Full-automatic oligonucleotide synthesis equipment and method

Abstract

The invention provides full-automatic oligonucleotide synthesis equipment which comprises a pipeline liquid feeding system, a reaction system and a control system, the pipeline liquid feeding system comprises a pumping module, a moving module and a liquid injection module, the liquid injection module is installed on the moving module, the moving module is constructed to enable the liquid injection module to move in a three-dimensional mode, and the pumping module is connected with the reaction system. Liquid is injected into the reaction system; the control system is in communication connection with the pipeline liquid feeding system and the reaction system and is used for controlling liquid feeding and reaction; the equipment preferably further comprises a non-pipeline liquid feeding system, the non-pipeline liquid feeding system comprises a liquid moving device, and the liquid moving device is preferably installed on the moving module and used for injecting liquid into the reaction system. The invention also provides a method for synthesizing oligonucleotide by using the full-automatic oligonucleotide synthesis equipment. The full-automatic oligonucleotide synthesis equipment is high in automation degree, suitable for synthesis of oligonucleotides of different scales, high in synthesis quality and high in efficiency.

Description

Technical Field

[0001] The present invention relates to the field of gene synthesis, and specifically relates to an automatic synthesis device and method for oligonucleotides, in particular to an automatic synthesis device and method for morpholino antisense oligonucleotides (phosphorodiamidate morpholino oligomer, referred to as PMO). Background Art

[0002] PMO has a high affinity for DNA and RNA, is resistant to various nucleases, is stable in the body, and has low toxicity. Therefore, it is a compound that has attracted much attention as an antisense oligonucleotide.

[0003] The synthesis of PMO is mainly based on artificial synthesis, and there is no mature PMO synthesizer on the current market. Chengxi Li et al. (Fully automated fast-flow synthesis of antisensephosphorodiamidate morpholino oligomers, Nature Communications, volume 12, Article number: 4396 (2021)) disclosed a PMO fast synthesis reactor that can complete the synthesis of a sequence within 3.5 hours, but the synthesis scale is small (<1umol) and cannot be synthesized in parallel batches. Chinese patent CN 217829968U discloses a laboratory oligonucleotide synthesizer, Chinese patent application CN 114984896A discloses an oligonucleotide synthesis device, and Chinese patent application CN 114985023A discloses a microarray chip and an oligonucleotide synthesis device including the microarray chip, but their synthesis scale is small and the degree of automation is poor, and it is impossible to achieve full automation of the production process.

[0004] The synthesis equipment currently used for PMO synthesis has the following defects: the entire production process cannot be automated, and human factors bring potential risks of failure to production; the synthesis quality is uneven, the repeatability is poor, the monomer consumption is large, and large-scale parallel synthesis is impossible; the synthesis equipment has no inert gas protection mode and a good exhaust system, the air tightness is poor, and the solvent is easy to evaporate, causing a strong odor in the production environment; the product is exposed during operation, the solvent has a strong odor, the product is easily spilled and lost, and there is a risk of foreign matter being introduced.

[0005] Therefore, there is a need in the art for improved fully automated oligonucleotide synthesis equipment and methods. Summary of the invention

[0006] The purpose of the present disclosure is to provide a new oligonucleotide automatic synthesis device to solve the above problems, which can achieve high-throughput, large-scale rapid synthesis of PMO.

[0007] On the one hand, the present disclosure provides a fully automatic oligonucleotide synthesis device, which includes: a pipeline liquid supply system, a reaction system and a control system, wherein the pipeline liquid supply system includes a pumping module, a moving module and an injection module, wherein the injection module is installed on the moving module, and the moving module is configured to enable the injection module to move in three dimensions, thereby injecting liquid into the reaction system; the control system is communicatively connected with the pipeline liquid supply system and the reaction system, and is used to control the liquid supply and reaction.

[0008] In some embodiments, the device further comprises a non-pipeline liquid supply system, the non-pipeline liquid supply system comprises a liquid transfer device, and the liquid transfer device is preferably mounted on the mobile module and is used to inject liquid into the reaction system.

[0009] In some embodiments, the reaction system includes at least 1 reaction module, preferably at least 2 reaction modules, for example 3, 4, 5 or 6 reaction modules; each reaction module includes at least 1 reactor, preferably at least 2 reactors, for example 3, 4, 5 or 6 reactors.

[0010] In some embodiments, each reactor is housed in a transition sleeve.

[0011] In some embodiments, the reaction module comprises a shaking mechanism, which preferably comprises a motion rail, more preferably comprises two sets of motion rails, which are configured to shake the reactor.

[0012] In some embodiments, the device comprises one or more injection modules, each injection module comprises an injection port for injecting liquid into the reactor, and preferably the number of injection ports of each injection module is equal to the number of reactors of each reaction module.

[0013] In some embodiments, the mobile module includes a first guide rail, a second guide rail, and a third guide rail that are perpendicular to each other, the second guide rail is configured to be able to move horizontally along the first guide rail; the third guide rail is installed on the second guide rail and is configured to be able to move horizontally along the second guide rail.

[0014] In some embodiments, the injection module is mounted on the third guide rail and is configured to be able to move vertically up and down along the third guide rail.

[0015] In some embodiments, the pumping module includes an electric syringe and a solenoid valve, wherein the electric syringe is connected to the solenoid valve through a pipeline, the solenoid valve includes an input port and an output port, the input port is connected to the reagent reservoir through a pipeline, and the output port is connected to the injection module through a pipeline.

[0016] In some embodiments, the device comprises a plurality of reagent reservoirs, wherein each reagent reservoir in at least a portion of the reagent reservoirs comprises one or more reagent outlets, and each reagent outlet is connected to an input port of a solenoid valve through a pipeline.

[0017] In some embodiments, at least one reagent reservoir includes only one reagent outlet, which is connected to the input port of an electromagnetic valve, and there is a diverter device between the output port of the electromagnetic valve and the injection module, the diverter device includes a diverter device inlet and multiple diverter device outlets, the diverter device inlet is connected to the output port of the electromagnetic valve through a pipeline, and each diverter device outlet is connected to an injection port through a pipeline.

[0018] In some embodiments, at least one reagent reservoir among the plurality of reagent reservoirs is configured to allow a pipetting device to pipette a reagent.

[0019] In another aspect, the present disclosure provides a method for synthesizing oligonucleotides using an oligonucleotide fully automatic synthesis device, comprising:

[0020] a. Cleaning the reactor;

[0021] b. adding a deprotecting agent to the reactor to carry out a deprotection reaction;

[0022] c. Cleaning the reactor;

[0023] d. adding a neutralizing agent to the reactor for a neutralization reaction;

[0024] e. Cleaning the reactor;

[0025] f. adding a coupling unit to the reactor;

[0026] g. Carry out coupling reaction,

[0027] Under the control of the control system, steps a to g are cyclically performed until the desired oligonucleotide is obtained, wherein the method includes, under the control of the control system, using a moving module to move the injection module to inject the reagent into the reactor.

[0028] In some embodiments, the method for synthesizing oligonucleotides further comprises, under the control of the control system, using a pipetting device to pipette reagents and inject them into the reactor, wherein the injection module and the pipetting device are used to inject different reagents.

[0029] In some embodiments, the fully automatic oligonucleotide synthesis device includes multiple reaction modules. After one reaction module completes step f and during step g, steps a to f are performed on the next reaction module until the required number of reaction modules perform steps a to f.

[0030] In some embodiments, one or more processes of cleaning, deprotection reaction, neutralization reaction and coupling reaction, preferably all processes involve one or more of the following operations under the control of a control system:

[0031] Shake the reactor;

[0032] heating the reactor to a predetermined temperature and maintaining the reactor at the predetermined temperature;

[0033] The reactor was purged with nitrogen to isolate it from the atmosphere.

[0034] In some embodiments, the oligonucleotide is a morpholino antisense oligonucleotide.

[0035] In some embodiments, in step a, washing with dichloromethane (DCM) and 30% trifluoroethanol (TFE) / dichloromethane (DCM) is performed successively, preferably by adding using an injection module, preferably for 30 seconds to 5 minutes, for example 1 minute, each washing.

[0036] In some embodiments, in step b, the deprotecting agent is a trifluoroacetic acid (TFA) and 4-cyanopyridine trifluoroethanol (TFE) / dichloromethane solution, preferably added using an injection module, and the reaction is preferably performed for 10 to 20 minutes, such as 15 minutes.

[0037] In some embodiments, in step c, washing with dichloromethane is preferably performed using an injection module, preferably for 30 seconds to 5 minutes, such as 1 minute.

[0038] In some embodiments, in step d, the neutralizing agent is a mixed solution of N,N-diisopropylethylamine (DIEA), isopropanol and dichloromethane, preferably added using an injection module, and preferably reacted for 30 seconds to 5 minutes, such as 1 minute.

[0039] In some embodiments, in step e, washing with dichloromethane and 1,3-dimethyl-2-imidazolidinone (DMI) is performed sequentially, preferably by adding using a liquid injection module, preferably for 30 seconds to 5 minutes each, for example 1 minute.

[0040] In some embodiments, in step f, a 10 volume % N-ethylmorpholine solution in 1,3-dimethyl-2-imidazolidinone (DMI) and a 0.1 M morpholino antisense oligonucleotide monomer solution in 1,3-dimethyl-2-imidazolidinone (DMI) are added to the reactor, preferably using a pipetting device.

[0041] In some embodiments, in step g, the reaction lasts for 2 to 4 hours, such as 3 hours. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] In order to more clearly illustrate the technical solution of the present application, the following briefly introduces the drawings involved in some embodiments of the present application. Obviously, the embodiments shown in the following drawings are only examples, and for ordinary technicians in this field, other drawings can be obtained based on the drawings without creative work.

[0043] Figure 1 is a partial schematic diagram of an apparatus for fully automated oligonucleotide synthesis according to some embodiments of the present disclosure.

[0044] Figure 2A is a schematic diagram of a pumping module according to some embodiments of the present disclosure.

[0045] Figure 2B is a partial schematic diagram of a pumping module according to some embodiments of the present disclosure.

[0046] Figure 3A is a schematic diagram of a mobile module according to some embodiments of the present disclosure.

[0047] Figure 3B is a schematic diagram of a pipette according to some embodiments of the present disclosure.

[0048] Figure 4A is a schematic diagram of a reaction module according to some embodiments of the present disclosure.

[0049] Figure 4B It is a schematic diagram of an exploded view of a reaction tube, a reaction tube transition sleeve and a reaction tube seat according to some embodiments of the present disclosure.

[0050] Figure 4C yes Figure 4B The schematic diagram of the assembly formed by the reaction tube, the reaction tube transition sleeve and the reaction tube seat is shown.

[0051] Figure 4D yes Figure 4C A perspective view of the assembly shown.

[0052] Figure 5A is a schematic diagram of liquid injection port distribution according to some embodiments of the present disclosure.

[0053] Figure 5B is a schematic diagram of an injection module according to some embodiments of the present disclosure.

[0054] Figure 6 is the test result of the morpholino antisense oligonucleotide prepared in the examples of the present disclosure.

[0055] Description of reference numerals:

[0056] 1-1: Pumping module; 1-2: Reaction module; 1-3: Injection module; 1-4: Moving module; 1-5: Pipette device / pipette gun; 2-1: Solenoid valve; 2-2: Electric syringe; 2-3: Screw rod; 2-4: Coupling; 2-5: Pumping motor; 2-6 to 2-8: Connecting pipe; 3-1: X-axis; 3-2: Y-axis; 3-3: Z-axis of injection module; 3-4: Z-axis of pipette device; 3-5: Pipette gun body; 3-6: Pipette gun tip; 4-1: Reactor / reaction tube; 4-2: Reaction tube transition sleeve; 4-3: Heat transfer fan; 4-4: Motion guide rail 1; 4-5: Motion guide rail 2; 4-6: Heating rod ; 4-7: reaction tube holder; 4-8: shaking motor; 4-9: first sealing ring; 4-10: second sealing ring; 4-11: waste liquid discharge port; 4-12: positive pressure air supply port; 5-1-1 to 5-1-10: openings No. 1 to No. 10 of liquid injection port No. 1 (corresponding to reaction tube No. 1); 5-2-1: opening No. 1 of liquid injection port No. 2 (corresponding to reaction tube No. 2); 5-2-10: opening No. 10 of liquid injection port No. 2 (corresponding to reaction tube No. 2); 5-6-1: opening No. 1 of liquid injection port No. 6 (corresponding to reaction tube No. 6); 5-6-10: opening No. 10 of liquid injection port No. 6 (corresponding to reaction tube No. 6). DETAILED DESCRIPTION

[0057] The specific implementation methods of the present application are further described in detail below. The following detailed description is used to exemplarily illustrate the content of the present application, but it should not be understood as limiting the scope of the present application.

[0058] " Scope " disclosed herein is limited in the form of lower limit and upper limit, and given range is limited by selecting a lower limit and / or an upper limit, and the selected lower limit and upper limit define the boundary of special range. The scope limited in this way can be including end value or not including end value, and can be combined arbitrarily, that is, any lower limit can form a scope with any upper limit combination. For example, if the scope of 60-120 and 80-110 is listed for a particular parameter, it is understood that the scope of 60-110 and 80-120 is also expected. In addition, if the minimum range value 1 and 2 listed, and if the maximum range value 3,4 and 5 are listed, the following scope can all be expected: 1-3, 1-4, 1-5, 2-3, 2-4 and 2-5. In this article, unless otherwise specified, numerical range " ab " represents the abbreviation of any real number combination between a and b, wherein a and b are real numbers. For example, the numerical range "0-5" means that all real numbers between "0-5" are listed in this document, and "0-5" is just an abbreviation of these numerical combinations. In addition, when a parameter is expressed as an integer ≥ 2, it is equivalent to disclosing that the parameter is, for example, an integer of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.

[0059] In this document, unless otherwise specified, all embodiments and preferred embodiments mentioned in this document can be combined with each other to form a new technical solution.

[0060] In this document, unless otherwise specified, all technical features and preferred features mentioned in this document can be combined with each other to form a new technical solution.

[0061] In this article, if there is no special explanation, the "include" and "comprising" mentioned in this article represent open-ended or closed-ended expressions. For example, the "include" and "comprising" may mean that other components not listed may also be included or included, or may only include or include the listed components.

[0062] In the description of this article, it should be noted that, unless otherwise specified, "above" and "below" are inclusive of the number itself, and "several" in "one or several" means two or more.

[0063] In the description herein, unless otherwise specified, the term "or" is inclusive. For example, the phrase "A or B" means "A, B, or both A and B". More specifically, any of the following conditions satisfies the condition "A or B": A is true (or exists) and B is false (or does not exist); A is false (or does not exist) and B is true (or exists); or both A and B are true (or exist).

[0064] The fully automatic oligonucleotide synthesis device disclosed in the present invention mainly includes: a pipeline liquid supply system, a reaction system and a control system. The pipeline liquid supply system is used to supply various reagents required for the oligonucleotide reaction from their respective storage devices such as reagent reservoirs (such as reagent bottles) to the reaction system through pipelines. These reagents include but are not limited to cleaning agents, deprotecting agents, neutralizing agents, coupling units, etc. Figure 1 As shown, the pipeline feeding system includes a pumping module 1-1, a liquid injection module 1-3 and a moving module 1-4. Figure 1 The pumping module 1-1 is connected to the injection module 1-3 through a pipeline. In order not to affect the observation, Figure 1 No pipelines are drawn. In some embodiments, in addition to the pipeline liquid supply system, the fully automatic oligonucleotide synthesis equipment disclosed in the present invention also includes a non-pipeline liquid supply system, and the non-pipeline liquid supply system includes a pipetting device, and the pipetting device directly transfers the reagent from the reagent storage device and injects it into the reaction system, such as injecting it into the reactor in the reaction system. That is to say, there is no pipeline connection between the pipetting device and the reaction system, which is particularly beneficial for reagents with smaller amounts (such as coupling units, etc.), avoiding reagent waste. For example, a specific form of the pipetting device can be a pipette gun, such as Figure 1 Pipettes 1-5 are shown in FIG. Figure 1 Also shown is a reaction system, which exemplarily includes five reaction modules 1-2. The pumping module 1-1, the reaction module 1-2, the injection module 1-3, the moving module 1-4 and the pipette 1-5 will be described in more detail below in conjunction with the corresponding drawings.

[0065] The control system (not shown) is connected to the pipeline feeding system, the non-pipeline feeding system and the reaction system in communication. The communication connection includes a wired connection and / or a wireless connection. Under the control of the control system, the pumping module 1-1 automatically extracts the reagent from the reagent reservoir and then delivers it to the injection module 1-3. The injection module 1-3 is connected to the mobile module 1-4, for example, it is installed on the mobile module 1-4. Under the control of the control system, the mobile module 1-4 automatically moves the injection module 1-3 to a position corresponding to the reaction module 1-2 where the reagent needs to be injected, for example, it moves to the top of the reaction module 1-2, so that the reagent is injected into the reactor in the reaction module 1-2. A liquid transfer device, such as a liquid transfer gun 1-5, is also connected to the mobile module 1-4. Under the control of the control system, the mobile module 1-4 automatically moves the liquid transfer device to the reagent reservoir, draws the reagent from the reagent reservoir, and then moves the mobile device to a position corresponding to the reaction module 1-2 where the reagent needs to be injected, for example, it moves to the top of the reaction module 1-2, so that the absorbed reagent is injected into the reactor in the reaction module 1-2.

[0066] Although Figure 110 pumping modules 1-1 are shown in the figure, but this is only for the purpose of illustration. The number of pumping modules 1-1 included in the fully automatic synthesis equipment for oligonucleotides disclosed in the present invention is not particularly limited, for example, it can be less than 10 pumping modules 1-1, or it can be more than 10 pumping modules 1-1. In some embodiments, the number of pumping modules 1-1 can be designed according to the number of reagent types required for synthesizing oligonucleotides, and each pumping module 1-1 only transports one reagent. For example, if 8 reagents are needed to synthesize oligonucleotides, 8 pumping modules 1-1 can be designed. If 2 of the 8 reagents need to be pipetted with a pipetting device, 6 pumping modules 1-1 can be designed. Although the fully automatic synthesis equipment for oligonucleotides disclosed in the present invention can customize the number of pumping modules and pipetting devices according to the specific oligonucleotides to be synthesized, a general number of pumping modules and pipetting devices can also be designed. For example, 12 pumping modules 1-1 can be designed. If only 6 reagents need to be pumped in a specific oligonucleotide synthesis reaction, only 6 pumping modules 1-1 out of 12 pumping modules 1-1 need to be activated. Similarly, multiple (for example 6) pipetting devices can also be designed. If only 2 reagents need to be transferred through a pipetting device in a specific oligonucleotide synthesis reaction, only 2 of the 6 pipetting devices need to be activated. Therefore, any number of pumping modules and any number of pipetting devices are included in the scope of protection requested in this application. In a preferred embodiment, each pumping module and each pipetting device each only transports one reagent, thereby avoiding cross contamination caused by the same pumping module or pipetting device transporting different reagents.

[0067] akin, Figure 15 reaction modules are shown in, and each reaction module includes 6 reactors (reaction tubes). However, the number of reaction modules included in the fully automatic synthesis equipment for oligonucleotides disclosed herein and the number of reactors included in each reaction module are not particularly limited. In some embodiments, the reaction system may include at least 1 reaction module, for example, 1-20 reaction modules, 2-15 reaction modules, 3-10 reaction modules, 4-9 reaction modules, 5-8 reaction modules, 6-7 reaction modules, such as 2, 3, 4, 5, 6 or more reaction modules. Similarly, each reaction module may include at least 1 reactor, for example, 1-20 reactor, 2-15 reactor, 3-10 reactor, 4-9 reactor, 5-8 reactor, 6-7 reactor, such as 2, 3, 4, 5, 6 or more reactors. Therefore, any number of reaction modules and any number of reactors are included in the scope of the present application. The more the number of reaction modules and reactors, the larger the synthesis scale. Therefore, the fully automatic oligonucleotide synthesis equipment disclosed in the present invention can customize the number of reaction modules and reactors according to actual synthesis needs, and can also design a universal number of reaction modules and reactors, so as to enable all or part of the reaction modules and reactors as needed. Since each pumping module and each liquid transfer device only transports one reagent each, different reaction modules and / or reactors can perform the same synthesis reaction, or different synthesis reactions. Therefore, the fully automatic oligonucleotide synthesis equipment disclosed in the present invention can realize fully automatic oligonucleotide synthesis from laboratory scale to industrial scale, thereby having great flexibility and greatly improving synthesis efficiency.

[0068] akin, Figure 1 One injection module 1-3 is shown in the figure, but in other embodiments, multiple injection modules can also be designed. Any number of injection modules is included in the scope of protection claimed in the present application.

[0069] In the fully automatic oligonucleotide synthesis device disclosed in the present invention, the pumping module 1-1, the reaction module 1-2, the injection module 1-3 and the liquid transfer device (such as a liquid transfer gun 1-5) can be fixed or detachable. In a detachable embodiment, only a part of the pumping module 1-1, the reaction module 1-2, the injection module 1-3 and / or the liquid transfer device (such as a liquid transfer gun 1-5) can be installed in the synthesis device as needed. The pumping module 1-1, the reaction module 1-2, the injection module 1-3, the moving module 1-4 and the liquid transfer device (such as a liquid transfer gun 1-5) are not limited to Figure 1 The arrangement shown is not limited to the arrangement shown in the figure, but there may be various arrangements as long as the purpose of the present disclosure can be achieved.

[0070] Figure 2AIt is a schematic diagram of a pumping module according to some embodiments of the present disclosure. The pumping module includes a solenoid valve 2-1 and an electric syringe 2-2. The electric syringe 2-2 is connected to the solenoid valve 2-1 through a pipeline. The solenoid valve 2-1 includes an input port and an output port (i.e., two pipeline connection ports at the top of the solenoid valve 2-1 in the figure), the input port is connected to the reagent reservoir through a pipeline, and the output port is connected to the injection module through a pipeline. The pumping module also includes a driving component for driving the electric syringe 2-2. For example, in the embodiment shown in Figure 2, the pumping module also includes a screw 2-3, a coupling 2-4 and a pumping motor 2-5. The pumping motor 2-5 drives the syringe 2-2 through the coupling 2-4 and the screw 2-3, first pulling the piston of the syringe 2-2 outward, so that the reagent is sucked into the inside of the syringe 2-2 through the input port of the solenoid valve 2-1. Then, the electromagnetic valve 2-1 switches the channel, and the pumping motor 2-5 drives the syringe 2-2 to push the piston of the syringe 2-2 inward, thereby pumping out through the output port of the electromagnetic valve 2-1, so that liquid suction and liquid supply can be achieved. The pumping module is connected to the control system in communication, for example, the electromagnetic valve 2-1 and the pumping motor 2-5 are connected to the control system to achieve automatic pumping.

[0071] Figure 2A Six solenoid valves 2-1 and six electric injectors 2-2 are shown. In order not to affect the observation, only one solenoid valve is shown to be connected to an electric injector through a pipeline, and only two pipelines on the upper part of a solenoid valve are shown, one of which leads to a reagent reservoir and the other leads to a liquid injection module. However, it should be understood that in some embodiments of the fully automatic synthesis equipment of oligonucleotides disclosed herein, more or less solenoid valves and electric injectors may be included. For example, 1-10, 2-9, 3-8, 4-7, 5-6 solenoid valves and electric injectors may be included. The fully automatic synthesis equipment of oligonucleotides disclosed herein can customize the number of solenoid valves and electric injectors according to actual synthesis needs, and can also design a general number of solenoid valves and electric injectors, so as to enable all or part of the solenoid valves and electric injectors as needed (when a certain solenoid valve is closed, its corresponding electric injector will also stop the effect of aspiration and liquid supply). Any number of solenoid valves and any number of electric injectors are included in the scope of the present application. Usually, a solenoid valve 2-1 is connected to an electric injector 2-2 by a pipeline. In a pumping module, the pumping motor 2-5 drives all the electric injectors 2-2 through the screw 2-3 and the coupling 2-4. Figure 2A 6 solenoid valves are shown in parallel, but they can also be arranged in any other suitable manner instead of in parallel. Figure 2A Six power injectors are shown arranged in a circle, but any other suitable arrangement may be used.

[0072] Figure 2B It is a partial schematic diagram of a pumping module according to some embodiments of the present disclosure, which more specifically shows the pipeline connection mode of the solenoid valve 2-1. In the illustrated embodiment, the connecting pipe 2-6 connects the syringe 2-2, the connecting pipe 2-8 connects the reagent reservoir, and the connecting pipe 2-7 connects the injection module. At this time, the solenoid valve connects the connecting pipe 2-8 to the connecting pipe 2-6, and the connecting pipe 2-6 is blocked from the connecting pipe 2-7. The pumping motor drives the syringe 2-2, pulls the piston of the syringe 2-2 outward, and causes the reagent to be sucked from the reagent reservoir through the connecting pipe 2-8 through the solenoid valve 2-1 and the connecting pipe 2-6 into the syringe 2-2. Then, the solenoid valve 2-1 switches the channel so that the connecting pipe 2-6 is connected to the connecting pipe 2-7, while the connecting pipe 2-6 is blocked from the connecting pipe 2-8. Then, the pumping motor 2-5 drives the syringe 2-2 again, so that the reagent sucked into the syringe 2-2 is pumped out from the connecting pipe 2-7 through the solenoid valve 2-1. In this way, liquid suction and liquid supply can be achieved.

[0073] Figure 3AIt is a schematic diagram of a mobile module according to some embodiments of the present disclosure. In some embodiments, the mobile module includes a rail system for making the injection module 1-3 and the pipetting device, such as a pipetting gun 1-5, move in three dimensions, so as to move to the injection position according to the control of the set program. The rail system may include a first rail, a second rail, and a third rail perpendicular to each other. For the purpose of convenience of description, the rail along the X-axis direction 3-1 in the figure may be referred to as the first rail, and the rail along the Y-axis direction 3-2 may be referred to as the second rail. The third rail along the Z-axis direction may include two sets of rails, namely, the third rail of the injection module along the Z-axis direction 3-3 of the injection module and the third rail of the pipetting device along the Z-axis direction 3-4 of the pipetting device. In some embodiments, the second rail may be configured to be able to move horizontally along the first rail (i.e., move along the X-axis direction); the third rail (including the third rail of the injection module and the third rail of the pipetting device) may be installed on the second rail and configured to be able to move horizontally along the second rail (i.e., move along the Y-axis direction). The injection module is installed on the third guide rail of the injection module, and is configured to be able to move vertically up and down along the third guide rail of the injection module (i.e., move along the Z-axis direction). Similarly, the pipetting device (such as the pipetting gun 1-5 in the figure) is installed on the third guide rail of the pipetting device, and is configured to be able to move vertically up and down along the third guide rail of the pipetting device (i.e., move along the Z-axis direction). The terms "first", "second", "third", "X-axis", "Y-axis" and "Z-axis" used above are only for the convenience of description and are not limited to a specific orientation. For example, the guide rail along the Y-axis direction can also be referred to as the "first guide rail", and the guide rails along the other two directions can be referred to as the "second guide rail" and the "third guide rail" respectively. The first guide rail, the second guide rail and the third guide rail can each include a set of guide rails, for example, each can include one or more guide rails. The form of these guide rails is not particularly limited, and can be guide rails known in the art, as long as the three-dimensional movement of the injection module and the pipetting device can be achieved. Figure 3A Only one set of first rails, one set of second rails, one set of third rails of the injection module and one set of third rails of the pipetting device are shown, but the embodiments of multiple sets of first rails, multiple sets of second rails, multiple sets of third rails of the injection module and / or multiple sets of third rails of the pipetting device are also included in the scope of protection requested by the present application. In the specification and claims of the present application, in the case where "injection module" or "pipetting device" is not used to limit, the term "third rail" refers to the third rail of the injection module and / or the third rail of the pipetting device.

[0074] The mobile module also includes a driving device (not shown), such as an electric driving device, for driving the guide rail, the injection module and the pipetting device to move, thereby realizing the three-dimensional movement of the injection module and the pipetting device. The mobile module is connected to the control system in communication, and under the control of the control system, the automatic three-dimensional movement of the injection module and the pipetting device is realized.

[0075] Figure 3B is a schematic diagram of a pipette according to some embodiments of the present disclosure, which is as follows Figure 3A The enlarged schematic diagram of the pipette 1-5 mounted on the mobile module is shown. As shown in the figure, the pipette 1-5 includes a pipette body 3-5 and a pipette tip 3-6. The pipette tip 3-6 is detachable and is tightly mounted on the pipette when in use, with no gap and no air leakage at the contact point. During pipetting, the pipette tip 3-6 is extended below the liquid surface to generate a vacuum in the pipette, and the liquid reagent is sucked into the pipette tip 3-6 and retained in the tip. The pipette is then moved to the injection position by the mobile module, and the liquid reagent in the pipette tip 3-6 is spit into the reactor, thereby completing the pipetting. The pipette can be a commercially available electronically controlled pipette, such as a pipette commonly used in automatic dispensers. The pipette tip can be placed in a tip box. When it is to be used, the mobile module automatically moves the pipette body to the tip box position under the control of the control system, and automatically takes the tip. The pipette can control the amount of liquid taken, and the amount of liquid taken at one time can be quantitatively added to one or more reactors. Figure 3A Three pipettes are shown, but the number of pipettes of the fully automated oligonucleotide synthesis apparatus disclosed herein is not particularly limited. A specific number of pipettes can be customized as needed; a general number of pipettes can also be designed, and all or part of the pipettes can be used as needed during the synthesis process. One pipette can be used to add liquid to multiple reactors in sequence, or multiple pipettes can be used to add liquid to multiple reactors at the same time. In order to avoid cross contamination, it is preferred that only one reagent is added to one pipette.

[0076] Figure 4A is a schematic diagram of a reaction module according to some embodiments of the present disclosure. The reaction module includes a reactor. Figure 4A In the embodiment, the reactor is in the form of a reaction tube 4-1, which is substantially cylindrical or cylindrical. It should be understood that the reactor may also be in other shapes, such as a cuboid, hexagon or other polygonal shapes, as long as it is suitable for the synthesis of oligonucleotides. In a preferred embodiment, the reactor is in the form of a reaction tube. Figure 4A As shown, the reaction tube 4-1 is installed in the reaction tube transition sleeve 4-2. The reaction tube 4-1 installed in the reaction tube transition sleeve 4-2 is placed on the reaction tube seat 4-7.

[0077] The reaction module may also include a shaking mechanism, which includes a first set of motion rails 4-4 and a second set of motion rails 4-5. In some embodiments, the two sets of motion rails are perpendicular to each other. Each set of motion rails includes at least one rail. Figure 4AIn the figure, the first group of motion guide rails 4-4 includes two guide rails, of which only a part of the right side guide rail is shown. The second group of motion guide rails 4-5 also includes two guide rails, of which only the guide rail on one side is shown, and the guide rail on the other side is not shown. It should be understood that the number of guide rails is not particularly limited, and any number of guide rails is included in the scope of protection requested in this application. A first support plate is provided on the first group of motion guide rails 4-4, a second support plate is provided on the second group of motion guide rails 4-5, the second group of motion guide rails 4-5 is located between the first support plate and the second support plate, and the reaction tube 4-1 is mounted on the second support plate. The shaking mechanism also includes a shaking motor 4-8. Under the control of the control system, the shaking motor 4-8 drives the first support plate and the second support plate to move along the first group of motion guide rails 4-4 and the second group of motion guide rails 4-5, so that the reaction tube 4-1 shakes regularly.

[0078] The reaction module may also include a heating and heat preservation device, which includes a heater (e.g. Figure 4A The heating rod 4-6 is used to heat the reactor (such as Figure 4A The reaction tube 4-1 shown is heated, and the heat transfer fan 4-3 makes the heat inside each reactor evenly distributed. The heating and heat preservation device also includes a temperature sensor for monitoring the temperature of the reactor. Under the control of the control system, the reaction module can automatically heat and keep warm, thereby realizing temperature closed-loop control.

[0079] The reaction module may also include an inert gas pipeline (not shown), which is connected in parallel with all reactors in the reaction module, and the inert gas leading to each reactor can be controlled separately. Inert gas may include nitrogen, argon, helium and other gases that can provide an inert atmosphere inside the reactor, and nitrogen is generally used. Dry nitrogen of a certain pressure is blown into the reaction tube, and the filled nitrogen will occupy the limited space of the reaction tube, and the limited air in the reaction tube is squeezed out of the reaction tube to achieve air isolation. The pressure of nitrogen cannot be too large, and the flow rate cannot be too fast, otherwise the solid inside the reaction tube will be blown over, and even blown onto the tube wall. Therefore, the pressure and flow rate of nitrogen are both set to be adjustable. Under the control of the control system, the pressure and flow rate of nitrogen can be automatically adjusted.

[0080] The reaction module may also include a negative pressure component (not shown). The negative pressure component is connected to the liquid outlet at the bottom of the reactor, and the other end is connected to a vacuum chamber with a vacuum pump. When the liquid is discharged from the reactor, the discharged liquid first goes to the vacuum chamber and then is discharged outside the device through the vacuum pump. The residual liquid and cleaning liquid after the reaction are pumped out of the reactor through the negative pressure component to achieve negative pressure discharge.

[0081] The present invention can create good reaction conditions through shaking, heating and heat preservation, air isolation, and negative pressure drainage of the above-mentioned components.

[0082] Figure 4B is an exploded schematic diagram of a reaction tube 4-1, a reaction tube transition sleeve 4-2 and a reaction tube seat 4-7 according to some embodiments of the present disclosure, Figure 4C It is a schematic diagram of an assembly formed by assembling a reaction tube 4-1, a reaction tube transition sleeve 4-2 and a reaction tube seat 4-7. Figure 4D 4-2 is a perspective view of the assembly. By designing the reaction tube transition sleeve 4-2, the reaction module of the present disclosure can be compatible with reaction tubes of different specifications. Specifically, the reaction module can be uniformly designed with a fixed specification chamber for accommodating the reaction tube, the outer shape and size of the reaction tube transition sleeve can be designed to fit exactly in the chamber, and the inner shape and size can be designed to different specifications to accommodate reaction tubes of different specifications. For example, Figure 1 and Figure 4A Three specifications of reaction tubes and three corresponding specifications of reaction tube transition sleeves are presented. Reaction tubes 4-1 of different specifications can be placed on the equipment for use with the aid of reaction tube transition sleeves 4-2, and the equipment can be compatible. Reaction tubes of different specifications have their own matching transition sleeves. A first sealing ring 4-9 is arranged in the transition sleeve. When the reaction tube is placed in the transition sleeve, the first sealing ring 4-9 plays a sealing role, so that the reaction tube and the transition sleeve are sealed as a whole. It should be understood that Figure 4B The reaction tube 4 - 1 and the reaction tube transition sleeve 4 - 2 are merely taken as examples, and a corresponding reactor transition sleeve can be designed for any reactor.

[0083] like Figure 4B and 4C As shown, the reaction tube seat 4-7 is provided with a positive pressure gas supply port 4-12 and a waste liquid discharge port 4-11, and the two ports are controlled to open and close by two solenoid valves. During the reaction, the solenoid valve of the waste liquid discharge port 4-11 is closed and blocked, and the gas supply port 4-12 is opened to allow positive pressure gas to enter the tube seat. A second sealing ring 4-10 is provided in the tube seat. Due to the action of the second sealing ring 4-10, the gas goes up into the transition sleeve of the reaction tube, and then due to the action of the first sealing ring 4-9, the gas smoothly enters the interior of the reaction tube. After the reaction is completed, the gas supply port 4-12 is closed, the waste liquid discharge port 4-11 connected to the vacuum is opened, and the waste liquid flows out from the waste liquid discharge port 4-11, and the waste liquid is drained and then closed.

[0084] Figure 5A is a schematic diagram of the distribution of injection ports according to some embodiments of the present disclosure. For illustrative purposes only, the figure shows 6 injection ports, each of which includes 10 openings. The 6 injection ports correspond to 6 reactors (reaction tubes) in a reaction module. Each injection port corresponds to the inlet of a reactor (reaction tube), so that the reagent passing through each opening in the injection port can enter the reactor (reaction tube) through the inlet. 10 openings can be used to inject 10 reagents. Using the illustrated injection module, the same reagent can be injected into up to 6 reactors at the same time. For example, Figure 5A As shown, if the 10 openings in the first injection port corresponding to the first reaction tube (reaction tube No. 1) are numbered 5-1-1 to 5-1-10 in sequence, they can be used to inject the first reagent (reagent No. 1) to the tenth reagent (reagent No. 10) respectively. Similarly, the openings 5-2-1 and 5-2-10 in the second injection port marked in the figure correspond to the second reaction tube (reaction tube No. 2), and the openings 5-6-1 and 5-6-10 in the sixth injection port correspond to the sixth reaction tube (reaction tube No. 6) (other openings can be numbered similarly, but in order not to interfere with observation, other opening numbers are not marked in the figure). Openings 5-2-1 and 5-6-1 can be used to inject reagent No. 1, and openings 5-2-10 and 5-6-10 can be used to inject reagent No. 10.

[0085] Figure 5B Schematic diagram of an injection module according to some embodiments of the present disclosure. As shown in the figure, an injection connector is inserted into each opening, the upper end of the connector is connected to the reagent delivery pipeline, and the lower end is a cylindrical or needle-shaped injection tube. It should be understood that an injection module may include more than 6 or less than 6 injection ports, each injection port may include more than 10 or less than 10 openings, and correspondingly may include more than 10 or less than 10 injection connectors. The openings in each injection port may not necessarily be as Figure 5A Arranged like that. The arrangement of the perforations is not critical and is not particularly limited, as long as it is ensured that the injection pipe of each injection joint can be inserted into the reactor inlet when the injection module is in the injection position. Any number of injection ports, any number of perforations and any arrangement of perforations are within the scope of the present application's request for protection. It should be understood that for specific oligonucleotide building-up reactions, not necessarily each injection port and / or each perforation need to be put into use. Therefore, with regard to the number of injection ports and perforations, it is possible to customize for specific oligonucleotide building-up reactions, or to design a general injection module.

[0086] In a preferred embodiment of the fully automated oligonucleotide synthesis apparatus disclosed herein, each reagent is delivered to the reactor via a separate path. For a non-pipeline liquid feeding system, such as using a pipette to transfer reagents, each reagent uses an independent pipette. For a pipeline liquid feeding system, for example, for a certain oligonucleotide synthesis reaction, if each reaction module uses a pipette, Figure 1 and 4A In the six reactors shown in FIG. 1 , each reactor performs the same synthesis reaction, and each reagent needs to be divided into six routes to enter the six reactors. In one embodiment, the six pipelines can be connected to the same reagent reservoir, and the other end of each pipeline is connected to the same reagent reservoir. Figure 2A A solenoid valve in the pumping module shown (eg Figure 2B The outlet pipeline of each solenoid valve is connected as shown in Figure 5B The liquid injection connector in one of the liquid injection ports shown in the figure enables the outlet pipelines of the six solenoid valves to be connected to the liquid injection connectors in the six liquid injection ports, respectively, so that the same reagent can be injected into the six reactors at the same time. In this embodiment, a reagent reservoir may include six outlets, each outlet connected to one pipeline; or the six pipelines may be gathered together and connected to one outlet of the reagent reservoir. Of course, other connection methods may also be used. These connection methods are all included in the scope of the present application.

[0087] For the synthetic reactions listed above, in another embodiment, one pipeline can be connected to a reagent reservoir, and the other end of the pipeline can be connected to a reagent reservoir. Figure 2A A solenoid valve in the pumping module shown (eg Figure 2B As shown), the outlet pipeline of the solenoid valve is connected to a diverter valve (such as a diverter solenoid valve, not shown), and the reagent is divided into 6 routes, each of which is connected as shown. Figure 5B An injection connector in an injection port is shown, so that the same reagent can be injected into 6 reactors at the same time.

[0088] The fully automatic oligonucleotide synthesis equipment of the present disclosure also includes a control system. The control system includes commercially available hardware (such as memory, monitor, processor, etc.) and software (such as programming software), etc. Those skilled in the art can program according to the control functions that the synthesis equipment of the present disclosure needs to realize.

[0089] The method for synthesizing oligonucleotides using an automatic oligonucleotide synthesis device disclosed herein comprises:

[0090] a. Cleaning the reactor;

[0091] b. adding a deprotecting agent to the reactor to carry out a deprotection reaction;

[0092] c. Cleaning the reactor;

[0093] d. adding a neutralizing agent to the reactor for a neutralization reaction;

[0094] e. Cleaning the reactor;

[0095] f. adding a coupling unit to the reactor;

[0096] g. Carry out coupling reaction,

[0097] Wherein, under the control of the control system, steps a to g are cyclically performed until the desired oligonucleotide is obtained, wherein the method includes, under the control of the control system, using the mobile module to automatically move the injection module to inject the reagent into the reactor. For reagents with small amounts or unstable reagents, the method for synthesizing oligonucleotides also includes, under the control of the control system, using a liquid transfer device to transfer the reagent into the reactor.

[0098] In the method for synthesizing oligonucleotides using the fully automatic oligonucleotide synthesis device disclosed in the present invention, the fully automatic oligonucleotide synthesis device includes multiple reaction modules, and after one reaction module completes step f and during step g, steps a to f are performed on the next reaction module until the required number of reaction modules perform steps a to f. This can significantly improve the synthesis efficiency.

[0099] One or more of the processes of cleaning, deprotection reaction, neutralization reaction and coupling reaction involve one or more of the following operations under the control of the control system:

[0100] Shake the reactor;

[0101] heating the reactor to a predetermined temperature and maintaining the reactor at the predetermined temperature;

[0102] The reactor was purged with nitrogen to isolate it from the atmosphere.

[0103] In the present disclosure, oligonucleotides include, but are not limited to, morpholino antisense oligonucleotides.

[0104] In step a, the sample may be first cleaned with dichloromethane (DCM) and then cleaned with 30% trifluoroethanol (TFE) / dichloromethane (DCM). These cleaning agents may be added using an injection module. In some embodiments, each of them is cleaned for 30 seconds to 5 minutes, such as 30 seconds to 4 minutes, 1 minute to 3 minutes.

[0105] In step b, the deprotecting agent can be trifluoroacetic acid (TFA) and 4-cyanopyridine in trifluoroethanol (TFE) / dichloromethane solution (CYTFA). The deprotecting agent can be added using an injection module. The deprotection reaction lasts for 10 to 20 minutes, such as 12 to 18 minutes, 14 to 16 minutes, 15 minutes.

[0106] In step c, washing with dichloromethane (DCM) may be used. The washing agent may be added using an injection module. Washing may be performed for 30 seconds to 5 minutes, such as 30 seconds to 4 minutes, 1 minute to 3 minutes.

[0107] In step d, the neutralizing agent is a mixed solution of N,N-diisopropylethylamine (DIEA), isopropanol and dichloromethane. The neutralizing agent can be added using an injection module. The neutralization reaction can last for 30 seconds to 5 minutes, such as 30 seconds to 4 minutes, 1 minute to 3 minutes.

[0108] In step e, the cleaning may be performed first with dichloromethane and then with 1,3-dimethyl-2-imidazolidinone (DMI). The cleaning agent may be added using an injection module. Each cleaning may be performed for 30 seconds to 5 minutes, for example, 30 seconds to 4 minutes, or 1 minute to 3 minutes.

[0109] In step f, a 10 vol% solution of N-ethylmorpholine (NEM) in 1,3-dimethyl-2-imidazolidinone (DMI) and a 0.1 M solution of morpholino antisense oligonucleotide (PMO) monomer in 1,3-dimethyl-2-imidazolidinone (DMI) can be added to the reactor. An exemplary PMO monomer is shown below:

[0110]

[0111] In some embodiments, PMO can be represented by the following general formula:

[0112]

[0113] Wherein: R1 represents a modified form of a natural purine and pyrimidine base (e.g., adenine, uracil, guanine, cytosine and thymine), its modified analogs and protected derivatives thereof;

[0114] R2 represents hydrogen or trityl, etc.;

[0115] R3 represents an amide or an imine, which can be represented by the following general formula:

[0116]

[0117] Wherein: X represents oxygen or sulfur; Y represents dialkylamino or alkoxy; Z represents chlorine.

[0118] They can both be added using a pipetting device.

[0119] In step g, the reaction may be continued for 2 hours to 4 hours, for example 3 hours.

[0120] The oligonucleotide synthesis device disclosed in the present invention has the following beneficial effects:

[0121] (1) The oligonucleotide synthesis device disclosed in the present invention improves the quality of chemical synthesis. The present invention proposes a technology for implementing multi-channel independent liquid feeding for a single reaction tube to prevent cross-contamination of residual reagents caused by shared pipelines, thereby comprehensively improving the synthesis quality in all reaction tubes. The present invention adopts a liquid transfer device (pipette gun) to transfer monomers, replacing the traditional steel needle liquid feeding method, fundamentally solving the problems of steel needle hanging liquid, steel needle crystallization, etc. that affect the synthesis quality.

[0122] (2) The oligonucleotide synthesis device disclosed in the present invention improves the reaction efficiency of chemical synthesis. The present invention realizes full stirring in all directions and without dead corners in the reaction tube through inert gas stirring, parallel shaking, and constant temperature gas bath functions throughout the reaction, fundamentally solving the problem of low reaction efficiency caused by uneven stirring of reaction reagents, deterioration of reagents due to air intrusion, and other factors in the prior art.

[0123] (3) The oligonucleotide synthesis device disclosed in the present invention achieves the consistency of reaction quality in multiple reaction tubes. By programming the entire production process, a high degree of automation is achieved, so that the entire process is carried out according to the established process, reducing the probability of human errors in the production process. The present invention uses high-precision liquid feeding and drainage modules and adjustable independent air intake pipelines to ensure that each reaction tube can obtain equal reaction reagents and inert gases, and the reaction reagents can enter each reaction tube evenly and simultaneously. In addition, the constant temperature air bath and shaking that exist at the same time ensure the equal contact time and stirring speed of the reaction reagents in each reaction tube, and also ensure the uniform synthesis quality of each reaction tube. The uniform synthesis quality in the reaction tube guarantees the integrity and accuracy of all fragments during downstream synthesis, thereby ensuring the quality and efficiency of the synthesis. The "full automatic" described herein refers to the various operations such as taking liquid from the reagent reservoir, delivering the reagent in the pipeline, injecting the reagent into the reactor, heating the reactor (including the operation of the heating rod and the fan) and shaking, conveying the inert gas, and discharging the waste liquid, which are automatically performed under the control of the control system, but does not exclude the necessary manual intervention such as manual preparation of reagents and manual input of parameters.

[0124] (4) The oligonucleotide synthesis device disclosed in the present invention achieves high-quality and high-efficiency oligonucleotide synthesis in terms of liquid feeding method, cavity size, liquid discharge method and stirring method, and has a breakthrough innovative value for high-throughput column synthesizers.

[0125] Example

[0126] The present disclosure is described in more detail below by specific examples. The following examples are for illustrative purposes only and are not intended to limit the present disclosure in any way. Those skilled in the art will readily recognize various non-critical parameters that can be changed or modified to produce substantially the same results.

[0127] In the following examples, the reagents and materials used, unless otherwise specified, can be obtained commercially or synthetically.

[0128] Example 1 Morpholino Antisense Oligonucleotide Fully Automatic Synthesis Equipment

[0129] The fully automatic synthesis equipment of morpholino antisense oligonucleotide in this embodiment includes 10 pumping modules, so it can pump up to 10 reagents. Each pumping module includes 6 solenoid valves and 6 electric injectors. The reaction system includes 5 reaction modules, each of which includes 6 reaction tubes, so a total of 30 reaction tubes can be used simultaneously. The equipment includes 3 pipettes and a gun tip box, in which the required gun tips are placed, one gun tip corresponds to one reagent bottle, and 3 pipettes can add liquid at the same time. The equipment includes an injection module, which includes 6 injection ports, each of which includes 10 openings and corresponding 10 injection connectors (such as Figure 5A and 5B As shown in the figure). The number of injection ports corresponds to (i.e. is equal to) the number of reaction tubes; the number of openings (number of injection joints) corresponds to (is equal to) the number of pumping modules, and also corresponds to (is equal to) the maximum number of reagent types that can be pumped. This ensures that each reagent is delivered to a reaction tube through an independent pipeline.

[0130] Example 2 Fully automated synthesis of morpholino antisense oligonucleotides

[0131] The morpholino antisense oligonucleotides were synthesized using the morpholino antisense oligonucleotide fully automatic synthesis device in Example 1. The specific steps are as follows:

[0132] (1) Set up the running program steps on the control system.

[0133] (2) The swollen resin is introduced into the reaction tube through the feed port and used as a carrier for the synthesis of morpholino antisense oligonucleotides; various reagents are prepared.

[0134] (3) Input the required parameters, including time, temperature, frequency and amplitude of shaking, and the amount of each reagent added.

[0135] (4) Input the oligonucleotide sequence information, main cycle length, and sub-cycle length to be synthesized, save as a synthesis plan, and click the start button to automatically work. At the same time, the system controls the execution of set parameters, equipment status tracking, and abnormal handling.

[0136] Run the program as follows:

[0137] a.DCM cleaning:

[0138] The mobile module drives the injection module to move, and DCM is added to the 6 reaction tubes of a reaction module at the same time. After the addition is completed, the mobile module rises, the reaction module starts, and shakes according to the preset parameters. At the same time, the heating device is turned on to heat to the set temperature (25°C), and nitrogen is blown to isolate the air. According to the preset time, the shaking continues for 1 minute, and the nitrogen is stopped. The vacuum pump generates negative pressure and the liquid in the reaction tube is discharged into the waste liquid bucket.

[0139] b.30% TFE / DCM cleaning:

[0140] The mobile module drives the injection module to move, and 30% TFE / DCM is added to the 6 reaction tubes of the reaction module at the same time. After the addition is completed, the mobile module rises, the reaction module starts, and shakes according to the preset parameters. At the same time, the heating device is turned on to heat to the set temperature (25°C), and nitrogen is blown to isolate the air. According to the preset time, continue shaking for 1 minute, stop blowing nitrogen, generate negative pressure through the vacuum pump, and drain the liquid in the reaction tube into the waste liquid bucket. Repeat this step twice.

[0141] c. Deprotection:

[0142] The mobile module drives the injection module to move, and CYTFA solution is added to the 6 reaction tubes of the reaction module at the same time. After the addition is completed, the mobile module rises, the reaction module starts, shakes according to the preset parameters, turns on the heating device to heat to the set temperature (25°C), and blows nitrogen to isolate the air. According to the preset time, continue shaking for 15 minutes, stop blowing nitrogen, generate negative pressure through the vacuum pump, and drain the liquid in the reaction tube into the waste liquid bucket. Repeat this step twice.

[0143] d.DCM cleaning:

[0144] The mobile module drives the injection module to move, and DCM is added to the 6 reaction tubes of the reaction module at the same time. After the addition is completed, the mobile module rises, the reaction module starts, shakes according to the preset parameters, turns on the heating device to heat to the set temperature (25°C), and blows nitrogen to isolate the air. According to the preset time, continue shaking for 1 minute, stop blowing nitrogen, generate negative pressure through the vacuum pump, and discharge the liquid in the reaction tube into the waste liquid bucket.

[0145] e. Neutralization:

[0146] The mobile module drives the injection module to move, and the neutralization solution is added to the 6 reaction tubes of the reaction module at the same time. (A mixed solution of N,N-diisopropylethylamine, isopropanol, and dichloromethane). After the addition is completed, the mobile module rises, the reaction module starts, shakes according to the preset parameters, turns on the heating device to heat to the set temperature (25°C), and blows nitrogen to isolate the air. According to the preset time, continue shaking for 1 minute, stop blowing nitrogen, generate negative pressure through the vacuum pump, and drain the liquid in the reaction tube into the waste liquid bucket. Repeat this step twice.

[0147] f.DCM cleaning:

[0148] The mobile module drives the injection module to move, and DCM is added to the 6 reaction tubes of the reaction module at the same time. After the addition is completed, the mobile module rises, the reaction module starts, shakes according to the preset parameters, turns on the heating device to heat to the set temperature (25°C), and blows nitrogen to isolate the air. According to the preset time, continue shaking for 1 minute, stop blowing nitrogen, generate negative pressure through the vacuum pump, and drain the liquid in the reaction tube into the waste liquid bucket. Repeat this step twice.

[0149] g.DMI cleaning:

[0150] The mobile module drives the injection module to move, and DMI is added to the 6 reaction tubes of the reaction module at the same time. After the addition is completed, the mobile module rises, the reaction module starts, shakes according to the preset parameters, turns on the heating device to heat to the set temperature (25°C), and blows nitrogen to isolate the air. According to the preset time, continue shaking for 1 minute, stop blowing nitrogen, generate negative pressure through the vacuum pump, and drain the liquid in the reaction tube into the waste liquid bucket.

[0151] h. Reaction:

[0152] The mobile module drives the pipette to transfer 10% NEM / DMI (V:V) solution and add it to the reaction tube. According to the synthesis scheme, different PMO monomer solutions are transferred with a pipette and added to the corresponding reaction tubes.

[0153] After the addition is completed, the mobile module rises, the reaction module starts, shakes according to the preset parameters, turns on the heating device to heat to the set temperature (35°C), and blows nitrogen to isolate the air. Shake for 3 hours according to the preset time. After the reaction is completed, stop blowing nitrogen, generate negative pressure through the vacuum pump, and drain the liquid in the reaction tube into the waste liquid bucket.

[0154] The above steps are one sub-cycle. After completing the set number of sub-cycles, you will be prompted to prepare the reagents. After manually preparing the required reagents, click the Continue button, and the reaction equipment will perform the set sub-cycle according to the input sequence information. Repeat the sub-cycle until the main cycle completes the synthesis, the display panel prompts that the synthesis is completed, and the sample is taken out.

[0155] The sample was taken out and washed with 1,3-dimethylpyrrolidone (NMP), and then the sample was placed in a reactor, and a 1,3-dimethylpyrrolidone (NMP) solution of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) and dithiothreitol (DTT) was added, and the reaction was carried out at 35°C for 5 hours to cut the morpholino antisense oligonucleotide synthesis from the carrier. Then filter and collect the filtrate. The filtrate was added to a jar containing 25% ammonia water and reacted at 55°C for 16 hours to remove the protective group on the morpholino antisense oligonucleotide synthesis.

[0156] After the reaction was completed, the temperature was restored to room temperature and samples were taken for chromatographic analysis. Figure 6 As shown, the main peak with a retention time of 11.253 minutes is morpholino antisense oligonucleotide. From the integration results, it can be seen that the purity of the obtained crude morpholino antisense oligonucleotide is 62-63%.

[0157] All documents mentioned in this disclosure are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of this disclosure, those skilled in the art may make various changes or modifications to this disclosure, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. An automatic oligonucleotide synthesis device, characterized in that: The device comprises: a pipeline liquid supply system, a reaction system and a control system, wherein the pipeline liquid supply system comprises a pumping module (1-1), a moving module (1-4) and a liquid injection module (1-3), wherein the liquid injection module (1-3) is installed on the moving module (1-4), and the moving module (1-4) is configured to enable the liquid injection module (1-3) to move in three dimensions, thereby injecting liquid into the reaction system; the control system is communicatively connected with the pipeline liquid supply system and the reaction system, and is used to control liquid supply and reaction; the device preferably also comprises a non-pipeline liquid supply system, and the non-pipeline liquid supply system comprises a liquid transfer device (1-5), and the liquid transfer device (1-5) is preferably installed on the moving module (1-4) and is used to inject liquid into the reaction system.

2. The fully automatic oligonucleotide synthesis device according to claim 1, characterized in that: The reaction system comprises at least one reaction module (1-2), preferably at least two reaction modules (1-2), for example, three, four, five or six reaction modules (1-2); each reaction module (1-2) comprises at least one reactor, preferably at least two reactors (4-1), for example, three, four, five or six reactors (4-1); each reactor (4-1) is optionally arranged in a transition sleeve (4-2); the reaction module (1-2) preferably comprises a shaking mechanism, and the shaking mechanism preferably comprises a motion guide rail, more preferably comprises two sets of motion guide rails (4-4, 4-5), which are configured to shake the reactor (4-1).

3. The fully automatic oligonucleotide synthesis device according to claim 2, characterized in that: The device comprises one or more injection modules (1-3), each injection module (1-3) comprises an injection port for injecting liquid into the reactor (4-1), and preferably the number of injection ports of each injection module (1-3) is equal to the number of reactors (4-1) of each reaction module (1-2).

4. The fully automatic oligonucleotide synthesis device according to claim 1 or 2, characterized in that: The moving module (1-4) comprises a first guide rail, a second guide rail and a third guide rail which are perpendicular to each other. The second guide rail is configured to be able to move horizontally along the first guide rail; the third guide rail is installed on the second guide rail and is configured to be able to move horizontally along the second guide rail; the injection module (1-3) is installed on the third guide rail and is configured to be able to move vertically up and down along the third guide rail.

5. The fully automatic oligonucleotide synthesis device according to claim 1 or 2, characterized in that: The pumping module (1-1) comprises an electric syringe (2-2) and a solenoid valve (2-1), wherein the electric syringe (2-2) is connected to the solenoid valve (2-1) via a pipeline, the solenoid valve (2-1) comprises an input port and an output port, the input port is connected to a reagent reservoir via a pipeline, and the output port is connected to a liquid injection module (1-3) via a pipeline; The device preferably comprises a plurality of reagent reservoirs, wherein each reagent reservoir in at least a portion of the reagent reservoirs comprises one or more reagent outlets, and each reagent outlet is connected to an input port of a solenoid valve (2-1) via a pipeline; Preferably, at least one reagent reservoir comprises only one reagent outlet, the reagent outlet is connected to an input port of a solenoid valve (2-1), and a flow dividing device is provided between the output port of the solenoid valve (2-1) and the injection module (1-3), the flow dividing device comprising a flow dividing device inlet and a plurality of flow dividing device outlets, the flow dividing device inlet is connected to the output port of the solenoid valve (2-1) via a pipeline, and each flow dividing device outlet is connected to an injection port via a pipeline; Preferably, at least one of the plurality of reagent reservoirs is configured to allow a pipetting device (1-5) to pipette a reagent.

6. A method for synthesizing oligonucleotides using an oligonucleotide fully automatic synthesis device, comprising: a. Cleaning the reactor (4-1); b. adding a deprotecting agent to the reactor (4-1) to carry out a deprotection reaction; c. Cleaning the reactor (4-1); d. adding a neutralizing agent to the reactor (4-1) to carry out a neutralization reaction; e. Cleaning the reactor (4-1); f. adding a coupling unit to the reactor (4-1); g. Carry out coupling reaction, Under the control of the control system, steps a to h are cyclically executed until the desired oligonucleotide is obtained, wherein the method includes, under the control of the control system, using a moving module (1-4) to move an injection module (1-3) to inject a reagent into a reactor (4-1), and preferably also includes, under the control of the control system, using a pipetting device (1-5) to transfer the reagent and inject it into the reactor (4-1), wherein the injection module (1-3) and the pipetting device (1-5) are used to inject different reagents.

7. The method as claimed in claim 6, wherein the fully automatic oligonucleotide synthesis equipment comprises a plurality of reaction modules (1-2), and after one reaction module (1-2) completes step f and during step g, steps a to f are performed on the next reaction module (1-2), until the required number of reaction modules (1-2) perform steps a to f.

8. The method of claim 7, wherein one or more of the cleaning, deprotection reaction, neutralization reaction and coupling reaction, preferably all of the processes involve one or more of the following operations under the control of a control system: Shaking the reactor (4-1); heating the reactor (4-1) to a predetermined temperature and maintaining the temperature at the predetermined temperature; The reactor (4-1) was blown with nitrogen to isolate it from the air.

9. The method of claim 6, wherein the oligonucleotide is a morpholino antisense oligonucleotide.

10. The method of claim 9, comprising at least one of the following: In step a, washing with dichloromethane and 30% trifluoroethanol / dichloromethane is performed successively, preferably using the injection module (1-3), preferably for 30 seconds to 5 minutes each, for example 1 minute; In step b, the deprotecting agent is a trifluoroacetic acid and 4-cyanopyridine trifluoroethanol / dichloromethane solution, preferably added using the injection module (1-3), preferably reacting for 10 to 20 minutes, for example 15 minutes; In step c, washing with dichloromethane, preferably using the injection module (1-3), preferably washing for 30 seconds to 5 minutes, for example 1 minute; In step d, the neutralizing agent is a mixed solution of N,N-diisopropylethylamine, isopropanol and dichloromethane, preferably added using the injection module (1-3), preferably reacting for 30 seconds to 5 minutes, for example 1 minute; In step e, washing with dichloromethane and 1,3-dimethyl-2-imidazolidinone is performed successively, preferably using the injection module (1-3), preferably for 30 seconds to 5 minutes each, for example 1 minute; In step f, a 10 volume % solution of N-ethylmorpholine in 1,3-dimethyl-2-imidazolidinone and a 0.1 M solution of morpholino antisense oligonucleotide monomer in 1,3-dimethyl-2-imidazolidinone are added to the reactor (4-1), preferably both are added using a pipetting device (1-5); In step g, the reaction is continued for 2 to 4 hours, for example 3 hours.

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