Single-point light-operated DNA synthesis device

Abstract

The invention provides a single-point light-operated DNA synthesis device which comprises a light path module, the light path module comprises at least one synthesis starting light source, a collimation and convergence lens group of the synthesis starting light source and a synthesis flow cell, and the collimation and convergence lens group is arranged to collimate and converge light emitted by the synthesis starting light source to the synthesis flow cell. And forming a light spot of a single DNA synthesis site. The single-point light-operated DNA synthesis device disclosed by the invention adopts a structural scheme that only one DNA synthesis site is arranged, so that the single-point light-operated DNA synthesis device is suitable for a research and development process, and various indexes of photosensitive molecules in a chemical reaction are obtained during a whole-process test; due to the fact that only one synthesis site is adopted, various indexes are reduced, the complexity of an instrument can be reduced, the cost of equipment can be reduced, the experimental process can be simplified, and various indexes and performance of photochemical synthesis DNA can be rapidly, efficiently and accurately obtained at low cost.

Description

Technical Field

[0001] The invention belongs to the field of DNA optical synthesis, and in particular relates to a single-point light-controlled DNA synthesis device, which realizes DNA synthesis based on light control. Background Art

[0002] In the field of nucleic acid chemistry, cutting-edge technology research areas include DNA information storage, DNA origami, supramolecular DNA and gene assembly, which all require the synthesis of large oligonucleotide libraries. Array-based parallel DNA synthesis can be performed in situ on a large scale, generating thousands to millions of unique sequences at a time. There are currently three types of array-based DNA synthesis pathways based on traditional phosphoamidite chemistry.

[0003] First, an inkjet printer was used to control the spotting of phosphoamidophosphotyrosine solutions on the surface for parallel oligonucleotide synthesis, where the CMYK color space was reused for the ACGT nucleotide alphabet, and the 5' acid-sensitive protecting group (hydroxydimethoxytriphenylphosphine, DMTr) that prevented the next binding reaction was simultaneously removed on the surface.

[0004] Second, this deprotection step is sterically restricted by using electrochemical or photogenerated acid so that phosphoamido conjugation occurs at the site where the DMTr group was removed.

[0005] Third, the DMTr protection strategy was replaced by a photosensitive group, and ultraviolet light (UV) at the 5-OH became the medium for regulating the next binding reaction. UV light irradiation can be controlled by a digital micromirror device (DMD), and each micromirror is individually addressable, so that UV light is patterned onto the reaction substrate. This photolithography process is called maskless array light-controlled DNA synthesis (MAS) when applied to nucleic acid synthesis.

[0006] The throughput of these in situ DNA array fabrication methods is a huge improvement over traditional solid-phase synthesis. However, throughput alone is not enough, and control of synthesis precision is also important, especially in applications such as in vivo gene delivery and digital DNA storage.

[0007] Compared with other array-based DNA synthesis technologies, light-controlled DNA synthesis has the following advantages: 1) It is independent of the material of the substrate. In theory, any substrate with hydroxyl, amino or epoxy groups on its surface can be used as a synthetic substrate. This technology has been successfully applied to a variety of surfaces such as diamond films, amorphous carbon, glassy carbon, carbon-metal composites and polymers to achieve DNA synthesis. 2) It can achieve high-density synthesis, with the size of the synthesis unit reaching 2.5 microns and the spacing being only 0.7 microns. 3) It is suitable for the synthesis of a variety of biological molecules. Currently, it has successfully synthesized peptides, RNA, peptide nucleic acids, L-DNA, etc., laying the foundation for subsequent DNA functionalization modification. 4) In addition to chemical synthesis methods, this technology also supports enzymatic synthesis, further broadening the scope of application.

[0008] Existing maskless array-type light-controlled DNA synthesis equipment needs to use a digital micromirror array device as the core encoding element, and with this as the center, it needs to be equipped with auxiliary facilities such as projection light path and encoding software, resulting in a complex structure and high price. The field of array-type DNA synthesis is still in a period of rapid growth, with new principles and synthesis functions being developed continuously, requiring a large number of experiments to test the function and performance of photosensitive molecules in photochemical reactions.

[0009] Related Literature:

[0010] 1.Singh-Gasson,S.,Green,R.,Yue,Y.et al.Maskless fabrication of light-directed oligonucleotide microarrays using a digital micromirror array.NatBiotechnol 17,974–978(1999).https: / / doi.org / 10.1038 / 13664

[0011] 2. Lietard J, Leger A, Erlich Y, et al. Chemical and photochemical errorrates in light-directed synthesis of complex DNA libraries [J]. Nucleic acidsresearch, 2021, 49(12): 6687-6701. Summary of the invention

[0012] The object of the present invention is to provide a single-point light-controlled DNA synthesis device to reduce the complexity and cost of the equipment.

[0013] In order to achieve the above-mentioned purpose, the present invention provides a single-point light-controlled DNA synthesis device, comprising an optical path module, wherein the optical path module includes at least one synthesis starting light source, a collimating and converging lens group of the synthesis starting light source, and a synthesis flow cell, wherein the collimating and converging lens group is configured to collimate and converge the light emitted by the synthesis starting light source to the synthesis flow cell to form a light spot of a single DNA synthesis site.

[0014] The synthetic starting light source includes a first synthetic starting light source and a second synthetic starting light source with different wavelengths.

[0015] The wavelength of the first synthetic starting light source is 405 nm, and the wavelength of the second synthetic starting light source is 365 nm.

[0016] The optical path module also includes a guiding light source for outputting a guiding light beam for the DNA synthesis site.

[0017] The collimating and converging lens group includes a first collimating lens corresponding to the first synthetic starting light source, a second collimating lens corresponding to the second synthetic starting light source, the third collimating lens, the first coupling mirror, the second coupling mirror and the reflecting mirror arranged in sequence along the first optical axis from the guiding light source, and a converging lens located between the reflecting mirror and the synthetic flow pool; the first coupling mirror is used to deflect the light of the first synthetic starting light source to coincide with the first optical axis and allow the light on the first optical axis to pass through; the second coupling mirror is used to deflect the light of the second synthetic starting light source to coincide with the first optical axis and allow the light on the first optical axis to pass through.

[0018] The collimating and converging lens group at least comprises a collimating lens and a converging lens, and the positions of the converging lens and the synthetic flow cell are adjustable.

[0019] The single-point light-controlled DNA synthesis device also includes an electric control device and a sample loading and washing module. The electric control module is configured to adjust the illumination time of the synthesis start light source and the sample loading and washing time of the sample loading and washing module.

[0020] The sample loading and flushing module comprises a multi-channel sample loading pump connected between a plurality of raw material solutions and flushing solutions and a synthesis circulation pool, and a waste liquid pool connected to the synthesis circulation pool through a flushing channel.

[0021] The electric control module includes a power adapter connected to the synthesis starting light source, and a controller connected to the power adapter and to the sample loading and washing module; the controller converts the DNA sequence into a synthesis task, which includes an electrical signal sent to the electric control module and a control signal sent to the sample loading and washing module, and then executes the synthesis task to achieve DNA synthesis.

[0022] The controller includes a function signal generator, and generates the waveform and switching time of the electrical signal of the synthetic starting light source through the function signal generator, so as to control the switching of the corresponding synthetic starting light source through the power adapter.

[0023] The single-point light-controlled DNA synthesis device of the present invention adopts a structural scheme of only setting one DNA synthesis site, so as to be suitable for the research and development process, and obtain various indicators of the photosensitive molecule in the chemical reaction during the full process test; since only one synthesis site is used, various indicators are reduced, so the complexity of the instrument can be reduced, the cost of the equipment can be reduced, and the experimental process can be simplified, and various indicators and performances of photochemically synthesized DNA can be obtained quickly, cheaply, efficiently and accurately, providing preliminary experimental data for the subsequent use of large-scale complex equipment for high-throughput synthesis of DNA, and indicating the direction and paving the way. In addition, the device of the present invention is provided with an electric control module and a controller, which can be fully automatically operated and has the characteristics of being simple and easy to use. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematic diagram of the structure of a fully automatic single-point light-controlled DNA synthesis device according to an embodiment of the present invention.

[0025] Figure 2 This is a structural diagram of a mechanical module of a fully automatic single-point light-controlled DNA synthesis device of the present invention.

[0026] Figure 3 The present invention is a flow chart of a controller of a fully automatic single-point light-controlled DNA synthesis device. DETAILED DESCRIPTION

[0027] The present invention is further described below in conjunction with specific examples. It should be understood that the following examples are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0028] The working principle of the single-point light-controlled DNA synthesis device of the present invention when used for light-controlled DNA synthesis is as follows:

[0029] In 1991, American scientist STEPHEN PAFodor first proposed a method for making DNA arrays, which is based on photolithography. Photolithography is a technology borrowed from the semiconductor industry, which reproduces the mask pattern onto a photosensitive surface by irradiating light onto a photolithography mask (similar to a photographic negative). Reagents used for making DNA arrays by photolithography are usually photosensitized phosphoramide reagents, in which the 5' hydroxyl group is usually replaced with a photoremovable protecting group (such as dimethoxytrityl, DMT). During the synthesis process, the protecting groups are removed from all areas where the nucleoside is to be extended by irradiation, and then the nucleoside is connected by exposing the entire array to an appropriate phosphoramide, and then the process is repeated for the next nucleoside after oxidation and washing steps. In order to make a DNA array of N bases in length, 4N cycles of deprotection and connection are required, each cycle corresponding to one of the four bases, multiplied by N base positions.

[0030] The working principle of the single-point light-controlled DNA synthesis device of the present invention is to construct a light path to form a light spot corresponding to the DNA synthesis site, and start the photochemical synthesis according to the principle of photochemical reaction.

[0031] like Figure 1 As shown, the single-point light-controlled DNA synthesis device of the present invention includes an optical path module, an electric control module, a mechanical module and a sample loading and washing module. Figure 1 The solid line represents light, the dotted line represents the power line and control line, and the double line is the liquid pipeline.

[0032] Among them, the optical path module includes at least one synthetic starting light source, a collimating and converging lens group of the synthetic starting light source, and a synthetic flow pool 30. The collimating and converging lens group is configured to collimate and converge the light emitted by the synthetic starting light source to the synthetic flow pool 30 to form a light spot of a single DNA synthesis site.

[0033] In this embodiment, the synthetic starting light source includes a first synthetic starting light source 11 and a second synthetic starting light source 12 with different wavelengths. The wavelength of the first synthetic starting light source is 405nm, and the wavelength of the second synthetic starting light source 12 is 365nm. The light of the first synthetic starting light source 11 and the second synthetic starting light source 12 are both used to remove the protecting group.

[0034] In other embodiments, the number of synthetic starting light sources can be increased or decreased as needed. It is also feasible to use only one synthetic starting light source to remove the protecting group, but the effect of the synthetic starting light source on removing different protecting groups will be different due to the different protecting groups.

[0035] In this embodiment, since the first synthetic starting light source 11 with a wavelength of 405nm and the second synthetic starting light source 12 with a wavelength of 365nm are used as synthetic starting light sources, and the wavelengths of light beams with wavelengths of 365nm and 405nm are difficult to observe, the optical path module further includes a guiding light source 13 for outputting a guiding light beam of the DNA synthesis site. In this embodiment, the wavelength of the light of the guiding light source 13 is 530nm.

[0036] The collimating and converging lens group includes a first collimating lens 21 corresponding to the first synthetic starting light source 11, a second collimating lens 22 corresponding to the second synthetic starting light source 12, the third collimating lens 23, the first coupling mirror 24, the second coupling mirror 25 and the reflector 26 arranged in sequence along the first optical axis from the guiding light source 13, and a converging lens 27 located between the reflector 26 and the synthetic circulation pool 30.

[0037] The collimating lens is used to convert the LED light into parallel light. The first coupling mirror 24 is used to deflect the light of the first synthetic starting light source 11 to coincide with the first optical axis and allow the light on the first optical axis to pass through; the second coupling mirror 25 is used to deflect the light of the second synthetic starting light source 12 to coincide with the first optical axis and allow the light on the first optical axis to pass through.

[0038] The positions of the converging lens 27 and the synthesis flow cell 30 can be adjusted to adjust the size of the light spot of the DNA synthesis site formed by the collimating and converging lens group at the synthesis flow cell 30. In this embodiment, the size of the light spot of the DNA synthesis site formed by the collimating and converging lens group at the synthesis flow cell 30 is about 5 mm.

[0039] In this embodiment, the reflector 26 and the converging lens 27 are located directly above the synthesis flow cell 30 .

[0040] Therefore, in the present invention, the postures of the first coupling mirror 24 and the second coupling mirror 25 are used to make the three beams of light from the first synthesis starting light source 11, the second synthesis starting light source 12 and the guiding light source 13 overlap to form a light spot of the DNA synthesis site at the synthesis circulation pool 30, and the posture of the reflecting mirror 26 is used to make the three overlapping beams of light hit the specific position of the synthesis circulation pool 30.

[0041] The electric control module is connected to the synthetic starting light source, and is configured to adjust the illumination time of the synthetic starting light source and the loading and flushing time of the loading and flushing module. In the present embodiment, the electric control module includes a first power adapter 41 connected to the first synthetic starting light source 11, a second power adapter 42 connected to the second synthetic starting light source 12, and a third power adapter 43 connected to the guide light source 13, so as to supply power to the first synthetic starting light source 11, the second synthetic starting light source 12 and the guide light source 13. The electric control module also includes a controller 44 connected to the power adapters of all the synthetic starting light sources and connected to the loading and flushing module. The first power adapter 41, the second power adapter 42 and the third power adapter 43 are all powered by a main power supply 45.

[0042] The controller includes a function signal generator, and generates the waveform and switching time of the electrical signal of the synthetic starting light source through the function signal generator, so as to control the switching of the corresponding first synthetic starting light source 11 and the second synthetic starting light source 12 through the first power adapter 41 and the second power adapter 42, which helps to start the encoded DNA synthesis reaction.

[0043] One of the research objectives of the device of the present invention is to determine the illumination duration corresponding to different protective groups, and the illumination duration is determined by the protective groups.

[0044] The mechanical module includes a fixing device for fixing all the collimating lenses, coupling mirrors, reflectors 26, converging lenses 27 and synthetic flow cell 30. In this embodiment, the specific structures of the collimating lenses, coupling mirrors, reflectors 26, converging lenses 27 and synthetic flow cell 30 and the mechanical module are as follows: Figure 2 As shown. Figure 2 As shown, the mechanical module includes a collimating and converging lens assembly mounting frame 51 for fixing all collimating lenses, coupling mirrors, reflectors 26, and converging lenses 27 together, a plurality of supporting columns 52 supported at the bottom of the converging lens assembly mounting frame, and a three-dimensional position adjustment frame 53 mounted on the side of the synthetic flow cell 30 to adjust the position and posture of the synthetic flow cell 30. The converging lens assembly mounting frame 51 can also play a role in dust and light protection.

[0045] The sample loading and flushing module is connected to the synthesis circulation pool 30. In this embodiment, the sample loading and flushing module includes a multi-channel sample loading pump 63 connected between the multiple raw material solutions 61 and the flushing solution 62 and the synthesis circulation pool 30, and a waste liquid pool 64 connected to the synthesis circulation pool 30 through a flushing channel.

[0046] Thus, the raw material solution 61 is pumped into the synthesis flow cell 30 by the loading pump, and after the reaction is completed, the washing liquid 62 is pumped into the synthesis flow cell 30 by the loading pump to wash away the unreacted raw material solution 61. In this embodiment, there are multiple raw material solutions 61, each of which includes only one nucleotide with a protective group, and the washing liquid uses a pure buffer.

[0047] The controller 44 is installed with software, so it is configured to convert the DNA sequence into a synthesis task, which includes an electrical signal sent to the electrical control module and a control signal sent to the sample loading and washing module, and then execute the synthesis task to achieve DNA synthesis.

[0048] Therefore, the synthesis time of each step can be controlled by the specific parameters of the synthesis task, and the synthesis reaction can be started or terminated automatically.

[0049] like Figure 3 As shown, the controller is configured to perform the following steps:

[0050] Step S1: Reading DNA sequence;

[0051] Step S2: performing sequence fragmentation to divide the DNA sequence into fragments according to one or more nucleotides for synthesis in subsequent steps;

[0052] In this embodiment, sequence fragmentation refers to splitting the DNA sequence into fragments according to groups of 5 (or other numbers) of nucleotides.

[0053] The purposes of sequence fragmentation include: 1. Preventing the amount of data of the generated waveform from being too large, causing the controller storage to overflow; 2. Preventing the sequence from being too long and the synthesis time from being too long, causing the interval time between the switching of the sample pump and the switching of the light to drift, resulting in inaccurate timing.

[0054] Step S3: creating a synthesis task, wherein the synthesis task is used to write a synthesis control waveform;

[0055] Creating a synthesis task specifically includes: using a code on a controller to create a synthesis task for synthesizing a single base. The created synthesis task is used to write a synthesis control waveform corresponding to only one base.

[0056] Step S4: creating a synthetic control waveform, wherein the synthetic control waveform includes an electrical signal converted from a single base (or a single fragment) and sent to the electrical control module and a control signal sent to the sample loading and washing module.

[0057] In a single created synthetic control waveform, the electrical signal sent to the electronic control module and the control signal sent to the sample loading and flushing module specifically include: the control signal of the multi-channel sample loading pump, the control signal of the flushing channel and the light LED electrical signal; the control signal of the multi-channel sample loading pump includes the selection of channels in the multi-channel sample loading pump and the switching time of the multi-channel sample loading pump, and the control signal of the flushing channel includes the switching event of the flushing channel; the electrical signal sent to the electronic control module includes the waveform and switching time of the electrical signal of each synthetic starting light source.

[0058] Since the purpose of the device of the present invention is mainly to study various photochemical parameters of photosensitive nucleotides (nucleotides with photosensitive protective groups) during photosynthesis, including the light activation time of the photosensitive nucleotides, the time of extension to the DNA chain, etc., these parameters contained in the created synthesis control waveform can be used as research objects to provide data support for subsequent large-scale parallel multi-channel photosensitivity synthesis.

[0059] Step S5: writing the synthesis control waveform into the synthesis task;

[0060] Step S6: executing the synthesis task to drive each synthesis start light source of the electric control module and the multi-channel loading pump and flushing channel of the loading and flushing module to complete the DNA synthesis reaction corresponding to one base.

[0061] Step S6 specifically includes: the controller drives the multi-channel sample loading pump to load samples, drives each synthesis start light source to irradiate respectively, and drives the flushing channel to flush, and then stops the synthesis task.

[0062] Step S7: Return to step S4 to repeat the above steps until the DNA sequence synthesis is completed.

[0063] For example, to synthesize a DNA sequence of AATAACGGG, the process is as follows: 1. The controller sends a control signal to the sample pump to open the sample pump channel corresponding to the A base, and couples the nucleotide A with a protective group to the glass substrate; 2. A control signal is sent to the sample pump to open the sample pump flushing channel to flush out the unreacted nucleotide A; 3. An electrical signal is sent to the synthesis start light source to turn on the 365LED (or 405LED) to start irradiation and deprotect the nucleotide A; 4. The above steps are repeated, except that the type of nucleotide is changed as needed until the DNA sequence is synthesized.

[0064] Among them, there are two optional light wavelengths, which can be adjusted by turning on a specific synthetic start light source through the controller. The light power is adjustable from 0-100%, which is adjusted through the power adapter. The illumination time is adjustable from ms to infinite length, which is adjusted through the electrical waveform of the controller. The flushing time is adjustable from seconds to infinite length, which is adjusted by controlling the sample pump to open and close through the controller.

[0065] The single-point light-controlled DNA synthesis device of the present invention adopts a structural scheme that only measures one DNA synthesis site, so as to be suitable for the research and development process, and obtain various indicators of the photosensitive molecule in the chemical reaction during the full process test; since only one synthesis site is used, various indicators are reduced, so the complexity of the instrument can be reduced, the cost of the equipment can be reduced, and the experimental process can be simplified, and various indicators and performances of photochemically synthesized DNA can be obtained quickly, cheaply, efficiently, and accurately, providing preliminary experimental data for the subsequent use of large-scale complex equipment for high-throughput synthesis of DNA, and indicating the direction and paving the way. In addition, the device of the present invention is provided with an electric control module and a controller, which can be fully automatically operated and has the characteristics of being simple and easy to use.

[0066] The above is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention. The above embodiments of the present invention can also be modified in various ways. All simple, equivalent changes and modifications made according to the claims and the contents of the specification of the present invention fall within the scope of protection of the claims of the present invention. The contents not described in detail in the present invention are all conventional technical contents.

Claims

1. A single-point light-controlled DNA synthesis device, characterized in that: The method comprises an optical path module, wherein the optical path module comprises at least one synthetic starting light source, a collimating and converging lens group of the synthetic starting light source, and a synthetic flow pool, wherein the collimating and converging lens group is configured to collimate and converge the light emitted by the synthetic starting light source to the synthetic flow pool to form a light spot of a single DNA synthesis site.

2. The single-point light-controlled DNA synthesis device according to claim 1, characterized in that: The synthetic starting light source includes a first synthetic starting light source and a second synthetic starting light source with different wavelengths.

3. The single-point light-controlled DNA synthesis device according to claim 2, characterized in that: The wavelength of the first synthetic starting light source is 405 nm, and the wavelength of the second synthetic starting light source is 365 nm.

4. The single-point light-controlled DNA synthesis device according to claim 2, characterized in that: The optical path module also includes a guiding light source for outputting a guiding light beam for the DNA synthesis site.

5. The single-point light-controlled DNA synthesis device according to claim 4, characterized in that: The collimating and converging lens group includes a first collimating lens corresponding to the first synthetic starting light source, a second collimating lens corresponding to the second synthetic starting light source, the third collimating lens, the first coupling mirror, the second coupling mirror and the reflecting mirror arranged in sequence along the first optical axis from the guiding light source, and a converging lens located between the reflecting mirror and the synthetic flow pool; the first coupling mirror is used to deflect the light of the first synthetic starting light source to coincide with the first optical axis and allow the light on the first optical axis to pass through; the second coupling mirror is used to deflect the light of the second synthetic starting light source to coincide with the first optical axis and allow the light on the first optical axis to pass through.

6. The single-point light-controlled DNA synthesis device according to claim 1, characterized in that: The collimating and converging lens group at least comprises a collimating lens and a converging lens, and the positions of the converging lens and the synthetic flow cell are adjustable.

7. The single-point light-controlled DNA synthesis device according to claim 1, characterized in that: It also includes an electric control device and a sample loading and flushing module, wherein the electric control module is configured to adjust the illumination time of the synthetic starting light source and the sample loading and flushing time of the sample loading and flushing module.

8. The single-point light-controlled DNA synthesis device according to claim 7, characterized in that: The sample loading and flushing module comprises a multi-channel sample loading pump connected between a plurality of raw material solutions and flushing solutions and a synthesis circulation pool, and a waste liquid pool connected to the synthesis circulation pool through a flushing channel.

9. The single-point light-controlled DNA synthesis device according to claim 7, characterized in that: The electric control module includes a power adapter connected to the synthetic starting light source, and a controller connected to the power adapter and to the sample loading and washing module; The controller converts the DNA sequence into a synthesis task, which includes an electrical signal sent to the electrical control module and a control signal sent to the sample loading and washing module, and then executes the synthesis task to achieve DNA synthesis.

10. The single-point light-controlled DNA synthesis device according to claim 9, characterized in that: The controller includes a function signal generator, and generates the waveform and switching time of the electrical signal of the synthetic starting light source through the function signal generator, so as to control the switching of the corresponding synthetic starting light source through the power adapter.

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