JP5100236B2 - Single-stranded nucleotide multimer extension immobilization substrate and method for producing the same - Google Patents

Description

  The present invention relates to a method for stably immobilizing nucleotide multimers such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) on a conductive substrate. More specifically, the present invention relates to a method for stably immobilizing a single-stranded nucleotide multimer as a monomolecular film on a conductive substrate such as graphite in a stretched state.

Deoxyribonucleic acid (DNA) is a molecule that carries the genetic information of living organisms, and information necessary for living organisms is encoded in its base sequence. Therefore, DNA sequencing [Non-Patent Document 1] is a basic means for analyzing genetic information. In DNA sequencing, methods for examining DNA fragment length, such as the Sanger method [Non-patent Document 2], are the mainstream.
However, since the above method involves DNA strand replication and further fragmentation, the DNA base sequence cannot be observed and analyzed as it is. In particular, there is a problem that the telomere existing at the end of the DNA is shortened by 50 to 200 bases every time of replication, and the base sequence of the telomere cannot be observed accurately.

On the other hand, if the single-stranded DNA is fixed to the substrate in a stretched state and the base sequence can be observed, the original DNA sequence can be observed.
Observation and analysis of single-molecule DNA as it is by scanning probe microscopy can be expected not only for single-molecule sequencing but also for application to manipulation of molecules in DNA.

  At present, the most widely used technique in scanning probe microscopy is atomic force microscopy (AFM), and this AFM has achieved a resolution for identifying a DNA helix [Non-Patent Documents 3 and 4]. Identification is difficult.

In contrast, scanning tunneling microscopy (STM) can obtain higher spatial resolution than AFM in DNA observation [Non-Patent Document 5], and a DNA sample prepared by vacuum spraying can be obtained under an ultrahigh vacuum. By observing at a liquid nitrogen temperature, a high spatial resolution capable of discriminating the base can be achieved [Non-Patent Document 6].
In order to obtain this high resolution at room temperature in a highly versatile environment such as in the air or a solid-liquid interface, it is necessary to stably fix the DNA to the substrate surface in a stretched state.

  Yamamoto et al. Developed a method for developing DNA stretched on a graphite surface [Non-Patent Document 8]. However, when the sample prepared by this technique was observed by STM, even the helical structure could be confirmed, but it was difficult to obtain a higher resolution because the DNA was not stably fixed to the substrate.

DNA and RNA are chain polymers having a phosphate group. A long-chain alkyl molecule having lattice matching with a substrate such as graphite, for example, octylamine is bonded to the phosphate group, thereby arranging a chain polymer on the substrate via the long-chain alkyl molecule. A technique has been developed [Non-Patent Document 7].
However, if this technique is simply applied to single-stranded DNA, a multilayer film can be obtained, but the single-stranded DNA cannot be arranged on the substrate as a monomolecular film.

  A technique has been reported in which a thiol group is introduced into one end of a 13-base DNA, a DNA self-assembled film is arranged on a gold substrate, and observed by STM [Non-patent Document 9]. According to this technique, the helical structure can be seen, but the DNA cannot be observed in the stretched state.

  Japanese Patent Application Laid-Open No. 2005-049176 [Patent Document 1] discloses a method of stretching and fixing a chain polymer such as DNA to a substrate. However, it is possible to identify bases in DNA in the air or at a solid-liquid interface. In order to perform STM observation with high resolution, the interaction between the DNA and the substrate was small, and the fixation could not be performed stably.

JP 2005-049176 A L. T. C. Franca, E. Carrilho, and T. B. L. Kist, Quart. Rev. Biophys. 35, 169 (2002). F. Sanger, S. Nicklen, and A. R. Coulson, Proc. Nat. Acad. Sci. USA 74, 5463 (1977). H. G. Hansma et al., Science 256, 1180 (1992). H. G. Hansma et al., Nucl. Acids Res. 20, 3585 (1992). R. Guckenberger et al., Science 266, 1538 (1994). H. Tanaka and T. Kawai, Surf. Sci. 539, L531 (2003). I. Yamamoto, T. Kanno, H. Tanaka and T. Kawai, Jpn. J. Appl. Phys. 42, L559 (2003). K. Ijiro and Y. Okahata, J. Chem. Soc., Chem. Commun. 1339 (1992). M. Gubb et al., J. Am. Chem. Soc., 7734 (2006).

  The present invention provides a method for easily and stably immobilizing single-stranded DNA or RNA (single-stranded nucleotide multimer) as a monomolecular film on a substrate, particularly a conductive substrate such as graphite, in a stretched state. The purpose is to do.

The present inventors apply the above-mentioned method [Non-Patent Document 8] in which a chain polymer is stretched and fixed on a substrate via a long-chain alkyl molecule, and an alkyl is added to the phosphate site of the single-stranded nucleotide multimer. We developed a method for fixing the monomolecular film to the substrate by producing a monomolecular film by performing the reaction for bonding the amine at the interface between water and an organic solvent. By observing the monolayer of the nucleotide multimer-alkylamine complex produced by this method with a high impedance STM of 1 teraohm or more, the spatial resolution observation that enables base discrimination without destroying the produced monomolecular film is achieved. did.
That is, in the present invention, in order to stably bond a single-stranded nucleotide multimer to a substrate in an extended state, an alkylamine is attached to the phosphate site of the single-stranded nucleotide multimer using a chemical reaction at the liquid interface. Combine to form a single stranded nucleotide multimer-alkylamine complex. Since the single-stranded nucleotide multimer-alkylamine complex is formed along the liquid interface, a monomolecular film of the single-stranded nucleotide multimer-alkylamine complex is formed. Next, utilizing the interaction between the alkylamine and the substrate, the single-stranded nucleotide multimer is immobilized as a monomolecular film on the substrate mechanically and electrically stably.

According to the method of the present invention, a single-stranded nucleotide multimer can be stably immobilized on a solid surface as a monomolecular film. Therefore, unlike the conventional method using ultra-high vacuum, one molecule under atmospheric pressure. Nucleotide multimers (DNA or RNA) can be sequenced.
Furthermore, the present invention improves the stability and reproducibility of the measurement of the intramolecular structure of DNA and molecular wires, which has been difficult in the past, and is considered to be one of the key technologies for realizing a single molecule device.

The present invention is a method for producing a conductive substrate on which a monomolecular film of a complex of one single-stranded nucleotide multimer and a plurality of alkylamines is immobilized,
(1) A step of allowing a water droplet of an aqueous solution of a single-stranded nucleotide multimer to stand on a conductive substrate;
(2) dropping an alkylamine solution in a first organic solvent incompatible with water so as to completely cover the water droplets to form a liquid film of the alkylamine solution;
(3) At the liquid interface between the aqueous solution of the single-stranded nucleotide multimer and the alkylamine solution, the plurality of alkylamines bind to the plurality of phosphate group sites of the single-stranded nucleotide multimer, And (4) a single-stranded nucleotide while heating the conductive substrate, and a step of allowing the single-stranded nucleotide multimer and a plurality of alkylamines to form a monomolecular film. Combining one single-stranded nucleotide multimer with a plurality of alkylamines by rinsing water droplets of the aqueous solution of the polymer and the liquid film of the alkylamine solution with a second organic solvent incompatible with water. There is provided a manufacturing method comprising a step of fixing a monomolecular film of a body on a conductive substrate.

A nucleotide is a substance in which a phosphate group is further bound to a nucleoside bound to a base and a sugar. A nucleotide whose sugar component is ribose is called a ribonucleotide, and a multimer of ribonucleotides is a ribonucleic acid (RNA). A nucleotide having a sugar component of deoxyribose is called deoxyribonucleotide, and a multimer of deoxyribonucleotide is deoxyribonucleic acid (DNA).
In the present invention, a nucleotide multimer is a chain polymer in which nucleotides are linked by a 5′-3 ′ phosphodiester bond, and is a concept including oligonucleotides and polynucleotides.

In DNA, complementary double strands usually form a double helix structure by hydrogen bonding between base pairs. On the other hand, RNA generally exists as a single strand, but there are exceptions.
In view of performing observation analysis of a nucleic acid base sequence as it is by a technique such as STM, the present invention is not a double strand in which a base is embedded in a double helix structure but a single-stranded nucleotide. For multimers.

An example of a single-stranded nucleotide multimer that is the subject of the present invention is shown in FIG. This nucleotide multimer is hexameric DNA (AAATTT; A = adenine, T = thymine).
The nucleotide multimer used in the present invention is water-soluble, but does not substantially dissolve in an organic solvent incompatible with water. In the present invention, the number of bases of the target nucleotide multimer is preferably 5 or more, more preferably 5 to 5000. When the number of bases exceeds 5000, it was difficult to obtain a monolayer of nucleotide multimers in an extended state from another study [Non-patent Document 7].

The alkylamine that can be used in the present invention is preferably a primary amine substituted with one alkyl group, and more preferably substituted with one alkyl group having 5 to 20 carbon atoms. If the primary amine is a primary amine, the polynucleotide multimer can be stably immobilized in direct contact with the substrate, but in the case of a higher amine, the polynucleotide multimer is separated from the substrate. The polynucleotide multimer could not be stably immobilized because it was immobilized in a state. Here, “to stably fix the polynucleotide multimer” means to fix the polynucleotide multimer so that the sequence state is not disturbed by the scanning of the probe in the STM analysis.

The alkylamine used in the present invention does not substantially dissolve in water but dissolves in an organic solvent incompatible with water.
An example of an alkylamine that can be used in the present invention is shown in FIG. This alkylamine is a primary amine substituted with an alkyl group having 8 carbon atoms (octylamine; CH ₃ (CH ₂ ) ₇ NH ₂ ).

  According to the present invention, a complex of a nucleotide multimer and an alkylamine in which an alkylamine is coordinated to each of a plurality of phosphate groups contained in one molecule of the nucleotide multimer is formed. An example of the complex of nucleotide multimer and alkylamine according to the present invention is shown in FIG.

The present invention is characterized in that this complex is immobilized on a conductive substrate as a monomolecular film in a stretched state.
Thereby, high spatial resolution observation using STM is enabled, and the sample for determining the base sequence of a nucleotide multimer can be prepared.
The conductive substrate used in the present invention may be any substrate that can be used for STM observation, and examples thereof include a graphite substrate and a metal substrate such as Au.
In particular, highly oriented sintered graphite (HOPG) is preferable. Since the interval between the hydrocarbon repeating units of the alkyl chain and the lattice constant of HOPG are almost the same, the alkyl chain is arranged on the HOPG substrate, and as a result, the polynucleotide multimer can be immobilized on the substrate in a stretched state. it can.

Next, the manufacturing method of the present invention will be described with reference to FIGS.
In step (1) of the production method of the present invention, an aqueous solution of a single-stranded nucleotide multimer is dropped on a conductive substrate, and the obtained water droplet is allowed to stand on the conductive substrate.
This process has two purposes. The first purpose is to separate polynucleotide multimers associated in solution into single molecules. The second purpose is to precipitate aggregated and entangled polynucleotide multimers. For this purpose, the preferred standing time is 1 to 60 minutes.
In the production method of the present invention, the aqueous solution concentration of the nucleotide multimer is preferably 10 pM / μl to 10 nM / μl. When the aqueous solution concentration is less than 10 pM / μl, nucleotide multimers are discretely immobilized on the substrate, and analysis by STM observation becomes insufficient. When the concentration exceeds 10 nM / μl, nucleotide multimers are aggregated. This is because it may be fixed to the substrate.
In the production method of the present invention, the dropping amount of the aqueous solution of nucleotide multimer is preferably 0.1 to 1 μl. This is because when the amount of the aqueous solution dropped is less than 0.1 μl, the absolute amount of nucleotides is insufficient, and analysis by STM observation becomes insufficient. Further, the upper limit of the dropping amount of the aqueous solution depends on the area of the substrate used for STM observation, and may be an amount that does not overflow from the substrate. A normal STM observation uses a 10 mm square substrate. Preferably does not exceed 1 μl.
That is, according to the method of the present invention, if a very small amount of nucleotide multimers are present, the base sequence can be observed and analyzed as it is.

In step (2) of the production method of the present invention, a solution of the alkylamine in the first organic solvent incompatible with water is dropped to completely cover the water droplets, thereby forming a liquid film of the alkylamine solution. Form.
The solution concentration of the alkylamine in the first organic solvent incompatible with water is preferably 1 nM to 10 mM.
As shown in FIG. 4, in this step, a two-layer structure of an aqueous solution of nucleotide multimers and an alkylamine solution is formed on the conductive substrate, and a liquid interface exists between the two solutions.

  In step (3) of the production method of the present invention, the alkylamine is considered to be arranged with the amine terminal side facing the liquid interface. Therefore, the phosphate group of the nucleotide multimer and the amine terminal of the alkylamine react only at a two-dimensionally limited liquid interface to form a complex, so that the monolayer of the complex follows the liquid interface. Formed. A preferable standing time for forming a monomolecular film of the complex is 30 to 180 minutes.

In the step (4) of the production method of the present invention, while heating the conductive substrate, a water droplet of an aqueous solution of a single-stranded nucleotide multimer and a liquid film of an alkylamine solution are converted into a second incompatible solution with water. By washing away with an organic solvent, the monomolecular film of the complex formed along the liquid interface is fixed on the conductive substrate.
When this step was performed at room temperature (about 25 ° C.), a monomolecular film was not formed on the substrate, and the composite was deposited three-dimensionally. When the process temperature was raised, a monomolecular film could be formed on the substrate at 35 ° C. or higher. In consideration of the influence on the nucleotide multimer, this step is preferably performed at 80 ° C. or lower.

  The first organic solvent used in step (2) and the second organic solvent used in step (4) are incompatible with water and can dissolve the nucleotide multimer that is the subject of the present invention. Those with low volatility are desirable. More specifically, the above organic solvents are the same or different and are selected from phenyloctane, toluene, hexane, octane, chloroform and the like.

  The present invention further relates to a complex of one single-stranded nucleotide multimer and a plurality of alkylamines, wherein the plurality of phosphate groups at the plurality of phosphate groups of the one single-stranded nucleotide multimer are provided. The present invention also provides a complex of a single-stranded nucleotide multimer and an alkylamine, to which the alkylamine is bound, and a conductive substrate on which such a complex is immobilized.

Example 1
0.1 μl of 100 pM / μl hexamer DNA (AAATTT, A = adenine, T = thymine) aqueous solution 2 was dropped on the surface of the cleaved graphite substrate 1 and allowed to stand for 10 minutes. A phenyloctane solution 3 of octylamine [CH ₃ (CH ₂ ) ₇ NH ₂ ], which is a primary amine, is gently dropped onto this aqueous solution, and a DNA-alkylamine complex is simply reacted by reaction at the liquid interface 4. A molecular film was produced. Then, while the substrate was heated to 60 ° C., phenyloctane was dropped onto the substrate and rinsed, whereby the generated monomolecular film was fixed to the graphite substrate 1.

  STM observation of the produced substrate was performed by controlling a Besocke type STM head (ATM300) manufactured by RHK Technology with a dedicated controller (SPM1000). In addition to general-purpose platinum iridium wires, the probe includes a platinum iridium probe fabricated by electropolishing, a probe in which a tungsten probe is coated with platinum or titanium, and a probe in which the surface of these probes is coated with a fluororesin. Needles were also used.

FIG. 5 shows an image obtained by observing the produced substrate with STM. During STM observation, the bias voltage was set to -1.0 V and the tunnel current was set to 0.2 pA.
The graphite substrate is twinned and has two crystal orientations <18 1 0> and <18 −1 0>, and the crossing angle between the two orientations is 5.4 °.
When the STM image is analyzed, the DNA-octylamine complex is arranged to form stripes in a plurality of directions, and the DNA-octylamine complex is formed on the surface of the graphite substrate from the arrangement direction of these stripes and the crossing angle of the stripes. It was confirmed that the films were epitaxially oriented in the <18 1 0> and <18 -10> orientations.

As a result of further detailed analysis of this image, the stripe interval was 4.6 nm. As shown in FIG. 6, when the crystal-crystal orientation was superposed and the DNA-octylamine complex formed dimers, At the graphite surface
I understood that I was taking From the results of this periodic structure analysis, it is considered that DNA is stretched by binding to octylamine and arranged on the graphite surface.

Example 2
The monomolecular film was fixed to the graphite substrate 1 in the same manner as in Example 1 except that octylamine was changed to primary amine dodecylamine [CH ₃ (CH ₂ ) ₁₁ NH ₂ ].
Although not shown, according to STM observation, the stripe interval was 5.6 nm, which increased reflecting the alkyl chain length when the octyl group having 8 carbon atoms was changed to a dodecyl group having 12 carbon atoms.
As in the case of using octylamine, DNA is considered to be stretched by binding to dodecylamine and arranged on the graphite surface.

Comparative Example 1
An attempt was made to immobilize the monomolecular film on the graphite substrate 1 in the same manner as in Example 1 except that octylamine was changed to dimethyldimyristylammonium bromide, which is a quaternary amine.
However, it could not be arranged on the graphite surface in the stretched state.
By binding a plurality of alkyl groups to the amine, it is considered that not all the alkyl groups are bonded on the substrate, and the DNA is in a floating state.
From the results of Examples 1 and 2 and Comparative Example 1, it was found that the alkylamine that can be used in the present invention is a primary amine.

Comparative Example 2
An attempt was made to immobilize the monomolecular film on the graphite 1 substrate 1 in the same manner as in Example 1 except that the DNA-alkylamine complex was not formed by dropping the phenyloctane solution 3 of octylamine.
By STM observation, as shown in FIG. 7, the DNA was randomly adsorbed on the substrate in a bent state.
From the results of Examples 1 and 2 and Comparative Example 2, it was confirmed that binding of alkylamine to DNA was necessary to stably fix the DNA on the substrate.

Comparative Example 3
According to the method of Patent Document 1, an aqueous solution 2 of hexamer DNA (AAATTT, A = adenine, T = thymine) having a concentration of 100 pM / μl is heated to 40 ° C. and dropped onto the cleaved graphite substrate 1 surface. After standing for 1 hour, DNA was fixed on the substrate by blowing off the aqueous DNA solution with air.
AFM observation revealed that DNA was fixed in a stretched state on the substrate. However, when STM observation was performed, the DNA fixed on the substrate moved due to the scanning of the STM probe. A good STM image could not be obtained.
From the results of Examples 1 and 2 and Comparative Example 3, it was confirmed that binding of alkylamine to DNA is necessary for stably fixing the DNA on the substrate.

A specific example (a) of a nucleotide multimer used in the present invention and a specific example (b) of an alkylamine. 1 is a specific example of a complex of a nucleotide multimer and an alkylamine according to the present invention. Schematic explaining the manufacturing method of the electroconductive board | substrate with which the monomolecular film of the composite_body | complex of the nucleotide multimer and alkylamine by this invention was fix | immobilized. Reaction of nucleotide multimers with alkylamines at the liquid interface. The STM image of the electroconductive board | substrate with which the monomolecular film of the composite_body | complex of the nucleotide multimer and alkylamine by this invention was fix | immobilized. The analysis result of the STM image of FIG. The STM image which shows the aspect on the electroconductive board | substrate of the nucleotide multimer of a comparative example.

Explanation of symbols

1 ... conductive substrate,
2 ... aqueous solution of nucleotide multimer,
3 ... alkylamine solution,
4 ... Liquid interface.

Claims (5)

A method for producing a conductive substrate in which a monomolecular film of a complex of one single-stranded nucleotide multimer and a plurality of alkylamines is immobilized,
(1) A step of allowing a water droplet of an aqueous solution of a single-stranded nucleotide multimer to stand on a conductive substrate;
(2) dropping an alkylamine solution in a first organic solvent incompatible with water so as to completely cover the water droplets to form a liquid film of the alkylamine solution;
(3) At the liquid interface between the aqueous solution of the single-stranded nucleotide multimer and the alkylamine solution, the plurality of alkylamines bind to the plurality of phosphate group sites of the single-stranded nucleotide multimer, And (4) a single-stranded nucleotide while heating the conductive substrate, and a step of allowing the single-stranded nucleotide multimer and a plurality of alkylamines to form a monomolecular film. Combining one single-stranded nucleotide multimer with a plurality of alkylamines by rinsing water droplets of the aqueous solution of the polymer and the liquid film of the alkylamine solution with a second organic solvent incompatible with water. The manufacturing method characterized by including the process of fixing the monomolecular film of a body on a conductive substrate.   A complex of one single-stranded nucleotide multimer and a plurality of alkylamines, wherein the plurality of alkylamines are bound to a plurality of phosphate groups of the one single-stranded nucleotide multimer. A complex of a single-stranded nucleotide multimer and an alkylamine.   The complex according to claim 2, wherein the single-stranded nucleotide multimer has 5 or more bases.   The composite according to claim 2, wherein the alkylamine is a primary amine substituted with one alkyl group having 5 to 20 carbon atoms.   A conductive substrate on which a monomolecular film of a complex of a single-stranded nucleotide multimer according to claim 2 and an alkylamine is immobilized.