WO2025053688A1 - High-precision dna synthesis method using atoliter nano-well array - Google Patents

Abstract

The present invention relates to a technology for an atoliter reaction chamber array that prevents unwanted light reflections, thereby reducing errors during the photochemical-based DNA synthesis process. Utilizing the array allows for a reduction in DNA synthesis errors and the consumption of samples.

Description

High-precision DNA synthesis method using an atotore nanowell array

The present invention relates to a technology for an atomizer nanowell array for DNA synthesis that can reduce errors in a photochemical-based DNA synthesis process by preventing unwanted reflection of light.

The amount of data generated is increasing very rapidly due to the increase in personal social network services (SNS) media that mainly use images and videos. In the future, as augmented reality (AR), virtual reality (VR), mixed reality (MR) devices, and 3D media such as holograms become widespread, the amount of data is expected to increase even more rapidly. Specifically, the amount of data stored worldwide in 2040 is expected to reach ¹⁰²⁴ to ¹⁰²⁹ bits (Summary report, Technology Working Group Meeting on future DNA synthesis technologies (September 14, 2017, Arlington, VA)).

Currently, the data storage medium of 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 required in 2040, 10 ¹⁴ kg of silicon wafers are required, but the supply of silicon wafers in 2040 is expected to be about 10 ⁸ kg, which is far short of demand. In addition, existing data storage media have a limitation that the data retention period is about 10 years.

Accordingly, attempts are continuously being made to develop new data storage media, one of which is DNA storage devices. Using DNA as a storage medium can overcome the data storage density, which is a shortcoming of existing storage media, and can store information stably even under physical shock. In addition, DNA can store ^2x1024 bits, which is equivalent to ¹⁰⁹ kg of flash memory, by synthesizing 1 kg, making it suitable for large-capacity data storage, and in particular, it has the advantage of a very long storage period for stored data. However, errors occurring during DNA synthesis cause a decrease in data storage density and information loss, which is an obstacle to the commercialization of DNA storage devices.

In particular, since the photochemical DNA synthesis method deprotects the protective group at the end of DNA by shining light, the light reaches undesired areas due to diffraction, scattering, and reflection of the light. In addition, there is a problem that there is an area where insufficient light reaches the area where DNA is to be synthesized. Due to these problems, deprotection occurs in unintended DNA and deprotection of DNA does not occur in the intended area, resulting in DNA synthesis errors.

In the above situation, the inventors of the present invention studied to improve the problem that, in the DNA deprotection step of a photochemical-based DNA synthesis process, i) light is formed as a blurry image on the opposite substrate in addition to the DNA synthesis surface, so that sufficient light is not provided to the edge, resulting in the failure to deprotect the protection group, or ii) light reflected from the opposite substrate reaches the synthesis substrate again, resulting in the deprotection of the protection group at a location where the light should not reach.

As a result, a nanowell array with a diameter of ~100 nm was formed by erecting a ~100 nm thick aluminum thin film on the surface of a DNA synthesis substrate, taking into account the characteristic that light cannot pass through an aperture narrower than the diffraction limit (~half wavelength). When the nanowell array is formed in this way, light irradiated from the bottom of the synthesis substrate exists as an evanescent field on the surface of the synthesis substrate and cannot pass through the nanowell. Therefore, the blurry image of the opposite substrate and the problem of DNA synthesis errors due to reflected light are fundamentally prevented. Since the photochemical reaction for DNA synthesis in the nanowell array is limited to a space of ~100x100x100 nm ³ (=~1 atto liter), the present inventors named the nanowell array an atotoliter nanowell array.

Accordingly, an object of the present invention is to provide the atotore nanowell array and a DNA synthesis device including the atotore nanowell array.

To achieve the above object, one aspect of the present invention provides an atotore nanowell array for DNA synthesis comprising the following composition:

Including a nanowell array formed on the above substrate,

The above nanowells are partitioned by a thin film and have a width, length, and height of 50 nm to 2 μm, respectively.

In the present invention, the width, length, and height of the nanowell may each be 50 nm to 2 μm, preferably 50 nm to 1 μm, more preferably 50 nm to 200 nm, and even more preferably 50 nm to 100 nm.

In the present invention, the substrate portion is made of a material selected from the group consisting of silicon, silicon nitride, silicon oxide, glass, and quartz.

In the present invention, the thin film is made of a material selected from the group consisting of aluminum (Al), chromium (Cr), titanium (Ti), copper (Cu), gold (Au), platinum (Pt), and silver (Ag).

Additionally, the thin film may have a thickness of 50 nm to 2 μm, preferably 50 nm to 200 nm, more preferably 50 nm to 100 nm.

Another object of the present invention is

The above nanowell array;

A light source unit that irradiates light in the direction of a thin film from the substrate unit of the above nanowell array; and

A DNA synthesis device is provided, comprising a light-space modulator that controls the light irradiation pattern to the nanowell array of the light source unit.

The above light-space modulator can be a photomask, a digital micromirror device (DMD), or a microLED.

In the present invention, the horizontal and vertical lengths of the nanowell may be shorter than the wavelength irradiated from the light source unit.

Using a nanowell array for DNA synthesis according to one example of the present invention, unnecessary reflection of light can be prevented during a photochemical-based DNA synthesis process, thereby reducing DNA synthesis errors and also reducing sample consumption.

Figure 1 schematically illustrates the causes of synthetic errors in the photochemical-based DNA synthesis process.

Figure 2 shows the results of measuring the amount of light irradiated to the synthesis site during a photochemical-based DNA synthesis process.

Figure 3 shows an example of an error occurring in a photochemical-based DNA synthesis process when light reaches the opposite substrate of the DNA synthesis substrate.

Figure 4 shows an example of the 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 the process in which DNA ends are deprotected and new nucleotides are added by a deprotecting molecule providing substance in a photochemical-based DNA synthesis process.

Figure 6 shows an example of an atolith nanowell array in which aluminum nanowells are formed on the surface of a DNA synthesis substrate.

Figure 7 shows the result of calculating the propagation profile when light having a UV wavelength of 365 nm is irradiated on a nanowell with a thickness of 20 nm and a diameter of 80 nm.

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 specific description of a related known function or configuration may unnecessarily obscure the gist of the present invention, the detailed description thereof will be omitted. In addition, the same reference numerals are used throughout the drawings for parts that perform similar functions and actions. Meanwhile, "including" a certain component does not exclude other components unless specifically stated otherwise, but rather means that other components may be further included.

Figure 1 schematically illustrates the causes of synthetic errors in the photochemical-based DNA synthesis process.

When light (UV or 100 to 500 nm wavelength) is selectively irradiated to the synthesis area of a DNA synthesis substrate using a photomask or a digital micromirror device (DMD), the light may be reflected, scattered, or spread by diffraction from the DNA synthesis substrate, so that the light may reach unwanted areas.

As shown in Figure 1, it can be seen that light diffracted from a DNA synthesis substrate deprotects DNA located outside the DNA synthesis region. In addition, light insufficiently reaches the edge of the synthesis region in the DNA synthesis substrate, and stochastic deprotection of protecting groups can occur at this edge.

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

It can also be confirmed through Figure 2 that there is an area where light insufficiently 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 a photochemical-based DNA synthesis process.

In Fig. 2, the left picture is a top view of light irradiating the DNA synthesis site, and the intensity of light at each point is expressed by color. The right picture is a drawing of the light intensity along the x-axis (blue) and the y-axis (green). When light is shined on the DNA synthesis site, the central area maintains a constant amount of light, but the amount of light reaching the edge decreases with a gradient. In other words, the contrast ratio of the light pattern illuminated on the substrate is not perfect.

As described in the description of Fig. 1, the edge portion (corresponding to the error-prone area in Fig. 1) cannot provide sufficient light for deprotection because the light intensity gradually decreases, and thus deprotection may occur at a specific location but not at another location. Since this probabilistic deprotection is very random, in each cycle of DNA synthesis, even if a DNA strand is not deprotected in the (n-1)th DNA synthesis, deprotection may occur in the nth DNA synthesis, and when a new nucleotide is added (coupled), a deletion error may occur for the (n-1)th sequence.

That is, whether or not deprotection occurs in the nth DNA synthesis is independent of whether or not deprotection occurred in the previous (n-1)th DNA synthesis. Because of deletion errors in such borders (error-prone regions), capping is required to block the ends of the DNA or nucleic acids being synthesized to prevent additional synthesis in each synthesis cycle, and a process to remove DNA shorter than the target length is required after synthesis is complete. The influence of errors in these borders increases as the length to surface ratio increases, that is, as the size of the area irradiated with light decreases, which makes it difficult to reduce the size of one spot to increase the number of possible simultaneous synthesis branches of microarray DNA synthesis.

Figure 3 shows an example of an error occurring in a photochemical-based DNA synthesis process when light reaches the opposite substrate of the DNA synthesis substrate.

When light irradiated from the bottom of the DNA synthesis substrate is focused on the opposite substrate, deprotection occurs in the DNA of that portion, and if additional light reflection occurs, the light can reach unwanted areas of the DNA synthesis substrate.

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

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

When the deprotecting group at the end of DNA is removed by light, the -OH group is exposed at the 5' end of the DNA, so that a new nucleotide can be attached to the end of the DNA strand being synthesized. Since the nucleotides supplied for synthesis have a protecting group attached to one end, additional DNA synthesis does not occur without a separate deprotection process.

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

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

When irradiated with light, the deprotecting molecule donor substance receives light and releases an active deprotecting molecule, which attacks and removes the protecting group. As a result, an -OH group is exposed at the 5' end of the DNA, so that a new nucleotide can be attached to the end of the DNA strand being synthesized.

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

Figure 6 shows an example of an atolith nanowell array in which nanowells are formed by an aluminum thin film on the surface of a DNA synthesis substrate.

Specifically, Fig. 6 shows the process of forming a circular aluminum nanowell array on the surface of a DNA synthesis substrate so that the nanowells have a thickness and diameter of ~100 nm, and then irradiating light to perform DNA synthesis.

In the present invention, the nanowell may have a width, length, and height of 50 nm to 1 μm, and the thin film may have a thickness of 50 nm to 2 μm. It is also possible to make the height of the nanowell and the thickness of the thin film the same.

The above aluminum nanowells can be created using laser interference lithography (LIL), and can also be processed using nanobead self-assembly lithography, electron beam lithography (EBL), and focused ion-beam lithography (FIB).

Referring to Figure 6, it can be seen that light irradiated from the bottom of the DNA synthesis substrate does not propagate due to the aluminum nanowells partitioned by the thin film, but is confined to the surface of the DNA synthesis substrate.

When the light irradiated from the light source is limited to the surface of the DNA synthesis substrate, a blurry image is formed on the opposite substrate, or the phenomenon of light being reflected is blocked, and the problem caused by light reflection during the DNA synthesis process can be fundamentally blocked with the atotorin nanowell array of the present invention. Therefore, by using the nanowell array according to an example of the present invention, the process of flowing a solution for refractive index matching, which is a method used in the past to prevent DNA synthesis errors caused by light reflection, into a flow cell or filling an anti-reflecting material into the back of the opposite substrate can be omitted.

Figure 7 shows the result of calculating the propagation profile using Matlab when irradiating a UV wavelength of 365 nm on a substrate having chromium nanowells with a thickness of 20 nm and a diameter of 80 nm.

It can be confirmed that the light irradiated from the bottom of the substrate is confined to the surface of the synthetic substrate.

Claims (7)

A substrate portion in which DNA synthesis occurs and which is made of a material that allows light to pass through; and Including a nanowell array formed on the above substrate, An atolytic nanowell array for DNA synthesis, wherein the nanowells are partitioned by a thin film and have a width, length, and height of 50 nm to 2 μm, respectively. In the first paragraph, the substrate portion is an atotorin nanowell array for DNA synthesis, made of a material selected from the group consisting of silicon, silicon nitride, silicon oxide, glass, and quartz. In the first paragraph, the thin film is a material selected from the group consisting of aluminum (Al), chromium (Cr), titanium (Ti), copper (Cu), gold (Au), platinum (Pt), and silver (Ag), an atolith nanowell array for DNA synthesis. In the first aspect, the thin film has a thickness of 50 nm to 2 μm, an atotore nanowell array for DNA synthesis. An atolytic nanowell array according to any one of claims 1 to 4, wherein DNA synthesis occurs; A light source unit that irradiates light in the direction of a thin film from the substrate unit of the above-mentioned atoritor nanowell array; and A DNA synthesis device, comprising a light-space modulator for controlling the light irradiation pattern to the atotolith nanowell array of the light source unit. A DNA synthesis device in claim 5, wherein the light-space modulator is a photomask, a digital micromirror device (DMD), or a microLED. A DNA synthesis device in claim 5, wherein the horizontal and vertical lengths of the nanowell are shorter than the wavelength irradiated by the light source.

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