CN110511973B - Solid phase carrier for nucleic acid in-situ synthesis and preparation method thereof - Google Patents
Solid phase carrier for nucleic acid in-situ synthesis and preparation method thereof Download PDFInfo
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Abstract
The invention provides a preparation method of a solid phase carrier for in-situ synthesis of nucleic acid, which comprises the steps of respectively carrying out first amination treatment, long-chain modification and second amination treatment on a solid phase substrate to form a long-chain organic joint on the surface of the solid phase substrate, and then grafting a cleavable linker to form the solid phase carrier. In addition, a solid phase carrier prepared by the above preparation method and a nucleic acid chip comprising the above solid phase carrier are also provided. By the preparation method, the effective selection of the solid phase substrate can be realized, and the efficiency of in-situ synthesis of nucleic acid and the purity of a nucleic acid product can be further improved.
Description
Technical Field
The invention relates to the field of gene chips, in particular to a solid phase carrier for in-situ synthesis of nucleic acid and a preparation method thereof.
Background
At present, oligonucleotide synthesis in situ by the phosphoramidite method has been widely used. In this method, it is necessary to load the nucleoside at the 3' -end of the oligonucleotide to be synthesized on a solid phase carrier via a cleavable linker in advance, and then to carry out automated synthesis of oligonucleotide by putting the carrier on a reaction column and mounting it on an automatic oligonucleotide synthesis apparatus. The synthesis steps are as follows:
(1) deprotecting the 5' -OH group of the nucleoside using a deprotection reagent;
(2) the phosphoramidite nucleoside monomer is subjected to coupling reaction with the exposed 5' -OH group in the presence of an activating agent;
(3) capping the unreacted 5' -OH groups with a capping reagent;
(4) oxidizing the phosphite with an oxidizing agent.
By repeating the above synthesis cycle, an oligonucleotide having a target sequence is synthesized, the cleavable linker is cleaved with concentrated ammonia water or the like, and the finally synthesized oligonucleotide is cleaved from the solid support to obtain a target oligonucleotide sequence.
At present, the commercial oligonucleotide synthesizer is a column synthesizer, and the synthesis method of the instrument is to fill a commercial solid phase inorganic particle carrier (such as a porous glass (CPG) carrier) in a reaction column and then perform automatic synthesis. With the need for in situ synthesis of oligonucleotide sequences, the disadvantages of such column synthesizers for oligonucleotides are becoming more and more pronounced, and there are two major problems with oligonucleotide column synthesizers: firstly, the flux of a synthesizer is small due to the limitation of a packed column, the types of oligonucleotide sequences synthesized in unit time are few, and the current market demand can not be met; on the other hand, the method is caused by the defects of the prior solid phase carrier CPG preparation technology.
The current commercial CPG carrier mainly comprises three parts: firstly, solid phase substrate material porous glass; secondly, a cleavable linker with a hydroxyl protecting group (which can be a monomer with a hydroxyl protecting group or other general structural vectors) for nucleic acid initial step synthesis; and thirdly, a linker (such as succinic anhydride and the like) for connecting the solid-phase substrate and the cleavable linker. Since the current commercial CPG is only realized on porous glass and is influenced by the adsorption effect of the porous glass on nucleic acid, the defects that the longer the oligonucleotide synthesis chain is, the lower the oligonucleotide synthesis capacity is (including the purity and synthesis efficiency of oligonucleotide synthesis) exist in the oligonucleotide synthesis process by using the current commercial CPG.
Disclosure of Invention
In view of the problems in the prior art, the present application provides a method for preparing a solid support for in situ synthesis of nucleic acid, and provides a corresponding solid support.
According to a first aspect of the present invention, there is provided a method for preparing a solid support for in situ synthesis of a nucleic acid, wherein the method comprises:
(1) carrying out first amination treatment on the solid phase substrate by using an amination modification reagent to obtain a first aminated solid phase substrate;
(2) modifying the first aminated solid phase substrate by using a long chain joint modification reagent to obtain a long chain grafted solid phase substrate;
(3) carrying out second amination treatment on the long-chain grafted solid-phase substrate by using a primary amination treatment reagent to obtain a second aminated solid-phase substrate; and
(4) and connecting the cleavable linker with the protecting group to the second aminated solid-phase substrate to obtain the solid-phase carrier.
In the above production method, the solid-phase substrate is a silicon material such as glass, quartz, ceramic, or polystyrene, or Fe3O4。
In the above preparation method, the concentration of the first amination modifying agent required per unit area of the solid substrate is 0.1% to 30%, preferably 1% to 10%.
In the above production method, the solid phase substrate is in the form of microspheres, and wherein the microspheres have a diameter of 1 μm to 1000 μm, preferably 50 μm to 150 μm.
In the above production method, the microspheres are porous microspheres, and wherein the pore diameter of the porous microspheres is
Preferably, it is
In the above preparation method, the linker modifying agent has the following structure:
wherein R is1Is a carboxyl group (-COOH), R2Is amino (-NH)2) Or halogen (-Cl, -Br, -I, etc.), in addition to R1And R2The number of carbon atoms n between the two is ≧ 6.
In the above production method, the amount of the linker-modifying agent to be used is 100. mu. mol/g to 100mmol/g, preferably 500. mu. mol/g to 5mmol/g, relative to the mass of the microspheroidal solid phase substrate.
In the above preparation method, the amount of linker-modifying reagent required per unit slide surface area for the sheet-like solid phase substrate is 10. mu. mol-10mmol, preferably 50. mu. mol-5mmol, where "unit slide area" refers to the surface area of a standard glass slide (25 mm. times.75 mm).
In the above preparation method, the amount of the cleavable linker is 1 to 150. mu. mol/g, preferably 1 to 100. mu. mol/g, relative to the mass of the microspheroidal solid-phase substrate.
According to a second aspect of the present invention, there is also provided a solid support prepared according to the preparation method of the first aspect of the present invention.
According to a third aspect of the present invention, there is also provided a nucleic acid chip comprising the solid carrier according to the second aspect of the present invention.
The invention provides a preparation method of a solid phase carrier for in-situ synthesis of nucleic acid, which comprises the steps of respectively carrying out first amination treatment, long-chain modification and second amination treatment on a solid phase substrate to form a long-chain organic joint on the surface of the solid phase substrate, and then grafting a cleavable linker to form the solid phase carrier.
In the present invention, on the one hand, the selection of the solid phase substrate is achieved by using the long-chain organic linker to link various solid phase substrates, thereby making it possible to efficiently select a suitable solid phase substrate, and thus to selectively optimize the efficiency of in situ synthesis of nucleic acid and the purity of nucleic acid product, and also to optimize further the efficiency of in situ synthesis of nucleic acid and the purity of nucleic acid by controlling the shape and pore size of the microspherical solid phase substrate; on the other hand, the long-chain organic linker of the solid phase substrate is optimized by selecting an amination reagent, a linker modification reagent and the like with appropriate concentration, so that the loading amount of the surface of the solid phase substrate is optimized, and the efficiency of in situ synthesis of nucleic acid and the purity of a nucleic acid product are further improved. Therefore, a set of reaction system capable of efficiently synthesizing nucleic acid in situ is obtained by the preparation method, so that the efficient synthesis of nucleic acid is realized.
The technical scheme of the invention overcomes the defects of the solid phase carrier preparation technology in the in-situ synthesis technology of nucleic acid in the prior art, for example, the solid phase substrate material connected with the cleavable linker is only limited to the porous glass beads, and the longer the synthesis chain of the nucleic acid is due to the influence of the adsorption effect of the porous glass on the nucleic acid, the lower the synthesis capacity of the nucleic acid is, and simultaneously, the lower the purity of the synthesized product is. According to the method, the solid phase substrate is modified by performing amination treatment, long-chain modification, second amination treatment and the like on the solid phase substrate by using a modification reagent, so that the selectivity of the solid phase substrate is improved, the efficiency of in-situ synthesis of nucleic acid is improved, and the purity of a nucleic acid product is improved.
Drawings
In order to more clearly illustrate the embodiments of the present invention, reference will now be made briefly to the accompanying drawings. It is obvious that the drawings in the following description are only some embodiments of the invention, and that for a person skilled in the art, other drawings can be derived from them without inventive effort.
FIG. 1 is a route for the preparation of solid supports for in situ synthesis of nucleic acids according to some embodiments of the invention.
Figure 2 is a mass spectrum of a nucleic acid product and corresponding purity data according to some embodiments of the invention.
FIG. 3 is a mass spectrum of a nucleic acid product synthesized by the CPG support of the comparative example and corresponding purity mass spectrum data.
FIG. 4 is a mass spectrum and corresponding purity data of nucleic acid products synthesized by commercial CPG supports.
Detailed Description
The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the described embodiments are only a few embodiments of the invention, and not all embodiments. All other embodiments that can be derived by one of ordinary skill in the art from the embodiments disclosed herein are within the scope of the present invention.
Currently, commercial solid supports for in situ synthesis of nucleic acids are composed of three main components: (1) solid phase substrate material porous glass; (2) a cleavable linker with a hydroxyl protecting group (which can be a monomer with a hydroxyl protecting group or other general structural supports, etc.) used for the synthesis of the nucleic acid initiation step; (3) and (3) a linker (such as succinic anhydride and the like) for connecting the solid-phase substrate and the cleavable linker. The current commercial solid phase carrier mainly uses a porous glass substrate, and is influenced by the adsorption effect of the porous glass on nucleic acid, and the in-situ synthesis of nucleic acid in the prior art has the defects of the reduction of the nucleic acid synthesis capability (including the purity and the synthesis efficiency of nucleic acid synthesis) of the longer nucleic acid synthesis chain.
Thus, according to a first aspect of the present invention, there is provided a method of preparing a solid support for in situ synthesis of a nucleic acid, wherein the method comprises:
(1) carrying out first amination treatment on the solid phase substrate by using a first amination modification reagent to obtain a first aminated solid phase substrate;
(2) modifying the first aminated solid phase substrate with a linker modification reagent to obtain a grafted solid phase substrate;
(3) carrying out second amination treatment on the grafted solid phase substrate by using a second amination modification reagent to obtain a second aminated solid phase substrate; and
(4) and connecting the cleavable linker with the protecting group to the second aminated solid-phase substrate to obtain the solid-phase carrier.
In a specific embodiment, a method for preparing a solid support for in situ synthesis of nucleic acids comprises:
(1) performing amination modification on the solid phase substrate by using an amination modification reagent to prepare an aminated solid phase substrate material, wherein the preparation route is shown as step S1 in FIG. 1;
(2) carrying out long-chain grafting modification on the aminated solid-phase substrate material, and utilizing a modification reagent with carboxyl at the tail end to realize the long-chain grafting of the aminated solid-phase substrate material through intermolecular dehydration condensation reaction, wherein the preparation route is shown as step S2 in figure 1;
(3) performing active primary amination treatment on the modified carrier, so as to change bromine on the long-chain grafting reagent into amino through primary amination reaction, wherein the preparation route is shown as step S3 in FIG. 1; and
(4) the cleavable linker with the protecting group is attached to the grafted solid support, and the preparation route is shown as step S4 in fig. 1.
In one embodiment, the solid phase substrate may be in the form of a sheet or a microsphere, preferably, the solid phase substrate is in the form of a porous microsphere.
In one embodiment, the solid phase substrate can be a silicon substrate or microsphere such as glass, quartz, ceramic, or polystyrene substrate or microsphere, or magnetic Fe3O4The substrate or the microsphere of (1), but the solid phase substrate is not limited thereto, and a solid phase substrate whose surface can be aminated can be used in the present invention.
In the invention, the amination modifying reagent is mainly used for amination treatment on the surface of the solid phase substrate so as to be used for further long-chain grafting. The first amination treatment with the first amination reagent is mainly used to functionalize the surface activity of the solid phase substrate so that it can react with the carboxyl group of the long-chain grafting reagent in the next step.
In one embodiment, the first amination modifying agent includes, but is not limited to, one or more of an amino group-containing silane coupling agent, chitosan, polyethyleneimine, polylysine, and like polyamino compounds. The source of the first amination reagent is not particularly limited in the present invention, and any commercially available product known to those skilled in the art to be useful for amination modification can be used in the present invention.
In one embodiment, the concentration of the first amination modifying agent required per unit area of solid phase substrate is 0.1% to 30%, preferably 1% to 10%, in an alcoholic solvent such as ethanol, methanol, and the like. When the concentration of the first amination modifying reagent is lower than 1%, the obtained first amination solid phase substrate has insufficient amino density modification, and the load capacity of the product is reduced; when the concentration of the first amination modifying reagent is higher than 10%, the amino density of the obtained first amination solid phase substrate is too high, so that the steric hindrance effect and the electronic effect of the surface are increased, the purity of the product is reduced, and the synthesis efficiency is reduced.
In one embodiment, the solid substrate is in the form of a sheet, preferably, the concentration of the first amination modifying reagent required per unit area of the solid substrate is 0.1% to 30%, preferably 1% to 10%, in an alcoholic solvent such as ethanol, methanol and the like.
In one embodiment, the solid substrate is in the form of microspheres, preferably having a diameter of from 1 μm to 1000 μm, preferably from 50 μm to 150 μm. When the microsphere particles are too small, the uniformity of the microspheres is not facilitated, and the efficiency of synthesizing products is reduced; when the size of the microspheroidal particle is too large, exchange of the reaction reagent is not facilitated, and the efficiency of synthesizing the product is reduced.
In one embodiment, the solid substrate is a porous microsphere having a pore size of
Preferably, it is
When the pore diameter of the porous micro-beads is too small, the synthesis of long-chain nucleic acid is not facilitated; when the pore size is too large, it is not favorable to increase the specific surface area of the porous microbead, which also results in a decrease in the loading amount of nucleic acid.
After the first amination treatment of the solid phase substrate, a further long chain grafting treatment is performed using a long chain modification reagent as a linker modification reagent. In one embodiment, the linker modifying agent has a structure represented by the following formula (1):
wherein R is1Is a carboxyl group (-COOH), R2Is amino (-NH)2) Or halogen (-Cl, -Br, -I, etc.), in addition to R1And R2The number of carbon atoms between n is more than or equal to 6. The source of the linker-modifying agent in the present invention is not particularly limited, and commercially available products having the above-mentioned structure, which are well known to those skilled in the art, can be used in the present invention.
In one embodiment, the amount of linker-modifying reagent used in the present invention is in the range of 100. mu. mol/g to 100mmol/g, preferably 500. mu. mol/g to 5mmol/g, relative to the mass of the microspheroidal solid phase substrate, wherein "g" refers to the mass of the solid phase substrate; for a sheet-like solid substrate, the amount of linker modifying reagent required per unit slide surface area is 10. mu. mol to 10mmol, preferably 50. mu. mol to 5mmol, where "unit slide area" refers to the surface area of a standard glass slide (25 mm. times.75 mm). When the amount of the linker-modifying agent is less than this range, the surface modification efficiency is not high, resulting in too low a loading amount of nucleic acid; when the amount of the linker-modifying agent is higher than this range, the surface nucleic acid-synthesizing ability is reduced and the product purity is lowered.
The invention uses the long-chain modifying reagent as the joint modifying reagent to carry out long-chain grafting on the surface of the solid phase substrate, thereby reducing the intermolecular electronic interference effect on the surface of the solid phase substrate material and reducing the steric hindrance effect of chemical reaction, thereby improving the purity and the synthesis efficiency of nucleic acid products. In addition, the nucleic acid synthesis effect superior to that of the commercial CPG carrier can be obtained by optimizing the amount of the reagent.
After the surface of the solid phase substrate is further subjected to a long chain grafting treatment, it is subjected to a second amination treatment using a second amination reagent, resulting in a second aminated solid phase substrate.
In one embodiment, the second amination is a primary amination, and in the present invention, the primary amination is performed by methods including, but not limited to, ammonolysis, Gabriel synthesis, or azide treatment, preferably ammonolysis, which is simple to operate and can be performed in one step.
In one embodiment, the second amination modifying reagent can be concentrated ammonia, wherein the concentration of concentrated ammonia is 25% to 28%. The source of the second amination reagent is not particularly limited in the present invention, and any commercially available product known to those skilled in the art to be useful for amination modification can be used in the present invention.
In the present invention, after the above-mentioned modification of the first amination, long-chain grafting, second amination is performed on the solid-phase carrier to obtain a second aminated solid-phase substrate, and then a cleavable linker is supported on the second aminated solid-phase substrate, in this step, the cleavable linker is supported on the second aminated solid-phase substrate by the reaction of the amino group obtained by the second amination and the carboxyl group of the cleavable linker, and then the resultant product is used as an origin compound of the nucleic acid synthesis reaction for the initiation step of nucleic acid synthesis, and the cleavable linker is cleavable under conditions of heating or alkali or the like, thereby making it possible to cleave the synthesized nucleic acid from the above-mentioned linker-modified carrier.
In one embodiment, the cleavable linker has a structure represented by the following formula (2):
wherein R is3Represents a base, and a nucleic acid base such as adenine, guanine, cytosine, thymine, uracil or the like can be used, and the above nucleic acid base may protect an amino group by a protective group such as an acetyl group, an isobutyryl group, a benzoyl group or the like; r4There may be used a hydrogen atom, a hydroxyl group, a halogen atom such as a fluorine atom, a chlorine atom, a bromine atom or the like, an alkoxy group having 1 to 20 carbon atoms such as a methoxy group or the like, a tert-butyl group, a di-substituted alkyl groupMethylsilyl and the like.
The cleavable linker as the starting compound for the nucleic acid synthesis reaction includes, but is not limited to, a nucleoside hydroxyl linker represented by the above formula (2), and nucleoside linkers to which various modification groups are bonded and common linkers that do not contain nucleosides can be used.
In one embodiment, the cleavable linker is loaded in an amount of 1 to 150. mu. mol/g, preferably 1 to 100. mu. mol/g, relative to the mass of the microspheroidal solid substrate, "g" referring to the mass of the solid substrate. When the loading amount of the cleavable linker is less than 1. mu. mol/g, the amount of nucleic acid that can be synthesized becomes small; when the loading amount of the cleavable linker exceeds 150. mu. mol/g, the synthesis ability of the nucleic acid does not show a difference compared to the solid support for nucleic acid synthesis which does not contain the long-chain organic support; relative to the sheet-shaped solid phase substrate, the number of the loaded molecules of the cleavable linker per unit area of the glass sheet is 104～1014Preferably 106～1012Here, the "unit slide area" refers to the surface area of a standard slide (25 mm. 75 mm). When the cleavable linker is loaded at less than 104At that time, the amount of nucleic acid that can be synthesized becomes small; when the loading of the cleavable linker exceeds 1012At all times, the ability of nucleic acid synthesis does not show a difference from that of a solid support for nucleic acid synthesis which does not contain a long-chain organic carrier
In the present invention, in the step (4), the cleavable linker may be supported on the solid-phase substrate by a dehydration condensation reaction, which is a reaction in which two or more organic molecules interact with each other in the presence of a condensing agent to covalently bond to form one macromolecule while water is lost.
In one embodiment, the solvent for carrying out the dehydration condensation reaction includes, but is not limited to, halogenated hydrocarbon solvents, aromatic solvents, aliphatic hydrocarbon solvents, nitrile solvents, and the like, and preferably aromatic solvents such as pyridine, which can function both as a solvent and a dehydrating agent to facilitate the condensation reaction of amino groups and carboxyl groups. Any inert solvent that can be used for the dehydration condensation reaction can be used in the present invention as long as the dehydration condensation reaction can be carried out, and the solvent for the dehydration condensation reaction is not limited in the present invention.
In one embodiment, the condensing agent includes one or more of Dicyclohexylcarbodiimide (DCC), 4-Dimethylaminopyridine (DMAP), Diisopropylcarbodiimide (DIC), N-ethyl-N' -3-dimethylaminopropylcarbodiimide or hydrochloride thereof (EDC. HC1), and the like, and Dicyclohexylcarbodiimide (DCC) and 4-Dimethylaminopyridine (DMAP) are preferable among them, and the catalytic reaction efficiency is high.
According to a second aspect of the present invention there is also provided a solid support for in situ synthesis of nucleic acids prepared by the method of the second aspect of the present invention.
According to a third aspect of the present invention, there is also provided a nucleic acid chip comprising: the solid support of the second aspect of the invention.
The invention provides a preparation method of a solid phase carrier for in-situ synthesis of nucleic acid, which comprises the steps of respectively carrying out first amination treatment, long-chain modification and second amination treatment on a solid phase substrate to form a long-chain organic joint on the surface of the solid phase substrate, and then grafting a cleavable linker to form the solid phase carrier. In the present invention, on the one hand, the selection of the solid phase substrate is achieved by using the long-chain organic linker to link various solid phase substrates, thereby making it possible to efficiently select a suitable solid phase substrate, and thus to selectively optimize the efficiency of in situ synthesis of nucleic acid and the purity of nucleic acid product, and also to optimize further the efficiency of in situ synthesis of nucleic acid and the purity of nucleic acid by controlling the shape and pore size of the microspherical solid phase substrate; on the other hand, the long-chain organic linker of the solid phase substrate is optimized by selecting an amination reagent, a linker modification reagent and the like with appropriate concentration, so that the loading amount of the surface of the solid phase substrate is optimized, and the efficiency of in situ synthesis of nucleic acid and the purity of a nucleic acid product are further improved. Therefore, a set of reaction system capable of efficiently synthesizing nucleic acid in situ is obtained by the preparation method, so that the efficient synthesis of nucleic acid is realized.
The technical solution of the present invention is described in more detail with reference to specific examples.
Example 1
This example prepares a nucleic acid chip using a quartz slide as a solid phase substrate, wherein the specification of the quartz slide is 25mm 75mm that of a standard slide.
First amination treatment of quartz slide: placing two quartz slides in a processed reaction vessel, adding 20ml (concentrated sulfuric acid: hydrogen peroxide: 7:3) of reaction solution, reacting for 3h under the ultrasonic condition of 70 ℃, cleaning with deionized water, drying with nitrogen, then adding 20ml of ethanol solution of 3-aminopropyltriethoxysilane with the concentration of 5%, reacting for 24h under the ultrasonic condition of 50 ℃, cleaning with deionized water after the reaction is finished, and placing in a 110 ℃ oven for overnight curing to obtain the first aminated slide.
Long-chain grafting of quartz slides: and (3) placing the activated aminated quartz slide into a reaction vessel, adding 600mg of 11-bromoundecanoic acid and 20ml of pyridine solution, carrying out ultrasonic reaction for 24 hours at room temperature, and cleaning with acetonitrile to obtain the long-chain grafted quartz slide.
Second amination treatment of long-chain grafted quartz slides: and (3) placing the long-chain grafted glass slide into a glass slide reaction vessel, adding 28% concentrated ammonia water into the glass slide reaction vessel, and performing ultrasonic treatment for 24 hours to obtain a second aminated long-chain grafted glass slide.
Cleavable linkers protect the thymidine linkage: and (3) placing the second aminated long-chain grafted glass slide into a glass slide reaction vessel, adding 500mg of 5' -O- (4,4' -dimethoxytriphenylmethyl) -thymidine-3 ' -O-succinic acid and 20ml of pyridine, carrying out ultrasonic treatment for 24h at room temperature, and washing with acetonitrile to obtain the thymidine-loaded chip.
Example 2
In this example, a nucleic acid chip was prepared using porous glass beads as a solid phase substrate.
First amination treatment of porous glass beads: placing 2g of porous glass beads in a 50ml centrifuge tube, wherein the diameter of the porous glass beads is about 90 μm to 110 μm and the pore diameter is
Adding 40ml of reaction solution (concentrated sulfuric acid: hydrogen peroxide: 7:3), reacting for 3h under the ultrasonic condition of 70 ℃, centrifugally cleaning by deionized water, drying by blowing nitrogen, then adding 40ml of ethanol solution of 3-aminopropyltriethoxysilane with the concentration of 5%, reacting for 24h under the ultrasonic condition of 50 ℃, centrifugally cleaning by deionized water after the reaction is finished, and placing in an oven at 110 ℃ for overnight curing to obtain the first aminated porous glass bead.
Long-chain grafting of porous glass beads: weighing 100mg of aminated porous glass beads, placing the beads in a 2ml centrifuge tube, adding 200mg of 11-bromoundecanoic acid and 1.5ml of pyridine as a solvent, reacting on a mixing instrument at room temperature for 24h, and washing with acetonitrile to obtain the long-chain grafted CPG.
Second amination treatment of long-chain grafted CPG: and (3) placing the long-chain grafted CPG particles into a 2ml centrifuge tube, adding 25% concentrated ammonia water into the centrifuge tube, placing the centrifuge tube in a constant-temperature mixing instrument at room temperature, and uniformly mixing the mixture for reaction for 24 hours to obtain the second aminated long-chain grafted CPG.
Cleavable linkers protect the thymidine linkage: placing the second aminated long-chain grafted CPG into a 2ml centrifuge tube, adding 136mg of 5' -O- (4,4' -dimethoxytriphenylmethyl) -thymidine-3 ' -O-succinic acid and 1.5ml of pyridine, carrying out ultrasonic treatment for 24h at room temperature, and washing with acetonitrile to obtain the CPG loaded with the protected thymidine.
Example 3
This example uses magnetic particles Fe3O4Prepared as a solid phase substrate.
Fe3O4Preparation of particles FeCl is weighed3·6H2O and FeCl2·4H2Dissolving O in 60ml deionized water, adding 40ml 2M ammonia water, stirring at room temperature for 1h, heating to 50 deg.C for 2h, heating to 90 deg.C for 1h, and deionizingAnd (5) performing magnetic separation and cleaning on water.
Fe3O4First amination treatment of the particles: mixing the above prepared 300mg Fe3O4The particles were placed in a three-necked flask, 2ml of ammonia water and 10ml of deionized water were added thereto and sonicated for 15min, then 100ml of isopropyl alcohol was added thereto, after further sonicating for 30min, mechanical stirring was carried out at 600rpm, 10g of polyethylene glycol 8000 was added thereto and stirred for 10min, then 5ml of tetraethyl orthosilicate (TEOS) was added thereto at a rate of 400ul/h, and magnetic separation and washing were carried out after reaction for 3 d. Cleaning, placing in 5% 3-aminopropyl triethoxysilane (APTES) ethanol solution, and reacting at 70 deg.C for 24 hr to obtain first aminated Fe3O4And (3) granules.
Fe3O4Long-chain grafting of the particles: 100mg of aminated Fe was weighed3O4Placing the particles in a 2ml centrifuge tube, adding 200mg of 11-bromoundecanoic acid and 1.5ml of pyridine as a solvent, reacting for 24h on a mixing instrument at room temperature, and washing with acetonitrile to obtain long-chain grafted Fe3O4And (3) granules.
Long chain Fe3O4Second amination treatment of the particles: fe grafted with the above long chain3O4Placing the particles in a 2ml centrifuge tube, adding 25% concentrated ammonia water, placing in a constant temperature mixing machine at room temperature, mixing uniformly, and reacting for 24h to obtain second aminated long-chain grafted Fe3O4And (3) granules.
Attachment of the cleavable linker: fe grafted with the second aminated Long chain3O4Placing the granules in a 2ml centrifuge tube, adding 136mg of 5' -O- (4,4' -dimethoxytriphenylmethyl) -thymidine-3 ' -O-succinic acid and 1.5ml of pyridine, performing ultrasonic treatment for 24h at room temperature, and washing with acetonitrile to obtain Fe loaded with protected thymidine3O4And (3) granules.
Comparative example 1
In this example, a nucleic acid chip was prepared using porous glass beads as a solid phase substrate.
First amination treatment of porous glass beads: placing 2g of porous glass beads in a 50ml centrifuge tube, wherein the diameter of the porous glass beads is about 90 μm to 110 μm and the pore diameter is
Adding 40ml of reaction solution (concentrated sulfuric acid: hydrogen peroxide: 7:3), reacting for 3h under the ultrasonic condition of 70 ℃, centrifugally cleaning by deionized water, drying by blowing nitrogen, then adding 40ml of ethanol solution of 3-aminopropyltriethoxysilane with the concentration of 5%, reacting for 24h under the ultrasonic condition of 50 ℃, centrifugally cleaning by deionized water after the reaction is finished, and placing in an oven at 110 ℃ for overnight curing to obtain the first aminated porous glass bead.
Long-chain grafting of porous glass beads: weighing 100mg of aminated porous glass beads, placing the beads in a 2ml centrifuge tube, adding 26mg of 11-bromoundecanoic acid and 1.5ml of pyridine as a solvent, reacting on a mixing instrument at room temperature for 24h, and washing with acetonitrile to obtain the long-chain grafted CPG.
Second amination treatment of long-chain CPG particles: and (3) placing the long-chain grafted CPG particles into a 2ml centrifuge tube, adding 25% concentrated ammonia water into the centrifuge tube, placing the centrifuge tube in a constant-temperature mixing instrument at room temperature, and uniformly mixing the mixture for reaction for 24 hours to obtain the second aminated long-chain grafted CPG.
Attachment of the cleavable linker: placing the second aminated long-chain grafted CPG into a 2ml centrifuge tube, adding 500mg of 5' -O- (4,4' -dimethoxytriphenylmethyl) -thymidine-3 ' -O-succinic acid and 1.5ml of pyridine, reacting for 24h on a mixing apparatus at room temperature, and washing with acetonitrile to obtain the CPG carrier loaded with protected thymidine.
Test example 1 ultraviolet spectrophotometer detection of nucleic acid load
The nucleic acid chips prepared in examples 1 to 3 and comparative example 1 and the commercial CPG carrier were subjected to dimethyl terephthalate (DMT) deprotection assay using an ultraviolet spectrophotometer according to the following specific procedures:
a. placing one glass slide prepared in example 1 into a glass slide reaction box, adding a commercial TCA reagent into the glass slide reaction box, soaking for 2h for DMT removal treatment, collecting soaking solution, placing the soaking solution into a 15ml centrifuge tube, then washing with acetonitrile for 3 times, collecting 3 times of washing solution into the 15ml centrifuge tube filled with the soaking solution, adding 1% acetonitrile solution of p-toluenesulfonic acid into the centrifuge tube, and fixing the volume to 5 ml.
b. 10mg of the CPG carrier prepared in the example 2 is taken and placed in a 2ml centrifuge tube, commercial TCA reagent is added into the CPG carrier and soaked for 2h for DMT removal treatment, soaking solution is collected and placed in a 15ml centrifuge tube, then acetonitrile is used for cleaning for 3 times, similarly, 3 times of cleaning solution is collected into a 15ml centrifuge tube filled with the soaking solution, 1% acetonitrile solution of p-toluenesulfonic acid is added into the centrifuge tube, and the volume is fixed to 5 ml.
c. Taking example 3Prepared magnetic beadsPutting 10mg in a 2ml centrifuge tube, adding a commercial TCA reagent into the centrifuge tube, soaking for 2h for DMT removal treatment, collecting soaking solution, putting the soaking solution in a 15ml centrifuge tube, then washing 3 times with acetonitrile, collecting 3 times of washing solution into the 15ml centrifuge tube filled with the soaking solution, adding 1% acetonitrile solution of p-toluenesulfonic acid into the centrifuge tube, and fixing the volume to 5 ml.
d. Putting 10mg of a commercial CPG carrier into a 2ml centrifuge tube, adding a commercial TCA reagent into the centrifuge tube, soaking for 2h for DMT removal treatment, collecting soaking solution, putting the soaking solution into a 15ml centrifuge tube, then washing with acetonitrile for 3 times, collecting 3 times of washing solution into the 15ml centrifuge tube filled with the soaking solution, adding 1% acetonitrile solution of p-toluenesulfonic acid into the centrifuge tube, and fixing the volume to 5 ml.
e. 10mg of the CPG carrier prepared in the comparative example 1 is taken and placed in a 2ml centrifuge tube, a commercial TCA reagent is added into the centrifuge tube to be soaked for 2h for DMT removal treatment, soaking liquid is collected and placed in a 15ml centrifuge tube, then acetonitrile is used for cleaning for 3 times, similarly 3 times of cleaning liquid is collected into a 15ml centrifuge tube filled with the soaking liquid, 1% acetonitrile solution of p-toluenesulfonic acid is added into the centrifuge tube, and the volume is fixed to 5 ml.
And (4) diluting the collected liquid b, c, d and e by 400 times to perform absorbance detection at 498nm of a spectrophotometer, wherein the collected liquid a can be directly used for detection. The results are shown in table 1 below:
TABLE 1 Spectrophotometer test results
| a | b | c | d | e | |
| Absorbance value | 0.64 | 1.83 | 2.43 | 1.35 | 1.26 |
From the above detection results, it can be seen that a, b, c, and e have absorbance at 498nm, which is the absorbance at 498nm of DMT as the protecting group on the modifier, and this shows that examples 1, 2, 3, and 1 are all prepared successfully.
For the microspherical solid substrate, it can be seen from the absorbance values of b, c, d of the same mass that the nucleic acids (i.e., b and c) prepared by examples 2 and 3 have larger absorbance values relative to the commercial CPG carrier d, which means that the carriers prepared by examples 2 and 3 have more nucleic acid loading, and wherein the carrier prepared by example 3 has the largest absorbance value, which is probably due to the magnetic particle Fe prepared by the present invention3O4With the largest specific surface.
As can be seen by a comprehensive comparison of the absorbance values of the same masses of a, b, c and d above, the absorbance value of a is minimal relative to b, c and d, probably because the solid substrate of a is a glass slide, has minimal surface area, and has fewer sites available for loading nucleic acids thereon, thus resulting in a smaller nucleic acid loading.
For the solid phase substrate of the porous glass beads, the absorbance of e is relatively small compared with that of b as shown by the comprehensive comparison of the absorbance of b and e with the same mass, which is mainly caused by that the nucleic acid loading is relatively small due to the small dosage and the small pore diameter of the linker modifying reagent used in the self-made CPG of comparative example 1.
Test example 2 detection of nucleic acid Loading amount and purity
1. Synthetic proof detection of nucleic acids
Nucleic acid synthesis (length 40 bp): 30mg of the CPG carrier prepared in example 2 of the present invention, 30mg of the CPG carrier prepared in comparative example 1 and 30mg of the commercial CPG carrier were weighed and filled in a column synthesizer for nucleic acid synthesis.
Shearing deprotection: placing synthesized CPG in a 1.5ml screw cap tube, treating with 800ml strong ammonia water at 65 ℃ for 4h, discarding CPG, taking supernatant, heating at 65 ℃ and vacuum concentrating to dry, adding 200 mu l of TE buffer solution (pH 7.5), standing at room temperature for 1h to fully dissolve the sheared oligo, taking the upper solution as completely as possible, placing in a new 2ml EP tube, and discarding the original tube and insoluble substances therein.
Desalting and purifying: to the DNA to be purified, sodium acetate (3mol/L pH 5.2) was added in a volume of 1/10 DNA solution (20. mu.l), and mixed well; add 220 ul of ice-precooled ethanol, mix well, after placing at-20 ℃ for at least 20min, 12000g centrifugate 10min, remove the supernatant, suck off the droplets of tube wall as much as possible, then add 1ml 70% ethanol, 12000g centrifugate 2min, remove the supernatant, suck off the droplets of tube wall as much as possible, open at room temperature, until it volatilizes to dry, add 100ml TE buffer to it to dissolve DNA precipitate (4 ℃, overnight).
Quantification: the nucleic acid product thus obtained was diluted 100-fold and quantified using NanoDrop2000, and the results are shown in Table 2 below.
TABLE 2 quantitative determination of nucleic acids
From the quantitative results, the capacity of the self-made CPG carrier is higher than that of the commercialized CPG carrier in the example 2, which is consistent with the detection result of a spectrophotometer; comparative example 1 the amount of linker reagent modified by self-made CPG was low, the pore size was small, resulting in a low loading.
2. Purity testing of nucleic acids
The mass spectrometer is used for carrying out quality detection analysis on the nucleic acid loading of the self-made CPG carrier in the example 2, and a mass spectrogram and corresponding purity data shown in the figure 2 are obtained.
And (3) carrying out quality inspection analysis on the nucleic acid load of the self-made CPG carrier in the comparative example 1 by using a mass spectrometer to obtain a mass spectrogram and corresponding purity data shown in figure 3.
The mass spectrometer was used to perform quality control analysis on the nucleic acid load of the commercial CPG carrier, and the obtained mass spectrum is shown in fig. 4 and the corresponding purity data.
As can be seen from the mass spectrograms and the corresponding purity data of fig. 2 and 3, the purity of the product obtained from the self-made CPG carrier of example 1 is 91%, the purity of the product obtained from the self-made CPG carrier of comparative example 1 is 35%, and the product obtained from the self-made CPG carrier of example 1 has lower and less peaks due to the mass spectrograms.
From the mass spectrograms and corresponding purity data of fig. 2 and 4, it can be seen that the purity of the product obtained from the self-made CPG carrier is 91%, the purity of the product obtained from the commercial CPG carrier is 63.36%, and the product obtained from the self-made CPG carrier has lower and less peaks from the mass spectrograms. Therefore, the novel CPG connection method of the invention is superior to the existing commercial CPG preparation method.
The present invention is not limited to the above preferred embodiments, and any modifications, equivalent substitutions, improvements, etc. within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (16)
1. A method for preparing a solid support for in situ synthesis of nucleic acid, wherein the method comprises the following steps:
(1) carrying out first amination treatment on the solid phase substrate by using an amination modification reagent to obtain a first aminated solid phase substrate;
(2) modifying the first aminated solid phase substrate with a long chain linker modifying agent to obtain a long chain grafted solid phase substrate, wherein the linker modifying agent has the following structure:
wherein R is1Is a carboxyl group, R2Is amino or halogen, additionally R1And R2N is not less than 6;
(3) uniformly mixing 25% -28% of concentrated ammonia water with the long-chain grafted solid-phase substrate at room temperature for reaction, and performing second amination treatment to obtain a second aminated solid-phase substrate; and
(4) connecting a cleavable linker with a protecting group to the second aminated solid-phase substrate through a dehydration condensation reaction to obtain the solid-phase carrier, wherein the cleavable linker has a structure shown in the following formula:
wherein R is3Represents a base group, R4Represents a hydrogen atom, a hydroxyl group, a halogen atom, an alkoxy group having 1 to 20 carbon atoms, a hydroxyl group protected by a t-butyldimethylsilyl group.
2. The method according to claim 1, wherein the solid substrate is made of silicon, polystyrene, or Fe3O4。
3. The method according to claim 2, wherein the silicon material is glass, quartz or ceramic.
4. The method according to any one of claims 1 to 3, wherein the concentration of the first amination modifying agent required per unit area of the solid substrate is 0.1% to 30%.
5. The method of claim 4, wherein the concentration of the first amination modifying agent required per unit area of the solid substrate is 1% to 10%.
6. The production method according to any one of claims 1 to 3, wherein the solid phase substrate is in the form of a microsphere, and wherein the microsphere has a diameter of 1 μm to 1000 μm.
7. The production method according to claim 6, wherein the diameter of the microsphere is 50 μm to 150 μm.
8. The production method according to claim 6, wherein the microspheres are porous microspheres, and wherein the pore diameters of the porous microspheres are
9. The production method according to claim 8, wherein the porous microspheres have a pore size of
10. The production method according to any one of claims 1 to 3, wherein the linker-modifying agent is used in an amount of 100 μmol/g to 100mmol/g relative to the mass of the microspheroidal solid phase substrate.
11. The method according to claim 10, wherein the linker-modifying agent is used in an amount of 500 μmol/g to 5mmol/g relative to the mass of the microspheroidal solid substrate.
12. The method of any one of claims 1-3, wherein the amount of linker modifying reagent required per unit slide surface area for the sheet-like solid phase substrate is 10 μmol-10mmol, where "unit slide area" refers to the surface area of a standard 25mm x 75mm slide.
13. The method of claim 12, wherein the amount of linker modifying reagent required per unit surface area of slide for the sheet-like solid phase substrate is 50 μmol to 5 mmol.
14. The production method according to any one of claims 1 to 3, wherein the loading amount of the cleavable linker is 1 to 150 μmol/g with respect to the mass of the microspheroidal solid-phase substrate.
15. The preparation method according to claim 14, wherein the loading amount of the cleavable linker is 1 to 100 μmol/g with respect to the mass of the microspheroidal solid-phase substrate.
16. A solid phase carrier produced by the production method according to any one of claims 1 to 15.
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| CN114106588B (en) * | 2020-08-25 | 2023-01-03 | 上海迪赢生物科技有限公司 | Functionalized surface treatment method for high-flux nucleic acid in-situ synthesis by 3D ink-jet method |
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