DNA-based digital information storage

Abstract

Provided herein are compositions, devices, systems and methods for generating and using biomolecule-based information for storage. Further provided are devices comprising addressable electrodes for controlling polynucleotide synthesis (deprotection, extension or cleavage, etc.). The compositions, devices, systems and methods described herein provide improved storage, density and retrieval of biomolecule-based information.

Description

[0001] Cross-references

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 613,728, filed on January 4, 2018, U.S. Provisional Patent Application No. 62 / 617,067, filed on January 12, 2018, and U.S. Provisional Patent Application No. 62 / 650,231, filed on March 29, 2018, all of which are incorporated herein by reference in their entirety. Background Art

[0003] Biomolecule-based information storage systems (e.g., DNA-based) have large storage capacity and stability over time. However, there is a need for a scalable, automated, highly accurate, and efficient system for generating biomolecules for information storage. Summary of the invention

[0004] Provided herein is a device for storing information, comprising: a solid support, wherein the solid support comprises a plurality of wells, wherein each of the wells comprises an addressable locus, wherein the addressable locus comprises: a synthetic surface located in a bottom region of each well; a bottom electrode in addressable communication with the synthetic surface; and at least one sidewall electrode located on a sidewall of each well, wherein the at least one sidewall electrode is 50 nm to 200 nm from the bottom region. Further provided herein is a device, wherein the solid support comprises a plurality of wells, wherein each of the wells comprises an addressable locus, wherein the addressable locus comprises: a synthetic surface located in a bottom region of each well; a bottom electrode in addressable communication with the synthetic surface; and at least one sidewall electrode located on a sidewall of each well, wherein the at least one sidewall electrode is 50 nm to 200 nm from the bottom region. 6 Addressable seats / cm 2 The present invention further provides such a device, wherein the solid support is 100x 10 6 Up to 100x 10 7 Addressable seats / cm 2 The apparatus further provides a device wherein the addressable loci have a diameter of at most about 750 nm. The apparatus further provides a device wherein each well has a depth of at most about 1000 nm. The apparatus further provides a device wherein each well has a depth of 100 nm to 1000 nm. The apparatus further provides a device wherein each well has a longest cross-sectional diameter of 100 nm to 800 nm. The apparatus further provides a device wherein each well is cylindrical. The apparatus further provides a device wherein the bottom electrode has a diameter of 10 4 nm 2 Up to 10 5 nm 2The longest cross-sectional area of ​​the device. Further provided herein is a device wherein the at least one sidewall electrode is 50nm to 200nm from the bottom region. Further provided herein is a device wherein the at least one sidewall electrode has a height of 5nm to 25nm. Further provided herein is a device comprising at least two sidewall electrodes. Further provided herein is a device wherein the at least one sidewall electrode and the bottom electrode are independently addressable.

[0005] Provided herein is a device for storing information, comprising: a solid support, wherein the solid support comprises a plurality of wells, wherein each of the wells comprises an addressable locus, the addressable locus comprising: a synthetic surface located in a bottom region of each well; a bottom electrode in addressable communication with the synthetic surface; at least one sidewall electrode located on a sidewall of each well, wherein the synthetic surface at each addressable locus comprises at least one polynucleotide extending from the synthetic surface, and wherein the polynucleotides comprising different sequences on the solid support are arranged in a pattern of at least 100×10 6 Polynucleotides / cm 2 There is further provided a device wherein the solid support is present at a density of at least 100 x 10 7 Polynucleotides / cm 2 The present invention further provides such a device, wherein the solid support is 100 x 10 6 Up to 100x 10 7 Polynucleotides / cm 2 The apparatus further provides a device wherein each well has a depth of at most about 1000 nm. The apparatus further provides a device wherein each well has a depth of 100 nm to 1000 nm. The apparatus further provides a device wherein the addressable wells have a diameter of at most about 750 nm. The apparatus further provides a device wherein each well has a longest cross-sectional diameter of 100 nm to 800 nm. The apparatus further provides a device wherein each well is cylindrical. The apparatus further provides a device wherein the bottom electrode has 10 4 nm 2 Up to 10 5 nm 2The longest cross-sectional area of ​​the substrate. Further provided herein is a device wherein the at least one sidewall electrode is 50 nm to 200 nm from the bottom region. Further provided herein is a device wherein the at least one sidewall electrode has a height of 5 nm to 25 nm. Further provided herein is a device comprising at least two sidewall electrodes. Further provided herein is a device wherein the at least one sidewall electrode and the base electrode are independently addressable.

[0006] Provided herein is a method for storing information, comprising: a) providing an apparatus as described herein; b) providing instructions for polynucleotide synthesis; c) depositing at least one nucleoside on a synthesis surface, wherein the at least one nucleoside is coupled to a polynucleotide attached to the synthesis surface; and d) repeating step c) to synthesize a plurality of polynucleotides on the synthesis surface, wherein the instructions comprise at least one sequence encoding the plurality of polynucleotides. Further provided herein is such a method, further comprising cutting at least one polynucleotide from the surface, wherein the polynucleotide is dissolved in a small droplet. Further provided herein is such a method, further comprising sequencing at least one polynucleotide from the surface. Further provided herein is such a method, wherein the nucleoside comprises a nucleoside phosphoramidite. Further provided herein is such a method, further comprising a cutting step, wherein the cutting step comprises applying an electric potential to the bottom electrode to generate a cutting reagent. Further provided herein is such a method, wherein the method further comprises drying the surface. Further provided herein is such a method, further comprising washing the nucleoside from the surface. Further provided herein is such a method, wherein the method further comprises a capping step. Further provided herein is such a method, wherein the method further comprises an oxidation step. Further provided herein is a method wherein the method further comprises a deblocking step, wherein the deblocking step comprises applying an electric potential to the at least one sidewall electrode to generate a deblocking reagent.

[0007] Provided herein is a method for storing information, comprising: a) providing a solid support comprising a surface; b) depositing at least one nucleoside on the surface, wherein the at least one nucleoside is coupled to a polynucleotide attached to the surface; and c) repeating step b) to synthesize a plurality of polynucleotides on the surface, wherein the polynucleotides with different sequences on the surface are arranged at a ratio of at least 100×10 6 polynucleotides / cm 2 Further provided herein is a method wherein the density of addressable loci on the solid support is at least 100 x 10 7 polynucleotides / cm 2 Further provided herein is a method wherein the density of addressable loci on the solid support is 100 x 106 Up to 100x 10 7 Polynucleotides / cm 2 . Further provided herein is such a method, wherein the method further comprises cutting at least one polynucleotide from the surface, wherein the polynucleotide is dissolved in the droplets. Further provided herein is such a method, wherein the method further comprises sequencing at least one polynucleotide from the surface. Further provided herein is such a method, wherein the nucleoside comprises a nucleoside phosphoramidite. Further provided herein is such a method, wherein the method further comprises drying the surface. Further provided herein is such a method, wherein the method further comprises washing the nucleoside from the surface. Further provided herein is such a method, wherein the method further comprises a capping step. Further provided herein is such a method, wherein the method further comprises an oxidation step. Further provided herein is such a method, wherein the method further comprises a deblocking step.

[0008] Provided herein is a method for storing information, comprising: a) providing a solid support comprising a surface; b) depositing droplets comprising at least one nucleoside on the surface, wherein the at least one nucleoside is coupled to a polynucleotide attached to the surface; and c) repeating step b) to synthesize a plurality of polynucleotides on the surface, wherein the volume of the droplets is less than about 100 femtoliters. Further provided herein is such a method, wherein the droplets have a volume of less than about 50 femtoliters. Further provided herein is such a method, wherein the droplets have a volume of less than about 25 femtoliters to 100 femtoliters. Further provided herein is such a method, wherein the method further comprises cutting at least one polynucleotide from the surface, wherein the polynucleotide is dissolved in the droplets. Further provided herein is such a method, wherein the method further comprises sequencing at least one polynucleotide from the surface. Further provided herein is such a method, wherein the nucleoside comprises a nucleoside phosphoramidite. Further provided herein is such a method, wherein the method further comprises drying the surface. Further provided herein is such a method, wherein the method further comprises washing the nucleoside from the surface. Further provided herein is such a method, wherein the method further comprises a capping step. Further provided herein is such a method, wherein the method further comprises an oxidation step. Further provided herein is such a method, wherein the method further comprises a deblocking step.

[0009] Provided herein is a method for storing information, comprising: a) providing a solid support comprising a surface; b) depositing at least one nucleoside on the surface, wherein the at least one nucleoside is coupled to a polynucleotide attached to the surface; and c) repeating step b) to synthesize a plurality of polynucleotides on the surface, wherein the time for repeating step b) using four different nucleotides is less than about 100 milliseconds. Further provided herein is such a method, wherein the time for repeating step b) using four different nucleotides is less than about 50 milliseconds. Further provided herein is such a method, wherein the time for repeating step b) using four different nucleotides is 25 milliseconds to 100 milliseconds. Further provided herein is such a method, wherein the method further comprises cutting at least one polynucleotide from the surface, wherein the polynucleotide is dissolved in a small droplet. Further provided herein is such a method, wherein the method further comprises sequencing at least one polynucleotide from the surface. Further provided herein is such a method, wherein the nucleoside comprises a nucleoside phosphoramidite. Further provided herein is such a method, wherein the method further comprises drying the surface. Further provided herein is such a method, wherein the method further comprises washing the nucleoside from the surface. Further provided herein is such a method, wherein the method further comprises a capping step. Further provided herein is such a method, wherein the method further comprises an oxidation step. Further provided herein is such a method, wherein the method further comprises a deblocking step.

[0010] Incorporation by reference

[0011] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The novel features of the present invention are particularly set forth in the appended claims. The features and advantages of the present invention will be better understood by referring to the following detailed description of illustrative embodiments utilizing the principles of the present invention and the accompanying drawings, in which:

[0013] Figure 1 An exemplary workflow for nucleic acid-based data storage is shown.

[0014] Figure 2 A plate configured for polynucleotide synthesis is shown, comprising 24 regions or subdomains, each having an array of 256 clusters.

[0015] Figure 3 Shows Figure 2 A close-up view of the subfield in , which has 16×16 clusters, each with 121 individual seats.

[0016] Figure 4 Shows Figure 2 Detailed view of a cluster in , where the cluster has 121 seats.

[0017] Figure 5A A front view of a plate with multiple channels is shown.

[0018] Figure 5B A cross-sectional view of a plate having a plurality of channels is shown.

[0019] Figure 6A-6B Continuous loop and reel-to-reel arrangements for flexible structures are depicted.

[0020] Figure 6C-6D A schematic diagram of the release and extraction of synthesized polynucleotides is depicted.

[0021] Figures 7A-7C Magnified views of flexible structures with spots, channels, or pores, respectively, are depicted.

[0022] Figure 8 An example of a computer system is shown.

[0023] Fig. 9 is a block diagram illustrating the architecture of a computer system.

[0024] Fig.10 is a diagram illustrating a network configured to incorporate multiple computer systems, multiple cellular telephones and personal data assistants, and network attached storage (NAS).

[0025] Fig.11 is a block diagram of a multiprocessor computer system that uses a shared virtual address storage space.

[0026] Fig. 12A is the front side of an example of a solid support array.

[0027] Fig. 12B is the back side of an example of a solid support array.

[0028] Fig.13 is a schematic diagram of a solid support comprising an active area and a fluidic interface.

[0029] Fig.14 is an example of a rack-style instrument.

[0030] Fig.15 Depicted are solid supports comprising addressable areas for nucleic acid synthesis or storage.

[0031] Fig.16A An array for synthesis using electrochemistry is depicted.

[0032] Fig. 16B An array for synthesis using electrochemistry is depicted.

[0033] Fig.17 Wells for nucleic acid synthesis or storage and the spacing between wells are depicted.

[0034] Fig.18 An example of a solid support comprising an addressable array is shown.

[0035] Fig.19 An example of a solid support array is shown where the spacing approximates the length of a 240-mer polynucleotide. DETAILED DESCRIPTION

[0036] As the amount of information generated and stored grows exponentially, larger storage systems are needed. Traditional storage media have limited capacity and require specialized techniques that change over time, so data needs to be constantly transferred to new media, which is often expensive. In contrast to traditional binary information encoding, biomolecules such as DNA molecules provide a host suitable for information storage, partly due to their stability over time and the ability to encode four bits of information. Therefore, a large amount of data is encoded in DNA with a relatively small amount of physical space compared to that used by commercially available information storage devices. This article provides a method for increasing DNA synthesis throughput by increasing sequence density and reducing turnaround time.

[0037] definition

[0038] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which these inventions belong.

[0039] Throughout the present disclosure, numerical features are given in range format. It should be understood that the description of the range format is only for convenience and simplicity, and should not be interpreted as a hard limit to the scope of any embodiment. Therefore, unless the context clearly stipulates otherwise, the description of the range should be considered to clearly disclose all possible sub-ranges and each numerical value accurate to one-tenth of the lower limit unit in the range. For example, the description of the range such as from 1 to 6 should be considered to have clearly disclosed such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, and each value in the range, for example, 1.1, 2, 2.3, 5 and 5.9. Regardless of the width of the range, this is applicable. The upper and lower limits of these intermediate ranges can be independently included in a smaller range, and are also included in the present invention, but are subject to any clearly excluded limit in the range. Unless the context clearly stipulates otherwise, in the case where the range includes one or both of the limits, the scope of excluding any or both of these included limits is also included in the present invention.

[0040] The terms used herein are only used for the purpose of describing specific embodiments and are not intended to limit any embodiment. Unless the context clearly stipulates otherwise, the singular forms "one", "a kind of" and "the" as used herein are also intended to include plural forms. It should be further understood that the terms "include" and / or "comprising" refer to the presence of the features, integers, steps, operations, elements and / or components when used in this specification, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components and / or their groups. As used herein, the term "and / or" includes any and all combinations of one or more related listed items.

[0041] Unless specifically stated or obvious from the context, as used herein, the term "about" with respect to a number or numerical range should be understood to mean the stated number and + / - 10% thereof, or, for values ​​listed in a range, 10% below the listed lower limit to 10% above the listed upper limit.

[0042] As used herein, the terms "preselected sequence", "predefined sequence" or "predetermined sequence" are used interchangeably. These terms mean that the sequence of a polymer is known and selected prior to the synthesis or assembly of the polymer. In particular, various aspects of the present invention are described herein primarily with respect to the preparation of nucleic acid molecules, and the sequence of the polynucleotide is known and selected prior to the synthesis or assembly of the nucleic acid molecules.

[0043] Provided herein are methods and compositions for producing synthetic (i.e., de novo synthesized or chemically synthesized) polynucleotides. Polynucleotides may also be referred to as oligonucleotides (oligonucleotide or oligo). Unless otherwise indicated, the polynucleotide sequences described herein may include DNA or RNA.

[0044] Solid support-based nucleic acid synthesis and storage

[0045] Devices, compositions, systems and methods for nucleic acid synthesis and storage based on chips are described herein. In some cases, polynucleotides are synthesized de novo using the methods based on solid supports as described herein. In some cases, polynucleotides are stored on solid supports after synthesis. In some cases, the methods based on solid supports as described herein are only used for storage.

[0046] This paper describes the device, composition, system and method for nucleic acid synthesis and storage based on solid support, wherein one or more nucleic acid synthesizer components are integrated into the solid support. The functional equivalent of the component or component can include a temperature control unit, an addressable electrode, a semiconductor surface, a fluid reservoir, a fluid control device, a synthesis surface, a power supply or other components for synthesizing polynucleotides. Any combination of integrated components is suitable for use with device, composition, system and method as described herein. In some cases, one or more components are outside the solid support (non-integrated).

[0047] The density of unique loci for polynucleotide synthesis on the surface is usually controlled by the spatial resolution achievable by the deposition of reagents. In addition, the deposition of reagents at unique sites requires the movement of the deposition device or the receiving surface to move the site of reagent deposition from one site to another. Alternatively, the coupling of bases is locally controlled at a defined site on the synthesis surface without the need for movement of the surface or the reagent deposition device. Local control is achieved by an array of addressable electrodes 1503, wherein each electrode electrochemically controls nucleosides (nucleoside phosphoramidites) coupling at a specific site on the surface 1505. In some cases, the electrode is a base electrode (or bottom electrode) located in a bottom area that is addressably communicated with the synthesis surface. In some cases, the electrode is a sidewall electrode located on the side of the hole. However, in some cases, conventional electrochemical processes on flat arrays are limited by spacing (see Fig.19). At high density (e.g., short spacing 1507), the length of the growing DNA oligonucleotide 1601 can reach an adjacent synthesis site from one synthesis site, thereby mixing discrete reaction products. To avoid mixing of adjacent reaction products, in some embodiments, the spacing 1507 is increased. Diffusion through the liquid reaction medium 1901 is another factor that affects the spatial control of polynucleotide synthesis. Therefore, it is advantageous to separate active sites to achieve higher array density. The coupling of nucleosides is controlled by local electrodes through many operations, such as the generation of local chemical reagents, local removal of reagents, repulsion of reagents, limitation of solvents, attraction of solvents, or other electrochemical or physical operations that affect one or more steps in base coupling. In some cases, a device 1500 comprising a plate 1501 having a hole 1502 is used to synthesize polynucleotides (see Fig.15 ). The bottom 1505 of each well comprises an electrode 1503 from which polynucleotides are synthesized. Each well has a cross-sectional diameter 1506, and a spacing 1507 between any two wells. In some cases, the sidewalls 1504 of the electrode comprise one or more sidewall electrodes (not shown).

[0048] In some cases, coupling is directly controlled by controlling the nucleoside addition step, the deprotection step, or other steps that affect the efficiency of the nucleoside coupling reaction. In some cases, a pattern of electrodes is charged to generate H at defined sites near the synthesis surface. + Ion gradient 1602 (see Fig.16A ); the polynucleotide 1601 at these sites is unblocked (where the polynucleotide is blocked with an acid-cleavable blocking group) and is available for coupling with a nucleoside.

[0049] Devices are described herein that include a solid support, wherein the solid support includes a plurality of wells, wherein each well includes an addressable locus, the addressable locus including: a synthetic surface located in a bottom region of each well; and at least one sidewall electrode located on a sidewall of each well, wherein electrochemical generation of a reagent is spatially separated from the point of attachment of a polynucleotide to the synthetic surface. In some cases, the devices described herein further include a bottom electrode in addressable communication with the synthetic surface. For example, sidewall electrodes 1603 can be used to control adhesion of a substrate or reagent (see Fig. 16B ). In some cases, the reagent comprises protons or other acid molecules. In some cases, the sidewall electrode 1603 is located at the hole surface of the hole having a depth 1604 (see Fig. 16B) at the position around the edge of the synthetic surface. In some cases, the sidewall electrode controls the chemical reaction that occurs near the synthetic surface. For example, if acid or other reagents are generated near the synthetic surface, the part of the polynucleotide 1601 bound to the surface will be in contact with a higher concentration of acid than the polynucleotide part away from the acid generation site. This may cause the degradation of the polynucleotide part exposed to the higher concentration of acid. In some cases, the sidewall electrode 1603 produces or controls a proton gradient 1602, which causes a part of the polynucleotide 1601 to be uniformly or targetedly exposed to acid. The site near the uncharged electrode is not coupled with the nucleoside deposited on the synthetic surface, and before adding the next nucleoside, the pattern of the charged electrode changes. By applying a series of electrode-controlled masks to the surface, the desired polynucleotide is synthesized at the exact position on the surface. In addition, in some cases, the local control of coupling reduces the synthesis step, reduces reagents / materials (due to higher polynucleotide density and reduced scale), and shortens the synthesis time (synthetic surface does not move). In some cases, the hole comprises one, two, three, four or more than four sidewall electrodes. In some cases, the hole comprises two sidewall electrodes. In some cases, each sidewall electrode is independently addressable. For example, different voltages are applied independently to two or more different sidewall electrodes. In some cases, such an arrangement is conducive to the diffusion of reagents or polynucleotides in the defined plane between the two sidewall electrodes. In some cases, such sidewall electrodes are annular or continuous around the circumference of the hole cross section. In some cases, the sidewall electrodes are discontinuous, or only partially cover a part of the sidewall surface. For example, the sidewall electrodes are continuous on about 5%, 10%, 15%, 30%, 50%, 75% or about 90% of the circumference of the hole cross section. In some cases, such sidewall electrodes have a height roughly equal to the hole height, or about 5%, 10%, 15%, 30%, 50%, 75% or about 90% of the hole height. In some cases, applying different voltages independently to two or more discontinuous sidewall electrodes can cause the diffusion of reagents or polynucleotides in the horizontal plane. In some cases, independently applying different voltages to two or more discrete sidewall electrodes can result in diffusion of reagents or polynucleotides in the vertical plane.

[0050] Polynucleotide synthesis usually needs to repeat deposition and remove liquid (fluidics) on the synthesis surface.In some cases, the overall movement of fluid causes fluid loss (volume of wetting, transport pipeline or reaction hole), which causes reagent use efficiency to be low and the cycle time of mobile fluid to be longer.An alternative method of overall fluidics in the synthesis process is to use digital fluidics, wherein reagent or reaction vessel are packaged as discrete droplets.In some cases, by operating or electrowetting on the surface comprising insulating (or coated with semiconductor) electrode, with discrete volume mixing, moving (merging, reaction), separate, store, add, remove or analyze droplets.Electrowetting allows local control of fluid-surface interaction; For example, energizing the electrode near the droplet causes the droplet to separate.In some cases, droplets as described herein have small volume.For example, the volume of the droplet is at most 10,20,50,75,100,125,150,200,300,500,800 or more than 1000 femtoliters. In some cases, the volume of the droplet is about 50 to about 200 femtoliters. In some cases, relative to overall fluidic control, digital fluidic control causes the cycle time to be shortened by at least 2,3,4,7,10 times or more than 10 times. In some cases, relative to overall fluidic control, digital fluidic control causes the cycle time to be shortened by about 2 to 10 times. In some cases, the time to complete a cycle (coupling successively of 4 bases, including washing) is about 1,2,3,5,7,10,12,15,17,20,30,50,100 or about 200 milliseconds (ms). In some cases, the time to complete a cycle is at most 1,2,3,5,7,10,12,15,17,20,30,50,100 or at most 200ms. In some cases, the time to complete a cycle is about 10 to about 50ms.

[0051] The movement of fluid inside and outside the surface described herein may include modifications or conditions that prevent undesirable fluid movement or other phenomena. For example, in some cases, the movement of fluid causes the formation of bubbles or air pockets, which limits the contact of fluid with components such as surfaces or polynucleotides. The methods, systems and compositions described herein cover various methods for controlling bubble formation or minimizing bubble formation. Such methods include controlling fluid pressure, the geometry of the hole or the surface material / coating. The geometry of the hole can be used to minimize bubbles. For example, taper the hole, channel or other surface to reduce or eliminate bubble formation during fluid flow. Surface materials with specific wetting properties can be used to reduce or eliminate bubble formation. For example, the surface described herein comprises a hydrophobic material. In some cases, the surface described herein comprises a hydrophilic material. Bubble formation during fluid movement can be controlled by pressure. In some cases, pressure is applied locally to components, surface areas, capillaries / channels, or to the entire system. In some cases, pressure is applied after or before the direction of fluid movement. In some cases, back pressure is applied to prevent bubble formation. The suitable pressure range for preventing bubble formation depends on the fluid, scale, flow geometry and materials used. For example, 5 to 10 atmospheres of pressure are maintained in the system. In some cases, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 20 or more than 50 atmospheres of pressure are applied. In some cases, up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 20 or up to 50 atmospheres of pressure are applied. In some cases, about 2 to about 10, about 2 to about 8, about 2 to about 5, about 4 to about 10, about 4 to about 12, about 5 to about 15, about 5 to about 7, about 7 to about 20, about 8 to about 15, or about 10 to about 20 atmospheres of pressure are applied.

[0052] Devices described herein can utilize control units for purposes of regulating environmental conditions such as temperature. Temperature control units are generally used to prepare or maintain conditions for storing solid supports containing polynucleotides. The storage conditions of nucleic acids can affect their long-term stability, which directly affects the quality of the retrieved digital storage information. Polynucleotides are optionally stored on solid supports at low temperatures (e.g., 10°C, 4°C, 0°C or lower), wherein the temperature control unit maintains the temperature of the solid support. The storage medium (such as solvated or dried) of the polynucleotides on the solid support also affects storage stability. In some cases, polynucleotides are stored in solutions in the form of droplets, such as aqueous solutions or buffers. In some cases, polynucleotides are freeze-dried (dried) for storage. In some cases, the temperature control unit increases the chip temperature to promote the drying of the polynucleotides attached thereto. In some cases, the temperature control unit also provides local control of the heating at the addressable position on the solid support. In some cases, after the droplets containing polynucleotides are added to the solid support, the solid support is dried. In some cases, the dried solid support is then resolvated. In some cases, the solid support is stored for later use. In some cases, the solid support further comprises an index map of the polynucleotides. In some cases, the solid support further comprises metadata.

[0053] Devices as described herein may include a power source for energizing each component of the device. The synthesis assembly in the solid support is optionally powered by an external power source or a power source integrated into the solid support. The power source may include a battery, a solar cell, a thermoelectric generator, an induction (wireless) power supply unit, a kinetic energy charger, a cellular phone, a tablet computer or other power sources suitable for synthesis assemblies or devices as described herein. In some cases, synthesis assemblies, surfaces or devices as described herein are portable.

[0054] Fluids comprising reagents, washing solvents or other synthetic components are deposited on the synthetic surface. In some cases, unused fluids (before contacting with the synthetic surface) or waste liquids (after contacting with the synthetic surface) are stored in one or more compartments that are integrated into the solid support. Alternatively or in combination, polynucleotides are moved in or out of the solid support for external analysis or storage. For example, the polynucleotides synthesized are cut from the seat on the solid support in the droplet, and the droplet of gained is moved from the outside to the synthesis region of the solid support. Optionally the droplet is dried for storage. In some cases, fluid is stored outside the solid support. In some cases, device as herein described comprises a solid support with a plurality of fluid ports, and the fluid ports allow fluid to move in and out of the solid support. In some cases, the port is oriented on the side of the solid support, and other configurations are also suitable for fluid transport to the synthetic surface. Such devices typically comprise, for example, at least 1, 2, 5, 10, 20, 50, 100, 200, 500, 1000, 2000, 5000, or at least 10,000 ports / mm solid support length. In some cases, the devices described herein comprise about 100 to about 5000 ports / mm solid support length.

[0055] The present invention describes addressable electrodes integrated into a solid support. The electrodes include, but are not limited to, conductors, insulators, or semiconductors, and are made of materials known in the art. The materials may include metals, non-metals, mixed metal oxides, nitrides, carbides, silicon-based materials, or other materials. In some cases, the metal oxides include TiO 2 、 2 O 5 , Nb 2 O 5 、Al 2 O 3 ,BaO,Y 2 O 3 , HfO 2 , SrO or other metal oxides known in the art. In some cases, the metal carbide includes TiC, WC, ThC 2 ,ThC,VC,W 2 C, ZrC, HfC, NbC, TaC, Ta 2 C or other metal carbides known in the art. In some cases, the metal nitrides include GaN, InN, BN, Be 3 N 2 Cr 2 N, MoN, Si 3 N 4 、TaN、Th 2 N 2, VN, ZrN, TiN, HfN, NbC, WN, TaN or other metal nitrides known in the art. In some cases, the devices disclosed herein are made using a combination of the materials listed herein or any other suitable materials known in the art.

[0056] The electrodes may have any shape, including disks, rods, holes, columns, substantially planar shapes, or any other form suitable for nucleic acid synthesis. The cross-sectional area of ​​each electrode varies depending on the size of the locus used for polynucleotide synthesis, but in some cases is up to 500 μm. 2 、200um 2 、100um 2 、75um 2 、50um 2 、25um 2 、10um 2 , less than 5um 2 In some cases, each electrode has a cross-sectional area of ​​about 500 μm. 2 To 10um 2 , about 100um 2 Up to 25um 2 or about 150um 2 Up to 50um 2 In some cases, each electrode has a cross-sectional area of ​​about 150 μm. 2 Up to 50um 2. The device provided herein includes an electrode having a diameter that varies according to the size of the seat for polynucleotide synthesis. Exemplary electrode diameters include, but are not limited to, at most 500um, 200um, 100um, 75um, 50um, 25um, 10um, less than 5um. In some cases, the diameter of each electrode is about 500um to 10um, about 100um to 25um, about 100um to about 200um, about 50um to about 200um, or about 150um to 50um. In some cases, the diameter of each electrode is about 200um to 50um. In some cases, the diameter of each electrode is about 200um to 100um. In some cases, the diameter of each electrode is at most 500nm, 200nm, 100nm, 75nm, 50nm, 25nm, 10nm, less than 5nm. In some cases, the diameter of each electrode is about 500nm to 10nm, about 100nm to 25nm, about 100nm to about 200nm, about 50nm to about 200nm, or about 150nm to 50nm. In some cases, the diameter of each electrode is about 200nm to 50nm. In some cases, the diameter of each electrode is about 200nm to 100nm. The thickness of each electrode is different according to the size of the seat used for polynucleotide synthesis, but is about 50nm, 100nm, 200nm, 500nm, 750nm, 1000nm, 1200nm, 1500nm, 2000nm, 2500nm, 3000nm, or is about 3500nm. In some cases, the thickness of the electrode is at least 50nm, 100nm, 200nm, 500nm, 750nm, 1000nm, 1200nm, 1500nm, 2000nm, 2500nm, 3000nm or at least 3500nm. In some cases, the thickness of the electrode is at least 1um, 2um, 3um, 5um, 10um, 15um, 20um, 30um, 50um or at least 75um. In some cases, the thickness of the electrode is about 1um, 2um, 3um, 5um, 10um, 15um, 20um, 30um, 50um or about 75um. In some cases, the thickness of the electrode is at most 1um, 2um, 3um, 5um, 10um, 15um, 20um, 30um, 50um or at most 75um. In some cases, the thickness of the electrode is at most 50 nm, 100 nm, 200 nm, 500 nm, 750 nm, 1000 nm, 1200 nm, 1500 nm, 2000 nm, 2500 nm, 3000 nm, or at most 3500 nm.In some cases, the thickness of the electrode is about 20nm to 3000nm, about 50nm to 2500, about 100nm to 750nm, about 400nm to 750nm, about 500nm to 3000nm or about 1000nm to 3000nm. In some cases, the thickness of the electrode is about 10um to about 20um. In some cases, the thickness of the electrode is about 5um to about 50um, about 10um to about 30um, about 15um to about 25um or about 30um to about 50um. In some cases, the electrode is coated with other materials, such as semiconductors or insulators. In some cases, the electrode is coated with materials for polynucleotide attachment and synthesis. In some cases, the size, shape, pattern or orientation of the electrode is selected to minimize the harmful side reactions caused by the reagent generated by electrochemistry. In some cases, a combination of electrodes is used, such as a grid of addressable electrodes and common electrodes. In some cases, the electrode is a cathode or an anode. The electrode or electrode array can be placed at any position on or near the polynucleotide surface. In some cases, the electrode is placed on the bottom of the seat for synthesis, such as the bottom of a hole or channel. In some cases, the electrode is placed in the sidewall of a hole or channel ("sidewall electrode"). In some cases, there are multiple sidewall electrodes on the side of the hole. The electrodes placed in different positions in the device can have different functions, and can be independently or simultaneously addressable. In some cases, the electrode in the hole bottom is used to cut polynucleotides from the surface at one or more seats, and the sidewall electrode is used to generate acid to deprotect the polynucleotides. In some cases, the electrode in the hole bottom is used to cut polynucleotides from the surface at one or more seats, and the first sidewall electrode is used to generate acid to deprotect the polynucleotides, and the second sidewall electrode is used to move the polynucleotides out of the hole after cutting. In an exemplary configuration, the sidewall electrode is located at about 10nm, about 25nm, about 50nm, about 75nm, about 100nm, about 125nm or about 200nm above the synthesis surface. In some cases, the sidewall electrodes are located about 10nm to about 100nm, about 50nm to about 150nm, about 40nm to 100nm, about 75nm to about 125nm, about 100 to 300nm above the synthetic surface. In some cases, a plurality of sidewall electrodes are located at different heights above the synthetic surface. For example, the seat comprises at least one sidewall electrode, at least 2 sidewall electrodes, at least 3 sidewall electrodes, or more than 3 sidewall electrodes. In an exemplary configuration, the height of the sidewall electrode is about 1nm, about 5nm, about 10nm, about 20nm, about 25nm, about 30nm, about 40nm, or about 50nm. In some cases, the height of the sidewall electrode is 1nm to 20nm, 2nm to 30nm, 5nm to 20nm, 10nm to 40nm, or 5nm to 25nm.

[0057] Electrode surface can support the movement, conformation, synthesis, growth and release of polynucleotide. In some cases, electrode is coated with one or more layers. In some cases, this layer is the monolayer that is conducive to the attachment of connector. In some cases, the electrode is charged to affect the region of the monolayer that will be functionalized by the connector. This allows masking of specific areas for chemical functionalization, such as modifying the surface with hydrophobic or hydrophilic chemical groups. In some cases, the electrode is charged to affect the region of the monolayer that will be extended with nucleoside monomer. In some cases, this includes generating reagents to promote or prevent the coupling of monomer and the synthetic surface near the electrode. In some cases, the electrode is charged to affect the region of the monolayer that releases polynucleotide. In some cases, this specific polynucleotide is used to control the monomer assembly of synthesis into larger polynucleotide with the controlled release of specific order. For example, the various combinations of polynucleotides assembled for overlapping PCR are optimized by exploring the release and allowing hybridization.

[0058] Each electrode can control one or more different loci for synthesis, wherein each locus for synthesis has a density of polynucleotides. In some cases, the density is at least 1 oligonucleotide / 10nm 2 , 20, 50, 100, 200, 500, 1,000, 2,000, 5,000 or at least 1 oligonucleotide / 10,000nm 2 In some cases, the density is about 1 oligonucleotide / 10 nm 2 About 1 oligonucleotide / 5,000nm 2 , about 1 oligonucleotide / 50nm 2 About 1 oligonucleotide / 500nm 2 or about 1 oligonucleotide / 25nm 2 About 1 oligonucleotide / 75nm 2 In some cases, the density of polynucleotides is about 1 oligonucleotide / 25 nm 2 About 1 oligonucleotide / 75nm 2 .

[0059] Provided herein is a device for polynucleotide synthesis with various types of electrodes.In some cases, the device comprises a reference electrode.The reference electrode is placed near the synthesis surface, or in the case of a hole or channel, for example, is placed above a hole or channel.In some cases, the reference electrode is about 1 to about 50um above the synthesis surface, about 2um to about 40um, about 3um to about 30um, about 5um to about 20um, about 10 to about 20um, about 15 to about 50um, about 30 to about 50um, about 5um to about 30um or about 7um to about 25um.In some cases, the reference electrode is about 1,2,5,7,8,9,10,12,14,16,18,20,22,24,26um or greater than 26um above the synthesis surface. In some cases, the reference electrode is at most 1, 2, 5, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26um or at most 26um above the synthetic surface. In some cases, the reference electrode is at least 1, 2, 5, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26um or at least 26um above the synthetic surface. In some cases, the reference electrode is adjacent to the synthetic surface, such as adjacent to a hole or channel. In some cases, each seat has a corresponding reference electrode. In some cases, each seat shares a common reference electrode with one or more adjacent seats. The device described herein may include any number of reference electrodes. In some cases, the reference electrode is a single uniform plate. In some cases, the device includes multiple reference electrodes. The reference electrode can address multiple seats, such as 2, 3, 4, 5, 10 or more seats.

[0060] Described herein are devices, compositions, systems and methods for nucleic acid synthesis and storage based on solid supports, wherein the solid supports have different sizes. In some cases, the size of the solid support is about 40 to 120 mm by about 25 to 100 mm. In some cases, the size of the solid support is about 80 mm by about 50 mm. In some cases, the width of the solid support is at least or about 10 mm, 20 mm, 40 mm, 60 mm, 80 mm, 100 mm, 150 mm, 200 mm, 300 mm, 400 mm, 500 mm or greater than 500 mm. In some cases, the height of the solid support is at least or about 10 mm, 20 mm, 40 mm, 60 mm, 80 mm, 100 mm, 150 mm, 200 mm, 300 mm, 400 mm, 500 mm or greater than 500 mm. In some cases, the planar surface area of ​​the solid support is at least or about 100 mm. 2 , 200mm 2 , 500mm 2 、1,000mm 2、2,000mm 2 、4,500mm 2 、5,000mm 2 、10,000mm 2 、12,000mm 2 、15,000mm 2 、20,000mm 2 、30,000mm 2 、40,000mm 2 、50,000mm 2 In some cases, the thickness of the solid support is about 50mm to about 2000mm, about 50mm to about 1000mm, about 100mm to about 1000mm, about 200mm to about 1000mm or about 250mm to about 1000mm. The limiting examples of the thickness of the solid support include 275mm, 375mm, 525mm, 625mm, 675mm, 725mm, 775mm and 925mm. In some cases, the thickness of the solid support is at least or about 0.5mm, 1.0mm, 1.5mm, 2.0mm, 2.5mm, 3.0mm, 3.5mm, 4.0mm or greater than 4.0mm.

[0061] Devices in which two or more solid supports are assembled are described herein. In some cases, the solid supports are interfaced together on a larger unit. The interface connection may include exchanging fluids, electrical signals, or other exchange media between the solid supports. The unit is capable of interfacing with any number of servers, computers, or networking devices. For example, multiple solid supports are integrated into a rack unit that is conveniently inserted into or removed from a server rack. A rack unit may contain any number of solid supports. In some cases, a rack unit contains at least 1, 2, 5, 10, 20, 50, 100, 200, 500, 1000, 2000, 5000, 10,000, 20,000, 50,000, 100,000, or more than 100,000 solid supports. In some cases, two or more solid supports are not interfaced with each other. Nucleic acids present on the solid supports (and the information stored therein) can be accessed from the rack unit. See, for example, Fig.14. Access includes removing polynucleotides from a solid support, directly analyzing the polynucleotides on a solid support, or any other method that allows the information stored in the nucleic acid to be manipulated or identified. In some cases, information is accessed from multiple racks, a single rack, a single solid support in a rack, a part of a solid support, or a single seat on a solid support. In various cases, access includes making nucleic acid and other devices such as mass spectrometers, HPLC, sequencing instruments, PCR thermal cyclers, or other device interfaces for operating nucleic acids. In some cases, access to nucleic acid information is achieved by cutting polynucleotides from the whole or part of a solid support. In some cases, cutting includes exposure to chemical reagents (ammonia or other reagents), potential, radiation, heat, light, acoustics, or other forms of energy capable of operating chemical bonds. In some cases, cutting occurs by charging one or more electrodes near the polynucleotide. In some cases, electromagnetic radiation in the form of ultraviolet rays is used to cut polynucleotides. In some cases, a lamp is used to cut polynucleotides, and a mask mediates ultraviolet exposure to the position of the surface. In some cases, a laser is used to cut polynucleotides, and a shutter open / closed state controls the exposure of ultraviolet rays to the surface. In some cases, access to nucleic acid information (including removal / addition of racks, solid supports, reagents, nucleic acids, or other components) is fully automated.

[0062] A solid support as described herein comprises an active region. In some cases, the active region comprises an addressable region or seat for nucleic acid synthesis. In some cases, the active region comprises an addressable region or seat for nucleic acid storage.

[0063] The active area has different sizes. For example, the size of the active area is about 1 mm to about 50 mm by about 1 mm to about 50 mm. In some cases, the active area has a width of at least or about 0.5, 1, 1.5, 2, 2.5, 3, 5, 5, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80 or more than 80 mm. In some cases, the active area has a height of at least or about 0.5, 1, 1.5, 2, 2.5, 3, 5, 5, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80 or more than 80 mm. Exemplary active areas within a solid support can be found at Fig.13 The package 1307 includes an active area 1305 within the solid support 1303. The package 1307 also includes a fluidic interface 1301.

[0064] Described herein are devices, compositions, systems and methods for nucleic acid synthesis and storage based on solid supports, wherein the solid support has a plurality of sites (e.g., spots) or positions for synthesis or storage. In some cases, the solid support comprises at most or approximately 10,000 times 10,000 positions in an area. In some cases, the solid support comprises about 1000 to 20,000 times about 1000 to 20,000 positions in an area. In some cases, the solid support comprises at least or approximately 10, 30, 50, 75, 100, 200, 300, 400, 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, 12,000, 14,000, 16,000, 18,000, 20,000, 25,000, 26,000, 27,000, 28,000, 29,000, 30,000, 31,000, 32,000, 33,000, 34,000, 35,000, 36,000, 37,000, 38,000, 39,000, 40,000, 41,000, 42,000, 43,000, 44,000, 45,000, 46,000, 47,000, 48,000, 49,000, 50, In some cases, the area is at most 0.25, 0.5, 0.75, 1.0, 1.25, 1.5, or 2.0 square inches. In some cases, the solid support comprises addressable loci that are at least or about 0.1, 0.2, 0.25, 0.3, 0.4, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5, 6, 7, 8, 9, 10, or more than 10 um in spacing. In some cases, the solid support comprises addressable loci that are about 5 um in spacing. In some cases, the solid support comprises addressable loci that are about 2 um in spacing. In some cases, the solid support comprises addressable loci that are about 1 um in spacing. In some cases, the solid support comprises addressable loci that are about 0.2 um in spacing. In some cases, the solid support comprises addressable loci with a spacing of about 0.2 um to about 10 um, about 0.2 to about 8 um, about 0.5 to about 10 um, about 1 um to about 10 um, about 2 um to about 8 um, about 3 um to about 5 um, about 1 um to about 3 um, or about 0.5 um to about 3 um. In some cases, the solid support comprises addressable loci with a spacing of about 0.1 um to about 3 um. See, e.g., Fig.15 , Fig.16A and Fig. 16B .

[0065] Solid supports for nucleic acid synthesis or storage as described herein have high data storage capacity. In some cases, the capacity of solid supports is at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or greater than 1000 petabytes (petabyte). In some cases, the capacity of solid supports is about 1 to about 10 petabytes or about 1 to about 100 petabytes. In some cases, the capacity of solid supports is about 100 petabytes. In some cases, data are stored as addressable data packets (packet) arrays in the form of droplets. In some cases, data are stored as addressable data packet arrays in the form of droplets on spots. In some cases, data are stored as addressable data packet arrays in the form of drying holes. In some cases, the addressable array contains at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 200, or more than 200 gigabytes of data. In some cases, the addressable array contains at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 200, or more than 200 terabytes of data. In some cases, the information item is stored in a data background. For example, the information item encodes about 10 to about 100 megabytes of data and is stored in 1 petabyte of background data. In some cases, the information item encodes at least or approximately 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, or more than 500 megabytes of data and is stored within 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, or more than 500 petabytes of background data.

[0066] Provided herein are devices, compositions, systems and methods for nucleic acid synthesis and storage based on solid supports, wherein after synthesis, polynucleotides are collected in a data packet in the form of one or more droplets. In some cases, polynucleotides are collected in a data packet in the form of one or more droplets and stored. In some cases, the number of droplets is at least or approximately 1, 10, 20, 50, 100, 200, 300, 500, 1000, 2500, 5000, 75000, 10,000, 25,000, 50,000, 75,000, 100,000, 1 million, 5 million, 10 million, 25 million, 50 million, 75 million, 100 million, 250 million, 50 million, 75 million, 100 million, 250 million, 500 million, 750 million or more than 750 million droplets. In some cases, the small droplet volume has a diameter of 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more than 100 um (micrometers). In some cases, the small droplet volume has a diameter of 1-100 um, 10-90 um, 20-80 um, 30-70 um or 40-50 um.

[0067] In some cases, the polynucleotides collected in the data packets comprise similar sequences. In some cases, the polynucleotides further comprise different sequences used as labels or barcodes. For example, different sequences are used to index the polynucleotides stored on the solid support, and later search for specific polynucleotides according to different sequences. Exemplary labels or barcode lengths include such barcode sequences, which include but are not limited to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more bases in length. In some cases, the label or barcode comprises at least or about 10, 50, 75, 100, 200, 300, 400 or more than 400 base pairs in length.

[0068] Provided herein are devices, compositions, systems and methods for nucleic acid synthesis and storage based on solid supports, wherein polynucleotides are collected in data packets comprising redundancy. For example, the data packet comprises about 100 to about 1000 copies of each polynucleotide. In some cases, the data packet comprises at least or about 50, 75, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1400, 1600, 1800, 2000 or more than 2000 copies of each polynucleotide. In some cases, the data packet comprises about 1000X to about 5000X synthetic redundancy. In some cases, the synthetic redundancy is at least or about 500X, 1000X, 1500X, 2000X, 2500X, 3000X, 3500X, 4000X, 5000X, 6000X, 7000X, 8000X or greater than 8000X. The polynucleotides synthesized using the method based on solid support as described herein comprise various lengths. In some cases, the polynucleotides are synthesized and further stored on a solid support. In some cases, the polynucleotide length is about 100 to about 1000 bases. In some cases, a polynucleotide comprises at least or about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, or more than 2000 bases in length.

[0069] Nucleic acid-based information storage

[0070] Provided herein are devices, compositions, systems and methods for nucleic acid-based information (data) storage. Figure 1An exemplary workflow is provided in . In the first step, a digital sequence of a coded information item (i.e., digital information in the form of binary code processed by a computer) is received (101). An encryption 103 scheme is applied to convert the digital sequence from a binary code into a nucleic acid sequence 105. The surface material for nucleic acid extension, the design of the seat (also referred to as an arrangement spot) for nucleic acid extension, and the reagent for nucleic acid synthesis (107) are selected. The surface of the structure is prepared for nucleic acid synthesis (108). De novo polynucleotide synthesis (109) is performed. The synthesized polynucleotide (111) is stored and the synthesized polynucleotide can be used in whole or in part for subsequent release (113). Once released, the polynucleotide is sequenced in whole or in part (115), and it is decrypted (117) to convert the nucleic acid sequence back into a digital sequence. The digital sequence (119) is then assembled to obtain the parallel encoding of the original information item.

[0071] Information Items

[0072] Optionally, the early steps of the data storage process disclosed herein include obtaining or receiving one or more information in the form of initial code. Information items include, but are not limited to, text, audio, and visual information. Exemplary sources of information items include, but are not limited to, books, journals, electronic databases, medical records, letters, tables, recordings, animal records, biological spectra, broadcasts, movies, short videos, emails, bookkeeping phone records, Internet activity logs, drawings, drawings, prints, photos, pixelated graphics, and software codes. Exemplary biological spectrum sources of information items include, but are not limited to, gene libraries, genomes, gene expression data, and protein activity data. Exemplary formats of information items include, but are not limited to, .txt, .PDF, .doc, .docx, .ppt, .pptx, .xls, .xlsx, .rtf, .jpg, .gif, .psd, .bmp, .tiff, .png, and .mpeg. The size of a single file encoding an information item in a digital format or the amount of multiple files encoding information items includes, but is not limited to, up to 1024 bytes (equal to 1 KB), 1024 KB (equal to 1 MB), 1024 MB (equal to 1 GB), 1024 GB (equal to 1 TB), 1024 TB (equal to 1 PB), 1 exabyte, 1 zettabyte, 1 yottabyte, 1 xenottabyte, or more. In some cases, the amount of digital information is at least 1 gigabyte (GB). In some cases, the amount of digital information is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or more than 1000 gigabytes. In some cases, the amount of digital information is at least 1 terabyte (TB). In some cases, the amount of digital information is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or more than 1000 terabytes. In some cases, the amount of digital information is at least 1 petabyte (PB). In some cases, the amount of digital information is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or more than 1000 petabytes.

[0073] Structures for polynucleotide synthesis

[0074] Provided herein are rigid or flexible structures for polynucleotide synthesis. In the case of a rigid structure, provided herein are devices with structures for generating polynucleotide libraries. In some cases, the structure comprises a plate. Figure 2An exemplary structure 200 is shown in , where the structure 200 has approximately the same size dimensions as a standard 96-well plate: 140 mm x 90 mm. The structure 200 contains clusters grouped in 24 regions or sub-domains 205, each sub-domain 205 containing an array of 256 clusters 210. Figure 3 An expanded view of an exemplary subdomain 205 is shown in FIG. In the expanded view of the four clusters ( Figure 3 ), the Y-axis cluster spacing (center-to-center distance between adjacent clusters) of a single cluster 210 is 1079.210um or 1142.694um, and the X-axis cluster spacing is 1125um. Figure 4 An illustrative cluster 210 is depicted in FIG, where the Y-axis seat pitch (center-to-center distance of adjacent seats) is 63.483 um, and the X-axis seat pitch is 75 um. The seat width at the longest portion (eg, the diameter of a circular seat) is 50 um, and the distance between seats is 24 um. Figure 4 The number of seats 405 in an exemplary cluster in is 121. The seats can be flat, holes or channels. Figure 5A-5B , which shows a plate 505 including a main channel 510 and a plurality of channels 515 connected to the main channel 510. The connection between the main channel 510 and the plurality of channels 515 provides fluid communication for a flow path from the main channel 510 to each of the plurality of channels 515. The plate 505 described herein may include a plurality of main channels 510. The plurality of channels 515 collectively form a cluster within the main channel 510.

[0075] In the case of a flexible structure, devices are provided herein where the flexible structure comprises a continuous loop 601 wrapped around one or more fixed structures (e.g., a pair of rollers 603) or a discontinuous flexible structure 607 wrapped around a separate fixed structure (e.g., a pair of rolls 605). Figure 6A-6B In some cases, the structure comprises multiple regions for polynucleotide synthesis. Figure 6C An exemplary structure is shown in , where the plate contains different regions 609 for polynucleotide synthesis. The different regions 609 can be separated (611) by breaking or cutting. Each different region can be further released, sequenced, decrypted and read (613) or stored (615). Fig.6D An alternative structure is shown in which the ribbon contains different regions 617 for polynucleotide synthesis. The different regions 617 can be separated (619) by breaking or cutting. Each different region can be further released, sequenced, decrypted and read (621) or stored (623). Provided herein is a flexible structure having a surface with multiple loci for polynucleotide extension. Figures 7A-7CAn enlarged view of a seat in a flexible structure is shown. Each seat in a portion of a flexible structure 701 can be a substantially planar spot 703 (e.g., flat), a channel 705, or a hole 707. In some cases, the width of each seat of the structure is about 10um, and the distance between the centers of each structure is about 21um. The seat may include, but is not limited to, a circular, rectangular, conical, or arcuate shape. Alternatively or in combination, the structure is rigid. In some cases, the rigid structure comprises a seat for polynucleotide synthesis. In some cases, the rigid structure comprises a substantially planar region, channel, or hole for polynucleotide synthesis.

[0076] In some cases, the ratio of the width of the hole described herein to the depth (or height) is 1 to 0.01, wherein the width is the measurement of the width at the narrowest section of the hole. In some cases, the ratio of the width of the hole described herein to the depth (or height) is 0.5 to 0.01, wherein the width is the measurement of the width at the narrowest section of the hole. In some cases, the ratio of the width of the hole described herein to the depth (or height) is about 0.01, 0.05, 0.1, 0.15, 0.16, 0.2, 0.5 or 1. Provided herein is a structure for polynucleotide synthesis, which comprises a plurality of discrete seats for polynucleotide synthesis. Exemplary structures for seats include, but are not limited to, substantially planar regions, channels, holes or projections. Structures described herein may include multiple clusters, each cluster comprising multiple holes, seats or channels. Alternatively, described herein may include a uniform arrangement of holes, seats or channels. The structure provided herein can comprise a hole that height or depth is about 5um to about 500um, about 5um to about 400um, about 5um to about 300um, about 5um to about 200um, about 5um to about 100um, about 5um to about 50um or about 10um to about 50um. In some cases, the height in the hole is less than 100um, less than 80um, less than 60um, less than 40um or less than 20um. In some cases, the height in the hole is about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500um or higher. In some cases, the height or depth in the hole is at least 10, 25, 50, 75, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or exceeds 1000nm. In some cases, the height or depth of the hole is in the range of about 10 nm to about 1000 nm, about 25 nm to about 900 nm, about 50 nm to about 800 nm, about 75 nm to about 700 nm, about 100 nm to about 600 nm, or about 200 nm to about 500 nm. In some cases, the height or depth of the hole is in the range of about 50 nm to about 1 um. In some cases, the height of the hole is about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 700, 800, 900, or about 1000 nm.

[0077] The structure for polynucleotide synthesis provided herein can include channel. The width of channel can be 1 to 0.01 with respect to the ratio of depth (or height), wherein width is the measured value of width at the narrowest section of microchannel. In some cases, the width of channel as described herein is 0.5 to 0.01 with respect to the ratio of depth (or height), wherein width is the measured value of width at the narrowest section of microchannel. In some cases, the width of channel as described herein is about 0.01, 0.05, 0.1, 0.15, 0.16, 0.2, 0.5 or 1 with respect to the ratio of depth (or height).

[0078] The structure for polynucleotide synthesis is described herein, and it comprises a plurality of discrete seats.Structure includes but is not limited to the substantially planar region, channel, projection or hole for polynucleotide synthesis.In some cases, structure described herein is provided, it comprises a plurality of channels, wherein the height or depth of channel is about 5um to about 500um, about 5um to about 400um, about 5um to about 300um, about 5um to about 200um, about 5um to about 100um, about 5um to about 50um or about 10um to about 50um.In some cases, the height of channel is less than 100um, less than 80um, less than 60um, less than 40um or less than 20um.In some cases, the height of channel is about 10,20,30,40,50,60,70,80,90,100,200,300,400,500um or higher. In some cases, the height or depth of the channel is at least 10, 25, 50, 75, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more than 1000 nm. In some cases, the height or depth of the channel is in the range of about 10 nm to about 1000 nm, about 25 nm to about 900 nm, about 50 nm to about 800 nm, about 75 nm to about 700 nm, about 100 nm to about 600 nm, or about 200 nm to about 500 nm. The channels described herein can be arranged on a surface in clusters or as uniform domains.

[0079] The width of the loci on the surface of the structures for polynucleotide synthesis described herein can be about 0.1um to about 500um, about 0.5um to about 500um, about 1um to about 200um, about 1um to about 100um, about 5um to about 100um, or about 0.1um to about 100um, for example, about 90um, 80um, 70um, 60um, 50um, 40um, 30um, 20um, 10um, 5um, 1um, or 0.5um. In some cases, the width of the loci is less than about 100um, 90um, 80um, 70um, 60um, 50um, 40um, 30um, 20um, or 10um. In some cases, the width of the loci is at least 10, 25, 50, 75, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or more than 1000nm. In some cases, the width of the locus is in the range of about 10 nm to about 1000 nm, about 25 nm to about 900 nm, about 50 nm to about 800 nm, about 75 nm to about 700 nm, about 100 nm to about 600 nm, or about 200 nm to about 500 nm. In some cases, the width of the locus is in the range of about 50 nm to about 1000 nm. In some cases, the distance between the centers of two adjacent loci is about 0.1 um to about 500 um, 0.5 um to about 500 um, about 1 um to about 200 um, about 1 um to about 100 um, about 5 um to about 200 um, about 5 um to about 100 um, about 5 um to about 50 um, or about 5 um to about 30 um, for example, about 20 um. In some cases, the total width of the locus is about 5 um, 10 um, 20 um, 30 um, 40 um, 50 um, 60 um, 70 um, 80 um, 90 um, or 100 um. In some cases, the total width of the seat is about 1um to 100um, 30um to 100um, or 50um to 70um. In some cases, the distance between the centers of two adjacent seats is about 0.5um to about 2um, 0.5um to about 2um, about 0.75um to about 2um, about 1um to about 2um, about 0.2um to about 1um, about 0.5um to about 1.5um, about 0.5um to about 0.8um, or about 0.5um to about 1um, for example, about 1um. In some cases, the total width of the seat is about 50nm, 0.1um, 0.2um, 0.3um, 0.4um, 0.5um, 0.6um, 0.7um, 0.8um, 0.9um, 1um, 1.1um, 1.2um, 1.3um, 1.4um, or 1.5um. In some cases, the total width of the seat is about 0.5um to 2um, 0.75um to 1um, or 0.9um to 2um.

[0080] In some cases, each seat supports the synthesis of a population of polynucleotides having a different sequence than the population of polynucleotides grown at another seat. Provided herein are surfaces comprising at least 10, 100, 256, 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000, 20000, 30000, 40000, 50000 or more clusters. Provided herein are surfaces comprising more than 2,000, 5,000, 10,000, 20,000, 30,000, 50,000, 100,000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, 1,000,000, 5,000,000, or 10,000,000 or more different seats. In some cases, each cluster comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 130, 150, 200, 500 or more seats. In some cases, each cluster contains 50 to 500, 50 to 200, 50 to 150, or 100 to 150 seats. In some cases, each cluster contains 100 to 150 seats. In some cases, each cluster contains 109, 121, 130, or 137 seats.

[0081] Provided herein are seats having a width of 5 to 100 um at the longest segment. In some cases, the seat has a width of about 30, 35, 40, 45, 50, 55, or 60 um at the longest segment. In some cases, the seat is a channel having multiple segments, wherein the center-to-center distance of each segment is 5 to 50 um. In some cases, the center-to-center distance of each segment is 5, 10, 15, 20, or 25 um.

[0082] In some cases, the number of different polynucleotides synthesized on the surface of a structure described herein depends on the number of different loci available in the substrate. In some cases, the density of loci within a cluster of the substrate is at least or about 1 locus / mm 2 , 10 seats / mm 2 , 25 seats / mm 2 , 50 seats / mm 2 、65 seats / mm 2 , 75 seats / mm 2 , 100 seats / mm 2 , 130 seats / mm 2 , 150 seats / mm2 , 175 seats / mm 2 , 200 seats / mm 2 , 300 seats / mm 2 , 400 seats / mm 2 , 500 seats / mm 2 , 1,000 seats / mm 2 , 10 4 Seats / mm 2 , 10 5 Seats / mm 2 , 10 6 Seats / mm 2 or greater. In some cases, the substrate comprises about 10 seats / mm 2 To about 500mm 2 , about 25 seats / mm 2 Up to about 400 seats / mm 2 , about 50 seats / mm 2 Up to about 500 seats / mm 2 , about 100 seats / mm 2 Up to about 500 seats / mm 2 , about 150 seats / mm 2 Up to about 500 seats / mm 2 , about 10 seats / mm 2 About 250 seats / mm 2 , about 50 seats / mm 2 To about 250 seats / mm 2 , about 10 seats / mm 2 Up to about 200 seats / mm 2 or about 50 seats / mm 2 Up to about 200 seats / mm 2 In some cases, the substrate comprises about 10 4 Seats / mm 2 to about 10 5 Seats / mm 2 In some cases, the substrate comprises about 10 5 Seats / mm 2 to about 10 7 Seats / mm 2 In some cases, the substrate comprises at least 10 5 Seats / mm 2 In some cases, the substrate comprises at least 10 6 Seats / mm 2 In some cases, the substrate comprises at least 10 7 Seats / mm 2In some cases, the substrate comprises about 10 4 Seats / mm 2 to about 10 5 Seats / mm 2 In some cases, the locus density within the cluster of the substrate is at least or about 1 locus / um 2 、10 seats / um 2 、25 seats / um 2 、50 seats / um 2 、65 seats / um 2 、75 seats / um 2 , 100 seats / um 2 、130 seats / um 2 , 150 seats / um 2 、175 seats / um 2 , 200 seats / um 2 、300 seats / um 2 , 400 seats / um 2 , 500 seats / um 2 , 1,000 seats / um 2 or greater. In some cases, the substrate comprises about 10 seats / um 2 About 500 seats / um 2 , about 25 seats / um 2 About 400 seats / um 2 、About 50 seats / um 2 About 500 seats / um 2 , about 100 seats / um 2 About 500 seats / um 2 , about 150 seats / um 2 About 500 seats / um 2 , about 10 seats / um 2 About 250 seats / um 2 、About 50 seats / um 2 About 250 seats / um 2 , about 10 seats / um 2 About 200 seats / um 2 or about 50 seats / um 2 About 200 seats / um 2 .

[0083] In some cases, the distance between the centers of two adjacent loci within a cluster is about 10 um to about 500 um, about 10 um to about 200 um, or about 10 um to about 100 um. In some cases, the distance between the centers of two adjacent loci is greater than about 10 um, 20 um, 30 um, 40 um, 50 um, 60 um, 70 um, 80 um, 90 um, or 100 um. In some cases, the distance between the centers of two adjacent loci is less than about 200 um, 150 um, 100 um, 80 um, 70 um, 60 um, 50 um, 40 um, 30 um, 20 um, or 10 um. In some cases, the distance between the centers of two adjacent loci is less than about 10000 nm, 8000 nm, 6000 nm, 4000 nm, 2000 nm, 1000 nm, 800 nm, 600 nm, 400 nm, 200 nm, 150 nm, 100 nm, 80 um, 70 nm, 60 nm, 50 nm, 40 nm, 30 nm, 20 nm, or 10 nm. In some embodiments, the structures described herein allow at least 10 7 , 10 8 , 10 9 , 10 10 , 10 11 loci, wherein each locus supports a polynucleotide. In some cases, in less than about 6, 5, 4, 3, 2, or 1 m 2 The structure described in this article supports 10 9 polynucleotides.

[0084] In some cases, the structures described herein are synthesized to produce more than 2,000, 5,000, 10,000, 20,000, 30,000, 50,000, 100,000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, 1,000,000 0, 1,200,000, 1,400,000, 1,600,000, 1,800,000, 2,000,000, 2,500,000, 3,000,000, 3,500,000, 4,000,000, 4,500,000, 5,000,000, 10,000,000 or more non-identical polynucleotides are provided. In some cases, the structures described herein are synthesized to produce more than 2,000, 5,000, 10,000, 20,000, 50,000, 100,000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, 1,000,000, 1,200,000, 0,000, 1,400,000, 1,600,000, 1,800,000, 2,000,000, 2,500,000, 3,000,000, 3,500,000, 4,000,000, 4,500,000, 5,000,000, 10,000,000 or more polynucleotides encoding non-identical sequences. In some cases, at least a portion of the polynucleotides have the same sequence or are configured to be synthesized with the same sequence. In some cases, the structure provides a surface environment for the growth of polynucleotides having at least 50, 60, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 bases or more. In some arrangements, the structures for polynucleotide synthesis described herein comprise uniformly arranged sites for polynucleotide synthesis.

[0085] In some cases, polynucleotides are synthesized at different loci of a structure, where each locus supports the synthesis of a population of polynucleotides. In some cases, each locus supports the synthesis of a population of polynucleotides having a different sequence from the population of polynucleotides growing at another locus. In some cases, the loci of the structure are within multiple clusters. In some cases, the structure contains at least 10, 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000, 20000, 30000, 40000, 50000 or more clusters. In some cases, the structure contains more than 2,000, 5,000, 10,000, 100,000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, 1,000,000, 1,100,000, 1,200,000, 1,300,000, 1,400,000, 1,500,000, 1,600,000, 1,700,000, 1,800,000, 1,900,000, 2,000,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, 1,000,000, 1,200,000, 1,400,000, 1,600,000, 1,800,000, 2,000,000, 2,500,000, 3,000,000, 3,500,000, 4,000,000, 4,500,000, 5,000,000 or 10,000,000 different loci. In some cases, each cluster contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 130, 150 or more loci. In some cases, each cluster contains 50 to 500, 100 to 150 or 100 to 200 loci. In some cases, each cluster contains 109, 121, 130 or 137 loci. In some cases, each cluster contains 5, 6, 7, 8, 9, 10, 11 or 12 loci. In some cases, the polynucleotides from different loci within a cluster have sequences that encode a continuous longer polynucleotide of a predetermined sequence upon assembly.

[0086] Structure size

[0087] In some cases, the structures described herein are approximately the size of a plate (e.g., a chip), such as about 40 to 120 mm by about 25 to 100 mm. In some cases, the diameter of the structures described herein is less than or equal to about 1000 mm, 500 mm, 450 mm, 400 mm, 300 mm, 250 mm, 200 mm, 150 mm, 100 mm, or 50 mm. In some cases, the diameter of the substrate is about 25 mm to 1000 mm, about 25 mm to about 800 mm, about 25 mm to about 600 mm, about 25 mm to about 500 mm, about 25 mm to about 400 mm, about 25 mm to about 300 mm, or about 25 mm to about 200 mm. Non-limiting examples of substrate size include about 300 mm, 200 mm, 150 mm, 130 mm, 100 mm, 84 mm, 76 mm, 54 mm, 51 mm, and 25 mm. In some cases, the planar surface area of ​​the substrate is at least 100 mm 2 , 200mm 2 , 500mm 2 、1,000mm 2 、2,000mm 2 、4,500mm 2 、5,000mm 2 、10,000mm 2 、12,000mm 2 、15,000mm 2 、20,000mm 2 、30,000mm 2 、40,000mm 2 、50,000mm 2 In some cases, the thickness is about 50mm to about 2000mm, about 50mm to about 1000mm, about 100mm to about 1000mm, about 200mm to about 1000mm or about 250mm to about 1000mm. Non-limiting examples of thickness include 275mm, 375mm, 525mm, 625mm, 675mm, 725mm, 775mm and 925mm. In some cases, the thickness is at least or about 0.5mm, 1.0mm, 1.5mm, 2.0mm, 2.5mm, 3.0mm, 3.5mm, 4.0mm or greater than 4.0mm. In some cases, the thickness varies with diameter and depends on the composition of substrate. For example, the structure comprising materials other than silicon can have a thickness different from the silicon structure of the same diameter. The structural thickness can depend on the mechanical strength of the material used, and the structure must be thick enough to support its own weight during operation without breaking. In some cases, the structure exceeds about 1, 2, 3, 4, 5, 10, 15, 30, 40, 50 feet in any one dimension.

[0088] Material

[0089] Provided herein is a device comprising a surface, wherein the surface is modified to support polynucleotide synthesis at a predetermined position, and has low error rate, low omission rate, high yield and high oligonucleotide presentation. In some cases, the surface of the device for polynucleotide synthesis provided herein is made of a variety of materials that can be modified to support de novo polynucleotide synthesis reactions. In some cases, the device has sufficient conductivity, for example, a uniform electric field can be formed across the entire device or a portion thereof. Devices described herein may include flexible materials. Exemplary flexible materials include but are not limited to modified nylon, unmodified nylon, nitrocellulose and polypropylene. Devices described herein may include rigid materials. Exemplary rigid materials include but are not limited to glass, fused quartz, silicon, silicon dioxide, silicon nitride, plastics (e.g., polytetrafluoroethylene, polypropylene, polystyrene, polycarbonate, and blends thereof) and metals (e.g., gold, platinum). Devices disclosed herein may be made of materials comprising silicon, polystyrene, agarose, dextran, cellulose polymers, polyacrylamide, polydimethylsiloxane (PDMS), glass or any combination thereof. In some cases, the devices disclosed herein are made using a combination of the materials listed herein or any other suitable materials known in the art.

[0090] Devices described herein may include materials with a certain range of tensile strengths. Exemplary materials with a certain range of tensile strengths include, but are not limited to, nylon (70 MPa), nitrocellulose (1.5 MPa), polypropylene (40 MPa), silicon (268 MPa), polystyrene (40 MPa), agarose (1-10 MPa), polyacrylamide (1-10 MPa), polydimethylsiloxane (PDMS) (3.9-10.8 MPa). The tensile strength of the solid support described herein may be 1 to 300, 1 to 40, 1 to 10, 1 to 5 or 3 to 11 MPa. The tensile strength of the solid support described herein may be about 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 20, 25, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 270 MPa or greater. In some cases, the devices described herein comprise a solid support for polynucleotide synthesis in the form of a flexible material such as a tape or flexible sheet that can be stored in a continuous loop or scroll.

[0091] Young's modulus measures the resistance of a material to elastic (recoverable) load deformation. Exemplary materials with a range of Young's modulus stiffness include, but are not limited to, nylon (3 GPa), nitrocellulose (1.5 GPa), polypropylene (2 GPa), silicon (150 GPa), polystyrene (3 GPa), agarose (1 - 10 GPa), polyacrylamide (1 - 10 GPa), polydimethylsiloxane (PDMS) (1 - 10 GPa). The Young's modulus of the solid support described herein can be 1 to 500, 1 to 40, 1 to 10, 1 to 5, or 3 to 11 GPa. The Young's modulus of the solid support described herein can be about 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 20, 25, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 400, 500 GPa or greater. Since the relationship between flexibility and stiffness is inverse to each other, flexible materials have a low Young's modulus and their shape changes significantly under load. In some cases, the solid support described herein has a surface that has at least the flexibility of nylon.

[0092] In some cases, the devices disclosed herein comprise a silica base and a silica surface layer. Alternatively, the device can have a silica base. The surface of the device provided herein can be textured, resulting in an increased total surface area for polynucleotide synthesis. In some cases, the devices disclosed herein comprise at least 5%, 10%, 25%, 50%, 80%, 90%, 95%, or 99% silicon. In some cases, the devices disclosed herein are made from silicon-on-insulator (SOI) wafers.

[0093] The structure can be made of a variety of materials suitable for the methods and compositions of the invention described herein. In certain cases, the materials used to fabricate the substrate / solid support of the present invention exhibit low levels of polynucleotide binding. In some cases, materials that are transparent to visible light and / or ultraviolet light can be employed. Materials with sufficient conductivity can be used, e.g., materials that can form a uniform electric field across the entire substrate / solid support described herein or a portion thereof. In some cases, such materials can be electrically grounded. In some cases, the substrate or solid support can be thermally conductive or thermally insulating. The materials can be chemical-resistant and heat-resistant to support chemical or biochemical reactions, such as a series of polynucleotide synthesis reactions. For flexible materials, materials of interest can include: modified and unmodified nylon, nitrocellulose, polypropylene, etc.

[0094] For rigid materials, specific materials of interest include: glass; fused quartz; silicon, plastics (e.g., polytetrafluoroethylene, polypropylene, polystyrene, polycarbonate, and blends thereof, etc.); metals (e.g., gold, platinum, etc.). The structure can be made of a material selected from silicon, polystyrene, agarose, dextran, cellulose polymers, polyacrylamide, polydimethylsiloxane (PDMS), and glass. The substrate / solid support or the microstructures therein, the reactor can be made using a combination of the materials listed herein or any other suitable materials known in the art.

[0095] In some cases, the substrate disclosed herein comprises computer readable material. Computer readable material includes, but is not limited to, magnetic media, reel-to-reel tape, cassette tape, cassette tape, floppy disk, paper media, film, microfilm, continuous tape (e.g., tape), and any medium suitable for storing electronic instructions. In some cases, the substrate comprises a magnetic reel-to-reel tape or a magnetic tape. In some cases, the substrate comprises a flexible printed circuit board.

[0096] The structures described herein can be transparent to visible light and / or ultraviolet light. In some cases, the structures described herein have sufficient conductivity to form a uniform electric field across the entire structure or a portion thereof. In some cases, the structures described herein are thermally conductive or heat-insulating. In some cases, the structure is chemically resistant and heat-resistant to support chemical reactions, such as polynucleotide synthesis reactions. In some cases, the substrate is magnetic. In some cases, the structure comprises a metal or a metal alloy.

[0097] The structures used for polynucleotide synthesis can be more than 1, 2, 5, 10, 30, 50 feet long or longer in any dimension. In the case of flexible structures, the flexible structures are optionally stored in a wound state, such as a roll. In the case of large rigid structures (e.g., greater than 1 foot in length), the rigid structures can be stored vertically or horizontally.

[0098] Surface preparation

[0099] Provided herein are methods for supporting the immobilization of biomolecules on substrates, wherein the surface of the structures described herein comprises a material and / or is coated with a material that promotes a coupling reaction with the biomolecule for attachment. To prepare structures for biomolecule immobilization, surface modification can be employed, which chemically and / or physically changes the surface of the substrate by an additive process or a subtractive process to change one or more chemical and / or physical properties of the substrate surface or selected sites or regions of the surface. For example, surface modification involves: (1) changing the wetting properties of the surface; (2) functionalizing the surface, i.e., providing, modifying or replacing surface functional groups; (3) defunctionalizing the surface, i.e., removing surface functional groups; (4) otherwise changing the chemical composition of the surface, such as by etching; (5) increasing or decreasing the surface roughness; (6) providing a coating on the surface, such as a coating that exhibits wetting properties different from the wetting properties of the surface; and / or (7) depositing microparticles on the surface. In some cases, the surface of a structure is selectively functionalized to produce two or more distinct regions on the structure, wherein at least one region has a different surface or chemical property than another region of the same structure. Such properties include, but are not limited to, surface energy, chemical termination, surface concentration of chemical moieties, and the like.

[0100] In some cases, the surface of the structures disclosed herein is modified to include one or more active functionalized surfaces configured to bind to both the substrate surface and biomolecules, thereby supporting coupling reactions to the surface. In some cases, the surface is also functionalized with a passivating material that is ineffective in binding biomolecules, thereby preventing attachment of biomolecules at sites where the passivating functionalizing agent binds. In some cases, the surface includes an active layer that defines only distinct loci for biomolecule support.

[0101] In some cases, the surface is contacted with a mixture of functionalized groups of any different ratios. In some cases, the mixture comprises at least 2, 3, 4, 5 or more different types of functionalizing agents. In some cases, the ratio of at least two types of surface functionalizing agents in the mixture is about 1: 1, 1: 2, 1: 5, 1: 10, 2: 10, 3: 10, 4: 10, 5: 10, 6: 10, 7: 10, 8: 10, 9: 10 or any other ratios presented in order to reach the desired surface of the two groups. In some cases, the desired surface tension, wettability, water contact angle and / or contact angle for other suitable solvents are achieved by providing a functionalizing agent of an appropriate ratio to the substrate surface. In some cases, the reagent in the mixture is selected from suitable reactive and inert parts, thereby diluting the surface density of the reactive groups to the desired level for downstream reactions. In some cases, the mixture of functionalizing agents comprises one or more reagents combined with biomolecules and one or more reagents not combined with biomolecules. Thus, modulation of the reagents allows control over the amount of biomolecule binding that occurs at different functionalized regions.

[0102] In some cases, the method for substrate functionalization includes depositing silane molecules onto the substrate surface.Silane molecules can be deposited on the high energy surface of the substrate.In some cases, the high surface energy region includes a passivating functionalizing agent.The method described herein provides a silane group to bind to the surface, and the rest of the molecule provides a distance from the surface and a free hydroxyl group at the end to which the biomolecule is attached.In some cases, the silane is an organofunctional alkoxysilane molecule.Non-limiting examples of organofunctional alkoxysilane molecules include dimethylchloro-octadecyl-silane, methyldichloro-octadecyl-silane, trichloro-octadecyl-silane, trimethyl-octadecyl-silane and triethyl-octadecyl-silane.In some cases, the silane is an aminosilane. The example of aminosilane includes but is not limited to 11-acetoxy undecyl triethoxysilane, n-decyl triethoxysilane, (3-aminopropyl) trimethoxysilane, (3-aminopropyl) triethoxysilane, glycidyloxypropyl / trimethoxysilane and N-(3-triethoxysilylpropyl)-4-hydroxybutyramide. In some cases, the silane includes 11-acetoxy undecyl triethoxysilane, n-decyl triethoxysilane, (3-aminopropyl) trimethoxysilane, (3-aminopropyl) triethoxysilane, glycidyloxypropyl / trimethoxysilane, N-(3-triethoxysilylpropyl)-4-hydroxybutyramide or any combination thereof. In some cases, the active functionalizing agent includes 11-acetoxy undecyl triethoxysilane. In some cases, the active functionalizing agent includes n-decyl triethoxysilane. In some cases, the active functionalizing agent includes glycidyloxypropyltriethoxysilane (GOPS). In some cases, the silane is a fluorosilane. In some cases, the silane is a hydrocarbon silane. In some cases, the silane is 3-iodo-propyltrimethoxysilane. In some cases, the silane is octylchlorosilane.

[0103] In some cases, silanization is carried out on the surface by self-assembly with organofunctional alkoxysilane molecules.Organofunctional alkoxysilanes are classified according to their organofunctionality.Non-limiting examples of siloxane functionalizing agents include: hydroxyalkyl siloxanes (silylated surface, functionalized with diborane, and oxidized with hydrogen peroxide to the alcohol), diol (dihydroxy alkyl) siloxanes (silylated surface, and hydrolyzed into diols), aminoalkyl siloxanes (amines do not require intermediate functionalization steps), glycidoxy silanes (3-glycidoxy propyl-dimethyl-ethoxy silane, glycidoxy-trimethoxy silane), mercapto silanes (3-mercaptopropyl-trimethoxy silane, 3-4-epoxycyclohexyl-ethyltrimethoxy silane or 3-mercaptopropyl-methyl-dimethoxy silane), bicycloheptyl-trichlorosilane, butyl-aldehyde-trimethoxy silane or dimeric secondary aminoalkyl siloxane. Exemplary hydroxyalkyl siloxanes include allyl trichlorosilane becoming 3-hydroxypropyl or 7-oct-1-enyl trichlorosilane becoming 8-hydroxyoctyl. Diol (dihydroxyalkyl) siloxanes include (2,3-dihydroxypropoxy) propyl (GOPS) derived from glycidyl trimethoxysilane. Aminoalkyl siloxanes include 3-aminopropyl trimethoxysilane (3-aminopropyl-triethoxysilane, 3-aminopropyl-diethoxy-methylsilane, 3-aminopropyl-dimethyl-ethoxysilane or 3-aminopropyl-trimethoxysilane) becoming 3-aminopropyl. In some cases, the dimeric secondary aminoalkyl siloxane is bis (3-trimethoxysilylpropyl) amine becoming bis (silyloxypropyl) amine.

[0104] The active functionalized region can contain one or more different types of silanes, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more silanes. In some cases, one of the one or more silanes is present in the functionalized composition in a higher amount than another silane. For example, a mixed silane solution with two silanes has a ratio of one silane to another silane of 99:1, 98:2, 97:3, 96:4, 95:5, 94:6, 93:7, 92:8, 91:9, 90:10, 89:11, 88:12, 87:13, 86:14, 85:15, 84:16, 83:17, 82:18, 81:19, 80:20, 75:25, 70:30, 65:35, 60:40, 55:45. In some cases, the reactive functionalizing agent comprises 11-acetoxy undecyl triethoxy silane and n-decyl triethoxy silane. In some cases, the reactive functionalizing agent comprises 11-acetoxy undecyl triethoxy silane and n-decyl triethoxy silane in a ratio of about 20:80 to about 1:99, or about 10:90 to about 2:98, or about 5:95.

[0105] In some cases, functionalization includes depositing a functionalizing agent onto the structure by any deposition technique, including but not limited to chemical vapor deposition (CVD), atomic layer deposition (ALD), plasma enhanced CVD (PECVD), plasma enhanced ALD (PEALD), metal organic CVD (MOCVD), hot filament CVD (HWCVD), initiated CVD (iCVD), modified CVD (MCVD), vapor axial deposition (VAD), outside vapor deposition (OVD), physical vapor deposition (e.g., sputtering deposition, evaporative deposition), and molecular layer deposition (MLD).

[0106] Depending on the desired properties of the final functionalized substrate, any step or component in the following functionalization process is omitted or changed. In some cases, additional components and / or process steps are added to the process workflow embodied herein. In some cases, the substrate is first cleaned, for example, using a piranha solution. An example of a cleaning process includes soaking the substrate in a piranha solution (e.g., 90% H2O2) at an elevated temperature (e.g., 120°C). 2 SO 4 , 10% H 2 O 2 ), and washing (e.g., water) and drying the substrate (e.g., nitrogen). The process optionally includes a post-piranha treatment, which includes immersing the piranha-treated substrate in an alkaline solution (e.g., NH 4 OH) followed by immersion in an aqueous wash (e.g., water). In some cases, optionally after the piranha solution immersion and optional post-piranha solution treatment, the surface of the structure is plasma cleaned. Examples of plasma cleaning processes include oxygen plasma etching. In some cases, the surface is deposited with an active functionalizing agent and then evaporated. In some cases, the substrate is reactively functionalized prior to cleaning, for example, by piranha solution treatment and / or plasma cleaning.

[0107] The process of surface functionalization optionally includes resist coating and resist stripping. In some cases, after the active surface functionalization, the substrate is spin-coated with a resist such as SPRTM3612 positive photoresist. In various cases, the process of surface functionalization includes photolithography with patterned functionalization. In some cases, photolithography is performed after resist coating. In some cases, after photolithography, the surface is visually inspected for photolithographic defects. In some cases, the surface functionalization process includes a cleaning step to remove residues of the substrate, for example, by plasma cleaning or etching. In some cases, plasma cleaning steps are performed in some steps after the photolithography step.

[0108] In some cases, for example, after functionalization and / or after photolithography, the surface coated with the resist is treated to remove the resist. In some cases, the resist is removed with a solvent, for example, with a stripping solution comprising N-methyl-2-pyrrolidone. In some cases, the resist stripping includes sonication or ultrasonication. In some cases, the resist is applied and stripped, and the exposed areas are then reactively functionalized to create the desired differentially functionalized pattern.

[0109] In some cases, the methods and compositions described herein relate to applying a photoresist to produce a surface property of change in a selective region, wherein the application of the photoresist depends on the fluid properties of the surface that limits the spatial distribution of the photoresist. Without being bound by theory, the surface tension effect associated with the applied fluid can limit the flow of the photoresist. For example, surface tension and / or capillary effects can contribute to drawing the photoresist into a small structure in a controlled manner before the resist solvent evaporates. In some cases, the resist contact point is limited by sharp edges, thereby controlling the advancement of the fluid. The infrastructure can be designed based on the desired flow pattern used to apply the photoresist during the manufacturing and functionalization process. The solid organic layer left after the solvent evaporates can be used to continue the subsequent steps of the manufacturing process. The structure can be designed to control the flow of the fluid by promoting or suppressing the wicking effect in the adjacent fluid path. For example, the design structure is to avoid the overlap between the top edge and the bottom edge, which is conducive to keeping the fluid in the top structure, thereby allowing the specific arrangement of the resist. In an alternative example, the top and bottom edges overlap, causing the applied fluid to wick into the underlying structure.Depending on the desired application of the resist, the appropriate design may be selected accordingly.

[0110] In some cases, the structures described herein have a surface comprising a material having a thickness of at least or at least 0.1 nm, 0.5 nm, 1 nm, 2 nm, 5 nm, 10 nm, or 25 nm, the material comprising a reactive group capable of binding nucleosides. Exemplary surfaces include, but are not limited to, glass and silicon, such as silicon dioxide and silicon nitride. In some cases, exemplary surfaces include nylon and PMMA.

[0111] In some cases, electromagnetic radiation in the form of ultraviolet rays is used for surface patterning. In some cases, lamps are used for surface patterning, and mask mediates ultraviolet exposure to the position of the surface. In some cases, lasers are used for surface patterning, and shutter open / closed state controls ultraviolet exposure to the surface. The laser arrangement can be used in combination with a flexible structure that can move. In such an arrangement, the coordination of laser exposure and flexible structure movement is used to create a pattern of one or more reagents with different nucleoside coupling abilities.

[0112] This paper describes a reusable surface for polynucleotide synthesis. After synthesis and / or cutting polynucleotide, the surface can be bathed, washed, cleaned, baked, etched or otherwise functionally restored to the conditions suitable for subsequent polynucleotide synthesis. The number of times of repeated use on the surface and the recycling / preparation method for the surface to be reused are different according to subsequent applications. In some cases, the surface prepared for reuse is reused at least 1, 2, 3, 5, 10, 20, 50, 100, 1,000 or more times. In some cases, the remaining "lifetime" or number of times that the surface is suitable for reuse is measured or predicted.

[0113] Material Deposition Systems

[0114] In some cases, the synthesized polynucleotides are stored on a substrate such as a solid support. Nucleic acid reagents can be deposited on the substrate surface in a discontinuous or on-demand droplet method. Examples of such methods include electromechanical transfer methods, electrothermal transfer methods, and electrostatic attraction methods. In the electromechanical transfer method, a piezoelectric element deformed by an electric pulse causes a small droplet to be ejected. In the electrothermal transfer method, bubbles are generated in the chamber of the device, and the expansion force of the bubbles causes the small droplets to be ejected. In the electrostatic attraction method, the small droplets are ejected onto the substrate using electrostatic attraction. In some cases, the droplet frequency is about 5KHz to about 500KHz; about 5KHz to about 100KHz; about 10KHz to about 500KHz; about 10KHz to about 100KHz; or about 50KHz to about 500KHz. In some cases, the frequency is less than about 500KHz, 200KHz, 100KHz, or 50KHz.

[0115] The size of the droplets distributed is related to the resolution of the device. In some cases, the device deposits droplets of reagents in a size of about 0.01 pl to about 20 pl, about 0.01 pl to about 10 pl, about 0.01 pl to about 1 pl, about 0.01 pl to about 0.5 pl, about 0.01 pl to about 0.01 pl, or about 0.05 pl to about 1 pl. In some cases, the size of the droplets is less than about 1 pl, 0.5 pl, 0.2 pl, 0.1 pl or 0.05 pl.

[0116] In some arrangements, the configuration of the polynucleotide synthesis system allows continuous polynucleotide synthesis process, which utilizes the flexibility of substrate to advance with a roll-to-roll process. This synthesis process is operated in a continuous production line mode, wherein the position of the substrate is rotated using one or more reels to advance the substrate through each stage of polynucleotide synthesis. In an exemplary case, the polynucleotide synthesis reaction comprises a rolling substrate: by a solvent bath below the deposition device for phosphoramidite deposition, by an oxidant bath, by an acetonitrile wash bath, and by a deblocking bath. Optionally, the band also passes through a capping bath. The roll-to-roll process allows the final product of the substrate comprising the synthesized polynucleotide to be easily gathered on the take-up reel, and the final product can be transported for further processing or storage at the take-up reel.

[0117] In some arrangements, polynucleotide synthesis is performed in a continuous process as the continuous flexible belt is transported along a conveyor belt system. Similar to a roll-to-roll process, polynucleotide synthesis on a continuous belt is operated in a production line manner, where the substrate travels through the various stages of polynucleotide synthesis during transport. However, in a conveyor belt process, the continuous belt returns to the polynucleotide synthesis step without rolling and unrolling the belt, as in a roll-to-roll process. In some arrangements, the polynucleotide synthesis step is divided into sections, and the continuous belt is transported through each section once or multiple times in a cycle. For example, a polynucleotide synthesis reaction may include (1) in a cycle, transporting the substrate through a solvent bath below a deposition device for phosphoramidite deposition, through an oxidant bath, through an acetonitrile wash bath, and through a closed bath; then (2) repeating the cycle to obtain a synthesized polynucleotide of a predetermined length. After the polynucleotide is synthesized, the flexible substrate is removed from the conveyor belt system and optionally rolled up for storage. It can be rolled around a roll for storage. In some cases, a flexible substrate comprising a thermoplastic material is coated with a nucleoside coupling agent. The coating is patterned into seats such that each seat has a diameter of about 10 um and the center-to-center distance between two adjacent seats is about 21 um. In this case, the seat size is sufficient to accommodate a 0.2 pl sessile drop volume during the polynucleotide synthesis deposition step. In some cases, the seat density is about 2.2 billion seats / m 2 (1 seat / 441x 10 -12 m 2 ). In some cases, 4.5m 2 The substrate contains about 10 billion seats, each seat has a diameter of 10um.

[0118] In some arrangements, the device for applying one or more reagents to substrate during building-up reaction is configured to deposit reagents and / or nucleoside monomers for the synthesis based on nucleoside phosphoramidites. The reagent for polynucleotide synthesis includes reagents and washing buffer for polynucleotide extension. As a non-limiting example, the device deposits cleaning reagents, coupling reagents, capping reagents, oxidants, deblocking agents, acetonitrile, gases such as nitrogen and any combination thereof. In addition, the device optionally deposits reagents for preparing and / or maintaining substrate integrity. In some cases, the polynucleotide synthesizer deposits a droplet less than about 200um, 100um or 50um in a volume less than about 1000, 500, 100, 50 or 20pl. In some cases, the polynucleotide synthesizer deposits about 1 to 10000, 1 to 5000, 100 to 5000 or 1000 to 5000 droplets per second.

[0119] Devices, methods, systems and compositions are described herein, wherein reagents for polynucleotide synthesis are recycled or reused. The recycling of reagents may include the collection, storage and use of unused reagents, or the purification / conversion of used reagents. For example, reagent baths are recycled and used for polynucleotide synthesis steps on the same or different surfaces. Reagents as described herein can be recycled 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 times or more. Alternatively or in combination, the reagent solution containing reaction byproducts is filtered to remove byproducts, and the reagent solution is used for other polynucleotide synthesis reactions.

[0120] Many integrated or non-integrated elements are used together with polynucleotide synthesis system usually.In some cases, polynucleotide synthesis system comprises one or more elements that can be used for the downstream processing of synthetic polynucleotide.As an example, this system comprises temperature control element, such as thermal cycler.In some cases, this temperature control element is used together with a plurality of analytical reactors, to carry out nucleic acid assembly such as PCA and / or nucleic acid amplification such as PCR.

[0121] De novo polynucleotide synthesis

[0122] Provided herein are systems and methods for synthesizing high density polynucleotides on a substrate in a short period of time. In some cases, the substrate is a flexible substrate. In some cases, at least 10 10 , 10 11 , 10 12 , 10 13 , 10 14 or 10 15 In some cases, at least 10 x 10 8 , 10x 10 9 , 10x 10 10 , 10x 1011 or 10x 10 12 polynucleotides. In some cases, each synthesized polynucleotide comprises at least 20, 50, 100, 200, 300, 400, or 500 nucleobases. In some cases, the overall average error rate in synthesizing these bases is less than about 1 / 100 base; 1 / 200 base; 1 / 300 base; 1 / 400 base; 1 / 500 base; 1 / 1000 base; 1 / 2000 base; 1 / 5000 base; 1 / 10000 base; 1 / 15000 base; 1 / 20000 base. In some cases, these error rates are at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.5% or more of the synthesized polynucleotides. In some cases, these at least 90%, 95%, 98%, 99%, 99.5% or more of the synthesized polynucleotides are indistinguishable from the predetermined sequence encoded therein. In some cases, the error rate of the polynucleotides synthesized on the substrate using the methods and systems described herein is less than about 1 / 200. In some cases, the error rate of the polynucleotides synthesized on the substrate using the methods and systems described herein is less than about 1 / 1,000. In some cases, the error rate of the polynucleotides synthesized on the substrate using the methods and systems described herein is less than about 1 / 2,000. In some cases, the error rate of the polynucleotides synthesized on the substrate using the methods and systems described herein is less than about 1 / 3,000. In some cases, the error rate of the polynucleotides synthesized on the substrate using the methods and systems described herein is less than about 1 / 5,000. Various types of error rates include mismatches, deletions, insertions and / or substitutions of the polynucleotides synthesized on the substrate. The term "error rate" refers to the comparison of the total amount of the synthesized polynucleotides with the sum of the predetermined polynucleotide sequences. In some cases, the synthesized polynucleotides disclosed herein include tethers of 12 to 25 bases. In some cases, the tether comprises 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more bases.

[0123] Methods, systems, devices and compositions are described herein, wherein the chemical reactions used in polynucleotide synthesis are controlled using electrochemistry. In some cases, electrochemical reactions are controlled by any energy source such as light, heat, radiation or electricity. For example, electrodes are used as all or part of discrete seats on the surface to control chemical reactions. In some cases, the electrodes are charged by applying an electric potential to the electrodes to control one or more chemical steps in polynucleotide synthesis. In some cases, these electrodes are addressable. In some cases, any number of chemical steps described herein are controlled with one or more electrodes. Electrochemical reactions may include oxidation, reduction, acid / base chemistry or other reactions controlled by electrodes. In some cases, electrodes generate electrons or protons used as chemical conversion reagents. In some cases, electrodes directly generate reagents, such as acids. In some cases, acids are protons. In some cases, electrodes directly generate reagents, such as alkalis. Acids or alkalis are generally used to cut protective groups, or, for example, by adjusting the pH of the reaction solution to affect the kinetics of various polynucleotide synthesis reactions. In some cases, electrochemically controlled polynucleotide synthesis reactions include redox-active metals or other redox-active organic materials. In some cases, metal or organic catalysts are used in these electrochemical reactions. In some cases, the acid is generated by oxidation of the quinone.

[0124] The control of chemical reactions is not limited to the electrochemical generation of reagents; the electric field (or gradient) generated by the electrode can indirectly affect the chemical reactivity through the biophysical changes of the substrate or reagent. In some cases, the substrate includes but is not limited to nucleic acids. In some cases, an electric field is generated that repels or attracts a specific reagent or substrate toward or away from the electrode or surface. In some cases, this type of field is generated by applying an electric potential to one or more electrodes. For example, negatively charged nucleic acids are repelled by negatively charged electrode surfaces. In some cases, this type of repulsion or attraction of polynucleotides or other reagents caused by local electric fields causes polynucleotides or other reagents to move into or out of the area of ​​a synthetic device or structure. In some cases, the electrode generates an electric field that repels polynucleotides away from the synthetic surface, structure or device. In some cases, the electrode generates an electric field that attracts polynucleotides to the synthetic surface, structure or device. In some cases, protons are repelled from the positively charged surface to limit the contact of protons with the substrate or its part. In some cases, repulsion or attraction is used to allow or prevent reagents or substrates from entering a specific area of ​​the synthetic surface. In some cases, nucleoside monomers are prevented from contacting with polynucleotide chains by applying an electric field near one or two components. Such an arrangement allows the gating of specific reagents, which can eliminate the need for blocking groups when controlling the concentration or rate of contact between reagents and / or substrates. In some cases, unprotected nucleoside monomers are used for polynucleotide synthesis. Alternatively, applying a field near one or two components promotes the contact of nucleoside monomers with polynucleotide chains. In addition, applying an electric field to a substrate can change substrate reactivity or conformation. In exemplary applications, the electric field generated by an electrode is used to prevent the polynucleotides at adjacent seats from interacting. In some cases, the substrate is a polynucleotide, optionally attached to a surface. In some cases, applying an electric field can change the three-dimensional structure of a polynucleotide. Such changes include the folding or unfolding of various structures such as spirals, hairpins, loops or other three-dimensional nucleic acid structures. Such changes are useful for manipulating nucleic acids inside holes, channels or other structures. In some cases, an electric field is applied to a nucleic acid substrate to prevent secondary structure. In some cases, an electric field eliminates the need for a connector or attachment to a solid support during polynucleotide synthesis.

[0125] The disclosed suitable method for synthesizing polynucleotide on substrate is phosphoramidite method, and it is included in the coupling step of forming phosphite triester bond between phosphoramidite structural unit and the nucleoside that is attached to substrate, and phosphoramidite structural unit (i.e. nucleoside phosphoramidite) is controlled to be added in the polynucleotide chain of growth.In some cases, nucleoside phosphoramidite is provided to the substrate of activation.In some cases, nucleoside phosphoramidite is provided to the substrate with activator.In some cases, nucleoside phosphoramidite is provided to substrate with 1.5,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,25,30,35,40,50,60,70,80,90,100 times or more times of excessive relative to the nucleoside that is attached to substrate.In some cases, the interpolation of nucleoside phosphoramidite is carried out in anhydrous environment (for example, in anhydrous acetonitrile). After adding and connecting nucleoside phosphoramidites in the coupling step, the substrate is optionally washed. In some cases, the coupling step is repeated once or more, and a washing step is optionally performed between adding nucleoside phosphoramidites to the substrate. In some cases, the polynucleotide synthesis method used herein includes 1, 2, 3 or more continuous coupling steps. In many cases, before coupling, the nucleoside bound to the substrate is deprotected by removing a protecting group, wherein the protecting group plays a role in preventing polymerization. The protecting group may include any chemical group that prevents the polynucleotide chain from extending. In some cases, the protecting group is cut (or removed) in the presence of an acid. In some cases, the protecting group is cut in the presence of a base. In some cases, the protecting group is removed with electromagnetic radiation such as light, heat or other energy sources. In some cases, the protecting group is removed by oxidation or reduction reaction. In some cases, the protecting group includes a triaryl methyl group. In some cases, the protecting group includes an aryl ether. In some cases, the protecting group includes a disulfide. In some cases, the protecting group includes an acid-labile silane. In some cases, the protecting group includes an acetal. In some cases, the protecting group includes ketal. In some cases, the protecting group includes enol ether. In some cases, the protecting group includes methoxybenzyl. In some cases, the protecting group includes azide. In some cases, the protecting group is 4,4'-dimethoxytrityl (DMT). In some cases, the protecting group is tert-butyl carbonate. In some cases, the protecting group is tert-butyl ester. In some cases, the protecting group includes a base-labile group.

[0126] After coupling, the phosphoramidite polynucleotide synthesis method optionally includes a capping step. In the capping step, the growing polynucleotide is treated with a capping agent. The capping step is generally used to block unreacted substrate-bound 5'-OH groups after coupling to prevent further chain extension, thereby preventing the formation of polynucleotides with internal base deletions. In addition, phosphoramidites activated with 1H-tetrazole generally react with the O6 position of guanosine to a small extent. Without being bound by theory, in the case of 1H-tetrazole, the phosphoramidite is activated with 1H-tetrazole. 2 / water oxidation, the byproduct (possibly via O6-N7 migration) undergoes depurination. Apurinic sites can terminate in the final deprotection process of polynucleotides and be cut, thereby reducing the yield of full-length products. The O6 modification can be removed by treating with a capping agent before oxidation with I2 / water. In some cases, including a capping step in the polynucleotide synthesis process reduces the error rate compared to synthesis without capping. As an example, the capping step includes treating the polynucleotide bound to the substrate with a mixture of acetic anhydride and 1-methylimidazole. After the capping step, the substrate is optionally washed.

[0127] After adding nucleoside phosphoramidites, and optionally after capping and one or more washing steps, the substrate described herein comprises the growing nucleic acid of the combination that can be oxidized. The oxidation step includes oxidation of phosphite triester to tetracoordinate phosphotriester-a protected precursor of naturally occurring phosphodiester internucleoside connection. In some cases, phosphite triester is electrochemically oxidized. In some cases, the oxidation of the growing polynucleotide is realized by optionally treating with iodine and water in the presence of weak bases such as pyridine, lutidine or collidine. Oxidation is sometimes carried out under anhydrous conditions using tert-butyl hydroperoxide or (1S)-(+)-(10-camphorsulfonyl)-oxaziridine (CSO). In some methods, capping step is carried out after oxidation. The second capping step allows the substrate to dry, because the residual water from oxidation that may persist can inhibit subsequent coupling. After oxidation, the substrate and the growing polynucleotide are optionally washed. In some cases, the oxidation step is replaced with a sulfurization step to obtain a polynucleotide phosphorothioate, wherein any capping step can be performed after sulfurization. Many reagents are capable of efficient sulfur transfer, including but not limited to 3-(dimethylaminomethylene)amino)-3H-1,2,4-dithiazole-3-thione, DDTT, 3H-1,2-benzodithiolan-3-one 1,1-dioxide (also known as Beaucage reagent) and N,N,N'N'-tetraethylthiuram disulfide (TETD).

[0128] In order to make subsequent nucleoside incorporation cycle occur by coupling, the protected 5' end (or 3' end, if synthesis is carried out in 5' to 3' direction) of the polynucleotide of growth combined with substrate is removed so that the primary hydroxyl can react with the next nucleoside phosphoramidite. In some cases, the blocking group is DMT, and the trichloroacetic acid in dichloromethane is used to deblock. In some cases, the blocking group is DMT, and the proton generated by electrochemistry is deblocked. Detritylation of extended time or detritylation using an acid solution stronger than the recommended acid solution can cause the depurination of the polynucleotide combined with solid support to increase, and therefore reduce the productive rate of required full-length product. The methods and compositions described herein provide controlled deblocking conditions, thereby limiting undesirable depurination reactions. In some cases, the polynucleotide combined with substrate is washed after deblocking. In some cases, the effective washing after deblocking helps to synthesize polynucleotides with low error rate.

[0129] The method of synthesizing polynucleotides on a substrate as described herein may include a series of repeated steps of applying a protected monomer to a surface of a substrate feature to connect to the surface, a linker, or to a previously deprotected monomer; deprotecting the applied monomer so that it can react with a subsequently applied protected monomer; and applying another protected monomer for connection. One or more intermediate steps include oxidation and / or sulfurization. In some cases, one or more washing steps are present before or after one or all of the steps.

[0130] The method of synthesizing polynucleotides on a substrate as described herein may include an oxidation step. For example, the method includes a series of repeated steps: applying a protected monomer to a surface of a substrate feature to connect to the surface, a linker, or to a previously deprotected monomer; deprotecting the applied monomer so that it can react with a subsequently applied protected monomer; applying another protected monomer for connection, and oxidation and / or sulfurization. In some cases, one or all of the steps are preceded or followed by one or more washing steps.

[0131] The method of synthesizing polynucleotides on a substrate as described herein may further comprise a series of repeated steps of: applying a protected monomer to a surface of a substrate feature to attach to the surface, a linker, or to a previously deprotected monomer; deprotecting the applied monomer so that it can react with a subsequently applied protected monomer; and oxidizing and / or sulfurizing. In some cases, one or more washing steps are present before or after one or all of the steps.

[0132] The method of synthesizing polynucleotides on a substrate as described herein may further comprise a series of repeated steps of: applying a protected monomer to the surface of a substrate feature for attachment to the surface, a linker, or a pre-deprotected monomer; and oxidizing and / or sulfurizing. In some cases, there is one or more washing steps before or after one or all of the steps.

[0133] The method of synthesizing polynucleotides on a substrate as described herein may further comprise a series of repeated steps of: applying a protected monomer to the surface of a substrate feature for attachment to the surface, a linker, or a pre-deprotected monomer; deprotecting the applied monomer to render it reactive with a subsequently applied protected monomer; and oxidizing and / or sulfurizing. In some cases, there is one or more washing steps before or after one or all of the steps.

[0134] In some cases, polynucleotides having photo-labile protecting groups are synthesized, where the hydroxyl groups generated on the surface are blocked by photo-labile protecting groups. When the surface is exposed to ultraviolet light, for example, through a photolithographic mask, a pattern of free hydroxyl groups can be generated on the surface. These hydroxyl groups can react with photo-protected nucleoside phosphoramidites according to phosphoramidite chemistry. A second photolithographic mask can be employed and the surface can be exposed to ultraviolet light to generate a second pattern of hydroxyl groups, which are then coupled to 5'-photo-protected nucleoside phosphoramidites. Similarly, patterns can be generated and the oligomer chain can be extended. Without being bound by theory, the instability of the photo-cleavable group depends on the wavelength and the polarity of the solvent employed, and the rate of photo-cleavage can be affected by the duration of exposure and the intensity of the light. The method can utilize a number of factors, such as the accuracy of mask alignment, the efficiency of photo-protecting group removal, and the yield of the phosphoramidite coupling step. In addition, unwanted light leakage to neighboring sites can be minimized. The density of oligomers synthesized per spot can be monitored by adjusting the loading of the lead nucleoside on the synthesis surface.

[0135] The surface of the substrate as described herein that supports polynucleotide synthesis can be chemically modified to allow cleavage of the synthesized polynucleotide chain from the surface. In some cases, the polynucleotide chain is cleaved while the polynucleotide is deprotected. In some cases, the polynucleotide chain is cleaved after the polynucleotide is deprotected. In an exemplary protocol, a trialkoxysilylamine such as (CH 3 CH 2 O) 3 Si-(CH 2 ) 2 -NH 2Reacts with the surface SiOH groups of the substrate and then reacts with succinic anhydride and amines to produce amide bonds and free OH that support nucleic acid chain growth. Cleavage includes gas cleavage with ammonia or methylamine. In some cases, cleavage includes linker cleavage with electrically generated reagents such as acids or bases. In some cases, once released from the surface, the polynucleotides are assembled into larger nucleic acids, which are sequenced and decoded to extract the stored information.

[0136] Surfaces as described herein can be reused after polynucleotide cutting to support additional cycles of polynucleotide synthesis. For example, connectors can be reused without additional treatment / chemical modification. In some cases, connectors are non-covalently bound to substrate surfaces or polynucleotides. In some embodiments, after cutting from the surface, connectors remain attached to polynucleotides. In some embodiments, connectors comprise reversible covalent bonds, such as esters, amides, ketals, β-substituted ketones, heterocycles or other groups that can be reversibly cut. In some cases, by adding or removing reagents, or by controlling such reversible cutting reactions by an electrochemical process controlled by an electrode. Optionally, after multiple cycles, chemical connectors or surface-bound chemical groups are regenerated to restore reactivity and remove undesirable byproduct formation on such connectors or surface-bound chemical groups.

[0137] assembly

[0138] Polynucleotides can be designed to jointly span large regions of a predetermined sequence encoding information. In some cases, synthetic polynucleotides are ligated by a ligation reaction to generate larger polynucleotides. An example of a ligation reaction is polymerase chain assembly (PCA). In some cases, at least a portion of the polynucleotide is designed to include an additional region that serves as a substrate for common primer binding. For the PCA reaction, the pre-synthesized polynucleotides include overlaps with each other (e.g., 4, 20, 40 or more bases with overlapping sequences). During the polymerase cycle, the polynucleotides anneal to complementary fragments and are then filled in by the polymerase. Thus, depending on which polynucleotides find each other, the length of the individual fragments is randomly increased in each cycle. Complementarity between the fragments allows the formation of a complete large-span double-stranded DNA. In some cases, after the PCA reaction is completed, a mismatch repair detection enzyme is used for an error correction step to remove mismatches in the sequence. Once the larger target sequence fragment is generated, the fragment can be amplified. For example, in some cases, a target sequence containing 5' and 3' end adapter sequences is amplified in a polymerase chain reaction (PCR) that includes modified primers that hybridize to the adapter sequences. In some cases, the modified primers contain one or more uracil bases. Using the modified primers allows the removal of the primers by enzymatic reactions that focus on the targeted modified bases and / or the nicks left by enzymes that cleave the modified base pairs from the fragments. What remains is a double-stranded amplification product lacking the adapter sequence remnants. In this way, multiple amplification products can be generated in parallel with the same set of primers to generate different double-stranded DNA fragments.

[0139] Error correction can be performed to the polynucleotide synthesized and / or the product of assembly. The exemplary strategy for error correction relates to site-directed mutagenesis to correct errors by overlap extension PCR, which is optionally combined with two or more rounds of cloning and sequencing. In some cases, double-stranded nucleic acids with mispairing, protrusion and small ring, chemically altered bases and / or other heteroduplexes are selectively removed from the correctly synthesized nucleic acid population. In some cases, error correction is performed using proteins / enzymes that recognize and combine or are next to mispairing or unpaired bases in double-stranded nucleic acids, to produce single-strand or double-strand breaks or to initiate chain transfer transposition events. Non-limiting examples of proteins / enzymes for error correction include endonucleases (T7 endonuclease I, Escherichia coli endonuclease V, T4 endonuclease VII, mung bean nuclease, cell Escherichia coli endonuclease IV, UVDE), restriction enzymes, glycosylases, ribonucleases, mispairing repair enzymes, resolvase, helicase, ligase, mispairing specific antibodies and variants thereof. The example of specific error correction enzyme includes T4 endonuclease 7, T7 endonuclease 1, S1, mung bean endonuclease, MutY, MutS, MutH, MutL, cleavage enzyme (cleavase), CELI and HINF1. In some cases, DNA mismatch binding protein MutS (Thermus aquaticus (Thermus aquaticus)) is used to remove the failed product from the synthetic product population. In some cases, error correction is carried out using a correction enzyme (Correctase). In some cases, error correction is carried out using SURVEYOR endonuclease (Transgenomic)-a kind of mismatch specific DNA endonuclease that scans known and unknown mutations and polymorphisms in heteroduplex DNA-is used.

[0140] Sequencing

[0141] After extracting and / or amplifying polynucleotide from the structural surface, suitable sequencing techniques can be adopted to order-check the polynucleotide. In some cases, the DNA sequence is read on substrate or in the feature of structure. In some cases, the polynucleotide stored on substrate is extracted and optionally assembled into longer nucleic acid, which is then ordered.

[0142] The polynucleotides synthesized and stored on the structures described herein encode data that can be interpreted by reading the sequence of the synthesized polynucleotide and converting the sequence into a computer-readable binary code. In some cases, the sequence needs to be assembled, and the assembly step may need to be at the nucleic acid sequence stage or the digital sequence stage.

[0143] Provided herein is a detection system, which includes a device that can directly sequence the stored polynucleotides on the structure and / or after being removed from the main structure. In the case where the structure is a roll of flexible material, the detection system includes a device for maintaining the structure and advancing the structure through a detection position, and is placed near the detection position for detecting the detector of the signal derived from the part when a part of the band is in the detection position. In some cases, the signal indicates the presence of polynucleotides. In some cases, the sequence (for example, fluorescent signal) of the signal indicates polynucleotides. In some cases, when the band is continuously transmitted through the detector operably connected to a computer, the computer reads the information encoded in the polynucleotides on the continuous band. In some cases, the detection system includes a computer system, and the computer system includes a polynucleotide sequencing device, a database for storing and retrieving data related to a polynucleotide sequence, a software for converting the DNA code of a polynucleotide sequence into a binary code, a computer for reading the binary code or its any combination.

[0144] This article provides a sequencing system that can be integrated into the device described herein. Various sequencing methods are well known in the art and include "base determination", wherein the identity of the base in the target polynucleotide is identified. In some cases, the polynucleotides synthesized using the methods, devices, compositions and systems described herein are sequenced after cutting from the synthesis surface. In some cases, sequencing is performed during polynucleotide synthesis or simultaneously with polynucleotide synthesis, wherein base determination is performed immediately after the nucleoside monomer is extended into the growing polynucleotide chain or before it. The base determination method includes measuring the current generated by the addition of the base catalyzed by the polymerase to the template chain. In some cases, the synthesis surface includes an enzyme, such as a polymerase. In some cases, such enzymes are tethered to an electrode or a synthesis surface.

[0145] Computer Systems

[0146] In various aspects, any system described herein is operably connected to a computer and optionally automated locally or remotely by a computer. In various cases, the methods and systems of the present invention further include a software program on a computer system and its use. Therefore, computerized control of synchronization of the distribution / vacuuming / refilling functions (such as choreographing and synchronizing material deposition device movement, distribution action and vacuum actuation) is within the scope of the present invention. In some cases, the computer system is programmed to engage between the base sequence specified by the user and the position of the material deposition device to deliver the correct reagent to the specified area of ​​the substrate.

[0147] Figure 8The computer system 800 shown in the figure can be understood as a logical device capable of reading instructions from a medium 811 and / or a network port 805, which can be optionally connected to a server 809 having a fixed medium 812. The system may include a CPU 801, a disk drive 803, an optional input device such as a keyboard 815 and / or a mouse 816, and an optional monitor 807. Data communication with a server at a local or remote location can be achieved through the communication medium shown. The communication medium may include any means of transmitting and / or receiving data. For example, the communication medium may be a network connection, a wireless connection, or an Internet connection. Such a connection may provide communication via the World Wide Web. It is contemplated that data related to the present disclosure may be transmitted via such a network or connection, thereby being received and / or viewed by a party 822.

[0148] Fig. 9 5 is a block diagram illustrating a first example architecture of a computer system that can be used in conjunction with an example embodiment of the present invention. As shown in Figure 5, the example computer system may include a processor 902 for processing instructions. Non-limiting examples of processors include: Intel Xeon™ processor, AMD Opteron™ processor, Samsung 32-bit RISC ARM 1176JZ (F) -S v1.0™ processor, ARM Cortex-A8 Samsung S5PC100™ processor, ARM Cortex-A8 AppleA4™ processor, Marvell PXA 930™ processor or functionally equivalent processor. Multiple execution threads can be used for parallel processing. In some cases, multiple processors or processors with multiple cores can also be used, whether in a single computer system, in a cluster, or distributed across a network system comprising multiple computers, cellular phones and / or personal data assistant devices.

[0149] like Fig. 9 As shown, cache memory 904 may be connected to or incorporated into processor 902 to provide high-speed storage of instructions or data that are recently or frequently used by processor 902. Processor 902 is connected to north bridge 906 via processor bus 908. North bridge 906 is connected to random access memory (RAM) 910 via memory bus 912 and manages access of processor 902 to RAM 910. North bridge 906 is also connected to south bridge 914 via chipset bus 916. South bridge 914 is in turn connected to peripheral bus 918. Peripheral bus may be, for example, PCI, PCI-X, PCI Express or other peripheral bus. North bridge and south bridge are generally referred to as processor chipsets and manage data transfer between processor, RAM and peripheral components on peripheral bus 918. In some alternative architectures, the functionality of north bridge may be incorporated into the processor instead of using a separate north bridge chip.

[0150] In some cases, the system 900 may include an accelerator card 922 attached to the peripheral bus 918. The accelerator may include a field programmable gate array (FPGA) or other hardware for accelerating a process. For example, the accelerator may be used for adaptive data reconstruction or for evaluating algebraic expressions used in extended set processing.

[0151] Software and data are stored in external memory 924 and may be loaded into RAM 910 and / or cache 904 for use by the processor. System 900 includes an operating system for managing system resources; non-limiting examples of operating systems include: Linux, Windows™, MACOS™, BlackBerry OS™, iOS™, and other functionally equivalent operating systems, and application software running on top of the operating system for managing data storage and optimization according to an exemplary embodiment of the present invention.

[0152] In this example, system 900 also includes network interface cards (NICs) 920 and 921 connected to the peripheral bus to provide a network interface to external storage such as network attached storage (NAS) and other computer systems that can be used for distributed parallel processing.

[0153] Fig.10 1 is a diagram showing a network 1000 having multiple computer systems 1002a and 1002b, multiple cell phones and personal data assistants 1002c, and network attached storage (NAS) 1004a and 1004b. In an exemplary embodiment, the systems 1002a, 1002b, and 1002c can manage data storage and optimize data access to data stored in the network attached storage (NAS) 1004a and 1004b. Mathematical models can be used for the data and evaluated using distributed parallel processing across the computer systems 1002a and 1002b and the cell phones and personal data assistant system 1002c. The computer systems 1002a and 1002b and the cell phones and personal data assistant system 1002c can also provide parallel processing for adaptive data reconstruction of data stored in the network attached storage (NAS) 1004a and 1004b. Fig.10 Only one example is shown, and a wide variety of other computer architectures and systems can be used with various embodiments of the present invention. For example, blade servers can be used to provide parallel processing. Processor blades can be connected via a backplane to provide parallel processing. Storage can also be connected to the backplane via a separate network interface or as a network attached storage (NAS).

[0154] In some example embodiments, a processor may maintain a separate memory space and transmit data through a network interface, backplane or other connector for parallel processing by other processors. In other cases, some or all processors may use a shared virtual address memory space.

[0155] Fig.11 1 is a block diagram of a multi-processor computer system 1100 using a shared virtual address storage space according to an exemplary embodiment. The system includes multiple processors 1102a-f that can access a shared memory subsystem 1104. In the system, multiple programmable hardware storage algorithm processors (MAPs) 1106a-f are incorporated into the memory subsystem 1104. Each MAP 1106a-f can include a memory 1108a-f and one or more field programmable gate arrays (FPGAs) 1110a-f. The MAP provides a configurable functional unit and can provide a specific algorithm or part of an algorithm to the FPGA 1110a-f for processing in close coordination with the corresponding processor. For example, in an exemplary embodiment, the MAP can be used to evaluate algebraic expressions related to a data model and to perform adaptive data reconstruction. In this example, each MAP can be globally accessed by all processors for these purposes. In one configuration, each MAP can use direct memory access (DMA) to access the associated memory 1108a-f, allowing it to perform tasks independently and asynchronously from the respective microprocessor 1102a-f. In this configuration, a MAP can feed results directly to another MAP for pipeline processing and parallel execution of algorithms.

[0156] The above computer architectures and systems are examples only, and a wide variety of other computer, cell phone, and personal data assistant architectures and systems may be used in conjunction with the example embodiments, including systems using any combination of general purpose processors, coprocessors, FPGAs and other programmable logic devices, systems on chips (SOCs), application specific integrated circuits (ASICs), and other processing and logic elements. In some embodiments, all or part of the computer system may be implemented in software or hardware. Any kind of data storage media may be used in conjunction with the example embodiments, including random access memory, hard drives, flash memory, tape drives, disk arrays, network attached storage (NAS), and other local or distributed data storage devices and systems.

[0157] In an example embodiment, the computer system can be implemented using software modules executed on any of the above or other computer architectures and systems. In other embodiments, the functions of the system can be partially or completely implemented in firmware, programmable logic devices such as field programmable gate arrays (FPGAs), systems on chips (SOCs), application specific integrated circuits (ASICs), or other processing and logic elements. For example, the set processor and optimizer can be implemented in a hardware accelerated manner by using a hardware accelerator card.

[0158] Implementation

[0159] Provided herein is a method for storing information, comprising: a) providing a structure comprising a surface; b) depositing at least one nucleotide on the surface, wherein the at least one nucleotide is coupled to a polynucleotide attached to the surface; and c) repeating step b) to synthesize a plurality of polynucleotides on the surface, wherein the storage density of unique polynucleotides on the surface is at least 100×10 6 Polynucleotides / cm 2 . Further provided herein are such methods, wherein the method further comprises cutting at least one polynucleotide from the surface, wherein the polynucleotide is dissolved in the droplets. Further provided herein are such methods, wherein the method further comprises sequencing at least one polynucleotide from the surface. Further provided herein are such methods, wherein the method further comprises drying the surface. Further provided herein are such methods, wherein the method further comprises washing the nucleotides from the surface. Further provided herein are such methods, wherein the surface is a solid support. Further provided herein are such methods, wherein the surface comprises glass, fused quartz, silicon, silicon dioxide, silicon nitride, plastic, metal, or a combination thereof.

[0160] Provided herein is a method for storing information, comprising: a) providing a structure comprising a surface; b) depositing droplets comprising at least one nucleotide on the surface, wherein the at least one nucleotide is coupled to a polynucleotide attached to the surface; and c) repeating step b) to synthesize a plurality of polynucleotides on the surface, wherein the volume of the droplets is less than about 100 femtoliters.

[0161] Provided herein is a method for storing information, comprising: a) providing a structure comprising a surface; b) depositing at least one nucleotide on the surface, wherein the at least one nucleotide is coupled to a polynucleotide attached to the surface; and c) repeating step b) to synthesize a plurality of polynucleotides on the surface, wherein the time for repeating step b) using four different nucleotides is less than about 100 milliseconds. Further provided herein is such a method, wherein the method further comprises one or more washing steps. Further provided herein is such a method, wherein the method further comprises unblocking, oxidation, washing, capping, or any combination thereof.

[0162] Provided herein is a device for storing information, comprising: a chip comprising an array of addressable loci, wherein one or more of the addressable loci comprises at least one electrode, a synthetic surface, and at least one fluid port, wherein the synthetic surface at each addressable locus comprises at least one polynucleotide extending from the surface, wherein the density of the addressable loci on the chip is at least 100×10 6 Polynucleotides / cm 2 . Further provided herein is such a device, wherein the at least one polynucleotide is about 150 to about 500 bases in length. Further provided herein is such a device, wherein the at least one polynucleotide is about 200 bases in length. Further provided herein is such a device, wherein the device further comprises a reagent reservoir. Further provided herein is such a device, wherein the device further comprises a heating or cooling unit. Further provided herein is such a device, wherein at least one addressable locus comprises a droplet. Further provided herein is such a device, wherein the droplet has a diameter of less than 50 microns.

[0163] Provided herein is a method for storing information, the method comprising: a) converting at least one item of information in the form of at least one digital sequence into at least one nucleic acid sequence; b) synthesizing a plurality of polynucleotides having a predetermined sequence, the predetermined sequences collectively encoding the at least one nucleic acid sequence; c) depositing droplets comprising at least one nucleotide on a surface, wherein the at least one nucleotide is coupled to a polynucleotide attached to the surface; d) repeating step c) to synthesize the plurality of polynucleotides on the surface; and e) storing the plurality of polynucleotides, wherein the volume of the droplets is less than about 100 femtoliters.

[0164] The present invention provides a method for storing information, which comprises: a) converting at least one item of information in the form of at least one digital sequence into at least one nucleic acid sequence; b) synthesizing a plurality of polynucleotides having a predetermined sequence, wherein the predetermined sequence collectively encodes the at least one nucleic acid sequence; c) depositing droplets comprising at least one nucleotide on a surface, wherein the at least one nucleotide is coupled to a polynucleotide attached to the surface; d) repeating step c) to synthesize the plurality of polynucleotides on the surface; and e) storing the plurality of polynucleotides, wherein the time for repeating step c) using four different nucleotides is less than about 100 milliseconds.

[0165] Provided herein is a method for synthesizing polynucleotides, comprising: a) providing a structure comprising a surface, wherein the surface comprises a plurality of loci for nucleotide extension; and b) synthesizing a plurality of polynucleotides extending from the surface, wherein the synthesis comprises depositing one or more reagents by applying a potential to the surface. Further provided is such a method, wherein the potential is an electric potential. Further provided is such a method, wherein the surface is a solid support.

[0166] The present invention provides an apparatus for storing information using any of the methods described herein. The present invention provides a system for storing information using any of the methods described herein.

[0167] The following examples are set forth to more clearly illustrate the principles and practices of the embodiments disclosed herein to those skilled in the art, and should not be construed as limiting the scope of any claimed embodiments. Unless otherwise specified, all parts and percentages are by weight.

[0168] Example

[0169] Example 1: Functionalization of device surfaces

[0170] The device was functionalized to support the attachment and synthesis of polynucleotide libraries. 2 SO 4 and 10% H 2 O 2 The device surface was wetted and cleaned with piranha solution for 20 minutes. The device was rinsed in several beakers containing deionized water, kept under a deionized water gooseneck stopcock for 5 minutes, and washed with N 2 The device was then dried in NH 4The device was then plasma cleaned by exposing the device surface to O2. O was performed using a SAMCO PC-300 instrument in downstream mode at 250 watts. 2 Plasma etching for 1 min.

[0171] The cleaned device surfaces were functionalized with a solution containing N-(3-triethoxysilylpropyl)-4-hydroxybutyramide using a YES-1224P vapor deposition oven system with the following parameters: 0.5 to 1 Torr, 60 min, 70°C, 135°C vaporizer. The device surfaces were resist coated using a Brewer Science 200X spin coater. The SPR TM 3612 photoresist was spin coated on the device at 2500rpm for 40sec. The device was pre-baked at 90°C for 30min on a Brewer hot plate. The device was photolithographically processed using a Karl Suss MA6 mask aligner. The device was exposed for 2.2sec and developed in MSF 26A for 1min. The remaining developer was rinsed with a handheld spray gun and the device was soaked in water for 5min. The device was baked in an oven at 100°C for 30min and then visually inspected for photolithographic defects using a Nikon L200. A clean process was performed using a SAMCO PC-300 instrument at 250W. 2 Plasma etching was performed for 1 min to remove the residual resist.

[0172] The device surface was passivated and functionalized with 100 μL perfluorooctyl trichlorosilane solution mixed with 10 μL light mineral oil. The device was placed in a chamber, pumped for 10 min, then the valve leading to the pump was closed and left to stand for 10 min. The chamber was vented to air. The device was stripped of the resist by soaking twice in 500 mL NMP at 70 ° C for 5 min and ultrasonically treated at maximum power (9 on the Crest system). The device was then soaked in 500 mL isopropanol for 5 min at room temperature and ultrasonically treated at maximum power. The device was immersed in 300 mL of 200 proof ethanol and N 2 Blow dry. Activate the functionalized surface to serve as a support for polynucleotide synthesis.

[0173] Example 2: Highly accurate DNA-based information storage and assembly

[0174] Digital information is selected in the form of binary data, totaling approximately 0.2GB, including the content of the "Universal Declaration of Human Rights" in more than 100 languages, the first 100 books of Project Guttenberg, and seed databases. The digital information is encrypted into a nucleic acid-based sequence and divided into strings. More than 10 million different polynucleotides are synthesized on a rigid silicon surface, each corresponding to a string. The length of each different polynucleotide is equal to or less than 200 bases. The synthesized polynucleotides are collected and sequenced, and decoded back into digital codes, which have 100% accuracy for the source digital information compared to the initial at least one digital sequence.

[0175] Example 3: High-density information storage system

[0176] Polynucleotides are synthesized de novo by the methods described herein. After synthesis, the polynucleotides are collected into individual droplets and transferred to a silicon solid support for storage. The solid support has dimensions of 86 mm x 54 mm and a thickness of 1–2 mm. The solid support has a capacity of 1-10 petabytes (PB), which is implemented in the form of an addressable array of 10-gigabyte packets. The physical partitioning is that the packets are redundantly encoded as leader sequences, and each polynucleotide within the packet shares a common initial sequence and any other sequence information for indexing or searching. The packets are implemented in the form of aqueous droplets with dissolved polynucleotides, each polynucleotide having a physical redundancy of 100-1000 copies. The polynucleotide length is in the range of 100-1000 bases. The droplet volume is equivalent to a sphere with a diameter of 40-50 μm. The solid support further contains up to 10,000 x 10,000 locations in an area less than one square inch.

[0177] Exemplary solid supports can be seen Figures 12A-12B . Fig. 12A The front side of the solid support is shown, which is made of glass and contains transparent windows for the array and fluid ports. Fig. 12B Shown is the back side of the solid support which is the circuit containing the electrical contacts (LGA 1 mm pitch) and thermal interface underneath the solid support area.

[0178] After the droplets are added to the solid support, the solid support can be dried and later resolvated for downstream applications. Alternatively, the solid support can be dried and stored. Because the droplets within each data packet contain sequence information for indexing and searching, a specific data packet is retrieved from multiple data packets based on the sequence information.

[0179] Embodiment 4: High-density information storage system for access

[0180] Polynucleotides are synthesized de novo by the methods described herein. After synthesis, the polynucleotides are collected into individual small droplets and transferred to a paper solid support for storage. The dimensions of the solid support are 3.5 inches by 2.5 inches. The capacity of the solid support is 1 petabyte, which is implemented in the form of an addressable 32 x 32 array containing 1024 spots. Each spot contains a 1 terabyte pool. See, for example, Fig.17 and Fig.18 .

[0181] 10-100 megabytes of at least one item of information is encoded in the DNA and stored in 1 petabyte of data. At a later time, the 10-100 megabytes of encoded DNA are retrieved by randomly accessing the encoded DNA and retrieving the encoded DNA from the 1 terabyte pool.

[0182] Example 5: Local Control of Polynucleotide Synthesis on a Solid Support

[0183] Polynucleotides of 240 bases in length were synthesized on a solid support using the methods described herein. The length of a polynucleotide comprising dsDNA was approximately 80 nm, while the length of a polynucleotide comprising ssDNA was approximately 160 nm. The solid support contained an array of 500 nm (depth) x 400 nm (diameter) wells (volume of approximately 0.628 femtoliters). Each well contained an addressable locus, an addressable electrode (area of ​​50,000 nm) in the sidewall of each well, and a 2 / electrode) and a 250nm(diameter) surface for polynucleotide synthesis / attachment in addressable communication with a 250nm(diameter) addressable bottom electrode. The electrodes are independently addressable.

[0184] Polynucleotides are 1 polynucleotide / 50nm 2 The density of H exists on the synthetic surface. The pore spacing is 1.0 μm. The 10 nm thick sidewall electrode (located about 100 nm above the polynucleotide surface) is charged to generate H + A gradient of ions is applied to remove protecting groups from the 5'OH groups of polynucleotides defined at positions on the synthesis surface (where the polynucleotides are blocked with acid-cleavable blocking groups). + ions. (See Fig. 16B ). Nucleoside phosphoramidite monomers are added, and the polynucleotide is at an unblocked site and is available for coupling with a nucleoside. The cycle of deprotection and coupling is repeated to synthesize the polynucleotide. By applying a series of electrode-controlled masks to the surface before adding each type of monomer, the desired polynucleotide is synthesized at the exact location on the surface in a controlled order.

[0185] Example 6: Local Control of Polynucleotide Synthesis on a Solid Support Using an Electric Field

[0186] Polynucleotides of 240 bases in length were synthesized on a solid support using the methods described herein. The solid support contained an array of 500 nm (depth) x 500 nm (diameter) wells. Each well contained an addressable locus, an addressable electrode (50,000 nm in area) in the sidewall of each well, and a 2 / electrode) and a 250nm (diameter) surface for polynucleotide synthesis / attachment, in addressable communication with a 250nm (diameter) addressable bottom electrode. The electrodes are independently addressable. Polynucleotides are addressed at 1 polynucleotide / 50nm 2 A density of is present on the synthesis surface. The spacing between the holes is 1.0um. Nucleoside phosphoramidite monomers are added, and the polynucleotide is at an unblocked site and is available for coupling with the nucleoside. The cycle of deprotection and coupling is repeated to synthesize the polynucleotide. By applying a series of electrode-controlled masks to the surface before adding each type of monomer, the desired polynucleotide is synthesized at the exact location on the surface in a controlled order. The bottom electrode at the bottom of the hole is activated, inducing the release of the polynucleotide attached to it. The sidewall electrodes are charged to generate an electric field, which moves the polynucleotide out of the hole. The nucleoside phosphoramidite monomer is then added, which extends from a reusable linker attached to the surface, and the synthesis is repeated.

[0187] Example 7: Local Control of Polynucleotide Synthesis on a Solid Support

[0188] Polynucleotides of 240 bases in length were synthesized on a solid support using the methods described herein. The length of a polynucleotide comprising dsDNA was approximately 80 nm, while the length of a polynucleotide comprising ssDNA was approximately 160 nm. The solid support contained an array of addressable electrodes of 100 nm in diameter on the surface for polynucleotide synthesis / attachment (see Fig.19 ).

[0189] Polynucleotides are 1 polynucleotide / 39.27nm 2 The density of H exists on the synthetic surface. The pore spacing is 0.25um. The electrodes are charged to generate H + A gradient of ions is applied to remove protecting groups from the 5'OH groups of polynucleotides defined at positions on the synthesis surface (where the polynucleotides are blocked with acid-cleavable blocking groups). + ions. Nucleoside phosphoramidite monomers are added and the polynucleotide is at an unblocked site and available for coupling with a nucleoside. The cycle of deprotection and coupling is repeated to synthesize the polynucleotide. By applying a series of electrode-controlled masks to the surface before adding each type of monomer, the desired polynucleotide is synthesized at the exact location on the surface in a controlled sequence.

[0190] Although preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that these embodiments are provided by way of example only. Without departing from the present invention, those skilled in the art will now appreciate that many variations, changes and substitutions may be employed in the process of implementing the present invention. It is intended that the scope of the present invention be limited to the appended claims, thereby encompassing methods and structures and their equivalents within the scope of these claims.

Claims

1. A device for storing DNA-based digital information, comprising: A solid support, wherein said solid support comprises a plurality of wells, wherein each of said wells comprises an addressable locus, said addressable locus comprising: a synthetic surface located in a bottom region of each of said wells; a bottom electrode in addressable communication with the synthetic surface; and At least one sidewall electrode is located on a sidewall of each of the holes, wherein the at least one sidewall electrode is 50 nm to 200 nm from the bottom region, and wherein the at least one sidewall electrode and the bottom electrode are independently addressable.

2. The device according to claim 1, wherein the solid support has a thickness of at least 100 x 10 6 Addressable seats / cm 2 The density contains addressable seats.

3. The device according to claim 1, wherein the solid support is 100 x 10 6 Up to 100x 10 7 Addressable seats / cm 2 The density contains addressable seats.

4. The device of any one of claims 1 to 3, wherein the addressable loci have a diameter of at most 750 nm.

5. The device according to any one of claims 1 to 3, wherein each of the holes has a depth of at most 1000 nm.

6. The device according to any one of claims 1 to 3, wherein each of the holes has a depth of 100 nm to 1000 nm.

7. The device according to any one of claims 1 to 3, wherein each of the pores has a longest cross-sectional diameter of 100 nm to 800 nm.

8. The device according to any one of claims 1 to 3, wherein each of the holes is cylindrical.

9. The device according to any one of claims 1 to 3, wherein the bottom electrode has 10 4 nm 2 Up to 10 5 nm 2 The longest cross-sectional area.

10. The device according to any one of claims 1 to 3, wherein the at least one sidewall electrode is 75 nm to 125 nm from the bottom region.

11. The device of any one of claims 1 to 3, wherein the at least one sidewall electrode has a height of 5 nm to 25 nm.

12. The device of any one of claims 1 to 3, comprising at least two sidewall electrodes.

13. A device for storing DNA-based digital information, comprising: A solid support, wherein said solid support comprises a plurality of wells, wherein each of said wells comprises an addressable locus, said addressable locus comprising: a synthetic surface located in a bottom region of each of said wells; a bottom electrode in addressable communication with the synthetic surface; at least one sidewall electrode located on a sidewall of each of said holes, wherein the synthetic surface at each addressable locus comprises at least one polynucleotide extending from said synthetic surface, and wherein the polynucleotides comprising different sequences are arranged in a pattern of at least 100×10 6 Polynucleotides / cm 2 A density exists wherein the at least one sidewall electrode and the bottom electrode are independently addressable.

14. The device according to claim 13, wherein the solid support has a thickness of at least 100 x 10 7 Polynucleotides / cm 2 The density contains polynucleotides of different sequences.

15. The device according to claim 13, wherein the solid support is 100 x 10 6 Up to 100x 10 7 Polynucleotides / cm 2 The density contains addressable seats.

16. A device according to any one of claims 13 to 15, wherein each of the holes has a depth of at most 1000 nm.

17. The device according to any one of claims 13 to 15, wherein each of the holes has a depth of 100 nm to 1000 nm.

18. The device of any one of claims 13 to 15, wherein the addressable loci have a diameter of at most 750 nm.

19. The device of any one of claims 13 to 15, wherein each of the pores has a longest cross-sectional diameter of 100 nm to 800 nm.

20. The device of any one of claims 13 to 15, wherein each of the holes is cylindrical.

21. The device according to any one of claims 13 to 15, wherein the bottom electrode has 10 4 nm 2 Up to 10 5 nm 2 The longest cross-sectional area.

22. The device of any one of claims 13 to 15, wherein the at least one sidewall electrode is 50 nm to 200 nm from the bottom region.

23. The device of any one of claims 13 to 15, wherein the at least one sidewall electrode has a height of 5 nm to 25 nm.

24. A device according to any one of claims 13 to 15, comprising at least two sidewall electrodes.

25. A method for storing DNA-based digital information, wherein include: a) providing a device according to any one of claims 1 to 24; b) providing instructions for polynucleotide synthesis; c) depositing at least one nucleoside on the synthesis surface, wherein the at least one nucleoside is coupled to a polynucleotide attached to the synthesis surface; as well as d) repeating step c) to synthesize a plurality of polynucleotides on the synthesis surface, wherein the instructions comprise at least one sequence encoding the plurality of polynucleotides.

26. The method of claim 25, wherein the method further comprises cleaving at least one polynucleotide from the surface, wherein the polynucleotide is dissolved in the droplets.

27. The method of claim 25, wherein the method further comprises sequencing at least one polynucleotide from the surface.

28. The method of any one of claims 25 to 27, wherein the nucleoside comprises a nucleoside phosphoramidite.

29. The method of any one of claims 25 to 27, wherein the method further comprises drying the surface.

30. The method of any one of claims 25 to 27, wherein the method further comprises a cleaving step, wherein the cleaving step comprises applying an electric potential to the bottom electrode to generate a cleaving agent.

31. The method according to any one of claims 25 to 27, wherein the method further comprises a capping step.

32. The method according to any one of claims 25 to 27, wherein the method further comprises an oxidation step.

33. The method of any one of claims 25 to 27, wherein the method further comprises a deblocking step, wherein the deblocking step comprises applying an electric potential to the at least one sidewall electrode to generate a deblocking reagent.

Images