JP5976463B2 - Oligonucleotide synthesis apparatus and synthesis method - Google Patents

Description

  The present invention relates to an oligonucleotide synthesis apparatus and synthesis method.

  Solid phase synthesis methods for oligonucleotides such as oligodeoxyribonucleotides and oligoribonucleotides have been known for over 20 years, and automatic synthesizers are also on the market. Many of the solid-phase synthesis methods for oligonucleotides are based on a synthesis scheme in which a nucleoside is bound on a solid-phase carrier, and nucleotides are sequentially bound to the nucleoside to obtain an extended oligonucleotide.

  More specifically, for example, in the case of a commonly used phosphoramidite method, an oligonucleotide is synthesized as follows. First, a nucleoside corresponding to the 3 'end of the oligonucleotide to be synthesized is supported on a solid phase carrier via a linker such as a succinyl group. Next, the solid phase carrier carrying the nucleoside linker is placed in a reaction vessel of an automatic nucleic acid synthesizer, and acetonitrile is poured as a solvent. Then, according to the synthesis program, the nucleic acid automatic synthesizer deprotects the 5′-OH group of the nucleoside, couples the nucleoside phosphoramidite to the 5′-OH group, and removes the unreacted 5′-OH group. By repeating a cycle comprising a capping reaction and an oxidation reaction of the produced phosphite, an oligonucleotide having the target sequence is synthesized. The finally synthesized oligonucleotide is cleaved from the solid phase carrier by hydrolysis of the linker using ammonia or the like.

  As solid phase carriers used for the synthesis of such oligonucleotides, particle carriers such as polystyrene resin beads and glass porous beads are known. In particular, inorganic particles such as CPG (Controlled Pore Glass) and silica gel are widely used as conventional solid phase carriers. The reason for this is, for example, when resin beads such as low cross-linkable polystyrene used for peptide solid phase synthesis are used as the solid phase carrier for oligonucleotide synthesis, a reagent for synthesis into the solid phase carrier that is frequently performed during the synthesis cycle This is probably because the resin beads swell with the entry / exit of the solvent, and as a result, it takes time to send the reagents and the oligonucleotide cannot be synthesized with high purity. On the other hand, highly cross-linked non-swellable porous polystyrene particles are also used as solid phase carriers for oligonucleotide synthesis in order to improve chemical stability against acids and alkalis used in oligonucleotide synthesis. (See Patent Documents 1 and 2).

  However, it has been pointed out that the synthesis of oligonucleotides using particulate solid phase carriers has a large variation in yield and purity between reaction lots, low reaction efficiency, and low reproducibility. When the above-mentioned CPG or non-swellable porous polystyrene particles are used as a solid support for oligonucleotide synthesis, it is generated when the amount of nucleoside linker supported as a base point is increased in order to increase the amount of synthesis per solid support. There is a problem that the purity of the oligonucleotide to be reduced significantly.

  Compared to DNA synthesis, when RNA is synthesized, it is necessary to synthesize using a phosphoramidite with a protecting group bonded to the 2′-OH group, which reduces the coupling efficiency of amidite. As a result, there is a problem that the purity of the obtained RNA is lowered. In order to synthesize RNA without reducing the purity as much as possible, it is necessary to reduce the amount of nucleoside / linker binding on the solid phase carrier that is the base of synthesis. A new problem arises in that the amount of synthesis per phase carrier is reduced.

Japanese Patent Laid-Open No. 3-68593 JP 2010-6888 A

  In view of the above-described conventional situation, the present invention provides a device for synthesizing an oligonucleotide using a reaction on a solid phase carrier, which has a small variation in yield and purity between reaction lots, a high reaction efficiency, and a high reproducibility. The object is to provide a synthesis method.

  As a result of intensive studies to solve the above problems, the present inventors have repeatedly sent reagents and solvents in a solid-phase synthesis column reaction vessel filled with particle carriers such as conventional inorganic beads and resin beads. When the carrier swells or the oligonucleotide synthesized on the carrier expands, the space of the flow path through which the reagent or solvent passes is narrowed. This causes pressure loss, and it takes time to feed the reagent or solvent. Furthermore, it was clarified that the fact that the synthesis of oligonucleotides on the solid phase carrier could not be performed in the intended sequence order due to the non-uniform distribution was the cause of the decrease in yield. Then, by allowing the synthesis reaction to be performed on the inner wall surface of the tubular microchannel having a minute inner diameter, it was found that a space through which reagents and solvents circulate was ensured and the reaction efficiency was increased, and the present invention was completed. .

  That is, the oligonucleotide synthesizer of the present invention includes a tubular microchannel, and any functional group selected from a hydroxy group, an amino group, a carboxyl group, a methoxy group, and a thiol group is provided on the inner wall surface of the microchannel. It includes a column reaction vessel for solid phase synthesis having a group.

  According to the present invention, it is possible to uniformly distribute reagents to a column reaction vessel for solid-phase synthesis without reducing the space in which the reagents and solvents circulate, and without causing pressure loss and taking time to feed the reagents and solvents. Supply is possible. Thereby, the synthesis | combination of the oligonucleotide on a solid-phase carrier can be performed in the sequence order intended, and the fall of a yield or synthetic | combination purity can be avoided. Problems, configurations, and effects other than those described above will be clarified by the following description of embodiments.

It is a figure which shows one Embodiment of the column reaction container for solid-phase synthesis in the synthesizer which concerns on this invention. It is a figure which shows one Embodiment of the column reaction container for solid-phase synthesis in the synthesizer which concerns on this invention. It is a figure which shows one Embodiment of the column reaction container for solid-phase synthesis in the synthesizer which concerns on this invention. It is a figure which shows one Embodiment of the column reaction container for solid-phase synthesis in the synthesizer which concerns on this invention. It is a figure which shows one Embodiment of the oligonucleotide synthesis apparatus which concerns on this invention. It is a figure which shows one Embodiment of the oligonucleotide synthesis apparatus which concerns on this invention. It is a figure which shows one Embodiment of the oligonucleotide synthesis apparatus which concerns on this invention.

  Hereinafter, an oligonucleotide synthesizer using the column reaction vessel for solid phase synthesis according to the present invention will be described in detail with reference to the drawings.

  As shown in FIG. 1, a column reaction vessel 1 for solid phase synthesis in the present invention includes a tubular microchannel 2 having a small inner diameter with an inlet and an outlet communicating with each other. The material of the column reaction vessel 1 for solid phase synthesis is applicable without particular limitation as long as it is resistant to the solvent and reaction reagent to be circulated and preferably not swelled by the solvent or the like. . In particular, those having resistance to organic solvents and strong alkalis and excellent shape processability are preferred. Specifically, in addition to polystyrene, polyvinyl chloride, polyvinylidene chloride, polyethylene, polypropylene, acrylic resin (for example, polymethyl methacrylate) and the like are used. A material having low resistance to strong alkalis such as polycarbonate is not preferable.

  The column reaction vessel 1 for solid phase synthesis in FIG. 1 can be formed by ordinary injection molding. Although FIG. 1 shows an example of a cylindrical column reaction vessel for solid phase synthesis, the reaction for solid phase synthesis of oligonucleotide is carried out using the inner wall surface of the microchannel 2 as a reaction field. The outer shape of the column reaction vessel may be any shape as long as it is suitable from the viewpoints of connectivity with pipes to be connected and handling properties, and it may be a cylindrical column or a polygonal column such as a square column. Also good. The cross-sectional shape of the microchannel 2 is not particularly limited as long as injection molding is not difficult, and may be a polygon such as a quadrangle or a hexagon other than a circle. Reaction reagents, washing solutions, and the like for oligonucleotide synthesis are sent to the microchannel 2, but the ratio of the inner circumference of the channel to the cross-sectional area of the microchannel is increased in terms of reagent utilization efficiency. It is desirable to be formed. That is, the inner diameter of the microchannel is 1 mm or less, desirably 1 to 100 μm. When the inner diameter of the microchannel is larger than this, the utilization efficiency of the reagent is lowered, and conversely, when the microchannel is smaller, the pressure loss when the reagent or the like is fed may increase.

  FIG. 1 shows the case where the column reaction vessel for solid-phase synthesis is composed of a single microchannel, but as another embodiment, a plurality of microchannels are provided, It is possible to increase the amount of oligonucleotides that can be synthesized at one time by collectively molding them together. In this case, it is preferable that the respective microchannels are molded so as not to cross each other from the entrance to the exit. If the flow paths intersect in the middle, the flow may be biased in one flow path due to the difference in pressure loss in the flow paths thereafter, which reduces the yield of the synthetic oligonucleotide.

Solid-phase synthesis of oligonucleotides consists of a hydroxy group (—OH), amino group (—NH ₂ ), carboxyl group (—COOH), methoxy group (—OCH ₃ ), thiol group ( -SH), etc., through a linker that binds to a functional group, etc., so that the total area of the inner wall of the microchannel per volume of the column reaction vessel for solid phase synthesis should be as large as possible to increase the amount of oligonucleotide synthesis. It is efficient to do. Specifically, the total number of microchannel inner circumferences per cross-sectional area of the solid-phase synthesis column reaction vessel orthogonal to the microchannels is increased, that is, the number of microchannels per unit area is increased. It is preferable.

  From this viewpoint, when a plurality of microchannels are integrally formed, the cross-sectional shape of each microchannel is preferably a polygon. Particularly for solid phase synthesis by forming a structure having a large number of axially through holes partitioned by partition walls, such as a honeycomb structure used as a catalyst carrier for purifying exhaust gas from internal combustion engines and boilers. It is preferable to use a column reaction vessel. FIG. 2 shows a column reaction vessel 1 for solid phase synthesis composed of a plurality of microchannels 2 having a quadrangular cross section, and FIG. 3 is for solid phase synthesis of a honeycomb structure integrally formed by injection molding. The column reaction container 1 is shown. Examples of the resin constituting the plurality of integrally formed micro flow paths include acrylic resin (for example, polymethyl methacrylate), polyethylene, polypropylene, polystyrene, polyethylene terephthalate, polyamide, polyimide, polyester, polyvinyl chloride, and the like. Is mentioned. In particular, acrylic resins (for example, polymethyl methacrylate), polystyrene, and polyethylene terephthalate are preferable.

  FIG. 4 shows another embodiment of the column reaction vessel for solid phase synthesis in the present invention. The column reaction vessel 1 for solid phase synthesis in FIG. 4 is configured by sequentially laminating resin substrates 3 having flow channel grooves formed on one surface and closing one side of the laminate with a lid member 5. The inside of the channel groove formed in the resin substrate 3 functions as the microchannel 2. As a method of manufacturing the resin substrate 3 in which the channel groove is formed, for example, a method of manufacturing by injection molding, a method of cutting the channel groove on a flat resin substrate, or the like can be given. In FIG. 4, the cross-sectional shape of the channel groove is a quadrangle, but there is no particular limitation, and the shape may be a polygon such as a triangle, a semicircle, or an ellipse. However, from the viewpoint of production, a triangle, a quadrangle, or a semicircle is preferable. As described above, the inner diameter of the microchannel is preferably 1 mm or less, particularly about 1 to 100 μm. When the inner diameter of the flow path is larger than this, the reagent utilization efficiency decreases, and when the flow path inner diameter is small, the pressure loss at the time of liquid feeding increases, which may not be preferable. When the cross-sectional shape of the microchannel is other than circular, the “inner diameter of the microchannel” indicates the longest inner diameter of the channels in various directions.

  Examples of the resin constituting the resin substrate 3 include acrylic resin (for example, polymethyl methacrylate), polyethylene, polypropylene, polystyrene, polyethylene terephthalate, polyamide, polyimide, polyester, polyvinyl chloride, and the like. In particular, acrylic resins (for example, polymethyl methacrylate), polystyrene, polyethylene terephthalate, and the like are preferably used. There is no particular limitation on the thickness of the partition wall of the resin substrate 3 in which the channel groove is formed. However, if the partition wall thickness is too thin, the strength of the structure is insufficient. This is not preferable because the amount of oligonucleotide synthesis per volume decreases. The partition wall thickness is desirably 50 μm to 2 mm. In the bonding process when another resin substrate 3 is laminated on the resin substrate 3, an adhesive 4 may be used in addition to thermocompression bonding and ultrasonic bonding in which pressure bonding is performed by heating. Examples of such adhesives include acrylic adhesives, epoxy adhesives, urethane adhesives, and polyester adhesives.

  The inner wall surface of the molded microchannel is derivatized so as to contain a functional group necessary for realizing a covalent bond via one of silicon substituents such as nucleoside silyl ether. Examples of functional groups that can be used for this purpose include hydroxy groups, carboxyl groups, amino groups, methoxy groups, and thiol groups, as shown in FIG. The hydroxy group can react with the chlorosilane functionality of the derivatized nucleoside. The amino group can also react with a carboxyl group or an epoxy group in the silicon substituent of the derivatized nucleoside. Those skilled in the art will readily understand the relationship between the functional groups on these inner walls and the derivatized nucleosides.

  There is no restriction | limiting in particular as a modification method of the inner wall face of a microchannel, A well-known method can be used. Examples of the method for introducing a carboxyl group, an amino group, and a hydroxy group include a method of graft polymerization of a polymer such as acrylic acid, methacrylic acid, acrylamide, hydroxylethyl acrylate, and hydroxyethyl methacrylate. A dendrimer (dendritic polymer) having a desired functional group introduced into the terminal group may be bonded to the surface of the channel substrate. In this case, in order to strengthen the bond between the dendrimer and the channel substrate, a method of bonding via a silane coupling agent can be mentioned. Further, a method of introducing an oxygen-containing functional group by surface treatment such as plasma treatment, corona discharge treatment, excimer laser treatment, flame treatment or the like is possible, but the present invention is not particularly limited by these treatment methods. .

The amount of the functional group such as hydroxy group, amino group, carboxyl group, methoxy group or thiol group on the inner wall surface of the microchannel is not particularly limited, but is 0.01 to 1 mmol / cm per unit area of the inner wall surface. ² is preferable. When the density is higher than 1 mmol / cm ² , the distance between adjacent functional groups is insufficient, so that oligonucleotide chains extending adjacently by a sequential synthesis reaction are sterically hindered from each other. An oligonucleotide having a sequence cannot be obtained, and as a result, the purity of the product oligonucleotide may be lowered.

  As described above, the inner diameter of the microchannel is preferably 1 to 100 μm. If the inner diameter is smaller than 1 μm, the oligonucleotide chain elongated by the sequential synthesis reaction or the reaction reagent dissolved in high concentration crystallizes and precipitates, resulting in clogging in the flow path and a decrease in the synthesis reaction yield. There is a risk of inviting. In addition, if the inner diameter exceeds 100 μm, excessive reagents are circulated for the synthesis reaction of oligonucleotides that extend from the inner wall surface of the microchannel, which may cause an excessive increase in reagent costs. Become.

  Oligonucleotide synthesis using the microchannel of the present invention can be performed by methods known to those skilled in the art. Specifically, a synthesizer having the same configuration as the conventional one as shown in FIG. 5 can be used. Instead of the conventional column reaction vessel for solid phase synthesis filled with inorganic beads or resin beads, the column reaction vessel 1 for solid phase synthesis provided with a microchannel is used. Then, the desired oligonucleotides can be extended and synthesized on the inner wall surface of the micro-channel by repeatedly feeding the monomer reagents 10 to 13 and the solvent one after another using the liquid feed pumps 20 to 23 as in the conventional case. it can.

  Oligonucleotide is synthesized by a conventional synthesis reaction such as H-phosphonate method, phosphoester method, solid phase phosphoramidite method and the like. In particular, the solid phase phosphoramidite method is preferably employed because of its high ability to synthesize oligonucleotides and high purity oligonucleotides can be obtained. This method includes the following four steps.

(1) A detritylation step of removing DMT (dimethoxytrityl group), which is a 5′-OH protecting group of a nucleoside succinyl linker bonded to a functional group on the inner wall surface of the microchannel, with a deprotecting agent such as dichloroacetic acid,
(2) a coupling step in which a nucleoside phosphoramidite (amidite reagent) activated by tetrazole or the like is bound by a nucleophilic substitution reaction with the deprotected 5′-OH,
(3) A capping step for acetylating and protecting 5′-OH, which was not bound to the amidite reagent, with acetic anhydride, etc., and (4) oxidizing the phosphite bond derived from the amidite reagent with an oxidizing agent such as iodine. Oxidation process to form phosphate triester.

  Oligonucleotide production can be automated by connecting the column reaction vessel for solid phase synthesis to an automatic synthesizer programmed with oligonucleotide synthesis reaction steps. For example, according to an instruction from the controller of the apparatus or a computer, the reagents are distributed in a predetermined order to the reaction vessel filled with the microchannel in the present invention, with the above (1) to (4) as one cycle. Each step is advanced by the reagent distribution system. After repeating this cycle as many times as necessary for the production of the target product, the microchannel to which the synthesized oligonucleotide is bound is taken out of the reaction vessel and collected in a vial. Concentrated ammonia or the like is added thereto and left at 55 ° C. for 8 hours, for example. Thereby, a succinyl linker is decomposed | disassembled and cut | disconnected, and the target oligonucleotide is isolate | separated from a microchannel. The target oligonucleotide is obtained by removing the cyanoethyl group and converting it to the phosphodiester form, further removing the amino group protecting group, and then isolating the oligonucleotide using a filter.

  Instead of the liquid feed pumps 20 to 23 used in FIG. 5, as shown in FIG. 6, a liquid feed system using an inert gas such as argon (Ar) gas can be configured. In order to further improve the efficiency of the oligonucleotide synthesis reaction in each liquid feeding stage and improve the utilization efficiency of the reagent, buffer tanks (Tank) are provided before and after the column reaction vessel 1 for solid phase synthesis, By switching the open / close operation, the unreacted reagent can be returned again to the solid-phase synthesis column reaction vessel 1 for reaction. FIG. 7 shows a synthesizer provided with a liquid feed pump for circulating a reagent in the solid phase synthesis column reaction vessel 1 in order to avoid an increase in the size of the apparatus by providing a buffer tank.

  In a conventional bead-packed column, the space through which reagents and solvents circulate becomes narrower as oligonucleotides extend, which causes pressure loss and takes time to deliver the reagents and solvents. The homogeneity of the oligonucleotides has a high risk that the synthesis of oligonucleotides on the solid support cannot be generated in the intended sequence order. On the other hand, in the synthesizer according to the present invention, for example, by using a reaction vessel having a large number of microchannels penetrating in the axial direction partitioned by a partition wall, the above problem can be solved. Efficiency can be improved.

  In addition, this invention is not limited to above-described embodiment, Various modifications are included. For example, with respect to a part of the configuration of the embodiment, it is possible to add, delete, or replace another configuration.

DESCRIPTION OF SYMBOLS 1 Column reaction container for solid phase synthesis 2 Micro flow path 3 Resin substrate 4 Adhesive 5 Lid member

Claims (7)

A column reaction vessel for solid phase synthesis comprising a tubular microchannel, and having any functional group selected from hydroxy group, amino group, carboxyl group, methoxy group and thiol group on the inner wall surface of the microchannel look including the amount of functional groups in the inner wall surface of the microchannel, oligonucleotide synthesizer is 0.01 to 1 mmol / cm ^2.   The oligonucleotide synthesizer according to claim 1, comprising a plurality of microchannels, wherein the plurality of microchannels are integrally formed.   The oligonucleotide synthesizer according to claim 1, wherein the cross-sectional shape of the microchannel is a polygon.   The oligonucleotide synthesizer according to claim 1, wherein the inner diameter of the microchannel is 1 to 100 µm. A tubular microchannel having an inner wall surface with any functional group selected from a hydroxy group, an amino group, a carboxyl group, a methoxy group, and a thiol group, wherein the amount of the functional group on the inner wall surface is 0.01 Using a microchannel that is ˜1 mmol / cm ² , and attaching a nucleoside to a functional group on the inner wall surface of the microchannel via a linker;
A step of sequentially binding and extending nucleotides based on the bound nucleoside;
Separating the synthesized oligonucleotide from the microchannel by decomposing the linker;
A method for synthesizing oligonucleotides. The method for synthesizing oligonucleotides according to claim 5 , wherein the step of sequentially binding and extending nucleotides is performed by sequentially supplying a deprotecting agent, an amidite reagent and an oxidizing agent to the microchannel. The method for synthesizing an oligonucleotide according to claim 6 , wherein the supply of the deprotecting agent, the amidite reagent and the oxidizing agent is carried out using the pressure of an inert gas.

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