KR20150019136A

KR20150019136A - Direct detecting method for target proteins using dna probe

Info

Publication number: KR20150019136A
Application number: KR20130095596A
Authority: KR
Inventors: 권영남; 한혁수; 변경은; 김희구; 이재우
Original assignee: 삼성전자주식회사
Priority date: 2013-08-12
Filing date: 2013-08-12
Publication date: 2015-02-25

Abstract

The present invention aims to provide: a DNA probe which directly and precisely measures interaction between a DNA and a protein; a method for measuring binding force of a target protein; and a method for detecting a target protein. The method for measuring interaction between a DNA and a protein includes the steps of: preparing a nucleic acid probe wherein nucleic acid is attached on a probe located on an end portion of a cantilever of an atomic force microscopy (AFM); measuring force generate while the nucleic acid probe approaches the target protein and then gets away from the target protein; and analyzing interaction between the nucleic acid and the target protein from a measured value of displacement of the cantilever.

Description

TECHNICAL FIELD [0001] The present invention relates to a method for directly detecting a target protein using a nucleic acid probe,

The present disclosure relates to a method for analyzing the interaction between a DNA probe and a target protein and a method for detecting a target protein, which can precisely measure the binding force and the number of binding sites between DNA and protein using an Atomic Force Microscopy (AFM) .

Atomic force microscopy has been used as an important means of understanding the various reaction mechanisms between biomolecules in vivo. Since atomic microscopy can analyze the binding between biomolecules, it is expected to be more useful in future nanotechnology and biotechnology, such as many studies carried out at the molecular level.

Specifically, the atomic force microscope senses the topography of the sample using a probe mounted on the end of the micro-machined cantilever. When the sample is scanned, the pivotal deflection of the cantilever occurs due to the interaction of the atomic force between the nano-meter sharp probe and the sample surface. The fine shape of the sample is determined by detecting such deflection.

Attempts have been made to observe inter-biomolecule binding using the operational principles and properties of the AFM mentioned above. However, this was largely limited to DNA-DNA interactions or protein-protein interactions.

Meanwhile, intracellular DNA plays an important role in the storage, transport, processing, and expression of genetic information. At this time, it is important to observe DNA-protein interactions because DNA performs its functions through interaction with proteins in cells. In other words, looking for a specific protein site that recognizes DNA or finding a specific DNA site that recognizes a protein is very important for understanding various biological processes.

Thus, attempts have been made to develop various measurement methods for observing protein-DNA interactions. Methods for detecting proteins that interact with DNA sequences include analytical methods such as radioisotope labeling, chemical staining, and indirect fluorescence staining.

However, there is a need to develop a method for evaluating the interaction in a more direct way through biomolecular interaction force (interaction force) measurement, such as prediction of a specific nucleotide sequence and binding site of DNA and binding propensity between protein and base .

In response to this need, the present disclosure relates to a method for measuring the binding force between a DNA probe and a target protein that directly and highly precisely measures the interaction between DNA and protein, particularly the interaction force (binding force) Thereby providing a protein detection method.

In particular, the present disclosure provides a method of detecting the binding specificity between DNA and protein by measuring the force of a specific protein binding to DNA using an AFM probe modified with DNA having a specific base sequence.

In one aspect, there is provided a method for preparing a nucleic acid probe, comprising: preparing a nucleic acid probe having a nucleic acid attached to a probe located at a cantilever end of an atomic force microscope (AFM); Measuring a force generated when the nucleic acid probe approaches and distances the target protein; And analyzing the interaction between the nucleic acid and the target protein from the measured value of the displacement of the cantilever.

The method includes preparing a nucleic acid probe having a nucleic acid attached to a probe located at a cantilever end of an atomic force microscope (AFM).

The step of preparing the nucleic acid probe may be to attach the nucleic acid to the solid surface. The attachment may be to activate the solid surface and bind the nucleic acid thereto. The nucleic acid may be a 5 'terminal, a 3' terminal, or both terminals modified to an active group. The activating group may be a thiol group, an aldehyde group, a carboxyl group, an activated ester group, or a combination thereof. Activation of the solid surface may be by coating an amino group, a reactive metal such as gold, or a combination thereof.

The nucleic acid may be labeled with a detectable label at the opposite end attached to a solid surface. The label may be an optical label, a radioactive label, an enzyme, or a combination thereof. The optical label may be a fluorescent material, a phosphorescent material, or a combination thereof. Wherein the detectable label is selected from the group consisting of biotin, rhodamine, cyanogen 3, cyanogen 5, pyrene, cyanogen 2, green fluorescent protein (GFP), calcein, fluorescein isothiocyanate (FITC) (HEX), 2 ', 7'-dichloro-6-carboxy-fluorene -Carboxy-4,7-dichlorofluorescein (TET), fluorescein chlorotriazinyl, fluorescein, Oregon green, magnesium green, calcium green, 6-carboxy- (JOE), tetramethylrhodamine, tetramethyl-rhodamine isothiocyanate (TRITC), carboxytetramethylrhodamine (TAMRA), rhodamine paloidine, pyrironine Y, lysamine , X-rhodamine (ROX), calcium cream line, Texas red, nile red and thiadicarbocyanine.

The nucleic acid may be a natural or a non-native nucleic acid. The nucleic acid may be DNA, RNA, PNA or a combination thereof. The nucleic acid may be double stranded, single stranded or a combination thereof. The nucleic acid may be 5 to 400 nt, 5 to 300 nt, 5 to 200 nt, 5 to 100 nt, 5 to 50 nt, 5 to 40 nt, 5 to 30 nt, 5 to 20 nt, 5 to 10 nt, 10 to 400 nt, , 25 to 100 nt, 10 to 50 nt, 15 to 40 nt, 10 to 30 nt, or 10 to 20 nt. The attachment may be to attach the 5 ' end, the 3 ' end, or both ends of the nucleic acid. The attachment may be accomplished by incubating the activated probe and the nucleic acid in a liquid solution. The incubation conditions can be appropriately selected depending on the activation method and the nucleic acid to be selected. The attachment may be made in the state where the probe is mounted on the AFM, or may be attached in a separated state and then attached to the AFM.

The probe located at the cantilevered end of the Atomic Force Microscopy (AFM) comprises a free end that is moved toward the target protein. The probe may be a surface exposed to the target protein when moved toward the target protein. The probe may be pyramidal or conical.

The AFM may include a cantilever with a tip at the tip so that it can be used to scan a specimen surface. A tip in this specification is used interchangeably with a probe. The cantilever may have one or more probes.

The probe attached to the distal end of the cantilever may have a radius of curvature on the order of nanometers. For example, the radius of curvature may be between 1 nm and 1000 nm, between 1 nm and 900 nm, and between 10 nm and 50 nm. The probe may be made of an insulating material. The probe may be comprised of silicon, silicon nitride, or a combination thereof.

The method also includes measuring the force between the nucleic acid probe and the target protein that occurs while the nucleic acid probe approaches and is away from the target protein.

The step of measuring the force may be performed while the nucleic acid probe approaches and moves away from the target protein under the condition that the nucleic acid and the protein can bind. By manipulating the AFM, the probe attached to the cantilever end can be directed to contact or adjacent to the protein.

The conditions under which the nucleic acid and the protein can bind include aqueous conditions. These aqueous conditions include MES, Bis-Tris, ADA, PIPES, MOPSO, BES, MOPS, TES, HEPES, DIPSO, MOBS, TEA, EPPS, Tricine, Bicine, HEPBS, TAPS, AMPD, TABS , AMPSO, CAPSO, AMP, CAPS, CABS or phosphate citrate glycine carbonate, or brine using some of the buffers. The saline solution may contain cations such as sodium, potassium, calcium, and magnesium. In addition, trace elements such as manganese, cobalt, nickel, copper, zinc and iron may be added to the solution. It may further contain surfactants or antioxidants to prevent protein denaturation. The surfactants include ionic and nonionic surfactants.

When the nucleic acid probe approaches the target protein, there may be an initial contact between the nucleic acid and the protein, or they may be adjacent to each other. Such initial contact or adjacency can be attributed to the affinity of the nucleic acid and protein, such as Van der Waals force, which is an attractive force between electrically neutral molecules. Defects may occur in the cantilevers as the nucleic acid probes attached with the nucleic acid probes are brought into contact with or close to the target protein while the nucleic acid and the protein are in contact or adjacent to each other. Therefore, the degree of the displacement of the cantilever may be proportional to the affinity between the nucleic acid and the target protein.

When the nucleic acid probe approaches and then retreats to the target protein, a force may be generated that interferes with the retraction of the nucleic acid probe. The force that prevents this return is also called the unbinding force. The unbinding force is due to the affinity or adhesive force between the nucleic acid and the protein. In other words, due to the affinity of the nucleic acid and the protein to be close to each other, an adhesive force for maintaining the contact state between the nucleic acid and the protein occurs, and due to the adhesive force, an unbinding force may be generated. The unbinding force can be measured through the displacement of the cantilever that occurs during and after the nucleic acid probe approaches the target protein. The principle of measuring the displacement of the cantilever is the law of the hook, and the law can be expressed by the following equation.

F = kx

Where F is the magnitude of the force, k is the spring constant, and x is the displacement length. The displacement length (degree of displacement) is proportional to the magnitude of the force externally applied. The spring constant may vary from 10 pN / nm to 20 pN / nm. The range of the force may vary from 100 nm / s to 600 nm / s.

Through the displacement of the cantilever, a force-distance curve showing the distance between the nucleic acid and the protein and the value of the force between the nucleic acid and the protein depending on the distance can be obtained by measuring the force between the nucleic acid and the protein. If the nucleic acid-protein binds and there is an unbinding force, when the nucleic acid-attached cantilever moves away from the target protein, the unbinding force increases from zero to the maximum unbinding force, and when the cantilever is completely away from the protein The force will be zero.

The time at which the tip of the cantilever with the nucleic acid probe attached thereto approaches the protein can be set. The time may be about 1 to 4 seconds, 1 to 3 seconds, 1 to 2 seconds, 0.5 to 1.5 seconds, and so on. The time at which the cantilever with the nucleic acid probe attached thereto moves away from the target protein, i.e., the retraction time, may be the same as or different from the approach time of the cantilever. In addition, the approach may be to move the tip relative to the target protein at a rate of about 1 [mu] m / s to 150 [mu] m /. The receding may be to move the tip from the target protein at a rate of about 1 [mu] m / s to 150 [mu] m / s. The approach may be to approach from 0 to about 1 nm for the target protein.

The step of analyzing the interaction between the nucleic acid and the target protein from the measured value of the displacement of the cantilever may include determining the binding strength between the nucleic acid and the protein, the number of binding sites, or the binding site.

The analysis of the interaction may be based on the correlation of the displacement of the cantilever with respect to the distance to the target protein of the cantilever tip. The displacement may be correlated with the interaction between the nucleic acid probe and the target protein. Thus, the analysis of the interaction may be based on the interaction forces of the nucleic acid probe and the target protein with respect to the distance to the target protein of the cantilever tip. The analysis of the interaction may also include comparing the force between the nucleic acid probe and the target protein measured while moving away the force between the nucleic acid probe and the target protein measured during the approach.

The binding strength between the nucleic acid and the protein can be determined from the max unbinding force (pN) obtained from the measurement of the displacement of the cantilever. The bond strength is proportional to the measured value of the displacement of the cantilever. When there is a bond between the nucleic acid and the protein, a high specific unbinding force can be measured.

Further, the number of binding sites between the nucleic acid and the protein can be determined based on the number of peak values obtained from the measurement of the displacement of the cantilever. The peak value means, for example, a measurement value of a portion having a larger value than the left and right values in the waveform of the graph showing the measured value of the displacement of the cantilever. The number of such peak values may be an integer, but may be a number having a decimal point. Even when the number of binding sites measured is not an integer, the number of binding sites can be determined through this to determine the binding pattern between the nucleic acid and the protein. Further, the position of the coupling portion can be determined based on the interval between the peak values obtained from the measurement of the displacement of the cantilever. The narrower the interval of the peak value, the closer the distance between the binding sites is.

Based on the measured value of the displacement of the cantilever, information on the binding strength, the number of binding sites and the location of the binding site can be determined, and the interaction between the nucleic acid and the protein can be analyzed. Information about the interaction between the nucleic acid and the protein can be used to detect the target protein. Therefore, the step of analyzing the interaction between the nucleic acid and the target protein may further include the step of detecting the target protein. The step of detecting the target protein includes a step of detecting a target protein in a sample by comparing the measured value of the displacement of the cantilever with a statistical value of data obtained through a method of analyzing interaction between a nucleic acid and a protein as a reference value .

The above-mentioned method for measuring the interaction between the nucleic acid probe and the target protein comprises the steps of: predicting the protein binding site by measuring the interaction force between a specific DNA base sequence and a site capable of binding with the protein; , The secondary structure of the protein, the length of the DNA sequence, and the frequency of binding, etc., can be made more easily and precisely under unlabeled conditions.

Through this, it is expected that it can be widely used in the fields of development of new drugs for proteomics research, diagnosis, and the like, such as protein expression and function studies, protein interaction studies and the like.

Figure 1 diagrammatically illustrates an experimental model implemented in an embodiment.
Figure 2 shows an experimental scheme for measuring biomolecular unbinding forces.
Figure 3 shows a typical force-distance graph measured by the AFM.
Fig. 4 shows a graph showing the maximum value of the unbinding force of the four experimental models measured in the embodiment as a percentage.
5A shows the result of measuring the unbinding force between the reference probe and the SSB protein.
FIG. 5B shows the result of measuring the unbinding force between ssDNA and SSB protein according to the embodiment.
FIG. 5C shows the result of measuring the unbinding force between ssDNA and BSA according to the embodiment.
FIG. 5D shows the result of measuring the unbinding force between the dsDNA and the SSB protein according to the embodiment.
FIG. 6 is a graph showing the results of measurement of unbinding force between ssDNA and SSB protein according to an embodiment in more detail.
Figure 7 shows the highest value of the unbinding force of the four experimental models measured in the example.
Figure 8 shows a graph showing the number of unbindings per curve of the four experimental models measured in the example.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example 1: Analysis of interaction between DNA and SSB using single strand DNA binding to SSB protein and AFM

In this embodiment, a single model system capable of showing a method for measuring the binding force between DNA and protein and a method for detecting a target protein is disclosed. Single-stranded DNA-binding proteins (SSB) The interaction between single stranded pyrimidine-rich sequences was analyzed. Single stranded pyrimidine-rich sequences were synthesized.

1. Materials

Single stranded DNA binding protein (SSB) was purified from Escherichia coli and purchased from Elpisbiotech Inc., South Korea. The size of the SSB is about 70 kDa and its diameter is about 5.76 nm.

Thiol modified single strand (SEQ ID NO: 1, oligomer, 45mer) and double stranded DNA (SEQ ID NO: 2, 45mer) were all commercially synthesized in Bioniar (Bioneer Co., Korea).

Thiol modified single strand (SEQ ID NO: 1):

5 'SH- CTC CCT CCC TCC CTC CCT CCC TCC CTC CCT CCT TCT CCC TCC CTC-FAM- 3' (oligo 1, 45mer)

Thiol modified double-stranded DNA (SEQ ID NO: 2):

5 'SH- CTC CCT CCC TCC CTC CCT CCC TCC CTC CCT CCT TCT CCC TCC CTC -FAM

GAG GGA GGG AGG GAG GGA GGG AGG GAG GGA GGA AGA GGG AGG GAG (45mer)

2. Tip Preparation

The tip material is silicon, the shape is tetrahedral shape, the height is 4-6 μm, gold coated, and the coating thickness is 30 nm +/- 5 nm. The force constant of the cantilever used is 0.045 N / m (Range 0.024 to 0.079 N / m) and the Resonance Frequency is 17 kHz (Range 14 to 21 kHz) (Applied NanoStructures, Inc. Lt; / RTI > A gold-coated AFM probe was attached to the end of a specific DNA sequence with an oligomer synthesized in the thiol (SH) group. In addition, a fluorescent substance called FAM was attached to the opposite side of the DNA sequence to confirm whether the base sequence was attached to the gold-coated AFM probe. At this time, the AFM probe was subjected to a passivation process using ODT to adjust the amount of DNA in the gold-coated AFM probe. The passivation process was carried out with a partial passivation process at room temperature for 1 minute with 1 uM ODT (1-octadecanethiol). Then, it was washed with ethanol for 10 seconds. For 1mM for 10 sec, for concentration, 3mM ODT may be used. Next, the obtained passivated gold-coated AFM probe was incubated at room temperature for 10 minutes after addition of 10 pmoles of pyrimidine-rich sequences aqueous solution (confirmation required), followed by washing with distilled water Demand).

SSB, which is a specific protein tested, preferentially reacts with ssDNA, so that the dsDNA-modified AFM probe was subjected to the same procedure for the control experiment in order to discriminate it from dsDNA.

3. Immobilization of protein on surface

SSB and BSA (Bovine serum albumin), which are specific proteins, were added to the cells in order to maintain the state of minimizing the external interference force to see how the ssDNA having the specific nucleotide sequence had a certain interaction force with the specific protein. And fixed on the surface of a substrate coated with a linker (Proteogen.co., Prolinker type A Subtrate). The concentration of the two proteins tested was spotted on the substrate at the same concentration of 100 ng / ul, incubated for 15 minutes at 37 ° C in humid conditions, and then washed three times with distilled water. In order to examine the reactivity with BSA as a comparative group, they were fixed on the surface in the same manner. The concentration of the two proteins tested was spotted on the amine substrate at the same concentration of 100 ng / μl, fixed in humid conditions at 37 ° C for 15 minutes, and washed three times with distilled water (see Table 1)

Input Analysis Results Glass type DOE Epoxide glass The solvent (glycerol amount) DOE 0% glycerol in PBS Incubation Time DOE 15 minutes Incubation temperature DOE 37 ℃

* DOE (Design of Experiment): You can check the interaction with the DOE variable (Variable).

4. AFM Analysis

AFM analysis was performed using a Bruker Catalyst, a force-distance measurement of the DNA immobilized on the AFM tip and the protein immobilized on the bottom in a PBS buffer solution.

5. Results

An experiment was conducted as shown in Fig. 2 to show an embodiment of a method of detecting a specific protein having an attractive force by using single-stranded DNA having a specific base sequence. Here, the attraction can be explained by the force when the ssDNA and the SSB come close to each other, that is, by measuring the unbinding force. Figure 1 diagrammatically illustrates this experimental model.

The molecular recognition ability and specificity of the ssDNA modified AFM probe were confirmed by measuring the force between ssDNA and SSB as described above. Using the AFM tip with the same spring constant, the tip was placed close to the sample. The amount of deflection change of the tip was measured by the amount of force and the displacement of the force when the tip was released to its original position. Statistics for each of the 25 values were obtained for each of the four experimental combinations. As shown in Fig. 2, four combinations of experiments were performed under the same measurement conditions.

A specific unbinding force ratio of ssDNA to SSB protein was detected to be 92% or more in the four combinations of tested conditions (see FIG. 4). The greater the specific unbinding force ratio, the more certain the strength of the specific interaction force between the two biomolecules can be. The graph of FIG. 5A shows the result of measuring the unbinding force between the gold-coated AFM probe and the SSB protein, in which the AFM probe was partially passivated with ODT. The graph of FIG. 5B shows a significant difference in unbinding force between single-stranded DNA and SSB protein as a measurement value in contrast to No. 1. FIG. 5C is a graph showing the unbinding force between single-stranded DNA and BSA, showing a similar reaction to the graph of FIG. 5A. The graph of FIG. 5D shows the results of measurement of unbinding force between dsDNA and SSB protein, To determine whether the SSB protein has unbinding specificity for ssDNA d. As a result, it was confirmed that it showed a similar pattern to the reference.

6 to 8 are the results of analyzing the measured values for the same experiment. Compared with the four combinations tested, the mean value of the highest unbinding force between ssDNA and SSB protein was 546.6 pN, which was significantly different from the other combinations (Reference: 230.5 pN, BSA: 89.6 pN, dsDNA: 158.9 pN) 7). The AFM tip with the same spring constant is used to position the tip close to the sample. When the unbinding force is measured when the sample is released and released to its original position, when the two molecules are unbinding, There are several unbinding force values. There may be several numbers with significant unbinding force values per unbinding force curve, such as when two sheets of paper are stuck together using an adhesive. As a result, single strand DNA and SSB protein were measured at 3.73 (Reference: 0.73, BSA: 0.3, dsDNA: 0.84) (see FIG. 8).

The above example exemplarily shows a method of selectively detecting a specific protein having an attraction by using a DNA having a single-stranded pyrimidine-rich sequence corresponding to 45-mer. In addition, through the above examples, it is possible to simplify the interaction between SSB protein and two template DNA single stranded DNAs solved by DNA helicase in DNA replication more easily than conventional methods, It can be confirmed that it can be measured.

As described above, using the method according to the present disclosure, it was confirmed that there is a specific interaction force between single-stranded DNA and SSB protein. The interaction force between two biomolecules can be expressed as unbinding force with the separation of two molecules. The average value of interaction force was measured under PBS buffer solution at 546.6 pN.

This provides reliability with consistent results in comparison with other experimental conditions.

<110> Samsung Electronics Co. Ltd <120> DIRECT DETECTING METHOD FOR TARGET PROTEINS USING DNA PROBE <130> PN100908 <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 45 <212> DNA <213> Artificial Sequence <220> Single stranded pyrimidine-rich sequence <400> 1 ctccctccct ccctccctcc ctccctccct ccttctccct ccctc 45 <210> 2 <211> 45 <212> DNA <213> Artificial Sequence <220> Double stranded pyrimidine-rich sequence <400> 2 ctccctccct ccctccctcc ctccctccct ccttctccct ccctc 45

Claims

Preparing a nucleic acid probe having a nucleic acid attached to a probe located at a cantilever end of an atomic force microscope (AFM);
Measuring a force generated when the nucleic acid probe approaches and distances the target protein; And
Analyzing the interaction between the nucleic acid and the target protein from a measured value of the displacement of the cantilever.

The method of claim 1, further comprising immobilizing the target protein to a solid support.

The method of claim 1, wherein analyzing the interaction between the nucleic acid and the target protein comprises: determining a binding strength between the nucleic acid and the target protein from a measured value of the displacement of the cantilever, Lt; / RTI >

2. The method of claim 1, wherein analyzing the interaction between the nucleic acid and the target protein comprises determining the number of binding sites between the nucleic acid and the target protein from the measurement of the displacement of the cantilever.

5. The method of claim 4, wherein the number of coupling sites is determined based on a number of peak values obtained from measurements of displacement of the cantilever.

The method of claim 1, wherein the target protein is a protein that binds to the nucleic acid.

6, wherein the protein that binds to the nucleic acid comprises a gene expression regulatory protein, a single stranded DNA binding protein (SSB), a protein involved in maintaining the stereostructure of the nucleic acid, a DNA-dependent ATPase, and a DNA rotavirus.

The method of claim 1, wherein the nucleic acid binds to the target protein.

9. The method of claim 8, wherein the nucleic acid comprises a promoter, an operator, or a combination thereof.

The method of claim 1, wherein the nucleic acid probe attached to the probe attached to the cantilever end has a thiol group at one end.

The method of claim 1, wherein attaching the nucleic acid probe to a probe attached to the cantilever end further comprises coating a probe attached to the cantilever end with gold.

The method of claim 1, wherein the opposite end of the nucleic acid probe end attached to the probe attached to the cantilever end is transformed into a detectable marker.

The method of claim 1, further comprising attaching alkane thiol to a probe attached to the cantilever end.

The method of claim 1, wherein the nucleic acid has a size of 55 nucleotides or less.

The method of claim 1, wherein the target protein is homogeneously or layer-fixed to the surface of a solid support coated with an amine linker.

The method of claim 1, further comprising analyzing the interaction between the nucleic acid and the target protein to detect the target protein.