Dna-based digital information storage

Abstract

The invention relates to DNA-based digital information storage. Provided herein are compositions, devices, systems, and methods for generating and using biomolecule-based information for storage. Further provided are devices comprising addressable electrodes that control 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] This application is a divisional application of an application filed on January 3, 2019, with application number 201980017154.2 and invention name “DNA-based digital information storage”.

[0002] Cross-references

[0003] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 613,728, filed January 4, 2018, U.S. Provisional Patent Application No. 62 / 617,067, filed January 12, 2018, and U.S. Provisional Patent Application No. 62 / 650,231, filed March 29, 2018, all of which are incorporated herein by reference in their entirety. Background Art

[0004] 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

[0005] Provided herein is a device for storing information, comprising: a solid support, wherein the solid support comprises a plurality of wells, wherein each well 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 well 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 Further provided herein is a device wherein the solid support comprises addressable loci at a density of 100 x 10 6 Up to 100x10 7 Addressable seats / cm 2 The density of the addressable loci is contained herein. Further provided herein is such a device, wherein the addressable loci have a diameter of at most about 750 nm. Further provided herein is such a device, wherein each well has a depth of at most about 1000 nm. Further provided herein is such a device, wherein each well has a depth of 100 nm to 1000 nm. Further provided herein is such a device, wherein each well has a longest cross-sectional diameter of 100 nm to 800 nm. Further provided herein is such a device, wherein each well is cylindrical. Further provided herein is such a device, wherein the bottom electrode has a diameter of 10 4 nm 2to 10 5 nm 2

[0014] Further provided herein are devices wherein the at least one sidewall electrode is 50 nm to 200 nm from the bottom region.

[0015] Further provided herein are devices wherein the at least one sidewall electrode has a height of 5 nm to 25 nm.

[0016] Further provided herein are devices comprising at least two sidewall electrodes.

[0017] Further provided herein are devices wherein the at least one sidewall electrode and the bottom electrode are independently addressable.

[0006] Provided herein is a device for storing information, comprising: a solid support, wherein the solid support comprises a plurality of wells, wherein each well 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; and 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 herein such a device, wherein the solid support is present at a density of at least 100x10 7 polynucleotides / cm 2 The present invention further provides a device wherein the solid support comprises polynucleotides of different sequences at a density of 100 x 10 6 Up to 100x 10 7 polynucleotides / cm 2 The density of the addressable loci is contained herein. Further provided herein is such a device wherein each pore has a depth of at most about 1000 nm. Further provided herein is such a device wherein each pore has a depth of 100 nm to 1000 nm. Further provided herein is such a device wherein the addressable loci have a diameter of at most about 750 nm. Further provided herein is such a device wherein each pore has a longest cross-sectional diameter of 100 nm to 800 nm. Further provided herein is such a device wherein each pore is cylindrical. Further provided herein is such a device wherein the bottom electrode has 10 4 nm 2 to 10 5 nm 2

[0014] Further provided herein are devices wherein the at least one sidewall electrode is 50 nm to 200 nm from the bottom region.

[0015] Further provided herein are devices wherein the at least one sidewall electrode has a height of 5 nm to 25 nm.

[0016] Further provided herein are devices comprising at least two sidewall electrodes.

[0017] Further provided herein are devices wherein the at least one sidewall electrode and the base electrode are independently addressable.

[0007] Provided herein are methods for storing information, comprising: a) providing a device 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 are methods further comprising cleaving at least one polynucleotide from the surface, wherein the polynucleotide is dissolved in a small droplet. Further provided herein are methods further comprising sequencing at least one polynucleotide from the surface. Further provided herein are methods wherein the nucleoside comprises a nucleoside phosphoramidite. Further provided herein are methods further comprising a cleavage step, wherein the cleavage step comprises applying an electric potential to the bottom electrode to generate a cleavage reagent. Further provided herein are methods wherein the method further comprises drying the surface. Further provided herein are methods wherein the method further comprises washing the nucleoside from the surface. Further provided herein are methods wherein the method further comprises a capping step. Further provided herein are methods wherein the method further comprises an oxidation step. Further provided herein are methods 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.

[0008] 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 present at a density 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 The present invention further provides such 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 are methods, wherein the method further comprises cleaving at least one polynucleotide from the surface, wherein the polynucleotide is dissolved in the droplets. Further provided herein are methods, wherein the method further comprises sequencing at least one polynucleotide from the surface. Further provided herein are methods, wherein the nucleoside comprises a nucleoside phosphoramidite. Further provided herein are methods, wherein the method further comprises drying the surface. Further provided herein are methods, wherein the method further comprises washing the nucleoside from the surface. Further provided herein are methods, wherein the method further comprises a capping step. Further provided herein are methods, wherein the method further comprises an oxidation step. Further provided herein are methods, wherein the method further comprises a deblocking step.

[0009] Provided herein are methods 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 are methods wherein the droplets have a volume of less than about 50 femtoliters. Further provided herein are methods wherein the droplets have a volume of less than about 25 femtoliters to 100 femtoliters. Further provided herein are methods wherein the method further comprises cleaving at least one polynucleotide from the surface, wherein the polynucleotide is dissolved in the droplets. Further provided herein are methods wherein the method further comprises sequencing at least one polynucleotide from the surface. Further provided herein are methods wherein the nucleoside comprises a nucleoside phosphoramidite. Further provided herein are methods wherein the method further comprises drying the surface. Further provided herein are methods wherein the method further comprises washing the nucleoside from the surface. Further provided herein are methods wherein the method further comprises a capping step. Further provided herein are methods wherein the method further comprises an oxidation step. Further provided herein are methods wherein the method further comprises a deblocking step.

[0010] Provided herein are methods 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 are methods wherein the time for repeating step b) using four different nucleotides is less than about 50 milliseconds. Further provided herein are methods wherein the time for repeating step b) using four different nucleotides is between 25 milliseconds and 100 milliseconds. Further provided herein are methods wherein the method further comprises cleaving at least one polynucleotide from the surface, wherein the polynucleotide is dissolved in a small droplet. Further provided herein are methods wherein the method further comprises sequencing at least one polynucleotide from the surface. Further provided herein are methods wherein the nucleoside comprises a nucleoside phosphoramidite. Further provided herein are methods wherein the method further comprises drying the surface. Further provided herein are methods wherein the method further comprises washing the nucleoside from the surface. Further provided herein are methods wherein the method further comprises a capping step. Further provided herein are methods wherein the method further comprises an oxidation step. Further provided herein are methods wherein the method further comprises a deblocking step.

[0011] Incorporation by reference

[0012] 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

[0013] The novel features of the present invention are particularly set forth in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by referring to the following detailed description which illustrates illustrative embodiments utilizing the principles of the present invention and the accompanying drawings, in which:

[0014] Figure 1 An exemplary workflow for nucleic acid-based data storage is shown.

[0015] Figure 2 A plate configured for polynucleotide synthesis is shown, comprising an array of 24 regions or subdomains, each having 256 clusters.

[0016] Figure 3 Shown Figure 2 A close-up view of the subfield in

[15] , which has 16×16 clusters, each with 121 individual seats.

[0017] Figure 4 Shown Figure 2 Detailed view of a cluster in , where the cluster has 121 seats.

[0018] Figure 5A Shown is a front view of a plate having multiple channels.

[0019] Figure 5B A cross-sectional view of a plate having multiple channels is shown.

[0020] Figures 6A-6B Continuous loop and reel-to-reel arrangements for flexible structures are depicted.

[0021] Figures 6C-6D A schematic diagram depicting the release and extraction of synthesized polynucleotides.

[0022] Figures 7A-7C Depicted are magnified views of flexible structures with spots, channels, or pores, respectively.

[0023] Figure 8 An example of a computer system is shown.

[0024] Figure 9 is a block diagram illustrating the architecture of a computer system.

[0025] Figure 10 is a diagram illustrating a network configured to incorporate multiple computer systems, multiple cellular phones and personal data assistants, and network attached storage (NAS).

[0026] Figure 11 is a block diagram of a multiprocessor computer system that uses a shared virtual address memory space.

[0027] Figure 12A is the front side of an example of a solid support array.

[0028] Figure 12B is the back side of an example of a solid support array.

[0029] Figure 13 Schematic diagram of a solid support containing an active area and a fluidic interface.

[0030] Figure 14 is an example of a rack-style instrument.

[0031] Figure 15 Depicted are solid supports comprising addressable areas for nucleic acid synthesis or storage.

[0032] Figure 16A Depicted is an array for synthesis using electrochemistry.

[0033] Figure 16B Depicted is an array for synthesis using electrochemistry.

[0034] Figure 17 The wells used for nucleic acid synthesis or storage and the spacing between the wells are depicted.

[0035] Figure 18 An example of a solid support comprising an addressable array is shown.

[0036] Figure 19 An example of a solid support array is shown where the spacing approximates the length of a 240-mer polynucleotide. DETAILED DESCRIPTION

[0037] 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 suitable host for information storage, partly due to their stability over time and ability to encode four bits of information. Therefore, compared with commercially available information storage devices, large amounts of data can be encoded in DNA in a relatively small amount of physical space. This article provides a method for increasing DNA synthesis throughput by increasing sequence density and reducing turnover time.

[0038] definition

[0039] 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.

[0040] Throughout this 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 within the range that is accurate to one-tenth of the lower limit unit. For example, the description of a range such as from 1 to 6 should be considered to have clearly disclosed sub-ranges 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 within 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 smaller ranges and are also encompassed in the present invention, but are subject to any clearly excluded limits in the range. Unless the context clearly stipulates otherwise, in the case where the range includes one or both of the limits, the range excluding any or both of these included limits is also encompassed in the present invention.

[0041] The terms used herein are only used for the purpose of describing specific embodiments and are not intended to limit any embodiments. Unless the context clearly dictates otherwise, the singular forms "a", "an", and "the" as used herein are also intended to include the plural forms. It should be further understood that the terms "include" and / or "comprise" when used in this specification refer to the presence of the features, integers, steps, operations, elements and / or components, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components and / or groups thereof. As used herein, the term "and / or" includes any and all combinations of one or more of the relevant listed items.

[0042] 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 lower limit listed to 10% above the upper limit listed.

[0043] 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. Specifically, various aspects of the present invention are primarily described herein with respect to the preparation of nucleic acid molecules, in which the sequence of the polynucleotide is known and selected prior to the synthesis or assembly of the nucleic acid molecules.

[0044] 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.

[0045] Solid support-based nucleic acid synthesis and storage

[0046] This paper describes devices, compositions, systems and methods for chip-based nucleic acid synthesis and storage. In some cases, polynucleotides are synthesized de novo using solid support-based methods as described herein. In some cases, polynucleotides are stored on a solid support after synthesis. In some cases, solid support-based methods as described herein are only used for storage.

[0047] 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 component or component can include temperature control unit, addressable electrode, semiconductor surface, fluid reservoir, fluid control device, synthesis surface, power supply or other components for synthesizing polynucleotide. Any combination of integrated components is suitable for use with device as herein described, composition, system and method. In some cases, one or more components are outside (non-integrated) of solid support.

[0048] The density of unique sites for polynucleotide synthesis on the surface is usually controlled by the spatial resolution that can be achieved 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 the coupling of nucleosides (nucleoside phosphoramidites) 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 in addressable communication 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 Figure 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 the active sites to achieve a 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 Figure 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).

[0049] 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 Figure 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 nucleosides.

[0050] Described herein are devices comprising a solid support, wherein the solid support comprises a plurality of wells, wherein each well comprises an addressable locus comprising: 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 comprise a bottom electrode in addressable communication with the synthetic surface. For example, the sidewall electrodes 1603 can be used to control adhesion of a substrate or reagent (see Figure 16B In some cases, the reagent comprises protons or other acid molecules. In some cases, the sidewall electrode 1603 is located at the surface of the well having a depth 1604 (see Figure 16B) at a position around the edge of the synthesis surface. In some cases, the sidewall electrodes control the chemical reactions that occur near the synthesis surface. For example, if acid or other reagents are generated near the synthesis surface, the portion of the polynucleotide 1601 bound to the surface will be exposed to a higher concentration of acid than the portion of the polynucleotide far from the acid generation site. This may result in degradation of the polynucleotide portion exposed to the higher concentration of acid. In some cases, the sidewall electrodes 1603 generate or control a proton gradient 1602, which causes a portion of the polynucleotide 1601 to be uniformly or targetedly exposed to acid. Sites near the uncharged electrodes are not coupled with the nucleosides deposited on the synthesis surface, and the pattern of the charged electrodes changes before adding the next nucleoside. 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, local control of coupling reduces synthesis steps, reduces reagents / materials (due to higher polynucleotide density and reduced scale), and shortens synthesis time (the synthesis 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 independently applied to two or more different sidewall electrodes. In some cases, such an arrangement facilitates 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 pore cross section. In some cases, the sidewall electrodes are discontinuous or only partially cover a portion of the sidewall surface. For example, the sidewall electrodes are continuous over approximately 5%, 10%, 15%, 30%, 50%, 75%, or approximately 90% of the circumference of the pore cross section. In some cases, such sidewall electrodes have a height that is approximately equal to the pore height, or approximately 5%, 10%, 15%, 30%, 50%, 75%, or approximately 90% of the pore height. In some cases, independently applying different voltages to two or more discontinuous sidewall electrodes can result in 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.

[0051] Polynucleotide synthesis usually needs to repeat deposition and remove liquid (fluidic control) on the synthesis surface.In some cases, the overall movement of fluid causes fluid loss (the volume of wetting, transport pipeline or reaction well), which causes the reagent use efficiency to be low and the cycle time of mobile fluid to be longer.The alternative method of overall fluidic control technology in the synthesis process is to use digital fluidic control technology, wherein reagent or reaction vessel are packaged as discrete droplets.In some cases, by operating or electrowetting on the surface comprising insulating (or being coated with semiconductor) electrode, with discrete volume mixing, moving (merging, reacting), separate, store, add, remove or analyze droplets.Electrowetting allows fluid-surface interaction to be carried out local control; For example, the electrode near the droplet is energized to cause the droplet to separate.In some cases, droplets as described herein have small volume.For example, the volume of droplets 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 small droplets is about 50 to about 200 femtoliters. In some cases, relative to overall fluidic control, digital fluidic control causes 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 cycle time to be shortened by about 2 to 10 times. In some cases, the time of completing a circulation (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 of completing a circulation is 1,2,3,5,7,10,12,15,17,20,30,50,100 or 200ms at the most. In some cases, the time of completing a circulation is about 10 to about 50ms.

[0052] The movement of fluids within and outside the surfaces described herein may include modifications or conditions that prevent undesirable fluid movement or other phenomena. For example, in some cases, the movement of fluids results in the formation of bubbles or air pockets, which limit the fluid's contact with components such as surfaces or polynucleotides. The methods, systems, and compositions described herein encompass various methods for controlling or minimizing bubble formation. Such methods include controlling fluid pressure, pore geometry, or surface materials / coatings. Pore geometry can be used to minimize bubbles. For example, tapering pores, channels, or other surfaces can 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 surfaces described herein include hydrophobic materials. In some cases, the surfaces described herein include hydrophilic materials. Pressure can be used to control bubble formation during fluid movement. In some cases, pressure is applied locally to a component, surface area, capillary / channel, 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 appropriate 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.

[0053] Devices described herein can utilize a control unit for regulating environmental conditions such as temperature. A temperature control unit is typically used to prepare or maintain conditions for storing a solid support comprising polynucleotides. The storage conditions of nucleic acids can affect their long-term stability, which directly affects the quality of the digital storage information retrieved. Polynucleotides are optionally stored on a solid support 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 (e.g., solvated or dried) of the polynucleotides on the solid support also affects storage stability. In some cases, polynucleotides are stored in a solution in the form of droplets, such as an aqueous solution or a buffer. 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 comprising the polynucleotides are added to the solid support, the solid support is dried. In some cases, the dried solid support is subsequently 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 polynucleotide. In some cases, the solid support further comprises metadata.

[0054] Device as herein described can comprise the power source for energizing each assembly of device.The synthesis assembly in the solid support is optionally powered by an external power source or the power source integrated into the solid support.The power source can 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 applicable to synthesis assembly as herein described or device.In some cases, synthesis assembly as herein described, surface or device are portable.

[0055] In some cases, the fluid comprising reagent, washing solvent or other synthetic components is deposited on the synthetic surface. In some cases, unused fluid (before contacting with the synthetic surface) or waste liquid (after contacting with the synthetic surface) are stored in one or more compartments that are integrated into the solid support. Alternatively or in combination, polynucleotide are moved in or out of the solid support to carry out external analysis or storage. For example, the polynucleotide synthesized is cut off from the seat on the solid support in the droplet, and the droplet of gained is moved to the synthesis region of the solid support from the outside. 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 described fluid ports allow fluid to move in and out of the solid support. In some cases, port orientation is 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 per mm of solid support length. In some cases, the devices described herein comprise from about 100 to about 5000 ports per mm of solid support length.

[0056] Described herein are addressable electrodes integrated into a solid support. Electrodes include, but are not limited to, conductors, insulators, or semiconductors and are made of materials known in the art. Materials can include metals, non-metals, mixed metal oxides, nitrides, carbides, silicon-based materials, or other materials. In some cases, metal oxides include TiO2, Ta2O5, Nb2O5, Al2O3, BaO, Y2O3, HfO2, SrO, or other metal oxides known in the art. In some cases, metal carbides include TiC, WC, ThC2, ThC, VC, W2C, ZrC, HfC, NbC, TaC, Ta2C, or other metal carbides known in the art. In some cases, metal nitrides include GaN, InN, BN, Be3N2, Cr2N, MoN, Si3N4, TaN, Th2N2, 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.

[0057] The electrodes can have any shape, including disks, rods, wells, posts, 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 、75um2 、50um 2 、25um 2 、10um 2 , less than 5um 2 In some cases, the cross-sectional area of each electrode is about 500 μm. 2 to 10um 2 , about 100um 2 to 25um 2 or about 150um 2 to 50um 2 In some cases, each electrode has a cross-sectional area of about 150 μm. 2 to 50um 2. The device provided herein includes electrodes 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 varies according to the size of the locus used for polynucleotide synthesis, but is about 50nm, 100nm, 200nm, 500nm, 750nm, 1000nm, 1200nm, 1500nm, 2000nm, 2500nm, 3000nm, or 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 are 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 anode. The electrode or electrode array can be placed on or near any position on 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 addressable independently or simultaneously. In some cases, the electrode in the hole bottom is used to cut polynucleotide from the surface at one or more seats, and the sidewall electrode is used to generate acid to deprotect the polynucleotide. In some cases, the electrode in the hole bottom is used to cut polynucleotide from the surface at one or more seats, and the first sidewall electrode is used to generate acid to deprotect the polynucleotide, and the second sidewall electrode is used to move the polynucleotide out of the hole after cutting. In an exemplary configuration, the sidewall electrode is located 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 10 nm to about 100 nm, about 50 nm to about 150 nm, about 40 nm to 100 nm, about 75 nm to about 125 nm, about 100 to 300 nm above the composite surface. In some cases, multiple sidewall electrodes are located at different heights above the composite surface. For example, the seat includes at least one sidewall electrode, at least 2 sidewall electrodes, at least 3 sidewall electrodes, or more than 3 sidewall electrodes. In exemplary configurations, the height of the sidewall electrodes is about 1 nm, about 5 nm, about 10 nm, about 20 nm, about 25 nm, about 30 nm, about 40 nm, or about 50 nm. In some cases, the height of the sidewall electrodes is 1 nm to 20 nm, 2 nm to 30 nm, 5 nm to 20 nm, 10 nm to 40 nm, or 5 nm to 25 nm.

[0058] 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 monomers. In some cases, this includes generating reagents to promote or prevent the coupling of monomers 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 iterative polynucleotide assembly process is optimized by exploring the various combinations of polynucleotides that are released and allowed to hybridize for overlapping PCR assembly.

[0059] 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 approximately 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 .

[0060] 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 the hole or channel.In some cases, the reference electrode is approximately 1 to approximately 50um above the synthesis surface, and approximately 2um is approximately 40um, and approximately 3um is approximately 30um, and approximately 5um is approximately 20um, and approximately 10 is approximately 20um, and approximately 15 is approximately 50um, and approximately 30 is approximately 50um, and approximately 5um is approximately 30um or approximately 7um is approximately 25um.In some cases, the reference electrode is approximately 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, 26 um or at most 26 um 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, 26 um or at least 26 um 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 devices 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.

[0061] 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 more 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 more 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 at least or approximately 0.5 mm, 1.0 mm, 1.5 mm, 2.0 mm, 2.5 mm, 3.0 mm, 3.5 mm, 4.0 mm or greater than 4.0 mm.

[0062] This article describes a device in which two or more solid supports are assembled. In some cases, the solid supports are interfaced together on a larger unit. The interface connection can include exchanging fluids, electrical signals or other exchange media between the solid supports. The unit can be interfaced 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 can 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. The nucleic acids present on the solid supports (and the information stored therein) can be accessed from the rack unit. See, for example, Figure 14. Access includes removing polynucleotides from the solid support, directly analyzing the polynucleotides on the solid support, or allowing any other methods for operating or identifying the information stored in the nucleic acid. In some cases, information is accessed from a single seat on a plurality of racks, a single rack, a single solid support in a rack, a part of a solid support, or 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 devices for operating nucleic acid interface connection. In some cases, access to nucleic acid information is achieved by cutting polynucleotides from the whole or part of the solid support. In some cases, cutting includes being exposed to chemical reagents (ammonia or other reagents), electric 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 light is used to cut polynucleotides. In some cases, a lamp is used to cut polynucleotides, and a mask mediates ultraviolet exposure to the position on the surface. In some cases, a laser is used to cut polynucleotides, and a shutter open / close state controls the exposure of ultraviolet light 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.

[0063] A solid support as described herein comprises an active area. In some cases, the active area comprises an addressable area or locus for nucleic acid synthesis. In some cases, the active area comprises an addressable area or locus for nucleic acid storage.

[0064] The active area has different sizes. For example, the size of the active area is about 1 mm to about 50 mm multiplied 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 Figure 13 The package 1307 comprises an active area 1305 within the solid support 1303. The package 1307 also comprises a fluidic interface 1301.

[0065] 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 a region. In some cases, the solid support comprises about 1000 to 20,000 times about 1000 to 20,000 positions in a region. 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, 50,000, 51,000, 52,000, 53,000, 54 000 locations by at least or about 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 locations. 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 spaced 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 μm apart. In some cases, the solid support comprises addressable loci that are spaced about 5 μm apart. In some cases, the solid support comprises addressable loci that are spaced about 2 μm apart. In some cases, the solid support comprises addressable loci that are spaced about 1 μm apart. In some cases, the solid support comprises addressable loci that are spaced about 0.2 μm apart. In some cases, the solid support comprises addressable loci spaced from about 0.2 μm to about 10 μm, from about 0.2 to about 8 μm, from about 0.5 to about 10 μm, from about 1 μm to about 10 μm, from about 2 μm to about 8 μm, from about 3 μm to about 5 μm, from about 1 μm to about 3 μm, or from about 0.5 μm to about 3 μm. In some cases, the solid support comprises addressable loci spaced from about 0.1 μm to about 3 μm. See, e.g., Figure 15 、 Figure 16A and Figure 16B .

[0066] In some cases, the solid support for nucleic acid synthesis or storage as described herein has a high data storage capacity. In some cases, the capacity of solid support is at least or approximately 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 support is approximately 1 to approximately 10 petabytes or approximately 1 to approximately 100 petabytes. In some cases, the capacity of solid support is approximately 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 wells. 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.

[0067] 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 data packets in the form of one or more droplets. In some cases, polynucleotides are collected in data packets 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, 1,000,000, 5,000,000, 10,000,000, 25,0 ...2500,000, 500,000, 750,000, 100,000, 2500,000, 500,000, 750,000, 100,000, 2500,000, 500,000, 750,000, 100,000, 25 In some cases, the 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 droplet volume has a diameter of 1-100 um, 10-90 um, 20-80 um, 30-70 um or 40-50 um.

[0068] 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.

[0069] 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 packets comprise about 100 to about 1000 copies of each polynucleotide. In some cases, the data packets comprise 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 packets comprise 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 methods based on solid supports 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 bases in length.

[0070] Nucleic acid-based information storage

[0071] 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 an encoded 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 for nucleic acid extension (also referred to as an arrangement spot), and the reagents for nucleic acid synthesis are selected (107). The surface of the structure is prepared for nucleic acid synthesis (108). De novo polynucleotide synthesis is performed (109). The synthesized polynucleotides are stored (111) and the synthesized polynucleotides can be used in whole or in part for subsequent release (113). Once released, the polynucleotides are sequenced in whole or in part (115) and decrypted (117) to convert the nucleic acid sequence back into a digital sequence. The digital sequence is then assembled (119) to obtain the parallel encoding of the original information item.

[0072] Information Item

[0073] Optionally, an early step in the data storage process disclosed herein includes 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, periodicals, electronic databases, medical records, letters, forms, recordings, animal records, biological profiles, broadcasts, movies, short videos, emails, bookkeeping phone records, internet activity logs, drawings, paintings, prints, photographs, pixelated graphics, and software codes. Exemplary biological profile 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 of an information item encoded in a digital format or the amount of multiple files of an information item encoded in a digital format 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.

[0074] Structures for polynucleotide synthesis

[0075] 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 FIG, wherein the structure 200 has approximately the same 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, with 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 μm and the X-axis seat pitch is 75 μm. The seat width at the longest portion (e.g., the diameter of a circular seat) is 50 μm, and the distance between seats is 24 μm. Figure 4 The number of seats 405 in an exemplary cluster in is 121. The seats can be flat, wells, or channels. Figures 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 can include a plurality of main channels 510. The plurality of channels 515 collectively form a cluster within the main channel 510.

[0076] 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). Figures 6A-6B In some cases, the structure comprises multiple regions for polynucleotide synthesis. Figure 6C An exemplary structure is shown in FIG, where a plate comprises 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). Figure 6D An alternative structure is shown in FIG, wherein the ribbon comprises distinct regions 617 for polynucleotide synthesis. The distinct regions 617 can be separated (619) by breaking or cleaving. Each distinct region can be further released, sequenced, decrypted and read (621) or stored (623). Provided herein are flexible structures having a surface with multiple loci for polynucleotide extension. Figures 7A-7CAn enlarged view of a locus in a flexible structure is shown. Each locus 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 locus of the structure is approximately 10 μm, and the distance between the centers of each structure is approximately 21 μm. The loci can include, but are not limited to, circular, rectangular, conical, or arcuate shapes. Alternatively, or in combination, the structure is rigid. In some cases, the rigid structure comprises a locus for polynucleotide synthesis. In some cases, the rigid structure comprises a substantially planar region, channel, or hole for polynucleotide synthesis.

[0077] 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 measured value 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 measured value 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 are structures for polynucleotide synthesis, comprising 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 comprise multiple clusters, each cluster comprising multiple holes, seats or channels. Alternatively, described herein may comprise a uniform arrangement of holes, seats or channels. 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 in the hole or the depth are at least 10,25,50,75,100,200,300,400,500,600,700,800,900,1000 or exceed 1000nm. In some cases, the height or depth of the pores 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 pores is in the range of about 50 nm to about 1 μm. In some cases, the height of the pores is about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 700, 800, 900, or about 1000 nm.

[0078] The structure for polynucleotide synthesis provided herein can comprise channel.The width of channel and the ratio of depth (or height) can be 1 to 0.01, and wherein width is the measured value of width at the narrowest section of microchannel.In some cases, the width of channel as herein described and the ratio of depth (or height) are 0.5 to 0.01, and wherein width is the measured value of width at the narrowest section of microchannel.In some cases, the width of channel as herein described and the ratio of depth (or height) are about 0.01,0.05,0.1,0.15,0.16,0.2,0.5 or 1.

[0079] Described herein is the structure for polynucleotide synthesis, and it comprises a plurality of discrete seats.Structure includes but is not limited to the substantially planar region, passage, projection or hole for polynucleotide synthesis.In some cases, provide structure described herein, it comprises a plurality of passages, wherein the height or the degree of depth in passage are 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 passage is less than 100um, less than 80um, less than 60um, less than 40um or less than 20um.In some cases, the height in passage 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.

[0080] The width of a locus on the surface of a structure for polynucleotide synthesis described herein can be from about 0.1 μm to about 500 μm, from about 0.5 μm to about 500 μm, from about 1 μm to about 200 μm, from about 1 μm to about 100 μm, from about 5 μm to about 100 μm, or from about 0.1 μm to about 100 μm, for example, about 90 μm, 80 μm, 70 μm, 60 μm, 50 μm, 40 μm, 30 μm, 20 μm, 10 μm, 5 μm, 1 μm, or 0.5 μm. In some cases, the width of the locus is less than about 100 μm, 90 μm, 80 μm, 70 μm, 60 μm, 50 μm, 40 μm, 30 μm, 20 μm, or 10 μm. In some cases, the width of the locus 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 width of the loci 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 loci 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 μm to about 500 μm, 0.5 μm to about 500 μm, about 1 μm to about 200 μm, about 1 μm to about 100 μm, about 5 μm to about 200 μm, about 5 μm to about 100 μm, about 5 μm to about 50 μm, or about 5 μm to about 30 μm, for example, about 20 μm. In some cases, the total width of the loci is about 5 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, or 100 μm. In some cases, the total width of the loci is about 1 μm to 100 μm, 30 μm to 100 μm, or 50 μm to 70 μm. In some cases, the distance between the centers of two adjacent loci is about 0.5 μm to about 2 μm, 0.5 μm to about 2 μm, about 0.75 μm to about 2 μm, about 1 μm to about 2 μm, about 0.2 μm to about 1 μm, about 0.5 μm to about 1.5 μm, about 0.5 μm to about 0.8 μm, or about 0.5 μm to about 1 μm, for example, about 1 μm. In some cases, the total width of the loci is about 50 nm, 0.1 μm, 0.2 μm, 0.3 μm, 0.4 μm, 0.5 μm, 0.6 μm, 0.7 μm, 0.8 μm, 0.9 μm, 1 μm, 1.1 μm, 1.2 μm, 1.3 μm, 1.4 μm, or 1.5 μm. In some cases, the total width of the loci is about 0.5 μm to 2 μm, 0.75 μm to 1 μm, or 0.9 μm to 2 μm.

[0081] 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.

[0082] Provided herein are loci having a width of 5 to 100 μm at the longest segment. In some cases, the loci have a width of about 30, 35, 40, 45, 50, 55, or 60 μm at the longest segment. In some cases, the loci are channels having multiple segments, wherein each segment has a center-to-center distance of 5 to 50 μm. In some cases, each segment has a center-to-center distance of 5, 10, 15, 20, or 25 μm. 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 loci density within a cluster of a 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 / mm 2 , 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 approximately 400 seats / mm 2 , about 50 seats / mm 2 Up to approximately 500 seats / mm 2 , about 100 seats / mm 2 Up to approximately 500 seats / mm 2 , about 150 seats / mm 2 Up to approximately 500 seats / mm 2 , about 10 seats / mm 2 About 250 seats / mm 2 , about 50 seats / mm 2 About 250 seats / mm 2 , about 10 seats / mm 2 Up to approximately 200 seats / mm 2 or about 50 seats / mm 2 Up to approximately 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 2 In some cases, the substrate comprises about 10 4 Seats / mm2 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 from about 10 μm to about 500 μm, from about 10 μm to about 200 μm, or from about 10 μm to about 100 μm. In some cases, the distance between the centers of two adjacent loci is greater than about 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, or 100 μm. In some cases, the distance between the centers of two adjacent loci is less than about 200 μm, 150 μm, 100 μm, 80 μm, 70 μm, 60 μm, 50 μm, 40 μm, 30 μm, 20 μm, or 10 μm. 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 μm, 70 nm, 60 nm, 50 nm, 40 nm, 30 nm, 20 nm, or 10 nm. In some embodiments, the structures described herein allow for 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 from 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. In some cases, the structures described herein are synthesized from 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, 1,20 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, polynucleotide is synthesized on the different seats of structure, and wherein each seat supports synthetic polynucleotide colony.In some cases, each seat supports synthetic polynucleotide colony with different sequence from the polynucleotide colony that grows on another seat.In some cases, the seat of structure is positioned at multiple clusters.In some cases, structure comprises 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 comprises 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 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 or more loci. In some cases, each cluster comprises 50 to 500, 100 to 150, or 100 to 200 loci. In some cases, each cluster comprises 109, 121, 130, or 137 loci. In some cases, each cluster comprises 5, 6, 7, 8, 9, 10, 11, or 12 loci. In some cases, polynucleotides from different loci within a cluster have sequences that, when assembled, encode a contiguous longer polynucleotide of a predetermined sequence.

[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 sizes 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 substrate has a planar surface area of 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, 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. The non-limiting examples of thickness include 275mm, 375mm, 525mm, 625mm, 675mm, 725mm, 775mm and 925mm. In some cases, 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, thickness varies with diameter and depends on the composition of substrate. For example, the structure comprising the material outside silicon can have a thickness different from the silicon structure of same diameter. Structure thickness can depend on the mechanical strength of materials used, and the structure must be thick enough to support its own weight and can not break during operation. 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 for supporting the synthesis of polynucleotides at predetermined positions, 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 enough conductivity, for example, it is possible to form a uniform electric field across the entire device or a part thereof. Device as described herein may comprise a flexible material. Exemplary flexible materials include but are not limited to modified nylon, unmodified nylon, nitrocellulose and polypropylene. Device as described herein may comprise a rigid material. Exemplary rigid materials include but are not limited to glass, fused quartz, silicon, silicon dioxide, silicon nitride, plastics (such as polytetrafluoroethylene, polypropylene, polystyrene, polycarbonate, and blends thereof) and metals (such as gold, platinum). Device disclosed herein may be made of a material 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 supports 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 supports 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 is a measure of a material's resistance to deformation under elastic (recoverable) loads. 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 supports 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 supports 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 more. Since the relationship between flexibility and stiffness is inverse to one another, flexible materials have a low Young's modulus and their shape changes significantly under load.In some cases, a solid support described herein has a surface that is at least as flexible as nylon.

[0092] In some cases, the device disclosed herein comprises a silicon dioxide base and a silicon oxide surface layer. Alternatively, the device can have a silicon oxide base. The surface of the device provided herein can be textured, thereby causing the total surface area for polynucleotide synthesis to increase. In some cases, the device disclosed herein comprises at least 5%, 10%, 25%, 50%, 80%, 90%, 95% or 99% silicon. In some cases, the device disclosed herein is made of silicon on insulator (SOI) wafer.

[0093] The structure can be made of a variety of materials suitable for the methods and compositions of the invention described herein. In some cases, the material for making the substrate / solid support of the present invention exhibits low levels of polynucleotide binding. In some cases, materials transparent to visible light and / or ultraviolet light can be adopted. Materials with sufficient electrical conductivity can be adopted, for example, 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 heat-insulating. The material can be chemically resistant and heat-resistant to support chemical reactions or biochemical reactions, such as a series of polynucleotide synthesis reactions. For flexible materials, materials of interest may include: modified nylon and unmodified nylon, nitrocellulose, polypropylene, etc.

[0094] Specific materials of interest for rigid materials include glass; fused silica; silicon, plastics (e.g., polytetrafluoroethylene, polypropylene, polystyrene, polycarbonate, and blends thereof); and 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, or 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, cartridge 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 magnetic reel-to-reel tape or 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 electrical 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 structures are chemically 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 alters the surface of the substrate by additive or subtractive processes 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) altering 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 altering the chemical composition of the surface, e.g., by etching; (5) increasing or decreasing the surface roughness; (6) providing a coating on the surface, e.g., a coating that exhibits wetting properties different from those of the surface; and / or (7) depositing microparticles on the surface. In some cases, the surface of a structure is selectively functionalized to create two or more distinct regions on the structure, wherein at least one region has different surface or chemical properties 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 on the surface. In some cases, the surface is also functionalized with a passivating material that is ineffective at binding biomolecules, thereby preventing the attachment of biomolecules at sites where the passivating functionalizing agent binds. In some cases, the surface includes an active layer that defines distinct sites for biomolecule support.

[0101] In some cases, the surface is contacted with a mixture of functional 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 ratio 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 the functionalizing agent of the appropriate ratio to the substrate surface. In some cases, the reagent in the mixture is selected from suitable reactivity and inert parts, thereby the surface density of the reactive groups is diluted to the desired level for downstream reactions. In some cases, the mixture of functionalizing agents comprises one or more reagents bound to biomolecules and one or more reagents not bound to 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. The 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, while 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 triethoxy silane, n-decyl triethoxy silane, (3-aminopropyl) trimethoxy silane, (3-aminopropyl) triethoxy silane, glycidyl oxypropyl / trimethoxy silane and N-(3-triethoxysilyl propyl)-4-hydroxy butanamide. In some cases, the silane includes 11-acetoxy undecyl triethoxy silane, n-decyl triethoxy silane, (3-aminopropyl) trimethoxy silane, (3-aminopropyl) triethoxy silane, glycidyl oxypropyl / trimethoxy silane, N-(3-triethoxysilyl propyl)-4-hydroxy butanamide or its any combination. In some cases, active functionalizing agent includes 11-acetoxy undecyl triethoxy silane. In some cases, active functionalizing agent includes n-decyl triethoxy silane. In some cases, the active functionalizing agent comprises 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 alkoxysilane is classified according to its organofunctional.The non-limiting example of siloxane functionalizing agent comprises: hydroxyalkyl siloxane (silylation surface, functionalized with diborane, and oxidizing this alcohol with hydrogen peroxide), glycol (dihydroxy alkyl) siloxane (silylation surface, and hydrolyzed into glycol), aminoalkyl siloxane (amine does not need intermediate functionalization step), glycidoxy silane (3-glycidoxypropyl-dimethyl-ethoxysilane, glycidoxy-trimethoxysilane), mercaptosilane (3-mercaptopropyl-trimethoxysilane, 3-4 epoxycyclohexyl-ethyltrimethoxysilane or 3-mercaptopropyl-methyl-dimethoxysilane), bicycloheptyl-trichlorosilane, butyl-aldehyde-trimethoxysilane or dipolymeric secondary aminoalkyl siloxane. Exemplary hydroxyalkyl siloxanes include allyl trichlorosilane converted to 3-hydroxypropyl or 7-oct-1-enyl trichlorosilane converted to 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) converted to 3-aminopropyl. In some cases, the dimeric secondary aminoalkyl siloxane is bis(3-trimethoxysilylpropyl)amine converted to bis(silyloxypropyl)amine.

[0104] The active functionalized region can comprise 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 the other silane. For example, a mixed silane solution having two silanes has a ratio of one silane to the other 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, or 55:45. In some cases, the reactive functionalizing agent comprises 11-acetoxyundecyltriethoxysilane and n-decyltriethoxysilane. In some cases, the reactive functionalizing agent comprises 11-acetoxyundecyltriethoxysilane and n-decyltriethoxysilane 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 comprises 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 steps or components in the following functionalization process may be omitted or modified. 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. Examples of cleaning processes include immersing the substrate in a piranha solution (e.g., 90% H2SO4, 10% H2O2) at an elevated temperature (e.g., 120°C), and washing (e.g., with water) and drying the substrate (e.g., with nitrogen). The process optionally includes a post-piranha treatment, which includes immersing the piranha-treated substrate in an alkaline solution (e.g., NH4OH) followed by immersion in an aqueous wash solution (e.g., water). In some cases, optionally after the piranha soak and optional post-piranha 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 is functionalized, the surface is coated with a resist such as SPR. TM 3612 positive photoresist is spin-coated onto the substrate. In various cases, the surface functionalization process includes photolithography with patterned functionalization. In some cases, photolithography is performed after the 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 from the substrate, for example, by plasma cleaning or etching. In some cases, a plasma cleaning step is performed at some step after the photolithography step.

[0108] In some cases, for example, after functionalization and / or after photolithography, the resist-coated surface is treated to remove the resist. In some cases, the resist is removed with a solvent, such as a stripping solution comprising N-methyl-2-pyrrolidone. In some cases, resist stripping includes sonication or ultrasonication. In some cases, the resist is applied and stripped, and the exposed areas are then subjected to reactive functionalization 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 that changes 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 action effects can help to draw 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 fluid by promoting or suppressing the wicking effect in the adjacent fluid path. For example, the design structure can 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, an 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 reactive groups 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 light is used for surface patterning. In some cases, a lamp is used for surface patterning, and a mask mediates the exposure of ultraviolet light to the surface. In some cases, a laser is used for surface patterning, and a shutter open / close state controls the exposure of ultraviolet light 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 the movement of the flexible structure 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 synthesizing and / or cutting polynucleotide, the surface can be bathed, washed, cleaned, baked, etched or otherwise functionally restored to the condition suitable for subsequent polynucleotide synthesis. The number of times of reuse on the surface and the recycling / preparation method for the reused surface 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, measurement or prediction surface is suitable for reuse of residual " life span " or number of times.

[0113] Material deposition system

[0114] In some cases, the synthesized polynucleotides are stored on a substrate such as a solid support. The nucleic acid reagent 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 small droplets 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 small droplets to be ejected. In the electrostatic attraction method, small droplets are ejected onto the substrate using electrostatic attraction. In some cases, the dripping 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 dispensed droplets is related to the resolution of the device. In some cases, the device deposits droplets of reagent 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 droplet size 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, and this process utilizes the flexibility of substrate to advance with roll-to-roll process.This synthesis process operates in a continuous production line mode, wherein uses one or more reels to rotate the position of substrate so that substrate advances through the various stages of polynucleotide synthesis.In exemplary cases, the polynucleotide building-up reaction comprises rolling substrate: by below the deposition device for phosphoramidite deposition, by oxidant bath, by acetonitrile wash bath, and by deblocking bath.Optionally, band also passes through 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 take-up reel place can be used for further processing or storage by transporting the final product.

[0117] In some arrangements, polynucleotide synthesis is carried out in a continuous process as a continuous flexible belt is conveyed along a conveyor belt system. Similar to a reel-to-reel process, polynucleotide synthesis on a continuous belt is operated in a production line manner, where the substrate progresses through the various stages of polynucleotide synthesis during conveyance. However, in a conveyor belt process, the continuous belt returns to the polynucleotide synthesis step without the need to roll and unroll the belt, as in a reel-to-reel process. In some arrangements, the polynucleotide synthesis step is divided into segments, and the continuous belt is conveyed through each segment once or multiple times in a cycle. For example, a polynucleotide synthesis reaction may include (1) conveying 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 in a cycle; and then (2) repeating the cycle to obtain a synthesized polynucleotide of a predetermined length. After polynucleotide synthesis, the flexible substrate is removed from the conveyor belt system and optionally rolled up for storage. It can be rolled around a reel for storage. In some cases, the flexible substrate comprising a thermoplastic material is coated with a nucleoside coupling agent. The coating was patterned into seats such that each seat had a diameter of approximately 10 μm and a center-to-center distance of approximately 21 μm between two adjacent seats. In this case, the seat size was sufficient to accommodate a 0.2 pl sessile drop volume during the polynucleotide synthesis deposition step. In some cases, the seat density was approximately 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 reactions is configured to deposit reagents and / or nucleoside monomers for the synthesis based on nucleoside phosphoramidites. The reagent for polynucleotide synthesis comprises reagent and wash buffer for polynucleotide extension. As non-limiting examples, 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 the preparation and / or maintenance of substrate integrity. In some cases, the polynucleotide synthesizer is less than the droplet of about 200um, 100um or 50um with a volume deposition diameter 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] Device, method, system and composition are described herein, and the reagent that is wherein used for polynucleotide synthesis is recycled or reused.The recycling of reagent can comprise the collection, storage and use of unused reagent, or the purification / conversion of used reagent.For example, reagent bath is recycled and is used for the polynucleotide synthesis step on identical or different surfaces.Reagent as herein described can recirculate 1,2,3,4,5,6,7,8,9,10 times or more times.Alternatively or in combination, filter the reagent solution that comprises reaction byproduct to remove byproduct, and this reagent solution is used for other polynucleotide building-up reactions.

[0120] Many integrated or non-integrated elements are used together with polynucleotide synthesis system conventionally.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 resolution 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 / 10,000 base; 1 / 15,000 base; 1 / 20,000 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, at least 90%, 95%, 98%, 99%, 99.5% or more of these synthesized polynucleotides are indistinguishable from the predetermined sequence encoded therefrom. In some cases, the error rate of the polynucleotides synthesized on a 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 a 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 a 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 a 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 a 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 a substrate. The term "error rate" refers to a comparison of the total amount of synthesized polynucleotides to the sum of the predetermined polynucleotide sequences. In some cases, the synthesized polynucleotides disclosed herein comprise 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] This article describes methods, systems, devices, and compositions in which 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 a surface to control chemical reactions. In some cases, 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 by one or more electrodes. Electrochemical reactions can include oxidation, reduction, acid / base chemistry, or other reactions controlled by electrodes. In some cases, electrodes generate electrons or protons that serve 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 bases. Acids or bases are typically used to cleave protecting groups, or, for example, to influence the kinetics of various polynucleotide synthesis reactions by adjusting the pH of the reaction solution. In some cases, electrochemically controlled polynucleotide synthesis reactions contain 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] Control of chemical reactions is not limited to the electrochemical generation of reagents; chemical reactivity can be indirectly affected by biophysical changes in substrates or reagents through the electric field (or gradient) generated by electrodes. In some cases, substrates include but are not limited to nucleic acids. In some cases, an electric field is generated that repels or attracts specific reagents or substrates toward or away from an electrode or surface. In some cases, such fields are 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, such repulsion or attraction of polynucleotides or other reagents caused by local electric fields causes polynucleotides or other reagents to move into or out of a region of a synthesis device or structure. In some cases, electrodes generate an electric field that repels polynucleotides away from a synthesis surface, structure, or device. In some cases, electrodes generate an electric field that attracts polynucleotides toward a synthesis surface, structure, or device. In some cases, protons are repelled from a positively charged surface to limit their contact with the substrate or a portion thereof. In some cases, repulsive or attractive forces are used to allow or prevent reagents or substrates from entering a specific region of a synthesis surface. In some cases, nucleoside monomers are prevented from contacting the polynucleotide chain by applying an electric field near one or two components. Such an arrangement allows for 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 contact of nucleoside monomers with the polynucleotide chain. In addition, applying an electric field to the substrate can change substrate reactivity or conformation. In exemplary applications, the electric field generated by the 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 the 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 the nucleic acid substrate to prevent secondary structure. In some cases, the electric field eliminates the need for connectors or attachment to solid supports during polynucleotide synthesis.

[0125] The disclosed suitable method for synthesizing polynucleotide on substrate is the phosphoramidite method, which is included in the coupling step of forming phosphite triester bond between the phosphoramidite structural unit and the nucleosides that are attached to substrate by the controlled addition of phosphoramidite structural unit (i.e. nucleoside phosphoramidite) into the polynucleotide chain of growth. In some cases, the nucleoside phosphoramidite is provided to the substrate of activation. In some cases, the nucleoside phosphoramidite is provided to the substrate with activator. In some cases, the nucleoside phosphoramidite is provided to the substrate with respect to 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 nucleosides bonded with substrate. In some cases, the intercalation of nucleoside phosphoramidite is carried out in anhydrous environment (for example, in anhydrous acetonitrile). In the coupling step, after adding and connecting nucleoside phosphoramidites, the substrate is optionally washed. In some cases, the coupling step is additionally repeated once or multiple times, optionally with a washing step between adding nucleoside phosphoramidites to the substrate. In some cases, the polynucleotide synthesis method used herein comprises 1, 2, 3 or more continuous coupling steps. In many cases, before coupling, the nucleoside bound to the substrate is deprotected by removing a blocking group, wherein the blocking group plays a role in preventing polymerization. The blocking group may include any chemical group that prevents the polynucleotide chain from extending. In some cases, the blocking group is cut (or removed) in the presence of an acid. In some cases, the blocking group is cut in the presence of a base. In some cases, the blocking group is removed with electromagnetic radiation such as light, heat or other energy sources. In some cases, the blocking group is removed by oxidation or reduction reaction. In some cases, the blocking group includes a triarylmethyl group. In some cases, the blocking group includes an aryl ether. In some cases, the blocking group includes a disulfide. In some cases, the blocking group includes an acid-unstable silane. In some cases, the blocking group includes an acetal. In some cases, the protecting group includes a ketal. In some cases, the protecting group includes an enol ether. In some cases, the protecting group includes a methoxybenzyl. In some cases, the protecting group includes an 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 5'-OH groups bound to the substrate after coupling to prevent further chain extension, thereby preventing the formation of polynucleotides with internal base deletions. In addition, phosphoramidites activated with 1H-tetrazole typically react with the O6 position of guanosine to a very small extent. Without being bound by theory, after oxidation with I2 / water, this byproduct (possibly via O6-N7 migration) undergoes depurination. Apurinic sites can end up being cleaved during the final deprotection process of the polynucleotide, thereby reducing the yield of the full-length product. 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 the nucleoside phosphoramidite, and optionally after capping and one or more washing steps, the substrate described herein comprises the bound growing nucleic acid that can be oxidized. The oxidation step comprises oxidizing a phosphite triester to a four-coordinated phosphotriester, a protected precursor of naturally occurring phosphodiester internucleoside connections. In some cases, the phosphite triester is electrochemically oxidized. In some cases, the oxidation of the growing polynucleotide is achieved by optionally treating with iodine and water in the presence of a weak base 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, a capping step is performed after oxidation. A second capping step allows the substrate to dry because any residual water from the oxidation that may persist can inhibit subsequent coupling. After oxidation, the substrate and growing polynucleotide are optionally washed. In some cases, the oxidation step is replaced by 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, remove the protected 5 ' end (or 3 ' end, if synthesis is carried out in 5 ' to 3 ' direction) of the polynucleotide of growth that is combined with substrate, so that primary hydroxyl can react with next nucleoside phosphoramidite.In some cases, blocking group is DMT, and the trichloroacetic acid in dichloromethane is used to deblock.In some cases, blocking group is DMT, and the proton generated by electrochemistry is deblocked.Carry out the detritylation of extending time or use the acid solution stronger than recommended acid solution to carry out detritylation and can cause the depurination of the polynucleotide combined with solid support to increase, and therefore reduce the productive rate of required full-length product.Method and composition described herein provide controlled deblocking condition, thereby limit undesirable depurination reaction.In some cases, the polynucleotide that is combined with substrate is washed after deblocking.In some cases, the effective washing after deblocking helps to synthesize polynucleotide with low error rate.

[0129] The methods for synthesizing polynucleotides on substrates described herein can include a series of repeated steps: applying a protected monomer to a surface of a substrate feature for attachment to the surface, a linker, or a previously deprotected monomer; deprotecting the applied monomer to allow it to react with a subsequently applied protected monomer; and applying another protected monomer for attachment. One or more intermediate steps may include oxidation and / or sulfurization. In some cases, one or all of the steps may be preceded or followed by one or more washing steps.

[0130] The methods for synthesizing polynucleotides on substrates described herein may include an oxidation step. For example, the methods may comprise a series of repeated steps: applying a protected monomer to a surface of a substrate feature for attachment to the surface, a linker, or a previously deprotected monomer; deprotecting the applied monomer to allow it to react with a subsequently applied protected monomer; applying another protected monomer for attachment, and oxidizing and / or sulfurizing. In some cases, one or all of these steps may be preceded or followed by one or more washing steps.

[0131] The methods for synthesizing polynucleotides on substrates described herein may further comprise a series of repeated steps of applying a protected monomer to a surface of a substrate feature for attachment to the surface, a linker, or a previously deprotected monomer; deprotecting the applied monomer to allow it to react with a subsequently applied protected monomer; and oxidizing and / or sulfurizing. In some cases, one or all of the steps are preceded or followed by one or more washing steps.

[0132] The methods for synthesizing polynucleotides on substrates 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; and oxidizing and / or sulfurizing. In some cases, one or all of the steps may be preceded or followed by one or more washing steps.

[0133] The methods for synthesizing polynucleotides on substrates described herein may further comprise a series of repeated steps of applying a protected monomer to a surface of a substrate feature for attachment to the surface, a linker, or a previously deprotected monomer; deprotecting the applied monomer to allow it to react with a subsequently applied protected monomer; and oxidizing and / or sulfurizing. In some cases, one or all of the steps are preceded or followed by one or more washing steps.

[0134] In some cases, a polynucleotide with a photolabile protective group is synthesized, wherein the hydroxyl groups generated on the surface are blocked by the photolabile protective group. When the surface is exposed to ultraviolet light, for example, through a photolithography mask, a pattern of free hydroxyl groups can be generated on the surface. These hydroxyl groups can react with photoprotected nucleoside phosphoramidites according to the phosphoramidite chemistry. A second photolithography mask can be used, and the surface can be exposed to ultraviolet light to generate a second pattern of hydroxyl groups, which is then coupled with a 5'-photoprotected nucleoside phosphoramidite. Similarly, a pattern can be generated and the oligomer chain can be extended. Without being bound by theory, the instability of the photocleavable group depends on the wavelength and the polarity of the solvent used, and the rate of photocleavage can be affected by the duration of exposure and the intensity of the light. This method can utilize many factors, for example, the accuracy of the mask alignment, the efficiency of the photoprotective group removal, and the yield of the phosphoramidite coupling step. In addition, the leakage of undesirable light to adjacent sites can be minimized. The density of each spot synthetic oligomer can be monitored by adjusting the load of the lead nucleoside on the synthesis surface.

[0135] The surface of the substrate described herein that provides support for polynucleotide synthesis can be chemically modified to allow to cut the synthetic polynucleotide chain from the surface. In some cases, the polynucleotide chain is cut when the polynucleotide is deprotected. In some cases, the polynucleotide chain is cut after the polynucleotide chain is deprotected. In an exemplary embodiment, trialkoxysilylamine such as (CH3CH2O)3Si-(CH2)2-NH2 reacts with the surface SiOH group of substrate, then with succinic anhydride and amine reaction to produce amide bond and the free OH that support nucleic acid chain growth. Cutting comprises that ammonia or methylamine are carried out gas cutting. In some cases, cutting comprises the connector cutting that the reagent such as acid or alkali that is generated with electricity. In some cases, once released from the surface, polynucleotide is just assembled into larger nucleic acid, it is sequenced and decoded to extract the information stored.

[0136] Surface as herein described can be reused after polynucleotide cutting, to support the extra circulation of polynucleotide synthesis.For example, can reuse connector when not carrying out additional treatment / chemical modification.In some cases, connector is non-covalently bound to substrate surface or polynucleotide.In some embodiments, after cutting from the surface, connector remains attached to polynucleotide.In some embodiments, connector comprises reversible covalent bond, as ester, amide, ketal, β-substituted ketone, heterocycle or other groups that can be reversibly cut.In some cases, by adding or removing reagent, or by the electrochemical process controlled by electrode to control this type of reversible cutting reaction.Optionally, after multiple cycles, chemical connector or surface-bound chemical group is regenerated, to recover reactivity and remove undesirable by-product formation on this type of connector or surface-bound chemical group.

[0137] assembly

[0138] Polynucleotide can be designed to span the large region of the predetermined sequence of coding information together. In some cases, the polynucleotide synthesized is connected by ligation to generate larger polynucleotide. An example of ligation is polymerase chain assembly (PCA). In some cases, at least a portion of polynucleotide is designed to include an additional region of the substrate bound as a universal primer. For PCA reactions, pre-synthesized polynucleotides include overlaps (e.g., 4, 20, 40 or more bases with overlapping sequences) with each other. During the polymerase cycle, polynucleotides anneal to complementary fragments and are then filled in by polymerase. Therefore, according to which polynucleotides are found to each other, each cycle randomly increases the length of each fragment. The complementarity between the fragments allows the formation of complete, large-span double-stranded DNA. In some cases, after the PCA reaction is complete, an error correction step is performed using a mismatch repair detection enzyme to remove the mismatch in the sequence. Once a larger target sequence fragment is generated, the fragment can be amplified. For example, in some cases, a target sequence comprising 5' and 3' end adapter sequences is amplified in polymerase chain reaction (PCR), and the PCR comprises a modified primer hybridized with the adapter sequence. In some cases, the modified primers include one or more uracil bases. The use of modified primers allows the removal of primers by an enzymatic reaction focused on the targeted modified bases and / or the gaps left by enzymes that cut the modified base pairs from the fragments. What remains is a double-stranded amplification product lacking the adaptor sequence residues. 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 carried out to the polynucleotide of synthesis and / or the product of assembly.Exemplary strategies for error correction relate to carrying out 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, the double-stranded nucleic acids with mispairing, protrusion and mini-ring, chemically altered base and / or other heteroduplexes are selectively removed from the correctly synthesized nucleic acid colony.In some cases, use identification and combine or carry out error correction next to the protein / enzyme of the mispairing or unpaired base in the double-stranded nucleic acid, to produce single-strand or double-strand break or start chain transfer transposition event.The limiting examples of the protein / enzyme for error correction include endonuclease (T7 endonuclease I, Escherichia coli endonuclease V, T4 endonuclease VII, mung bean nuclease, cell Escherichia coli endonuclease IV, UVDE), restriction enzyme, glycosylase, ribonuclease, mispairing repair enzyme, resolvase, helicase, ligase, mispairing specific antibody and variant thereof. In some cases, error correction is performed using a correction enzyme. In some cases, error correction is performed using a correction enzyme. In some cases, error correction is performed using a SURVEYOR endonuclease (Transgenomic)--a mismatch-specific DNA endonuclease that scans for known and unknown mutations and polymorphisms in heteroduplex DNA.

[0140] Sequencing

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

[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 comprises a device that can directly order-check the polynucleotide stored in 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 comprises a device for keeping the structure and advancing the structure by a detection position, and is placed near the detection position for detecting the signal derived from the part when the part of the band is in the detection position. In some cases, the signal indicates the presence of polynucleotide. In some cases, the sequence (for example, fluorescent signal) of the signal indicates the polynucleotide. In some cases, when the band is continuously transmitted by the detector that is operably connected to a computer, the computer reads the information encoded in the polynucleotide on the continuous band. In some cases, the detection system comprises a computer system, and the computer system comprises a polynucleotide sequencing device, a database for storing and retrieving the data relevant to the polynucleotide sequence, the software for converting the DNA code of the polynucleotide sequence into a binary code, the computer for reading the binary code or its arbitrary 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," which identifies the identity of bases in a target polynucleotide. In some cases, polynucleotides synthesized using the methods, devices, compositions, and systems described herein are sequenced after cutting from a synthesis surface. In some cases, sequencing is performed during polynucleotide synthesis or simultaneously with polynucleotide synthesis, wherein base determination is performed immediately after or before the extension of nucleoside monomers into a growing polynucleotide chain. Base determination methods include measuring the current generated by the addition of bases catalyzed by a polymerase to a 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 synthesis surface.

[0145] Computer system

[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 the synchronization of the distribution / vacuuming / refilling functions (such as arranging and synchronizing the movement of the material deposition device, the distribution action and the 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 FIG. 8 can be understood as a logic device capable of reading instructions from a medium 811 and / or a network port 805, which can optionally be connected to a server 809 having a fixed medium 812. The system can include a CPU 801, a disk drive 803, optional input devices 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 via the communication medium shown. The communication medium can include any means of transmitting and / or receiving data. For example, the communication medium can be a network connection, a wireless connection, or an Internet connection. Such a connection can provide communication via the World Wide Web. It is contemplated that data related to the present disclosure can be transmitted via such a network or connection and thereby received and / or viewed by a party 822.

[0148] Figure 9 is a block diagram illustrating a first example architecture of a computer system that may be used in conjunction with example embodiments of the present invention. Figure 9 As shown, 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 processors. 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 comprising multiple computers, cellular phones, and / or personal data assistant devices.

[0149] like Figure 9As shown, a cache memory 904 may be connected to or incorporated into the processor 902 to provide high-speed storage for instructions or data that are recently or frequently used by the processor 902. The processor 902 is connected to a north bridge 906 via a processor bus 908. The north bridge 906 is connected to a random access memory (RAM) 910 via a memory bus 912 and manages access to the RAM 910 by the processor 902. The north bridge 906 is also connected to a south bridge 914 via a chipset bus 916. The south bridge 914 is in turn connected to a peripheral bus 918. The peripheral bus may be, for example, PCI, PCI-X, PCI Express, or other peripheral bus. The north bridge and south bridge are commonly referred to as a processor chipset and manage data transfer between the processor, RAM, and peripheral components on the peripheral bus 918. In some alternative architectures, the functionality of the north bridge may be incorporated into the processor rather than 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 example embodiments 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] Figure 101 is a diagram illustrating 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 applied to the data and evaluated using distributed parallel processing across the computer systems 1002a and 1002b and the cell phone and personal data assistant system 1002c. The computer systems 1002a and 1002b and the cell phone and personal data assistant system 1002c can also provide parallel processing for adaptive data reconstruction of the data stored in the network attached storage (NAS) 1004a and 1004b. Figure 10 This is just one example, 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 network attached storage (NAS).

[0154] In some example embodiments, a processor may maintain a separate memory space and transmit data via 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] Figure 111 is a block diagram of a multi-processor computer system 1100 using a shared virtual address memory space, according to an exemplary embodiment. The system includes multiple processors 1102a-f that can access a shared memory subsystem 1104. The system incorporates multiple programmable hardware memory algorithm processors (MAPs) 1106a-f within the memory subsystem 1104. Each MAP 1106a-f can contain memory 1108a-f and one or more field programmable gate arrays (FPGAs) 1110a-f. The MAPs provide configurable functional units and can provide specific algorithms or portions of algorithms to the FPGAs 1110a-f for processing in close coordination with the respective processors. For example, in an exemplary embodiment, the MAPs can be used to evaluate algebraic expressions associated with data models and to perform adaptive data reconstruction. In this example, each MAP is globally accessible to all processors used for these purposes. In one configuration, each MAP can use direct memory access (DMA) to access its associated memory 1108a-f, enabling it to perform tasks independently and asynchronously from its 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 a chip (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 type of data storage medium 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 exemplary embodiment, the computer system may be implemented using software modules executed on any of the above or other computer architectures and systems. In other embodiments, the functionality of the system may be implemented partially or completely 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 may be implemented in a hardware accelerated manner using a hardware accelerator card.

[0158] Implementation Plan

[0159] Provided herein are methods 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 methods, wherein the method further comprises cleaving at least one polynucleotide from the surface, wherein the polynucleotide is dissolved in the droplets. Further provided herein are methods, wherein the method further comprises sequencing at least one polynucleotide from the surface. Further provided herein are methods, wherein the method further comprises drying the surface. Further provided herein are methods, wherein the method further comprises washing the nucleotides from the surface. Further provided herein are methods, wherein the surface is a solid support. Further provided herein are methods, wherein the surface comprises glass, fused quartz, silicon, silicon dioxide, silicon nitride, plastic, metal, or a combination thereof.

[0160] Provided herein are methods 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 are methods 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 are methods, wherein the method further comprises one or more washing steps. Further provided herein are methods, 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 fluidic 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 predetermined sequences, 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] 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 predetermined sequences, 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 time for repeating step c) using four different nucleotides is less than about 100 milliseconds.

[0165] Provided herein are methods 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 are methods wherein the potential is an electric potential. Further provided are methods 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 indicated, all parts and percentages are by weight.

[0168] Example

[0169] Example 1: Functionalization of device surfaces

[0170] The device is functionalized to support the attachment and synthesis of the polynucleotide library. First, the device surface is wet-cleaned for 20 minutes using a piranha solution containing 90% H2SO4 and 10% H2O2. The device is rinsed in several beakers containing deionized water, kept under a deionized water gooseneck stopcock for 5 minutes, and dried with N2. The device is then immersed in NH4OH (1:100; 3mL:300mL) for 5 minutes, rinsed with deionized water using a handgun, immersed in three consecutive beakers containing deionized water for 1 minute each, and then rinsed with deionized water using a handgun. The device is then plasma cleaned by exposing the device surface to O2. O2 plasma etching is performed for 1 minute at 250 watts using a SAMCO PC-300 instrument in downstream mode.

[0171] The cleaned device surface was activated and functionalized with a solution containing N-(3-triethoxysilylpropyl)-4-hydroxybutanamide 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 surface was resist coated using a Brewer Science 200X spin coater. TM 3612 photoresist was spin-coated onto the device at 2500 rpm for 40 seconds. The device was pre-baked on a Brewer hot plate at 90°C for 30 minutes. The device was photolithographically processed using a Karl Suss MA6 mask aligner. The device was exposed for 2.2 seconds and developed in MSF 26A for 1 minute. The remaining developer was rinsed with a handheld spray gun, and the device was immersed in water for 5 minutes. The device was baked in an oven at 100°C for 30 minutes and then visually inspected for photolithographic defects using a Nikon L200. A clean process was used to remove residual resist using a SAMCO PC-300 instrument with an O2 plasma etch at 250 watts for 1 minute.

[0172] The device surface is passivated functionalized with 100 μL perfluorooctyl trichlorosilane solution mixed with 10 μL light mineral oil. The device is placed in a chamber, pumped for 10 min, then the valve leading to the pump is closed and allowed to stand for 10 min. The chamber is exhausted to air. The device is subjected to ultrasonic treatment to peel off the etching resist by soaking twice for 5 min in 500 mL NMP at 70 ° C and simultaneously with maximum power (9 on the Crest system). The device is then soaked for 5 min at room temperature in 500 mL isopropanol and simultaneously with maximum power. The device is immersed in 300 mL of 200 proof alcohol (proof) ethanol and blown dry with N2. The functionalized surface is activated to serve as the support for polynucleotide synthesis.

[0173] Example 2: Highly Accurate DNA-Based Information Storage and Assembly

[0174] Digital information in the form of binary data, totaling approximately 0.2 GB, was selected, including the text of the Universal Declaration of Human Rights in over 100 languages, the first 100 books from Project Guttenberg, and a seed database. The digital information was encrypted into a nucleic acid-based sequence and divided into strings. Over 10 million unique polynucleotides, each corresponding to a string, were synthesized on a rigid silicon surface. Each unique polynucleotide was 200 bases or less in length. The synthesized polynucleotides were collected, sequenced, and decoded back into a digital code that was 100% accurate for the source digital information compared to at least one initial digital sequence.

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

[0176] Polynucleotides are synthesized de novo using 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), implemented as an addressable array of 10-gigabyte packets. Physical partitioning is such 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 used 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. Polynucleotide lengths range from 100-1000 bases. The droplet volume is equivalent to a sphere with a diameter of 40-50 μm. The solid support further comprises 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 . Figure 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. Figure 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 can be retrieved from multiple data packets based on this sequence information.

[0179] Example 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 solid support measures 3.5 inches by 2.5 inches. The solid support has a capacity of 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, Figure 17 and Figure 18 .

[0181] 10-100 megabytes of at least one item of information is encoded in DNA and stored in 1 petabyte of data. At a later time, the 10-100 megabytes of encoded DNA can be 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 the polynucleotides comprising dsDNA was approximately 80 nm, while the length of the polynucleotides comprising ssDNA was approximately 160 nm. The solid support contained an array of 500 nm (depth) x 400 nm (diameter) wells (volume approximately 0.628 femtoliters). Each well contained an addressable locus, an addressable electrode (area 50,000 nm) in the sidewalls of each well, and a 240 base polynucleotide. 2 / electrode) and a 250 nm (diameter) surface for polynucleotide synthesis / attachment, in addressable communication with a 250 nm (diameter) addressable bottom electrode. The electrodes are independently addressable.

[0184] Polynucleotides are 1 polynucleotide / 50nm 2 The density of the pores is present on the synthesized surface. The pore spacing is 1.0 μm. The 10 nm thick sidewall electrodes (located approximately 100 nm above the polynucleotide surface) are charged to generate H + A gradient of ions is applied to remove protecting groups from the 5' OH groups of polynucleotides defined at sites on the synthesis surface (where the polynucleotides are blocked with acid-cleavable blocking groups). + ions. (See Figure 16B ). 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.

[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 sidewalls of each well, and a 50,000 nm (diameter) well. 2 / electrode) and a 250 nm (diameter) surface for polynucleotide synthesis / attachment, in addressable communication with a 250 nm (diameter) addressable bottom electrode. The electrodes are independently addressable.

[0187] Polynucleotides are 1 polynucleotide / 50nm 2 The density is present on the synthesis surface. The spacing between the holes is 1.0 μm. Nucleoside phosphoramidite monomers are added, and the polynucleotide is at an unblocked site and can be used 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, thereby 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 the reusable linker attached to the surface, and the synthesis is repeated.

[0188] Example 7: Local Control of Polynucleotide Synthesis on a Solid Support

[0189] 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 with a diameter of 100 nm on the surface for polynucleotide synthesis / attachment (see Figure 19 ).

[0190] Polynucleotides are 1 polynucleotide / 39.27nm 2 The density of ions exists on the synthetic surface. The pore spacing is 0.25 μm. 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 sites 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, 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.

[0191] The present invention also provides the following items:

[0192] 1. A device for storing information, comprising:

[0193] 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:

[0194] a synthetic surface located in a bottom region of each of said wells;

[0195] a bottom electrode in addressable communication with the composite surface; and

[0196] 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 away from the bottom region.

[0197] 2. The device according to item 1, wherein the solid support has a thickness of at least 100 x 10 6 Addressable seats / cm 2 The density includes addressable seating.

[0198] 3. The device according to item 1, wherein the solid support is 100 x 10 6 Up to 100x 10 7 Addressable seats / cm 2 The density includes addressable seating.

[0199] 4. The device of any one of items 1 to 3, wherein the addressable loci have a diameter of at most about 750 nm.

[0200] 5. The device of any one of items 1 to 4, wherein each of the holes has a depth of at most about 1000 nm.

[0201] 6. The device according to any one of items 1 to 4, wherein each of the holes has a depth of 100 nm to 1000 nm.

[0202] 7. The device according to any one of items 1 to 6, wherein each of the pores has a longest cross-sectional diameter of 100 nm to 800 nm.

[0203] 8. The device according to any one of items 1 to 7, wherein each of the holes is cylindrical.

[0204] 9. The device according to any one of items 1 to 8, wherein the bottom electrode has 10 4 nm 2 to 10 5 nm 2 The longest cross-sectional area.

[0205] 10. The device of any one of items 1 to 9, wherein the at least one sidewall electrode is 75 nm to 125 nm from the bottom region.

[0206] 11. The device of any one of items 1 to 10, wherein the at least one sidewall electrode has a height of 5 nm to 25 nm.

[0207] 12. The device of any one of items 1 to 11, comprising at least two sidewall electrodes.

[0208] 13. The device of any one of items 1 to 12, wherein the at least one sidewall electrode and the bottom electrode are independently addressable.

[0209] 14. A device for storing information, comprising:

[0210] 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:

[0211] a synthetic surface located in a bottom region of each of said wells;

[0212] a bottom electrode in addressable communication with the synthetic surface;

[0213] at least one sidewall electrode located on a sidewall of each of said holes,

[0214] wherein the synthetic surface at each addressable locus comprises at least one polynucleotide extending from said synthetic surface, and wherein polynucleotides comprising different sequences are arranged on said solid support at a density of at least 100 x 10 6 polynucleotides / cm 2 The density exists.

[0215] 15. The device according to item 14, wherein the solid support has a thickness of at least 100 x 10 7 polynucleotides / cm 2 The density contains polynucleotides of different sequences.

[0216] 16. The device according to item 14, wherein the solid support is 100 x 10 6 Up to 100x10 7 polynucleotides / cm 2 The density includes addressable seating.

[0217] 17. The device of any one of items 14 to 16, wherein each of the holes has a depth of at most about 1000 nm.

[0218] 18. The device according to any one of items 14 to 16, wherein each of the holes has a depth of 100 nm to 1000 nm.

[0219] 19. The device of any one of items 14 to 18, wherein the addressable loci have a diameter of at most about 750 nm.

[0220] 20. The device of any one of items 14 to 18, wherein each of the pores has a longest cross-sectional diameter of 100 nm to 800 nm.

[0221] 21. The device according to any one of items 14 to 20, wherein each of the holes is cylindrical.

[0222] 22. The device according to any one of items 14 to 21, wherein the bottom electrode has 10 4 nm 2 to 10 5 nm 2 The longest cross-sectional area.

[0223] 23. The device of any one of items 14 to 22, wherein the at least one sidewall electrode is 50 nm to 200 nm from the bottom region.

[0224] 24. The device of any one of items 14 to 23, wherein the at least one sidewall electrode has a height of 5 nm to 25 nm.

[0225] 25. The device of any one of items 14 to 24, comprising at least two sidewall electrodes.

[0226] 26. The device of any one of items 14 to 25, wherein the at least one sidewall electrode and the base electrode are independently addressable.

[0227] 27. A method for storing information, comprising:

[0228] a) Providing the device of any one of items 1 to 26;

[0229] b) providing instructions for polynucleotide synthesis;

[0230] 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; and

[0231] 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.

[0232] 28. The method according to item 27, wherein the method further comprises cleaving at least one polynucleotide from the surface, wherein the polynucleotide is dissolved in the droplets.

[0233] 29. The method according to item 27 or 28, wherein the method further comprises sequencing at least one polynucleotide from the surface.

[0234] 30. The method of any one of items 27 to 29, wherein the nucleoside comprises a nucleoside phosphoramidite.

[0235] 31. The method of any one of items 27 to 30, wherein the method further comprises drying the surface.

[0236] 32. A method according to any one of items 27 to 31, wherein the method further comprises a cutting step, wherein the cutting step comprises applying an electric potential to the bottom electrode to generate a cutting agent.

[0237] 33. The method according to any one of items 27 to 32, wherein the method further comprises a capping step.

[0238] 34. The method according to items 27 to 33, wherein the method further comprises an oxidation step.

[0239] 35. A method according to items 27 to 34, 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.

[0240] 36. A method for storing information, comprising:

[0241] a) providing a solid support comprising a surface;

[0242] 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

[0243] 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 density of at least 100×10 6 polynucleotides / cm 2 The density exists.

[0244] 37. The method according to item 36, wherein the density of addressable loci on the solid support is at least 100 x 10 7 polynucleotides / cm 2 .

[0245] 38. The method according to item 36, wherein the density of addressable loci on the solid support is 100x10 6 Up to 100x 10 7 polynucleotides / cm 2 .

[0246] 39. The method according to any one of items 36 to 38, wherein the method further comprises cleaving at least one polynucleotide from the surface, wherein the polynucleotide is dissolved in the droplets.

[0247] 40. The method of any one of items 36 or 39, wherein the method further comprises sequencing at least one polynucleotide from the surface.

[0248] 41. The method of any one of items 36 to 40, wherein the nucleoside comprises a nucleoside phosphoramidite.

[0249] 42. The method of any one of items 36 to 41, wherein the method further comprises drying the surface.

[0250] 43. The method of any one of items 36 to 42, wherein the method further comprises washing the nucleoside from the surface.

[0251] 44. The method according to any one of items 36 to 43, wherein the method further comprises a capping step.

[0252] 45. The method according to any one of items 36 to 44, wherein the method further comprises an oxidation step.

[0253] 46. The method according to any one of items 36 to 45, wherein the method further comprises a deblocking step.

[0254] 47. A method for storing information, comprising:

[0255] a) providing a solid support comprising a surface;

[0256] 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

[0257] c) repeating step b) to synthesize a plurality of polynucleotides on the surface, wherein the volume of the droplet is less than about 100 femtoliters.

[0258] 48. The method of item 47, wherein the droplets have a volume of less than about 50 femtoliters.

[0259] 49. The method of item 47, wherein the droplets have a volume of less than about 25 femtoliters to 100 femtoliters.

[0260] 50. The method according to any one of items 47 to 49, wherein the method further comprises cleaving at least one polynucleotide from the surface, wherein the polynucleotide is dissolved in the droplets.

[0261] 51. The method of any one of items 47 or 50, wherein the method further comprises sequencing at least one polynucleotide from the surface.

[0262] 52. The method of any one of items 47 to 51, wherein the nucleoside comprises a nucleoside phosphoramidite.

[0263] 53. The method of any one of items 47 to 52, wherein the method further comprises drying the surface.

[0264] 54. The method of any one of items 47 to 53, wherein the method further comprises washing the nucleoside from the surface.

[0265] 55. The method according to any one of items 47 to 54, wherein the method further comprises a capping step.

[0266] 56. The method according to any one of items 47 to 55, wherein the method further comprises an oxidation step.

[0267] 57. The method according to any one of items 47 to 56, wherein the method further comprises a deblocking step.

[0268] 58. A method for storing information, comprising:

[0269] a) providing a solid support comprising a surface;

[0270] 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

[0271] 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.

[0272] 59. The method of item 58, wherein the time for repeating step b) using four different nucleotides is less than about 50 milliseconds.

[0273] 60. The method according to item 58, wherein the time for repeating step b) using four different nucleotides is 25 milliseconds to 100 milliseconds.

[0274] 61. The method according to any one of items 58 to 60, wherein the method further comprises cleaving at least one polynucleotide from the surface, wherein the polynucleotide is dissolved in the droplets.

[0275] 62. A method according to any one of items 58 to 61, wherein the method further comprises sequencing at least one polynucleotide from the surface.

[0276] 63. The method of any one of items 58 to 62, wherein the nucleoside comprises a nucleoside phosphoramidite.

[0277] 64. The method of any one of items 58 to 63, wherein the method further comprises drying the surface.

[0278] 65. The method of any one of items 58 to 64, wherein the method further comprises washing the nucleoside from the surface.

[0279] 66. The method according to any one of items 58 to 65, wherein the method further comprises a capping step.

[0280] 67. The method according to any one of items 58 to 66, wherein the method further comprises an oxidation step.

[0281] 68. The method according to any one of items 58 to 67, wherein the method further comprises a deblocking step.

[0282] 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 implementing the present invention. It is to be understood that various alternatives to the embodiments of the present invention described herein may be employed. It is intended that the scope of the present invention be defined by the appended claims, and that methods and structures and their equivalents within the scope of these claims be encompassed thereby.

Claims

1. A method for storing nucleic acid-based digital information, comprising: An apparatus is provided, comprising: A solid support comprising a plurality of wells, wherein each well comprises: a synthetic surface located in a bottom region of each of said wells; bottom electrode; and a sidewall electrode located on a sidewall of each of the holes; coupling blocked nucleosides to polynucleotides on the synthetic surface attached to wells in the plurality of wells; as well as An electric potential is applied to the sidewall electrodes of the pore, thereby deblocking the blocked nucleosides.

2. The method of claim 1, further comprising cleaving the polynucleotide from the synthesis surface and dissolving the polynucleotide in small droplets.

3. The method of claim 1, further comprising applying an electric potential to the bottom electrode of the well to generate a cleavage reagent. The method of claim 2 , further comprising sequencing the polynucleotide.

5. The method of claim 1, wherein the blocked nucleoside comprises a nucleoside phosphoramidite. 6 . The method of claim 1 , further comprising applying a capping agent to the pore, wherein the capping agent blocks unreacted 5′-OH groups of the polynucleotide.

7. The method of claim 6, wherein the capping agent comprises acetic anhydride, 1-methylimidazole, or a combination thereof.

8. The method of claim 1, further comprising applying an oxidizing agent to the pore to oxidize the phosphite triester of the polynucleotide to a tetracoordinated phosphotriester.

9. The method of claim 8, wherein the oxidizing agent comprises tert-butyl hydroperoxide, (1S)-(+)-(10-camphorsulfonyl)-oxaziridine, or a combination thereof.

10. The method of claim 1, wherein applying an electric potential to the sidewall electrode generates a deprotecting reagent in the vicinity of the blocked nucleoside.

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