WO2009093558A1 - Nucleic acid construction member, nucleic acid construct using the same and method of producing the nucleic acid construct - Google Patents

Abstract

Provided is a nucleic acid construction member available as a construction unit in forming a stable nanonucleic acid construct, which has an elevated spatial resolution, is rich in diversity and shows a reduced strain, and the nanonucleic acid construct as described above. A nucleic acid construction member characterized in that the base sequence at the free end of a single strand carried by a first nucleic acid molecule having a double helix structure is connected via a hydrogen bond to a base sequence located at a definite position in the non-terminal region of one nucleotide strand of a second nucleic acid molecule having a double helix structure which is a base sequence being non-complementary to the other nucleotide strand but complementary to the base sequence at the free end carried by the preceding first nucleic acid molecule.

Description

Nucleic acid structure, nucleic acid structure using the nucleic acid structure, and method for producing the nucleic acid structure

The present invention relates to a novel nucleic acid structure and a nucleic acid structure produced by self-assembling the nucleic acid structure as a constituent element.
The present invention also relates to a novel method for producing a nucleic acid structure.

In recent years, nanotechnology that constructs nanoscale functional structures and devices by using artificially synthesized biomolecules as self-assembly materials for the application of nanodevices and nanorobotics has attracted attention. Among them, a technique using a nucleic acid molecule such as DNA as a material is called DNA nanotechnology.

There are several advantages of using nucleic acid molecules such as DNA as biomolecules. As one of them, first, a DNA molecule composed of sugar, phosphoric acid, and four types of bases of adenine (A), thymine (T), guanine (G), and cytosine (C) is Watson- A double helix structure is formed based on Crick complementarity, and the base sequence can be used as a programmable information carrier / structure-forming molecule having selective binding properties. In addition, many years of research in various fields such as biology and chemistry have developed many results that can be used to construct nanostructured materials and dynamic systems, such as enzymatic manipulation and artificial intelligent nucleic acids. Can be mentioned. Although research on DNA nanotechnology is still less than 10 years since the start of full-scale research, many achievements have already been reported, and this technology is expected to play a central role in the bio-nanotechnology field in the near future. (For example, see Non-Patent Documents 1, 2, 3, 7, and 11).

To date, many motifs for DNA nanonucleic acid structures have been developed, most of which are based on structural units of DNA branches called crossovers as shown in FIGS. 15 (A) to (C). (For example, see Non-Patent Documents 7, 8, 9, 10, and 11).

The crossover structural unit, which is a branched structure of this DNA double helix, is Holday.
It was developed from a structure called Junction that appears at the time of homologous recombination of a gene in vivo (for example, see Non-Patent Document 4). In general, this crossover structural unit used in a nanonucleic acid structure forms a form in which two DNA double helices are arranged in parallel as shown in FIG. Although it is known that such a structure is taken in a solution containing magnesium ions (see, for example, Non-Patent Document 5), in reality, the two double helices are not strictly parallel. Therefore, a double crossover molecule as shown in FIGS. 16A and 16B, in which the structure is stabilized by combining two or more thereof, is widely used as a basic unit (for example, Non-Patent Document 6). reference).

When such a crossover-based structural motif is used as a structural unit, the structure that can be finally created is a shape in which double spirals are spread in parallel. For example, DNA nanonucleic acid structures such as DNA tiles and DNA origami.

More specifically, the DNA tile has a planar assembly structure with tiles in which double spirals are arranged in parallel by having a crossover structure inside (for example, see Non-Patent Document 7). Are folded in parallel and connected with a number of staple base sequences to form a crossover structure, thereby creating an arbitrary planar structure. In addition, 4 × 4 tiles (black style) in which double crossover molecules are connected in a cross shape have been reported (see, for example, Non-Patent Document 10), but this is also essentially based on double crossover molecules. It is a structure.

On the other hand, production of various DNA nanonucleic acid structures has been attempted.
For example, a two-dimensional structure in which three hexagons are joined by forming one side of a structure with a single double helix, and connecting the triangular structures to each other at the sides to form a tetrahedron or octahedron structure. Thus, there is a report that a side is made of a single double helix (see Non-Patent Document 12).

In addition, there is a description of a structure in which a plurality of triangular structures are joined at the side portion, and there is a report that a planar structure can be formed from a basic triangular structure (see Patent Document 1). .

In addition, we created a rhombus motif consisting of four vertices with crossover junctions, and combined this rhombus motif to achieve a one-dimensional structure (ladder) and two-dimensional structure (plane). There is a report that the planar structure has also been successful (see Non-Patent Document 13).

In addition, there is a report that a triangular structure is formed by a crossover junction and a so-called kagome lattice is produced, and that the resulting structure is changed to a square lattice by binding protein RuvA thereto (Non-Patent Document 14). reference).

In addition, there is a report that a plane structure with a large triangle is produced by a motif in which the vertices of a triangle are formed by a crossover junction, and that the void is larger than the conventional DNA plane structure (see Non-Patent Document 15).

In addition, there is a report that a structure in which a structure is formed by attaching a plurality of bulges with T inside or outside the three vertices of a triangular structure composed of a single double helix (see Non-Patent Document 16). .

The inventor has disclosed the technical contents related to the present invention (see Non-Patent Documents 17 and 18).

JP 2005-263745 A

However, since the crossover structure, which is a structural unit used in nano-nucleic acid structures such as DNA tiles and DNA origami that has been developed so far, can only connect adjacent DNA double helices in parallel, The nano-nucleic acid structure that can be produced is basically a combination of spirally arranged structures. Although a nano-nucleic acid structure consisting of a plane can be produced, examples of successful production of a three-dimensional crystal structure, etc. Not yet reported.

In addition, since this crossover structure cannot form a DNA branch structure unless the phases of adjacent double helices coincide with each other, the spatial resolution (1 base = The length of the double helix can be adjusted in units of 0.3 (nm)), and the size of the structural motif becomes larger. In particular, the structure is stabilized by including two crossovers. In the double crossover molecule, the length in the direction of the helix axis is about four times the radial direction in order to satisfy the geometric constraint of the helix, and a large anisotropy is generated in the spatial resolution. For this reason, it is very difficult to reduce the size of a nanonucleic acid structure produced using a crossover structure as a structural unit, which limits the size and density of the nanonucleic acid structure that can be designed. There are limitations on the structure that can be designed in that only motifs based on the shapes arranged in the above can be produced, and there is a big problem in terms of diversity.

Furthermore, as long as nucleic acid molecules such as DNA are used as materials, electrostatic repulsion between negatively charged backbones is unavoidable. However, the crossover structure has the property of electrically repelling each other. Due to the structure in which the double helices are arranged in parallel, there is a problem that a large strain in the structure is generated in the prepared nanonucleic acid structure, which makes it difficult to manufacture a high-dimensional crystal structure.

On the other hand, in the method of Non-Patent Document 12, a structure in which a triangular structure is connected by a side is an independent single structure, not a plane, and a motif in which three hexagons are connected exists as a single element, and a repetitive structure is not possible. Moreover, in patent document 1, there is no specific description about the Example (experimental result) of a planar structure. Moreover, in the nonpatent literature 13, the magnitude | size of a planar structure is limited and clear data are not disclosed. In Non-Patent Document 14, it is stated that both planar crystals were observed by TEM up to a size of 2 μm, but they are not described in detail, and wide-area photographs are not published. In Non-Patent Document 15, the planar structure does not grow greatly in the width direction, and only a ribbon-like structure having a constant width can be formed. Further, Non-Patent Document 16 does not aim to produce a planar lattice structure, but only a structure having an appearance with a triangle and a small triangle attached thereto is actually produced.

The present invention has been proposed in view of such circumstances, and is a nucleic acid structure that is a structural unit of a nanoscale nucleic acid structure, and has higher spatial resolution, rich variety, and reduced strain. An object of the present invention is to provide a nucleic acid structure that is a structural unit for producing a stable nucleic acid structure, a nucleic acid structure that can be produced using the nucleic acid structure as a structural unit, and a method for producing the nucleic acid structure.
Another object of the present invention is to provide a novel method for producing a nucleic acid structure.

That is, the nucleic acid structure according to the present invention includes a single-stranded free-end base sequence of the first nucleic acid molecule having a double helix structure and one nucleotide chain of the second nucleic acid molecule having a double helix structure. Is a base sequence that is non-complementary to the corresponding nucleotide chain at a predetermined position of the non-terminal part, and is characterized in that the free end base sequence and the complementary base sequence are hydrogen-bonded.

The nucleic acid structure according to the present invention includes a single-stranded free-end base sequence of the first nucleic acid molecule having a double helix structure and one nucleotide chain of the second nucleic acid molecule having a double helix structure. Has a nucleotide sequence that is non-complementary to the other nucleotide chain at a predetermined position of the non-terminal part, and has a nucleic acid structure formed by hydrogen bonding between the free-end base sequence and the complementary base sequence. It is characterized by.

In addition, the method for producing a nucleic acid structure according to the present invention includes a first nucleic acid molecule having a double helix structure having a single-stranded free-end base sequence and a non-complementary to the other nucleotide chain of a pair of nucleotide chains. Forming a second nucleic acid molecule having a double helix structure having a base sequence complementary to the free end base sequence at a predetermined position of the non-terminal portion of one of the nucleotide chains. A hydrogen bond is formed between the end base sequence and a base sequence complementary to the free end base sequence of the second nucleic acid molecule.

In addition, the method for producing a nucleic acid structure according to the present invention comprises a double helix structure with a pair of nucleotide chains having a base sequence that is non-complementary to the other nucleotide chain at a predetermined position of the non-terminal part of one nucleotide chain. A nucleotide chain having a free-end base sequence complementary to the base sequence and the nucleic acid molecule are hydrogen-bonded between the complementary base sequences.

In addition, the method for producing a nucleic acid structure according to the present invention is a method for producing a nucleic acid structure by annealing a solution containing a plurality of oligonucleotides, and immersing a substrate having a surface charge in the solution. It is characterized by that.

In addition, the nucleic acid structure according to the present invention is configured by combining a plurality of repeating structures, and the plurality of repeating structures have a planar structure and a double helix simple structure, The nucleotide chain of one of the repeating structures has a base sequence complementary to the nucleotide sequence of the nucleotide chain of another adjacent repeating structure.

According to the present invention, it becomes possible to form a branch at an arbitrary phase of a double helix, and a stable nanoscale nucleic acid structure with higher spatial resolution, diversity, and no distortion is produced. This nanoscale nucleic acid structure makes it possible to produce small, high-density, high-definition functional nanodevices.
Further, according to the present invention, it is possible to provide a novel method for producing a nucleic acid structure.

It is the schematic model which represented the nucleic acid structure which concerns on this Embodiment three-dimensionally. It is the schematic which represented the nucleic acid structure which concerns on this Embodiment planarly. It is a figure for demonstrating the formation method of the nucleic acid structure which concerns on this Embodiment, (A) has four oligonucleotides synthesize | combined in order to form the T-shaped nucleic acid structure which concerns on this Embodiment It is a figure which shows a base sequence, (B) is a hydrogen bond state between bases of the T-shaped nucleic acid structure formed by self-assembling four types of oligonucleotides which have the base sequence described in (A). It is the schematic expressed clearly. It is a schematic diagram of a DNA double helix structure. It is a figure for demonstrating the phase difference in the main groove side and subgroove side of a DNA double helix structure. It is the schematic of the nucleic acid structure which the base sequence of the oligonucleotide used in order to form the nucleic acid structure which has a branched structure, and the base sequence can form theoretically. It is the electrophoresis result figure which electrophoresed the reaction solution made to react using the oligonucleotide which has a base sequence shown in FIG. 1 is a schematic diagram of a unit structure of a nanonucleic acid structure according to a first embodiment and a part of the nanonucleic acid structure according to the first embodiment in which the nucleic acid structure according to the present embodiment is a structural unit. It is a figure which shows the AFM observation image of the nanonucleic acid structure which concerns on 1st Embodiment. It is the schematic of the unit structure of the nanonucleic acid structure which concerns on 2nd Embodiment which uses the nucleic acid structure which concerns on this Embodiment as a structural unit, and a part of said nanonucleic acid structure. It is a figure which shows the AFM observation image of the nanonucleic acid structure which concerns on 2nd Embodiment. It is the schematic of the two-dimensional polar coordinate structure which is a nanonucleic acid structure which uses the nucleic acid structure which concerns on this Embodiment as a structural unit. It is the schematic of the three-dimensional crystal structure which is a nanonucleic acid structure which uses the nucleic acid structure which concerns on this Embodiment as a structural unit. It is the schematic of the three-dimensional curved surface structure which is a nanonucleic acid structure which uses the nucleic acid structure which concerns on this Embodiment as a structural unit. FIG. 2 is a schematic view showing a crossover structure which is a conventional nucleic acid structure, and (A) to (C) are views showing various crossover structures. It is the schematic which shows the double crossover structure which is the conventional nucleic acid structure. It is the schematic of the unit structure of the nucleic acid structure which concerns on embodiment, and a part of said nucleic acid structure. It is a figure which shows the AFM observation image of the nucleic acid structure which concerns on embodiment. It is the schematic of the unit structure of the nucleic acid structure which concerns on embodiment, and a part of said nucleic acid structure. It is a figure which shows the AFM observation image of the nucleic acid structure which concerns on embodiment. It is the schematic of the unit structure of the nucleic acid structure which concerns on embodiment, and a part of said nucleic acid structure. It is a figure which shows the AFM observation image of the nucleic acid structure which concerns on embodiment. It is the schematic of the unit structure of the nucleic acid structure which concerns on embodiment, and a part of said nucleic acid structure. It is a figure which shows the AFM observation image of the nucleic acid structure which concerns on embodiment. It is the schematic of the unit structure of the nucleic acid structure which concerns on embodiment, and a part of said nucleic acid structure. It is a figure which shows the AFM observation image of the nucleic acid structure which concerns on embodiment. It is a figure which shows the basic structure of a triangular structure single-piece | unit. It is a figure which shows a motif shape and its DNA base sequence. It is an image figure (for four motifs) of a plane formed by combining motifs. It is a figure which shows the preparation procedure of DNA planar structure. It is a figure which shows the AFM observation image of the product produced in the free solution. It is a figure which shows the AFM observation image of the DNA structure produced on the board | substrate. It is a figure which shows the basic structure of a triangular structure single-piece | unit. It is a figure which shows a motif shape and its DNA base sequence. It is an image figure (for seven motifs) of a plane formed by combining motifs. It is a figure which shows the preparation procedure of DNA planar structure. It is a figure which shows the AFM observation image of the product produced in the free solution. It is a figure which shows the AFM observation image of the DNA structure produced on the board | substrate. It is an enlarged view which shows the AFM observation image of the DNA structure produced on the board | substrate. It is a figure which shows the shape of a cross motif. It is a figure which shows the sequence example of a cross motif. It is a figure which shows the planar structure comprised by a cross motif. It is a figure (10x10 micrometer) which shows the AFM observation image of a DNA structure when annealed in a free solution. It is a figure (6x6micrometer) which shows the AFM observation image of the DNA planar structure by the cross motif created on the board | substrate. FIG. 45 is an enlarged view (1 × 1 μm) of an AFM observation image of a DNA structure created on a substrate, corresponding to the lower right part of FIG. 44.

Explanation of symbols

11 1st oligonucleotide, 12 2nd oligonucleotide, 13 3rd oligonucleotide, 14 4th oligonucleotide

The present invention is particularly based on nucleic acid structures formed by self-assembly of oligonucleotides composed of artificially designed and synthesized sugars, phosphates, and bases, and this suitable design. The present invention relates to a nanometer-scale nucleic acid structure produced using the prepared nucleic acid structure as a structural unit. Hereinafter, specific embodiments to which the present invention is applied will be described in detail with reference to the drawings. In the following description, a complementary base sequence refers to a base sequence in a binding relationship that can form a base pair based on Watson-Crick complementarity.

FIG. 1 is a schematic diagram three-dimensionally showing the nucleic acid structure according to the present embodiment. As shown in FIG. 1, this nucleic acid structure is bent at any phase of the double helix of nucleic acid so that the double helix is branched in the vertical direction, and another double helix is connected to the bent portion. Thus, a branched structure is formed.

Here, in the nucleic acid structure according to the present embodiment, nucleic acid molecules such as DNA, RNA, and PNA can be used as the nucleic acid. Hereinafter, an example in which DNA is used as a nucleic acid molecule will be described specifically, but the nucleic acid molecule used in the nucleic acid structure according to the present embodiment is not limited to DNA.

FIG. 2 is a schematic diagram schematically showing the nucleic acid structure according to the present embodiment in a plan view. The four arrows in FIG. 2 represent oligonucleotides constituting a double helix, and the direction of the arrows indicates the direction from the 5 'end to the 3' end of each oligonucleotide. As shown in FIG. 2, the nucleic acid structure according to the present embodiment includes a first DNA molecule having a double helix structure having a single-stranded free-end base sequence, and the other oligo of a pair of oligonucleotides. A second DNA molecule having a double-stranded structure having a nucleotide sequence that is non-complementary to the nucleotide and having a nucleotide sequence complementary to the free-end nucleotide sequence in one oligonucleotide, It is formed by hydrogen bonding with the base sequence having the complementary binding relationship of the second DNA molecule.

More specifically, the first DNA molecule is formed by hydrogen bonding between the first oligonucleotide 11 and the second oligonucleotide 12 between the bases having the complementary binding relationship, This DNA molecule is formed by hydrogen bonding between the third oligonucleotide 13 and the fourth oligonucleotide 14 between the bases having the complementary binding relationship.

The first oligonucleotide 11 constituting the first nucleic acid molecule has a hydrogen complementary to the second oligonucleotide 12 that forms a double helix at the 5 ′ end and constitutes the first nucleic acid molecule together. It has a free-end base sequence consisting of 5 bases that cannot form a bond.

Further, the third oligonucleotide 13 constituting the second nucleic acid molecule forms a double helix and forms a complementary hydrogen bond with the fourth oligonucleotide 14 constituting the second nucleic acid molecule together. A non-complementary base sequence that cannot be obtained, and a base sequence consisting of 5 bases forming a hydrogen bond complementary to the free end base sequence of the first oligonucleotide 11 at its 5 'end is It is in a part that is not a part, that is, in a non-terminal part.

The nucleic acid structure according to the present embodiment has a double helical structure by forming a hydrogen bond between the bases having the complementary binding relationship between the first oligonucleotide 11 and the second oligonucleotide 12. A second nucleic acid having a double helical structure by generating a first nucleic acid molecule and forming a hydrogen bond between bases having a complementary relationship between the third oligonucleotide 13 and the fourth oligonucleotide 14 A molecule is generated, and then the free-end base sequence of the first oligonucleotide 11 constituting the first nucleic acid molecule and the third oligonucleotide 13 constituting the second nucleic acid molecule have at the non-terminal part. It is formed by hydrogen bonding between the base sequence having a complementary relationship with the free end base sequence.

In that case, the nucleic acid structure according to the present embodiment has a base sequence that is non-complementary to the fourth oligonucleotide 14 of the third oligonucleotide 13 and has a complementary relationship with the free-end base sequence. Is bent at the base sequence portion that forms the structure, and the second nucleic acid molecule is connected to the bent portion to form a branched structure, and also forms a T-shape. Yes.

Hereinafter, the nucleic acid structure according to the present embodiment will be described in detail from the viewpoint of the method for forming the nucleic acid structure and the basis for the formation of the T-shaped structure, and then the T-shaped structure. A nanoscale nucleic acid structure that can be prepared using a nucleic acid structure as a structural unit will be described. In this description, the nucleic acid structure is formed by hydrogen bonding between a first nucleic acid molecule and a second nucleic acid molecule as shown in FIG. 2 at one site having a complementary binding relationship. The nucleic acid complex means a structure formed by including the nucleic acid structure in the structure or by using the nucleic acid structure as a structural unit.

<Method of forming nucleic acid structure>
First, a method for forming a nucleic acid structure having a T-shape according to this embodiment will be described. In the description regarding this formation method, it demonstrates using the oligonucleotide which designed the specific base sequence.

FIG. 3 (A) is a diagram showing base sequences (SEQ ID NOs: 1 to 4) possessed by four oligonucleotides synthesized to form a T-shaped nucleic acid structure according to the present embodiment. When determining the base sequence of this oligonucleotide, the base sequence is determined in consideration of information on the base sequence length, that is, information on the base sequence length so that the target structure is formed in the intended order. Specifically, first, the base sequence so that the sequence length of its complementary base sequence portion is increased so that the oligonucleotide constituting the motif forms a hydrogen bond to form a double helix. To design. Then, after the double helix molecule is formed, the DNA molecules are connected by hydrogen bonding to form a nucleic acid structure in which the components are combined. The sequence portion is designed with a short base sequence length.

Thus, in order to form a nucleic acid structure consisting of a T-shaped oligonucleotide by forming a hydrogen bond in its complementary base sequence portion in the intended order, the oligonucleotide is sequenced in consideration of the base sequence length of the binding portion. Make a decision. This is because the longer the base sequence length of the oligonucleotide to be hybridized, the higher the temperature environment, the higher the temperature environment, and the shorter the base sequence length, the lower the temperature environment, the lower the temperature environment. This is for self-assembly.

A detailed description of the operation for causing the specific self-assembly will be described later. First, an oligonucleotide having a long base sequence length is obtained by shifting the solution containing the oligonucleotide from a high temperature to a low temperature. A DNA molecule is formed by hydrogen bonding, and subsequently, at a low temperature, the formed DNA molecules are hydrogen-bonded and connected at a shorter base sequence portion to form a nucleic acid structure having a T-shape. .

As described above, the nucleic acid structure according to the present embodiment has a base sequence in consideration of information on the base sequence length in order to form hydrogen bonds at complementary base sequence portions in the intended order under appropriate temperature control. To decide. In fact, using a base sequence design program, comprehensively examine possible hybridization between all base sequences to be used, and unless the target structure is formed, an unintended base sequence at a high temperature The sequence is determined so that no double helix formation occurs between them. Based on the determined base sequence, an oligonucleotide is artificially synthesized by a known method such as a solid phase synthesis method.

FIG. 3 (B) shows a nucleic acid structure having a T-shape formed by self-assembling four types of oligonucleotides having the base sequences shown in FIG. 3 (A) designed as described above. FIG. 2 is a schematic schematic view clearly showing a hydrogen bonding state between bases. In this figure, the direction of the arrow indicates the direction from the N-terminal (5′-end) to the C-terminal (3′-end) of the oligonucleotide. As shown in FIG. 3B, this T-shaped nucleic acid structure is formed by complementary binding of the first oligonucleotide (α−) and the second oligonucleotide (α +). Second double-stranded DNA molecule formed by complementary binding of the first double-stranded DNA molecule to be prepared, the third oligonucleotide (β +) and the fourth oligonucleotide (β-) It consists of and.

The first oligonucleotide (α−) and the second oligonucleotide (α +) are composed of a free-end base sequence CCCCC (FIG. 3 (A) α−) that the first oligonucleotide (α−) has at its 5 ′ end. : A hydrogen bond is formed between the base sequences having a complementary binding relationship except for the square box portion in the base sequence of the oligonucleotide) to form a first double-stranded DNA molecule. That is, the formed first double-stranded DNA molecule has a single-stranded free-end base sequence CCCCC, and that portion becomes a portion connected to the second double-stranded DNA molecule. ing.

On the other hand, the third oligonucleotide (β +) and the fourth oligonucleotide (β−) are composed of a base sequence GGGGG (β + in FIG. 3 (A): base of oligonucleotide) that the third oligonucleotide has at its non-terminal portion. A hydrogen bond is formed between the base sequences having a complementary binding relationship except for a square box portion in the sequence to form a second double helix DNA molecule. That is, the third oligonucleotide (β +) has a base sequence GGGGGG that does not form a complementary binding relationship with the fourth oligonucleotide (β−) in the middle of the series of base sequences. Each base of the oligonucleotide (β−) skips the base sequence portion GGGGGG that does not have its complementary binding relationship, and forms a hydrogen bond with the third oligonucleotide (β +) in the subsequent complementary base sequence portion. Forming a second double-stranded DNA molecule.

The second double helix DNA molecule formed in this way has a free base sequence portion not forming a hydrogen bond on the DNA double helix, and as a result, the double helix structure has a double helix structure. A bent structure is formed at that portion. Further, the bent structure is a structure bent in a substantially vertical direction with respect to the cylindrical shape when the double helical structure is assumed to be virtually cylindrical. Although the detailed reason for forming such a structure will be described later, the distance between the 6 bases (bp) of the DNA double helix is the diameter of the DNA itself, in other words, between the opposing complementary base pairs (opposing bases). The phase difference on the main groove side (Major Groove side) at that time is very small, and can be formed.

The second double-stranded DNA molecule formed with such structural features is connected to the first double-stranded DNA molecule by hydrogen bonding at the free base sequence GGGGGG portion. It has become. That is, as shown by the X part in FIG. 3B, the free-end base sequence CCCCC consisting of the single strand of the first double-stranded DNA molecule described above and the freeness of the second double-stranded DNA molecule The base sequence GGGGGG is designed so as to have a complementary binding relationship, and by forming a hydrogen bond with each other at this site, the first double helix DNA molecule and the second double helix A DNA molecule is connected to construct a T-shaped nucleic acid structure according to the present embodiment.

In addition, as shown in FIG. 3A, the first duplex (α−) and the third oligonucleotide (β +) have a first duplex as compared to the base sequence length of each oligonucleotide. Since the base sequence length that becomes the connecting part between the helical DNA molecule and the second double helical DNA molecule is designed to be extremely short, the nucleic acid structure is self-assembled by controlling the temperature from a high temperature to a low temperature. By forming, each DNA molecule is first formed, and then, the formed DNA molecules are connected by hydrogen bonding to construct a T-shaped nucleic acid structure. As a result, hydrogen bonds are formed at unintended sites, and an unintended nucleic acid structure is not formed.

Thus, regarding the nucleotide sequence determination of the oligonucleotide for forming the T-shaped nucleic acid structure according to the present embodiment, considering that the double helix of DNA bends at an appropriate site, Comprehensive possible hybridization between all base sequences used, considering information on base sequence length to form hydrogen bonds in complementary base sequence parts in the intended order under appropriate temperature control Except for the case where the desired structure is formed, the sequence is determined so that unintentional double helix formation does not occur between the base sequences at high temperatures.

Specifically, an oligonucleotide having a free-end base sequence consisting of 5 bases at the 5 ′ end and an oligonucleotide having a base sequence that forms a hydrogen bond complementary to the oligonucleotide form a double-stranded DNA molecule. On the other hand, an oligonucleotide having a base sequence consisting of 5 bases having a complementary binding relationship with the above-mentioned free end base sequence at a site other than the terminal portion, and hydrogen complementary to the oligonucleotide The nucleotide sequence is determined so that the oligonucleotide having the nucleotide sequence that forms a bond forms a double helix DNA molecule. A T-shaped nucleic acid structure having a structure branched in the vertical direction by self-assembling four kinds of oligonucleotides synthesized based on these designs from a high temperature to a low temperature under temperature control Can be formed.

By the way, the T-shaped nucleic acid structure according to the present embodiment formed as described above is perpendicular to the cylindrical shape when the DNA double helix structure is assumed to be virtually cylindrical. The structure is branched in the direction. This is because the distance between 6 base pairs (bp) of the double helix structure is the diameter of the DNA double helix structure itself, in other words, the length between the opposing complementary base pairs. This is because the phase difference on the main groove (Major 溝 Groove) side at that time is extremely small. Therefore, as shown in FIG. 1 to FIG. 3, a connecting portion corresponding to 6 bp which is the number of bases constituting the oligonucleotide having a length corresponding to the length between the opposing complementary base pairs is formed (particularly, FIG. By forming a base sequence consisting of 5 bases obtained by subtracting 1 from 6 bp as a sticky part, it becomes possible to extend the double helix in the vertical direction, and A T-shaped nucleic acid structure can be formed by hydrogen bonding with a second double-stranded DNA molecule having a free-end base sequence composed of the same five bases. Hereinafter, this point will be described in detail.

<Verification from the characteristics of DNA double helix structure>
DNA in an aqueous solution takes a conformation called B-type DNA. FIG. 4 is a structural schematic diagram of the B-type DNA. As shown in FIG. 4, B-type DNA has a right-handed double helix structure, and is rotated by 34.3 ° per base pair (bp), that is, has a structure that makes one round (360 °) at 10.5 bp. Yes. The distance in the double helix direction between the base pairs in the B-type DNA is about 0.34 nm (3.4 angstroms), and the diameter of the DNA double helix structure is about 2.37 nm (23. 23 nm). 7 angstroms). In the following, first, the nucleic acid structure according to the present embodiment will be explained by calculation based on each distance and phase difference of the DNA double helix structure, with respect to forming a T-shaped shape by extending branches in the vertical direction. To do.

As described above, it is generally known that the diameter of the DNA double helix structure is approximately 2.37 nm (23.7 angstroms). However, the opposite base of DNA does not exist so as to constitute a positional relationship having completely opposite phases as in the DNA double helix structure schematically shown in FIG. The bases are in a positional relationship such that the angle between the opposing base backbones is about 135 ° to 140 ° with respect to the center of the double helix. Therefore, considering the angle between the opposing base backbones and calculating the angle between the opposing base backbones to 135 ° as in the formula (1), the diameter of the DNA double helix structure is:
2 × 2.37 / 2 × sin (135 ° / 2) ≈2.19 (nm) (1)
It becomes.

Based on the diameter of this DNA double helix structure, when this double helix structure is assumed to be a cylindrical shape, let us consider branching and extending the double helix in a direction perpendicular to the cylindrical shape. Since the value of the diameter of the DNA double helix structure is 2.19 nm as described above, from the calculation formula (2) below,
2.19 / 0.34≈6.44 (bp) (2)
That is, the length of about 6 to 7 bp corresponds to the length of the diameter of the DNA double helix structure, in other words, the length between opposing complementary base pairs, and therefore, the distance of 6 or 7 bp. A free-end base sequence consisting of the same 5 or 6 bases was formed by forming a connecting portion with a gap and forming a base sequence consisting of 5 or 6 bases obtained by subtracting 1 from this 6 or 7 bp as an adhesive portion. Hydrogen bonds with the second double helix DNA molecule, allowing the double helix to extend vertically as described above.

Next, the phase of the DNA double helix structure in the nucleic acid structure according to the present embodiment will be described with reference to FIG. As shown in the structural schematic diagram of the DNA double helix structure in FIG. 3, the DNA double helix structure has a main groove that is a large groove (Major 大 き な Groove) and a minor groove that is a small groove (Minor Groove). The structure is repeated alternately. At this time, when an attempt is made to extend the DNA double helix structure in the vertical direction with an interval of 6 or 7 bp as described above, the sites that can have an interval of 6 or 7 bp are the main groove side and the secondary groove. There are two locations on the groove side.

Here, considering the phase difference of 6 bp in the DNA double helix structure, since the DNA double helix structure has a structure that makes one round at 10.5 bp, the rotation angle of the double helix per 1 bp is 360 ° / 10.5≈34.3 °. Then, using the fact that the angle between the opposing base backbones is 135 °, from the following formulas (3) and (4):
Major Groove: 360 °-(34.3 ° × 6 + 135 °) = 19.2 ° (3)
Minor Groove: 34.3 ° × 6-135 ° = 70.8 ° (4)
Thus, the phase difference between the main groove side and the sub groove side can be obtained, and it can be seen that the phase difference at the main groove side is smaller than the phase difference at the sub groove side. In addition, when the phase difference at 7 bp is considered in the same manner, from the following formulas (5) and (6),
Major Groove: 360 °-(34.3 ° × 7 + 135 °) = − 15.1 ° (5)
Minor Groove: 34.3 ° × 6-135 ° = 105.1 ° (6)
In this case also, it is understood that the phase difference on the main groove side is smaller than the phase difference on the sub groove side.

As described above, since the phase difference on the main groove side is smaller than the phase difference on the sub groove side, the extension of the DNA double helix on the main groove side is longer than the extension of the DNA double helix on the sub groove side. More preferably, it can be said that the DNA double helix is branched in the vertical direction on the main groove side to be suitable for forming a nucleic acid structure having a T-shape.

As shown in the above specific examples, the number of bases constituting an oligonucleotide of 6 to 7 bp on the main groove side of the DNA double helix structure, that is, the length corresponding to the length between opposing complementary base pairs It is possible to extend the DNA double helix in the vertical direction as described above by forming a connecting portion with an interval of minutes (forming an adhesive portion consisting of 6 or 7 bp-1 = 5 or 6 bases). Become. In addition, since this T-shaped shape has an appearance in which a branch structure is connected by a single double helix, it is possible to eliminate distortion (including twist) of the double helix part generated in the branch part to some extent. . Therefore, even if a slight design deviation occurs, it can be resolved. Furthermore, the free-end base sequence length is not limited to 5 and 6, but can be prepared with 7 bases, for example, as long as distortion caused thereby can be eliminated.

<Structural demonstration experiment based on electrophoresis>
Based on the relationship between the structural characteristics and phase of the DNA double helix structure described above, regarding the extension on the main groove side and the extension on the sub groove side, in the structure formation of the T-shaped nucleic acid structure according to this embodiment The occurrence of the difference will be described based on the results of a demonstration experiment using electrophoresis.

In this demonstration experiment, each nucleotide was hybridized and self-assembled in the intended order, and a comprehensive T-shaped nucleic acid structure was formed using a base sequence design program. A nucleotide sequence was designed, and four types of oligonucleotides represented by the following (I) were artificially synthesized, and a structure was formed using these four types of oligonucleotides. The oligonucleotide having the base sequence (SEQ ID NO: 1 to 4) shown in the following (I) has a structure in which the DNA double helix branches in the main groove side (Major （Groove side), that is, extends in the vertical direction. In order to form a structure so as to extend to the minor groove side (Minor Groove side), the oligonucleotide (I) described in the following 5 ′ end → 3 ′ end direction On the other hand, it was formed by using an oligonucleotide having the base sequence (SEQ ID NO: 5 to 8) described in (II) in which the direction from the 5 ′ end to the 3 ′ end was reversed.

FIG. 6 is a schematic diagram of a T-shaped nucleic acid structure that can be theoretically obtained using the oligonucleotides described in (I) and (II) above.

In addition, the structure formation procedure of the said nucleic acid structure was performed by the one-pot annealing method which is a well-known method. Specifically, first, each oligonucleotide artificially synthesized by determining the base sequence was dissolved in TAE buffer (1 × TAE / Mg ²⁺ (12.5 mM)) so as to have a concentration of 1 μM, Dissolved oligonucleotides were dispensed and mixed in equal amounts in a single reaction tube. Next, the mixed solution was allowed to hybridize by slowly cooling to about 4 ° C. starting from about 95 ° C. This annealing operation was performed by continuously cooling for about 24 hours using a thermal cycler (manufactured by Eppendorf). The sample thus annealed was placed in a thermostatic bath and stored at about 3 ° C.

In this annealing operation, oligonucleotides having a long base sequence length are hydrogen-bonded between bases in a complementary relationship at a high temperature. For example, the above-mentioned synthesis was performed for the purpose of extending in the vertical direction on the main groove side. In the oligonucleotide having the base sequence shown in (I), α +: oligonucleotide base sequence 5′-TATATTATATAGAATAATAAGAGACTATACTAGAA-3 ′ (SEQ ID NO: 1) and α− : Base sequence of oligonucleotide 5'-CCCCCTTCTAGTATAGTCTCTTATTATTCTATATAATATA-3 '(SEQ ID NO: 2) forms hydrogen bonds between its complementary bases to form a double helix, then β +: oligonucleotide base sequence 5' -TGGGCAGCGGGGGGATTAAAACTATAATTC-3 '(SEQ ID NO: 3) and β-: oligonucleotide base sequence 5'-GAATTATAGT TTTAATCGCTGCCCA-3 '(SEQ ID NO: 4) forms a double helix by hydrogen bonding between its complementary bases. Then, between the DNA double helix formed by α + and α− and the DNA double helix formed by β + and β−, between the nucleotide sequences having the free complementary relationship Hydrogen bonds to form the target T-shaped nucleic acid structure.

FIG. 7 shows the experimental results of electrophoresis of the reaction solution hybridized as described above. In FIG. 6, the sample in lane 2 contains α +, the sample in lane 3 contains α−, the sample in lane 4 contains β +, the sample in lane 5 contains β−, Sample 6 contains α + and α−, sample in lane 7 contains β + and β−, and sample in lane 8 contains all nucleotides (α +, α−, The sample in lane 9 contains all nucleotides (α +, α−, β +, β−) for the purpose of branch extension on the main groove side. The electrophoresis was performed using a 12% polyacrylamide gel and a TAE buffer solution (1 × TAE / Mg ²⁺ (12.5 mM)) having a Mg ²⁺ concentration of 12.5 mM.

As is apparent from the electrophoretic results shown in FIG. 7, the sample contains all oligonucleotides (α +, α−, β +, β−) for the purpose of extending the branch on the minor groove side of the double helix structure. In Lane 8, a band indicating a double helix formed by hydrogen bonding of α + and α− and a double helix formed by hydrogen bonding of β + and β− was confirmed, It can be seen that hydrogen bonds are not formed between the base sequences having the free complementary relationship in each double helix and are completely separated.

In contrast, in Lane 9, which is a sample containing all oligonucleotides (α +, α−, β +, β−) for the purpose of extending branches on the main groove side of the double helix structure, α + It can be seen that a band showing a larger structure is observed on a band showing a double helix composed of α-. Here, in the experiment using the oligonucleotide having the base sequence shown in (I) above, the structure larger than the double helix composed of α + and α− The structures involved, ie, the double helix formed by hydrogen bonding of α + and α−, and the double helix formed by hydrogen bonding of β + and β− are in each double helix. There is only a T-shaped nucleic acid structure formed by hydrogen bonding between the base sequences having the free complementary relationship. Therefore, it was found that the sample in lane 9 was formed with a nucleic acid structure that formed a T-shape by extending vertically in an arbitrary phase of the DNA double helix structure. It was demonstrated that a T-shaped nucleic acid structure was formed only by extending the double helix in the vertical direction on the groove side.

The oligonucleotide for forming the T-shaped nucleic acid structure according to the present embodiment is not limited to the oligonucleotide sequence described in (I) above. For example, in the oligonucleotide according to (I), the bases of cytosine (C) and guanine (G) are used as complementary base sequence parts in which two double-stranded DNAs form a hydrogen bond to form a nucleic acid structure. However, the present invention is not limited to this, and bases of adenine (A) and thymine (T) may be arranged. Naturally, adenine (A), thymine (T), guanine (G), The base sequence may be a combination of cytosine (C).

In the above, specific examples using DNA as a nucleic acid molecule have been described. However, the nucleic acid molecule is not limited to DNA, and the interval formed at the connecting portion is limited to the interval corresponding to the main groove side 6 or 7 bp. It is not something. That is, the nucleic acid structure according to this embodiment can be formed using nucleic acid molecules such as RNA and PNA. In the example using the above DNA, specifically, on the main groove side of the DNA double helix structure, it corresponds to the length of the diameter of the double helix, in other words, the length between the opposing complementary base pairs. The DNA double helix can be extended in the vertical direction by forming a connecting portion with an interval of 5 or 6 bp that is the number of bases to be the length of the nucleic acid molecule. Even in the case of using RNA, PNA, etc., in the double helical structure of RNA or PNA, a connecting portion is formed with an interval corresponding to the number of bases corresponding to the length between opposing complementary base pairs. This makes it possible to extend the double helix in the vertical direction. That is, in the double helix structure, as described above, a base consisting of the number of bases obtained by subtracting 1 from the number of bases constituting an oligonucleotide having a length corresponding to the length between the opposing complementary base pairs. By forming the array as an adhesive portion, the double helix can be extended in the vertical direction. Then, in the adhesive portion, another double helix having the same base number as that of the adhesive portion and having a complementary binding relationship as a free-end base sequence is hydrogen-bonded at the site, so that T A character-shaped nucleic acid structure can be formed. Therefore, the nucleic acid is not limited to DNA, and RNA or PNA may be used. Therefore, the number of bases corresponding to the length between the complementary base pairs facing each other is naturally 5 or 6 bases. Not limited.

In addition, even when DNA is used as a nucleic acid molecule, the length of the double helix diameter may vary depending on the base sequence of the connecting portion. By forming the connecting portions spaced by the number of bases corresponding to the length of the double helix, the double helix can be extended in the vertical direction.

As described above, when DNA is used, the phase difference on the main groove side of the double helix structure is extremely small, and on the main groove side, the length of the diameter of the double helix, in other words, the opposite complementary Bases constituting an oligonucleotide having a length corresponding to the length between the opposing complementary base pairs, forming a connecting portion with an interval corresponding to the number of bases corresponding to the length corresponding to the length between base pairs A first oligonucleotide having a free-end base sequence consisting of the number of bases obtained by subtracting 1 from the number at the 5 ′ end, and a second oligonucleotide having a base sequence having a complementary relationship with the first oligonucleotide, And a base sequence comprising the number of bases obtained by subtracting 1 from the number of bases constituting the oligonucleotide having a length corresponding to the length between the above-mentioned complementary base pairs opposed to each other complementary to the free-end base sequence By reacting a solution containing a third oligonucleotide having a non-terminal portion and a fourth oligonucleotide having a base sequence having a complementary relationship with the third oligonucleotide by a well-known annealing operation A nucleic acid structure having a structure branched in the vertical direction and having a T-shape can be formed.

Similarly, even when RNA, PNA, or the like is used as a nucleic acid, in the double helical structure of RNA or PNA, an interval corresponding to the number of bases corresponding to the length between opposing complementary base pairs By forming the connecting portion with a gap, it is possible to extend the double helix in the vertical direction, and to form a T-shaped nucleic acid structure having a structure that branches in the vertical direction. It has become.

A conventional nucleic acid structure such as a crossover cannot form a branched structure of a DNA molecule except in a region where the phases of adjacent double helices are aligned, which is compared with the spatial resolution of the DNA molecule itself. Thus, there is a problem that the size of the structural motif that can be produced using the nucleic acid structure becomes large. However, according to the nucleic acid structure according to the present embodiment, the main groove side of the DNA double helix structure is A branched structure can be formed anywhere as long as it is in phase. By using this nucleic acid structure as a structural unit, a nucleic acid structure with high spatial resolution can be produced. Specifically, when the branch is extended in the same phase on the main groove side, the branch may be formed at approximately every 10.5 × n (bp) (n = 1, 2, 3,...). When a branch is extended to the opposite phase on the main groove side, a branch structure is formed at approximately every 10.5 × m + 5.25 (bp) (m = 0, 1, 2,...). It becomes possible.

In addition, since it is possible to form a branch structure at an arbitrary phase, more structures orthogonal to the vertical direction can be formed and used, and various structures thus formed can be combined. Thus, various nanoscale nucleic acid structures can be produced. Furthermore, it can be combined with an existing structure such as a crossover, and more structures can be designed in terms of diversity.

Hereinafter, a nanoscale nucleic acid structure produced using the above-described T-shaped nucleic acid structure as a structural unit will be described. In the following, DNA is used as a nucleic acid, and a connecting portion is formed with an interval of 6 bp, which is the number of bases constituting an oligonucleotide having a length corresponding to the length between opposing complementary base pairs ( A specific example of extending the DNA double helix in the vertical direction by forming an adhesive part consisting of 6 bp-1 = 5 bases) will be described. However, the nucleic acid molecule is not limited to DNA, but RNA or PNA Even in the case of using, etc., it is necessary to form a connection portion with an interval corresponding to the number of bases corresponding to the length corresponding to the length between opposing complementary base pairs, and in the intended order. It goes without saying that a nanoscale nucleic acid structure as described below can be produced by designing an appropriate base sequence so as to be hybridized.

<Method for producing nanoscale nucleic acid structure>
When a nanoscale nucleic acid structure (hereinafter referred to as “nanonucleic acid structure”) is produced using the T-shaped nucleic acid structure according to the present embodiment as a structural unit, a DNA double helix structure is hypothesized. It is important to properly design the relative rotation angle (phase) and length (sequence length) of the bases at both ends thereof, and to appropriately control the temperature during the reaction.

The production of nanonucleic acid structures by self-assembly using DNA molecules, all materials are put in a tube, and then slowly cooled from high temperature to low temperature by a temperature drop method called annealing, and a structure is created in the process. This can be done by using a so-called one-pot manufacturing method. During annealing, the oligonucleotide basically forms a double helix from a base sequence having a long complementary binding relationship, but at this time, the base sequence of the base sequence is formed so that the intended structure can be formed in the desired order. It is necessary to design the sequence length and to design a base sequence to prevent unwanted double helix formation. This will be specifically described below.

First, the nano-nucleic acid structure to be produced is designed, and the unit structure that is repeated in the structure is determined. In determining the unit structure, consideration is given to rotation and inversion, and the structure to be created is adjusted so that it matches the conditions related to the phase and length of the DNA double helix structure. It is also preferable to consider.

Once the target nanonucleic acid structure is designed and the unit structure in the structure is determined, if any site has a sequence with complementary binding between bases, then in the intended order Hybridization between bases will be investigated to see if they will self-assemble into the target structure, and the base sequences of multiple oligonucleotides will be designed. The base sequence of the oligonucleotide is a one-dimensional sequence consisting of four combinations of adenine (A), thymine (T), guanine (G), and cytosine (C). In designing the base sequence, the base sequences of the oligonucleotides that bind to each other inside the unit are designed to have a sufficiently long sequence length than the base sequences that bind to each other between the units. Thus, by making the sequence lengths of the nucleotide sequences of the oligonucleotides different, it is possible to bind the target sequences in the intended order and form the target nanonucleic acid structure. In other words, the longer the base sequence length, the longer the base sequence length, the higher the temperature environment, the higher the temperature environment, the higher the temperature, the complementary base sequence part has the property of forming a hydrogen bond. The target nano-nucleic acid structure can be obtained by binding oligonucleotides in the complementary sequence sequence in the intended order by performing a temperature control operation that changes the reaction solution containing the synthesized oligonucleotide from high to low temperatures. An object can be formed.

Specifically, it is designed so that the base sequence possessed by the oligonucleotide bound inside the unit has a sufficiently longer sequence length than the base sequence bound between the units. As a result, first, a unit structure that is a repetitive structure portion of the nanonucleic acid structure to be designed is formed by a plurality of oligonucleotides, and then the target nanonucleic acid structure is formed by the formed units at low temperature conditions. Will come to be.

Even within the unit, there is a difference in the order of hybridization between the oligonucleotides forming the unit structure. For example, when only one T-shaped nucleic acid structure is set as the unit structure, As described with reference to FIG. 3, two double helices constituting a T-shaped nucleic acid structure are first formed by hydrogen bonding between complementary base sequences between oligonucleotides, followed by Two double helices are hybridized at a complementary base sequence portion consisting of 5 bases to form a T-shaped nucleic acid structure as a unit structure. When the unit structure in the target nanonucleic acid structure is formed first as described above, next, bonding between units is started under a low temperature environment, and a plurality of units are hydrogen-bonded. As a result, a nanonucleic acid structure is formed.

When the base sequence is designed so that the intended nano-nucleic acid structure is formed in the intended order in this way, the oligonucleotide consisting of sugar, phosphate, and base is actually used based on the base sequence. Combine multiple. The oligonucleotide can be synthesized using a known method such as solid phase synthesis.

When a plurality of oligonucleotides are synthesized, these oligonucleotides are then dissolved in an appropriate buffer, and each solution is dispensed in equal amounts to obtain an oligonucleotide mixed solution containing these solutions. Specifically, for example, it is dissolved in TAE buffer (1 × TAE / Mg ²⁺ (12.5 mM)) so that the concentration becomes 1 μM, and each solution dissolved in the buffer is dispensed and dispensed. Mix all solutions in equal volumes in a single reaction tube. Note that these conditions are merely examples, and the type and concentration of the buffer are not limited thereto.

Next, the mixed solution containing the plurality of oligonucleotides is annealed and hybridized in the desired order. The annealing operation can be performed by a well-known method. Specifically, for example, the purified oligonucleotide mixed solution is gradually cooled to about 4 ° C. starting from about 95 ° C. over about 24 hours. The plurality of oligonucleotides in the mixed solution are annealed, and the reacted mixed solution is stored at a predetermined temperature. The temperature setting and time in the annealing operation are not particularly limited to the above conditions. For example, when the temperature is sequentially lowered, an operation of leaving the reaction solution at a predetermined temperature may be performed. Absent.

As described above, paying attention to the unit structure in the target nano-nucleic acid structure, the oligo nucleotides can be appropriately self-assembled by hybridization in the intended order in the complementary base sequence part inside and between the units. A complex nano-nucleic acid structure in which a nucleotide sequence of nucleotides is designed and annealed under appropriate temperature control, so that a T-shaped nucleic acid structure is used as a structural unit and a plurality of the nucleic acid structures are hydrogen-bonded. Can be produced.

In the preparation of the nano-nucleic acid structure described above, the number of types of oligonucleotides used is not limited, and the nucleic acid structure may be prepared by annealing four kinds of oligonucleotides. Alternatively, a nucleic acid structure may be prepared by reacting two kinds of oligonucleotides.

Since the nano-nucleic acid structure produced in this way is produced by using the T-shaped nucleic acid structure according to the present embodiment as a structural unit, there is a problem in the conventional crossover structure. There is no dependency on the direction of forming the branched structure and restrictions on the structure, and it is possible to form a DNA branched structure at an arbitrary phase as long as it is a phase on the main groove side of the DNA double helix structure. It will have a higher spatial resolution.

In addition, a DNA double helix structure having electrostatic repulsion characteristics compared to the conventional technique in which a nano nucleic acid structure is formed by arranging double helixes such as a crossover structure in parallel, Since it is possible to form a space between adjacent double helixes by forming a structure orthogonal to each other, this nano-nucleic acid structure produced using the T-shaped nucleic acid structure as a structural unit is The strain generated in the structure itself is greatly reduced, and a high-dimensional structure such as a three-dimensional structure can be created as a stable structure.

Furthermore, since the nanonucleic acid structure produced by using the T-shaped nucleic acid structure according to the present embodiment as a structural unit is formed by combining orthogonal double spirals as described above, a T-shape is further formed. It is possible to create nano-nucleic acid structures in combination with existing motifs by using a nucleic acid structure in a mold shape, and design more structures in terms of diversity compared to existing motifs. It becomes possible. And according to the nanoscale nucleic acid structure produced by using the T-shaped nucleic acid structure according to the present embodiment as a structural unit in this way, a smaller, high-density and high-definition functional nanodevice Can be produced.

Hereinafter, specific examples of nano-nucleic acid structures will be described.

<Specific Embodiment of Nano-Nucleic Acid Structure>
Example 1
FIG. 8 is a diagram schematically showing a specific unit structure of a nano-nucleic acid structure and a part of a ladder-type structure that is a nano-nucleic acid structure that is produced by repeating the unit structure. As shown in FIG. 8, the ladder structure according to this embodiment is formed by connecting a T-shaped nucleic acid structure as a unit structure and connecting a plurality of the T-shaped nucleic acid structures as repeating structures. To do. The numbers shown in the structure shown in FIG. 8 indicate the number of base pairs (bp), and α−, β +, and β− indicate the positions of oligonucleotides having the base sequences shown below. Yes.

This ladder-type structure is based on a shape in which double helix DNA is vertically connected between double helix DNAs arranged in parallel, and is produced by forming a branched structure in the same phase of the double helix structure. can do. When a branched structure is formed in the same phase as seen from the double helix structure of DNA, a branched structure is formed at approximately every 10.5 × n (bp) (n = 1, 2, 3,...). Therefore, in the case of the ladder structure in this embodiment, a T-shaped nucleic acid is formed by forming a branched structure in the vertical direction every 4 turns of the DNA double helix. A ladder-type structure having a structure as a repeating structure can be created.

The three base sequences (SEQ ID NOs: 9 to 11) shown in (III) below are designed so that hybridization occurs in the intended order by determining the unit structure that becomes the above-mentioned repeating structure and examining the phase. This is the base sequence possessed by the oligonucleotide. The base sequence of this oligonucleotide shows the base sequence in the direction from the 5 'end to the 3' end.

As a method for forming a nanonucleic acid structure using these oligonucleotides, a known one-pot annealing method can be used.

In the ladder structure according to this embodiment, among the oligonucleotides having the base sequence shown in (III) above, under a high temperature condition, β +: oligonucleotide base sequence 5′-CCCTGGGCAGCGGGAGGATTAAAACTATAATTCTGCGTGATCATTAAATCTCTA-3 ′ (SEQ ID NO: 10) and β-: oligonucleotide base sequence 5′-CACGCAGAATTATAGTTTTAATCGCTGCCCAGGGTTCGAA-3 ′ (SEQ ID NO: 11) form a hydrogen bond in the complementary base sequence portion, forming a double helix DNA. . In this case, the base sequence GGAGG (β + in the above (III): in the oligonucleotide) cannot form a complementary hydrogen bond with β-, which forms a double-stranded DNA, together with β + described in (III) above. In this base sequence part, hydrogen bonds are not formed with each other, while the corresponding β-s forming base pairs skip base sequences without their complementary binding relationship. Thus, the base portion having the next complementary bond relationship continues to form hydrogen bonds. Therefore, the double helix DNA is bent at the portion where the base sequence GGAGG is formed, and a structure branched in a substantially vertical direction is formed.

When the annealing operation is continued, α-: oligonucleotide base sequence 5′-CCTCCTAGAGATTTAATGAT-3 ′ (SEQ ID NO: 9) having the base sequence shown in (III) above is the base sequence shown in the X part in FIG. And the part constituted by the base sequence shown in the X ′ part in FIG. 8 of the base sequence of β + are connected by forming a hydrogen bond.

Then, when the annealing operation is further continued, the base sequence CCTCC consisting of 5 bases designed at the 5 ′ end of the base sequence of α- having the base sequence shown in (III) above (the α in base of (III) above) -: Underlined portion in the oligonucleotide) forms a complementary hydrogen bond with the free base sequence portion in which the hydrogen bond of the double helix DNA previously formed between β + and β- is not formed. Since it is designed to have a relationship, at the lower temperature, a hydrogen bond is finally formed in the complementary base sequence portion consisting of 5 bases indicated by a Y portion in FIG. A shaped structure is formed.

Thus, the base sequence that binds at the site indicated by Y in FIG. 8 is designed to be extremely shorter than the base sequence that binds at the sites indicated by X and X ′ in FIG. In FIG. 8, a hydrogen bond is formed between complementary bases at the site indicated by Y, so that a ladder-type structure is formed.

The formation operation and confirmation observation of this ladder-shaped nucleic acid structure by the one-pot annealing method were performed as follows.

First, each oligonucleotide synthesized by determining a base sequence by a program based on the knowledge as described above is dissolved in TAE buffer (1 × TAE / Mg ²⁺ (12.5 mM)) so as to have a concentration of 1 μM. The oligonucleotide solution was purified. Then, each of the solutions was dispensed, and all the dispensed solutions were mixed in equal amounts in one reaction tube to purify the oligonucleotide mixed solution.

Next, the oligonucleotide mixed solution is annealed by using a thermal cycler (Eppendorf) and slowly cooling to about 4 ° C. starting from about 95 ° C. Hybridization was performed at the target nucleotide sequence. The annealing operation was continuously performed for about 24 hours. The annealed sample was placed in a thermostatic bath and stored at about 3 ° C.

FIG. 9 is an atomic force microscope (AFM) observation image of a ladder structure specifically manufactured by the above operation. In the confirmation observation of nanostructure formation by this AFM, the sample to be observed using a pipette as appropriate from the sample that has been continuously cooled and annealed for about 24 hours and then stored in a thermostatic bath The sample was taken out each time and was observed in a liquid tapping mode using NanoScope® III manufactured by Beco.

As is apparent from the AFM observation image of FIG. 9, it can be seen that a ladder type structure of about 100 nm is formed. Then, a double helix DNA is vertically connected between the double helix DNAs arranged substantially in parallel in each ladder type structure, and a so-called ladder step portion is formed. It can also be clearly seen that a space of about 10 to 15 nm is formed between the ladders of this ladder structure.

As described above, according to the T-shaped nucleic acid structure according to the present embodiment, a branched structure can be formed at an arbitrary phase of the double helical structure of DNA, and the nano-nucleic acid structure with higher resolution can be formed. It is possible to create.

(Example 2)
FIG. 10 is a diagram schematically showing a specific unit structure of a nano-nucleic acid structure and a partial structure of a planar structure that is a nano-nucleic acid structure that is produced using the unit structure as a repetitive structure. As shown in FIG. 10, in the planar structure according to this embodiment, a structure in which two T-shaped nucleic acid structures are combined in opposite directions is determined as a unit structure, and a plurality of unit structures are connected as a repeating structure. It is a lattice structure formed by making it. The numbers described in the structure shown in FIG. 10 indicate the number of base pairs (bp), and + and − indicate the arrangement site of the oligonucleotide having the base sequence shown below.

This planar structure is based on a shape in which double helix DNAs are vertically connected between double helix DNAs arranged in parallel, and is created by forming a branched structure in the reverse phase of the DNA double helix structure. can do. In view of the double helix structure of DNA, when a branched structure is formed in an opposite phase, a branched structure is formed approximately every 10.5 × m + 5.25 (bp) (m = 0, 1, 2,...). In the case of the planar structure in this embodiment, an antiphase branching structure is formed in the vertical direction every 1.5 turns of the double helix of DNA. A planar structure having two T-shaped nucleic acid structures as repeating structures can be prepared.

The two base sequences (SEQ ID NOs: 12 and 13) shown in (IV) below are designed so that hybridization occurs in the intended order by determining the unit structure to be a repetitive structure and examining the phase. The nucleotide sequence of the oligonucleotide. The base sequence of this oligonucleotide indicates the base sequence in the direction from the 5 'end to the 3' end, respectively.

As a method for forming a planar structure using this oligonucleotide, it can be prepared by a one-pot annealing method, similarly to the method for forming a ladder structure described in the first embodiment.

In the planar structure according to this embodiment, under a high temperature environment, first, the nucleotide sequence 5′-AGCCCTTGTGGTAGTTGGCACCAGAAGCCAACCGAAGACCACGGTGGGCTTAACACCATC-3 ′ (SEQ ID NO: 12) shown in the above (IV) and −: oligonucleotide The base sequence 5′-CGACGGATGGTGTTAACCGTGGTCTTCGGTTGGCTTCTGGTGCCACGTCGACTACCACAA-3 ′ (SEQ ID NO: 13) forms a hydrogen bond in the complementary base sequence part (underlined part of each oligonucleotide in the above (IV)), and a double helix comprising these forms It is formed. At that time, the +: oligonucleotide described in the above (IV) includes the base sequence GGGCT that cannot form a complementary hydrogen bond with the oligonucleotide (+: the non-underline in the non-terminal portion of the oligonucleotide (IV) above). In this base sequence portion, hydrogen bonds are not formed with each other, while the corresponding-: oligonucleotide skips the base sequence without its complementary binding relationship and the next complement It continues to form hydrogen bonds with the base moiety having a chemical bond relationship. Therefore, as shown in the X part in the unit schematic diagram of FIG. 10, the double helix is bent at the part where the base sequence GGGCT of the oligonucleotide is formed, so as to form a structure branched in the vertical direction. Become.

On the other hand, −: oligonucleotide also has +: base sequence CGTCG that cannot form a complementary hydrogen bond with the oligonucleotide (in the above (IV), −: non-underlined portion of the non-terminal portion of the oligonucleotide). In this base sequence portion, hydrogen bonds are not formed with each other, while the corresponding +: oligonucleotide skips the base sequence without its complementary binding relationship and has the following complementary binding relationship. Continue to form hydrogen bonds with the base moiety it has. Therefore, as shown in the X ′ part in the unit schematic diagram of FIG. 10, the double helix is bent even in the part where the base sequence CGTCG of the oligonucleotide is formed to form a structure branched in the vertical direction. It becomes like this.

In the unit structure of the planar structure, each of the two kinds of oligonucleotides forming the unit structure has a base sequence for folding the double helix and branching in the vertical direction, and the branched structure. Are designed in the opposite phase on the main groove side of the DNA double helix structure to thereby form two T-shaped nucleic acid structures having a unit structure as shown in FIG. It is possible to form a complex nanonucleic acid structure having the unit structure as a repeating structure.

In this manner, after the start of the annealing operation, in a high temperature environment, the +: and −: oligonucleotides form hydrogen bonds at their complementary base sequence portions, thereby forming a T-shaped nucleic acid structure. A unit structure having two connected structures is formed.

Then, by connecting these units under a lower temperature environment, it is possible to create a planar structure having the unit structure as a repeated structure. In the unit-to-unit connection, the +: and −: free end base sequence portions designed at the 5 ′ ends of the oligonucleotides shown in the above (IV) are branched structure portions in the double helix DNA of other unit structures. Units are connected by forming a hydrogen bond with a base sequence that does not form a hydrogen bond. Specifically, the free end base sequence portion of the Y portion in the unit schematic diagram of FIG. 10 is connected in a complementary manner to form a hydrogen bond with the branched portion indicated by X of the other unit, and the unit of FIG. The free end base sequence portion of the Y ′ portion of the schematic diagram is further connected by forming a hydrogen bond in a complementary manner with the branched portion indicated by X ′ of another unit. At that time, the units are connected while being reversed.

In this way, between the units, while the units are inverted, the free end base sequence portion and the portion forming the branched structure are hydrogen-bonded, so that the units are connected, and finally the target lattice-like shape A planar structure is formed.

The formation operation and confirmation observation of the nucleic acid structure having a lattice-like planar structure by the one-pot annealing method were performed according to the following procedure.

First, each oligonucleotide synthesized by determining the base sequence by a program based on the above-described knowledge was dissolved in TAE buffer (1 × TAE / Mg ²⁺ (12.5 mM)) so as to have a concentration of 1 μM. Then, each of the solutions was dispensed, and each of the dispensed solutions was mixed in an equal amount in one reaction tube.

Next, the oligonucleotide mixed solution is annealed by using a thermal cycler (Eppendorf) and slowly cooling to about 4 ° C. starting from about 95 ° C. Hybridization was performed at the target nucleotide sequence. The annealing operation was continuously performed for about 24 hours. The annealed sample was placed in a thermostatic bath and stored at about 3 ° C.

FIG. 11 is an atomic force microscope (AFM) observation image of the planar structure specifically created by the above operation. In the confirmation observation of nanostructure formation by this AFM, the sample to be observed using a pipette as appropriate from the sample that has been continuously cooled and annealed for about 24 hours and then stored in a thermostatic bath The sample was taken out each time and was observed in a liquid tapping mode using NanoScope® III manufactured by Beco.

As is apparent from the AFM observation image shown in FIG. 11, it can be seen that a lattice-like planar structure having holes of about 10 to 15 nm regularly and in steps is formed.

In addition, the planar structure according to the second embodiment has a DNA double helix structure without changing the basic design guideline of the ladder structure described in the first embodiment of the nanonucleic acid structure. It was proved that it can be produced only by reversing the phase forming the branched structure.

Thus, by producing a nanonucleic acid structure using the T-shaped nucleic acid structure according to the present embodiment as a basic unit, the phase at the time of forming a branched structure, that is, branching to the same phase or vice versa It is possible to easily design a wide variety of nano-nucleic acid structures having completely different shapes without changing the basic specific design guideline only by considering whether the phase is branched or not.

Furthermore, by designing the base sequence so that the units having the branched structure composed of the T-shaped nucleic acid structures according to the present embodiment are connected while being inverted, more structures can be easily formed. Can be created.

(Example 3)
As described above, specific nano-nucleic acid structures that can be produced by using the T-shaped nucleic acid structure according to the present embodiment as the structural unit have been described. It is not intended to limit. That is, using the T-shaped nucleic acid structure according to the present embodiment as a structural unit and using the same method as the above-described method for producing a nanonucleic acid structure, the following nanonucleic acid structure is further produced. Is also possible.

FIG. 12 is a schematic diagram schematically showing a two-dimensional polar coordinate structure capable of creating the nucleic acid structure according to this embodiment as a structural unit. FIG. 13 is a schematic view schematically showing a three-dimensional crystal structure that can be prepared using the nucleic acid structure according to the present embodiment as a structural unit. Furthermore, FIG. 14 is a schematic diagram schematically showing a three-dimensional curved surface structure that can be created using the nucleic acid structure according to the present embodiment as a structural unit.

As shown in FIGS. 12 to 14, according to the T-shaped nucleic acid structure according to the present embodiment, the dependence on the direction in which the branched structure that has been a problem in the crossover structure of the prior art is formed. Since there is no restriction in terms of structure and structure, and a phase on the main groove side of the DNA double helix structure, it is possible to form a DNA branched structure at an arbitrary phase. With a body as a structural unit, a wide variety of structures having higher spatial resolution can be produced.

In addition, the nucleic acid structure according to the present embodiment is electrostatically repelled compared to the conventional technique in which a nanonucleic acid structure is formed by arranging double spirals like a crossover structure in parallel. Since the DNA double helix structure having the property is orthogonalized to form a structure, it is possible to provide a space between adjacent double helixes. Therefore, this T-shaped nucleic acid structure is a structural unit. By forming a nanonucleic acid structure as described above, it becomes possible to greatly reduce the strain generated in the created structure itself, and a three-dimensional structure as shown in FIGS. 13 and 14 is also created as a stable structure. It becomes possible.

Furthermore, since the structure produced using the nucleic acid structure according to the present embodiment as a structural unit is formed by combining orthogonal double helices as described above, a T-shaped nucleic acid structure is further formed. By utilizing this, it becomes possible to produce nano-nucleic acid structures combined with existing motifs, and it is possible to design more structures in terms of diversity compared to existing motifs.

<Application to nanodevices with nanonucleic acid structures as basic structure>
As described above, according to the nucleic acid structure according to the present embodiment, this nucleic acid structure is used as a structural unit, and the spatial resolution is higher and higher than any known nano-nucleic acid structure. It is possible to realize high-density and high-definition nanonucleic acid structures. In addition to the listed two-dimensional structures and three-dimensional structures, it is possible to produce various complicated and diverse structures. It is possible to realize a structure in which the electrostatic repulsive force that is inherently reduced can be realized, to reduce strain generated in the structure, and to create a more stable structure.

Then, according to the nanoscale nucleic acid structure produced using the nucleic acid structure according to the present embodiment as a structural unit in this way, for example, by aligning and fixing functional particles in the structure, It becomes possible to fabricate functional nanodevices that are smaller and have higher density and higher definition. The specific application example is given below.

For example, by using a high-density nano-nucleic acid structure produced by using the T-shaped nucleic acid structure according to the present embodiment as its structural unit, the magnetic capacity with dramatically improved storage capacity per unit area A storage medium can be realized. Specifically, regarding the ladder-type structure or planar structure shown in FIGS. 8 and 10, the nano-nucleic acid structure is used as a template on the surface of a hard disk as a storage medium, and the T-shaped shape of these structures is used. By aligning and fixing magnetic nanoparticles in space portions formed at predetermined intervals by the branched structure of the nucleic acid structure, it is possible to arrange magnetic nanoparticles more finely on a nanoscale, and 100 terabytes / square inch. The ultra-high density magnetic storage medium can be realized.

Moreover, it can also be applied to lithography by self-assembling oligonucleotides in various shapes based on the T-shaped nucleic acid structure according to the present embodiment. As the downsizing of electronic equipment has further accelerated and the demand for higher density and higher definition of electronic components has increased, oligonucleotides can be self-assembled using the nucleic acid structure according to this embodiment. By forming a complicated and high-density shape, it is possible to create a high-density and high-definition circuit pattern. Specifically, for example, the base sequence of an oligonucleotide is designed based on a T-shaped nucleic acid structure so as to have a target structure, and a RecA protein is bound to a predetermined site, which is then used as a Si substrate. When the substrate is self-assembled and the substrate is treated with AgNO ₃ or the like, the RecA protein acts as a resist, and a complicated and high-density circuit pattern can be formed.

In addition, by using the T-shaped nucleic acid structure according to the present embodiment, high accuracy and mass production of nano-optical / electrical circuits such as nano-gap electrodes, nano-LCR circuits, and photonic crystals are realized. Is possible.

In addition, according to the T-shaped nucleic acid structure according to the present embodiment, complicated and diverse nano-nucleic acid structures can be stably produced, so the three-dimensional curved surface structure shown in FIG. A three-dimensional lattice structure can also be produced, and by using these three-dimensional structures, it can be used for crystal structure analysis of proteins. For example, a three-dimensional lattice structure is created using the T-shaped nucleic acid structure according to the present embodiment, and proteins to be analyzed are arranged and bound along the three-dimensional lattice structure. By performing X-ray analysis, it becomes possible to examine the crystal structure of a complex protein quickly, with high accuracy, at low cost.

In addition, since the structure is formed by using nucleic acid molecules such as DNA, which is a biomolecule, as a constituent element, it has high affinity with respect to the living body, and it becomes possible to easily bind enzymes, physiologically active substances, and the like. Therefore, it is used as a research material to analyze the function of the enzyme, etc. by binding the enzyme or bioactive substance to the prepared nanonucleic acid structure and substituting these reaction pathways with physical fields. It is possible to realize a complex chemical reaction system involving many enzymes, for example, a nano-reaction field that implements a reaction system such as photosynthesis or cellulose decomposition reaction, which can be realized by existing chemical engineering methods. The efficiency of difficult useful reaction analysis can be realized.

Also, it is possible to use a two-dimensional structure such as a ladder structure or a planar structure described with reference to FIGS. 8 and 10 as a filter. Specifically, according to the T-shaped nucleic acid structure according to the present embodiment, it is possible to form a branched structure at an arbitrary phase in the double helix structure. By creating a ladder-type structure or a lattice-like planar structure using the nucleic acid structure as a structural unit, the size of the space portion provided in the structure can be arbitrarily adjusted. A filter is prepared by loading a chemical substance on the part, and a desired substance that specifically binds to the chemical substance carried in the space is separated by passing a solution containing a predetermined compound through the filter. It can be used for such processing.

Similarly, since a nucleic acid structure is prepared using an oligonucleotide as a material, and various nanonucleic acid structures are prepared using the nucleic acid structure as a structural unit, there is an advantage that chemical modification is easy. Therefore, for example, the three-dimensional curved surface structure shown in FIG. 14 can be used for nanomaterials and nanodevices such as drug delivery systems.

It should be noted that application to these devices is one example, and application thereof is not limited only by the method specifically described. Further, as described above, the nucleic acid to be used is not limited to DNA, and it goes without saying that application to functional nanodevices is possible even when RNA, PNA, or the like is used as the nucleic acid.

As described above in detail, the nucleic acid structure according to the present embodiment includes the first nucleic acid molecule having a double helix structure having a single-stranded free end base sequence and the free end base thereof. A second nucleic acid molecule having a double helix structure having a base sequence complementary to the sequence in one nucleotide chain, the free end base sequence, and the free end base sequence of the second nucleic acid molecule; The nucleic acid structure is characterized in that a complementary base sequence is hydrogen-bonded, and this nucleic acid structure is an arbitrary on the main groove side of the double helix when the double helix structure is assumed to be virtually cylindrical. In this phase, a branching structure that branches in a direction perpendicular to the cylindrical shape is formed.

Therefore, according to the nucleic acid structure according to the present embodiment, it is possible to form a branched structure at an arbitrary phase as long as the phase is on the main groove side of the double helix. Compared to conventional technologies such as a crossover structure that has restrictions when forming a branched structure that can only be configured by different parts, it is possible to form a wide variety of structures with higher spatial resolution. It becomes.

In addition, according to the nucleic acid structure according to the present embodiment, electrostatic repulsion is achieved in contrast to the conventional technique in which a nanonucleic acid structure is formed by arranging double spirals such as a crossover structure in parallel. By forming the structure by making the double helix having the property to intersect perpendicularly, it becomes possible to make a space between the adjacent double helix, so that the strain generated in the manufactured structure itself is greatly reduced. Therefore, it is possible to stably produce a high-dimensional structure in which a strain such as a three-dimensional structure easily affects the stability.

Furthermore, since the structure produced using the nucleic acid structure according to the present embodiment as a structural unit is formed by combining orthogonal double helices as described above, a T-shaped nucleic acid structure is further formed. Utilizing it, it becomes possible to produce nano-nucleic acid structures combined with existing motifs, and it is possible to design more structures in terms of diversity compared to existing motifs.

As described above, the nucleic acid structure according to the present embodiment has a problem in a conventionally developed nucleic acid structure such as a crossover, and production of a nano-nucleic acid structure that can be produced using the structure. All the above restrictions and problems can be solved.

The embodiment to which the present invention is applied has been described above. However, the present invention is not limited to the above-described embodiment, and various modifications can be made without departing from the present invention. .

For example, in the embodiment described above, specific examples using DNA as a nucleic acid have been mainly described. However, the nucleic acid is not limited to DNA, and a nucleic acid such as RNA or PNA may be used. The nucleic acid structure and the nano nucleic acid structure according to the present embodiment may be formed by combining different nucleic acids such as RNA, DNA and PNA and hybridizing them.

Further, in the above-described embodiment, an example in which a nucleic acid structure or a nanonucleic acid structure is produced by synthesizing an oligonucleotide and self-assembling the oligonucleotide has been described. However, a base designed based on the above description is used. A polynucleotide having a sequence may be synthesized and self-assembled.

Next, a mode for carrying out the invention relating to a nucleic acid structure and a method for producing the nucleic acid structure will be described.

The method for producing a nucleic acid structure is a method of immersing a substrate having a surface charge in a solution in a method for producing a nucleic acid structure by annealing a solution containing a plurality of oligonucleotides.

Here, the substrate preferably has a negative surface charge.
The nucleic acid structure preferably has a planar structure. In addition, the nucleic acid structure preferably has a double helix simple substance structure. Further, the nucleic acid structure is preferably constituted by combining a plurality of repetitive structures.

In addition, the nucleic acid structure is configured by combining a plurality of repeating structures, the plurality of repeating structures have a planar structure and a double helix single structure, and one repeating structure among the plurality of repeating structures The nucleotide chain of the product preferably has a base sequence complementary to the nucleotide sequence of the nucleotide chain of another adjacent repeating structure.

The nucleic acid structure is configured by combining a plurality of repetitive structures. The plurality of repetitive structures have a planar structure and a double helix single structure, and one of the repetitive structures is a repetitive structure. The nucleotide chain has a base sequence complementary to the nucleotide sequence of the nucleotide chain of another adjacent repeating structure.

Here, the nucleotide chain existing on the outer periphery of one of the plurality of repeating structures has a base sequence complementary to the nucleotide sequence of the nucleotide chain existing on the outer periphery of another adjacent repeating structure. It is preferable to have.

The concentration of each oligonucleotide is preferably in the range of 0.001 μM to 10 μM units.

As the substrate, any flat plate having an appropriate surface charge density with respect to the motif may be used, and substrates such as mica, silicon, glass, gold, and graphite, and these surfaces are chemically modified with a self-assembled monolayer or the like. A board | substrate etc. can be employ | adopted. Further, it is also possible to use a substrate whose electrode surface is used as an electrode and in which a potential difference is generated entirely or locally. In the case of mica, mica is negatively charged and DNA is also negatively charged, and magnesium is used as a divalent ion that connects these. However, DNA may be directly adsorbed on a positively charged substrate.

Buffers that do not adversely affect nucleic acid hybridization such as TAE buffer, TBE buffer, phosphate buffer, SSC buffer, and TE buffer can be basically used.

As the cation, not only Mg ²⁺ but also divalent or higher ions and combinations thereof can be used. The cation may be a metal or an organic molecule. Further, complex ions such as hexaamminecobalt (III) ions can also be used.

A preferable range of the concentration of the cation having a valence of 2 or more will be described. First, substrate adsorption limited to mica will be described. The concentration of the cation having a valence of 2 or more is desirably in the range of 2 mM to 1 M. For example, in the case of Mg ²⁺ ions, it is known that adsorption occurs if it is about 1 M or less. The lower limit varies depending on the concentration of monovalent ions such as Na ⁺ in the solution, and the lower the monovalent ion concentration is known to cause adsorption at lower concentrations. I have confirmed.

This value increases or decreases depending on the metal species and ionic species, the concentration of monovalent cations in the solution, and the surface charge density determined by the type of substrate, and thus the possibility of production at concentrations outside this range is not denied ( References: David Pastre, Loic Hamon, Fabrice Landousy, Isabelle Sorel, Marie-Odile David, Alain Zozime, Eric Le Cam, Olivier Pietrement. Anionic polyelectrolyte adsorption on mica mediated high ionic strengths. Langmuir. (2006). 22 (15) 6651-60.).

Hereinafter, specific examples of the nucleic acid structure and a method for producing the nucleic acid structure will be described.

<Ladder type structure (1)>
FIG. 17 schematically shows a specific structure of a nucleic acid structure unit structure (a1) and a part of a ladder structure that is a nucleic acid structure (a2) that is produced by repeating the unit structure. FIG. As shown in FIG. 17, the ladder structure according to this embodiment is formed by connecting a T-shaped nucleic acid structure as a unit structure and connecting a plurality of the T-shaped nucleic acid structures as a repeating structure. To do. The numbers described in the structure shown in FIG. 17 indicate the number of base pairs (bp).

The two base sequences (SEQ ID NOs: 14 and 15) shown in (V) below are designed so that hybridization occurs in the intended order by determining the unit structure that becomes the repetitive structure and examining the phase. The nucleotide sequence of the oligonucleotide. The base sequence of this oligonucleotide shows the base sequence in the direction from the 5 'end to the 3' end.

After the start of annealing, first, the sequence 14 as the + sequence and the sequence 15 as the − sequence form hydrogen bonds at the complementary base sequence portion at a high temperature to form double-stranded DNA. At that time, both of these sequences have two base sequences that cannot form complementary hydrogen bonds (+ CGACAC inside the sequence, ACTGCA inside the -sequence), and in this base sequence part, On the other hand, the corresponding sequence forming the base pair does not form a hydrogen bond, skips the base sequence having no complementary binding relationship, and continues to form a hydrogen bond with the base portion having the next complementary binding relationship. . Further, both sequences have a complementary sequence of the base sequence that cannot form a complementary hydrogen bond in the + sequence of the-sequence if it is a + sequence, and in the case of a + sequence. Does not form hydrogen bonds at this stage. Therefore, double helical DNA bends at two parts of the base sequence (CGACAC in sequence, ACTGCA in sequence) that cannot form complementary hydrogen bonds within the sequence, forming a U-shaped structure. To come. When the temperature is further lowered, the base sequences that have not undergone hydrogen bonding function as sticky ends and hydrogen bond. At this time, since the X portion and the Y portion shown in FIG. 17 (a1) and the X ′ portion and the Y ′ portion are designed to have complementary sequences, as a result, after cooling, the ladder type shown in FIG. 17 (a2) is obtained. A structure is obtained.

Example 4
The formation operation and confirmation observation of this ladder-shaped nucleic acid structure by the one-pot annealing method were performed in the following procedure.

First, each oligonucleotide synthesized by determining a base sequence by a program based on the knowledge as described above is dissolved in TAE buffer (1 × TAE / Mg ²⁺ (12.5 mM)) so as to have a concentration of 1 μM. The oligonucleotide solution was purified. Then, each of the solutions was dispensed, and all the dispensed solutions were mixed in equal amounts in one reaction tube to purify the oligonucleotide mixed solution.

Next, the oligonucleotide mixed solution is annealed by using a thermal cycler (Eppendorf) and slowly cooling to about 4 ° C. starting from about 95 ° C. Hybridization was performed at the target nucleotide sequence. The annealing operation was continuously performed over about 2 days. The annealed sample was placed in a thermostatic bath and stored at about 3 ° C.

FIG. 18 (a3) is an atomic force microscope (AFM) observation image of a ladder structure specifically manufactured by the above operation. In the confirmation observation of the nanostructure formation by this AFM, the sample to be observed using a pipette is appropriately measured from the sample that has been continuously cooled and annealed for about 2 days and then stored in a thermostat. The sample was taken out each time and was observed in a liquid tapping mode using NanoScope® III manufactured by Beco.

As is clear from the AFM observation image of FIG. 18 (a3), a ladder structure as designed was obtained. However, the length was about several hundred nanometers to 1 micrometer.

(Example 5)
The formation operation and confirmation observation of this ladder-shaped nucleic acid structure by a one-pot annealing method were carried out by the following procedure using a method different from Example 4.

First, each oligonucleotide synthesized by determining a base sequence by a program based on the above-described knowledge is dissolved in TAE buffer (1 × TAE / Mg ²⁺ (12.5 mM)) so as to have a concentration of 0.1 μM. The oligonucleotide solution was purified. Then, each of the solutions was dispensed, and all the dispensed solutions were mixed in equal amounts in one reaction tube to purify the oligonucleotide mixed solution. When mixing the solution, mica pieces (manufactured by Niraco, natural mica (muscovite)) cleaved after being simultaneously cut into about 6 mm square were put into a tube. The solution volume was 200 μl so that the entire mica piece could touch the solution.

Next, the oligonucleotide mixed solution is annealed by using a thermal cycler (Eppendorf) and slowly cooling to about 4 ° C. starting from about 95 ° C. Hybridization was performed at the target nucleotide sequence. The annealing operation was continuously performed over about 2 days. The annealed sample was placed in a thermostatic bath and stored at about 3 ° C.

FIG. 18 (a4, a5) is an atomic force microscope (AFM) observation image of a ladder structure specifically manufactured by the above operation. In the confirmation observation of the nanostructure formation by this AFM, the sample to be observed using a pipette is appropriately measured from the sample that has been continuously cooled and annealed for about 2 days and then stored in a thermostat. The sample was taken out each time and was observed in a liquid tapping mode using NanoScope® III manufactured by Beco.

As is clear from the AFM observation images of FIGS. 18 (a4, a5), it is clear that the structure length is greatly increased by the growth of the substrate as compared with Example 4, and the structures are aligned on the substrate. I understand. The phenomenon that the structures are aligned is thought to be due to the fact that it is desirable to adsorb as much DNA as possible through counter ions in order to cancel the charge of the substrate. In order to place as many structures as possible in a limited plane, it is considered that the structures move while receiving restraints that move only on the plane, and finally are densely packed.

<Ladder type structure (2)>
FIG. 19 schematically shows the structure of a part of a ladder structure, which is a specific nucleic acid structure unit structure (b1) and a nucleic acid structure (b2) that is produced by repeating the unit structure as a repetitive structure. FIG. As shown in FIG. 19, the ladder-type structure according to this embodiment is formed by connecting a T-shaped nucleic acid structure as a unit structure and connecting a plurality of T-shaped nucleic acid structures as repeating structures. To do. Note that the numbers described in the structure shown in FIG. 19 indicate the number of base pairs (bp).

The two base sequences shown in (VI) below (SEQ ID NOs: 16 and 17) are designed so that hybridization occurs in the intended order by determining the unit structure to be a repetitive structure and examining the phase. The nucleotide sequence of the oligonucleotide. The base sequence of this oligonucleotide shows the base sequence in the direction from the 5 'end to the 3' end.

After the start of annealing, firstly, the sequence 16 having the + sequence and the sequence 17 having the − sequence form a hydrogen bond at the complementary base sequence portion at a high temperature to form double-stranded DNA. At that time, both of these sequences have two base sequences that cannot form complementary hydrogen bonds (+ CGACAC inside the sequence, ACTGCA inside the -sequence), and in this base sequence part, On the other hand, the corresponding sequence forming the base pair does not form a hydrogen bond, skips the base sequence having no complementary binding relationship, and continues to form a hydrogen bond with the base portion having the next complementary binding relationship. . Further, both sequences have a complementary sequence of the base sequence that cannot form a complementary hydrogen bond in the + sequence of the-sequence if it is a + sequence, and in the case of a + sequence. Does not form hydrogen bonds at this stage. Therefore, double-stranded DNA bends at two parts of the base sequence (+ CGACAC in the sequence, ACTGCA in the sequence) that cannot form complementary hydrogen bonds inside the sequence, and a double-shaped structure Will come to form. When the temperature is further lowered, the base sequences that have not undergone hydrogen bonding function as sticky ends and hydrogen bond. At this time, the X portion and the Y portion shown in FIG. 19 (b1) and the X ′ portion and the Y ′ portion are designed to have complementary sequences. As a result, after cooling, the ladder type shown in FIG. 19 (b2) is obtained. A structure is obtained.

(Example 6)
The formation operation and confirmation observation of this ladder-shaped nucleic acid structure by the one-pot annealing method were performed in the following procedure.

The operation of forming the nucleic acid structure by the one-pot annealing method is the same as in Example 4.
FIG. 20 (b3) is an atomic force microscope (AFM) observation image of the nucleic acid structure specifically produced by the above operation. The method for the atomic force microscope observation of the nucleic acid structure is the same as in Example 4.

As is apparent from the AFM observation image of FIG. 20 (b3), a ladder structure as designed was obtained. However, the length was about several hundred nanometers to 1 micrometer.

(Example 7)
The formation operation and confirmation observation of this ladder-shaped nucleic acid structure by a one-pot annealing method were carried out by the following procedure using a method different from that in Example 6.

The operation of forming the nucleic acid structure by the one-pot annealing method is the same as in Example 5.
FIG. 20 (b4, b5) is an atomic force microscope (AFM) observation image of the nucleic acid structure specifically produced by the above operation. The method for the atomic force microscope observation of the nucleic acid structure is the same as in Example 5.

As is clear from the AFM observation images of FIGS. 20 (b4, b5), compared with Example 6, the structure length is significantly increased by the growth of the substrate, and it is clear that the structures are aligned on the substrate. I understand. The phenomenon that the structures are aligned is thought to be due to the fact that it is desirable to adsorb as much DNA as possible through counter ions in order to cancel the charge of the substrate. In order to place as many structures as possible in a limited plane, it is considered that the structures move while receiving restraints that move only on the plane, and finally are densely packed.

<Plane structure (1)>
FIG. 21 is a diagram schematically showing a part of the structure of a nucleic acid structure which is a specific nucleic acid structure unit structure (c1) and a nucleic acid structure (c2) produced by repeating the unit structure as a repetitive structure. It is. As shown in FIG. 21, the nucleic acid structure according to this embodiment is formed by connecting a plurality of T-shaped nucleic acid structures as unit structures and connecting a plurality of T-shaped nucleic acid structures as repeating structures. . The numbers described in the structure shown in FIG. 21 indicate the number of base pairs (bp).

The two types of base sequences shown in (VII) below (SEQ ID NO: 18, 19), the two types of base sequences shown in (VIII) below (SEQ ID NO: 20, 21), and the following (IX) The two types of base sequences (SEQ ID NOs: 22 and 23) are the base sequences possessed by the oligonucleotides designed so that hybridization occurs in the intended order by determining the unit structure to be a repetitive structure and examining the phase. It is. The base sequence of this oligonucleotide shows the base sequence in the direction from the 5 'end to the 3' end. The following sequences (VII), (VIII) and (IX) are all designed based on the same guidelines, and the same structure can be produced. This indicates that the T-shaped structure is not reproduced only by a specific (one kind of) array, and can be widely produced if the array is based on the design guidelines.

After the start of annealing, first, the sequence 18 having the + sequence and the sequence 19 having the − sequence form a hydrogen bond at the complementary base sequence portion at a high temperature to form a double helix DNA. At that time, both of these sequences have two base sequences that cannot form complementary hydrogen bonds (+ GGGCT inside the sequence, -CGTCG inside the -sequence). On the other hand, the corresponding sequence forming the base pair does not form a hydrogen bond, skips the base sequence having no complementary binding relationship, and continues to form a hydrogen bond with the base portion having the next complementary binding relationship. . In addition, both sequences have a complementary sequence of a base sequence that cannot form a complementary hydrogen bond inside the + sequence if it is a + sequence, or if it is a − sequence, on the 5 ′ end side. Does not form hydrogen bonds at this stage. Therefore, double-stranded DNA is folded at two parts of the base sequence (+ GGGCT in the sequence, -CGTCG in the sequence) that cannot form a complementary hydrogen bond inside the sequence, and a double-shaped structure Will come to form. When the temperature is further lowered, the base sequences that have not undergone hydrogen bonding function as sticky ends and hydrogen bond. At this time, the X portion and the Y portion shown in FIG. 21 (c1) and the X ′ portion and the Y ′ portion are designed to have complementary sequences. As a result, after cooling, the planar type shown in FIG. 21 (c2) is obtained. A structure is obtained.

In the case of the arrays 20 and 21, the above-described array 18 is replaced with 20 and the array 19 is replaced with 21. Similarly, in the case of the arrays 22 and 23, the array 18 is replaced with 22 and the array 19 is replaced with 23. . At this time, the base sequences that cannot form complementary hydrogen bonds within the sequence are the GGGCT in the + sequence and the CGTCG in the − sequence in the case of 20, 21 respectively, and the + sequence in the case of 22, 23 Within the CCACA and-within the sequence ATCCG.

(Example 8)
The formation operation and confirmation observation of this nucleic acid structure by the one-pot annealing method were performed according to the following procedure.

The operation of forming the nucleic acid structure by the one-pot annealing method is the same as in Example 4.
FIG. 22 (c3) is an atomic force microscope (AFM) observation image of the nucleic acid structure specifically produced by the above operation. The method for the atomic force microscope observation of the nucleic acid structure is the same as in Example 4.

As is clear from the AFM observation image of FIG. 22 (c3), a planar structure as designed was obtained, but the growth direction has peculiarities. This is considered to be mainly due to the tube-like structure in the growth in free solution.

Example 9
The formation procedure and confirmation observation of this nucleic acid structure by a one-pot annealing method were carried out by the following procedure using a method different from that in Example 8.

The operation of forming the nucleic acid structure by the one-pot annealing method is the same as in Example 5.
FIG. 22 (c4, c5) is an atomic force microscope (AFM) observation image of the nucleic acid structure specifically produced by the above operation. The method for the atomic force microscope observation of the nucleic acid structure is the same as in Example 5.

As is apparent from the AFM observation image of FIG. 22 (c4, c5), the tube-like growth was shown in Example 8, but in Example 9, it grew in a planar shape due to the growth effect on the substrate. Yes. A planar single crystal structure on the order of μm covers the entire surface of the substrate. Since this single crystal region can be expanded by devising the growth process, large-scale crystal growth can be realized.

<Plane structure (2)>
FIG. 23 is a diagram schematically showing a part of the structure of a nucleic acid structure which is a specific nucleic acid structure unit structure (d1) and a nucleic acid structure (d2) which is produced by repeating the unit structure as a repetitive structure. It is. As shown in FIG. 23, the nucleic acid structure according to this embodiment is formed by using a T-shaped nucleic acid structure as a unit structure and connecting a plurality of the T-shaped nucleic acid structures as repeating structures. . Note that the numbers described in the structure shown in FIG. 23 indicate the number of base pairs (bp).

The two base sequences (SEQ ID NOs: 24, 25) shown in (X) below are designed so that hybridization occurs in the intended order by determining the unit structure that becomes the above-mentioned repeating structure and examining the phase. The nucleotide sequence of the oligonucleotide. The base sequence of this oligonucleotide shows the base sequence in the direction from the 5 'end to the 3' end.

After the start of annealing, first, the sequence 24 having the + sequence and the sequence 25 having the − sequence form a hydrogen bond at a complementary base sequence portion at a high temperature to form a double helix DNA. At that time, both of these sequences have two base sequences that cannot form complementary hydrogen bonds (+ CTCTT inside the sequence, -GATGA inside the -sequence). A corresponding sequence that forms a base pair does not form a hydrogen bond, but skips a base sequence that does not have a complementary binding relationship, and continues to form a hydrogen bond with a base portion that has the next complementary binding relationship. . Further, both sequences have a complementary sequence of the base sequence that cannot form a complementary hydrogen bond in the + sequence of the-sequence if it is a + sequence, and in the case of a + sequence. Does not form hydrogen bonds at this stage. Therefore, double-stranded DNA bends at two parts of the base sequence (+ CTCTT in the sequence, GATGA in the sequence) that cannot form complementary hydrogen bonds within the sequence, and a double-shaped structure Will come to form. When the temperature is further lowered, the base sequences that have not undergone hydrogen bonding function as sticky ends and hydrogen bond. At this time, the X portion and the Y portion shown in FIG. 23 (d1) and the X ′ portion and the Y ′ portion are designed to have complementary sequences. As a result, after cooling, the planar type shown in FIG. 23 (d2) is obtained. A structure is obtained.

(Example 10)
The formation operation and confirmation observation of this nucleic acid structure by the one-pot annealing method were performed according to the following procedure.

The operation of forming the nucleic acid structure by the one-pot annealing method is the same as in Example 4.
FIG. 24 (d3) is an atomic force microscope (AFM) observation image of the nucleic acid structure specifically produced by the above operation. The method for the atomic force microscope observation of the nucleic acid structure is the same as in Example 4.

As is clear from the AFM observation image of FIG. 24 (d3), a planar structure as designed was obtained, but the growth size is limited to about several hundreds × several hundred nanometers.

(Example 11)
The formation procedure and confirmation observation of this nucleic acid structure by a one-pot annealing method were carried out by the following procedure using a method different from Example 10.

The operation of forming the nucleic acid structure by the one-pot annealing method is the same as in Example 5.
FIG. 24 (d4, d5) is an atomic force microscope (AFM) observation image of the nucleic acid structure specifically produced by the above operation. The method for the atomic force microscope observation of the nucleic acid structure is the same as in Example 5.

As is apparent from the AFM observation image of FIG. 24 (d4, d5), almost no growth was observed in Example 10, but in Example 11, a planar structure can be produced by growing on the substrate. it can.

<Two-dimensional polar coordinate structure>
FIG. 25 is a diagram schematically showing a part of the structure of a nucleic acid structure which is a specific nucleic acid structure unit structure (e1) and a nucleic acid structure (e2) produced as a repeating structure of the unit structure. It is. As shown in FIG. 25, the nucleic acid structure according to this embodiment is formed by forming a T-shaped nucleic acid structure as a unit structure and connecting a plurality of the T-shaped nucleic acid structures as repeating structures. . The numbers shown in the structure shown in FIG. 25 indicate the number of base pairs (bp).

The two types of base sequences (SEQ ID NOs: 26 and 27) shown in (XI) below are designed so that hybridization occurs in the intended order by determining the unit structure to be a repetitive structure and examining the phase. The nucleotide sequence of the oligonucleotide. The base sequence of this oligonucleotide indicates the base sequence in the direction from the 5 'end to the 3' end.

After the start of annealing, first, the sequence 26 having the + sequence and the sequence 27 having the − sequence form a hydrogen bond at a complementary base sequence portion at a high temperature to form a double helix DNA. At that time, both of these sequences have two base sequences that cannot form complementary hydrogen bonds (+ AAGCG inside the sequence, -GTGGA inside the -sequence), and in this base sequence part, On the other hand, the corresponding sequence forming the base pair does not form a hydrogen bond, skips the base sequence having no complementary binding relationship, and continues to form a hydrogen bond with the base portion having the next complementary binding relationship. . Further, both sequences have a complementary sequence of the base sequence that cannot form a complementary hydrogen bond in the + sequence of the-sequence if it is a + sequence, and in the case of a + sequence. Does not form hydrogen bonds at this stage. Therefore, double-stranded DNA is folded at two parts of the base sequence (+ AAGCG in the sequence, GTGGA in the sequence) that cannot form a complementary hydrogen bond inside the sequence, and a double-shaped structure Will come to form. When the temperature is further lowered, the base sequences that have not undergone hydrogen bonding function as sticky ends and hydrogen bond. At this time, the X portion and the Y portion shown in FIG. 25 (e1) and the X ′ portion and the Y ′ portion are designed to have complementary sequences. As a result, after cooling, the two-dimensional portion shown in FIG. 25 (e2) is obtained. A polar coordinate structure is obtained.

Example 12
The formation operation and confirmation observation of this nucleic acid structure by the one-pot annealing method were performed according to the following procedure.

The operation of forming the nucleic acid structure by the one-pot annealing method is the same as in Example 4.
FIG. 26 (e3) is an atomic force microscope (AFM) observation image of the nucleic acid structure specifically produced by the above operation. The method for the atomic force microscope observation of the nucleic acid structure is the same as in Example 4.

As is apparent from the AFM observation image of FIG. 26 (e3), a two-dimensional polar coordinate structure as designed was obtained. Further, at this time, it is adsorbed randomly on the mica substrate.

(Example 13)
The formation procedure and confirmation observation of this nucleic acid structure by a one-pot annealing method were carried out by the following procedure using a method different from that in Example 12.

The operation of forming the nucleic acid structure by the one-pot annealing method is the same as in Example 5.
FIG. 26 (e4, e5) is an atomic force microscope (AFM) observation image of the nucleic acid structure specifically produced by the above operation. The method for the atomic force microscope observation of the nucleic acid structure is the same as in Example 5.

In Example 13, the yield increases sharply compared to Example 12. What was around 20% in Example 12 is greatly improved to almost 100% in the growth on the substrate in Example 13. In addition, as is apparent from the AFM observation image of FIG. 26 (e4, e5), the nucleic acid structures are aligned. In this case, since it is a circular structure, it can be confirmed that it is arranged in a hexagonal close-packed structure on a plane. The phenomenon that the structures are aligned is thought to be due to the fact that it is desirable to adsorb as much DNA as possible through counter ions in order to cancel the charge of the substrate. In order to place as many structures as possible in a limited plane, it is considered that the structures move while receiving restraints that move only on the plane, and finally close packed in hexagonal form.

<Plane structure (3)>
(Comparative Example 1)
The basic structure of the repetitive structure will be described with reference to FIGS. The basic structure is composed of eight single-stranded DNAs a to h in FIG. After the six DNA molecules c to h in FIG. 28 are combined to form a hexamer, the two DNA molecules a and b connect the hexamers to each other to form a planar structure as shown in FIG. Form.

Only two of a and b in FIG. 28 are circular, that is, a closed single-stranded DNA structure. The reason why two rings a and b in FIG. 28 are annular is as follows. DNA molecules are basically bound in order from a portion of a long continuous complementary base sequence. However, if the sequences of a and b are not circular and a portion corresponding to one side of a triangular structure is open, the DNA molecules are continuous. Since the binding force due to the complementary base sequence is weak in the part of the side that is not, the side becomes difficult to bind. When the arrangement of a and b is not circular and the structure corresponding to one vertex of the triangular structure is open, the motifs composed of hexamers are fixed in a form in which the arrangement of a and b is fixed. It becomes impossible to connect and the possibility of becoming a structure different from the purpose increases.

One side of the triangular structure is a very small structure with 10 bases corresponding to about one rotation of the DNA double helix. In addition, as shown in FIG. 27, since there are a plurality of nucleobases that do not bind at the hinge portion at each vertex of the triangular structure, the motif is highly flexible and structural distortion does not accumulate. This motif has two thymines (T).

The conditions for producing the nucleic acid structure will be described. All eight DNAs as materials were put in a tube, and a nucleic acid structure was prepared by slowly cooling from high temperature to low temperature by a temperature drop method called annealing. This is a general method for preparing a DNA structure, called a one-pot annealing method. Thereafter, all the structures shown here were formed by this procedure.

As shown in FIG. 30, the structure was designed so that the structure formation temperature of the motif alone was around 50 ° C., and the growth temperature of the planar crystal structure was around 20 ° C. Therefore, annealing was performed by first raising the temperature to 90 ° C. and then gradually cooling to 4 ° C. over 2 days. The amount of the sample solution was 100 μl, and the concentration of each DNA molecule was equimolar conditions of 0.04 μM.

Explain the experimental results. In the assembly in the free solution, the planar structure could not be confirmed. Instead, an infinite number of clustered small structures as shown in FIG. 31 were observed by AFM (atomic force microscope).

(Example 14)
As the basic structure of the repetitive structure, the same structure as that prepared in Comparative Example 1 was used.

The conditions for producing the nucleic acid structure will be described. Although it is almost the same as the conditions prepared in Comparative Example 1, this time, since a DNA nucleic acid structure is directly formed on the substrate, a mica substrate having a negative surface charge is placed in the same tube, unlike the conventional annealing of only the sample solution. Was immersed in the sample solution for annealing.

As the substrate, mica (mica) was used, and the size was about 5 mm × 10 mm, and the size fits in the used tube. Since the amount of the sample solution also needs to be in a state in which the substrate is sufficiently immersed in the solution, the amount of the sample solution was twice the amount of the sample (100 μl) in the free solution of Comparative Example 1. Specifically, the sample amount was 200 μl, and the concentration of each DNA molecule was equimolar conditions of 0.04 μM. The concentration is the same as that in the free solution of Comparative Example 1.

Explain the experimental results. In the assembly on the substrate, as shown in FIG. 32, a large planar structure with holes in some places was confirmed by AFM. The whole is covered with a DNA structure of uniform thickness.

<Plane structure (4)>
(Comparative Example 2)
The basic structure of the repetitive structure will be described with reference to FIGS. The basic structure is composed of six single-stranded DNAs a to f in FIG. After the DNA molecules a to f in FIG. 34 are bonded to form a hexamer, the hexamers are directly bonded to each other at the sticky end portion on the side located outside the structure, thereby forming a planar structure as shown in FIG. Form.

One side of the triangular structure is a small structure with 21 bases corresponding to about two rotations of the DNA double helix. As shown in FIG. 33, since there are a plurality of nucleobases that do not bind at the hinge portion at each vertex of the triangular structure, the motif has high flexibility and structural distortion does not accumulate. This motif has two thymines (T).

The conditions for producing the nucleic acid structure will be described. As shown in FIG. 36, the structure was designed so that the structure formation temperature of the motif alone was around 70 ° C., and the growth temperature of the planar crystal structure was around 40 ° C. Annealing was performed by raising the temperature to 90 ° C. and then gradually cooling to 20 ° C. over 2 days. The amount of the sample solution was 100 μl, and the concentration of each DNA molecule was 0.01 μM.

Explain the experimental results. In the assembly in the free solution, the planar structure could not be confirmed. Instead, an infinite number of clustered small structures as shown in FIG. 37 were observed by AFM (atomic force microscope).

(Example 15)
As the basic structure of the repetitive structure, the same structure as that prepared in Comparative Example 2 was used.

The conditions for producing the nucleic acid structure will be described. Although it is almost the same as the conditions prepared in Comparative Example 2, this time, since a DNA nanostructure is directly formed on a substrate, a mica substrate having a negative surface charge is placed in the same tube unlike conventional annealing of only a sample solution. Was immersed in the sample solution for annealing.

As the substrate, mica (mica) was used, and the size was about 5 mm × 10 mm, and the size fits in the used tube. Since the amount of the sample solution also needs to be in a state in which the substrate is sufficiently immersed in the solution, the amount was twice as much as the sample amount (100 μl) in the free solution. Specifically, the sample amount was 200 μl, and the concentration of each DNA molecule was 0.01 μM. The concentration is the same as that in Comparative Example 2 when prepared in a free solution.

Explain the experimental results. In the assembly on the substrate, as shown in FIG. 38, a large and uniform planar structure was confirmed by AFM. Further, as shown in FIG. 39, a hexagonal lattice structure, which is a structural unit of the structure, was confirmed by AFM.

<Plane structure (5)>
The basic structure of the cross motif will be described. As shown in FIG. 40, the basic structure consists of four single-stranded DNAs, and the arrow indicates the direction from the 5 ′ end to the 3 ′ end of the DNA base sequence. Each single-stranded DNA binds to the other three single-stranded DNAs at three points. Corresponding arrangement parts (α and α ′, etc.) of the Greek letters shown in FIG. 40 are complementary and joined together.

In the formation of the structure, the base sequence is designed so that a cross-shaped cross motif is first formed at a high temperature, and then the motifs are combined with each other after taking a low temperature to form a wide planar structure. A specific arrangement is as shown in FIG. Among the binding portions of each single-stranded DNA, the bonds forming a cross shape, that is, the bonds of α, β, δ, and ζ are bonded at around 60 ° C. The remaining two bonds, γ and ε, are bonded to each other around 22 ° C. Therefore, when forming the structure, a large number of cruciform structures as shown in FIG. 40 are first formed at around 60 ° C., and the cruciform structures are coupled to each other around 22 ° C. to form a planar structure as shown in FIG. To do.

As shown in FIG. 40, each single-stranded DNA has two bases of thymine (T) at a position corresponding to a corner when taking a cross shape. Since this thymine dinucleotide does not have a corresponding complementary sequence, it remains unbound to other sequences. Therefore, this TT portion is soft and can be arranged relatively freely. Therefore, this TT2 base plays a role of eliminating structural distortion that occurs when a planar structure is formed, and enables a wide planar structure.

(Comparative Example 3)
Manufacturing conditions will be described. 4 single-stranded DNAs as materials are mixed in one tube, dissolved in 1 × TAE buffer (Mg ²⁺ (12.5 mM)) solvent so that the final product concentration is 0.1 μM, Purify the solution. The solution volume was 50 μl. This tube was immersed in hot water at 90 ° C. and allowed to cool naturally to room temperature (24 ° C.) over 20 hours for annealing.

The experimental results will be described. After the annealing, 1 μl of the solution in the tube was placed on the clean surface of mica, 40 μl of 1 × TAE buffer (Mg ²⁺ (12.5 mM)) solvent was added from above, and observation by AFM was performed. FIG. 43 shows that, but a planar structure was not formed, and DNA fragments and objects were confirmed. What appears white are random agglomerates of DNA, resulting in measurement noise, and the lower half of the image is disturbed.

(Example 16)
Manufacturing conditions will be described. 4 single-stranded DNAs as materials are mixed in one tube, dissolved in 1 × TAE buffer (Mg ²⁺ (12.5 mM)) solvent so that the final product concentration is 0.1 μM, Purify the solution. The volume of the solution was 200 μl. Place a piece of mica with a clean surface exposed in the tube and immerse it in the solution. This tube was immersed in hot water at 90 ° C. and allowed to cool naturally to room temperature (24 ° C.) over 20 hours for annealing.

Explain the experimental results. After the completion of annealing, the mica pieces taken out from the tube were observed by AFM. In FIG. 44, a wide planar structure can be seen. In the lower right, you can see the part peeled off by the AFM cantilever. Since the difference in height between the peeled portion and the other portion is almost equal to the DNA layer, it can be seen that a wide planar structure is visible.

FIG. 45 is an enlarged view of the broken part with the planar structure peeled off. In some places, a square lattice structure can be seen. This appears to be a portion where the structure remains when the planar structure is broken. From this result, it can be said that DNA has a lattice-like planar structure.

From the above, the following has been clarified by the present invention.
Conventionally, many DNA nanostructures have been proposed, but they all self-assemble in a free solution and cannot be crystallized on a large scale on an interface. The reason is presumed as follows.

(1) A conventional motif (a unit of self-assembly) includes a plurality of joint constraints (a structure called a crossover) in the motif so that the rigidity of the motif itself is as large as possible. This facilitates the formation of small crystals (1 micron or less) in free solution. On the other hand, since the minute geometric distortion of the motif cannot be resolved, it is difficult to make it larger.

(2) The conventional motif includes a structure in which a plurality of double helices are bundled in parallel. DNA has a negatively charged backbone, and looks like it is forcibly placed next to each other. For this reason, the negative charge density on the motif surface is high, the interaction with the charge on the substrate becomes too strong, and free movement on the interface of the motif necessary for crystallization is hindered.

(3) If the motif is larger than a certain level, the reverse of the motif is not allowed due to the adsorption effect, so that the situation is the same as if there are a large amount of impurities.

The present invention solves the problems of conventional DNA motifs as follows.

(1) A certain softness is ensured by losing the accuracy of the geometrical shape of the motif by making the motif structure a structure that does not bind the double helix (double helix simple structure).

(2) Since the structure in which the motif is assembled has a mesh-like void, the DNA backbones do not come close to each other, and the strain due to electrostatic repulsion can be alleviated. In addition, since the surface charge density of the motif is small, it does not hinder the planar motion in the interface.

(3) Since the motif is small (the number of constituents is small and the length is short), it can be inverted in the electric double layer on the interface, and all molecules can be crystallized.

The principle that the effect of the present invention appears is considered as follows. When a buffer solution is deposited on a negatively charged substrate such as mica, an electric double layer is formed at the solid-liquid interface by cations contained in the buffer. On the other hand, DNA molecules have a negative charge on the phosphate group of the backbone. Due to the interaction between the electric double layer and the DNA molecule, the DNA molecule is attracted to the vicinity of the substrate. When a divalent cation enters between DNA and mica, DNA and mica are slowly bonded. In this state, the DNA molecules self-assemble (self-assembly), so that a two-dimensional DNA nanostructure can be grown in a self-organized manner. Unlike self-assembly in free solution where the molecules move in three dimensions, here the movement of the DNA molecules is considered to be constrained in a nearly two-dimensional plane.

Conventional DNA nanostructures are all assembled in a free solution, and after annealing the solution, the solution is dropped onto the substrate together to fix the nanostructure. Therefore, the quality of the nanostructures (crystal defects) cannot be arranged because the fabricated nanostructures cannot be arranged in the determined position and direction, and the crystallization conditions change midway (concentration changes and temperature changes in the test tube). Rate), it is difficult to form a large structure, three-dimensional crystallization cannot be performed, and the yield is poor, that is, a large amount of DNA that does not crystallize in the solution remains and is wasteful. there were.

The present invention solves these problems and can produce a large area, low defect rate DNA nano two-dimensional crystal directly on a substrate. This is a breakthrough for applying nanotechnology.
(1) Since it can be directly produced on a substrate, it is easy to combine with top-down nanotechnology (lithography). DNA nanostructures can be self-organized according to the pattern created by lithography. Application fields include ultra-high density storage media, nanoelectronic circuit devices, biosensors, and the like.

(2) Almost all DNA in the solution can be crystallized, and there is no waste.
(3) Hierarchical assembly is possible by solution replacement on the substrate, and self-assembly of a much more complicated one, for example, a three-dimensional crystal, is possible compared to the conventional assembly in a single solution. Application fields include photonic crystals and protein crystal analysis.

(4) DNA microcapsules can be produced by assembling on soluble spherical beads. After creating the DNA nanostructure, it is possible to make a functional container by melting the gel of the bead material. This technique can be developed into a technique for creating a well-controlled DNA nanostructure on a charged free-form surface, and may have a completely new application. Applications include intelligent drug delivery systems.

In the embodiment described above, specific examples using DNA as a nucleic acid have been mainly described. However, the nucleic acid is not limited to DNA, and a nucleic acid such as RNA or PNA may be used. The nucleic acid structure and the nano nucleic acid structure according to the present embodiment may be formed by combining different nucleic acids such as RNA, DNA and PNA and hybridizing them.

Further, the present invention is not limited to the above-described embodiment, and it is needless to say that various other configurations can be adopted without departing from the gist of the present invention.

Claims (30)

1. A single-stranded free-end base sequence of the first nucleic acid molecule having a double helix structure;
   One nucleotide chain of the second nucleic acid molecule having a double helix structure has a base sequence that is non-complementary to the other nucleotide chain at a predetermined position of the non-terminal portion, and is complementary to the above-mentioned free-end base sequence The base sequence is
   A nucleic acid structure formed by hydrogen bonding.
2. The free-end base sequence is composed of the number of bases obtained by subtracting 1 from the number of bases constituting a nucleotide chain having a length corresponding to the length between complementary base pairs in the second nucleic acid molecule. The nucleic acid structure according to claim 1.
3. The nucleic acid structure according to claim 1 or 2, wherein the nucleic acid structure has a substantially branched T-shaped structure.
4. A single-stranded free-end base sequence of the first nucleic acid molecule having a double helix structure;
   One nucleotide chain of the second nucleic acid molecule having a double helix structure has a base sequence that is non-complementary to the other nucleotide chain at a predetermined position of the non-terminal portion, and is complementary to the above-mentioned free-end base sequence A nucleic acid structure comprising a nucleic acid structure formed by hydrogen bonding with a base sequence.
5. The free-end base sequence is composed of the number of bases obtained by subtracting 1 from the number of bases constituting a nucleotide chain having a length corresponding to the length between complementary base pairs in the second nucleic acid molecule. The nucleic acid structure according to claim 4.
6. The nucleic acid structure according to claim 4 or 5, wherein the nucleic acid structure has a substantially T-shaped branched structure.
7. The nucleic acid structure according to claim 6, wherein the nucleic acid structure has a ladder structure.
8. The nucleic acid structure according to claim 6, wherein the nucleic acid structure has a planar structure.
9. The nucleic acid structure according to claim 8, wherein the nucleic acid structure has a lattice structure.
10. The nucleic acid structure according to claim 6, wherein the nucleic acid structure has a two-dimensional polar coordinate structure.
11. The nucleic acid structure according to claim 6, wherein the nucleic acid structure has a three-dimensional crystal structure.
12. The nucleic acid structure according to claim 6, wherein the nucleic acid structure has a three-dimensional curved surface structure.
13. A first nucleic acid molecule having a double helix structure having a single-stranded free-end base sequence, and a base sequence that is non-complementary to the other nucleotide chain of a pair of nucleotide chains, and complementary to the free-end base sequence A second nucleic acid molecule having a double helix structure having a specific base sequence at a predetermined position of the non-terminal part of one nucleotide chain,
    A method for producing a nucleic acid structure, wherein a hydrogen bond is formed between the free end base sequence and a base sequence complementary to the free end base sequence of the second nucleic acid molecule.
14. The method for producing a nucleic acid structure according to claim 13, wherein the nucleic acid structure forms a branched structure at a hydrogen bonding site between the first nucleic acid molecule and the second nucleic acid molecule.
15. The method for producing a nucleic acid structure according to claim 14, wherein the branched structure is branched at an arbitrary phase on the main groove side of the double helical structure of the second nucleic acid molecule.
16. A nucleic acid molecule having a double helix structure is formed by a pair of nucleotide chains having a base sequence that is non-complementary to the other nucleotide chain at a predetermined position of the non-terminal part of one nucleotide chain,
    A method for producing a nucleic acid structure, wherein a nucleotide chain having a free-end base sequence complementary to the base sequence and the nucleic acid molecule are hydrogen-bonded between the complementary base sequences.
17. The method for producing a nucleic acid structure according to claim 16, wherein the nucleic acid structure forms a branched structure at a hydrogen bonding site between the nucleic acid molecule and the nucleotide chain.
18. The free-end base sequence is composed of the number of bases obtained by subtracting 1 from the number of bases constituting a nucleotide chain having a length corresponding to the length between complementary base pairs in the nucleic acid molecule. Or a method for producing the nucleic acid structure according to 16;
19. The method for producing a nucleic acid structure according to claim 14 or 17, wherein the branched structure is substantially T-shaped.
20. The method for producing a nucleic acid structure according to claim 17, wherein the branched structure is branched at an arbitrary phase on the main groove side of the double helix structure of the nucleic acid molecule.
21. In a method for producing a nucleic acid structure, a solution containing a plurality of oligonucleotides is annealed to produce a nucleic acid structure.
    A method for producing a nucleic acid structure, which comprises immersing a substrate having a surface charge in a solution.
22. The method for producing a nucleic acid structure according to claim 21, wherein the substrate has a negative surface charge.
23. The method for producing a nucleic acid structure according to claim 22, wherein the substrate is mica.
24. The method for producing a nucleic acid structure according to claim 21, 22 or 23, wherein the nucleic acid structure has a planar structure.
25. The method for producing a nucleic acid structure according to claim 21, 22 or 23, wherein the nucleic acid structure has a double helix simplex structure.
26. The method for producing a nucleic acid structure according to claim 21, 22 or 23, wherein the nucleic acid structure is constituted by combining a plurality of repetitive structures.
27. The nucleic acid structure is
    A single-stranded free-end base sequence of the first nucleic acid molecule having a double helix structure;
    One nucleotide chain of the second nucleic acid molecule having a double helix structure has a base sequence that is non-complementary to the other nucleotide chain at a predetermined position of the non-terminal portion, and is complementary to the above-mentioned free-end base sequence 24. The method for producing a nucleic acid structure according to claim 21, 22 or 23, wherein the nucleic acid structure is formed by hydrogen bonding with the base sequence.
28. A nucleic acid structure is formed by combining a plurality of repetitive structures,
    The plurality of repeating structures have a planar structure and a double helix single structure,
    The nucleotide chain of one repeating structure among the plurality of repeating structures has a base sequence complementary to the nucleotide sequence of the nucleotide chain of another adjacent repeating structure. A method for producing a nucleic acid structure according to claim 22 or 23.
29. Combining multiple repeating structures,
    The plurality of repeating structures have a planar structure and a double helix single structure,
    A nucleotide chain of one repeating structure among the plurality of repeating structures has a nucleotide sequence complementary to the nucleotide sequence of the nucleotide chain of another adjacent repeating structure. .
30. The nucleotide chain existing on the outer periphery of one of the plurality of repeating structures has a base sequence complementary to the nucleotide sequence of the nucleotide chain existing on the outer periphery of another adjacent repeating structure. 30. Nucleic acid structure according to claim 29, characterized in that

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