WO2023200259A1 - System and method for precise optical synthesis of dna - Google Patents

Abstract

The present invention relates to a technique which can reduce errors that occur during photochemical DNA synthesis, and which can prevent the deprotection of undesired DNA by irradiating desired DNA synthesis sites with a first wavelength for DNA deprotection, and then irradiating undesired sites with a second wavelength longer than the first wavelength, and thus can reduce DNA synthesis errors.

Description

Systems and methods for precise DNA optical synthesis

The present invention relates to a system and method for precise DNA optical synthesis. Specifically, it is a technology that reduces DNA synthesis errors by preventing light from deprotecting DNA in unwanted regions during the photochemical-based DNA synthesis process.

The amount of data generated is increasing very rapidly due to the increase in personal social network service (SNS) media, which mainly focuses on images and videos. In the future, as 3D media such as AR (augmented reality), VR (virtual reality), MR (mixed reality) devices, and holograms become popular, the amount of data is expected to increase rapidly. Specifically, the amount of data stored worldwide is expected to reach 10 ²⁹ bits to 10 ²⁹ bits in 2040 (Summary report, Technology Working Group Meeting on future DNA synthesis technologies; September 14, 2017, Arlington, VA).

Currently, the data storage medium for major devices such as computers and smartphones is flash memory, and flash memory has a data density of about 1 bit per 1 pg. In order to supply flash memory corresponding to the amount of data needed in 2040, 10 ¹⁴ kg of silicon wafers are needed, but the supply of silicon wafers in 2040 is expected to be about 10 ⁸ kg, which falls far short of demand. Additionally, existing data storage media have a limitation of data retention period of approximately 10 years.

Accordingly, attempts are continuing to develop new data storage media, one of which is DNA storage. Using DNA as a storage medium can overcome the data storage density, which is a disadvantage of existing storage media, and can store information stably even when subjected to physical shock. In addition, DNA can store 2x10 ²⁴ bits, equivalent to 10 ⁹ kg of flash memory, by synthesizing 1 kg, making it suitable for large-capacity data storage. In particular, it has the advantage of having a very long storage period for the stored data. However, errors that occur during DNA synthesis cause a decrease in data storage density and information loss, acting as an obstacle to the commercialization of DNA storage devices.

In particular, in the photochemical DNA synthesis method, when light is shined to deprotect the protecting groups at the ends of DNA, the light reaches unwanted areas due to light diffraction, scattering, and reflection. This ultimately causes deprotection at the unintended ends of the DNA, resulting in errors in DNA synthesis.

In the above situation, the present inventors studied a method to reduce diffraction, scattering, and reflection of light during the photochemical DNA synthesis process, irradiated the desired DNA synthesis area with a first wavelength for DNA deprotection, and A method of irradiating a second wavelength, which is longer than the first wavelength, to areas where DNA synthesis is not desired was devised at the same time as the irradiation.

Using the above method, the protecting group at the end of the DNA absorbs energy from the first wavelength in the region where DNA synthesis is not desired, and the absorbed energy is stimulated emission by irradiation of the second wavelength before breaking the chemical bond, thereby preventing unwanted DNA synthesis. It can prevent deprotection of DNA.

Therefore, the purpose of the present invention is to provide a DNA synthesis method using the above principles and a DNA synthesis system for implementing the method.

To achieve the above object, one aspect of the present invention provides a DNA synthesis system comprising the following components:

DNA synthesis unit where DNA synthesis occurs;

a first wavelength light source that irradiates a first wavelength to the synthesis area of the DNA synthesis unit;

a second wavelength light source that irradiates a second wavelength to the synthesis area of the DNA synthesis unit;

a first light-space modulator that controls the irradiation pattern of the first wavelength light source to the DNA synthesis unit; and

A second light-space modulator that controls the irradiation pattern of the second wavelength light source to the DNA synthesis unit.

To achieve the above object, another aspect of the present invention provides a DNA synthesis system comprising the following components:

A DNA synthesis unit including a transparent electrode and where DNA synthesis occurs;

A first wavelength light source that irradiates a first wavelength to the target site of the DNA synthesis unit;

a first light-space modulator that controls the irradiation pattern of the first wavelength light source to the DNA synthesis unit; and

A power supply unit that supplies power to the electrodes of the DNA synthesis unit.

Another aspect of the present invention provides a DNA synthesis method using the DNA synthesis system.

The above method is

(a) irradiating a first wavelength through a first light-spatial modulator to the site where DNA is to be synthesized in the DNA synthesis unit;

(b-1) Simultaneously with irradiation of the first wavelength, irradiating a second wavelength longer than the first wavelength through a second light-space modulator to the remaining portions of the DNA synthesis unit excluding the portion where DNA is to be synthesized; or

(b-2) applying voltage to the DNA synthesis unit simultaneously with irradiation of the first wavelength; and

It includes the step of supplying a nucleotide solution to be synthesized.

By using the DNA synthesis method according to an example of the present invention, errors occurring in the photochemical-based DNA synthesis process can be significantly reduced.

Figure 1 schematically shows the structure of a DNA synthesis system using a first wavelength (λe) and a second wavelength (λp).

Figure 2 outlines the causes of synthesis errors in the photochemical-based DNA synthesis process.

Figure 3 shows the results of measuring the amount of light irradiated to the synthesis site during the photochemical-based DNA synthesis process.

Figure 4 shows an example of a process in which DNA ends are deprotected and new nucleotides are added in a photochemical-based DNA synthesis method.

Figure 5 shows an example of a process in which DNA ends are deprotected by a deprotection molecule-providing substance and new nucleotides are added during the photochemical-based DNA synthesis process.

Figure 6 shows an example of a method of irradiating light to the DNA synthesis site and simultaneously applying voltage to increase the spatial resolution of the site when synthesizing DNA based on photochemistry using a DNA synthesis substrate containing a transparent electrode. It shows.

Hereinafter, with reference to the attached drawings, preferred embodiments will be described in detail so that those skilled in the art can easily practice the present invention. However, when describing preferred embodiments of the present invention in detail, if it is determined that a detailed description of a related known function or configuration may unnecessarily obscure the gist of the present invention, the detailed description will be omitted. In addition, the same symbols are used throughout the drawings for parts that perform similar functions and actions. On the other hand, 'including' a certain component does not mean excluding other components, but may further include other components, unless specifically stated to the contrary.

Photochemistry-based DNA synthesis method uses a method of selectively deprotecting the protecting group attached to the end of the newly added nucleotide by applying light.

Figure 2 outlines the causes of synthesis errors in the photochemical-based DNA synthesis process.

When selectively irradiating light (UV or 100 to 500 nm wavelength) for deprotection onto the synthesis area of the DNA synthesis substrate, whether using a photomask or a DMD (digital micromirror device), the light is applied to the DNA synthesis substrate. Light may be reflected, dispersed, or spread by diffraction and reach unwanted areas.

Looking at Figure 1, it can be seen that the light diffracted from the DNA synthesis substrate deprotects DNA located in a place other than the DNA synthesis area. In addition, insufficient light reaches the border of the synthesis region in the DNA synthesis substrate, where stochastic deprotection of protecting groups may occur.

The area where insufficient light reaches is indicated in FIG. 1 as an error prone zone. Since the light reaching the error-prone region does not reach the threshold required for deprotection of the protecting group, some protecting groups are deprotected, but some protecting groups are not removed and remain as is.

It can also be confirmed through Figure 3 that there is a region where insufficient light reaches the target location of the DNA synthesis substrate.

Figure 2 shows the results of measuring the amount of light irradiated to the target location (DNA synthesis area) of the DNA synthesis substrate during the photochemical-based DNA synthesis process.

The picture on the left in Figure 2 is a top view of light being irradiated to the DNA synthesis site, and the intensity of light at each point is expressed in color. The picture on the right depicts the intensity of light on the ), it can be seen that it decreases. In other words, the contrast ratio of the light pattern shining on the substrate is not perfect.

As described in the description of Figure 2, the edge portion (corresponding to the error-prone area in Figure 2) cannot provide sufficient light for deprotection because the light intensity gradually decreases, and as a result, deprotection occurs at a specific location. Me, it may not happen in other locations. Because this stochastic deprotection is very random, in each cycle of DNA synthesis, even if the DNA strand is not deprotected in the (n-1)th DNA synthesis, deprotection occurs in the nth DNA synthesis, and a new nucleotide is generated. If (coupling) is added, a deletion error may occur for the (n-1)th sequence.

In other words, whether or not it is deprotected in the nth DNA synthesis is independent of whether it is deprotected in the previous (n-1)th DNA synthesis. Because of deletion errors at the border (error-prone area), capping is required to block the ends of the DNA or nucleic acid being synthesized to prevent additional synthesis from occurring in each synthesis cycle, and after synthesis is completed. requires a process to remove DNA shorter than the target length. Errors at these edges have a greater impact as the length to surface ratio increases, i.e., as the size of the area illuminated by light decreases, which is due to the simultaneous microarray DNA synthesis. It makes it difficult to reduce the size of a spot in order to increase the number of possible composites.

Figures 4 and 5 explain the deprotection and coupling mechanisms during DNA synthesis.

Figure 4 shows an example of a process in which DNA ends are deprotected and new nucleotides are added in a photochemical-based DNA synthesis method.

When light removes the deprotecting group at the end of the DNA, the -OH group at the 5' end of the DNA is exposed, allowing a new nucleotide to bind to the end of the DNA strand being synthesized. Since the nucleotide supplied for synthesis is bound to a protection device at one end, additional DNA synthesis does not occur without a separate deprotection process.

Protecting groups that fall off by reacting directly with light include BzNPPOC, NPPOC, and SPh-NOOPC.

Figure 5 shows an example of a process in which DNA ends are deprotected by a deprotecting molecule supplier and new nucleotides are added during the photochemical-based DNA synthesis process.

When light is irradiated, the deprotecting molecule-providing substance receives the light and releases an active deprotecting molecule, which attacks and removes the protecting group. As a result, the -OH group is exposed at the 5' end of the DNA, allowing new nucleotides to bind to the end of the DNA strand being synthesized.

Hydroquinone may be used as the deprotection molecule providing material, hydrogen ion (H ⁺ ) may be used as the active deprotection molecule, and DMT may be used as the protecting group.

In order to solve the problems described in Figures 2 and 3, the present inventors irradiated the DNA synthesis area with a first wavelength for DNA deprotection, and simultaneously irradiated the first wavelength with a second wavelength longer than the first wavelength for DNA synthesis. A method was designed to irradiate unwanted areas (areas other than the DNA synthesis area).

Figure 1 schematically shows the structure of a DNA synthesis system using a first wavelength (λe) and a second wavelength (λp) to implement the method.

The DNA synthesis system 100 of the present invention is

DNA synthesis unit (110) where DNA synthesis occurs;

A first wavelength light source 120 that radiates a first wavelength to the DNA synthesis unit;

A second wavelength light source 140 that radiates a second wavelength to the DNA synthesis unit;

A first light-space modulator (130) that controls the irradiation pattern of the first wavelength light source to the DNA synthesis unit; and

It includes a second light-space modulator 150 that controls the irradiation pattern of the second wavelength light source to the DNA synthesis unit.

The term 'light-space modulator' used in the present invention is a device that controls light emitted from a light source so that it can be irradiated to a specific area of the DNA synthesis region. The first light-space modulator and the second light-space modulator may independently be a photomask or a digital micromirror device (DMD).

DNA synthesis methods using the above system include:

Irradiating a first wavelength to the irradiation area of the DNA synthesis unit through a first light-spatial modulator;

Simultaneously with the irradiation of the first wavelength, irradiating a second wavelength longer than the first wavelength through a second light-spatial modulator to the inversion irradiation area of the DNA synthesis unit; and

Step of supplying a nucleotide solution to be synthesized.

As used in the present invention, the term 'irradiation area' refers to the area where DNA is to be synthesized in the DNA synthesis unit, and the 'inversion irradiation area' refers to the remaining area other than the area where DNA is to be synthesized in the DNA synthesis unit.

In order to selectively deprotect the DNA in the irradiated area, light λe of the first wavelength is reflected by DMD1, the first light-space modulator, and is illuminated on the substrate. At this time, light of λe can reach the inversion irradiation area due to diffraction, refraction, reflection, etc. of light. Before the photo-labile protection group energized by this light breaks the chemical bond or the deprotecting molecule donor releases the active deprotecting molecule, a second wavelength of light λp (λp) is applied to the reverse irradiation area. >λe), energy is released through stimulated emission, preventing deprotection from occurring in the reverse irradiation area.

The second wavelength of light may be irradiated simultaneously with the first wavelength or sequentially with a short delay (eg, <10 ns). Meanwhile, the first and second wavelengths may be independently irradiated to the DNA synthesis unit for 0.1 nanoseconds to 10 minutes.

The DNA synthesis method using the DNA synthesis system of FIG. 1 may further include the step of supplying a DNA synthesis site selection material to the DNA synthesis unit before irradiating the first wavelength to the irradiation area.

In the present invention, the term 'DNA synthesis site selection material' refers to a material necessary to synthesize DNA only at a desired location during the DNA synthesis process, and may be a material that generates hydrogen ions or hydroxide ions or provides divalent cations by light. there is. The divalent cation-providing material may be a material that generates divalent cations by light, or it may be a material that traps divalent cations and then releases the divalent cations by light.

DMNP-EDTA is a divalent cation donor, hydroquinone is a hydrogen ion generator, and quinone is a hydroxide ion generator.

The DNA synthesis method of the present invention described above can be used to introduce a desired monomer into a desired region in the form of a microarray by selectively deprotecting protecting groups such as DMT and BzNPPOC at high resolution, but the present invention is not limited to this. .

The DNA synthesis described in Figures 1 to 5 is all DNA synthesis using phosphoramidite using an organic solvent, and the present invention can also be applied to a DNA polymerase-based method.

Among DNA synthesis enzymes, TdT (Terminal deoxynucleotidyl transferase) is an exception, as it can synthesize single-stranded DNA of arbitrary sequence without a template DNA and requires a divalent cation to be activated. The most important issue in DNA synthesis using TdT is how to introduce the desired monomer among A, G, T, and C, and the present invention can solve this issue.

When light of the first wavelength is irradiated to the irradiation area, divalent cations caged by DMNP-EDTA are released, but even in this case, unwanted light may reach the reverse irradiation area. If the second wavelength is irradiated to the reverse irradiation area almost simultaneously with the irradiation of the first wavelength, the energy absorbed by DMNP-EDTA in the reverse irradiation area is released, thereby preventing TdT from being activated in the reverse irradiation area.

Meanwhile, another aspect of the present invention provides a DNA synthesis system using an electrode instead of a second wavelength.

The system is

A DNA synthesis unit including a transparent electrode and where DNA synthesis occurs;

A first wavelength light source that irradiates a first wavelength to the target site of the DNA synthesis unit;

a first light-space modulator that controls the irradiation pattern of the first wavelength light source to the DNA synthesis unit; and

It includes a power supply unit that supplies power to the electrodes of the DNA synthesis unit.

The DNA synthesis method using the above system is

Irradiating a first wavelength to the irradiation area of the DNA synthesis unit through a first light-spatial modulator;

Applying a voltage to the DNA synthesis unit simultaneously with irradiation of the first wavelength; and

It includes the step of supplying a nucleotide solution to be synthesized.

Referring to Figure 6, the H ⁺ ions released from hydroquinone by light diffuse very quickly and can reach the DNA of the spot located in the reverse irradiation area as shown in Figure 6A, causing deprotection. there is.

However, as shown in Figure 6B, a transparent electrode (e.g., ITO, IZO) is placed on a transparent substrate, and a first voltage (e.g., a negative voltage with respect to the reference electrode) is applied to deactivate the active deprotection material by an electrochemical reaction on the transparent electrode. The active deprotecting substance can be removed by generating an active deprotecting molecule quencher (e.g. OH ^- ion) (e.g. adding OH ^- ion to H ⁺ ion to form H ₂ O). As a result, the area where light of the first wavelength reaches can be limited, and an effective H ⁺ concentration can be maintained only in that limited area.

In Figure 6, DNA is shown directly attached to the transparent electrode, but an additional layer may be included between the transparent electrode and the DNA. For example, i) a self-assembled monolayer to facilitate the bonding between the transparent electrode and DNA, ii) a porous glass layer to expand the area to which DNA can attach, etc. Additional information may be added, but is not limited to this. This enables high-resolution parallel DNA synthesis in photochemical-based DNA synthesis.

[Explanation of symbols]

100: DNA synthesis system;

110: DNA synthesis section;

120: first wavelength light source;

130: first light-space modulator;

140: second wavelength light source; and

150: First light-space modulator.

Claims (13)

DNA synthesis unit where DNA synthesis occurs; a first wavelength light source that radiates a first wavelength to the DNA synthesis unit; a second wavelength light source that irradiates a second wavelength to the DNA synthesis unit; a first light-space modulator that controls the irradiation pattern of the first wavelength light source to the DNA synthesis unit; and A DNA synthesis system comprising a second light-spatial modulator that controls the irradiation pattern of the second wavelength light source to the DNA synthesis unit. The DNA synthesis system according to claim 1, wherein the first light-space modulator and the second light-space modulator are independently a photomask or a digital micromirror device (DMD). A DNA synthesis unit including a transparent electrode and where DNA synthesis occurs; a first wavelength light source that radiates a first wavelength to the DNA synthesis unit; a first light-space modulator that controls the irradiation pattern of the first wavelength light source to the DNA synthesis unit; and A DNA synthesis system comprising a power supply unit that supplies power to the electrodes of the DNA synthesis unit. 4. The DNA synthesis system of claim 3, wherein the first light-space modulator is a photomask or a DMD. A DNA synthesis method using the DNA synthesis system of claim 1 and comprising the following steps: Irradiating a first wavelength to the irradiation area of the DNA synthesis unit through a first light-spatial modulator; Simultaneously with the irradiation of the first wavelength, irradiating a second wavelength longer than the first wavelength through a second light-spatial modulator to the inversion irradiation area of the DNA synthesis unit; and Step of supplying a nucleotide solution to be synthesized. A DNA synthesis method using the DNA synthesis system of claim 3 and comprising the following steps: Irradiating a first wavelength to the irradiation area of the DNA synthesis unit through a first light-spatial modulator; Applying a voltage to the DNA synthesis unit simultaneously with irradiation of the first wavelength; and Step of supplying a nucleotide solution to be synthesized. The DNA synthesis method according to claim 5 or 6, further comprising supplying a DNA synthesis site selection material to the DNA synthesis unit before the step of irradiating the first wavelength. The method of claim 7, wherein the DNA synthesis site selection material is a hydrogen ion generating material, a hydroxide ion generating material, or a divalent cation providing material. The method of claim 8, wherein the divalent cation donor is DMNP-EDTA. The method of claim 8, wherein the hydrogen ion generating substance is hydroquinone. The method of claim 8, wherein the hydroxide ion generating substance is quinone. The method of claim 5 or 6, wherein the step of irradiating the first wavelength is performed for 0.1 nanoseconds to 10 minutes. The method of claim 5, wherein the step of irradiating the second wavelength is performed for 0.1 nanoseconds to 10 minutes.

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