Method and apparatus for identifying enhancer activity in living cells

Abstract

This disclosure provides a method and apparatus for identifying enhancer activity in live cells, enabling sensitive detection of enhancer activity within live cells. The method utilizes a nano-electroporation biochip to perform highly focused perforation of the cell membrane and provides the driving force to accelerate the delivery of enhancer probes to the cell nucleus. The enhancer probes are designed using a cyclic amplification strategy, increasing the fluorescence signal by more than 100-fold within one hour. In the embodiments, the method and apparatus successfully detected the activity of CCAT1 enhancer in live cells without affecting cell viability, demonstrating the robustness and reliability of the proposed method and apparatus in detecting enhancer activity in live cells. This disclosure provides a convenient and universal platform for research on enhancer gene regulatory mechanisms in live cells and for clinical diagnostic applications.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of cell biology analysis, and particularly relates to a method and device for identifying enhancer activity in living cells. BACKGROUND

[0002] Enhancers are a class of non-coding regulatory elements in the genome in the nucleus, which are activated by transcription factors interacting with promoters and can significantly increase the expression of specific genes. Controllable enhancer activation plays an important role in maintaining the evolution of genes in mammalian cells. Conversely, uncontrolled enhancer activation can lead to a series of abnormal cell behaviors, such as tumor occurrence and excessive proliferation. Identifying the activity of enhancers, especially in living cells, can provide important clues for exploring the potential genetic regulation mechanisms of cancer.

[0003] Traditional gene sequencing methods, such as chromatin immunoprecipitation sequencing (ChIP-seq), have been applied to study enhancer activity. In the ChIP-seq method, antibodies are used to capture transcription factors from fragmented genomes, and by sequencing the enhancers and promoters bound to the transcription factors, the activation state of the target enhancer can be examined. It is worth noting that this method requires time-consuming follow-up validation experiments to exclude artificial interference from genomic fragmentation. In addition, since the genome needs to be extracted from lysed cells, such gene sequencing-based methods cannot provide real-time feedback on the status of enhancers.

[0004] There are two major obstacles in studying enhancer activity in living cells: (1) The double biological barriers of the cell plasma membrane and the nuclear membrane. Most existing molecular delivery techniques, by loading probes on carriers such as nanoparticles, liposomes, etc., use the endocytosis of cells to deliver probes into cells, which can only make the probes cross the barrier of the cell plasma membrane, but cannot continue to bring the probes into the nucleus, which means that the identification of enhancer activity in the nucleus cannot be achieved. (2) High sensitivity of detection methods. Since enhancers are activated by interacting with promoters, detecting the activity of target enhancers is to identify this interaction. Due to the uniqueness of the interaction site between enhancers and promoters, the detection scheme requires extremely high sensitivity. According to the previous research on existing methods by the present disclosure, there is no reported method that can simultaneously solve the above two major obstacles to achieve the detection of enhancer activity in living cells. SUMMARY

[0005] The application specifically provides a method and device for identifying enhancer activity in living cells, solves the above two obstacles, and realizes sensitive detection of the activity of nuclear enhancers in living cells.

[0006] The technical scheme adopted by the application is a device for identifying enhancer activity in living cells, which comprises an enhancer probe and a nanoelectroporation biochip.

[0007] The enhancer probe specifically feeds back the activity state of the enhancer through amplified signals.

[0008] The nanoelectroporation biochip is used for delivering the enhancer probe into the nucleus of living cells.

[0009] Further, the enhancer probe is composed of a group of DNA sequences, and the amplification of the detection signal is realized through a cyclic fluorescent signal amplification strategy.

[0010] Further, the nanoelectroporation biochip is composed of five parts, from bottom to top: a lower electrode, a probe storage chamber, a nanopore layer, a cell culture chamber and an upper electrode. The lower electrode is connected to the negative electrode of the electroporation instrument; the probe storage chamber is used for storing the probe solution to be delivered; the nanopore layer is used for focusing the electric field and providing a channel for the probe to move from the probe storage chamber into the cell; the cell culture chamber is used for adherent culture of cells; and the upper electrode is connected to the positive electrode of the electroporation instrument and forms a path with the lower electrode.

[0011] Further, the DNA sequence comprises four functional sequences P1, P2, H1 and H2.

[0012] P1 is composed of three parts, including a part combined with the enhancer sequence, a part combined with the promoter sequence, and a connecting part between the two parts;

[0013] P2 is complementary to the sequence part combined with the promoter sequence in P1;

[0014] H1: partially complementary to itself, with a "hairpin" structure containing one sticky end, so that the fluorescent group of the label is quenched by the proximity of the quencher group of the label;

[0015] H2: partially complementary to itself, with a "hairpin" structure containing one sticky end.

[0016] Further, the cycle fluorescent signal amplification strategy is as follows: sequence P2 can bind to sequence H1, and open the "hairpin" structure of H1, restore the fluorescent signal on H1, and then H2 can bind to sequence H1, and replace the sequence P2 bound on H1, and the replaced P2 can continue to bind to sequence H1, and cycle to trigger the fluorescent recovery reaction.

[0017] Further, the nanometer pore layer has a through hole with a nanometer size (1-1000 nanometers), and is made of polycarbonate, silicon or the like, and has a focusing electric field effect.

[0018] Further, the nanometer electroporation biochip has the function of perforating the cell membrane and generating a driving force to promote the enhancer probe into the nucleus.

[0019] A method for identifying enhancer activity in living cells, and the process for realizing the method is as follows: the lower electrode of the device is connected to the negative electrode of the electroporator, and the upper electrode is connected to the positive electrode of the electroporator, and a square wave pulse voltage is applied through the electroporator, at this time, the nanometer pore layer plays a focusing electric field effect, and safe and reversible perforation of the cell membrane can be realized, and at the same time, electrophoretic force is generated to make the probe below the nanometer pore layer accelerate through the nanometer pore layer and enter the perforated cell, and the probe entering the cell continues to enter the cell nucleus under the action of the electrophoretic force. The probe entering the cell nucleus first specifically binds to the enhancer sequence by P1, and when the enhancer is in an activated state and interacts with the promoter, the other end of P1 binds to the promoter sequence, so that P2 originally bound on P1 is replaced. The replaced P2 binds to the hairpin-shaped H1, so that the hairpin-shaped H1 is opened, so that the fluorescent group is away from the quencher group to emit fluorescence. Subsequently, H2 is complementary to H1, so that the P2 sequence is replaced. The replaced P2 probe can continue to bind to the hairpin-shaped H1, thereby starting a new cycle of fluorescent recovery and sequence replacement. After multiple cycles, the P2 replaced by only one enhancer interaction realizes multiple fluorescent signal amplification, greatly increasing the detection sensitivity.

[0020] Compared with the prior art, the present application first proposes the function of nanometer electroporation to promote the delivery of probes into the nucleus, and first realizes the sensitive detection of enhancer activity in living cells, which provides a powerful research tool for studying gene interactions in the nucleus of living cells. BRIEF DESCRIPTION OF DRAWINGS

[0021] For a more complete understanding of the present disclosure and its advantages, reference is now made to the following description taken in conjunction with the accompanying drawings, in which the device structures in the drawings are not necessarily drawn to scale, and in which like reference numerals designate similar structural elements throughout the several views. In the drawings:

[0022] Figure 1 Principle diagram of the method and device for identifying enhancer activity in living cells proposed in the present disclosure.

[0023] Figure 2 Evaluation of the recognition ability of the enhancer probe for detecting target sequences.

[0024] Figure 3 In the table, A is the response time of the enhancer probe for detecting target sequences; B is the sensitivity of the enhancer probe for detecting target sequences.

[0025] Figure 4 In the table, A is the physical diagram of the method and device for identifying enhancer activity in living cells proposed in the present disclosure; B is the physical diagram of the nanoelectroporation biochip; C is the through-hole electron microscope image on the polymer nanomembrane.

[0026] Figure 5 In the table, A is the simulated electric field distribution of cells in the initial state of nanoelectroporation; B is the simulated electric field distribution after the cell membrane near the nanomembrane is perforated; C is the simulated electric field distribution of the cell membrane near the nanomembrane and the cell membrane at the top after perforation; D is an enlarged image of the electric field distribution near the nucleus in the C diagram; E is the internal and external potential difference of the nucleus near the nanomembrane in D.

[0027] Figure 6 In the table, A is the cell fluorescence image for identifying CCAT1 enhancer activity in living cells; B is the cell fluorescence statistical result for identifying CCAT1 enhancer activity in living cells; C is the flow cytometry detection result of cell fluorescence intensity. Among them, Control is untreated cells, Incubation is to deliver enhancer probes into living cells by incubation, H1+(P1+P2) is to deliver probes without signal amplification function into living cells by nanoelectroporation biochip, and H1+H2+(P1+P2) is to deliver enhancer probes into living cells by nanoelectroporation biochip.

[0028] Figure 7 In the table, A is the gene expression change of SOX2 and CCAT1 after SOX2 is knocked down in cells; B is the cell fluorescence intensity statistical result of enhancer activity in SOX2-knocked-down cells detected by the method and device for identifying enhancer activity in living cells proposed in the present disclosure.

[0029] Figure 8In Figure A, the cell proliferation is compared before and after detection using the live-cell enhancer activity identification method and device proposed in this disclosure; in Figure B, the cell migration behavior is compared before and after detection using the live-cell enhancer activity identification method and device proposed in this disclosure. Detailed Implementation

[0030] The present disclosure will now be described in detail with reference to the accompanying drawings and embodiments to assist those skilled in the art in fully understanding the purpose, features, and effects of the present disclosure. The accompanying drawings illustrate exemplary embodiments of the present disclosure; however, it should be understood that the present application can be implemented in various other forms and should not be limited to the embodiments described herein. Furthermore, descriptions of well-known structures and technologies are omitted in the following description to avoid unnecessarily obscuring the concepts of the present disclosure. Additionally, the various embodiments and technical features provided below can be combined with each other in any manner.

[0031] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit this disclosure. Furthermore, the terms “comprising,” “including,” “having,” etc., as used herein indicate the presence of said features, steps, operations, and / or components, but do not exclude the presence or addition of one or more other features, steps, operations, or components. All terms used herein (including technical and scientific terms) have the meanings commonly understood by those skilled in the art, unless otherwise defined. It should be noted that the terms used herein should be interpreted in a manner consistent with the context of this specification and should not be interpreted in an idealized or overly rigid manner.

[0032] Example 1: Identification of CCAT1 enhancer activity in live cells

[0033] CCAT1 is a long non-coding RNA that promotes tumor invasion and metastasis. Modulating the activity of CCAT1 enhancers can significantly increase CCAT1 gene expression. This embodiment aims to detect CCAT1 enhancer activity by designing and preparing enhancer probes and fabricating a nano-electroporation biochip for sensitive detection of CCAT1 enhancer activity in living cells. Figure 1 The specific steps are as follows:

[0034] Step 1: Enhancer probe design and fabrication

[0035] Step 1.1: Based on the CCAT1 enhancer sequence and promoter sequence, design and prepare the sequences of enhancer probe components H1, H2, P1, and P2, as follows:

[0036] H1:

[0037] 5'-BHQ1-ATTTTAGTAGGTCTCTCAGGTCACCTACTAAAA / i6FAMdT / AAACACAG-3'

[0038] H2:

[0039] 5'-CTCTCAGGTCCTGTGTTTATTTTAGTAGGTGACCTGAGAGACCTACT-3'

[0040] P1:

[0041] 5'-ACCTACTAAAATAAACACAGAATGGAGCCCATGCGTTAGTCGTACCTACCTTTCGATATGCTAGTCTCATAACTTAGAACACCATTCAGC-3'

[0042] P2:

[0043] 5'-CTGTGTTTATTTTAGTAGGT-3'

[0044] Step 1.2: Dissolve the sequences of H1, H2, P1 and P2 in Tris-HCl buffer solution (containing 1 mM MgCl2, pH = 8.0) with a concentration of 10 μM, respectively;

[0045] Step 1.3: Mix the sequences of P1 and P2 at a ratio of 1:1;

[0046] Step 1.4: Anneal H1, H2 and the mixture of P1 and P2 in step 1.3 in a water bath at 95 °C for 10 minutes, and then slowly cool to room temperature;

[0047] Step 1.5: Before use, mix H1, H2 and the mixture of P1 and P2 at a volume ratio of 1:1:2 to prepare an enhancer probe (the final concentration of each sequence is 2.5 μM).

[0048] Step 1.6: In order to verify the function of the enhancer probe, mix the sequences H1 and H2 of the enhancer probe in PBS buffer solution at a ratio of 1:1 (the final concentration is 2.5 μM), and then add the sequence P2 as the target sequence to the above mixture, and react at 37 °C. The fluorescence intensity of the mixed solution was determined using a multifunctional enzyme marker.

[0049] Since P2 can open the hairpin structure of H1 and initiate subsequent signal amplification, in Example 1, the ability of H1 and H2 probes to recognize the sequence P2 in the buffer solution was first verified. According to the low fluorescence intensity of the H1 sequence itself, and the fluorescence signal recovered in the presence of P2 (H1+P2), it was confirmed that H1 could respond to the P2 sequenceFigure 2 ) For the mixture of H1 and H2 (H1+H2), the fluorescence intensity of the mixture was significantly enhanced after mixing with P2 (H1+H2+P2), which proved that the fluorescence signal was amplified. In addition, when P2 was combined with P1 in advance, the fluorescence intensity values of H1 (H1+(P1+P2)) and the mixture of H1 and H2 (H1+H2+(P1+P2)) were very low, which indicated that P2 could not bind with H1 to produce a fluorescence signal after P2 was combined with P1. Further, it was identified that the reaction time of H1 and H2 to detect sequence P2 was 50 minutes Figure 3 A), and the detection limit reached 5nM Figure 3 B).

[0050] Step 2: Preparation of nanoelectroporation biochip

[0051] Step 2.1: Mix the base and curing agent of polydimethylsiloxane (PDMS) uniformly at a mass ratio of 10:1;

[0052] Step 2.2: Pour the mixed glue into a flat mold, and the height of the PDMS is 3 millimeters;

[0053] Step 2.3: Vacuumize the PDMS in step 2.2 for 30 minutes;

[0054] Step 2.4: Cure the PDMS in step 2.3 at 80°C for 1 hour;

[0055] Step 2.5: Peel the cured PDMS layer in step 2.4 from the mold;

[0056] Step 2.6: Prepare the cell culture chamber and probe storage chamber (diameter 7 millimeters) by punching holes on the cured PDMS layer;

[0057] Step 2.7: Use a commercially available polycarbonate membrane with a pore size of 800 nanometers as a nanopore membrane to provide a nanopore for nanoelectroporation;

[0058] Step 2.8: Assemble the cell culture layer and the nanopore membrane by oxygen plasma bonding;

[0059] Step 2.9: Assemble the probe storage layer and the ITO glass electrode by oxygen plasma bonding.

[0060] Through the above steps, the nanoelectroporation biochip shown in Figure 4 is prepared. Among them, Figure 4 The nanopore layer shown in C plays a key role in accelerating the entry of enhancer probes into cells: the electric field is concentrated on the loaded cells through the nanopore Figure 5A), at a safe voltage (15V), the potential difference between the inside and outside of the cell membrane exceeds the transmembrane potential of the cell (about 0.2-1.0V). At this time, the cell membrane attached to the nanopore forms a metastable hydrophilic opening (>0.5 nanometers), which establishes a channel for the enhancer probe to enter Figure 5 B) At the same time, the high potential difference has an electrophoretic effect on the enhancer probe, which can accelerate the probe through the nanopore and the cell membrane. Subsequently, due to the enhancement of the potential difference, new perforations are generated in other parts of the cell membrane, and further lead to the rearrangement of the electric field around the nuclear membrane Figure 5 C-5D) The driving force of the positive potential difference between the inside and outside of the cell nucleus increases the opportunity of the enhancer probe to pass through the nuclear pore (80-120 nanometers), thereby promoting the aggregation of the enhancer probe in the cell nucleus Figure 5 E).

[0061] Step 3: Detection of enhancer activity in living cells

[0062] In order to test the ability of the method and device in the present disclosure to detect enhancer activity in living cells, in this embodiment, tumor cell lines A549 and H1975 are selected as high CCAT1 enhancer activity models, and fibroblast cell line MRC5 is selected as a low CCAT1 enhancer activity model, to detect enhancer activity in living cells.

[0063] Step 3.1: Before cell seeding, irradiate the nanoelectroporation biochip with ultraviolet light for 30 minutes;

[0064] Step 3.2: Seed 1×10 5 cells into the cell culture chamber, and then culture overnight;

[0065] Step 3.3: Replace the cell culture solution in the cell culture chamber with PBS buffer, add freshly prepared enhancer probe (60 μL) in the probe storage chamber, and then place the cell culture layer on the probe storage layer;

[0066] Step 3.4: Connect the upper silver electrode to the positive electrode of the electroporator, and connect the lower ITO glass electrode to the negative electrode;

[0067] Step 3.5: Apply a pulsed electric field, with the following specific conditions: voltage 15V, pulses 100, interval time 0.1 seconds, and pulse duration 3 milliseconds;

[0068] Step 3.6: After probe delivery, culture the cells in the cell culture medium at 37°C for 50 minutes;

[0069] Step 3.7: After digesting the cells with 0.25% trypsin, collect the cells;

[0070] Step 3.8: Wash the cells twice by centrifugation with PBS, and finally resuspend the cells in PBS;

[0071] Step 3.9: Fluorescent signal detection of cells by confocal microscope and flow cytometry, and cell images were analyzed by Image J software.

[0072] Through the above operation steps, green fluorescence was observed in the nucleus of A549 and H1975 cells, which indicated that the enhancer probe was successfully delivered into the nucleus of the cells Figure 6 A-6B). At the same time, MRC5 cells showed lower fluorescence signals, which could be attributed to their low CCAT1 enhancer activity. In contrast, no fluorescence signal was observed in cells incubated with the enhancer probe, indicating that the probe could not enter the nucleus by passive molecular diffusion Figure 6 C). In addition, the fluorescence signal of cells delivered with the enhancer probe (H1+H2+(P1+P2)) was significantly enhanced compared to that of cells delivered with H1+(P1+P2) probe (i.e. probe without signal amplification function) Figure 6 C), which proved that the enhancer probe completed the amplification of the circulating fluorescence signal. Further, after knocking down the expression level of SOX2, a transcription factor mediating the interaction between CCAT1 enhancer and CCAT1 promoter, the fluorescence signal in the cells was significantly reduced Figure 7 A-7B), indicating the specificity of the enhancer probe in detecting CCAT1 enhancer activity. In addition, according to the consistent growth state and migration ability of the cells before and after the detection, it can be determined that the behavior of the cells was not disturbed by the method and device of the present disclosure Figure 8 A-8B).

[0073] It should be noted that although the present disclosure has been shown and described with reference to specific exemplary embodiments thereof, it should be understood by those skilled in the art that the present disclosure is not limited to the above-described embodiments, and various modifications or variations of the present disclosure can be made without departing from the spirit and scope of the present disclosure, provided that such modifications and variations fall within the scope of the claims and equivalent technology of the present disclosure. The present disclosure also means to include such modifications and variations.

[0074] In particular, the features recited in the various embodiments and / or claims of the present disclosure can be combined and / or integrated in various combinations and / or combinations, even if such combinations or combinations are not explicitly recited in the present disclosure. All such combinations and / or combinations are within the scope of the present disclosure. Therefore, the scope of the present disclosure is not only determined by the appended claims, but also by the equivalents of the appended claims.

Claims

1. An apparatus for identifying enhancer activity in living cells, characterized by: The enhancer probe and the nanoelectroporation biochip, wherein the enhancer probe is arranged inside the nanoelectroporation biochip; The enhancer probe specifically feedbacks the activity state of the enhancer through amplified signals; The nanoelectroporation biochip is used to deliver the enhancer probe into the living cell nucleus; The enhancer probe is composed of a group of DNA sequences, and the detection signal is amplified through a cyclic fluorescent signal amplification strategy; The nanoelectroporation biochip is composed of five parts from bottom to top, which are lower electrode, probe storage chamber, nanopore layer, cell culture chamber and upper electrode; wherein the lower electrode is connected to the negative electrode of the electroporator; the probe storage chamber is used to store the probe solution to be delivered; the nanopore layer is used to focus the electric field and provide a channel for the probe to move from the probe storage chamber into the cell; the cell culture chamber is used for cell adhesion culture; the upper electrode is connected to the positive electrode of the electroporator and forms a path with the lower electrode; The DNA sequence comprises four functional sequences P1, P2, H1 and H2: P1: composed of three parts, including the part combined with the enhancer sequence, the part combined with the promoter sequence, and the connecting part between the two parts; P2: complementary to the sequence part combined with the promoter sequence in P1; H1: the sequence part is complementary to itself, has a "hairpin" structure containing a sticky end, so that the labeled fluorescent group is quenched due to the proximity of the quenched group; H2: the sequence part is complementary to itself, has a "hairpin" structure containing a sticky end; The cyclic fluorescent signal amplification strategy is as follows: sequence P2 can bind to sequence H1 and open the "hairpin" structure of H1, restore the fluorescent signal on H1, and then H2 can bind to sequence H1 and replace sequence P2 combined with H1, the replaced P2 can continue to bind to sequence H1, and the cyclic fluorescent recovery reaction is triggered; The nanopore layer has a through hole of 1-1000 nanometers, and the material is polycarbonate or silicon; The nanoelectroporation biochip has a cytoplasmic membrane perforation and generates a pushing force to promote the enhancer probe into the nucleus.

2. A method for identifying enhancer activity in living cells using the device of any one of claims 1, wherein the method is implemented by connecting the lower electrode of the device to the negative pole of an electroporator and the upper electrode to the positive pole of the electroporator, applying a square wave voltage pulse through the electroporator, and using the nanohole layer to generate a focusing electric field to safely and reversibly perforate the cell membrane and generate an electrophoretic force to accelerate the probes below the nanohole layer through the nanohole layer and into the perforated cell, and to accelerate the probes in the cell through the nuclear pores and into the nucleus; the probes in the nucleus first bind to the enhancer sequence via P1, and when the enhancer is in an activated state and interacts with the promoter, the other end of P1 binds to the promoter sequence, replacing P2 originally bound to P1; the replaced P2 binds to H1 in a hairpin shape, opening the hairpin shape of H1 and causing the fluorescent group to emit fluorescence away from the quenching group; subsequently, H2 binds to H1 in a complementary manner, replacing P2; the replaced P2 probe continues to bind to H1 in a hairpin shape, thereby starting a new cycle of fluorescence recovery and sequence replacement; after multiple cycles, the P2 replaced by the interaction of only one enhancer is amplified by multiple fluorescence signals, increasing the detection sensitivity.

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