CN104991090B - A kind of method of atomic force microscope detection single molecules level intermolecular interaction - Google Patents

Abstract

The invention discloses a method for atomic force microscopy to detect the interaction between molecules at the single-molecule level. DNA1 is modified on the substrate surface and DNA2 is modified on the AFM probe; when the AFM probe is close to the substrate surface, the DNA1 and DNA2 Complementary hybridization to form a DNA1/DNA2 double-stranded structure; use a nicking endonuclease to specifically recognize a specific sequence in the double-stranded DNA1/DNA2, and cut off the single-stranded DNA1; then use an atomic force microscope to detect the force between the DNA, thereby The change of the interaction force between the DNA before and after the cut of the DNA double strand by the nicking endonuclease is detected.

Description

An Atomic Force Microscopy Method for Detecting Intermolecular Interactions at the Single Molecule Level

technical field

The invention belongs to the field of biotechnology, and in particular relates to a method for detecting the interaction between molecules at the single-molecule level by an atomic force microscope.

Background technique

Single molecular detection (SMD) is an ultra-sensitive detection technology developed rapidly in the past decade. Conventional molecular detection techniques can only obtain the overall properties of general substances, and cannot accurately and effectively express individual molecules and the interactions between individual molecules. However, many biomolecules (such as proteins, DNA, RNA, etc.) have multiple conformations, and there are interactions between molecules. Therefore, detection of biomolecules at the single-molecule level is of great significance for identifying, classifying, and quantitatively describing biomolecules, monitoring chemical reaction pathways in real time, probing biomolecules under physiological conditions, and providing information between molecular structure and function.

Single-molecule detection is the detection of a finite number of molecules or molecular interaction events at the nanoscale spatial scale. Enzymatic reactions using atomic force microscopy enable controllable immobilization of target molecules on the surface of solid supports with nanometer or micrometer precision. Etching techniques for enzymatic nanofabrication can provide information on the morphology of enzymatic reaction products as they redeposit as the AFM probe moves. For example, negative images can be obtained by immobilizing active enzyme molecules on AFM probes to remove pre-existing biomolecules or convert them to small molecules. In order to achieve high-throughput and flexible molecular printing, dip-pennanolithography (DPN) and microcontact printing technology have been applied to multi-component DPN and polymer pen processing and etching technology, and can be verified by atomic force microscopy. or fluorescence imaging visualization. Since these techniques involve nanoscale manipulations, it is necessary to establish controllable methods to achieve the immobilization of individual molecules at specific locations on the substrate surface. Atomic force microscopy can fix individual molecules at designated positions, and has been applied to the study of single molecules and other single-molecule mechanical spectroscopy techniques.

In the prior art, there is no direct measurement of the interaction force between DNAs by using atomic force microscopy to monitor the shearing reaction of DNA double strands under the action of nicking endonucleases at the single molecule level. This situation needs to be resolved urgently.

Contents of the invention

The purpose of the present invention is to provide a method for atomic force microscopy to detect molecular interactions at the single-molecule level. By using the atomic force microscope to directly measure the interaction force between DNA, it is possible to monitor DNA double strands at the single-molecule level. The cleavage reaction under the action of endonuclease provides a new method for studying enzymatic reactions at the single-molecule level.

To achieve the above object, the present invention is achieved through the following technical solutions:

A method for atomic force microscopy to detect intermolecular interactions at the single-molecule level. DNA1 is modified on the substrate surface and DNA2 is modified on the AFM probe; when the AFM probe is close to the substrate surface, DNA1 and DNA2 are complementary hybridized to form DNA1/DNA2 double-stranded structure; use nicking endonuclease to specifically recognize a specific sequence in double-stranded DNA1/DNA2, and cut single-stranded DNA1; then use atomic force microscope to detect the force between DNA.

Include the following steps:

(1) AFM probe modified DNA2: carry out hydroxylation modification on the AFM probe to obtain a hydroxyl-modified AFM probe, then activate the hydroxyl-modified AFM probe, and immobilize the activated AFM probe with the amino-modified DNA2 , take out the AFM probe, and perform post-processing on the AFM probe.

(2) Immobilizing DNA1 on the gold substrate: Pretreating the gold substrate, performing immobilization reaction with the treated gold substrate and the sulfhydryl-modified DNA1, and reacting at 37° C. for more than 3 hours.

(3) Atomic force microscope force measurement: 3 drops of buffer solution on the gold substrate obtained in step (2), fixed the droplet on the scanner of the atomic microscope, and measured the interaction force F1 between DNA; For endonuclease, add the mixed solution containing 1× nicking endonuclease buffer, nicking endonuclease and buffer solution 3 dropwise onto the gold substrate, and fix the droplet on the scanner of the atomic microscope , to measure the interaction force F2 between DNAs; thereby detecting the change of the interaction force between DNAs before and after the nicking endonuclease cuts the DNA double strands.

In step (1), the process of the hydroxylation modification is: soak the AFM probe in a mixed solution of concentrated sulfuric acid and hydrogen peroxide (concentrated sulfuric acid (mass fraction 95-98%): hydrogen peroxide (mass fraction 30%)=7 : 3 (volume ratio)) for 30 minutes, washed with deionized water to obtain a hydroxyl-modified AFM probe.

In step (1), the activation process is as follows: immerse the hydroxyl-modified AFM probe in 200 μl of 100 mM N-hydroxysuccinimide (NHS) solution (note: the NHS solution is prepared now), protected from light After standing for 30 minutes, rinse with deionized water.

In step (1), the post-treatment process is as follows: first wash with buffer solution 2 three times, then wash with deionized water, and dry in air for 30 minutes. The composition of the buffer 2: 300mM NaCl, 20mM Na ₂ HPO ₄ , 2mM ethylenediaminetetraacetic acid, 7mM sodium dodecyl sulfate, pH=7.4; function: used for cleaning the AFM probe.

In step (1), the process of the immobilization reaction is: soak the AFM probe modified with DNA2 in 200 μl of amino-modified DNA2 solution with a concentration of 1 μM, and react at 25° C. for more than 12 hours, preferably 12 to 24 hours, and at most 16 hours is preferred.

In step (2), the process of pretreating the gold substrate is: cleaning and drying the gold substrate. The further specific process is: immerse in cleaning solution for 10 minutes in ultrasonic, rinse with deionized water, and blow dry with nitrogen. The cleaning solution is: a mixed solution of ammonia water and hydrogen peroxide, wherein the volume ratio of water:ammonia water:hydrogen peroxide is 5:1:1.

In step (2), the fixation reaction process is as follows: the treated gold substrate is soaked in 1 ml of sulfhydryl-modified DNA1 with a concentration of 1 μM to carry out the fixation reaction.

In step (2), the reaction time is 3 to 24 hours.

In step (3), the process of measuring the interaction force between DNAs is as follows: after putting the AFM probe into the liquid phase probe frame, the probe frame is loaded into the atomic force scanning microscope. Turn on the switch of the instrument, adjust the probe frame to make it descend, and contact with the liquid droplet on the gold substrate. After ensuring that there are no air bubbles between the gold substrate and the glass slide of the probe holder, adjust the light spot. Select the contact mode, enter the needle, dwell time: 10000ms. Start to measure the interaction force between DNA, so as to detect the change of the interaction force between DNA double strands before and after nicking endonuclease cutting.

In step (3), the composition of the buffer 3 is: 20mM Tris-HCl, 0.1M MgCl ₂ , pH=8.0, function: a detection system for atomic force microscope force measurement.

In step (3), the nicking endonuclease is a type of endonuclease that recognizes a specific DNA sequence and cuts a DNA strand in a double-stranded DNA substrate at or around the recognition site. The nicking endonuclease is nicking endonuclease Nb.BbvCI; the composition of the 1× nicking endonuclease Nb.BbvCI buffer is potassium acetate, Tris-acetic acid, magnesium acetate and bovine serum albumin (BSA) , wherein the concentration of each component is: 50mM potassium acetate, 20mM Tris-acetic acid, 10mM magnesium acetate, 100μg/ml BSA, pH7.9.

The DNA1 and DNA2 are two base-complementary strands to be tested.

The beneficial effects of the present invention are: the present invention establishes an atomic force microscopy (AFM) method for studying the cleavage reaction of a DNA double strand under the action of a nicking endonuclease at the single-molecule level. A gold sheet was selected as the substrate, and DNA1 was immobilized on the gold substrate through the Au-S covalent bond to make it complementary hybridize with the DNA2 immobilized on the AFM probe through the amide reaction, thereby forming a fully complementary hybridized DNA1/DNA2 double on the surface of the substrate. strand structure, and the double strand contains specific recognition sites for nicking endonucleases. Under the action of nicking endonuclease, the single-stranded DNA1 immobilized on the gold substrate was cut into DNA1' and DNA1", wherein DNA1' was immobilized on the gold substrate. Compared with the double-stranded DNA1/DNA2, since DNA1' and DNA2 The number of bases for complementary hybridization decreases, so the DNA interaction force decreases. The present invention realizes the monitoring of DNA double strands under the action of nick endonuclease on the single molecule level by directly measuring the interaction force between DNAs by using an atomic force microscope. The shearing reaction that occurs provides a new method for studying enzymatic reactions at the single-molecule level, and can accurately and effectively express single molecules and the interactions between single molecules, providing a theory for revealing important processes of life phenomena It has extensive practical application value in the fields of analytical chemistry, clinical diagnosis and bioprocess dynamics.

Description of drawings

Figure 1: Schematic diagram of atomic force microscopy to study DNA double strand cleavage reaction under the action of nicking endonuclease at the single-molecule level.

detailed description

The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

In the present invention, the AFM probe comes from Yuanyuan Nano Instrument Co., Ltd. (contact mode), the gold substrate comes from BioNavis Company of Finland, DNA1 and DNA2 come from Sangon Bioengineering (Shanghai) Co., Ltd., and the atomic force microscope comes from Yuanyuan Nano Instrument Co., Ltd. CSPM5500 scanning probe microscope (SPM) system, other reagents are from commercial sources unless otherwise specified.

In the present invention, M means mol/L, mM means mmol/L, and μM means μmol/L.

Example 1

In this example, the nicking endonuclease Nb.BbvCI was selected to study the cleavage reaction of DNA double strands at the single-molecule level under the action of the nicking endonuclease (the experimental principle is shown in FIG. 1 ). It mainly includes the following steps:

1. Modified DNA1 on the AFM probe:

1. First prepare buffer solution 1, buffer solution 2 and buffer solution 3 respectively.

The composition of buffer solution 1: 25 mM NaHCO ₃ , 5 mM MgCl ₂ , 10% (v/v) dimethyl sulfoxide, pH=8.5. Function: used to prepare DNA solution.

The composition of buffer solution 2: 300 mM NaCl, 20 mM Na ₂ HPO ₄ , 2 mM EDTA, 7 mM sodium dodecyl sulfate, pH=7.4. Function: Used to clean the AFM probe.

The composition of buffer solution 3: 20mM Tris-HCl, 0.1M MgCl ₂ , pH=8.0. Function: The detection system of atomic force microscope force measurement.

2. Soak the AFM probe in the mixed solution of concentrated sulfuric acid and hydrogen peroxide (concentrated sulfuric acid (mass fraction 95%): hydrogen peroxide (mass fraction 30%) = 7:3 (volume ratio)) for 30 minutes, then wash it with deionized water , to obtain a hydroxyl-modified AFM probe with a probe force constant of 0.2N/m.

3. Soak the hydroxyl-modified AFM probe in 200 μl of 100 mM N-hydroxysuccinimide (NHS) solution (note: the NHS solution is ready for use), activate the AFM probe, and place it in the dark for 30 minutes before removing it. Rinse with ionized water.

4. Soak the obtained AFM probe in 200 μl of amino-modified DNA2 solution with a concentration of 1 μM, react at 25° C. for 16 hours, and immobilize the DNA2 on the AFM probe through amide reaction.

Wherein, the nucleotide sequence of DNA2 is, as shown in SEQ ID NO: 2:

5'-GGTTACTATCACGCCTCAGCCTGAGGCATCAGCACGATTAGTAGTCATATTAGGTAGGG-3', 3' terminal amino modification.

5. Take out the AFM probe, wash it three times with buffer solution 2, then wash it with deionized water, and dry it in the air for 30 minutes.

2. Immobilizing DNA2 on the gold substrate:

1. Before using the gold substrate, soak it in ultrasonic for 10 minutes with cleaning solution (composition of cleaning solution: water: ammonia water: hydrogen peroxide volume ratio is 5:1:1), rinse with deionized water, and blow dry with nitrogen.

2. Soak the treated gold substrate in 1ml of thiol-modified DNA1 with a concentration of 1μM, and react at 37°C for 3 hours.

Wherein, the nucleotide sequence of the DNA1 is, as shown in SEQ ID NO: 1:

5'-CCCTACCTAATATGACTACTAATCGTGCTGATGCCTCAGGCTGAGGCGTGATAGTAACC-3', 3' end thiol modification.

3. Atomic force microscope force measurement

1. Three drops of 100 μl buffer solution were placed on the gold substrate immobilized with DNA1 obtained in step 2 to form a large droplet. Fix the large droplet on the scanner of the atomic force microscope.

2. After putting the AFM probe into the liquid phase probe rack, put the probe rack into the atomic force scanning microscope. Turn on the switch of the instrument, adjust the probe frame to make it descend, and contact with the liquid droplet on the gold substrate. Since DNA1 and DNA2 sequences are completely complementary and contain specific recognition sites for the nick endonuclease Nb.BbvCI (▲ represents the cutting position). When the AFM probe approaches the substrate surface, DNA1 and DNA2 hybridize complementary to form a DNA1/DNA2 double-stranded structure.

After ensuring that there are no air bubbles between the gold substrate and the glass slide of the probe holder, adjust the light spot.

3. Select the contact mode, insert the needle, and dwell time: 10000ms. Start measuring. The force at 5 different positions was measured by atomic force microscope (50 parallel measurements at each position), and the measured interaction force between double-stranded DNA1/DNA2 was 30.5±3.7nN.

4. Using the above steps to obtain the DNA2 modified AFM probe and the DNA1 modified gold substrate. Add 10 μl of 10× nicking endonuclease buffer, 10 U of nicking endonuclease, add buffer 3 to supplement the volume to 100 μl, and add dropwise onto the gold substrate modified by DNA1. Make the nicking endonuclease Nb.BbvCI recognize the cleavage site of DNA1 in double-stranded DNA1/DNA2, and cut DNA1 into DNA1' and DNA1", wherein DNA1' is immobilized on the substrate. After the reaction, take back the AFM probe, At this time, the force between the DNAs was measured to be 10.2±3.7nN. It shows that the nicking endonuclease Nb.BbvCI specifically recognizes the cleavage site of double-stranded DNA1/DNA2, and then fixes the single-stranded DNA1 on the gold substrate. Cut into two parts DNA1' (18 bases) and DNA1" (41 bases). Since DNA1' is immobilized on the gold substrate, compared with double-stranded DNA1/DNA2 (59 base pairs) before shearing, the number of bases that DNA1' hybridizes complementary to DNA2 is reduced to 18 base pairs, so the DNA The interaction force is reduced, and the detection of enzymatic reactions at the single-molecule level is realized. This method can directly provide information on intermolecular interactions at the single-molecule level, providing a theoretical basis for revealing important processes of life phenomena, and has extensive practical application value in the fields of analytical chemistry, clinical diagnosis, and biological process dynamics.

Wherein, the composition of the 1×nicking endonuclease Nb.BbvCI buffer solution is potassium acetate, Tris-acetic acid, magnesium acetate, BSA, wherein the concentration of each component is respectively: 50mM potassium acetate, 20mM Tris-acetic acid, 10mM Magnesium acetate, 100 μg/ml BSA, pH 7.9.

In this example, an atomic force microscopy technique is used to establish a method for studying the cleavage reaction of a DNA double strand under the action of a nicking endonuclease at the single-molecule level. DNA1 and DNA2 were modified on the substrate surface and the AFM probe, respectively. When the AFM probe is close to the substrate surface, a DNA1/DNA2 double-stranded structure is formed through the complementary hybridization of DNA1 and DNA2. The nicking endonuclease can specifically recognize a specific sequence in double-stranded DNA1/DNA2 and cut single-stranded DNA1. Since the length of hybridization between the DNA on the substrate and the DNA on the AFM probe becomes shorter after cleavage, when the AFM probe is lifted up, the interaction force between DNA measured by atomic force microscopy decreases.

Although the specific implementation of the present invention has been described above in conjunction with the accompanying drawings, it does not limit the protection scope of the present invention. Those skilled in the art should understand that on the basis of the technical solution of the present invention, those skilled in the art do not need to pay creative work Various modifications or variations that can be made are still within the protection scope of the present invention.

Claims (8)

1. atomic force microscope is for the method detecting single molecules level intermolecular interaction, it is characterized in that: in substrate Surface modifying DNA 1 and on AFM probe modifying DNA 2, the concrete grammar of described AFM probe modifying DNA 2 is: entered by AFM probe Row hydroxylating is modified, and obtains the AFM probe of hydroxyl modified, the AFM probe that then activated hydroxyl groups is modified, is visited by the AFM after activation Pin and amido modified DNA2 are fixed reaction, take out AFM probe, AFM probe is carried out post processing；When AFM probe and base Basal surface near time, by the Complementary hybridization of DNA1 Yu DNA2, form DNA1/DNA2 duplex structure, wherein, described substrate surface The concrete grammar of upper modifying DNA 1 is: substrate uses gold substrate, first pretreatment gold substrate, then by the gold substrate handled well with The DNA1 of sulfydryl modification is fixed reaction, and 37^oC reacts more than 3 hours；Use nicking restriction endonuclease specific recognition double-strand Particular sequence in DNA1/DNA2, and single stranded DNA 1 is cut off；Then the effect between atomic force microscope detection DNA is used Power, thus detect the change of the Interaction Force of DNA before and after nicking restriction endonuclease is sheared of DNA double chain.

2. the method for claim 1, is characterized in that, the active force between described employing atomic force microscope detection DNA Concrete grammar be: buffer solution 3 is dropped in the gold substrate being modified with DNA1, drop is fixed on sweeping of atomic force microscope Retouch on device, survey the interaction force F1 between DNA；Use nicking restriction endonuclease time, will containing 1 × nicking restriction endonuclease buffer, cut Carve restriction endonuclease and the mixed solution of described buffer solution 3, be added drop-wise in gold substrate, drop is fixed on sweeping of atomic force microscope Retouch on device, survey the interaction force F2 between DNA；Thus detect DNA double chain phase between DNA before and after nicking restriction endonuclease is sheared The change of interreaction force.

3. the method for claim 1, is characterized in that: the process that described hydroxylating is modified is: be immersed in by AFM probe dense In the mixed solution of sulphuric acid and hydrogen peroxide 30 minutes, deionized water was cleaned, and obtains hydroxyl modified AFM probe.

4. the method for claim 1, is characterized in that: described activation process is: be immersed in by the AFM probe of hydroxyl modified 200 μ l concentration are in 100 mM N-hydroxy-succinamide (NHS) solution, after lucifuge is placed 30 minutes, and deionized water rinsing Totally.

5. the method for claim 1, is characterized in that: the process of post processing is: first flush three times with buffer solution 2, then It is washed with deionized water clean, air drying 30 minutes；The composition of described buffer 2: 300 mM NaCl, 20 mM Na₂HPO₄, 2 MM ethylenediaminetetraacetic acid, 7 mM sodium lauryl sulphates, pH=7.4；Effect: be used for cleaning AFM probe.

6. the method for claim 1, is characterized in that: described will activation after AFM probe enter with amido modified DNA2 The process of the fixing reaction of row is: the AFM probe being modified with DNA2 is immersed in the amido modified DNA2 that 200 μ l concentration are 1 μM In solution, 25^oC reacts more than 12 hours.

7. the method for claim 1, is characterized in that: the process of described pretreatment gold substrate is: gold substrate cleaned, do Dry；The most concrete process is: soak ultrasonic 10 minutes with cleanout fluid, and deionized water rinsing is clean, and nitrogen dries up；Described clearly Washing liquid is: ammonia and the mixed solution of hydrogen peroxide, Qi Zhongshui: ammonia: the volume ratio of hydrogen peroxide is 5:1:1.

8. the method for claim 1, is characterized in that: the described DNA1 by the gold substrate handled well with sulfydryl modification is carried out The process of fixing reaction is: the gold substrate handled well is immersed in the sulfydryl modification DNA1 that 1 ml concentration is 1 μM and is fixed Reaction.