGF. (C) The probes were analyzed by flow cytometry for detection of HepG2 cells treated under different conditions (Control: no HGF and probe; i: no HGF; ii: 10ng/ml HGF; iii: 50ng/ml HGF; iv: 100ng/ml HGF).

FIG. 9 is CLSM images (25 μm scale) of probe against HepG2 cells under different treatment conditions in example 1 (A). (B) Western blot analysis of Met (monomeric Met) and p-Met (homodimeric Met) in HepG2 cells under different treatment conditions. (C) Flow cytometry probes were tested against HepG2 cells treated under different conditions (Control: no HGF and probe; i: no HGF; ii: 100ng/ml HGF; iii: 100ng/ml HGF plus 2. mu.g/ml anti-HGF antibody).

Fig. 10 is CLSM imaging of HGF treated HepG2 cells with probes and control probes in example 1.

FIG. 11 is the change over time of fluorescence in HGF-stimulated HepG2 cells of example 1 (A). (B) Fluorescence changes over time in HepG2 cells not stimulated with HGF. The scale bar is 25 μm.

Detailed Description

It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.

As described in the background section, the present invention addresses the problem of FRET signaling and protein homodimerization events in the prior art There is a mismatch therebetween, resulting in a problem that signal loss is serious. The invention proposesAn i-motif recombination-mediated FRET probe comprising: executing a chain Half-i @ Apt and a prebraked chain packer;

The invention establishes a method capable of converting protein homodimerization behaviors into highly matched optical signals, and realizes accurate visualization of protein homodimerization.

In some embodiments, the sequence of the executive chain Half-i @ Apt is Cy5-TCCCCCCTCCCCCCATTGATGATCTATTTTTTTTGGATGGTAGCTCGGTCGGGGTGGGTGGGTTGGCAAGTCTTTTTTTT-Cy3-TAGATCATCAATGTGG (SEQ ID No. 1). Herein, we propose an i-motif recombination-mediated FRET strategy for in situ imaging of protein homodimerization on the surface of live cancer cells, using the structural symmetry and acid-responsive allosteric properties of i-motif (FIG. 1). The probe consists of an execution chain (Half-i @ Apt) and a pre-blocking chain (Blocker). In the Half-i @ Apt, the formation sequence of i-motif (C) is first sequenced₆TC₆TC₆TC₆) Split into two parts (C) which are completely symmetrical₆TC₆) Known as Half-i. The Half-i is integrated with the Linker chain, the aptamer of the target protein, and the Linker-x chain complementary to the Linker and a partial sequence of the Half-i in sequence. Cy5 and Cy3 were labeled at the 5' ends of Half-i and Linker, respectively, for use as acceptors and donors for FRET. The entire Linker, part of the Half-i and part of the protein aptamer were pre-primed by the Blocker chainBlocking was performed to prevent the recombination of Half-i to i-motif in the free state, while also maintaining a distance between the donor (Cy3) and acceptor (Cy5) to prevent their FRET effect. The imaging procedure of the probe is here explained in model of the Met receptor: the binding of the aptamer to the Met receptor releases the hybridization between the aptamer strand and the Blocker strand, resulting in unstable assembly of the Blocker strand with other parts and eventually dissociation of the Blocker strand from all of its complementary sequences. The released Linker and a portion of Half-i form duplexes with the Linker to continue to maintain the distance between Cy3 and Cy5 such that FRET cannot occur. Met monomer is activated to form homodimers when stimulated by its ligand (HGF). The homodimerization monomers lead the two probes to approach each other, so that the two Half-i sequences approach each other and can be recombined into a bimolecular i-motif quadruplex in an acidic cancer extracellular microenvironment, and further, the FRET donor-acceptor pair spacing reaches the effective distance for generating the FRET effect. Finally, Cy5 was lit up and Cy3 was reduced to represent the level of Met homodimer and monomer, respectively, so that monomer and homodimer could be well distinguished by two different fluorescences. More importantly, the signal of the protein homodimer in this design is induced by the close proximity of two identical probes, which is highly matched to the pattern of protein homodimerization, enabling accurate and dynamic in situ imaging of protein homodimerization on the surface of living cancer cells, as shown in fig. 1.

In some embodiments, the sequence of the pre-Blocker strand Blocker is AACCCACCCACCCCGACCGAGCTACCATCCAAAAAAAATAGATTATCAATGGGG (seq id No. 2). To prevent the recombination of Half-i in the free state to i-motif, while also enabling the maintenance of a distance between the donor (Cy3) and the acceptor (Cy5) to prevent their FRET effect.

The invention also relates toProvided is a method for preparing a nucleic acid probe based on an i-motif recombination mediated FRET strategy, comprising:

The preparation method of the probe of the present invention can adopt the conventional preparation method in the field to improve the preparation efficiency and the stability of the probe.

In some embodiments, the concentrated solutions of the Half-i @ Apt chain and the Blocker chain are mixed in a ratio of 1: 1 to 1.5.

In some embodiments, the solvent of the concentrated solution is a TE buffer to effectively dissolve the DNA powder.

The invention also provides the use of any of the above probes for in situ imaging of homodimerization of a live cancer cell surface protein.

In some embodiments, the method comprises:

treating HepG2 cells with different concentrations of HGF to induce different levels of homodimerization of Met receptor;

after the cells and HGF are incubated for 25-35 minutes, removing the culture medium and cleaning the cells, then adding a probe, and incubating for 10-17 minutes in a dark place; then, cells were washed 2-3 times with PBS for CLSM imaging; to image different levels of homodimerization of the target protein.

To obtain the optimal time for the probe to interact with the cells, a quenching probe Q-probe was designed, indicating binding of the probe to the target protein by the recovery of fluorescence from Cy 3. Therefore, in some embodiments, the incubation time is 10-17 minutes away from light, so that the probe can be efficiently bound to the cell.

In some embodiments, the confocal scanning imaging parameters are as follows: excitation was performed with a 525nm laser at 10% intensity, emission fluorescence of Cy3 was collected in the range of 550nm to 600nm using a HyD detector at a gain value of 200% and fluorescence of Cy5 was collected in the wavelength range of 650nm to 700nm using a PMT detector at a gain value of 700, so that Cy3 and FRET-excited Cy5 of the same concentration exhibited the same intensity, facilitating comparison of image fluorescence intensities.

The present invention is described in further detail below with reference to specific examples, which are intended to be illustrative of the invention and not limiting.

Example 1:

1. experimental part

1.1 materials and reagents

All DNA oligonucleotides were synthesized and purified by Biotechnology engineering, Inc. (Shanghai, China). The DNA sequences and modifications are listed in table 1. DMEM medium, RPMI 1640, Fetal Bovine Serum (FBS), penicillin streptomycin (PS, 100U/ml), trypsin and phosphate buffered saline (PBS, pH 7.4) for cell culture were all purchased from Biological Industries (Israel). Recombinant human hepatocyte growth factor (HGF, #100-39) was purchased from PeproTech (usa). anti-HGF antibody [ ab83760] was purchased from Abcam (UK). RIPA lysis buffer (P0013D), protease and phosphatase inhibitor cocktail (P1050) were purchased from bi yunnan biotechnology limited (shanghai, china). Primary antibody to β -Actin (AB0035) was purchased from Abways Technology (Shanghai, China). Primary antibodies for Met (#8198) and phosphorus-Met (Tyr1234/1235, #3077) were purchased from Cell Signaling Technology (USA). The secondary antibody, Goat Anti-Rabbit IgG (H + L) HRP, was purchased from Abways Technology (Shanghai, China). LumiQ universal luminophore solution was purchased from santa aurantium (shanghai, china). Protein sample loading buffer (5 ×) and multi-color pre-stained protein markers were purchased from yase biotechnology limited (shanghai, china). All aqueous solutions were prepared using ultrapure water (18.25 M.OMEGA.. multidot.cm).

TABLE 1 oligonucleotide sequences and modifications used in this work

^[a]In the Half-i @ Apt, underlined portions indicate an aptamer sequence of a Met receptor, double-underlined portions indicate a Half-i sequence, dotted underlined portions indicate a Linker, wavy line portions indicate a Linker, bases with asterisks indicate mismatched bases between the Half-i @ Apt and a Blocker chain, and bases with a pound sign (#) indicate mismatched bases between the Half-i @ Apt and the Linker.

^[b]The Half-i-linker and the exact-i-linker sequences used in the PAGE experiments.

^[c]The sequence used to construct Q-probe was optimized for the duration of probe incubation with cells.

^[d]Control probes were constructed using the Arbitrary strand.

^[e]Cy3-Aptamer and Cy5-complementary strand were used to determine instrument parameters in CLSM imaging.

1.2 instruments

Non-denaturing polyacrylamide gels for DNA analysis by GelDocTM XR⁺Imaging systems (Bio-RAD Laboratories Inc., USA) perform imaging. SDS-polyacrylamide gels for Western blot analysis were imaged by Amersham Imager 600 imaging System (GE Healthcare, USA). The UV-visible absorption spectrum was measured by a U-2910 spectrophotometer (Hitachi, Japan). Circular dichroism spectra were determined by a Chirascan V100 circular dichroism spectrometer (Applied Photophysics, UK). Cytotoxicity experiments the absorbance was determined by a SPARK microplate reader (Tecan Austria GmbH, Austria). Flow cytometry measurements were performed by NovoCyte3130 flow cytometer (ACEA Biosciences inc., UAS). Confocal Laser Scanning Microscopy (CLSM) imaging was performed by Leica TCS SP8 STED confocal microscope (Leica, germany) with 63x objective lens selected.

1.3 cells and cell cultures

All cell lines were at 37 ℃ and 5% CO₂The wet incubator of (1). Human liver cancer cell line (HepG2) was cultured in DMEM medium containing 10% FBS and 1% PS. Human prostate adenocarcinoma cell line (LNCaP) was cultured in RPMI 1640 containing 10% FBS.

1.4 preparation of Probe

First, TE buffer (10mM Tris, 1mM Na) was used₂EDTA, pH 8.0) to dissolve the DNA powder to obtain a concentrated solution of DNA. The two strands were hybridized thoroughly by mixing the concentrated stock solutions of Half-i @ Apt strand and Blocker strand at a ratio of 1: 1, heating to 95 ℃ for 5 minutes, slowly cooling to room temperature, and then leaving in the dark at 4 ℃ for 12 hours. PBS buffers (10mM NaH) at different pH values were used₂PO₄、10mM Na₂HPO₄、130mM NaCl、4.6mM KCl、5.0mM MgCl₂) The probe stock solution was diluted to the appropriate concentration for subsequent experiments.

1.5 pH-responsive reconstitution studies of i-motif in solution.

Native polyacrylamide gel electrophoresis (Native PAGE), ultraviolet-visible (UV-vis) spectroscopy and Circular Dichroism (CD) spectroscopy were used to verify the ability of Half-i to recombine into i-motif in an environment of pH6.5 (corresponding to acidity of the microenvironment outside the cancer cells).

Native PAGE analysis. The sequence of the Half-i-linker was used for PAGE analysis, and the exact-i-linker sequence containing twice the Half-Half-i sequence was used as a control. Concentrated solutions (100. mu.M) of Half-i-linker and exact-i-linker were diluted to 2. mu.M using PBS at pH 7.4 and pH6.5 and incubated for 30 min. Samples diluted with pH 7.4PBS were treated with 12% non-denaturing polyacrylamide gel TAE buffer (40mM Tris, 2mM Na) at pH 7.4₂EDTA，20mM CH₃COOH) at a constant temperature of 15 ℃ for 1 hour at a constant current of 30 mA. Samples diluted in PBS pH6.5 were run on 12% non-denaturing polyacrylamide gel in TAE buffer (40mM Tris, 2mM Na) pH6.5₂EDTA，20mM CH₃COOH) at a constant temperature of 15 ℃ for 1.5 hours at a constant current of 30 mA. After electrophoresis, the gel was stained with 1x SYBR Gold for 40 minutes in the dark and then imaged by an imaging system.

Ultraviolet-visible spectrum. Concentrated solutions of Half-i (200. mu.M) were diluted to 8. mu.M with PBS pH 7.4 and pH6.5, respectively, for ultraviolet-visible (UV-vis) spectroscopy. The spectra were recorded at room temperature in the range 220nm to 320 nm.

CD spectrum. Stock solutions of Half-i (200. mu.M) were diluted to 15. mu.M with PBS pH 7.4 and pH6.5, respectively, for CD measurement. The spectra were recorded at room temperature in the range 220nm to 320 nm.

1.6 kinetic study of i-motif recombination

To analyze the kinetics of recombination of Half-i into bimolecular i-motif, we added a concentrated stock solution of Half-i (200 μ M) in situ to PBS pH6.5 and mixed rapidly to a concentration of 15 μ M. The ellipticity at 287nm was measured at 25 ℃ and every 90 seconds. The ellipticity of Half-i in PBS pH 7.4 was used as the starting point for the change.

1.7 thermal stability of recombinant i-motif

A15. mu.M concentration of Half-i solution was prepared in PBS pH6.5 and the ellipticity of the Half-i solution at 287nm at different temperatures was determined by gradually increasing the temperature at a rate of 1 ℃/min by means of a CD spectrometer equipped with a temperature-controlled water bath. Plotting the ellipticity against temperature, the temperature at which 50% of the recombinant i-motif structures are dissociated being taken as T_mThe value is obtained. The background spectrum of the buffer was subtracted from the sample assay data.

1.8 cytotoxicity of probes

The cytotoxicity of the probes was assessed by MTT assay. HepG2 and LNCaP cells were plated at 5000 cell per well for 24 hours in 96-well plates to allow adherence, and the medium was replaced with medium containing different concentrations of probe (0nM, 10nM, 50nM, 100nM, 250nM, 500 nM). After 24 hours of co-incubation of the cells with the probe at 37 ℃,10 μ l of MTT (5mg/ml) was added to each well, and then the cells were incubated at 37 ℃ for 4 hours. Then, the culture medium was removed and 150. mu.l of DMSO was added to each well to dissolve formazan crystals therein, and after shaking for 4 to 5 minutes, absorbance at 490nm was measured to calculate cell activity. Cells without probe treatment served as control. 6 replicate wells were set for each probe concentration.

1.9Western blot experiment

Cells were cultured in 6-well plates to a confluency of 75% to 85%, and different treatments were applied to the cells for different studies. After treatment, cells were washed 3 times with warm physiological saline to remove the medium and then lysed on ice for 15 minutes in RIPA lysis buffer containing a mixture of protease and phosphatase inhibitors. The cells were scraped from the well plate with a cell scraper, and then the separated cells were centrifuged at 12000rpm at 4 ℃ for 15 minutes, and the supernatant was collected. To the supernatant was added a protein sample loading buffer and the protein was denatured by heating at 100 ℃ for 5 minutes. Electrophoresis analysis was performed on an 8% SDS-polyacrylamide gel, and 30. mu.g of total protein extract was loaded per lane. After the electrophoresis was completed, the proteins were transferred to PVDF membrane (MilliporeImmobilon-P, 0.45 μm), and after the completion of the membrane transfer, non-specific binding sites were blocked with 5% skim milk powder solubilized with TBST (10mM Tris, 150mM NaCl, 0.05% (v/v) Tween-20, pH 7.2-7.5). The PVDF membrane was then washed 3 times with TBST. Primary antibodies to Met, p-Met and β -Actin were incubated with the protein on PVDF membrane overnight at 4 ℃. After washing 3 times with TBST, the membrane was incubated with secondary antibody for 2 hours at room temperature with gentle shaking. After washing 3 times with TBST, PVDF membranes were imaged by ECL. The grey scale of the image was analyzed by ImageJ software.

1.10 interference of probes on expression and homodimerization of target proteins

To ensure that the probe reflects the native state of the target protein, we evaluated the probe for interference with Met receptor expression and homodimerization. HepG2 cells in six-well plates were grown to 80% confluence. To investigate whether the probe interfered with Met receptor expression, 1ml of probe dissolved in PBS at pH6.5 at a concentration of 100nM was applied to HGF-untreated cells and incubated for 15 minutes; to investigate whether the probe interfered with Met receptor homodimerization, cells were first incubated with 1ml HGF at a concentration of 100ng/ml for 30 minutes, and then 1ml probe dissolved in PBS at pH6.5 at a concentration of 100n M was applied to the cells and incubated for 15 minutes. After treatment, cells were lysed and total protein was extracted for Western blotting analysis.

1.11 optimization of incubation time

Too long incubation time of the probe with the cell may result in degradation or endocytosis of the probe into the cell, and too short incubation time may result in insufficient binding of the probe to the target protein, both of which are detrimental to imaging accuracy. In order to explore the optimal incubation time of the probe and the cells, a fluorescence-quenched probe (Q-probe) is constructed by designing a sequence Q-blocker which is labeled with a fluorescence quenching group BHQ2 and is partially complementary with a Half-i @ Apt chain, and hybridizing the sequence Q-blocker with the Half-i @ Apt chain to quench Cy3 fluorescence. After finding a clear focal plane for imaging the cells under a confocal microscope, 100. mu.l of 100nM Q-probe was applied to the cells, and the binding of the probe to the target protein removed the blocker chain and BHQ2, thereby recovering the fluorescence of Cy3, and indicating the binding of the probe to the target protein by the recovery of the fluorescence of Cy 3. In the experimental process, the laser with the wavelength of 525nm is used for exciting fluorescence, and the emitted fluorescence within the range of 550-600nm is received.

1.12 Confocal Laser Scanning Microscope (CLSM) imaging

To be able to more directly compare the fluorescence intensities obtained by imaging, we first adjusted the imaging parameters of the microscope so that Cy3 and FRET-activated Cy5 show the same intensity at the same concentration. HepG2 cells were plated on a confocal dish and cultured for 24 hours to adhere to the wall. Labeled aptamers of Cy3 (Cy3-Aptamer) and Cy3/Cy5 pairs (assembled from Cy3-Aptamer and Cy5-complementary strand) were mixed in a ratio of 1: 1, and incubated for 15 minutes in the dark. The cells were then gently washed 2-3 times with pH6.5 PBS to remove excess probe and imaged by confocal laser scanning microscopy. Fluorescence was excited with a laser at 525nm, and the gain values of Cy3 and Cy5 channels were adjusted so that Cy3 and Cy5 showed the same intensity. The confocal scanning imaging parameters were finally determined as follows: excitation was performed with a 525nm laser at 10% intensity, emission fluorescence of Cy3 was collected at a gain value of 200% in the 550nm to 600nm range using a HyD detector, and fluorescence of Cy5 was collected at a gain value of 700 in the 650nm to 700nm wavelength range using a PMT detector. All treatments for CLSM imaging were performed in a volume of 100 μ l, unless otherwise specified. The intensity of the CLSM image was analyzed by a quantitative tool equipped with the microscope system.

1.12.1 image different levels of homodimerization of the protein of interest. HepG2 cells were treated with different concentrations of HGF (0ng/ml, 10ng/ml, 50ng/ml and 100ng/ml) to induce different levels of homodimerization of the Met receptor. After incubation of cells with HGF for 30 min, the medium was removed and the cells washed, then 100nM probe (dissolved in PBS pH 6.5) was added and incubated for 15 min in the dark. Then, the cells were washed 2-3 times with PBS pH6.5 for CLSM imaging.

2.12.2 inhibitor experiments. Inhibitor experiments were performed to further verify the specificity and accuracy of the probes. 100ng/ml HGF was first incubated with 2ug/ml of Anti-HGF antibody for 1 hour to occupy the binding site of HGF, and then HGF was applied to HepG2 cells for another 30 minutes. The medium was then removed and the cells washed, 100nM probe (dissolved in PBS pH 6.5) added and incubated for 15 minutes in the absence of light. Cells were washed 2-3 times with PBS pH6.5 for CLSM imaging.

1.12.3 control probe assay. To explore the imaging mechanism of the probe, we constructed a control probe by replacing the Half-i sequence in the probe with an arbitrary sequence without acid-responsive ability. The HepG2 cells were incubated for 30 minutes with medium containing 100ng/ml HGF. The probe and control probe (dissolved in PBS pH 6.5) were added to the cells at a concentration of 100nM, respectively, and incubated for 15 minutes in the absence of light. Cells were washed 2-3 times with PBS pH6.5 for CLSM imaging.

1.12.4 real-time imaging of protein homodimerization. HepG2 cells were first incubated with 100nM probe in PBS at pH6.5, protected from light for 15 minutes. The cells were washed with PBS pH6.5 to remove excess probe. After determination of a well defined focal plane under a confocal laser scanning microscope, 100. mu.l of HGF at a concentration of 100ng/ml were added to the cells and fluorescence images were taken immediately with an xyt scanning mode at time intervals of 1 minute and 1 time.

1.13 investigation of the specificity of the Probe for the protein of interest

HepG2 cells expressing the target protein are used as positive cells, LNCaP cells not expressing the target protein are used as negative cells, and the specificity of the probe on the target protein is checked in a contrast mode. HepG2 cells and LNCaP cells were cultured separately in confocal dishes for 24 hours of adherence. The probes were applied to both cells separately and after 15 minutes of incubation, washed 2-3 times with PBS for CLSM imaging. The emission fluorescence of Cy3 in the range of 550-600nm is received by laser excitation at 525nm during the experiment.

1.14 flow cytometry

Cells were cultured in six-well plates to 75% to 85% confluence, with different treatments applied for different experiments. After the treatment, 100nM probe (dissolved in PBS pH 6.5) was applied to the cells, incubated for 15 minutes in the dark, washed 2-3 times with PBS pH6.5, and the cells were scraped off with a cell scraper for flow cytometry analysis. For different levels of protein homodimerization analysis, cells were incubated for 30 minutes with media containing 0,10,50,100ng/ml HGF, respectively. For inhibitor experiments, 100ng/ml HGF was incubated with 2ug/ml inhibitor for 1 hour prior to applying to the cells for 30 minutes.

2. Results and discussion

2.1 pH responsive reconstitution Studies of i-motif in solution

Because of the ability of Half-i to be at pH6.5 (consistent with the acidity of the cancer extracellular microenvironment)^[9]) Recombination into intermolecular i-motif is one of the keys to the feasibility of this scheme design. We examined the acid-responsive recombination capability of Half-i by polyacrylamide gel electrophoresis (PAGE), ultraviolet-visible absorption (UV-vis) spectroscopy, and Circular Dichroism (CD) spectroscopy. As shown by the PAGE results in FIG. 2A, at pH 7.4, the Half-i-linker showed only a single band at the lower position, and did not form a band having a molecular weight similar to that of the control exact-i-linker; under the condition of pH6.5, the Half-i-linker forms a brighter band with the molecular weight similar to that of the integer-i-linker and a very weaker band with the same position as that of the buffer solution with pH 7.4, which indicates that the Half-i-linker can form a bimolecular structure with higher efficiency in the environment with pH6.5^[8b]. The Uv-vis spectrum (FIG. 2B) shows a significant increase in absorbance at 295nm (characteristic absorption of the i-motif quadruplex structure) of the Half-i solution at pH6.5 compared to pH 7.4, indicating that Half-i forms the i-motif structure at pH 6.5; the CD spectrum (FIG. 2C) shows that in the pH 7.4 buffer, the negative and positive peaks of Half-i appear at 232nm and 277nm, respectively, whereas in the pH6.5 buffer, the negative and positive peaks are red-shifted to 265nm and 287nm, respectively, and their values are also significantly increased, consistent with the spectral characteristics of i-motif, indicating the formation of i-motif structure. All the above results show that Half-i can form an intermolecular i-motif quadruplex structure in an environment of pH6.5, and the feasibility of the design proposed by us is preliminarily confirmed.

2.2 kinetics of i-motif recombination

The kinetics of recombination of halof-i into i-motif was determined by circular dichroism spectroscopy, and the amount of i-motif formed was indicated by the ellipticity of the halof-i at 287 nm. As shown in FIG. 3, the ellipticity rapidly increases within the first 90 seconds, the rising speed significantly decreases within 90-180 seconds, and slightly increases within 180-630 seconds, and tends to be stable after 630 seconds. This pattern of change indicates that Half-is can recombine rapidly to i-motif within 90 seconds, to completion in 630 seconds.

2.3 thermal stability of recombinant i-motif

The thermal stability of recombinant i-motif was assessed by measuring the ellipticity of Half-i at 287nm in pH6.5 buffer as a function of temperature by CD spectrometer with temperature as abscissa and ellipticity as ordinate, and all points were fitted with Boltzmann function. T of i-motif obtained by recombination of Half-i as shown in FIG. 4_mThe value was 46 degrees celsius. This indicates that recombinant i-motifs can exist stably at room temperature or 37 ℃ and supports the use of the probe for live cell imaging.

2.4 interference of the Probe with the cell's native State

In order to enable imaging results to reveal the monomer and homodimer levels of the target protein in the native state, we expect that the probe does not interfere with the native state of the cell. Firstly, the cytotoxicity examination of the probe is carried out, as shown in fig. 5A, the MTT measurement result shows that the survival rate of the cells treated by the probe with different concentrations is more than 85%, which indicates that the probe has no obvious cytotoxicity to the cells and lays a foundation for live cell imaging. We then evaluated the interference of this probe on the expression of the target protein and homodimerization. After incubating HepG2 cells with the probe for a certain period of time, the cells were analyzed by western blot for monomers and homodimers of the target Met receptor. Results as shown in fig. 5B, the cells treated with the probe showed a consistent trend in Met monomer and homodimer expression as compared to the cells not treated with the probe (fig. 8B). It was confirmed that the probe hardly interferes with the cell activity and the expression and homodimerization of the target protein, and thus images obtained from the probe can reliably reveal the natural level of homodimerization of the target protein.

2.5 optimization of Probe and cell incubation time

To obtain the optimal time for the probe to interact with the cells, a quenching probe Q-probe was designed, indicating binding of the probe to the target protein by the recovery of fluorescence from Cy 3. As shown in fig. 6, Cy3 fluorescence rapidly increased within 7.5 minutes after the probe was added to the cells, indicating that the probe bound to the target protein rapidly because of the high concentration of both probe and exposed protein; from 7.5 to 17.5 minutes, the Cy3 fluorescence intensity continued to increase, but the rate of increase decreased significantly due to the gradual decrease in the concentration of the probe as it bound to the target protein. After 17.5 minutes, the fluorescence intensity began to decrease, probably because the fluorescence was bleached to some extent by the laser irradiation or affected by the complicated cellular environment. The final incubation time of the needle with the cells was chosen to be 15 minutes, taking into account the time required for the imaging procedure and the recombination of the halof-i into i-motif.

2.6 specificity of the probes

The specificity of the probes for the target molecules was assessed by Confocal Laser Scanning Microscopy (CLSM) imaging. As can be seen in fig. 7B, LNCaP cells that do not express the Mer receptor show little fluorescence after probe treatment, indicating that the probe does not bind to non-target molecules. For HepG2 cells expressing Met receptor, the images showed significant Cy3 fluorescence, indicating that the probe binds to Met receptor on the cell membrane. The result shows that the probe can eliminate the interference of non-target molecules on the imaging result, has good specificity on the target molecules, and has potential application to imaging of homodimerization of specific target proteins.

2.7 imaging of probes on different levels of homodimerization

HepG2 cells were treated with different concentrations of HGF to induce different levels of homodimerization of the target protein, followed by CLSM imaging, Western blot analysis, and flow cytometry analysis. As shown in fig. 8A, when HGF was not added, the cell membrane showed very bright Cy3 fluorescence representing Met monomer, the Cy5 fluorescence intensity representing Met homodimer was almost zero, the phenomenon was similar to that of HGF-free treatment when HGF was added at 10ng/ml, the Cy3 fluorescence intensity was still high, and the Cy5 intensity was weak, indicating that the target protein was hosted in monomer form and the homodimer level was low under these two conditions; when HGF is treated at 50ng/ml and 100ng/ml, the obvious Cy5 fluorescence appears on the surface of the cell membrane, and the fluorescence intensity of Cy3 is reduced, which indicates that the homodimer of the target protein is increased and the existence of the homodimer of the target protein is reduced. Confocal fluorescence imaging results were consistent with western blots (fig. 8B), and flow cytometry results (fig. 8C) also showed that the probe was able to sensitively respond to different levels of protein homodimerization, indicating that the probe was able to image protein homodimerization in situ with high sensitivity and accuracy.

2.8 inhibitor assay

To further validate the specificity and accuracy of the probes, we performed inhibitor experiments and CLSM imaging results are shown in fig. 9A. Only HGF-stimulated cells exhibited simultaneous Cy3 and Cy5 fluorescence, indicating the simultaneous presence of monomeric and homodimeric Met receptors. Inhibitor treated cells showed high intensity of Cy3 fluorescence, whereas Cy5 fluorescence was very weak, representing higher levels of protein monomer and low levels of dimer, since HGF co-incubated with inhibitor did not induce homodimerization of Met receptor. The result of the CLSM image is consistent with the detection trends of western blotting (figure 9B) and flow cytometry (figure 9C), and the probe has good specificity and accuracy on the homodimerization of the target protein.

2.9 control Probe experiment

To further explore the mechanism of homodimerization of target proteins imaged by the probe, we replaced Half-i in the probe with an arbitrary sequence without acid-responsive properties as a control probe. CLSM imaging studies were performed on HGF-stimulated HepG2 cells. As shown in fig. 10, cells imaged with our designed probe showed both significant Cy3 fluorescence representing protein monomers and Cy5 fluorescence representing homodimers. Cells imaged with the control probe showed only a strong Cy3 fluorescence signal, with a very weak Cy5 signal. This result demonstrates that accurate imaging of protein homodimerization is possible because of the acid-responsive recombinant allosteric conformation of Half-i, which is a key part of probe imaging protein homodimerization.

2.10 immediate imaging capability of the Probe

CLSM was used to examine the real-time imaging capabilities of the probe in xyt scan mode. As shown in fig. 11A, in HGF-stimulated cells, the probe exhibited strong Cy3 fluorescence upon addition of HGF, whereas Cy5 fluorescence was almost zero; weak Cy5 fluorescence appeared at 5 min; the fluorescence of Cy5 gradually increased and the fluorescence intensity of Cy3 slightly decreased in 5 to 15 minutes; the two fluorescence intensities remained essentially stable for 15 to 30 minutes. In cells not stimulated with HGF (fig. 11B), the fluorescence of Cy3 was stably maintained at a higher intensity within 30 minutes; cy5 fluorescence was always at very weak intensity. The probe was demonstrated to be able to image protein homodimerization in real time with high accuracy. This will help the probe to be used for rapid detection of protein interactions.

3. Conclusion

We propose an i-motif recombination mediated fluorescence resonance energy transfer strategy for in situ imaging of homodimerization of live cancer cell surface proteins. The probe exhibits satisfactory stability, specificity and real-time imaging capabilities. Importantly, the FRET signal used in this strategy to indicate protein homodimers is generated by the recombination of two identical Half-i into i-motif in close proximity, which is highly matched to the mode of homodimerization, thereby making the strategy more fidelity in imaging homodimerization events. Using this strategy, we achieved accurate, dynamic in situ imaging of Met receptor homodimerization on the surface of living cancer cells. Since aptamers to various membrane proteins can be obtained by in vitro screening, this strategy can be extended to monitoring of other proteins of interest for homodimerization by simply replacing the recognition sequence. Given that membrane protein homodimerization is involved in multiple signaling pathways and plays an important role in the development and progression of cancer, this strategy will provide a powerful tool for relevant signaling networks and pathological studies.

It should be noted that the above-mentioned embodiments are only preferred embodiments of the present invention, and the present invention is not limited thereto, and although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications and equivalents can be made in the technical solutions described in the foregoing embodiments, or equivalents thereof. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention. Although the embodiments of the present invention have been described with reference to the accompanying drawings, it is not intended to limit the scope of the present invention, and it should be understood by those skilled in the art that various modifications and variations can be made without inventive efforts by those skilled in the art based on the technical solution of the present invention.

SEQUENCE LISTING

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<120> application of FRET probe mediated by I-motif recombination and cancer cell surface protein homodimerization in-situ imaging thereof

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Claims

1. a FRET probe mediated by the i-motif recombination of the homodimerization of proteins on the in situ imaging cancer cell membrane, is characterized in that: comprises execution chain Half-i@Apt and pre-blocking chain Blocker; The execution chain Half-i @Apt contains the Half-i sequence, the Linker sequence, the aptamer sequence of the target protein, and the Linker* complementary to the partial sequence of the Linker and Half-i, and Cy5 and Cy3 are respectively labeled in Half-i And the 5' end of Linker* is used as the acceptor and donor of FRET; the entire Linker, part of Half-i and part of the protein aptamer sequence are blocked in advance by the Blocker chain; The Half-i is formed by the i-motif sequence Completely symmetrical two parts obtained by splitting; the sequence SEQIDNo.1 of the execution chain Half-i@Apt is

Cy5-TCCCCCCTCCCCCCATTGATGATCTATTTTTTTTGGATGGTAGCTCGGTCGGGGTGGGTGGGTTGGCAAGTCTTTTTTTT-Cy3-TAGATCATCAATGTGG.

2. The i-motif recombination-mediated FRET probe for in situ imaging of homodimerization of proteins on cancer cell membranes as claimed in claim 1, wherein the sequence of the pre-blocking chain Blocker is SEQ ID No. .2 for

AACCCACCCACCCCGACCGAGCTACCATCCAAAAAAAATAGATTATCAATG

ggg.

3. The i-motif recombination-mediated FRET probe used for in situ imaging of homodimerization of proteins on cancer cell membranes according to claim 1, wherein the preparation method is as follows:

Mix the concentrated stock solution of Half-i@Apt chain and Blocker chain, heat it to 90~95℃ for 4~5 minutes, then slowly cool to room temperature, then place it in the dark at 3~5℃ for more than 8 hours, so that the two The strands are fully hybridized and obtained.

4. The i-motif recombination-mediated FRET probe for in situ imaging of the homodimerization of proteins on cancer cell membranes as claimed in claim 3, wherein the Half-i@Apt chain and the Blocker chain The mixing ratio of concentrated stock solution is 1:1~1.5.

5. The i-motif recombination-mediated FRET probe for in situ imaging of homodimerization of proteins on cancer cell membranes as claimed in claim 3, wherein the solvent of the concentrated stock solution is TE buffer liquid.

6. The application of the probe according to claim 1 or 2 in the in situ imaging of protein homodimerization on the surface of cancer cells.

7. The application according to claim 6, characterized in that, comprising: treating HepG2 cells with different concentrations of HGF to induce homodimerization of Met receptors at different levels; after the cells are incubated with HGF for 25-35 minutes , remove the medium and wash the cells, then add the probes and incubate in the dark for 10-17 min; then, wash the cells 2-3 times with PBS for CLSM imaging.

8. The application according to claim 7, wherein the incubation time in the dark is 15-16 minutes.

9. The application of claim 7, wherein the confocal scanning imaging parameters are as follows: excitation with a 525 nm laser at 10% intensity, using a HyD detector with a gain value of 200% in the range of 550 nm to 600 nm The emitted fluorescence of Cy3 was collected within the 650 nm to 700 nm wavelength range using a PMT detector with a gain value of 700.