JP6667896B2 - Ribonucleic acid-immobilized carrier and method for separating and recovering ribonucleic acid using the same - Google Patents

Description

  The present invention relates to a ribonucleic acid-immobilized carrier and a method for separating and recovering ribonucleic acid using the same, in particular, using mesoporous silica particles as a ribonucleic acid-immobilized carrier, and adsorbing ribonucleic acid from a liquid containing ribonucleic acid. The present invention relates to a method for selectively separating and recovering ribonucleic acid.

For extraction and purification of nucleic acids (DNA (deoxyribonucleic acid) and RNA (ribonucleic acid)) present in cell lysates of animal cells and plant cells, inorganic particles such as silica particles are mainly used.
For example, unmodified silica particles have been used as an adsorbent for separating nucleic acids (DNA and RNA).
In this case, the cell lysate or the nucleic acid lysate is contained in the liquid by adding sodium perchlorate or guanidine thiocyanate as a chaotropic substance, or polyethylene glycol lauryl ether as a surfactant. Several methods have been reported for separating and recovering nucleic acids by adsorbing them on an adsorbing member (Non-Patent Document 1, Patent Documents 1 and 2, etc.).

However, these methods require the mixing of very high concentrations of chaotropic substances and surfactants, for example, chaotropic substances; 5 mol / L, surfactant; Structural stability is not guaranteed. For example, Non-Patent Document 2 reports that the addition of a chaotropic substance to a solution of double-stranded DNA reduces the thermal stability of the double-stranded DNA.
Further, in these methods, magnetic silica beads (Patent Document 1) and glass fiber filters (Patent Document 2) are used as the adsorbing member, and the specific surface area of these members is 100 m ² / g or less at the maximum. Therefore, it is necessary to further increase the specific surface area of the adsorption member for efficiently capturing the nucleic acid molecules.

In the future, research and development will be activated to efficiently separate and adsorb RNAs such as messenger RNA, transfer RNA, and ribosomal RNA existing in cells, and to analyze the reduction of diseases occurring in cells. It is expected.
Therefore, a column carrier that efficiently separates only ribonucleic acid from a solution in which biopolymers such as proteins and saccharides as well as other types of nucleic acids such as DNA are mixed is required. In addition, there is a demand for an adsorbent carrier which can be selectively separated.

  A method for selectively separating and purifying RNA is reported in Patent Document 3. One of the features of this patent document is to use silica gel having a double pore structure in which a silica skeleton having micrometer-sized pores (through pores) and nanometer-sized pores (mesopores) connected to each other is intertwined. However, the patent document is also characterized in that the solution containing RNA contains a high concentration of 1M to 8M of a chaotropic agent or a water-soluble organic solvent such as ethanol, as described above.

In Non-patent Document 3, polyadenylation (poly A chain) is formed during transcription of messenger RNA from DNA, and this specific poly A chain is used as affinity binding chromatography to convert RNA from a cell lysate or the like. As a column for selective separation and purification, a chromatography column in which a carrier surface ligand: oligothymidine (oligo dT) is immobilized on the surface of cellulose beads has been reported.
However, it is difficult to modify oligo dT on the surface of organic or inorganic carriers at high density, and it requires very high cost surface treatment. Is needed.

  As described above, in the conventional nucleic acid separation method using silica particles or the like, there is an example in which various solutions are complicatedly mixed with a nucleic acid extract to improve the nucleic acid separation amount and the selective adsorption of DNA / RNA. There are many.

On the other hand, there have been reported some examples in which the silica particles themselves were examined, such as the shape, specific surface area, nanoporosity, and surface modification of the silica particles (Patent Documents 4 to 7).
For example, Patent Documents 4 and 5 describe that an acceptor group is introduced into the surface of a silica particle to form a nonporous silica particle containing a functional substance in a surface layer or inside.
Patent Document 6 describes that a functional group that reversibly binds to a pore of mesoporous silica is introduced, and a functional substance is included in the pore.
Further, Patent Document 7 describes a DNA synthase-silica-based nanoparticle pore material composite in which a DNA synthase having a DNA synthesizing ability is adsorbed and fixed to pores of mesoporous silica. In the literature, silica-based porosity materials modified with amino groups (central pore diameter: 6.2 nm) adsorb λ-DNA molecules, but unmodified silica-based porosity materials (central pore diameter: 7. 0 nm). 2 nm) describes that DNA molecules are not adsorbed and that DNA synthase is selectively immobilized (see Example 5 and FIG. 5).
However, none of these patent documents mentions the selective adsorption of ribonucleic acid.

JP 2010-193814 A JP 2011-67150 A JP 2007-244375 A JP-A-2007-142316 JP-A-2006-070582 JP 2004-26636 A JP 2014-103924 A

R. BOOM, C. J.A.SOL, M.M.M.SALIMANS, C.L. Hamaguchi, K. and Geiduschek, E.P., The Effect of Electrolytes on the Stability of the Deoxyribonucleate Helix1, J. Am. Chem. Soc., 84, 1329-1338 (1962). JOHN A. BANTLE, IAN H. MAXWELL, AND WILLIAM E. HAHN, Specificity of Oligo (dT) -Cellulose Chromatography in the isolation of Polyadenylated RNA, ANALYTICAL BIOCHEMISTRY 72.413-427 (1976). WERNER STOBER, ARTHUR FINKand ERNST BOHN, Controlled Growth of Monodisperse Silica Spheres in the MicronSize Range, J. Colloid Interface Sci. 26, 62-69 (1968).

  As described above, as a carrier that stably separates and adsorbs nucleic acids, particularly RNA, it is a material that can be stably used without adding chaotropic substances, surfactants, alcohols, and the like. There is a need for a material that has a larger amount of ribonucleic acid adsorbed per adsorption column carrier and that can selectively adsorb and separate ribonucleic acid as compared with the adsorption member used in the technique.

  The present invention has been made in view of the above situation, and has excellent various characteristics, and separates and recovers ribonucleic acid by a simple adsorption method without requiring a complicated method using a conventional chaotropic agent or the like. It is an object of the present invention to provide a carrier for immobilizing ribonucleic acid, and a method for separating and recovering ribonucleic acid using the carrier for immobilizing ribonucleic acid.

  The present inventors have conducted intensive studies to achieve the above object, and as a result, aminated mesoporous silica particles having mesopores are excellent in various characteristics, and are suitably used as a carrier for nucleic acid separation, particularly ribonucleic acid separation and recovery. It has been found that it can be used. Further, as a result of the study, it was found that by controlling the particle size, pore size, and amination amount of the aminated mesoporous silica, ribonucleic acid can be specifically adsorbed from a liquid containing deoxyribonucleic acid and ribonucleic acid. There was found.

The present invention has been completed based on these findings, and according to the present invention, the following inventions are provided.
[1] A carrier for immobilizing ribonucleic acid, comprising aminated mesoporous silica particles having mesopores capable of selectively adsorbing ribonucleic acid.
[2] The ribonucleic acid immobilization according to [1], wherein the aminated mesoporous silica is formed by a sol-gel reaction from a mixture of a silane compound having an amino group and a silane compound having no amino group. Carrier.
[3] When the total of the silane compound having an amino group and the silane compound having no amino group in the mixture is 100 mol%, the silane compound having an amino group is 5 to 40 mol%. The carrier for immobilizing ribonucleic acid according to [2].
[4] The carrier for immobilizing ribonucleic acid according to any one of [1] to [3], wherein the pore diameter of the aminated mesoporous silica particles is in a range of 2 to 20 nm.
[5] The carrier for immobilizing ribonucleic acid according to any one of [1] to [4], wherein the aminated mesoporous silica particles have a particle diameter in a range of 50 nm to 300 nm.
[6] A method for separating and recovering ribonucleic acid from a liquid containing ribonucleic acid by adsorbing the ribonucleic acid onto a carrier for immobilization without using a chaotropic agent, wherein the solidified carrier is aminated mesoporous silica. A method for separating and recovering ribonucleic acid, comprising using particles.
[7] A ribonucleic acid is specifically adsorbed from a liquid containing deoxyribonucleic acid and ribonucleic acid by controlling the particle diameter, pore diameter, and amination amount of the aminated mesoporous silica [6]. ] The method for separating and recovering ribonucleic acid according to [1].
[8] The aminated mesoporous silica is formed by a sol-gel reaction from a mixture of a silane compound having an amino group and a silane compound having no amino group, and the silane compound having an amino group and the amino group The method for separating and recovering ribonucleic acid according to [6] or [7], wherein the silane compound having an amino group is 5 to 40% by mole, assuming that the total of the silane compounds having no amino acid is 100 mol%. .
[9] The method for separating and recovering ribonucleic acid according to any one of [6] to [8], wherein the pore diameter of the aminated mesoporous silica particles is in a range of 2 to 20 nm.
[10] The method for separating and recovering ribonucleic acid according to any one of [6] to [9], wherein the aminated mesoporous silica particles have a particle diameter in a range of 50 nm to 300 nm.
[11] a step (1) of mixing water and an organic compound for forming mesopores to form micelles and prepare a micelle-containing liquid;
A step (2) of adding a silica precursor obtained by mixing a silane compound having an amino group and a silane compound having no amino group to the micelle-containing liquid obtained in the previous step;
A step (3) of forming an organic-silica intermediate by subjecting the micelle-containing liquid to which the silica precursor has been added to a sol-gel reaction;
Washing the organic substance-silica intermediate (4);
The method for producing a carrier for immobilizing RNA according to any one of [1] to [5], comprising:
[12] When the total of the silane compound having an amino group and the silane compound having no amino group in the silica precursor is 100 mol%, the silane compound having an amino group is 5 to 40 mol%. The method for producing a carrier for immobilizing RNA according to [11].

  According to the present invention, by using aminated mesoporous silica particles as a carrier for immobilizing RNA, it is possible to stably adsorb and recover RNA without adding chaotropic substances, surfactants, alcohols, and the like. Becomes possible. When the carrier for immobilizing RNA according to the present invention is used as an RNA separation / adsorption column, it has advantages such as high strength, chemical resistance and recyclable properties of silica.

Diagram showing an example of a method for producing aminated mesoporous silica particles The figure which shows an example of this invention typically Type diagram of synthesized aminated mesoporous silica particles Scanning electron microscope (SEM) images of aminated mesoporous silica particles synthesized under various conditions Fourier transform infrared spectroscopy (FT-IR) analysis of aminated mesoporous silica particles synthesized under various conditions Diagram showing the effect of silica particles on nucleic acid (DNA and RNA) adsorption with and without mesopores Diagram showing the effect of silica particle size on nucleic acid (DNA and RNA) adsorption The figure which shows the influence which the amount of amino groups and the pore size of aminated mesoporous silica particle have on nucleic acid (DNA and RNA) adsorption. Diagram showing nitrogen adsorption and desorption curves before and after adsorption of nucleic acids (DNA and RNA) on aminated mesoporous silica particles, and pore volume and average pore diameter Figure showing the effect of aminated mesoporous silica particles to concentrate RNA from a DNA / RNA mixed solution Agarose electrophoresis diagram of RNA after adsorption and recovery on aminated mesoporous silica particles

The ribonucleic acid-immobilizing carrier of the present invention is characterized by comprising aminated mesoporous silica particles having mesopores capable of selectively adsorbing ribonucleic acid.
Further, the method of the present invention uses the ribonucleic acid-immobilizing carrier, and separates the ribonucleic acid by adsorbing the ribonucleic acid onto the immobilizing carrier from the liquid containing the ribonucleic acid without using a chaotropic agent. It is characterized by being collected.

  First, the RNA-immobilized carrier of the present invention and the method for producing the same will be described in detail.

1. Aminated Mesoporous Silica Particles The aminated mesoporous silica particles of the present invention are mesoporous silica particles having a selective adsorption property of adsorbing a large amount of RNA and minimizing the adsorption of DNA having a similar structure.

(1) Nucleic acids (DNA and RNA)
In the present invention, “nucleic acid” means deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Nucleic acid is a chain molecule consisting of nucleobases, sugars, and phosphate groups in the molecule. Usually, DNA forms a double strand by intramolecular hydrogen bonding, while RNA exists as a single strand. I do. In an aqueous solution under neutral conditions, nucleic acids have a strong negative charge due to the influence of phosphate groups.

(2) Adsorption In the present invention, “adsorption” means chemical adsorption. That is, adsorption is performed on the electrostatic interaction between the positive charge of the amino group present in the aminated mesoporous silica particles and the negative charge of the phosphate group in the nucleic acid.

(3) Aminated mesoporous silica particles In the present invention, the “aminated mesoporous silica particles” have a structure derived from a silane compound having an amino group in the basic skeleton, and have a meso region (2 to 50 nm). It means particles with pores.

Although the mesopore size of the aminated mesoporous silica particles by the BET method is not particularly limited, as described above, it is 2 to 50 nm, preferably 2 to 20 nm, and more preferably 5 to 10 nm.
The molecular size of the ribonucleic acid adsorbed on the aminated mesoporous silica particles is about 5 to 10 nm. In other words, silica having mesopores of 5 to 10 nm has the same size as RNA, and RNA can be efficiently adsorbed to amino groups on the surface of mesopores.

  The particle size of the aminated mesoporous silica particles by SEM observation is not particularly limited. The particle diameter is preferably from 50 to 1000 nm, more preferably from 50 to 500 nm, and still more preferably from 50 to 150 nm. When the content is within this range, the dispersibility of the particles becomes high, and the adsorbability of RNA also increases. That is, the amount of RNA adsorbed on the aminated mesoporous silica particles per unit weight increases.

The specific surface area of the aminated mesoporous silica particles by the BET method is not particularly limited, but is usually 15 to 1000 m ² / g, preferably 50 to 1000 m ² / g, and more preferably 100 to 1000 m ² / g. It is. This is because if the amount is within this range, the amount of RNA adsorbed on the aminated mesoporous silica particles per unit weight increases significantly.
The volume of pore volume of the aminated mesoporous silica particles by the BET method is not particularly limited, but is usually 0.01 to ² cm ³ / g, preferably 0.1 to ² m ³ / g, and more preferably. Is from 0.5 to ² cm ³ / g. This is because if the amount is within this range, the amount of RNA having a small molecular size adsorbed in the mesopores of the aminated mesoporous silica particles increases, and the ability to selectively separate RNA from DNA increases.

2. Method for Producing Aminated Mesoporous Silica Particles The aminated mesoporous silica particles of the present invention have a structure derived from a silane compound having an amino group in the basic skeleton.
Usually, as a method for obtaining mesoporous silica having an amino group, there are a method of introducing an amino group after synthesizing the mesoporous silica, and a method of simultaneously introducing an amino group during the synthesis. In the present invention, the latter method is used. More specifically, a method in which a silane compound containing an amino group and a silane compound not containing an amino group are mixed in a required ratio to start a sol-gel reaction and to contain an amino group in a SiO ₂ skeleton. Is preferably used.
As described below, in the present invention, high nucleic acid selective adsorption characteristics (RNA / DNA value) can be obtained by optimizing the particle size, pore size, and amination amount of the aminated mesoporous silica. This is because it is difficult to control the particle size, the pore size, and the amount of amination by the former method.

  The method for producing aminated mesoporous silica particles according to the present invention comprises a micelle-containing liquid preparation step (1) in which water and an organic compound for forming mesopores are mixed to form micelles, and a micelle-containing liquid is prepared. A silica precursor addition step (2) of adding a silica precursor to the micelle-containing liquid obtained in the step, and a sol-gel reaction of the micelle-containing liquid to which the silica precursor has been added to form an organic-silica intermediate (3) washing the organic matter from the organic matter-silica intermediate to form mesopores (4).

FIG. 1 shows an example of a preferred method for producing the aminated mesoporous silica particles of the present invention.
Hereinafter, each step will be described.
(1) Micelle-Containing Liquid Preparation Step In this step, as a micelle-containing liquid preparation step, an organic compound for forming mesopores is dissolved and dispersed in an aqueous solution to form uniform micelles.
The organic compound for forming mesopores is not particularly limited as long as it is an organic compound, but a surfactant is preferable.
Examples of the organic compound for forming mesopores include ionic surfactants such as alkylammonium trimethyl chloride and alkylammonium trimethyl bromide, and triblock copolymers of polyethylene oxide and polypropylene oxide having a molecular weight of 6,000 to 30,000 (P123 , F108, F127, etc., available from Aldrich-Sigma), and polyethylene glycol monocetyl ether-based nonionic surfactants having a molecular weight of 1,000 to 20,000 (Brij30, Brij58, etc., manufactured by Aldrich-Sigma) It is. The organic compounds can be used alone or in combination of two or more.
Among these organic compounds, ionic surfactants such as alkyl ammonium trimethyl chloride and alkyl ammonium trimethyl bromide are preferable.
Alkyl ammonium trimethyl chloride has high solubility in an aqueous solution, and micelles are easily formed. For this reason, mesopores of 2 to 5 nm are often obtained with a relatively uniform size.

In this step, if necessary, a solvent (organic swelling agent) for swelling the micelles and adjusting the pore size of the mesopores is mixed.
The solvent is not particularly limited, but includes, for example, 1,2-dimethylbenzene, 1,3-dimethylbenzene, 1,4-dimethylbenzene, 1,2,3-trimethylbenzene, 1,3,5-trimethylbenzene , 1,2,4-trimethylbenzene, 1,2,5-trimethylbenzene, 1,3,4-trimethylbenzene, 1,2,3-triisopropylbenzene, 1,3,5-tripropylbenzene and other alkyl Examples include a substituted benzene-based aromatic compound and an alkane-based compound such as hexane, octane, decane, and hexadecane. The solvents can be used alone or in combination of two or more.
Among these solvents, 1,3,5-trimethylbenzene is preferred. Since 1,3,5-trimethylbenzene has high hydrophobicity and a uniform molecular structure, it can be easily mixed with the above-mentioned triblock copolymer (P123) and the like, and the mesopore size can be easily adjusted.

  The mixing ratio of the organic compound for forming mesopores and the solvent is not particularly limited. When the amount of the organic compound for forming mesopores is 100 parts by weight, the amount of the solvent is usually 10 to 500 parts by weight, preferably 10 to 100 parts by weight, and more preferably 10 to 50 parts by weight. This is because if the mixing ratio is within this range, mesopores (3 to 15 nm) of the same size as the nucleic acid size (4 to 10 nm) are easily formed.

(2) Silica Precursor Addition Step In this step, in order to produce aminated mesoporous silica particles, a silica precursor is added to the micelle-containing liquid prepared in the previous step.
As the “silica precursor”, a mixture of a silane compound having an amino group (aminated silane compound) and a silane compound having no amino group is used, and any of the silane compounds is used as a skeleton raw material of mesoporous silica particles. A well-known silicon alkoxide can be suitably used.

  Although it does not specifically limit as a silicon alkoxide, tetramethoxysilane (TMOS), tetraethoxysilane (TEOS), tetrapropoxysilane, methyltrimethoxysilane, methyltriethoxysilane, n-propylethoxysilane, n-propyltrimethoxysilane , Dimethyldiethoxysilane or the like is used, preferably tetramethoxysilane (TMOS), and more preferably tetraethoxysilane (TEOS). Silicon alkoxides can be used alone or in combination of two or more.

As the amino group raw material, aminated silicon alkoxide mixed with silicon alkoxide includes 3-aminopropyltriethoxysilane, N- (2-aminoethyl) -3-aminopropyltriethoxysilane, N- (6-aminohexyl) ) Aminopropyltriethoxysila, (3-triethoxysilylpropyl-diethylenetriamine) and the like are used, and preferred is 3-aminopropyltriethoxysilane.
The aminated silicon alkoxide can be used alone or in combination of two or more.

The mixing ratio of the silane compound having an amino group (aminated silane compound) and the silane compound having no amino group, which is the “silica precursor”, is not particularly limited, but the silane compound having an amino group and the silane compound having no amino group are included. When the total of the compounds is 100 mol%, the silane compound having an amino group is 5 to 40 mol%, preferably 5 to 30 mol%, more preferably 5 to 20 mol%.
When the mixing ratio is in this range, the amount of nucleic acid, particularly ribonucleic acid, adsorbed to the synthesized aminated mesoporous particles is maximized. However, when the mixing ratio exceeds 40 mol%, the synthesis of the aminated mesoporous silica particles itself becomes difficult, and If the amount is less than 5 mol%, a sufficient amount of nucleic acid, particularly ribonucleic acid, adsorbed on the synthesized aminated mesoporous silica particles cannot be obtained.
Further, as the mixing ratio increases, the adsorption amount of ribonucleic acid (RNA) does not change significantly, but the adsorption amount of deoxyribonucleic acid (DNA) increases. As a result, the nucleic acid selective adsorption characteristics (RNA / DNA value) Decrease. Therefore, from the viewpoint of the nucleic acid-selective adsorption characteristics, the mixing ratio is preferably 5 to 30 mol%, more preferably 5 to 20 mol%.

In the above description, the (2) silica precursor addition step is performed after the (1) micelle-containing liquid preparation step, but the order of these steps, that is, the mesopore forming organic compound, the solvent, and the silica precursor The order of mixing the bodies is not particularly limited.
For example, an organic compound for forming a mesopore and a solvent may be mixed in advance, and a silica precursor may be further mixed with the mixed solution, or the organic compound for forming a mesopore, a solvent, and a silica precursor may be substantially mixed. You may mix simultaneously.
However, since the silica precursor has high hygroscopicity and decomposability, in the present production method, the mesopore forming organic compound and the solvent are preliminarily mixed and sufficiently stirred, and then the silica precursor is added to the mixed solution. Further mixing is preferred.

(3) Intermediate Forming Step This step is a step in which a mixture of the organic compound for forming mesopores and the silica precursor obtained in the previous step is subjected to a sol-gel reaction to form an organic-silica intermediate.
That is, this is a step of converting a mixture of a silane compound having an amino group and a silane carbohydrate having no amino group used as a silica precursor into aminated silica.
In this step, first, an acid or a base needs to be added to a mixed solution of the silica precursor and the organic compound for forming mesopores in order to promote the sol-gel reaction of the used silica precursor. By the addition of an acid or a base, a sol-gel reaction is induced around micelles formed by the organic compound for forming mesopores, and silicon hydroxide is formed.
The acid or base is not particularly limited. Examples of the acid include hydrochloric acid, sulfuric acid, and nitric acid. Examples of the base include sodium hydroxide, potassium hydroxide, and aqueous ammonia. Among these solvents, hydrochloric acid is preferred as the acid, and sodium hydroxide is preferred as the base. These two can not only promote the sol-gel reaction but also increase the synthesis yield of the target aminated mesoporous silica.

  Next, in order to convert the silicon hydroxide formed by the sol-gel reaction into silica, heating is performed under a temperature condition of 10 ° C to 70 ° C for 1 to 48 hours. Preferably, the reaction is carried out at room temperature (around 25 ° C.) for 12 hours. Under these conditions, complexation with the organic substance in the solution proceeds efficiently, and an organic substance-silica intermediate is formed.

(4) Cleaning Step This step is performed to remove organic substances and form mesopores.
In the production of ordinary mesoporous silica particles, it is common to remove organic substances and form mesopores by firing, but in order to prevent the amino groups introduced into the silane from being decomposed by firing, In the present invention, the cleaning is performed.
Specifically, in order to remove the organic substance of the organic substance-silica intermediate synthesized in the above section (0037), the organic substance is heated and refluxed in an organic solvent to obtain mesoporous silica particles as a target substance.
The organic solvent used is not particularly limited, but methanol, ethanol, alcohols such as n-propanol, ketones such as acetone, amides such as N, N'-dimethylamide, and the like are preferably used. Ethanol, more preferably ethanol. Ethanol can efficiently remove an organic substance from an organic substance-silica intermediate in order to sufficiently dissolve an ionic surfactant and a triblock copolymer which are organic compounds for forming mesopores.

  The heating time is preferably from 1 to 48 hours, more preferably 48 hours. Under these conditions, almost 100% of the organic substance is dissolved and removed from the organic substance-silica intermediate.

According to the above method, having a pore diameter of 2 to 50 nm, the specific surface area and the pore volume calculated by the nitrogen adsorption / desorption method are 100 to 1000 m ² / g, and 0.5 to 3.0 cm ³ / g, Aminated mesoporous silica particles having a particle diameter of about 50 to 300 nm can be obtained.

3. Aminated mesoporous silica particles to which RNA is adsorbed The aminated mesoporous silica particles to which the ribonucleic acid (RNA) of the present invention is adsorbed are such that RNA is adsorbed to amino groups present on the silica surface by electrostatic interaction. It is characterized by being.
FIG. 2 is a schematic view of a ribonucleic acid-immobilizing carrier used in the present invention, which is obtained by mixing an amino group-containing alkylsilane in a mesoporous silica particle skeleton having mesopores having an average pore diameter of 2 to 5 nm. 4 schematically shows a state in which RNA is adsorbed on the surface of the obtained silica particles.
The “nucleic acid”, “adsorption”, and “aminated mesoporous silica particles” are described in 1. above. Can be applied as it is.

Next, separation and recovery of RNA using the above-described carrier for solidifying RNA will be described below.
Nucleic acid (DNA or RNA) is converted to phosphate buffer, Tris buffer, acetate buffer, HEPES (4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid), MOPS (2-Hydroxy-3- morpholino-1-propanesulfonic acid) A) Dissolved in a buffer such as a buffer and mixed with the aminated mesoporous silica particles synthesized in the solution. The mixture is stirred overnight at room temperature to allow the nucleic acid to be adsorbed on the silica surface by electrostatic bonding utilizing the positive charge of the amino group and the negative charge of the nucleic acid. Thereafter, the silica-nucleic acid complex is recovered by centrifugation. As a buffer for dissolving nucleic acids, a phosphate buffer or Tris buffer near neutrality is desirable. Particularly, Tris buffer is preferable. This is because the structure of the dissolved nucleic acid is most stable in the neutral Tris buffer. The nucleic acid molecule bound to the amino group can be separated by suspending again in the salt solution.

The recovery of nucleic acids from aminated mesoporous silica particles to which nucleic acids have been adsorbed is performed as follows.
The nucleic acid-adsorbed aminated mesoporous silica particles are suspended in a solution containing a salt such as sodium chloride, potassium chloride, and sodium sulfate. The nucleic acid can be recovered in a salt solution by stirring at room temperature for 1 to 12 hours. The salt solution used at this time is preferably sodium chloride or potassium chloride. Among them, sodium chloride is preferred. This is because sodium chloride is inexpensive and can achieve a nucleic acid recovery of 100%.

  The collected aminated mesoporous silica particles can be reused as an RNA separation adsorbent again by the method described above.

  Hereinafter, the present invention will be described based on examples, but the present invention is not limited to these examples.

(Example 1: Synthesis of aminated mesoporous silica particles)
2.5 g of hexadecyltrimethylammonium chloride is sufficiently dissolved in a mixture of ethanol (10 mL) and distilled water (76 mL) at 60 ° C. for about 1 hour. Then, it was mixed at room temperature overnight. Next, 2 mL of triethanolamine was added to make the inside of the solution basic, and immediately after stirring at 60 ° C. for 30 minutes, a mixed solution of tetraethoxysilane / 3-aminopropyltriethoxysilane in a molar ratio of 11/2 was used. Over about 3 minutes. Thereafter, the mixture was stirred at 50 ° C. for 20 minutes and at room temperature for 12 hours. The resulting precipitate was collected by centrifugation at 6,000 rpm for 30 minutes, and the precipitate was washed with distilled water and ethanol. Further, the precipitate was dispersed in 200 mL of ethanol and heated under reflux for 12 hours to dissolve and remove hexadecyltrimethylammonium chloride as an organic template. The aminated mesoporous silica particle dispersion is collected by centrifugation at 6000 rpm for 30 minutes and dried at room temperature. Completed the object.

Further, silica spherical particles having no mesopores (so-called Stober silica, see Non-Patent Document 4) were synthesized in the same manner as in the above example except that hexadecyltrimethylammonium chloride, which is an organic compound for forming mesopores, was not added. did.
Also, after 2.5 g of hexadecyltrimethylammonium chloride was sufficiently dissolved in a mixture of ethanol (10 mL) and distilled water (76 mL) at 60 ° C. for about 1 hour, the pore size of the mesopores was reduced. Silica having a pore size increased to 5 nm under the same synthesis conditions as described in the above example, except that 2.5 g of 1,3,5-trimethylbenzene was added as a solvent (organic swelling agent) for adjustment. The particles were synthesized.
Further, under the conditions of the method for synthesizing the mesoporous silica particles described in Example 1, mainly by changing the mixing ratio of ethanol and distilled water, to control the particle size of the silica particles to be synthesized, Silica particles having particle diameters of 150 nm and 300 nm were synthesized.

FIG. 3 describes the nomenclature of silica particles synthesized under various conditions.
The 50 nm particle diameter (without mesopores) TEOS / aminopropyl ratio = 11/2 (84.6% / 15.4% molar ratio) was defined as Stober.
The particle diameter of 50 nm (with mesopores) TEOS / aminopropyl ratio = 11/2 (84.6% / 15.4% molar ratio) was set to MCM-41c 11/2, and the amino group mixing amount of the silica was 9/9. 4 (69.2% / 30.8% molar ratio) and 12/1 (92.3% / 7.7% molar ratio) silica were changed to MCM-41c 9/4 and MCM-41c 12, respectively. / 1.
The silica in which the pore size of MCM-41c 12/1 was increased to 5 mn was named MCM-41c 12/1 5 nm.
Further, MCM-41s 0.15 and MCM-41s 0.30 were silicas whose particle diameters of MCM-41c 11/2 were expanded to 150 nm or 300 nm, respectively.

FIG. 4 shows SEM observation images of aminated mesoporous silica particles synthesized under various conditions.
From the SEM observation image in FIG. 4, it was found that the synthesized mesoporous silica particles were spherical and had a particle size of about 50 to 300 nm.

The obtained silica particles were analyzed using a Fourier transform infrared spectrophotometer (FT-IR) (JASCO MFT-2000 FT-IR spectrophotometer manufactured by JASCO).
FIG. 5 is an FT-IR diagram of the aminated silica particles obtained in the examples. In each case, the peaks derived from silica (1100 cm ⁻¹ and 3400 cm ⁻¹ ) and the peak derived from the amino group (1550 cm ⁻¹ ) were obtained. ^-1 ) and a peak derived from a propyl group (around 2980 cm ^-1 ), indicating that all silica particles contain an aminopropyl chain.

(Example 2: Adsorption of nucleic acid to various aminated mesoporous silica particles)
1.5 mg of each aminated silica was mixed well with 500 μL of 10 mM Tris-HCl buffer pH 7.0. A solution of a nucleic acid (DNA or RNA: both manufactured by Sigma-Aldrich) 500 μg / 500 μL of 10 mM Tris-HCl buffer pH 7.0 was added thereto, and the mixture was stirred for 6 hours to adsorb the nucleic acid on the surface of the aminated silica. I let it. Thereafter, the mixture was centrifuged at 12,000 rpm for 10 minutes to precipitate and separate silica, and the amount of unadsorbed nucleic acid remaining in the supernatant solution was measured at a wavelength of 260 nm using an ultraviolet-visible spectrophotometer (V-560 manufactured by JASCO Corporation). .

FIG. 6 shows the results on the effect of silica particles on nucleic acid (DNA and RNA) adsorption with and without mesopores.
Referring to Non-Patent Document 1, as a result of measuring the amount of nucleic acid adsorption in a solution containing sodium perchlorate (chaotropic agent), the amount of adsorption to Stober silica was about 5 μg / mg for DNA and about 10 μg / mg for RNA. mg.
Next, as a result of an attempt to adsorb nucleic acid in a Tris-HCl buffer solution containing no chaotropic agent, the amount of adsorption to Stober silica was up to 13.7 μg / mg for DNA and up to 63.2 μg / mg for RNA, respectively. Was found to increase.
Further, in the case of silica MCM-41c 11/2 having a mesopore (pore diameter of 2.7 nm) having a similar particle diameter of 50 nm, the amount of adsorption to the silica was 54.1 μg / mg for DNA and 249.2 μg / mg for RNA. mg, indicating that the presence of pores (increase in surface area) increases the amount of adsorbed nucleic acid about four-fold.
In addition, it was found that the nucleic acid-selective adsorption characteristics of both aminated silicas were about RNA / DNA = 4.6, and no significant difference was caused.

FIG. 7 describes the effect of silica particle size on nucleic acid (DNA and RNA) adsorption.
The amount of amino groups contained in the silica particles was kept constant (TEOS / aminopropyl ratio = 11/2) and the particle size was increased from 50 nm to 150 nm: MCM-41s 0.15 and 300 nm: MCM-41s 0.30. As a result, no significant change was observed in the amount of adsorbed DNA, but the amount of adsorbed RNA slightly decreased.
This value is a result that the adsorption amount of DNA and RNA is about 1.5 to 2 times higher than that of Stober silica having no pores, but the nucleic acid selective adsorption characteristic is MCM-41s 0.30 having a particle size of 300 nm. In the case of the above, RNA / DNA = 2.33, and it was found that the ratio was reduced to about half with 4.61 in the case of silica MCM-41c 11/2.
From these results, it can be seen that aminated mesoporous silica particles exhibit high adsorption characteristics up to a particle size of about 300 nm with respect to both DNA and RNA nucleic acids, but the optimum nucleic acid selective adsorption characteristic is a silica particle size of about 50 nm. I understood.

Furthermore, in order to increase the amount of adsorbed nucleic acid and improve the nucleic acid-selective adsorption characteristics, the amount of aminopropyl group and the pore size in the silica particles were changed.
FIG. 8 shows the effect of the amount of amino groups or pore size of the aminated mesoporous silica particles on nucleic acid (DNA and RNA) adsorption.
As shown in FIG. 8, when the amount of the aminopropyl group was decreased (MCM-41c 12/1) or increased (MCM-41c 9/4), the amount was smaller than that of the silica (MCM-41c 11/2). In each case, the same high RNA adsorption amount (about 280 μg (RNA) / mg silica) was obtained.
In the case of MCM-41c 12/1 having a low amino group content, the amount of DNA adsorbed was reduced while maintaining a high RNA adsorbed amount, so that the RNA / DNA value, which is a nucleic acid selective adsorption property, was 11.08. It was clear that it would rise. Conversely, silica containing a large amount of amino groups (MCM-41c 9/4) maintained a high RNA adsorption amount, but also greatly increased the DNA adsorption amount. As a result, it was found that the nucleic acid-selective adsorption characteristics were significantly reduced (RNA / DNA = 2.89).
Although not shown in FIG. 8, no DNA or RNA was adsorbed to mesoporous silica composed of silicon having no amino group (results below the detection limit).

  Next, when the pore diameter was increased from 2.7 nm to 5.3 nm (MCM-41c 12/15 nm), almost no change in the amount of adsorbed RNA was observed (258 μg (RNA) / mg silica). However, since the amount of adsorbed DNA was greatly increased (99 μg (DNA) / mg silica), it was found that the nucleic acid-selective adsorption characteristics were slightly lowered (RNA / DNA value = 5.07).

(Example 3: Evaluation of pore structure of aminated silica before and after nucleic acid adsorption by nitrogen adsorption desorption method)
In order to elucidate the expression mechanism of the selective adsorption characteristics (RNA / DNA value = 11.08) of RNA having high aminated silica MCM-41c 12/1, DNA and RNA were separately converted to aminated silica MCM-41c. It was adsorbed on December 1 and its pore structure was measured by a nitrogen adsorption / desorption method. From the analysis results, the specific surface area, pore volume, and average pore diameter were calculated.

FIG. 9 shows the result.
The specific surface area and pore volume of the aminated silica before nucleic acid adsorption were 651 m ² / g and 1.28 cm ³ / g, respectively. In the case of silica after RNA adsorption, the values were 162 m ² / g and 0.74 cm ³ / g, and it was found that both values were greatly reduced. This indicates that the RNA molecules are adsorbed and fixed in the pores of the silica. However, in the case of DNA adsorption, the specific surface area and the pore volume were 530 m ² / g and 1.27 cm ³ / g, which were equivalent to those before the nucleic acid adsorption described above. This result suggests that there is almost no entry into the pores in the adsorption of DNA on the silica surface.
As described above, it was found that the manner of adsorbing nucleic acids on aminated silica having mesopores was significantly different between DNA and RNA.

(Example 4: RNA concentration effect from DNA and RNA mixed solution)
It is generally said that RNA has a larger mass ratio between DNA and RNA present in yeasts and cells. Then, MCM-41c 12/1, which is an aminated silica exhibiting high nucleic acid-selective adsorption characteristics (RNA / DNA value = 11.08) and obtained as a result of the examination in Example 2, was prepared with an RNA / DNA ratio of 6 The solution was mixed in a solution of / 1, and the RNA concentration effect was examined.

  First, a solution of RNA / DNA = 300 μg / 50 μg was prepared, and aminated silica MCM-41c 12/1 1.5 mg was put into the nucleic acid solution. Then, after stirring at room temperature for 6 hours, the nucleic acid adsorption-silica was recovered by centrifugation at 12,000 rpm for 10 minutes. The precipitate was mixed with 600 mM sodium chloride and stirred for 3 hours at room temperature. Again, the silica was removed by centrifugation, and the amount of recovered nucleic acid remaining in the supernatant solution was quantified. Quantification of RNA in the mixed solution of RNA and DNA was performed using the QuantiFluor (registered trademark) RNA system of Promega. The DNA in the above mixture was quantified using QuantiFluor (registered trademark) dsDNA system manufactured by Promega.

The result is shown in FIG. A solution (Mixture) of RNA / DNA = 300 μg / 50 μg (RNA / DNA = 6.0) was mixed with 1.5 mg of aminated silica MCM-41c 12/1, and then centrifuged to separate silica. Quantification of the nucleic acid remaining in the supernatant revealed that 148.4 μg of RNA and 4.0 μg of DNA (adsorption RNA / DNA = 37.1) remained, that is, 151.6 μg of RNA and 56.0 μg of RNA. It was found that the DNA was adsorbed on the silica.
As a result, when the RNA adsorption amount of the aminated silica (MCM-41c 12/1) shown in FIG. 8 was compared with (280 μg (RNA) / mg silica), the RNA adsorption amount was reduced Was found to slightly affect the adsorption of silica and RNA.

  Next, as described above, the separated nucleic acid-adsorbed silica was resuspended in a 600 mM sodium chloride solution to elute the adsorbed nucleic acid. As a result of quantifying the eluted amount, 70.8 μg RNA and 6.0 μg DNA could be collected. In other words, finally, RNA is selectively extracted from a solution of RNA / DNA = 300 μg / 50 μg = 6/1 to RNA / DNA = 70.8 μg / 6 μg = 11.8 / 1 using silica particles as a separation carrier. Concentration was achieved.

(Example 5: Electrophoretic evaluation of RNA eluted from RNA-adsorbed aminated silica)
Whether the RNA adsorbed on the aminated silica was cleaved or degraded, etc., was confirmed by agarose gel electrophoresis. A gel for electrophoresis was prepared from the 6% agarose solution. The RNA was dissolved in a gel loading buffer, loaded on an agarose gel, and electrophoresed at 100 V for 30 minutes using 1 × TAE buffer as a running buffer. Thereafter, the RNA was stained with SYBR (registered trademark) GreenI Nucleic Acid Gel Stain (Bio-Rad), and the electrophoresis image was observed under ultraviolet irradiation.

  FIG. 11 shows an agarose electrophoresis diagram of RNA after adsorption and recovery on aminated mesoporous silica particles. The left lane shows the RNA standard before adsorption, and the right lane shows the RNA collected after RNA adsorption and separation. As can be determined from the figure, the recovered RNA shows an electrophoretic image with the same molecular weight as the standard RNA, and it can be determined that no cleavage or degradation of the RNA molecule has occurred.

  According to the present invention, a carrier has been developed which can rapidly and safely carry out separation / recovery work without using a solvent or the like which may damage RNA when separating nucleic acids, particularly RNA. RNA obtained by this method can be applied to various analyzes by subsequent amplification by RT-PCR or Northern blotting. The use of this formulation is expected to greatly contribute to the development of biotechnology using RNA, such as disease and allergy analysis.

Claims (6)

A carrier for ribonucleic acid immobilization for separation and recovery of ribonucleic acid without using a chaotropic agent ,
The carrier for immobilizing ribonucleic acid is composed of aminated mesoporous silica particles having mesopores capable of selectively adsorbing ribonucleic acid,
Yes it said aminated mesoporous silica, which has been made mixture or we form the silane compound having an amino group and an amino group having no silane compound, a silane compound and an amino group having an amino group該記mixture When the total of the silane compounds not used is 100 mol%, the silane compound having an amino group is 5 to 30 mol%,
A carrier for immobilizing ribonucleic acid, wherein the pore diameter of the aminated mesoporous silica particles is in the range of 2 to 5 nm. The carrier for immobilizing ribonucleic acid according to claim 1 , wherein the aminated mesoporous silica particles have a particle size in a range of 50 nm to 300 nm. From a solution containing ribonucleic acid, without using a chaotropic agent, a method for separating and recovering ribonucleic acid by adsorbing ribonucleic acid on a carrier for immobilization,
Using aminated mesoporous silica particles as the solidification carrier,
Without the amination mesoporous silica, which has been made mixture or we form not having a silane compound of a silane compound and an amino group having an amino group, a silane compound and an amino group having an amino group in the mixture When the total of the silane compounds is 100 mol%, the silane compound having an amino group is 5 to 30 mol%,
A method for separating and recovering ribonucleic acid, wherein the pore diameter of the aminated mesoporous silica particles is in the range of 2 to 5 nm. Particle diameter of the aminated mesoporous silica, pore size, and by controlling the aminated amount, according to claim 3, characterized in that to specifically adsorb the ribonucleic acid from the liquid containing deoxyribonucleic acid and ribonucleic acid Method for separating and recovering ribonucleic acid. The method for separating and recovering ribonucleic acid according to claim 3 or 4 , wherein the particle diameter of the aminated mesoporous silica particles is in a range of 50 nm to 300 nm. Mixing water and the organic compound for forming mesopores to form micelles and prepare a micelle-containing liquid (1);
A step (2) of adding a silica precursor obtained by mixing a silane compound having an amino group and a silane compound having no amino group to the micelle-containing liquid obtained in the previous step;
A step (3) of forming an organic-silica intermediate by subjecting the micelle-containing liquid to which the silica precursor has been added to a sol-gel reaction;
Washing the organic substance-silica intermediate (4);
The method for producing a carrier for immobilizing ribonucleic acid according to claim 1 or 2 , comprising :