JPH0638799A - Method for recognizing molecule having specific nucleic acid base sequence - Google Patents

Abstract

(57) [Summary] [Object] To provide a method for easily recognizing a molecule having a specific nucleobase sequence, which is suitable as a method for analyzing a DNA sensor or an oligonucleotide. [Structure] The first molecule 1 is formed on a monomolecular film formed by using the first molecule 1 having a specific nucleobase sequence in the molecule.
The second molecule 2 having a nucleobase sequence complementary to the nucleobase sequence of is adsorbed. [Effect] The monomolecular film of the first molecule having a specific nucleobase sequence in the molecule selectively adsorbs the second molecule having the nucleobase sequence complementary to the nucleobase sequence. As a result, a molecule having a specific nucleobase sequence can be recognized, and a DNA sensor or an oligonucleotide can be easily and efficiently analyzed.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for recognizing a molecule having a specific nucleic acid base sequence, and more particularly to a method for recognizing a molecule having a specific nucleic acid base sequence suitable as a DNA sensor or an oligonucleotide analysis method. .

[0002]

2. Description of the Related Art As a method for analyzing the sequence of nucleic acid bases, a method utilizing a restriction endonuclease or a DNA polymerase reaction, and further, Sanger which has developed these methods.
-Coulson method, Maxam-Gilbert method and the like are known.

[0003]

However, all of the above-mentioned conventional analysis methods have the disadvantages that complicated operations are required and expensive equipment is required for automation.

The present invention solves the above-mentioned conventional problems, and
It is an object of the present invention to provide a method for easily recognizing a molecule having a specific nucleobase sequence, which is suitable as an A sensor or an oligonucleotide analysis method.

[0005]

A method for recognizing a molecule having a specific nucleobase sequence according to claim 1 is a single molecule formed on a substrate using a first molecule having a specific nucleobase sequence. By adsorbing a second molecule having a nucleobase sequence complementary to the nucleobase sequence onto the molecular film, the first
It is characterized in that it recognizes the nucleobase sequence of the molecule or the second molecule.

The method for recognizing a molecule having a specific nucleic acid base sequence according to claim 2 is the method according to claim 1, wherein the first molecule having the specific nucleic acid base sequence has an oligonucleotide structure in the molecule. A monomolecular film is formed by bonding to the surface of the metal substrate via sulfur atoms.

The present invention will be described in detail below with reference to the drawings.

First, the molecular recognition mechanism of the present invention will be described with reference to FIG. 1, which is a schematic diagram showing an adsorption mechanism according to an embodiment of the method for recognizing a molecule having a specific nucleic acid base sequence of the present invention.

In FIG. 1, reference numeral 10 indicates the surface of a metal substrate, and the first molecule 1 has a specific nucleobase sequence (adenine A oligonucleotide unit in this example) in the molecule.
Is formed on the surface 10 of the metal substrate via the sulfur atom S at the terminal.

As shown in FIG. 1 (a), a second molecule 2 having a sequence of thymine T, which is a nucleic acid base complementary to adenine A, and a non-complementary adenine A are added to the first molecule 1. When a third molecule 3 having a sequence of guanine G, which is a typical nucleic acid base, is brought into contact with the third molecule 3, as shown in FIG. 1B, only the second molecule 2 is present on the monomolecular film of the first molecule 1. , Adsorbed by a hydrogen bond between adenine A and thymine T shown by a broken line,
Adsorption of molecule 3 of does not occur.

As a result, if the nucleobase sequence of either the first molecule 1 or the second molecule 2 is known in advance, the nucleobase sequence of the other one can be recognized.

In the present invention, such a monomolecular film of a molecule having a specific nucleobase sequence in the molecule has, for example, an oligonucleotide unit in one molecule and an -SR group (R
Represents a hydrogen or thiol protecting group. Can be easily formed by immersing the surface of the heavy metal substrate in a solution containing the compound having a).

Here, an example of the compound having an oligonucleotide unit in one molecule and a --SR group (R represents a hydrogen or thiol protecting group) at the end is represented by, for example, the following general formula [I]. An oligonucleotide is mentioned.

[0014]

[Chemical 1]

In the above general formula [I], the nucleobase of E includes a nucleobase appropriately selected from the group consisting of adenine, guanine, thymine, cytosine and the like.

Examples of the thiol protecting group for R include an acetyl group, a 2-tetrahydropyranyl group, and a triphenylmethyl group which may have a substituent such as an alkoxy group.

Although n is an integer of 5 or more, if n is too large, for example, 20 or more, it is difficult to obtain the reagent.

Among such oligonucleotides, R
Can be produced according to the following process, for example.

[0019]

[Chemical 2]

[0020]

[Chemical 3]

Of the above steps, step A is carried out in a solvent such as methylene chloride in the presence of diisopropylamine or the like at a temperature of 20 to 25 ° C.
The steps B, C and D are usually performed in an automatic DNA synthesizer. The step B is performed in acetonitrile, for example. The step C is carried out using tetraethylthiuram disulfide as a reagent when X is S and iodine as a reagent when X is O. Step D is performed using a base such as ammonia.

Further, in the above-mentioned general formula [I], the oligonucleotide in which R is hydrogen can be obtained by removing the protecting group from the above-obtained oligonucleotide by a conventional method.

Such an oligonucleotide can be synthesized relatively easily by an automatic DNA synthesizer and with a desired nucleobase sequence, and a monolayer can be easily obtained for the detection of various nucleobase sequences. You can

The monomolecular film formed of such an oligonucleotide and used in the present invention has the same structure as described in J. Am. Chem. So
c. , 111, 321 to 335 (1989). In addition, the general formula [I]
In the case where R is a thiol protecting group, a monomolecular film can be prepared as follows.

That is, the oligonucleotide represented by the general formula [I] according to the present invention is dissolved in a solvent such as ethanol, ethanol / water (buffer), acetonitrile / water (buffer), and dichloroacetic acid is added to the solvent. Acids such as methanesulfonic acid and p-toluenesulfonic acid pyridine salt are added to remove the thiol protecting group in the solution, and the thiol is taken out, or without taking out and purifying the solution, clean Au, Ag or Forming the monomolecular film according to the present invention on the substrate by immersing the substrate having a heavy metal surface such as Cu, leaving it at 10 to 50 ° C. for about 1 hour to 3 days, and then pulling up the substrate. You can In addition,
In this case, phenols such as phenol and cresol can also be used as the acceptor of the protective group.

The monomolecular film used in the present invention can be formed as a mixed monomolecular film containing the above-mentioned oligonucleotide and another alkylthiol derivative. Examples of the alkylthiol derivative used here include those represented by the following general formula [VIII].

[0027]

[Chemical 4]

In the present invention, the monomolecular film thus obtained has a nucleobase sequence complementary to a specific nucleobase sequence held in the first molecule constituting the monolayer. By immersing in a solution of 2 molecules, only the second molecule having this complementary nucleobase sequence can be selectively adsorbed to the nucleobase sequence of the monolayer by hydrogen bonding.

By utilizing this adsorption mechanism, the monomolecular film can be used as a sensor for a molecule having a specific nucleic acid base sequence, and can be preferably used as a method for analyzing such a molecule. As a method for detecting adsorbed molecules,
For example, a method of measuring the thickness change of the monomolecular film by ellipsometry or the like, or a method of detecting by fluorescence analysis, radiation analysis or the like when the substance to be analyzed is labeled with a fluorescent substance or radioisotope. Etc. can be adopted.

[0030]

The monomolecular film of the first molecule having a specific nucleobase sequence in the molecule selectively adsorbs the second molecule having the nucleobase sequence complementary to the nucleobase sequence. As a result, a molecule having a specific nucleobase sequence can be recognized, and a DNA sensor or an oligonucleotide can be easily and efficiently analyzed.

[0031]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to the following examples unless it exceeds the gist.

Example 1 In the above general formula [I], R = H, R ¹ = H, E =
Adenine, X = S, Y = H, m = 4, n = 11
Synthesis of Rigonucleotide [Ia] A: In the general formula [II], R = acetyl group, R ¹ =
246 mg of a compound [IIa] of H, n = 11, the structural formula [I
II] compound 355 mg and diisopropylamine 386 mg are dissolved in methylene chloride 8 ml, and 20 to 25
The reaction was carried out at 0 ° C for 1 hour.

The reaction solution was extracted with ethyl acetate and subjected to column chromatography using silica gel as a carrier and n-hexane-chloroform-triethylamine (4.5: 4.5: 1 volume ratio) as a developing solution. The general formula
In [IV], 397 mg of a compound [IVa] in which R = acetyl group, R ¹ = H, and n = 11 was obtained. The analysis results of this product are as follows.

¹ H NMR (CDCl ₃ ), TMS standard, 300 MHz 1.18 (m, 12H), 1.27 (m, 14H),
1.58 (m, 4H), 2.32 (s, 3H), 2.6
4 (t of d, 2H), 2.86 (t, 2H),
3.60 (m, 4H), 3.80 (m, 2H) ¹³ C NMR (CDCl ₃ ), 75 Hz 20.34, 24.58, 25.91, 28.78, 2
9.00, 29.12, 29.28, 29.42, 2
9.48, 29.52, 30.62, 31.19, 4
2.95, 58.29, 63.71, 117.66, 1
96.00 ³¹ P NMR (60% H ₃ PO ₄ , external) 109.25
Hz-147.7 BD: The reaction was carried out in a DNA synthesizer.

In the DNA synthesizer, a compound [Va] in which E is adenine, Y is H and m is 4 in the above general formula [V] is reacted with the above compound [IVa] in acetonitrile. Thus, a compound [VIa] in which R = acetyl group, R ¹ = H, n = 11, m = 4, Y = H, E = adenine in the general formula [VI] was obtained. This compound [VIa] is reacted with tetraethylthiuram disulfide to give the compound of the general formula [V
II], R = acetyl group, R ¹ = H, n = 11,
m = 4, X = S, Y = H, E = adenine compound [VIIa]
Was synthesized. When this compound [VIIa] was treated with aqueous ammonia, the acetyl group of the protecting group was removed, and the desired oligonucleotide [Ia] was obtained.

Incidentally, these series of reactions are applied.
Biosystems User Bulletin
58-2 (1991).

The analysis results of the obtained oligonucleotide [Ia] by high performance liquid chromatography are as follows.

Column C-18 Reversed phase column Gradient (straight line) Solution A: 0.05 M ammonium acetate Solution B: acetonitrile Gradient program Start: A 95% + B 5% After 30 minutes: A 40% + B 60% After 37.66 minutes: A 0% + B100% Detection wavelength 260 nm Temperature 25 ° C. The retention time under the above conditions was 20 minutes.

First portion having a specific nucleobase sequence
Preparation of monolayer of child (oligonucleotide [Ia]) Oligonucleotide [Ia] synthesized above in ethanol
0.05 mM and 0.5 μM of dodecanethiol were dissolved, and a substrate having an Au surface of 1.2 cm × 1.2 cm was dissolved in this mixed solution (a Cr film having a thickness of 250 Å on a silicon wafer of 1.2 cm × 1.2 cm, Furthermore, Au is vapor-deposited thereon to a thickness of 15000Å) at 25 ° C for 24 hours.
Soak for hours. Then, the substrate was pulled up and washed with ethanol to obtain a mixed monolayer of oligonucleotide [Ia] and dodecanethiol.

The analytical values of the obtained monomolecular film are as follows. Film thickness (measured by ellipsometry): 19Å Contact angle (water): 6 ° Recognition of second molecule having complementary nucleobase sequence R = in the above general formula [I] synthesized in the same manner as above
H, R ¹ = H, n = 11, X = S, E = thymine, Y =
H, m = 4 oligonucleotide [Ib] 1.0MN
It was dissolved in an aCl aqueous solution (0.05 mM), and the substrate having the monomolecular film prepared above was immersed in this solution at 25 ° C. for 1 hour. After that, the substrate was pulled up, washed with ethanol, and the film thickness was measured again. The results were as follows.

Film thickness (measured by ellipsometry): 42
Å The extent of this increase in film thickness is due to the fact that the adenine oligonucleotide of the monolayer of the oligonucleotide [Ia] recognizes the oligonucleotide [Ib] having thymine, which is a nucleic acid base complementary to it, and bonds via hydrogen bond. It shows that it did.

Confirmation Experiment The above recognition is further supported by the following three experiments.

After rinsing the monomolecular film obtained in Experiment 1 with ethanol / water, the film thickness was measured to be 21Å, which was equal to the thickness of the monomolecular film of the oligonucleotide [Ia] within the experimental error range. became. This indicates that the hydrogen bond between the oligonucleotide [Ia] and the oligonucleotide [Ib] was destroyed by ethanol / water and returned to the monolayer composed of only the oligonucleotide [Ia].

Experiment 2 : Oligonucleotide [Ia] was added to 1.0
Dissolved in M NaCl aqueous solution (0.05 mM), Experiment 1
The substrate having the monomolecular film after the treatment in 1 was immersed at 25 ° C. for 1 hour. Then, the substrate was pulled up, washed with ethanol, and the film thickness was measured. The film thickness is 38 Å, which is the same as the film thickness obtained within the range of experimental error, and again the adenine oligonucleotide of the monolayer of the oligonucleotide [Ia] has the complementary nucleobase thymine [I].
b] is recognized, and it is shown that they are bound via hydrogen bonds.

Experiment 3 : In the above general formula [I], R = H,
R ¹ = H, n = 11, X = S, E = adenine, m = 4
The oligonucleotide in which Y = H is 1.0 M NaCl
The substrate having a monomolecular film obtained by being dissolved in an aqueous solution (0.05 mM) and treated in Experiment 1 was immersed at 25 ° C. for 1 hour. Then, the substrate was pulled up, washed with ethanol, and the film thickness was measured. The film thickness is 22Å, and the experiment 1
The film thickness is the same as that shown in, indicating that it does not bind to an oligonucleotide containing a nucleic acid base having no complementary relationship.

[0046]

As described in detail above, according to the method for recognizing a molecule having a specific nucleobase sequence of the present invention, a molecule having a specific nucleobase sequence can be easily and efficiently recognized. The present invention is a DNA sensor or
It is industrially very useful as a method for analyzing oligonucleotides.

[Brief description of drawings]

FIG. 1 is a schematic diagram showing an adsorption mechanism according to an embodiment of a method for recognizing a molecule having a specific nucleobase sequence of the present invention.

[Explanation of symbols]

 1 1st molecule 2 2nd molecule 10 Metal substrate surface A Adenine G Guanine S Sulfur atom T Thymine

Claims (2)

[Claims] 1. A second molecule having a nucleobase sequence complementary to the nucleobase sequence on a monomolecular film formed by using a first molecule having a specific nucleobase sequence in the molecule on a substrate. A method for recognizing a molecule having a specific nucleobase sequence, which comprises recognizing the nucleobase sequence of the first molecule or the second molecule by adsorbing 2. The first molecule having the specific nucleobase sequence has an oligonucleotide structure in the molecule and is bound to the surface of a metal substrate through a sulfur atom to form a monomolecular film. The method for recognizing a molecule having a specific nucleobase sequence according to claim 1, characterized in that