Nucleotide synthesis reagent suitable for ink jet synthesis

Abstract

The invention relates to a coupling reagent for nucleotide synthesis, comprising: a plurality of phosphamide monomer solutions wherein the mass volume ratio of phosphamide monomers to a solvent is (2-4): 30; the concentration of an activating agent in the activating solution ranges from 0.5 M to 1.25 M. The synthetic reagent prepared according to the formula of the DNA synthetic reagent suitable for ink-jet printing can be used for stable ink jet by using ink-jet printing, and meanwhile, relatively high product concentration and purity during oligonucleotide synthesis are ensured.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to a nucleotide synthesis reagent suitable for inkjet synthesis. Background Art

[0002] The nucleotide synthesis process based on inkjet printing technology requires that the oligonucleotide synthesis reagents be sprayed onto the preset target points on the glass slide or silicon-based chip in a highly accurate and stable manner. The synthesis flux of this process is high, reaching millions, while the existing column-based nucleotide synthesis flux is generally only 96-1536.

[0003] The nucleotide synthesis process gradually extends the length of the oligonucleotide chain by sequentially adding four nucleotide monomers, activators and other chemical reagents. In order to achieve high-throughput and low-cost synthesis, inkjet print heads are usually designed with high-density, small-caliber nozzles that can generate droplets with micron-level accuracy. The ejected droplets need to fall precisely on the corresponding points, so the choice of solvent for the synthetic reagent is crucial. It is necessary to ensure that the solvent has sufficient viscosity and fluidity to support stable injection and precise control of the inkjet head.

[0004] In the traditional column-based oligonucleotide synthesis process, acetonitrile (ACN) is usually used as a solvent. However, acetonitrile is not suitable for inkjet printing technology because its low viscosity and high permeability will cause the solution at the nozzle of the inkjet head to seep out without negative pressure control, thus affecting the formation and injection accuracy of the droplets. During the addition of synthetic reagents, the instability of the droplets will lead to deviations in the flight trajectory, making it impossible to achieve precise injection at the micron level, thereby reducing the synthesis efficiency of the oligonucleotide. In addition, acetonitrile is highly volatile and may evaporate before reaching the target point. Summary of the invention

[0005] In view of the technical problems existing in the prior art, the present invention proposes a coupling reagent for nucleotide synthesis, comprising: a plurality of phosphoramide monomer solutions, wherein the mass volume ratio of the phosphoramide monomer to the solvent is (2-4):30; and an activation solution, wherein the concentration of the activator in the activation solution is 0.5M-1.25M.

[0006] In some embodiments, the mass volume ratio of the phosphoramide monomer to the solvent in the phosphoramide monomer solution is (2-4):30 or (3-4):30; preferably, the mass volume ratio of the phosphoramide monomer to the solvent in the phosphoramide monomer solution is 4:30.

[0007] In some embodiments, the concentration of the activator is 0.50M-1.00M; more preferably, the concentration of the activator is 0.75M-1.00M; further preferably, the concentration of the activator is 0.75M.

[0008] In some embodiments, the plurality of phosphoramidite monomers are modified or unmodified base monomers.

[0009] In some embodiments, the base monomer is dA, dG, dC, dT or dU.

[0010] In some embodiments, the activator is selected from tetrazole compounds.

[0011] In some embodiments, the tetrazole compound is 5-ethylthiotetrazolium (ETT).

[0012] In some embodiments, the solvent in the plurality of phosphoramide monomer solutions and / or the activation solution is propylene carbonate.

[0013] The present application relates to a composite reagent for nucleotide synthesis, which comprises any coupling reagent as described above.

[0014] In some embodiments, the complex reagent further comprises a deprotection reagent; preferably, the deprotection reagent comprises trichloroacetic acid; more preferably, the mass volume ratio of the trichloroacetic acid to the deprotection reagent is (1-10) w / t%.

[0015] In some embodiments, the complexing reagent further comprises a capping reagent; preferably, the capping reagent is selected from one or more of isobutyric anhydride, N-methylimidazole, pyridine and tetrahydrofuran.

[0016] In some embodiments, the capping reagent includes: A solution, wherein the A solution is an isobutyric acid solution, wherein the volume ratio of isobutyric anhydride to its solvent is 1:(2-6); preferably, the volume ratio of isobutyric anhydride to its solvent is 1:4; and / or B solution, wherein the B solution includes N-methylimidazole, pyridine and tetrahydrofuran in a volume ratio of 10:(10-20):(60-100); preferably, the volume ratio of N-methylimidazole, pyridine and tetrahydrofuran is about 10:14:76.

[0017] In some embodiments, the complex reagent further comprises an oxidizing agent; preferably, the oxidizing agent is selected from iodine, pyridine, tetrahydrofuran (ie, THF) or a combination thereof; preferably, the oxidizing agent is an iodine solution comprising 0.01-0.1 M iodine.

[0018] The present application also relates to a method for synthesizing nucleotides using any of the above-described composite reagents, comprising: a deprotection step: adding a deprotection reagent to a reaction vessel; a coupling step: adding one of a plurality of phosphoramide monomer solutions in the coupling reagent; adding an activation solution in the coupling reagent; a capping step: adding a capping reagent; and an oxidation step: adding an oxidizing reagent.

[0019] In some embodiments, the method for synthesizing nucleotides further comprises cyclically repeating a deprotection step, a coupling step, a capping step, and an oxidation step until a specific nucleotide sequence is obtained.

[0020] In some embodiments, the method for synthesizing nucleotides further comprises a washing step, wherein a washing reagent is added after each step of the method for synthesizing nucleotides as described above is completed, or after the oxidation step is completed.

[0021] A nucleotide synthesizer comprises a plurality of liquid adding devices, wherein the plurality of liquid adding devices are configured to respectively add one of the above-mentioned composite reagents; wherein the same liquid adding device adds the same reagent.

[0022] In the nucleotide synthesizer as described above, the liquid adding device is configured to eject reagents; preferably, the liquid adding device comprises an inkjet head or a piezoelectric valve.

[0023] In the nucleotide synthesizer as described above, the diameter of the reagents sprayed by the multiple liquid adding devices is 1-1000 microns; preferably, the diameter of the reagents sprayed by the multiple liquid adding devices is 600-800 microns; or preferably, the diameter of the reagents sprayed by the multiple liquid adding devices is 20-80 microns; more preferably, the diameter of the reagents sprayed by the multiple liquid adding devices is 30-50 microns.

[0024] A method for operating a nucleotide synthesizer comprises: sequentially spraying any of the above-mentioned composite reagents through a liquid adding device.

[0025] The synthetic reagents configured in the nucleotide synthesis reagent formula suitable for inkjet printing of the present application can be stably jetted using inkjet printing, while ensuring a higher monomer coupling efficiency in the case of oligonucleotides, and having a higher product concentration and purity. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The preferred embodiments of the present invention will be further described in detail below with reference to the accompanying drawings, wherein:

[0027] Figure 1 The formation and falling state of ink droplets using acetonitrile as a solvent in a coupling step based on an inkjet head according to one embodiment of the present invention; and

[0028] Figure 2 The figure shows the formation and falling state of ink droplets using propylene carbonate as a solvent in a coupling step based on an inkjet head according to one embodiment of the present invention. DETAILED DESCRIPTION

[0029] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0030] In the following detailed description, reference may be made to the various specification drawings that are part of the present application and are used to illustrate specific embodiments of the present application. In the accompanying drawings, similar reference numerals describe substantially similar components in different figures. The various specific embodiments of the present application are described below in sufficient detail so that a person of ordinary skill in the art with relevant knowledge and skills in the art can implement the technical solutions of the present application. It should be understood that other embodiments may also be used or structural, logical or electrical changes may be made to the embodiments of the present application.

[0031] Inkjet printing-based nucleotides, i.e., using inkjet technology to achieve nucleotide oligonucleotide chain synthesis, is an innovative biotechnology that combines inkjet printing technology with nucleotide synthesis, spraying nucleotide synthesis reagents containing specific base monomers onto a substrate in the form of tiny droplets to synthesize a specific oligonucleotide sequence. In some embodiments, the nucleotide sequence can be, for example, a DNA sequence, an RNA sequence, etc.

[0032] The nucleotide synthesizer mentioned in the present application is an instrument that can synthesize oligonucleotide fragments. In certain embodiments, the nucleotide synthesizer can be a nucleotide synthesizer based on inkjet printing technology or a nucleotide synthesizer based on traditional column method. The nucleotide synthesizer adds reagents to the reaction vessel through a liquid adding device.

[0033] In some embodiments, the liquid adding device can be an inkjet head; further, the inkjet head includes a liquid inlet and a plurality of inkjet nozzles; the reagent enters the inkjet head from the liquid inlet and is ejected from the inkjet nozzles. In some embodiments, different inkjet heads will eject different reagents; in some embodiments, different inkjet nozzles of the same inkjet head can eject the same reagent. In some embodiments, the diameter of the reagent ejected by the inkjet nozzle is about 20-80 microns, preferably 30-50 microns.

[0034] In other embodiments, the liquid adding device can be a piezoelectric valve. The piezoelectric valve includes an inkjet nozzle configured to eject reagents. In some embodiments, different piezoelectric valves will eject different reagents. In some embodiments, the diameter of the reagent ejected by the piezoelectric valve is about 500-1000 microns, preferably 600-800 microns.

[0035] The nucleotide synthesizer based on inkjet printing technology (hereinafter referred to as inkjet synthesizer) includes an inkjet head, or an inkjet head and a piezoelectric valve. In some embodiments, the inkjet synthesizer ejects coupling reagents through the inkjet head, and ejects deprotection reagents, capping reagents, oxidation reagents and other reagents through the piezoelectric valve. In other embodiments, the inkjet synthesizer ejects deprotection reagents, coupling reagents, capping reagents, oxidation reagents and other reagents through the inkjet head.

[0036] The nucleotide synthesizer based on the traditional column method (hereinafter referred to as the traditional synthesizer) is a commonly used synthesizer for nucleotide synthesis. In some embodiments, the traditional synthesizer does not include an inkjet head, and the reagent is not ejected in the form of tiny droplets by the inkjet head to achieve nucleotide synthesis, but a drop of larger reagent is ejected onto the substrate through the inkjet nozzle of the piezoelectric valve for nucleotide synthesis. Compared with the inkjet synthesizer, the traditional synthesizer requires more reagents, is costly, and has a smaller nucleotide synthesis flux. Since the two are different liquid addition methods, and the droplet size is different, the reagents used in the traditional synthesizer cannot be directly used in the inkjet synthesizer. As shown in the embodiments of the present application, the reagents used for the nucleotide synthesis process of the inkjet synthesizer can be applied to the traditional synthesizer.

[0037] In some embodiments, the "conventional synthesizer" referred to in the present application is a synthesizer of model LK-192 / P4 produced by Jiangsu Lingkun Biotechnology Co., Ltd. In some embodiments, the diameter of droplets ejected by the conventional synthesizer is 600-800 microns.

[0038] Nucleotide synthesis proceeds from the 3' end to the 5' end of the target product. The 3'-OH of the initial nucleotide (e.g., deoxyribonucleotide) is covalently linked to a solid support (e.g., porous glass beads CPG) via a long alkyl arm. The "deprotection", "coupling", "capping", and "oxidation" mentioned in this application refer to the four-step chemical synthesis of nucleotides. These four steps form the basis of chemical synthesis of nucleotides and can be applied to the inkjet synthesis process of nucleotides.

[0039] Among them, "deprotection" is also called deprotection, which refers to the removal of chemically active protecting groups at the 3' and / or 5' positions of nucleotides; the terminal nucleotides with protecting groups pre-connected to a solid support are treated with dichloroacetic acid or trichloroacetic acid solution, for example, the protecting group DMT of the 5'-OH of the deoxyribose ring of DNA is removed to release the 5'-OH.

[0040] "Coupling" refers to a coupling reaction, whereby oligonucleotide chains are extended one by one on a solid support; for example, the raw materials for synthesizing DNA (phosphoramidite-protected nucleotide monomers) are mixed with the activator tetrazole to obtain a highly reactive nucleoside phosphite-activated intermediate. The activated intermediate undergoes a coupling reaction with the free 5'-OH on the terminal nucleotide to rapidly form a phosphite triester bond.

[0041] In some embodiments, the coupling process includes five phosphoramide monomers (e.g., A, G, C, T required for DNA synthesis and A, G, C, U required for RNA synthesis) and an activator. Among them, the concentrations of the five phosphoramide monomers are the same or not much different. In some embodiments, in principle, there is no particular restriction on the five phosphoramide monomers, and one of the modified or unmodified base monomers A, T, C, G and U can be used as needed, wherein the modified base monomers include but are not limited to modified bases formed by methylation, acetylation, hydrogenation, fluorination, sulfurization modification, phosphorylation modification, etc. In some embodiments, the five phosphoramide monomers are dA, dG, dC, dT and dU, respectively.

[0042] In the inkjet synthesis process of nucleotides, coupling activators play a vital role. They can enhance the connection efficiency between nucleotide molecules and ensure the accuracy and stability of nucleotide synthesis. In some embodiments, the activator is configured to give better reactivity to the phosphoramide monomer. In some embodiments, the activator can be a tetrazole compound. Further, in some embodiments, the activator is ETT or ETT solution.

[0043] The "ETT" mentioned in this application refers to 5-(Ethylthio)-1H-tetrazole, also known as 5-ethylthiotetrazolyl, which is an intermediate in the synthesis of medicines and pesticides and is often used as an activator for nucleotide synthesis. The ETT mentioned in this application and the solution of ETT have the same or similar meanings.

[0044] In some embodiments, the solvent in which the phosphoramide monomer and the activator are dissolved is the same or different. In some embodiments, the solvent is a polar aprotic solvent with a certain viscosity that is conducive to the distribution of droplets during nucleotide synthesis. In some embodiments, the solvent is a polar aprotic solvent with a boiling point above 80°C; preferably, the organic solvent can include one of acetonitrile, propylene carbonate (PC), etc., or a mixture thereof.

[0045] Among them, acetonitrile (ACN) is a commonly used solvent in nucleotide synthesis because of its strong solubility, low viscosity, strong permeability, and strong fluidity between solution molecules, which makes the reaction faster and more complete. However, due to its volatility and low viscosity, it is not suitable for nucleotide synthesis achieved by inkjet printing technology.

[0046] In the coupling process, after the four different phosphoramide monomers for synthesizing DNA or RNA are added to the predetermined synthesis reaction wells, an activator needs to be added immediately to activate the phosphoramide monomers. Since the synthesis chip is composed of many microwells, if the activator is added in excess, the liquid activator may cause the phosphoramide monomers to diffuse to other microwells with it, resulting in a "string hole" phenomenon, affecting the synthesis of phosphoramide monomers in adjacent wells, and thus affecting the accuracy of the entire synthesis process. Therefore, in order to ensure the accurate addition of phosphoramide monomers and activators in each synthesis unit and avoid the string hole phenomenon, only these four phosphoramide monomers and activators need to be precisely controlled by the inkjet head.

[0047] "Capping", also known as capping reaction, refers to preventing unreacted nucleotides from continuing to participate in subsequent reactions; in the coupling reaction, there may be very few (such as less than 2%) 5'-OH that do not participate in the reaction. The unreacted 5'-OH is acetylated so that the oligonucleotide chain in which it is located cannot continue to extend. This short fragment can be separated and removed during purification, which is beneficial to reduce synthesis errors and synthesize full-length nucleotide fragments.

[0048] "Oxidation" refers to an oxidation reaction that promotes the formation of phosphodiester bonds between nucleotides; the newly generated 3',5'-trivalent phosphite bonds are very unstable and easily break under acidic or alkaline conditions, so an oxidant is needed in the reaction to oxidize the trivalent phosphite bonds into stable pentavalent phosphite bonds.

[0049] Each time the above four steps are cycled, the oligonucleotide chain is extended by one nucleotide in the 5'-OH direction until the oligonucleotide chain of a specific sequence length is completely synthesized. The extended oligonucleotide chain is always fixed on the solid phase carrier, and the excess reaction reagents or intermediates are removed by solvent washing.

[0050] Among the above reagents involved in this application, PC is provided by Anhui Zesheng Technology Co., Ltd., and the remaining reagents are provided by Hebei Dinaxingke Biotechnology Co., Ltd.

[0051] The "stepwise coupling efficiency" mentioned in this application refers to the efficiency of each extension of a phosphoramidite monomer during DNA synthesis.

[0052] In some embodiments, the method of nucleotide synthesis comprises:

[0053] A deprotection agent is added to the reaction vessel to remove the protecting group of the terminal nucleotide 5'-OH. In some embodiments, the reaction vessel is a substrate applicable to inkjet printing technology, a groove that can be used for nucleotide synthesis, etc., to synthesize nucleotides on its surface. In some embodiments, the deprotection agent is trichloroacetic acid. Further, in some embodiments, trichloroacetic acid is dissolved in dichloromethane. In some embodiments, the concentration of trichloroacetic acid is (1-10) w / t% by mass volume ratio; preferably, the concentration of trichloroacetic acid is (1.5-8) w / t% by mass volume ratio; more preferably, the concentration of trichloroacetic acid is (2-6) w / t% by mass volume ratio; further preferably, the concentration of trichloroacetic acid is (2.5-4) w / t% by mass volume ratio; further preferably, the concentration of trichloroacetic acid is about 3w / t% by mass volume ratio.

[0054] Add one of a plurality of phosphoramide monomer solutions; wherein the plurality of phosphoramide monomers are base monomers. Further, in some embodiments, the base monomer is dA, dG, dC, dT or dU. In some embodiments, the phosphoramide monomer dA or dG or dC or dT or dU is dissolved in a solvent, respectively, and the volume of the monomer is 1:30 w / v, or 1:15 w / v, or 1:10 w / v, or 2:15 w / v, or 1:6 w / v. Wherein, w / v refers to the mass volume ratio, for example, 1:30 w / v means that 1 gram of phosphoramide monomer is dissolved in 30 ml of solvent.

[0055] An activator solution is added, which is configured to enhance the connection efficiency between nucleotide molecules and ensure the accuracy and stability of nucleotide synthesis. In some embodiments, the activator is configured to impart better reactivity to the phosphoramide monomer. In some embodiments, the activator can be a tetrazole compound. Further, in some embodiments, the activator is ETT or an ETT solution. In the present application, the ETT solution is a solution obtained by dissolving ETT in a solvent. Further, the concentration of ETT in the ETT solution is 0.1-1M; preferably, the concentration of ETT in the ETT solution is 0.2-0.8M; further preferably, the concentration of ETT in the ETT solution is 0.25-0.75M.

[0056] A capping agent is added, which is configured to prevent the phosphoramide monomers that are not involved in the reaction from participating in the subsequent nucleotide synthesis. In some embodiments, the capping agent includes isobutyric anhydride, tetrahydrofuran, pyridine, acetic anhydride, N-methylimidazole or a combination thereof. Preferably, the capping agent may include both capping agent A and / or capping agent B, and capping agent A may include isobutyric anhydride, which is dissolved in an organic solvent, and the organic solvent is one or both of acetonitrile or propylene carbonate. In some embodiments, the volume ratio (v / v) of isobutyric anhydride to the organic solvent is 1:(2-6); preferably, the volume ratio of isobutyric anhydride to the organic solvent is 1:4. In some embodiments, capping agent B may include N-methylimidazole, pyridine, tetrahydrofuran or a combination thereof. In some embodiments, capping agent B includes N-methylimidazole, pyridine, and tetrahydrofuran, and the volume ratio (v / v / v) of the three is 10:(10-20):(60-100); preferably, the volume ratio of the three is about 10:14:76. In some embodiments, capping reagent A and capping reagent B are used in combination.

[0057] An oxidizing agent is added. In some embodiments, the oxidizing agent may include iodine, pyridine, tetrahydrofuran (ie, THF) or a combination thereof; more preferably, the oxidizing agent includes iodine, as well as pyridine, tetrahydrofuran or a combination thereof. Further preferably, the oxidizing agent may be used in the form of an aqueous solution. In a more preferred embodiment, the oxidizing agent is an iodine solution, wherein the concentration of iodine is 0.01-0.1M, preferably, the iodine concentration is 0.05. The solvent is an aqueous solution of pyridine and THF, wherein the volume ratio (v / v / v) of water, pyridine, and THF is 10:(15-25):(60-80); preferably, the volume ratio of water, pyridine, and THF is 10:20:70.

[0058] In some embodiments, the method for nucleotide synthesis also includes a cleaning step to remove unwanted residues or spills that may be present in or around the reaction vessel. In some embodiments, the cleaning step may be performed throughout the entire nucleotide synthesis process, that is, after each process (such as deprotection, coupling, capping, or oxidation step) is completed, a cleaning solution is added for cleaning; or after a cycle is completed (that is, after the oxidation step is completed), a cleaning solution is added for cleaning. In some embodiments, the clear and dilute solution is an organic solvent. In some embodiments, the organic solvent may have good compatibility with the organic solvent in the reagent described above, or may be the same or similar to the organic solvent in the reagent. In some embodiments, the cleaning solution is a solvent for the reagent added in each step. In other embodiments, the cleaning solution is acetonitrile; in other embodiments, the cleaning solution is propylene carbonate.

[0059] In some embodiments, except for the coupling step, the reagents used in other steps (deprotection, capping, oxidation) and the cleaning process are the same for different synthesis sites. Therefore, a liquid addition pump can be used to add excess cleaning reagents, while cleaning each oligonucleotide chain being synthesized, and the reagents can be sucked in and discharged through a negative pressure system.

[0060] Those skilled in the art should understand that the synthesis process of nucleotides should be a cyclic process, and each cycle will extend the length of the nucleotide sequence by 1 bp, that is, extend one nucleotide at the end of the nucleotide; then enter the next cycle and continue to extend the nucleotide sequence until the DNA synthesis of a specific sequence length is completed. The extended nucleotide chain is always fixed on the solid phase carrier, and the excess reaction reagents or intermediates are removed by solvent washing.

[0061] The invention provides a coupling reagent for nucleotide synthesis, comprising: a plurality of phosphoramide monomer solutions and an activation solution.

[0062] In some embodiments, the plurality of phosphoramide monomers are modified or unmodified base monomers; further, in some embodiments, the base monomers are dA, dG, dC, dT or dU. In some embodiments, the mass volume ratio of phosphoramide monomer to solvent is (2-4):30; in some embodiments, the mass volume ratio of phosphoramide monomer to solvent in the phosphoramide monomer solution is (2-4):30 or (3-4):30; preferably, the mass volume ratio of phosphoramide monomer to solvent in the phosphoramide monomer solution is 4:30.

[0063] In some embodiments, the activator is selected from tetrazole compounds. In some embodiments, the tetrazole compound is 5-ethylthiotetrazolyl (ETT). In some embodiments, the concentration of the activator in the activation solution is 0.5M-1.25M; preferably, the concentration of the activator is 0.50M-1.00M; more preferably, the concentration of the activator is 0.75M-1.00M; further preferably, the concentration of the activator is 0.75M.

[0064] In some embodiments, the solvent in the plurality of phosphoramide monomer solutions and / or activation solutions is an organic solvent. Further, in some embodiments, the solvent is acetonitrile or propylene carbonate.

[0065] The present application relates to a composite reagent for nucleotide synthesis, which includes, in addition to the coupling reagent described above, a deprotection reagent, a capping reagent and an oxidation reagent. In some embodiments, the composite reagent also includes a cleaning reagent.

[0066] In some embodiments, the deprotecting agent includes trichloroacetic acid (TCA); preferably, the deprotecting agent solution is a solution obtained by dissolving trichloroacetic acid in dichloromethane (DCM). Preferably, the mass volume ratio of trichloroacetic acid to the deprotecting agent is (1-10) w / t%. More preferably, the mass volume ratio of trichloroacetic acid to the deprotecting agent is 3 w / t%.

[0067] In some embodiments, the capping agent is selected from one or more of isobutyric anhydride, N-methylimidazole, pyridine and tetrahydrofuran. In some embodiments, the capping agent comprises: A solution, A solution is an isobutyric acid solution, and its solvent is acetonitrile or propylene carbonate; wherein the volume ratio of isobutyric anhydride to its solvent is 1:

[0068] (2-6); preferably, the volume ratio of isobutyric anhydride to its solvent is 1:4; and / or B solution, B solution includes N-methylimidazole, pyridine and tetrahydrofuran in a volume ratio of 10:(10-20):(60-100); preferably, the volume ratio of N-methylimidazole, pyridine and tetrahydrofuran is about 10:14:76. In some embodiments, A solution and B solution.

[0069] In some embodiments, the oxidizing agent is selected from iodine, pyridine, tetrahydrofuran (THF) or a combination thereof; in some embodiments, the oxidizing agent is an iodine solution comprising 0.01-0.1M iodine; in some embodiments, the oxidizing agent is an iodine solution comprising 0.05M iodine. Further, in some embodiments, the solvent in the iodine solution comprises water, pyridine and tetrahydrofuran (THF); preferably, the volume ratio of water, pyridine and tetrahydrofuran is 10: (10-40): (50-80); more preferably, the ratio of water, pyridine and tetrahydrofuran is 10: 20: 70.

[0070] The present application further relates to a nucleotide synthesizer, comprising a plurality of liquid adding devices, wherein the heads of the plurality of liquid adding devices are configured to respectively eject one of the composite reagents described above; wherein the same liquid adding device ejects the same reagent.

[0071] For example, the deprotection reagent is sprayed out through the first liquid-adding device; the dA reagent is sprayed out through the second liquid-adding device; the dG reagent is sprayed out through the third liquid-adding device; the dC reagent is sprayed out through the fourth liquid-adding device; the dT reagent is sprayed out through the fifth liquid-adding device or the dU reagent is sprayed out through the sixth liquid-adding device; the activation reagent is sprayed out through the seventh liquid-adding device; the cap A reagent is sprayed out through the eighth liquid-adding device; the cap B reagent is sprayed out through the ninth liquid-adding device and the oxidizing reagent is sprayed out through the tenth nozzle. This paragraph describes only the spraying relationship between the liquid-adding device and the composite reagent, and does not limit the technical solution of the present application. In some embodiments, each liquid-adding device is an inkjet head. In some embodiments, the second to sixth liquid-adding devices are inkjet heads, and the first and seventh to ninth liquid-adding devices are piezoelectric valves. In some embodiments, each liquid-adding device is a piezoelectric valve.

[0072] In some embodiments, the complex reagent is sprayed into a reaction vessel. Depending on different reaction requirements, the reaction vessel can have different shapes or include different components. For example, the reaction vessel can be a substrate including a groove, which includes a solid carrier, which can fix the phosphoramide monomer.

[0073] In some embodiments, the diameter of the small ink droplets of reagent sprayed by the multiple liquid adding devices is 1-1000 microns; preferably, the diameter of the small ink droplets of reagent sprayed by the multiple liquid adding devices is 600-800 microns or 20-80 microns; more preferably, the diameter of the small ink droplets of reagent sprayed by the multiple liquid adding devices is 30-50 microns.

[0074] The present application further relates to an operating method of a nucleotide synthesizer, comprising: sequentially ejecting the aforementioned composite reagents through a liquid adding device. For example, when synthesizing DNA, first eject the deprotection reagent through the first liquid adding device, then eject the amide monomer reagent through one of the second to fifth liquid adding devices as required; then eject the activation reagent through the seventh liquid adding device; then eject the capping A reagent through the eighth liquid adding device, and eject the capping B reagent through the ninth liquid adding device, and finally eject the oxidizing reagent through the tenth nozzle.

[0075] The following will take the synthesis of oligonucleotide sequences of DNA by a nucleotide synthesizer as an example to illustrate the technical solution of the present application. Those skilled in the art should understand that the following specific examples are only for illustrating the implementation scheme of the present application, and are not intended to limit the protection scope of the present application.

[0076] Example 1 Effect of phosphoramidite monomer concentration on DNA synthesis

[0077] In this embodiment, the deprotection reagent is 3% trichloroacetic acid (TCA) dissolved in dichloromethane (DCM). The capping reagent is isobutyric anhydride / ACN (1:4, v / v) and N-methylimidazole / pyridine / tetrahydrofuran (10 / 14 / 76, v / v / v). The oxidizing reagent is iodine solution (water / pyridine / THF, 0.05M, 10 / 20 / 70, v / v / v). The applicant further verified the effect of the concentration of phosphoramide monomers in the coupling reagent on DNA synthesis.

[0078] Experimental groups 1-5 were prepared with PC as solvent and the concentration of phosphoramide monomer and activator increased to 1, 2, 3, 4 and 5 times of the normal concentration. By designing the synthesis experiment of the 20nt target product, keeping the activator concentration at 5 times the normal concentration, gradually increasing the concentration of phosphoramide monomer, measuring the concentration and coupling efficiency of the product, the optimal concentration of phosphoramide monomer was determined. Among them, the conventional concentration of phosphoramide monomer (1 times) is 1:30 mass volume ratio of monomer to solvent, w / t; 2 times is 2:30 mass volume ratio of monomer to solvent (i.e. 1:15), w / t; 3 times is 3:30 mass volume ratio of monomer to solvent (i.e. 1:10), w / t; 4 times is 4:30 mass volume ratio of monomer to solvent (i.e. 2:15), w / t; 5 times is 5:30 mass volume ratio of monomer to solvent (i.e. 1:6), w / t; in conventional use, the concentration of activator ETT in solvent is 0.25M, and the conventional concentration of 5 times is 0.75M; 6 times the conventional concentration of ETT has exceeded its solubility limit, and 5 times the conventional concentration is the highest solubility of ETT. Among them, the target sequence of the experimental group is: AGCTAGCTAGCTAGCTAGCTAGCT.

[0079] Control group 1 is a phosphoramide monomer and an activator prepared with PC as a solvent, and the concentrations are normal. The target sequence of control group 1 is: AGCT.

[0080] Control group 2 is a conventional concentration of phosphoramide monomer and activator prepared with ACN as solvent, i.e., the mass volume ratio of phosphoramide monomer to solvent is 1:30, w / t; the concentration of activator in ETT solvent is 0.25 M. The target sequence of control group 2 is: AGCT.

[0081] In this embodiment, the ambient temperature is room temperature, about 20-30 degrees Celsius, and the relative humidity is about 45%. The experiment and results are shown in Table 1:

[0082] Table 1 Synthesis sequence of PC-configured activators and phosphoramide monomers with different concentrations

[0083]

[0084]

[0085] Note: Each experiment was repeated 8 times and the results were averaged.

[0086] The slash in experimental group 1 indicates that no target sequence was synthesized under this concentration ratio. The results showed that during DNA synthesis, the ACN group had better activity than the PC group, and the DNA synthesis efficiency and yield were much higher than those of the PC as solvent group. When PC was used as a solvent, the concentration of the activator configured with PC was kept at 5 times the normal concentration, and the concentration of the phosphoramide monomer configured with PC was kept at 4 times the normal concentration, the concentration and coupling efficiency of the synthetic product were the highest, which were 2.71 μM and 98.70%, respectively.

[0087] Example 2 Effect of activator concentration on DNA synthesis

[0088] Based on the results of Experimental Group 4 of Example 1, the concentration of the activator was further adjusted to detect the effect of the concentration of the activator on DNA synthesis.

[0089] In this embodiment, the ambient temperature is room temperature, about 20-30 degrees Celsius, and the relative humidity is about 30%. PC is used as a solvent, the phosphoramide monomer concentration is about 4:30, w / v, and the concentration of the activator is gradually increased to verify its effect on the concentration of the synthetic product and the coupling efficiency, and finally determine the optimal concentration of the activator in the PC solvent.

[0090] The conventional concentration of the activator (1 times concentration) is about 0.25 M; the 2 times concentration of the activator is about 0.5 M; the 3 times concentration of the activator is about 0.75 M; the 4 times concentration of the activator is about 1.00 M; and the 5 times concentration of the activator is about 1.25 M. The target sequence in this embodiment is: AGCTAGCTAGCTAGCTAGCTAGCT.

[0091] The experiments and results are shown in Table 2:

[0092] Table 2 Synthesis sequence of PC-configured phosphoramide monomers and activators of different concentrations

[0093]

[0094]

[0095] Note: Each experiment was repeated 8 times and the results were averaged.

[0096] The results showed that when the concentration of phosphoramide monomer in PC configuration was kept at 4 times of the conventional concentration and the concentration of activator in PC configuration was kept at 3 times of the conventional concentration, the concentration and coupling efficiency of the synthesized product were the highest, which were 4.77 μM and 99.41%, respectively.

[0097] Both Table 1 and Table 2 include the results when the phosphoramide monomer concentration is 4:30, w / v and the activator concentration is 1.25M, but the concentrations and step-coupling efficiencies of the two are different. Those skilled in the art should understand that the ambient temperature and relative humidity have a great influence on the ink droplets and DNA synthesis during the DNA synthesis process. The reagents used in Table 1 and Table 2 are the same, but the relative humidity of the environment during the experiment is not the same. It can be seen from Example 1 and Example 2 that a relative humidity of no more than about 45% can be used for DNA synthesis. Preferably, when the relative humidity is no more than about 30%, the DNA synthesis efficiency can be effectively improved. It can be inferred that the lower the ambient humidity, the higher the DNA synthesis efficiency.

[0098] Example 3 Effect of solvent on inkjet printing

[0099] In this embodiment, the applicant measured the inkjet state of acetonitrile and propylene carbonate as organic solvents in the coupling step. Among them, the instrument for observing the state of ink droplets is an ink droplet observation instrument, and the instrument for adding reagents is an inkjet print head. After the inkjet print head ejects the ink droplets, the ink droplet observation instrument observes. Among them, the inkjet print head can be used for DNA synthesis using inkjet printing technology. The measurement method is to use a high-speed camera to continuously record the formation and falling state of ink droplets from the side of the inkjet head ejecting ink. Among them, the two parallel photos are continuously recorded photos.

[0100] Figure 1 The formation and falling state of ink droplets using acetonitrile as the solvent in the coupling step according to one embodiment of the present invention are shown. Acetonitrile is the stock solution, and the concentrations of phosphoramide monomer and ETT in acetonitrile are the concentrations conventionally used in the DNA synthesis process, that is, the mass volume ratio of phosphoramide monomer and acetonitrile is 1:30, w / v, and the concentration of ETT in acetonitrile is about 0.25M.

[0101] Figure 2 The formation and falling state of ink droplets using propylene carbonate as a solvent in the coupling step according to an embodiment of the present invention are shown. Propylene carbonate is its stock solution, and the concentrations of phosphoramide monomer and ETT in propylene carbonate are both concentrations adjusted by the experiment of this application, that is, the mass volume ratio of phosphoramide monomer and propylene carbonate is 2:15, w / v, and the concentration of ETT in propylene carbonate is about 0.55M.

[0102] in, Figure 1 and Figure 2 In the recording environment, the temperature is normal temperature, about 20-30 degrees Celsius; the relative humidity is about 25%-50%.

[0103] like Figure 1As shown, when acetonitrile is used as the solvent, the ink droplets fall in a chaotic manner, the ink droplets are of different sizes, the flow speeds are uneven, and the ink droplet flow rate is fast. The ink droplets in each nozzle cannot fall synchronously, so it is difficult to control the flow rate and flow rate of the ink droplets.

[0104] like Figure 2 As shown, when propylene carbonate is used as a solvent, PC-based phosphoramide monomers and activators all exhibit good droplet formation and vertical inkjet performance, and the ink droplets in each nozzle can be ejected synchronously, with uniform droplet size and consistent falling speed, stable inkjet, etc. Therefore, propylene carbonate is more suitable for the inkjet synthesis process of DNA.

[0105] Through the above optimization process, the synthesis efficiency of phosphoramide monomers and activators prepared by PC was significantly improved, and an optimized formula of phosphoramide monomers and activators based on PC was obtained: the concentration of phosphoramide monomers was 4 times the conventional concentration, and the concentration of activators was 3 times the conventional concentration, that is, four phosphoramide monomers (dA, dG, dC, dT), dissolved in propylene carbonate (PC, 2:15, w / v), and the activator was 5-(Ethylthio)-1H-tetrazole (ETT, 0.75M, dissolved in propylene carbonate). Furthermore, the reagents of the present application are not only suitable for the inkjet printing synthesis method of nucleotides, but also for other printing synthesis methods of nucleotides. Furthermore, the reagents of the present application are suitable for the synthesis of nucleotides based on printing technology, wherein the nucleotides can be DNA or RNA.

[0106] The above embodiments are only used to illustrate the present invention, but not to limit the present invention. Ordinary technicians in the relevant technical field can make various changes and modifications without departing from the scope of the present invention. Therefore, all equivalent technical solutions should also fall within the scope of the present invention.

Claims

1. A coupling reagent for nucleotide synthesis, comprising: A plurality of phosphoramide monomer solutions, wherein the mass volume ratio of the phosphoramide monomer to the solvent is (2-4):30; as well as The activation solution has an activating agent concentration of 0.5M-1.25M.

2. The coupling reagent according to claim 1, wherein the plurality of phosphoramide monomers are modified or unmodified base monomers.

3. The coupling reagent according to claim 2, wherein the base monomer is dA, dG, dC, dT or dU. The coupling reagent according to claim 1 , wherein the activator is selected from tetrazole compounds. The coupling reagent according to claim 4 , wherein the tetrazole compound is 5-ethylthiotetrazolium.

6. The coupling reagent according to claim 1, wherein the solvent in the multiple phosphoramide monomer solutions and / or the activation solution is propylene carbonate.

7. A complex reagent for nucleotide synthesis, comprising the coupling reagent as described in any one of claims 1 to 6.

8. The composite reagent according to claim 7, further comprising a deprotecting agent; preferably, the deprotecting agent comprises trichloroacetic acid; more preferably, the mass volume ratio of the trichloroacetic acid to the deprotecting agent is (1-10) w / t%.

9. The composite reagent according to claim 7, further comprising a capping agent; preferably, the capping agent is selected from one or more of isobutyric anhydride, N-methylimidazole, pyridine and tetrahydrofuran.

10. The composite reagent according to claim 9, wherein the capping reagent comprises: A solution, wherein the A solution is an isobutyric acid solution, wherein the volume ratio of isobutyric anhydride to its solvent is 1:(2-6); preferably, the volume ratio of isobutyric anhydride to its solvent is 1:4; and / or Solution B, wherein the solution B comprises N-methylimidazole, pyridine and tetrahydrofuran in a volume ratio of 10:(10-20):(60-100); preferably, the volume ratio of N-methylimidazole, pyridine and tetrahydrofuran is about 10:14:

76.

11. The composite reagent according to claim 7, further comprising an oxidizing agent; preferably, the oxidizing agent is selected from iodine, pyridine, tetrahydrofuran (ie, THF) or a combination thereof; preferably, the oxidizing agent is an iodine solution comprising 0.01-0.1 M iodine.

12. A method for synthesizing nucleotides using the composite reagent according to any one of claims 7 to 11, comprising: Deprotection step: add deprotection reagent to the reaction vessel; Coupling steps: adding one of the multiple phosphoramide monomer solutions in the coupling reagent; adding the activation solution in the coupling reagent; Capping step: adding capping reagent; as well as Oxidation step: Add oxidizing reagent.

13. The method for synthesizing nucleotides according to claim 12, further comprising cyclically repeating a deprotection step, a coupling step, a capping step, and an oxidation step until a specific nucleotide sequence is obtained.

14. The method for synthesizing nucleotides according to claim 12, further comprising: A washing step, wherein a washing reagent is added after each step of the method for synthesizing nucleotides as claimed in claim 12 is completed, or after the oxidation step is completed.

15. A nucleotide synthesizer, comprising a plurality of liquid adding devices, wherein the plurality of liquid adding devices are configured to respectively add one of the complex reagents as described in any one of claims 7 to 11; wherein the same liquid adding device adds the same reagent.

16. The nucleotide synthesizer according to claim 15, wherein the liquid adding device is configured to eject reagents; preferably, the liquid adding device comprises an inkjet head or a piezoelectric valve.

17. According to the nucleotide synthesizer of claim 16, the diameter of the reagents sprayed by the multiple liquid adding devices is 1-1000 microns; preferably, the diameter of the reagents sprayed by the multiple liquid adding devices is 600-800 microns; or preferably, the diameter of the reagents sprayed by the multiple liquid adding devices is 20-80 microns; more preferably, the diameter of the reagents sprayed by the multiple liquid adding devices is 30-50 microns.

18. A method for operating a nucleotide synthesizer, comprising: The composite reagent as described in any one of claims 7 to 11 is sprayed out in sequence through a liquid adding device.

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