WO2010010862A1 - Method for construction of artificial rnp nano-structure by utilizing rna-protein complex interaction motif - Google Patents

Abstract

Disclosed is a method for constructing an artificial RNP nano-structure by utilizing an RNA-protein complex interaction motif.  Also disclosed is a method for inducing the change in structure of an RNA molecule with a protein.  Specifically disclosed is a method for inducing the specific change in structure of an RNA molecule with a protein molecule.  The method is characterized by adding a protein molecule which comprises an amino acid sequence capable of binding specifically to a nucleotide sequence derived from an RNA-protein complex interaction motif in a non-naturally occurring RNA molecule comprising the nucleotide sequence to the RNA molecule.

Description

Method for constructing artificial RNP nanostructure using RNA-protein complex interaction motif

The present invention relates to a method for constructing an artificial RNP nanostructure using an RNA-protein complex interaction motif, and to induce a specific structural change of an RNA molecule using an RNA-protein complex interaction motif with a protein molecule. And a RNA molecule for use in this method.

With the progress of post-genomic science, information on the structure and function of biomolecules such as proteins and RNA has been accumulated. In contrast to conventional reductive and analytical biology, such an increasing amount of information is used to understand life systems through “synthesis”. Is growing. In particular, artificially constructing or reconfiguring biomolecules and genetic circuits has received much attention not only from life science research but also from the viewpoint of industrial applications.

In the field of nanotechnology, nanoscale structures using biomolecules are being constructed, and in the field of protein engineering, new creation of enzymes by molecular design is being attempted. The “material” used in these attempts is a nucleic acid or protein.

Prior art of nanoscale structure construction using biomolecules is known. In this technique, formation of a DNA polyhedron combining three types of DNA strands has been reported (see Non-Patent Document 1). However, DNA has a simple structure and the design of the structure is easy, but there is a problem that there are few variations as a part for creating the structure.

In addition, the creation of an RNA jigsaw puzzle using double strand formation between the kissing-loop and complementary strand of RNA has been reported (see Non-Patent Document 2). RNA has a variety of structures and functions as compared to DNA, and the structure of the molecule itself is relatively simple. Therefore, RNA is more suitable for designing and synthesizing structures. However, it cannot be said that variations as a part for creating a structure are sufficient.

Protein is also considered as a biomolecular material, but there is a problem that it is difficult to design a molecule because the protein has a complicated structure.

There are no reports of useful biomolecular materials that can be expected to undergo desired structural changes that are useful as materials for artificially constructing or reconfiguring biomolecules and genetic circuits.
He Y, Nature. 2008 Mar 13; 452 (7184): 198-201. Chworos A, Science. 2004 Dec 17; 306 (5704): 2068-72.

An object of the present invention is to provide a method for designing and constructing an artificial RNA-protein nanostructure and inducing a structural change of an RNA molecule with a protein.

The present invention has been made to solve the above problems. That is, according to one embodiment of the present invention, there is provided a method for inducing a specific structural change of an RNA molecule with a protein molecule, which comprises a base sequence derived from an RNA-protein complex interaction motif. This is a method characterized in that a protein molecule comprising an amino acid sequence that specifically binds to a base sequence derived from the RNA-protein complex interaction motif of the RNA molecule is added to a natural RNA molecule. Here, the amino acid sequence that specifically binds to the base sequence derived from the RNA-protein complex interaction motif of the RNA molecule refers to the protein derived from the RNA-protein complex interaction motif introduced into the RNA molecule. The amino acid sequence which comprises comprises the amino acid sequence which introduce | transduced some variation | mutation into this amino acid sequence. The slight mutation refers to a mutation that does not affect the RNA-protein complex interaction.

According to another embodiment, the present invention is a non-natural RNA molecule comprising a base sequence derived from an RNA-protein complex interaction motif, which is an RNA molecule that specifically changes its structure.

According to another embodiment of the present invention, there is provided a method for producing an RNA molecule having a specific structure change as described above, wherein an RNA-protein complex interaction motif having a known structure change is selected. And determining the base sequence of the RNA molecule by arranging the base sequence derived from the RNA-protein complex interaction motif and the scaffold base sequence using a computer molecular modeling method. And a step of producing an RNA molecule based on the base sequence obtained by the determining step. The scaffold base sequence refers to a base sequence other than a base sequence derived from an RNA-protein complex interaction motif that causes a specific structural change.

According to still another embodiment of the present invention, there is provided a kit for inducing a specific structural change of an RNA molecule with a protein molecule, wherein the RNA molecule specifically changes its structure as described above, A protein comprising a non-natural RNA molecule comprising a base sequence derived from an RNA-protein complex interaction motif and an amino acid sequence that specifically binds to the base sequence derived from the RNA-protein complex interaction motif of the RNA molecule A molecule.

It is preferable that the kit further includes an RNA molecule that competes with the formation of the RNA-protein complex. Here, the competing RNA has a base sequence derived from an RNA-protein complex interaction motif.

According to still another embodiment of the present invention, there is provided a functional RNA-protein complex, which is an RNA molecule having a specific structure change as described above, which is derived from an RNA-protein complex interaction motif. A non-natural RNA molecule comprising a base sequence, an amino acid sequence that specifically binds to the base sequence derived from the RNA-protein complex interaction motif of the RNA molecule, and a protein molecule comprising a functional protein; including.

The functional protein is preferably one or more selected from a marker protein, a membrane-permeable protein, a protein specific to a tumor, a protein having a therapeutic effect, and an antibody that recognizes a specific cell surface.

According to yet another embodiment of the present invention, there is provided a method for producing an RNA-protein complex nanostructure, which comprises a non-naturally occurring sequence comprising a base sequence derived from an RNA-protein complex interaction motif. A protein molecule comprising an amino acid sequence that specifically binds to a base sequence derived from an RNA-protein complex interaction motif of the RNA molecule and optionally a metal ion are added to the RNA molecule. To do.

As an effect of the present invention, structural changes in RNA molecules can be induced by proteins. In addition, RNA molecules that can be induced by proteins and structures composed of the RNA molecules can be constructed. Furthermore, an RNA molecule that can be induced by a protein and a functional molecule that is a complex of the protein can be provided. Any of these methods or molecules are useful as materials for artificially constructing or reconfiguring biomolecules, multifunctional nanostructures, or genetic circuits.

FIG. 1 is a diagram schematically showing a method for inducing a structural change of an RNA molecule 1 with a protein molecule 2 according to the first embodiment. FIG. 2 is a ribbon diagram showing a composite formed by L7Ae and Box C / D. In FIG. 3, (a) is a diagram schematically showing the state of an unfixed Box C / D arrangement, and (b) is a Box C / D fixed to a structure bent at 60 ° C. by L7Ae. It is a figure which shows the state of an arrangement | sequence roughly. FIG. 4 is an enlarged schematic view of a bent portion of the Box C / D arrangement. FIG. 5 is a schematic diagram showing a regular octahedron structure formed by inducing RNA molecule 1 with protein molecule 2. FIG. 6 is a diagram showing an RNA-protein complex in which functional protein molecules are arranged in RNA molecules. FIG. 7 is a diagram schematically illustrating a method for arranging a plurality of different functional protein molecules on an RNA molecule according to the fourth embodiment. FIG. 8 is a diagram showing the secondary structure of an RNA molecule depicted using Discovery Studio 2.0. FIG. 9 is a diagram showing a primary sequence of an RNA molecule designed by a computer molecule in Example 1. (a) shows a primary sequence of Long chain, and (b) shows a primary sequence of Short chain. FIG. 10 is a diagram showing the secondary structure of an RNA molecule designed by a computer molecule in Example 1. FIG. 11 is a diagram showing the results of confirming the formation of the RNP structure by gel shift assay. FIG. 12 is a photomicrograph showing the results of observing a solution containing only the buffer using an atomic force microscope. FIG. 13 is a photomicrograph showing the result of observing a solution containing only RNA molecules using an atomic force microscope, and it can be seen that the RNA molecules exist in a circular shape. FIG. 14 is a photomicrograph showing the result of observing a solution containing only L7Ae using an atomic force microscope, and L7Ae can be observed in the form of dots. FIG. 15 is a photomicrograph showing the result of observing a solution containing an RNA molecule and L7Ae using an atomic force microscope, and it can be seen that a triangular structure is formed by the RNA molecule and L7Ae. FIG. 16 is a diagram of a large L7-3kaku drawn using Discovery Studio 2.0, which is a triangle in which three L7Ae-BoxC / Ds are linked by 48 base pair RNA. FIG. 17 is an L7-3kis ribbon diagram drawn using Discovery Studio 2.0, where one side of the triangle is 10 nm. FIG. 18 is a diagram showing a hypothetical scheme of triangle formation by the L7Ae-BoxC / D motif and the Kissing-loop motif. FIG. 19 is a diagram of Delivery-3kaku drawn using Discovery Studio 2.0, where a fluorescent protein GFP is bound to one corner and a polyarginine motif is bound to two corners. FIG. 20 is a diagram showing secondary structures of RNA molecules constituting divided Delivery-3kaku designed by computer molecules in Example 7.

1 RNA molecule 2 Protein molecule 2a Fusion protein molecule 2b Fusion protein molecule 2c Fusion protein molecule 3 Functional protein molecule 4 Functional protein molecule 5 Functional protein molecule

Hereinafter, the present invention will be described in detail with reference to embodiments. However, the following description does not limit the present invention.

The rapid development of molecular biology from the second half of the 20th century to the present has identified an enormous number of genes, and the functions of various biopolymers have been clarified, centering on the proteins encoded by them. Furthermore, detailed three-dimensional structures of DNA, RNA and proteins were elucidated, and it was proved at the atomic level that they function by intermolecular interactions and selective chemical reactions. Therefore, if these interactions and chemical reactions can be freely controlled, a new disease treatment method and a method for solving energy problems should be developed.

As a method for realizing this, it is possible to design and create a new molecule having a function of directly controlling the function of the target molecule by intermolecular interaction, thereby controlling cells and tissues. RNA can form various three-dimensional structures. In addition to proteins, RNA has an enzyme function, and the correlation between the function and structure has been elucidated in detail through the analysis of the three-dimensional structure. In addition, RNA made of four basic units (bases) is formed by a simple construction principle. Therefore, RNA can be widely used for the design and construction of molecules having a highly three-dimensional structure as a nanoblock. However, RNA alone has a limited number of motifs, and the structures that can be created are limited. On the other hand, a protein made up of 20 types of basic units (amino acids) has much more various and complicated three-dimensional structures and functions than RNA. At present, the structure of natural proteins has been analyzed with a large number and high resolution, but its molecular design and construction is difficult, and it is limited to those having simple structures. Therefore, at present, it is realistic to design and construct RNP (RNA-protein complex) as a 3D object with a complex function and structure at the nanoscale. Combining the “artificial RNA created by” and “naturally known protein” is a highly feasible method for developing molecules with functions based on molecular design.

The present inventors considered using an RNA-protein interaction motif (RNP) as a material for designing and constructing a functional molecule such as a nanoscale structure or enzyme, and an artificial material using this material. The present invention has been completed by constructing specific molecules and designing and creating nanostructures. RNPs are structurally diverse compared to nucleic acids, and it is considered that complex structures that cannot be created only with nucleic acids can be designed and constructed relatively easily by designing RNA that is easy to design as a basic skeleton.

The present invention, according to the first embodiment, is a method for inducing a structural change of an RNA molecule with a protein, wherein the RNA molecule having a base sequence derived from an RNA-protein complex interaction motif is specific for the base sequence. A step of adding a molecule containing a protein that binds automatically.

The scheme of the method according to the present embodiment is schematically shown in FIG. According to the scheme shown in FIG. 1, in the absence of a protein, by adding protein 2 to RNA molecule 1 that exists in a circular shape, a triangular RNA molecule in which one protein 2 is bound to one corner is formed. Can be induced specifically. In addition, FIG. 1 is schematic and this invention is not limited to the induction | guidance | derivation from another specific shape to another specific shape. Hereinafter, the structure of an RNA molecule in which structural changes are induced by the present invention, a preparation method thereof, and a method for inducing structural changes of RNA molecules with proteins will be described in detail.

[Configuration of RNA molecule having base sequence derived from RNA-protein complex interaction motif]
The RNA molecule whose structural change is induced by a protein used in the method according to the present embodiment is an RNA molecule having a base sequence derived from an RNA-protein complex interaction motif. More specifically, the RNA molecule in which the structural change is induced according to the present embodiment is a non-sequence comprising a base sequence derived from an RNA-protein complex interaction motif and a scaffold base sequence that is another base sequence. It is a natural molecule. Each of the scaffold base sequence and the RNA-protein interaction motif can be extracted from the sequence of a naturally occurring molecule. The base sequence derived from the RNA-protein complex interaction motif functions as a part that causes a structural change in the RNA molecule upon binding to the protein. The scaffold base sequence is a part that acts as a skeleton of the nanostructure in the RNA molecule and does not cause a structural change. The RNA molecule according to the present embodiment only needs to include a base sequence derived from an RNA-protein complex interaction motif, but preferably does not include a portion such as an RNA base sequence that binds nonspecifically to a protein.

[1. Base sequence derived from RNA-protein complex interaction motif]
Here, the base sequence derived from the RNA-protein complex interaction motif is the base known as the sequence on the RNA side of the interaction motif between RNA and protein in a natural and known RNA-protein complex. A sequence and a base sequence that is a sequence on the RNA side in an artificial RNA-protein complex interaction motif obtained by an in vitro selection method (in vitro selection method) are included. An RNA-protein complex is an association of protein and RNA that has been confirmed in large numbers in a living body, and is a 3D object having a complicated structure.

A base sequence derived from a natural RNA-protein complex interaction motif is usually composed of about 5 to 30 bases, and non-covalently, that is, by hydrogen bonding, with a specific amino acid sequence of a specific protein. It is known to form specific bonds. The nucleotide sequences derived from such natural RNA-protein complex interaction motifs are shown in Tables 1 and 2 below, and a database available on the website: http: // gibk26. bse. kyutech. ac. jp / jouhou / image / dna-protein / rna / rna. From html, a motif that produces the desired structural change can be selected. The RNA-protein interaction motif preferably used in this embodiment is a motif whose X-ray crystal structure analysis or NMR structural analysis has already been performed, or a three-dimensional structure estimated from the three-dimensional structure of a homologous protein that has been subjected to structural analysis. It is a possible motif. Furthermore, it is desirable that the protein is a motif that specifically recognizes the secondary structure and base sequence of RNA.

The base sequence derived from an artificial RNA-protein complex interaction motif is a base sequence on the RNA side of an RNA-protein interaction motif in an artificially designed RNA-protein complex. Such a base sequence is usually composed of about 5 to 30 bases, and forms a specific bond with a specific amino acid sequence of a specific protein non-covalently, that is, by hydrogen bonding. design. The base sequences listed in Table 3 below are also known, and these can also be used as base sequences derived from the RNA-protein complex interaction motif of the present invention.

An artificial RNA-protein complex can be prepared by using a molecular design method, an in vitro evolution method, or a combination of both. In the in vitro evolution method, aptamers and ribozymes can be obtained by repeating functional reactions such as selecting functional RNA from a molecular library with various sequence diversity and amplifying and transcribing the gene (DNA). . Therefore, an RNA-protein interaction motif adapted to RNP having a target functional structure in advance in molecular design can be extracted from natural RNP molecules or artificially created by in vitro evolution.

In the present embodiment, the base sequence derived from the RNA-protein complex interaction motif preferably has a dissociation constant Kd of the RNA-protein complex from which the base sequence is derived from about 0.1 nM to about 1 μM. . This is because the RNA-protein interaction has a high affinity, and the state can be maintained even after the structural change is induced.

As a specific base sequence derived from an RNA-protein complex interaction motif, L7Ae derived from ultra-high heat sulfate-reducing archaea, which is known to be involved in RNA modification such as RNA methylation or pseudouridine formation (SEQ ID NO: 1) ) (Moore T et al., Structure Vol. 12, pp. 807-818 (2004)) is a base sequence to which 5′GGGGGUGAUGCGGAAAGCUGACCCC3 ′ (SEQ ID NO: 2), an enzyme that performs aminoacylation Escherichia coli-derived Threonyl-tRNA synthetase (Cell (Cambridge, Mass.) V97, pp.371-381 (19), which is known to have feedback inhibition that inhibits translation and inhibits translation. 9)) is a nucleotide sequence that binds, 5'JijishijiyueiyujiyujieiyuCUUUCGUGUGGGUCACCACUGCGCC3 'including but nucleotide sequences, such as (SEQ ID NO: 3), but is not limited to.

When the nucleotide sequences derived from these RNA-protein complex interaction motifs are determined, amino acid sequences derived from the same RNA-protein complex interaction motif are simultaneously determined, and these are specific under physiological conditions. And non-covalent binding occurs.

By the way, such a natural or artificial RNA-protein complex interaction motif is known to change the RNA structure before and after complex formation between RNA and protein. ing. Regarding the structural change during the formation of the complex, taking as an example a complex of 5′GGGCGUGAUGCGAAAGCUGACCCC3 ′ (SEQ ID NO: 4) (hereinafter referred to as BoxC / D), which is the base sequence to which L7Ae binds, explain. L7Ae and BoxC / D are known to form a complex as shown in FIG. 2 (Moore T et al., Structure Vol. 12, pp. 807-818 (2004)). In particular, L7Ae is known to specifically bind to a Box C / D sequence in which the complementary sequence portion forms a double strand.

The Box C / D sequence in the form of a double strand is a molecule having a flexible, unfixed structure before L7Ae binds. Such a state of the Box C / D arrangement is schematically shown in FIG. Here, when L7Ae is added to the BoxC / D sequence forming a double strand, the BoxC / D sequence and L7Ae interact to form a complex. Then, this Box C / D arrangement is fixed to a structure bent at 60 ° C. The BoxC / D array and L7Ae in such a state are schematically shown in FIG. In more detail, the structure bent at 60 ° C. is formed around the base U of the sixth strand site from the 5 ′ side in the sequence. The schematic diagram which expanded the bending part is shown in FIG. This is a specific structural change known for the RNA-protein complex interaction motif between the Box C / D sequence and L7Ae.

The RNA-protein complex interaction motif between the Box C / D sequence and L7Ae is an example, and the other RNA-protein complex interaction motifs listed in the above table may each have specific structural changes. Are known. For example, taking a 3-way junction (3- way over junction) structure, Bacillus subtilis -derived S15 (RNA: GGGCGGCCUUCGGGCUAGACGGUGGGAGAGGCUUCGGCUGGUCCACCCGUGACGCUC (SEQ ID NO: 5)) (Protein: EmuPiaishikeiiikeikyukeibuiaikyuiefueiaruefuPijiditijiesutiibuikyubuieieruerutieruaruaienuarueruesuieichierukeibuieichikeikeidieichieichiesueichiarujierueruemuemubuijikyuRRRLLRYLQREDPERYRALIEKLGI (SEQ ID NO: 6)), an angle of 90 degrees by binding proteins A highly thermophilic bacterium-derived L1 protein (RNA: GGGAGUGAAGGAGGCUUCGGCCGCGAAACUUCACUCCC (SEQ ID NO: 7)) (protein: PKGHGRYRALLEKVD) EnukeiaiwaitiaidiieieieichierubuikeiierueitieikeiefudiitibuiibuieichieikeierujiaidiPiaruaruesudikyuenubuiarujitibuiesueruPieichijierujikeikyubuiarubuierueiaieikeijiikeiaikeiieiiieijieidiwaibuijijiiiaiaikyukeiaierudijidaburyuemudiefudieibuibuieitiPidibuiemujieibuijiesukeierujiaruaieruJipiarujierueruPienupikeieijitibuijiefuenuaijiiaiaiaruiaikeieijiaruaiiefuaruenudikeitijieiaieichieiPibuijikeieiesuefuPiPiikeierueidienuaiarueiefuaiarueieruieieichikeiPiijiAKGTFLRSVYVTTTMGPSVRINPHS (SEQ ID NO: 8)) (Nevskaya, N., Tishchenko, S., Volchkov, S., Kljashtorny, V., Nikonova, E., Nikonov, O., Nikulin, A., Kohrer, C., Piendl W., Zimmermann, R., Stockley, P., Garber, M., Nikonov, S. (2006) New insights into the interaction protein.7.Jul. Human-derived U1A (RNA: GGCAGAGCUCUCUCGGGACAUUGCACCUGCC (CC: SEQ ID NO: 9)) (protein: AVPETRPNHTIYINNLNEKIKKKDHALKSFLDQKLDHALKGSQQLDDIFSSRKMRGDQ Such as FV (SEQ ID NO: 10)) is known.

In this embodiment, one RNA molecule may be arranged with a plurality of identical nucleotide sequences derived from the RNA-protein complex interaction motif. Alternatively, base sequences derived from different types of RNA-protein complex interaction motifs may be arranged in one RNA molecule. By arranging base sequences derived from different RNA-protein complex interaction motifs in one RNA molecule, multiple types of structural changes can be introduced into one RNA molecule, and multiple types of structural changes can be made. It is possible to guide.

[Base sequence other than base sequence derived from RNA-protein complex interaction motif]
The base sequence other than the base sequence derived from the RNA-protein complex interaction motif that constitutes the RNA molecule according to the present embodiment is a portion that does not cause a structural change. As another expression, it can also be said to be a part that serves as a scaffold for arranging a part that causes a structural change. The base sequence that does not cause such a structural change may be an artificial base sequence or a naturally-derived base sequence. Preferred sequences that do not cause structural changes include, for example, sequences that do not have a complicated three-dimensional structure, double strands formed by Watson-Crick base pairing, tRNA, 3-way junction, kissing-loop, and loop-receptor. Examples of such RNA-RNA interaction motifs include, but are not limited to. The length, arrangement position, etc. of the base sequence that does not cause a structural change can be appropriately determined in relation to the base sequence derived from the RNA-protein complex interaction motif. In addition, the manufacturing method of the RNA molecule used for this embodiment including determination of length, arrangement | positioning position, etc. is mentioned later.

[Protein molecules that induce structural changes]
The protein molecule that induces structural changes used in the method according to the present embodiment has an amino acid sequence derived from the same RNA-protein complex interaction motif as the base sequence derived from the RNA-protein complex interaction motif of the RNA molecule. Contains protein molecules. Here, the amino acid sequence derived from the RNA-protein complex interaction motif specifically interacts with the base sequence derived from the RNA-protein complex interaction motif of the RNA molecule in which the structural change is induced. . That is, the RNA molecule and the protein molecule that induces a structural change are selected so as to form an RNA-protein complex.
In this embodiment, when selecting a base sequence derived from an RNA-protein complex interaction motif and an amino acid sequence derived from an RNA-protein complex interaction motif, a known RNA-protein complex interaction is selected. A mutation may be introduced into the motif-derived sequence. Those skilled in the art can appropriately determine and introduce mutations in the RNA-protein complex interaction motif that do not change the interaction characteristics. For example, a mutation of about 1 to 5 bases may be introduced only into the base sequence derived from the RNA-protein complex interaction motif, and 1 to 5 only into the amino acid sequence derived from the RNA-protein complex interaction motif. Mutations as large as peptides may be introduced, and mutations may be introduced into both. The number of such mutations varies depending on the RNA-protein complex interaction motif to be used, and is not limited to these values.

As long as the protein molecule has such an amino acid sequence, it may be a fusion protein molecule containing a functional protein. Examples of functional proteins include, but are not limited to, fluorescent proteins having a marker function, apoptosis-inducing proteins having a therapeutic effect, and the like. Such a protein molecule can be appropriately produced by those skilled in the art using a vector or the like based on the known DNA sequence information of the desired protein.

If the RNA molecule from which the structural change is induced is a single RNA molecule with a plurality of different types of RNA-protein complex interaction motifs, the corresponding sequence Thus, protein molecules having amino acid sequences derived from different types of RNA-protein complex interaction motifs are required.

[Induction of structural changes in RNA molecules]
Induction of the structural change of the RNA molecule can be performed in a liquid. More specifically, it can be carried out as long as it is a liquid under physiological conditions of 4 to 80 ° C. and pH 3.5 to 10.5 under atmospheric pressure. The method according to the present embodiment can be performed by mixing the protein molecule with a liquid under physiological conditions containing the RNA molecule. The mixing amount can be determined by the number of base sequences derived from interacting RNA-protein complex interaction motifs on the RNA molecule, the molar ratio during interaction, and the like. Note that the molar ratio at the time of interaction of the RNA-protein complex is specific to each RNA-protein complex interaction motif.

[Confirmation of induction of structural changes in RNA molecules]
In the method according to the present embodiment, it can be confirmed whether the induction of the structural change of the RNA molecule has actually been performed. Confirmation is performed by mixing the solution containing RNA molecules before and after mixing the protein molecules, using a predetermined method known to those skilled in the art, and observing with a gel electrophoresis method or an atomic force microscope. can do. Atomic force microscopy allows the structure to be visualized at the atomic level, so it is easy to determine whether there is a structural change predicted from the RNA-protein complex interaction motif introduced into the RNA molecule. can do.

[Induction of structural changes in RNA molecules]
Next, an example of inducing a specific structural change of an RNA molecule by a protein will be described.
As an example, a circular double-stranded RNA molecule having three BoxC / D sequences at regular intervals can be designed as an RNA molecule. In this case, in the absence of protein, it exists as a circular RNA double-stranded molecule in a liquid under physiological conditions. Here, the L7Ae molecule is added in a molar amount of 3 times or more of the RNA molecule. As a result, three L7Ae molecules bind to the double-stranded RNA molecule, and a structure having three 60-degree angles at equal intervals, that is, an equilateral triangle, is induced.

As another example, a circular double-stranded RNA molecule having three BoxC / D sequences at regular intervals and an L7Ae dimer in which two L7Ae molecules as proteins are fused can be designed as RNA molecules. In this case, in the absence of the L7Ae dimer, the RNA molecule is present in the fluid under physiological conditions as a circular RNA double-stranded molecule. Here, the L7Ae dimer is added in a molar amount so as to be 6 or more L7Ae dimers with respect to 4 molecules of double-stranded RNA molecules. As a result, six L7Ae dimers bind to four RNA molecules, and a regular octahedral structure is induced. A schematic diagram in this case is shown in FIG. In FIG. 5, it is RNA molecules that constitute each side, and an L7Ae dimer is located at the apex. The formation of such an octahedron is performed spontaneously only by mixing the RNA molecule and the L7Ae dimer.

Also, square RNP can be designed. An RNA motif that binds to protein L1 is inserted into the vertices of the four sides of double-stranded RNA. In the presence of a protein ratio, it is expected to have a circular structure, but it can be expected that a square RNP molecule can be formed by binding the L1 protein to each vertex.

According to the method of the first embodiment of the present invention, a specific structural change of a specific RNA molecule can be induced in the protein by designing the specific non-natural RNA molecule or protein molecule. Control of unnatural RNA molecular structure using such RNA-protein complex interaction motifs has not been reported so far, and it is used as a material for artificially constructing or reconfiguring biomolecules and genetic circuits. Promising as a tool for

Next, a method for producing an RNA molecular material will be described as a second embodiment of the present invention. According to the second embodiment, the above-described method for producing an RNA molecule having a specific structural change, the step of selecting an RNA-protein complex interaction motif whose desired structural change is known, and the RNA molecule Determining a base sequence derived from the RNA-protein complex interaction motif and a scaffold base sequence by using a computer molecular modeling method, and determining the above And a step of producing RNA based on the base sequence obtained by the step.

The RNA molecule produced in this embodiment is an RNA molecule having the characteristics described in the first embodiment and whose structural change is induced by a protein molecule.

In the step of selecting an RNA-protein complex interaction motif whose desired structural change is known, an RNA-protein complex interaction motif capable of realizing the structural change to be introduced is selected from the aforementioned table or database. For example, structural changes of RNA molecules that can be introduced with the RNA-protein complex interaction motif include 60 degree bend, 90 degree bend, change from double stranded RNA to single stranded RNA, single stranded RNA Examples include, but are not limited to, changes to double-stranded RNA. A plurality of structural changes can be realized for one RNA molecule, and in this case, a plurality of different RNA-protein complex interaction motifs can be selected in combination. RNA-protein complex interaction motifs can be introduced.

The step of determining the base sequence of the RNA molecular material can be performed by a computer molecular modeling method. Commercially available molecular modeling software can be used for this. In computer molecular modeling, first, a three-dimensional structure of a desired RNA-protein complex interaction motif is obtained. Then, one or a plurality of RNA-protein complex interaction motif-derived base sequences and RNA-protein complex interaction so that the RNA-protein complex after introduction of structural change forms a desired structure. The base sequence other than the base sequence derived from the motif is determined. Base sequences other than the base sequence derived from the RNA-protein complex interaction motif serve as a scaffold for locating the base sequence derived from the RNA-protein complex interaction motif. A sequence known not to form such a secondary structure or the like can be selected so as not to form a structure or a tertiary structure. Thereafter, the secondary structure that can be taken by the RNA having the designed sequence is predicted, and it is confirmed that the designed secondary structure is the most stable structure and does not take any other stable secondary structure. For this, the secondary structure prediction program mfold (http://mfold.bioinfo.rpi.edu/cgi-bin/rna-form1.cgi) of the nucleic acid used on the web can be suitably used.

In the step of producing an RNA molecule based on the base sequence obtained by the determining step, an RNA molecule is produced from the obtained base sequence by a known method. If a predetermined RNA sequence is given, it is common practice for those skilled in the art to produce RNA molecules based on this sequence. For example, template DNA is synthesized by PCR and transcription using RNA synthase is performed. RNA molecules can be produced by the reaction. In addition, RNA having a short chain length of about 30 bases can be directly synthesized by chemical synthesis. RNA molecules can be produced by such a method.

The RNA molecule that induces a structural change thus produced can be used in the first embodiment, and can be used as a molecular material together with a protein molecule that induces a structural change as a kit described later. it can.

According to the second embodiment of the present invention, an RNA molecule that induces a structural change is designed and manufactured by combining a database of RNA-protein complex interaction motifs, a computer molecular modeling method, and a genetic engineering method. Can do. According to such a method, it is possible to design a molecule with high accuracy, and thus it is possible to easily and accurately design an RNA molecule in which a desired structural change is induced. Moreover, such RNA molecules can actually be produced in large quantities by genetic engineering methods. The method according to the second embodiment is very useful in producing a desired RNA molecule.

According to the third embodiment, the present invention provides a kit for inducing a structural change of an RNA molecule with a protein molecule, wherein the RNA molecule specifically changes in structure as described above, and the RNA-protein that the RNA molecule has And a protein molecule comprising an amino acid sequence that specifically binds to a base sequence derived from a complex interaction motif.

In such a kit, the RNA molecule that specifically changes its structure and the protein molecule containing the amino acid sequence derived from the RNA-protein complex interaction motif may be any of those described in the first embodiment.

The kit according to the present embodiment may include an RNA molecule that competes for RNA-protein complex formation as an optional component. A specific RNA base sequence competing for RNA-protein complex formation can have a base sequence derived from an RNA-protein complex interaction motif.

In the present embodiment, the RNA molecule specifically changing in structure and the protein molecule containing the amino acid sequence derived from the RNA-protein complex interaction motif included in the kit form a complex when mixed under predetermined conditions. This bond is a stable and specific non-covalent bond by hydrogen bonding. However, by adding an RNA molecule that competes for RNA-protein complex formation to this solution, binding can be easily suppressed and formation of the RNP structure can be controlled.

The kit according to the third embodiment of the present invention can induce specific structural changes of RNA molecules under physiological conditions. In addition, the inclusion of RNA molecules that compete for RNA-protein complex formation, which is an optional component, enables reversible structural changes in the RNP nanostructure.

According to the fourth embodiment of the present invention, the functional RNA-protein complex described above is derived from an RNA molecule that specifically changes its structure, and an RNA-protein complex interaction motif possessed by the RNA molecule. A protein molecule comprising an amino acid sequence that specifically binds to a base sequence and a functional protein.

FIG. 6 schematically shows a functional RNA-protein complex according to the fourth embodiment. The complex shown in the figure has three vertices of a triangle composed of RNA molecules 1 each having a base sequence derived from an RNA-protein complex interaction motif in which the structural changes described in the first embodiment are induced on each side. In addition, a fusion protein molecule comprising the amino acid sequence portion 2 that specifically binds to the base sequence and functional protein molecules 3, 4, and 5 is bound. All three functional protein molecules 3, 4 and 5 are different.

Here, the functional protein refers to a protein having a predetermined function, for example, fluorescent proteins such as GFP and YFP, proteins having a membrane permeation function such as polyarginine motif, and tissue-specific functions. Examples include, but are not limited to, antibodies that specifically bind to the surface of cancer cells, such as proteins that bind to expressed membrane proteins, apoptosis-inducing proteins, and proteins that have a therapeutic effect on specific diseases. .

Such a complex is an RNA molecule designed as described in the first embodiment, and an amino acid sequence that specifically binds to a base sequence derived from an RNA-protein complex interaction motif introduced into the RNA molecule. It can be obtained by producing a fusion protein molecule in which any functional protein molecule is bound to the protein molecule to be contained, and mixing the RNA molecule and the fusion protein molecule. For example, when L7Ae-BoxC / D exemplified in the first embodiment is used as an RNA-protein complex interaction motif, a fusion protein molecule of an L7Ae protein and an arbitrary protein or peptide is prepared. An RNA-protein complex having any of a plurality of functions can be easily prepared simply by mixing with the RNA strand exemplified in one embodiment. Thus, when multiple types of functional protein molecules are bound to one RNA molecule, the number of functional protein molecules to be bound is changed by changing the molar ratio of the multiple types of functional protein molecules to be added. And can control the type. For example, the complex shown in FIG. 6 can be formed by adding a functional protein molecule to a circular RNA molecule 1 having three Box C / D sequences at regular intervals. It is obtained by adding sex protein molecules 3, 4, 5 in equimolar amounts. Although not shown in the figure, even when the same RNA molecule as in FIG. 6 is used, if it is desired to form a complex in which only functional protein molecules 3 and 4 are bound at a ratio of 1: 2, By adding molecule 3 and functional protein molecule 4 at a ratio of 1: 2, functional protein molecule 3 is bound to one corner of the triangle and functional protein molecule 4 is bound to the other two corners of the triangle. A complex can be formed.

Fig. 7 shows a schematic scheme for constructing a functional RNA-protein complex. FIG. 7 is a scheme in the case of adding three different types of functional protein molecules 2a, 2b, and 2c to RNA molecules. In this way, each time one kind of protein molecule derived from an RNA-protein complex interaction motif fused with a functional protein molecule is added, the structure of the RNA molecule is changed, and finally the functional RNA of the desired shape— Protein complexes can be created.

In the production of a functional RNA-protein complex, for example, if a protein that binds to a membrane-permeable peptide or a tissue-specific membrane protein is used, the complex can be delivered to specific cells. Can be used. In addition, when a fluorescent protein such as GFP or YFP is used, there is an advantage that the delivery of the complex can be visualized and observed. Furthermore, it can be used as a therapeutic agent for delivering an apoptosis-inducing protein to cancer cells. Like the complex shown in FIG. 6, a complex in which a plurality of proteins having different functions are combined has all these advantages. Furthermore, since RNA and protein are bound by specific and non-covalent bonds, they can be made into a complex with a flexible binding mode as compared with conventional covalent bonds. Therefore, any protein having an RNA binding motif can be freely exchanged (installed), and modular engineering of a functional RNA-protein complex becomes possible. In addition, RNA molecules designed by this method can form a stable double strand and protect the hydroxyl group of the terminal nucleotide from RNase present in the cell, as shown in the examples described later and FIG. become. For this reason, it can be expected to exist stably in the cell.

The present invention provides a method for producing an RNA-protein complex nanostructure according to the fifth embodiment, wherein an RNA molecule having a base sequence derived from an RNA-protein complex interaction motif is added to the RNA molecule. A protein molecule containing an amino acid sequence that specifically binds to a base sequence derived from an RNA-protein complex interaction motif and, if necessary, a metal ion such as magnesium or calcium are added.

In this embodiment, as in the first embodiment, a specific structural change of an RNA molecule is induced by a protein molecule to create an RNA-protein complex nanostructure. A desired structure can be obtained by designing an RNA molecule, and specific structures include, but are not limited to, the equilateral triangle, regular octahedron, and square exemplified in the first embodiment.

Another example is a method for preparing an RNA-protein complex nanostructure by adding an RNA molecule, a protein molecule, and a metal ion. This method is preferably used when a Kissing-loop sequence is introduced as a scaffold base sequence into an RNA molecule. At this time, a structural change is introduced into one RNA molecule by a protein molecule to form one RNA-protein complex. By adding metal ions, one RNA-protein complex interacts with another RNA-protein complex. When such a Kissing-loop sequence is used, the metal ion can link RNA molecules non-covalently. Thereby, an RNA-protein complex nanostructure in which a plurality of RNA molecules are bound can be produced. Such a substance that binds a plurality of molecules depends on the introduced motif or the like, but also includes an RNA loop receptor motif.

Also in this case, as described in the fourth embodiment, the protein molecule can be a fusion protein in which a functional protein is fused.

According to the fifth embodiment of the present invention, it is possible not only to introduce a structural change into an RNA molecule to form a complex, but also to provide a method for creating a nanostructure by combining several complexes. it can. According to such a method, it is possible to create a larger number of desired nanostructures, and it can be said that it is easier to apply as a tool for artificially constructing biomolecules and genetic circuits.

[Example]
A method of creating a nanostructure using RNP is shown. This technique is a technique that makes it easier to create nanoscale structures and functional molecules than when using only proteins or RNA alone. Specific experimental examples are given below.

[Computer molecular design of RNP nanostructures]
For the molecular design of the RNP nanostructure, Discovery Studio 2.0 (Accelrys), which is molecular modeling software, was used. First, as a three-dimensional structure of the RNP motif used for molecular design, a pdb file in which the structure of L7Ae-BoxC / D is described is The RCSB Protein Data Bank (http://www.rcsb.org/pdb/home/home/ Do) (PDB ID: 1RLG). The 3 ′, 5th and 21st base 5 ′ bromouridines of Box C / D contained in the obtained file were substituted with uridine in accordance with the RNA to be actually synthesized. Generate a molecular model of linear double-stranded A helix RNA of any sequence, and adjust the length of the double strand so that the three L7Ae-Box C / Ds at the top are arranged in the same plane Inserted in the side part. The length of the duplex connecting Box C / D is 26 base pairs. A similar attempt was made so that the double-stranded sequences of the sides do not form unwanted secondary structures. P. It was replaced with the sequence of the sides of the DNA tetrahedron of Goodman et al. (Science, 2005, 310, 1661-1665).

Fig. 8 (a) is a view of L7-3 kaku し た depicted using Discovery Studio 2.0 from the top, and Fig. 8 (b) is a triangular side view of Fig. 8 (a) rotated 90 degrees. FIG. The RNA consists of 114 nt long chain (Fig. 9 (a)) and 150 nt short chain (Fig. 9 (b)). By complementary pairing of these RNAs, circular RNA with three BoxC / Ds A chain is formed. By binding L7Ae to these BoxC / D sequences, it is expected that three positions of the circular RNA will be fixed at 60 degrees to form a triangle. The length of one side of the formed RNA triangle is about 10 nm (FIG. 10). After designing the molecular model, energy minimization calculation was performed using Minimization Protocol to evaluate the validity of the model structure. When the structure of the designed molecular model and the structure after the energy minimization calculation were superimposed, there was almost no structural difference, so it was confirmed that the designed molecular model was stable in terms of energy.

[Synthesis and construction of RNP nanostructures]
[Production of L7Ae]
The protein of the RNA-protein complex interaction motif, L7Ae, was expressed using a plasmid (SEQ ID NO: 11) received from Alexander Huttenhofer. The plasmid is A.I. Fulgidus was amplified using a restriction enzyme N using primers of L7Ae Fwd (5′-CTGACATATGTTACGTGAGATTTGAGGTTC-3 ′) (SEQ ID NO: 12), L7Ae Rev (5′-CTGACTCGAGTTACTTCTGGAGCCTTTTATC-3 ′) and SEQ ID NO: 13 It was prepared by integrating into a pET-28b + vector (Novagen) cut with XhoI. The expression purification method is shown below. First, E. coli BL21 (DE3) pLysS was transformed. The obtained colonies were inoculated into 5 mL of LB medium containing 25 μg / mL kanamycin and 100 μg / mL chloramphenicol, and cultured with shaking at 37 ° C. overnight. Subsequently, the entire culture was inoculated into 500 mL of LB medium containing 25 μg / mL kanamycin and 100 μg / mL chloramphenicol. O. D. _The culture was shaken at 37 ° C. until ₆₀₀ reached 0.6 to 0.7, and then 500 μL of 1M IPTG was added (final concentration 1 mM) to induce expression, followed by overnight shaking at 30 ° C. The cells were collected by centrifugation (4 ° C., 6000 rpm, 20 minutes), 5 mL of a sonication buffer (50 mM Na phosphate, 0.3 M NaCl, pH 8.0) was added, and sonication was performed to disrupt the cells. . In the ultrasonic treatment, the operation of applying ultrasonic waves for 15 seconds after cooling on ice was repeated 6 times. Thereafter, the impure protein was denatured at 80 ° C. for 15 minutes. Centrifugation (4 ° C., 6000 rpm, 20 minutes) was performed, the supernatant was collected, and the protein with a histidine tag was purified by a batch method using a Ni-NTA column (Qiagen). Specifically, first, the supernatant and 1 mL of Ni-NTA were mixed and stirred at 4 ° C. for 1 hour. Thereafter, the column was packed and washed twice with 4 mL of wash buffer (50 mM Na phosphate, 0.3 M NaCl, 20 mM imidazole, pH 8.0). An elution buffer containing 50 mM, 100 mM, 200 mM, and 300 mM imidazole (prepared by adding imidazole to 50 mM Naphosphate, 0.3 M NaCl, pH 8.0) was eluted stepwise in 1 mL steps. Confirmation was performed by 17% SDS-PAGE. Subsequently, the protein was concentrated using Microcon YM-3 (Millipore) and replaced with a dialysis buffer (20 mM Hepes-KOH, 1.5 mM MgCl ₂ , 150 mM KCl, 5% glycerol, pH 7.5). The protein concentration was determined by the Bradford method using a protein assay (BIO-RAD).

[Synthesis of RNA]
The template DNA of long chain is L Fwd (10 μM, 5′-CTAATACGACTCACTATAGGGCGCAAAGGCCTGTAATCGGCGGTGATGCTGCTGCTGCTGCCTGCCTGCCTGCTGCTGCCTGCCTGCCTGCTGCTGCCTGCCTGCCTGCTGCTGCCTGC 1ng / μL, 5'-GGCCTGTAATCGGCGTGATGAGCCATGCGAGGAGGAAATGAAGTCCAATGGCGTGATGAGCCTCTACGGGAAGAGC- 3 ' SEQ ID NO: ^{16) 1μL, KOD + (TOYOBO} ) 1μL, 10 × KOD + buffer 5μL, 25mM MgSO 4 1.6μL, 2.5mM dNT s 4 [mu] L, mixed ultrapure water 34.4μL, 94 ℃ 15 seconds, 55 ° C. 30 seconds, to prepare perform 15 cycles of PCR in three steps of 68 ° C. 60 seconds. Template DNA synthesis short chain, first S template (1ng / μL, 5'-GCCTTTGCGCCTTGCTACGCTCTGACCCGGATGGGCATGCTCTTCCCGTAGAGGCTCTGACCATTGGACTTC-3 'SEQ ID NO: 17) 1μL, S Rev (10μM, 5'-TGTAATCGGTCAGAGCCATGCGAGGAGGAAATGAAGTCCAATGGTCAGAG-3' SEQ ID NO: 18) 5μL, Ex Taq ( Takara) 1 μL, 10 × Ex Taq buffer 10 μL, 2.5 mM dNTPs 8 μL, and ultrapure water 75 μL were mixed, and 5-cycle PCR was performed in 3 steps of 94 ° C. for 30 seconds, 55 ° C. for 30 seconds, and 68 ° C. for 30 seconds. The synthesized DNA strand was purified by phenol extraction, diethyl ether extraction, and ethanol precipitation.

Next, using 1 μL of this DNA (1 ng / μL) as a template, 1.5 μL of S Fwd (10 μM, 5′-CTAATACGACTCACTATAGGCCTTTGCGCCCTGCTACCG-3 ′ SEQ ID NO: 19), S new Rev (10 μM, 5′-TGTAATCGGTCAGAGCCATGCGGAG sequence 20) 1.5 μL, KOD ⁺ (TOYOBO) 1 μL, 10 × KOD ⁺ buffer 5 μL, 25 mM MgSO ₄ 1.6 μL, 2.5 mM dNTPs 4 μL, ultrapure water 34.4 μL, 94 ° C. 15 seconds, 55 ° C. PCR was performed in 15 cycles of 3 steps of 30 seconds at 68 ° C. for 60 seconds to synthesize template DNA. Each template DNA was confirmed to be synthesized by 4% agarose gel electrophoresis, and purified by phenol extraction, diethyl ether extraction, and ethanol precipitation. The purified template DNA was dissolved in 8 μL of ultrapure water and used for transcription. For transcription, MEGAshortscript (trademark) (Ambion) was used as follows. Template DNA 8 μL, T7 10 × Reaction Buffer 2 μL, T7 ATP Solution (75 mM) 2 μL (same for CTP, GTP, UTP) and T7 Enzyme Mix 2 μL were mixed overnight at 37 ° C. After the reaction, 1 μL of TURBO DNase was added and incubated at 37 ° C. for 1 hour to decompose the template DNA. 115 μL of ultrapure water and 15 μL ammonium acetate stop solution were added to the reaction solution, and further purified by phenol treatment, diethyl ether extraction, and ethanol precipitation. The precipitate was dissolved in 20 μL of a denaturing dye (80% formamide, 0.17% XC, 0.27% BPB) and separated by 10% polyacrylamide (1/30 bisacrylamide) denaturing gel electrophoresis. A band of the desired size was cut out, 500 μL of elution buffer (0.3 M sodium acetate, pH 7.0) was added, and incubated at 37 ° C. overnight to elute. The eluted RNA was purified again by phenol extraction, diethyl ether extraction and ethanol precipitation, dissolved in ultrapure water and used in the subsequent experiments.

[Confirmation of RNP structure formation by gel shift assay]
Confirmation of the binding of RNA and protein was performed under the reaction conditions of 2 ng / μL, 20 mM Hepes-KOH, 150 mM KCl, 1.5 mM MgCl ₂ , 2 mM DTT, 3% glycerol, and protein 100-1000 nM, respectively. The following was performed. 2 μL of 5 × binding buffer (100 mM Hepes-KOH (pH 7.5), 750 mM KCl, 7.5 mM MgCl ₂ , 10 mM DTT, 15% glycerol) and 2 μL of short chain and long chain (both 10 ng / μL) After heating at 80 ° C. for 3 minutes to denature the RNA, it was placed on ice and rapidly cooled to fold. L7Ae was added, the reaction solution volume was adjusted to 10 μL with ultrapure water, and the mixture was allowed to stand on ice for 15 minutes to bind RNA and L7Ae (100 nM, 300 nM, 1000 nM). 1 μL of dye (0.25% BPB, 0.25% XC, 30% glycerol) was added, layered on a non-denaturing 5% polyacrylamide gel, and subjected to electrophoresis at 4 ° C. for 3 to 4 hours. After electrophoresis, the gel was stained with SYBR Green, and the band was confirmed with FLA-7000 (FUJI FILM). The results are shown in FIG. As a result, it was suggested that three L7Ae were bound to RNA in which short chain and long chain were bound.

[Observation of structure with atomic force microscope]
A sample for observation with an atomic force microscope was prepared as follows. Mix 2 × 5 × binding buffer (100 mM Hepes-KOH (pH 7.5), 750 mM KCl, 7.5 mM MgCl ₂ , 10 mM DTT, 15% glycerol) 2 μL and short chain and long chain (both 10 ng / μL) 2 μL each The RNA was denatured by heating at 80 ° C. for 3 minutes, and then placed on ice to rapidly cool and fold. After mixing 3 μL of ultrapure water and 1 μL of 10 μM L7Ae (final concentration 1000 nM), the mixture was allowed to stand on ice for 15 minutes to bind RNA and L7Ae. 18 μL of 10 mM HEPES-KOH (pH 7.5) was added to 2 μL of the reaction solution to dilute it 10 times, and 10 μL of this diluted solution was used for one measurement. The sample was immobilized on a mica substrate and observed. First, 20 μL of 10 mM spermidine was dropped on the surface of the mica substrate and allowed to stand at room temperature for 10 minutes. After spermidine was blown off with nitrogen gas and dried, 10 μL of the sample was dropped and allowed to stand at room temperature for 10 minutes to adhere the sample to the mica piece surface. The surface of the mica piece was washed away with 1 mL of ultrapure water, and dried by blowing nitrogen gas. The mica substrate was set on an atomic force microscope (manufactured by Veeco), scanned, and imaged on a scale of 500 nm × 500 nm. Nanoscope (manufactured by Veeco) was used for control of the atomic force microscope and image processing. The results are shown in FIG. 12, FIG. 13, FIG. 14, and FIG. As a result, it can be observed that RNA molecules (FIG. 13) that existed alone in an oval shape form a triangular structure when protein L7Ae and RNA molecules coexist (FIG. 15), and in the presence of protein L7Ae. Only in, a triangular structure as designed was confirmed.

Further, based on the obtained image, the particle size and height distribution of the observed RNA or RNP structure were measured and analyzed. As a result, in the case of RNA alone (number of samples N = 121, average value of particle size = 26.15 nm, standard deviation σ = 4.62, average value of height = 1.80 nm, standard deviation σ = 0.66) In comparison with L7Ae, the dispersion (standard deviation) of the distribution was smaller, and both the particle size and height converged to a certain value (number of samples N = 122, average value of particle size = 24). .26 nm, standard deviation σ = 2.68, average height = 1.24 nm, standard deviation σ = 0.20). From this experimental analysis, it was clarified that RNA having a flexible structure, which cannot take a strong single structure by itself, stably formed a triangular structure as designed by binding to the L7Ae protein.

[Large L7-3 kaku computer molecular design]
In the same manner as in Example 1, two 48 bp RNAs were prepared using Discovery Studio 2.0 (Accelrys), which is molecular modeling software, so that the three L7Ae-BoxC / D motifs form a regular triangle in the same plane. A large L7-3 kaku connected by a chain was designed. FIG. 16 is a diagram of a large L7-3 kaku depicted using Discovery Studio 2.0. In this large L7-3 kaku, the triangle was composed of RNA double strands, and one side of the triangle formed by RNA was about 17 nm and 48 bp.

Results of computer molecular design, arrangement of long chain is, JijishijishieieieijijishishiyujiyueieiyushijijijishijishishijieijishijijishijiyujieiyujieijishiyujiyuyushieishishijijishieiyushijiyujishishishieiyushieiyujishijieijijieijijieieieiyujieieijiyushishieieiyujijishijiyujieiyujieijishishiyushiyueishijijijieieijieijishieiyujishishishieiyushishijishishiyujieiyushijieijishiyujieieiyujijishijieiyujijishijiyuGAUGAGCAUGGUGAGCAAGUAGCAA (SEQ ID NO: 21), the sequence of short chain is, JijishishiyuyuyujishijishishiyuyujishiyueishiyuyujishiyushieishishieiyujishiyushiyujieishishieiyushijishishieiyuyushieijishiyushijieiyushieijijishijijieiyujijijishieiyujiCUCUUCCCGUAGAGGCUCUGACCAU It was determined to JijieishiyuyushieiyuyuyushishiyushishiyushijishieiyujieiyujijijishieishijieiyujishishijijiyujieieishieijishiUCUGACCGCUCGGCGCCCGAUUACA (SEQ ID NO: 22).

Also, a large L7-3 kaku composed of a combination of different sequences was designed and created. The arrangement of long chain and short chain used at that time is as follows. Sequence of long chain is, 5'-GGACGAGCUGUACACCAUGGUGACCGCCGCCGGGCGUGAUGAGCUCCAAGGACCCCAACGAGAAGCGCGAUCACAUGAUCUACUUCGGCUUCGGCGUGAUGAGCCCUGUGCUGCUGCCCGAUAACCACUACCUGCCAUCACCCACGGCCCUGGGCGUGAUGAGCAUUCCACCCAGAGCGC-3 '(SEQ ID NO: 28, Artificial, 180nt), the sequence of short chain may, 5'-GGUGUACAGCUCGUCCGCGCUCUGGGUGGAAUGCUCUGACCCAGGGCCGUGGGUGAUGGCAGGUAGUGGUUAUCGGGCAGCAGCAC GGGCUCUGACCGAAGCCGAAGUAGAUCAUGUGAUCGCGCUUCUCGUUGGGGUCCUUGGAGCUCUGACCCGGCGGCGGUCACCAU-3 '(SEQ ID NO: 29, Artificial, 171nt) was created.

[Synthesis of RNA]
Template DNA long chain is, L Fwd (2μM, 5'-CTAATACGACTCACTATAGGACGAGCTGTACACCATGGTGACCGCCGCCGGGCGTGATGAGCTCCAAGGACCCCAACGAGAAGCGCGATCACATGATCTACTTCGGCTT-3 ', SEQ ID NO: 30, Artificial, 109nt) 10μL, L Rev (2μM, 5'-GCGCTCTGGGTGGAATGCTCATCACGCCCAGGGCCGTGGGTGATGGCAGGTAGTGGTTATCGGGCAGCAGCACAGGGCTCATCACGCCGAAGCCGAAGTAGATCATGTG-3', SEQ ID NO: 31, Artificial, 109nt ) 10 μL, Ex Taq (Takar a) 0.5 μL, 10 × Ex Taq buffer 5 μL, 2.5 mM dNTPs 4 μL, and ultrapure water 21.5 μL are mixed, and 5 cycles PCR in 3 steps of 94 ° C. for 30 seconds, 48 ° C. for 1 minute, 72 ° C. for 30 seconds It produced by doing.

Template DNA short chain is, S Fwd (2μM, 5'-CTAATACGACTCACTATAGGTGTACAGCTCGTCCGCGCTCTGGGTGGAATGCTCTGACCCAGGGCCGTGGGTGATGGCAGGTAGTGGTTATCGGGCAGCAGCACA-3 '(SEQ ID NO: 32, Artificial, 105nt)) 10μL, S Rev (2μM, 5'-ATGGTGACCGCCGCCGGGTCAGAGCTCCAAGGACCCCAACGAGAAGCGCGATCACATGATCTACTTCGGCTTCGGTCAGAGCCCTGTGCTGCTGCCCGATAACC-3' (SEQ ID NO: 33, Artificial, 104 nt)) 10 μL, Ex Taq (Taka a) 0.5 μL, 10 × Ex Taq buffer 5 μL, 2.5 mM dNTPs 4 μL, and ultrapure water 21.5 μL are mixed, and 5-cycle PCR is performed in 3 steps of 94 ° C. for 30 seconds, 56 ° C. for 1 minute, and 72 ° C. for 30 seconds. It produced by doing. The synthesized template DNA strand was subjected to electrophoresis on a non-denaturing 6% acrylamide gel (1/30 bisacrylamide) to cut out a band of the desired size. To the excised gel piece, 500 μL of elution buffer (0.1% SDS, 0.3 M sodium acetate, pH 7.0) was added and incubated overnight at 37 ° C. to elute the DNA. Thereafter, phenol extraction, diethyl ether extraction and ethanol precipitation were performed, dissolved in 20 μL of ultrapure water, and 8 μL was used for the following transcription reaction.

The transfer was performed as follows using MEGAshortscript (trademark) (Ambion). A total of 20 μL of template DNA 8 μL, T7 10 × Reaction Buffer 2 μL, T7 ATP Solution (75 mM) 2 μL (same for CTP, GTP, UTP) and T7 Enzyme Mix 2 μL were reacted at 37 ° C. overnight. After the reaction, 1 μL of TURBO DNase was added and incubated at 37 ° C. for 15 minutes to decompose the template DNA. 20 μL of a denaturing dye (80% formamide, 0.17% XC, 0.27% BPB) was added to the reaction solution and separated by 10% polyacrylamide (1/30 bisacrylamide) denaturing gel electrophoresis. A band of the desired size was cut out, 500 μL of elution buffer (0.1% SDS, 0.3 M sodium acetate, pH 7.0) was added, and the mixture was incubated overnight at 37 ° C. for elution. The eluted RNA was subjected to phenol extraction, diethyl ether extraction and ethanol precipitation, dissolved in ultrapure water, and used in the subsequent experiments.

[Confirmation of RNP structure formation by gel shift assay]
As in Example 3, the binding between RNA and protein was confirmed by gel shift assay. The final concentrations of the two kinds of RNA were 50 fmol / μL, 20 mM Hepes-KOH, 150 mM KCl, 1.5 mM MgCl ₂ , 2 mM DTT, 3% glycerol, and 100 to 1000 nM protein as follows. 5 × binding buffer (100 mM Hepes-KOH (pH 7.5), 750 mM KCl, 7.5 mM MgCl ₂ , 10 mM DTT, 15% glycerol) 2 μL and ultrapure water 6 μL, short chain and long chain (both 1 pmol / μL) 0 Each 5 μL was mixed and heated at 80 ° C. for 3 minutes to denature the RNA, then placed on ice and rapidly cooled to fold. 1 μL of L7Ae was added and allowed to stand on ice for 15 minutes to bind RNA and L7Ae (final concentrations 100 nM, 300 nM, 500 nM, 1000 nM). 1 μL of dye (0.25% BPB, 0.25% XC, 30% glycerol) was added, layered on a non-denaturing 5% polyacrylamide gel, and subjected to electrophoresis at room temperature for 3 to 4 hours. After electrophoresis, the gel was stained with SYBR Green II, and the band was confirmed with FLA-7000 (FUJI FILM). As a result, it was suggested that three L7Ae were bound to the RNA in which long chain and short chain were bound.

[Observation of structure with atomic force microscope]
Similar to Example 4, a sample was prepared as follows and observed with an atomic force microscope. 5 × binding buffer (100 mM Hepes-KOH (pH 7.5), 750 mM KCl, 7.5 mM MgCl ₂ , 10 mM DTT, 15% glycerol) 2 μL and ultrapure water 6 μL, short chain and long chain (both 1 pmol / μL) 0 Each 5 μL was mixed and heated at 80 ° C. for 3 minutes to denature the RNA, and then placed at room temperature for 10 minutes for folding. After mixing 1 μL of 7.5 μM L7Ae (final concentration 750 nM), the mixture was allowed to stand at room temperature for 15 minutes to bind RNA and L7Ae. 18 μL of 10 mM HEPES-KOH (pH 7.5) was added to 2 μL of the reaction solution to dilute it 10 times, and 10 μL of this diluted solution was used for one measurement. The sample was immobilized on a mica substrate and observed. First, 20 μL of 10 mM spermidine was dropped on the surface of the mica substrate and allowed to stand at room temperature for 10 minutes. After spermidine was blown off with nitrogen gas and dried, 10 μL of the sample was dropped and allowed to stand at room temperature for 10 minutes to adhere the sample to the mica piece surface. The surface of the mica piece was washed away with 1 mL of ultrapure water, and dried by blowing nitrogen gas. The mica substrate was set on an atomic force microscope (Veeco) and scanned. Nanoscope (manufactured by Veeco) was used for control of the atomic force microscope and image processing. As a result, in the case of RNA alone, large circular structures and linear structures that were considered to be self-assembled or associated with each other were observed. On the other hand, in the presence of L7Ae, a decrease in those structures and formation of a triangular structure as designed by computer were observed, and it was confirmed that the formation of RNP structure was performed as designed in the presence of L7Ae.

[Computer molecular design of L7-3kiss]
Similarly to Example 1, Discovery Studio 2.0 (Accelrys), which is molecular modeling software, was used to design L7-3kiss, which is a triangle formed by connecting three L7Ae-BoxC / D motifs with a Kissing-loop motif. did. Kissing-loop motif, Crystal structures of coaxially stacked kissing complexes of the HIV-1 RNA dimerization initiation site, Eric Ennifar, Philippe Walter, Bernard Ehresmann, Chantal Ehresmann and Philippe Dumas, Nature Structural Biology, Vol. 8, Number 12, 1064-1068, 2001, which is a motif in which two loops having mutually opposite complementary sequences are stably bound by base pairing and stacking between the loops. This L7-3kiss was designed as an example of structural change control using the RNP motif and the RNA-RNA interaction motif.

As a design premise, FIG. 18 shows a hypothetical scheme of triangle formation by the L7Ae-BoxC / D motif and the Kissing-loop motif. A loop-shaped RNA having a Box C / D motif at the center and forming a double strand is considered to exist in a loop shape in the absence of L7Ae or Mg ²⁺ ions. When L7Ae is added to this loop-shaped RNA, it is considered that the structure changes specifically to the bent-loop RNA exhibiting a 60-degree bent structure. Further, when Mg ²⁺ ions are added, it is considered that a triangle is formed by the Kissing-loop motif. In addition, when only Mg ²⁺ ions are added to loop-shaped RNA without adding L7Ae, the loop-shaped RNA remains as it is and interacts with the kissing-loop motif to form various non-specific shapes. Conceivable. Unlike the previous design, this method has the advantage that a structure can be formed by self-assembly just by mixing short RNA fragments.

FIG. 17 is a ribbon diagram of L7-3kiss drawn using Discovery Studio 2.0. In this L7-3kiss, one triangle consists of three RNA strands, and the three RNA strands have the same sequence. One side of the triangle formed by RNA was about 10 nm. The three RNA strands exhibited a bent structure of 60 degrees by L7Ae, formed homotrimers by kissing-loop interaction, and became triangular.

As a result of computer molecular design, the sequence of three identical RNAs was determined as CUACGGGAAGCGCGGCACCCCUAGAGGGCUCUGACCCCGAUGGGCACAGCGCGCAGCCCAUCCCGGGCGUGAUGAGCU (SEQ ID NO: 23).

[Computer molecular design of Delivery-3kaku]
Similarly to Example 1, two 26 bp RNAs were prepared using Discovery Studio 2.0 (Accelrys), which is molecular modeling software, so that the three L7Ae-BoxC / D motifs are coplanar and form an equilateral triangle. Delivery-3 kaku was designed in which three chains of L7Ae were linked together, one of which was linked to the fluorescent protein GFP and the other was linked to the polyarginine motif. FIG. 19 is a diagram of Delivery-3kaku drawn using Discovery Studio 2.0. In this Delivery-3kaku, L7Ae fused with the polyarginine motif is bound to the upper left corner and upper right corner, and L7Ae fused to the fluorescent protein GFP is bound to the lower left corner. Was composed of RNA duplex, and one side of the triangle formed by RNA was about 10 nm.

Results of computer molecular design, arrangement of long chain is, 5'-GGCGCAAAGGCCUGUAAUCGGCGUGAUGAGCCAUGCGAGGAGGAAAUGAAGUCCAAUGGCGUGAUGAGCCUCUACGGGAAGAGCAUGCCCAUCCGGGCGUGAUGAGCGUAGCAA-3 '(SEQ ID NO: 24), the sequence of short chain may, 5'-GGCCUUUGCGCCUUGCUACGCUCUGACCCGGAUGGGCAUGCUCUUCCCGUAGAGGCUCUGACCAUUGGACUUCAUUUCCUCCUCGCAUGGCUCUGACCGAUUACA-3' (SEQ ID NO: 25), L7Ae- fluorescent protein GFP The peptide sequence is VPEDMQNEALSLLEKV ESGKVKKGTNETTKAVERGLAKLVYIAEDVDPPEIVAHLPLLCEEKNVPYIYVKSKNDLGRAVGIEVPCASAAIINEGELRKELGSLVEKIKGLQKPFTVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLEFVTAAGITLGMDE Y (SEQ ID NO: 26), L7Ae- polyarginine motif peptide sequences were determined with IdiemukyuenuieieruesuerueruikeibuiaruiesujikeibuikeikeijitienuititikeieibuiiarujierueikeierubuiwaiaieiidibuidiPiPiiaibuieieichieruPieruerushiiikeienubuiPiwaiaiwaibuikeiesukeienudierujiarueibuijiaiibuiPishieiesueieiaiaienuijiieruarukeiELGSLVEKIKGLQKRRRRRRRRRRR (SEQ ID NO: 27).

In addition, in order to bind three different L7Ae fusion proteins to the three apex sites without fail, a divided Delivery-3kaku was also designed. RNA is divided into three at the sides of the triangle, and a triangle is constructed by base pairing between the sticky ends of the side. After binding the L7Ae fusion protein for each vertex, the three types of RNA-protein complexes are mixed to obtain Delivery-3kaku having one of three different proteins. Each of the three parts consists of two RNA strands, and the entire triangle consists of a total of six types of RNA strands. FIG. 20 is a diagram showing secondary structures of RNA molecules constituting divided Delivery-3kaku designed by computer molecules in Example 7.

The six RNAs constituting the divided Delivery-3kaku are the chain A-Long sequence, 5′-GGCGCAAAGGCCUGUAAUCGGUGCGAUGAGCCCAUGCGA-3 ′ (SEQ ID NO: 34, Artificial, 38nt), and the chain A-Shor sequence, respectively. -GGCUUCAUUCUCCUCCUCCGCAUGGCUCUGACCGAUUACA-3 '(SEQ ID NO: 35, Artificial, 38nt), chain B-Long sequence is 5'-GGAGGAAAUGAGCCCAAUGGCGUGAUCCCArCUt h36, t-36 -GGCAUGCUCUUCCCGU GAGGCUCUGACCAUG-3 ′ (SEQ ID NO: 37, Artificial, 33 nt), chain C-Long is 5′-GGAAGAGCAUGCCCAUCCGGGUGCGGAUGAGCGGUAGCAA-3 ′ (SEQ ID NO: 38, Artificial, 38nt), Shain-Cin It was determined as GGCCUUUGCGCCUUGCUACGCUCUGACCCCGAUG-3 ′ (SEQ ID NO: 39, Artificial, 34 nt).

Claims (8)

1. A method for inducing specific structural changes in RNA molecules with protein molecules,
   A protein comprising a non-natural RNA molecule comprising a base sequence derived from an RNA-protein complex interaction motif and an amino acid sequence that specifically binds to the base sequence derived from the RNA-protein complex interaction motif of the RNA molecule Adding a molecule.
2. An RNA molecule that changes its structure specifically and is a non-natural RNA molecule comprising a base sequence derived from an RNA-protein complex interaction motif.
3. A method for producing a specifically structurally changing RNA molecule according to claim 2, comprising:
   Selecting an RNA-protein complex interaction motif whose known structural change is known;
   Determining a base sequence of an RNA molecule, wherein the base sequence derived from the RNA-protein complex interaction motif and a scaffold base sequence are arranged by using a computer molecular modeling method; and
   And a step of producing an RNA molecule based on the base sequence obtained by the determining step.
4. A non-natural RNA molecule comprising a base sequence derived from an RNA-protein complex interaction motif, wherein the RNA molecule specifically changes in structure according to claim 2;
   A protein molecule comprising an amino acid sequence that specifically binds to a base sequence derived from the RNA-protein complex interaction motif of the RNA molecule, for inducing a specific structural change of the RNA molecule with the protein molecule kit.
5. The kit according to claim 4, further comprising an RNA molecule that competes with the RNA-protein complex formation.
6. A non-natural RNA molecule comprising a base sequence derived from an RNA-protein complex interaction motif, wherein the RNA molecule specifically changes in structure according to claim 2;
   A functional RNA-protein complex comprising an amino acid sequence that specifically binds to a base sequence derived from the RNA-protein complex interaction motif of the RNA molecule, and a protein molecule comprising a functional protein.
7. The function according to claim 7, wherein the functional protein is one or more selected from a marker protein, a membrane-permeable protein, a protein specific to a tumor, a protein having a therapeutic effect, and an antibody that recognizes a specific cell surface. Sex RNA-protein complex.
8. A method for producing an RNA-protein complex nanostructure comprising:
   A non-natural RNA molecule comprising a base sequence derived from an RNA-protein complex interaction motif contains an amino acid sequence that specifically binds to the base sequence derived from the RNA-protein complex interaction motif of the RNA molecule. A method comprising adding a protein molecule and optionally a metal ion.

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