EA039806B1 - Dna-based digital information storage - Google Patents

Abstract

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

Description

State of the art

Information storage systems based on biomolecules, such as DNA, are characterized by high storage capacity and stability over time. However, there is a need for scalable, automated, high-precision and high-performance systems for obtaining biomolecules for the purpose of storing information.

The essence of the invention

Provided herein are information storage devices comprising a solid support, the solid support comprising a plurality of wells, each of the wells comprising an addressable locus comprising a synthesis surface located in the lower region of each of the wells; a bottom electrode in addressable communication with the synthesis surface; and at least one side electrode located on the side wall of each of the wells, with at least one side electrode located at a distance of 50 to 200 nm from the lower region. Also provided herein are devices where the solid support contains addressable loci at a density of at least 100x106 addressable loci per cm ² . Also provided herein are devices where the solid support contains addressable loci at a density of 100x106 to 100x107 addressable loci per cm ² . Also provided herein are devices where the targeted locus contains a diameter of up to about 750 nm. Also provided herein are devices where each of the wells contains a depth of up to about 1000 nm. Also provided herein are devices where each of the wells contains a depth of 100 to 1000 nm. In addition, devices are presented herein, where each of the wells contains the largest cross-sectional diameter from 100 to 800 nm. In addition, devices are presented herein where each of the wells is cylindrical. In addition, this document presents devices where the bottom electrode contains the largest cross-sectional area from 10 ⁴ to 105 nm ² . Also provided herein are devices where at least one side electrode is between 50 and 200 nm from the bottom region. Also provided herein are devices where at least one side electrode comprises a height of 5 to 25 nm. Also provided herein are devices comprising at least two ground electrodes. Also provided herein are devices where at least one side electrode and bottom electrode are independently addressable.

Provided herein are information storage devices comprising a solid support, the solid support comprising a plurality of wells, each of the wells comprising an addressable locus comprising a synthesis surface located in the lower region of each of the wells; a bottom electrode in addressable communication with the synthesis surface; at least one side electrode located on the side wall of each of the wells, and the surface for synthesis in each addressable locus contains at least one polynucleotide extending from the surface for synthesis, and moreover, polynucleotides containing different sequences on a solid support are present with a density of at least measure 100x106 polynucleotides per 1 cm ² . Also provided herein are devices wherein the solid support contains polynucleotides of different sequences at a density of at least 100 x 107 polynucleotides per cm ² . Also provided herein are devices where the solid support contains addressable loci at a density of 100x106 to 100x107 polynucleotides/cm ² . Also provided herein are devices where each of the wells contains a depth of up to about 1000 nm. Also provided herein are devices where each of the wells contains a depth of 100 to 1000 nm. Also provided herein are devices where the targeted locus contains a diameter of up to about 750 nm. Also provided herein are devices where each of the wells has a largest cross-sectional diameter of 100 nm to 800 nm. In addition, devices are presented herein where each of the wells is cylindrical. In addition, this document presents devices where the bottom electrode contains the largest cross-sectional area from 10 ⁴ to 105 nm ² . Also provided herein are devices where at least one side electrode is between 50 and 200 nm from the bottom region. Also provided herein are devices where at least one ground electrode has a height of 5 to 25 nm. Also provided herein are devices comprising at least two ground electrodes. Also provided herein are devices where at least one side electrode and the main electrode are independently addressable.

- 1 039806

This document presents methods for storing information, including (a) providing the device described herein; (b) providing instructions for the synthesis of polynucleotides; (c) introducing at least one nucleoside onto the synthesis surface, the at least one nucleoside being coupled to the polynucleotide attached to the synthesis surface; and (d) repeating step c) to synthesize a plurality of polynucleotides on a synthesis surface, the instructions providing for at least one sequence encoding the plurality of polynucleotides. Also provided herein are methods further comprising cleaving at least one polynucleotide from a surface, wherein the polynucleotide is dissolved in the droplet. Also provided herein are methods further comprising sequencing at least one polynucleotide from a surface. Also provided herein are methods in which the nucleoside provides for a nucleoside amidophosphite. Also provided herein are methods further comprising a cleavage step, wherein the cleavage step comprises applying an electrical potential to the bottom electrode to generate a cleavage reagent. In addition, methods are presented herein, said method further comprising drying the surface. Also provided herein are methods further comprising washing nucleosides from a surface. In addition, methods are presented herein, said method further comprising a capping step. In addition, methods are provided herein, said method further comprising an oxidation step. Also provided herein are methods, said method further comprising a deblocking step, the deblocking step comprising applying an electrical potential to at least one side electrode to generate a deprotectant.

This document presents methods for storing information, including (a) providing a solid substrate containing a surface; (b) introducing at least one nucleoside onto the surface, the at least one nucleoside being coupled to the polynucleotide attached to the surface; and (c) repeating step b) to synthesize a plurality of polynucleotides on the surface, wherein polynucleotides having different sequences are present on the surface at a density of at least 100 x 10 ⁶ polynucleotides/cm ² . Also provided herein are methods in which the density of addressable loci on a solid support is at least 100 x 10 ⁷ polynucleotides/cm ² . Also provided herein are methods in which the density of addressable loci on a solid support is from 100x10 ⁶ to 100x10 ⁷ polynucleotides/cm ² . Also provided herein are methods, said method further comprising cleaving at least one polynucleotide from a surface, the polynucleotide being dissolved in the droplet. Also provided herein are methods, said method further comprising sequencing at least one polynucleotide from a surface. Also provided herein are methods in which the nucleoside provides for a nucleoside amidophosphite. In addition, methods are presented herein, said method further comprising drying the surface. In addition, methods are provided herein, said method further comprising flushing nucleosides from a surface. In addition, methods are presented herein, said method further comprising a capping step. In addition, methods are presented herein, said method further comprising an oxidation step. In addition, methods are presented herein, said method further comprising a deblocking step.

This document presents methods for storing information, including (a) providing a solid substrate containing a surface; (b) applying to the surface of a droplet containing at least one nucleoside, wherein at least one nucleoside reacts with a polynucleotide attached to the surface; and (c) repeating step b) to effect the synthesis of a plurality of polynucleotides on the surface, wherein the droplet volume is less than about 100 femtoliters. Also provided herein are methods in which the droplet volume is less than about 50 femtoliters. Also provided herein are methods in which the droplet volume is less than about 25 to 100 femtoliters. Also provided herein are methods, said method further comprising cleaving at least one polynucleotide from a surface, the polynucleotide being dissolved in the droplet. Also provided herein are methods, said method further comprising sequencing at least one polynucleotide from a surface. Also provided herein are methods in which the nucleoside provides for a nucleoside amidophosphite. In addition, methods are presented herein, said method further comprising drying the surface. In addition, methods are provided herein, said method further comprising rinsing nucleosides from a surface. Besides, in

- 2 039806 the present document presents the methods, and the specified method further includes the step of capping. In addition, methods are provided herein, said method further comprising an oxidation step. In addition, methods are presented herein, said method further comprising a deblocking step.

This document presents methods for storing information, including (a) providing a solid substrate containing a surface; (b) introducing at least one nucleoside onto the surface, the at least one nucleoside being coupled to the polynucleotide attached to the surface; and (c) repeating step b) to synthesize a plurality of surface polynucleotides, wherein the time to repeat step b) using four different nucleotides is less than about 100 ms. Also provided herein are methods in which the time to repeat step b) using four different nucleotides is less than about 50 ms. Also provided herein are methods in which the time to repeat step b) using four different nucleotides is between 25 and 100 ms. Also provided herein are methods, said method further comprising cleaving at least one polynucleotide from a surface, the polynucleotide being dissolved in the droplet. Also provided herein are methods further comprising sequencing at least one polynucleotide from a surface. Also provided herein are methods in which the nucleoside provides for a nucleoside amidophosphite. In addition, methods are presented herein, said method further comprising drying the surface. In addition, methods are provided herein, said method further comprising rinsing nucleosides from a surface. In addition, methods are presented herein, said method further comprising a capping step. In addition, methods are provided herein, said method further comprising an oxidation step. In addition, methods are presented herein, said method further comprising a deblocking step.

Inclusion by reference

All publications, patents, and patent applications referred to in this specification are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application were specifically and individually designated for inclusion by reference.

Brief description of the drawings

New features of the present invention are detailed in the appended claims. A better understanding of the features and advantages of the present invention will be provided with reference to the following detailed description, which sets forth illustrative embodiments that use the principles of the present invention and the accompanying drawings, including:

in fig. 1 shows an exemplary workflow for storing nucleic acid data;

in fig. 2 shows a plate capable of synthesizing polynucleotides containing 24 regions, or subfields, each containing an array of 256 clusters;

in fig. 3 shows an enlarged view of the subfields of FIG. 2 with 16x16 clusters, with each cluster containing 121 distinct loci;

in fig. 4 shows a detailed view of the cluster from FIG. 2, where the cluster contains 121 loci;

in fig. 5A is a front view of a tablet with multiple channels;

in fig. 5B is a sectional view of a plate with multiple channels;

in fig. 6A, 6B show continuous loop and coil formats for flexible structures;

in fig. 6C, 6D are schemes for the release and extraction of the synthesized polynucleotides;

in fig. 7A-7C are enlarged views of a flexible structure containing spots, channels, or pits, respectively;

in fig. 8 shows an example computer system;

fig. 9 is a block diagram showing the architecture of a computer system;

fig. 10 is a diagram showing a network for connecting a plurality of computer systems, a plurality of mobile phones and personal digital assistants, and a network attached storage (NAS);

fig. 11 is a block diagram of a multiprocessor computer system using a shared virtual address memory space;

fig. 12A is the front side of an exemplary array on a solid substrate;

fig. 12B is the back of an exemplary array on a solid substrate;

fig. 13 is a diagram of a solid substrate containing an active region and a microfluidic contact surface;

in fig. 14 shows an example of an instrument in rack format;

in fig. 15 shows a solid support containing addressable regions for the synthesis or storage of nucleic acids;

in fig. 16A shows an array for synthesis using electrochemical processes;

in fig. 16B shows an array for synthesis using electrochemical processes;

in fig. 17 shows wells for the synthesis or storage of nucleic acids and the spacing between wells;

in fig. 18 shows an example of a hard substrate containing an addressable array;

in fig. 19 shows an example of an array on a solid support, where the pitch is approximately equal to the length of a 240-mer polynucleotide.

Detailed description of the invention

There is a need for larger memory systems as the amount of information generated and stored increases exponentially. Traditional storage media have limited capacity and require specialized technologies that change over time, requiring constant transfer of data to new media, often at great cost. A biomolecule, such as DNA, provides a suitable host for information storage, in part due to its stability over time and its ability to encode information in four bits, as opposed to traditional binary encoding of information. Thus, large amounts of data are encoded in DNA in a relatively smaller amount of physical space compared to that used by commercially available information storage devices. This document provides methods for improving the throughput of DNA synthesis by increasing sequence density and reducing turnaround time.

Definitions

Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as would be commonly understood by one of ordinary skill in the art to which the present inventions relate.

Throughout the present disclosure, quantitative characteristics are presented in range format. It should be understood that the description in range format is for convenience and brevity only and should not be construed as an inflexible limitation on the scope of any embodiments. Accordingly, a description of a range should be construed as specifically disclosing all possible subranges, as well as individual numerical values within a range up to a tenth of a lower limit unit, unless the context clearly indicates otherwise. For example, a description of a range such as 1 to 6 should be considered as specifically disclosing subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6. and the like, as well as individual values within such a range, such as 1.1, 2, 2.3, 5, and 5.9. This principle applies regardless of the range width. The upper and lower limits of such intermediate ranges can be independently included in the smaller ranges and are also covered by the present invention, subject to any specifically excluded limit in the specified range. In cases where the specified range includes one or both of the limits, ranges excluding one or both of such included limits are also included in the present invention, unless the context clearly indicates otherwise.

The terminology used herein is only intended to describe specific embodiments and is not intended to limit any embodiment. In the context of this document, the singular is intended to include the plural as well, unless the context clearly indicates otherwise. In addition, it should be understood that the terms includes (contains, provides for) and / or including (containing, providing) when used in the present description determine the presence of the specified features, integers, steps, actions, elements and / or components, but do not exclude the presence or adding one or more other features, integers, steps, actions, elements, components, and/or groups thereof. In the context of this document, the term and/or includes any combination of one or more of its associated listed items.

Unless specifically stated or obvious from the context, in the context of this document, the term approximately in relation to a number or range of numbers should be understood to mean the specified number and numbers ± 10% of them, or 10% less than the lower specified limit and 10% greater than the upper the specified limit for the values listed in the range.

In the context of this document, the terms preselected sequence, predetermined sequence, or predetermined sequence are used interchangeably. These terms mean that the sequence of the polymer is known and chosen before the synthesis or assembly of the polymer. In particular, various aspects of the present invention are described herein primarily with respect to the production of nucleic acid molecules, wherein the sequence of the polynucleotide is known and selected prior to the synthesis or assembly of the nucleic acid molecules.

Provided herein are methods and compositions for the production of synthetic (ie, de novo synthesized or chemically synthesized) polynucleotides. Polynucleotides may also be referred to as oligonucleotides or oligos. Polynucleotide sequences described in

- 4 039806 herein, unless otherwise indicated, may include DNA or RNA.

Synthesis and storage of nucleic acids on a solid substrate.

This document describes devices, compositions, systems and methods for synthesizing and storing nucleic acids on a chip. In some instances, polynucleotides are synthesized de novo using solid support methods as described herein. In some cases, the polynucleotides are stored on a solid support after synthesis. In some instances, the solid support methods described herein are used solely for storage.

The present document describes devices, compositions, systems, and methods for synthesizing and storing nucleic acids on a solid support, wherein one or more nucleic acid synthesizer components are integrated into the solid support. The components or functional equivalents of the components may include temperature controllers, addressable electrodes, semi-conductive surfaces, fluid reservoirs, inkjet elements, synthesis surfaces, power supplies, or other component used for polynucleotide synthesis. Any combination of integrated components is suitable for use with the devices, compositions, systems, and methods described herein. In some cases, one or more components are outside (not integrated) of the solid support.

The density of unique loci for polynucleotide synthesis on the surface is often controlled by the spatial resolution provided by the addition of reagents. In addition, applying reagents to unique sites requires moving either the reagent application device or the receiving surface to move the reagent application site from one site to another. Alternatively, the combination of bases is locally controlled at certain sites on the surface for synthesis without moving the surface or the device for introducing reagents. Local control is achieved through an array of addressable electrodes 1503, with each electrode controlling a combination of nucleosides (nucleoside amidophosphites) through electrochemical processes at specific loci on the surface 1505. communication with the surface for synthesis. In some cases, the electrodes are side electrodes located on the side wall of the well. However, the traditional electrochemical method on a two-dimensional matrix is in some cases limited by the step size (see Fig. 19). At high density values (for example, in the case of a small step 1507), the length of growing DNA oligonucleotides 1601 can reach a distance from one synthesis site to adjacent synthesis sites, which leads to mixing of discrete reaction products. In order to avoid mixing of adjacent reaction products, step size 1507 is increased in some embodiments. Diffusion through the 1901 reaction medium is an additional factor that affects the spatial control of polynucleotide synthesis. Thus, it is advisable to isolate active sites in order to achieve higher array density values. Nucleoside coupling is controlled by local electrodes through any number of manipulations, such as generation of local reactants, local removal of reactants, reactant repulsion, solvent volume limitation, solvent attraction, or other electrochemical or physical manipulation that affects one or more steps in the coupling process. grounds. In some cases, a device 1500 is used for the synthesis of polynucleotides, providing a plate 1501 with wells 1502 (see Fig. 15). The bottom 1505 of each well contains an electrode 1503 from which polynucleotides are synthesized. Each well is characterized by a cross-sectional diameter 1506 and a step size 1507 between any two wells. The side walls 1504 of the well, in some cases, contain one or more side electrodes (not shown).

Coupling control is in some cases carried out directly by controlling the nucleoside addition step, the deprotection step, or another step that affects the efficiency of the nucleoside coupling reaction. In some cases, the pattern of electrodes is charged to create a concentration gradient of H ⁺ 1602 ions at certain sites near the surface for synthesis (see Fig. 16A); the 1601 polynucleotides at such sites are unblocked (where the polynucleotide is blocked with an acid-cleavable blocking group) and will be available for coupling with nucleosides.

Devices are described herein that provide a solid support, the solid support comprising a plurality of wells, each of the wells containing an addressable locus comprising: a synthesis surface located in the lower region of each of the wells; and at least one side electrode located on the side wall of each of the wells, and the electrochemical generation of reagents is separated in space from the point of attachment of the polynucleotide to the synthesis surface. In some instances, the devices described herein also include a bottom electrode in addressable communication with the synthesis surface. For example, ground electrodes 1603 can be used to control adhesion of substrates or reagents (see FIG. 16B). In some cases, the reactants provide for protons or another acidic molecule. In some services

In some cases, the ground electrodes 1603 are located at specific locations along the edges of the well surface (see FIG. 16B) in the case of a well having a depth of 1604. In some cases, the ground electrodes provide control of the chemical reactions occurring near the synthesis surface. For example, if an acid or other reagent is generated near a synthesis surface, the portion of the 1601 polynucleotide bound to that surface will contact a higher concentration of acid than the portion of the polynucleotide that is distal to the site of acid generation. This may result in degradation of the portion of the polynucleotide that is exposed to higher concentrations of acid. The side electrodes 1603 in some cases create or control a proton concentration gradient 1602, resulting in a uniform or directed effect of the acid on a portion of the 1601 polynucleotide. before adding the next nucleoside. By applying a series of electrode-controlled masks to the surface, the desired polynucleotides are synthesized at precise locations on the surface. In addition, local coupling control in some cases allows for shorter synthesis steps, fewer reagents/materials (due to higher polynucleotide density and reduced scale), and reduced synthesis time (no surface movement for synthesis). The wells in some cases contain one, two, three, four or more than four ground electrodes. In some cases, the wells contain two side electrodes. In some cases, each side electrode is independently addressable. For example, different voltages are applied to two or more different side electrodes. Such schemes, in some cases, facilitate the diffusion of reagents or polynucleotides in a certain plane between two side electrodes. Such ground electrodes are in some cases annular or arranged continuously around the circumference of the well's cross section. In some cases, the side electrodes are discontinuous or only partially cover part of the side wall surface. For example, the side electrode is located continuously over about 5%, 10%, 10%, 30%, 50%, 75%, or about 90% of the circumference of the well's cross section. Such ground electrodes are in some cases characterized by a height about equal to the height of the well, or about 5, 10 15, 30, 50, 75, or about 90% of the height of the well. In some cases, applying different voltages independently to two or more discontinuous ground electrodes causes the reagents or polynucleotides to diffuse in the horizontal plane. In some cases, applying different voltages independently to two or more discontinuous side electrodes causes the reagents or polynucleotides to diffuse in the vertical plane.

The synthesis of polynucleotides in general requires the repeated application of liquids (fluid elements) to and from the synthesis surface. In some instances, bulk fluid movement results in fluid loss (loss by wetting, volume flow, or reaction wells), resulting in low reagent efficiency and increased cycle times due to fluid movement. An alternative to jet elements that provide mass movement of fluids in the synthesis process is the use of jet elements with digital control, and the reagents or reaction tanks are presented in the form of discrete drops. Droplets are sometimes mixed, moved (dipped, reacted), separated, stored, added, removed, or analyzed in discrete volumes by manipulation through a surface containing insulated (or semiconductor coated) electrodes, or by electrochemical wetting. Electrochemical wetting provides local control of the fluid-surface interaction; for example, activation of an electrode near a drop results in separation of the drop. In some cases, the droplets described herein have a small volume. For example, the drop volume is up to 10, 20, 50, 75, 100, 125, 150, 200, 300, 500, 800 or more than 1000 femtoliters. In some cases, the droplet volume is from about 50 to about 200 femtoliters. In some instances, digitally controlled fluid elements result in a cycle time reduction of at least 2, 3, 4, 7, 10, or more than 10 times compared to bulk fluid fluid elements. In some instances, digitally controlled fluid elements result in a cycle time reduction of at least 2 to 10 times compared to fluid mass transfer fluid elements. In some cases, the execution time of one cycle (sequential combination of 4 bases, including washes) is about 1, 2, 3, 5, 7, 10, 12, 15, 17, 20, 30, 50, 100, or about 200 ms. In some cases, the execution time of one cycle is up to 1, 2, 3, 5, 7, 10, 12, 15, 17, 20, 30, 50, 100, or up to 200 ms. In some cases, the execution time of one cycle is from about 10 to about 50 ms.

The movement of fluids on or off the surfaces described herein may include modifications or conditions that prevent undesirable fluid movement or other phenomena. For example, the movement of a fluid in some cases results in the formation of bubbles or accumulations of gas, which limits the contact of fluids with components such as surfaces or polynucleotides. Described in this document

- 6 039806 methods, systems and compositions provide various ways to control or minimize the formation of bubbles. Such methods include fluid pressure control, well geometry, or surface materials/coatings. The specific geometry of the wells can minimize the formation of bubbles. For example, constriction of the wells, channels, or other surface may reduce or prevent the formation of bubbles during fluid movement. Surface materials having specific wettability properties can reduce or prevent bubbling. For example, the surfaces described herein contain hydrophobic materials. In some cases, the surfaces described herein contain hydrophilic materials. Pressure can be applied to control the formation of bubbles during the movement of the fluid. The pressure is in some cases locally applied to the component, surface area, capillary/channel, or it is applied to the entire system. In some cases, pressure is applied either behind or in front of the fluid transfer region. In some cases, back pressure is applied to prevent bubble formation. Suitable pressures used to prevent bubble formation may vary depending on the fluid, scale, flow geometry, and materials used. For example, the system maintains a pressure of 5 to 10 atm. In some cases, a pressure of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 20 or more than 50 atm is applied. In some cases, pressures up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 20 or up to 50 atm are applied. In some cases, pressure is applied from about 2 to about 10, from about 2 to about 8, from about 2 to about 5, from about 4 to about 10, from about 4 to about 12, from about 5 to about 15, from about 5 to about 7, from about 7 to about 20, from about 8 to about 15, or from about 10 to about 20 atm.

The devices described herein may use controllers to control environmental conditions such as temperature. Temperature controllers are often used to create or maintain storage conditions for solid supports containing polynucleotides. Storage conditions for nucleic acids can affect their long-term stability, which directly affects the quality of retrieved information stored in digital form. The polynucleotides are optionally stored at a low temperature (eg, 10, 4, 0° C. or below) on a solid support, with a temperature controller maintaining the temperature of the solid support. The storage medium for polynucleotides on a solid support, such as in solvated or dry form, also affects storage stability. In some cases, the polynucleotides are stored in a solution, such as an aqueous solution or buffer, in the form of drops. In some cases, the polynucleotides are stored in lyophilized (dry) form. Temperature controllers in some cases raise the temperature of the chip to help dry the polynucleotides attached to it. In some cases, the temperature controllers also provide local control of heating at targeted locations on the solid substrate. In some cases, after addition of droplets containing polynucleotides to the solid support, the solid support is dried. In some cases, the contents of the dried solid support are subsequently resolvated. In some cases, the solid support is stored for later use. In some cases, the solid support further comprises a polynucleotide map. In some cases, the solid support further provides metadata.

The devices described in this document may include power supplies used to supply power to various components of the device. The components for synthesis on a solid substrate are optionally powered by an external power supply, or the power supply is integrated into the solid substrate. Power sources may include batteries, solar panels, thermoelectric generators, induction (wireless) power supplies, kinetic charger, cell phones, tablets, or other power source suitable for use with the synthesis components or devices described herein. In some instances, the synthesis components, surfaces, or devices described herein are portable.

Fluids containing reagents, wash solvents, or other synthesis components are applied to the synthesis surface. Unused fluid (before contact with the synthesis surface) or spent fluid (after contact with the synthesis surface) is in some cases stored in one or more cells integrated into the solid support. Alternatively, or in combination, polynucleotides are placed on or removed from a solid support for external analysis or storage. For example, synthesized polynucleotides are cleaved from loci on a solid support in the form of a drop, and the resulting drop is moved outside the synthesis site on the solid support. If stored, the drop is optionally dried. In some cases, fluids are stored outside of the solid support. In some instances, the apparatus described herein includes a solid support with a plurality of fluid ports that allow fluids to move into or out of the solid support. In some cases, the holes are arranged on the side walls of the solid support, however, other configurations are also suitable for delivering fluids to the surface for synthesis. Such a device often contains, for example, at least 1, 2, 5, 10, 20, 50, 100, 200, 500, 1000, 2000, 5000 or at least

- 7 039806 measure 10,000 holes/mm of the length of the solid substrate. In some cases, the device described herein contains from about 100 to about 5000 holes/mm of the length of the solid substrate.

This document describes addressable electrodes integrated into a solid substrate. The electrodes, without limitation, include conductors, insulators, or semiconductors, and are made from materials well known in the art. The materials may include metals, non-metals, mixed metal oxides, nitrides, carbides, silicon-based materials, or other material. In some cases, metal oxides include TiO2, Ta ₂ O ₅ , Nb ₂ O ₅ , Al ₂ O ₃ , BaO, Y ₂ O ₃ , HfO ₂ , SrO or other metal oxide known in the art. In some cases, metal carbides include TiC, WC, ThC ₂ , ThC, VC, W2C, ZrC, HfC, NbC, TaC, Ta ₂ C, or other metal carbide known in the art. In some cases, metal nitrides include GaN, InN, BN, Be ₃ N2, Cr2N, MoN, Si ₃ N ₄ , TaN, Th ₂ N ₂ , VN, ZrN, TiN, HfN, NbC, WN, TaN or other known in this metal nitride region. In some instances, the device disclosed herein is made using a combination of the materials listed herein or any other suitable material known in the art.

The electrodes may be of any shape, including discs, rods, wells, posts, a substantially flat shape, or any other shape suitable for nucleic acid synthesis. The cross-sectional area of each electrode varies depending on the size of the loci for the synthesis of polynucleotides, while in some cases it is up to 500, 200, 100, 75, 50, 25, 10, less than 5 μm ² . In some cases, the cross-sectional area of each electrode is from about 500 to 10 µm ² , from about 100 to 25 µm ² , or from about 150 to 50 µm ² . In some cases, the cross-sectional area of each electrode is from about 150 to 50 µm ² . The devices provided herein include electrodes with a diameter that varies depending on the size of the loci for polynucleotide synthesis. Exemplary electrode diameters include, without limitation, values up to 500, 200, 100, 75, 50, 25, 10, less than 5 microns. In some cases, the diameter of each electrode is about 500 to 10 microns, about 100 to 25 microns, about 100 to about 200 microns, about 50 to about 200 microns, or about 150 to 50 microns. In some cases, the diameter of each electrode is from about 200 to 50 microns. In some cases, the diameter of each electrode is from about 200 to 100 microns. In some cases, the diameter of each electrode is up to 500, 200, 100, 75, 50, 25, 10, less than 5 nm. In some cases, the diameter of each electrode is about 500 to 10 nm, about 100 to 25 nm, about 100 to about 200 nm, about 50 to about 200 nm, or about 150 to 50 nm. In some cases, the diameter of each electrode is from about 200 to 50 nm. In some cases, the diameter of each electrode is from about 200 to 100 nm. The thickness of each electrode varies depending on the size of the polynucleotide synthesis loci, being about 50, 100, 200, 500, 750, 1000, 1200, 1500, 2000, 2500, 3000 or about 3500 nm in some cases. In some cases, the thickness of the electrode is at least 50, 100, 200, 500, 750, 1000, 1200, 1500, 2000, 2500, 3000 or at least 3500 nm. In some cases, the thickness of the electrode is at least 1, 2, 3, 5, 10, 15, 20, 30, 50 or at least 75 microns. In some cases, the thickness of the electrode is about 1, 2, 3, 5, 10, 15, 20, 30, 50, or about 75 microns. In some cases, the thickness of the electrode is up to 1, 2, 3, 5, 10, 15, 20, 30, 50 or up to 75 microns. In some cases, the thickness of the electrode is up to 50, 100, 200, 500, 750, 1000, 1200, 1500, 2000, 2500, 3000 or up to 3500 nm. In some cases, the electrode thickness is about 20 to 3000 nm, about 50 to 2500, about 100 to 750 nm, about 400 to 750 nm, about 500 to 3000 nm, or about 1000 to 3000 nm. In some cases, the thickness of the electrode is from about 10 to about 20 microns. In some instances, the thickness of the electrode is about 5 to about 50 microns, about 10 to about 30 microns, about 15 to about 25 microns, or about 30 to about 50 microns. In some cases, the electrodes are coated with additional materials such as semiconductors or insulators. In some cases, the electrodes are coated with materials for the attachment and synthesis of polynucleotides. In some cases, the size, shape, pattern, or orientation of the electrodes is chosen to minimize harmful side reactions caused by electrochemically generated reagents. In some cases, combinations of electrodes are used, such as a grid of addressable electrodes and a conventional electrode. In some cases, the electrodes are cathodes or anodes. The electrodes or electrode arrays can be located anywhere on or near the polynucleotide surface. In some cases, electrodes are placed at the bottom of the synthesis loci, such as at the bottom of a well or channel. In some cases, the electrodes are placed on the side walls of the well or channel (side electrodes). In some cases, a plurality of side electrodes are located on the walls of the well. The electrodes located in different locations in the device may perform different functions and may be independently or simultaneously addressable. In some cases, an electrode at the bottom of the well is used to cleave polynucleotides from the surface at one or more loci, and a side electrode is used to generate acid to deprotect the polynucleotides. In some cases, the electrode at the bottom of the well is used to cleave polynucleotides from the surface at one or more loci, and the first side electrode is used to generate acid to deprotect the polynucleotides, and the second side electrode is used to move the polynucleotides out of the well after cleavage. splitting. In exemplary configurations, the side electrodes are located about 10, about 25, about 50, about 75, about 100, about 125, or about 200 nm above the synthesis surface. In some cases, the side electrodes are located at a distance of about 10 to about 100 nm, about 50 to about 150 nm, about 40 to 100 nm, about 75 to about 125 nm, about 100 to 300 nm above the synthesis surface. In some cases, several side electrodes are located at different heights above the synthesis surface. For example, the locus contains at least one ground electrode, at least 2 ground electrodes, at least 3 ground electrodes, or more than 3 ground electrodes. In exemplary configurations, the side electrodes are about 1, about 5, about 10, about 20, about 25, about 30, about 40, or about 50 nm high. In some cases, the side electrodes are 1 to 20 nm, 2 to 30 nm, 5 to 20 nm, 10 to 40 nm, or 5 to 25 nm in height.

The electrode surfaces can assist in the movement, conformation, synthesis, growth, and release of polynucleotides. In some cases, the electrodes are coated with one or more layers. In some cases, the layer is a monolayer, which facilitates the attachment of the linker. In some cases, the electrode is charged to affect the portion of the monolayer to be functionalized with the linker. This allows certain areas to be masked for chemical functionalization, such as surface modification with hydrophobic or hydrophilic chemical groups. In some cases, the electrode is charged to affect the area of the monolayer where the extension will be carried out using the nucleoside as the monomer. In some cases, such action includes providing the generation of reagents to promote or prevent the combination of monomers with surfaces for synthesis near the electrode. In some cases, the electrode is charged to affect the area of the monolayer from which the polynucleotides are released. In some cases, such controlled release of certain polynucleotides in a certain order is used to control the assembly of synthesized monomers into larger polynucleotides. For example, the process of iterative assembly of polynucleotides is optimized by examining different combinations of polynucleotides that are released and can hybridize for assembly using overlap PCR.

Each electrode can control one or a plurality of different synthesis loci, with each synthesis locus having a specific density of polynucleotides. In some cases, the density is at least 1 oligonucleotide per 10 nm ² , 20, 50, 100, 200, 500, 1000, 2000, 5000, or at least 1 oligonucleotide per 10,000 nm ² . In some cases, the density is from about 1 oligonucleotide per 10 nm ² to about 1 oligonucleotide per 5000 nm ² , from about 1 oligonucleotide per 50 nm ² to about 1 oligonucleotide per 500 nm ² , or from about 1 oligonucleotide per 25 nm ² to about 1 oligonucleotide at 75 nm ² . In some cases, the density of polynucleotides is from about 1 oligonucleotide per 25 nm ² to about 1 oligonucleotide per 75 nm ² .

This document presents devices for the synthesis of polynucleotides with various types of electrodes. In some cases, the device contains a reference electrode. The reference electrodes are placed near the synthesis surface or, in the case of a well or channel, eg above the well or channel. In some instances, the reference electrode is at a distance of about 1 to about 50 µm, about 2 to about 40 µm, about 3 to about 30 µm, about 5 to about 20 µm, about 10 to about 20 µm, about 15 to about 50 microns, about 30 to about 50 microns, about 5 to about 30 microns, or about 7 to about 25 microns above the synthesis surface. In some cases, the reference electrode is about 1, 2, 5, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, or more than 26 µm above the synthesis surface. In some cases, the reference electrode is up to 1, 2, 5, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, or up to 26 µm above the synthesis 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, or at least 26 µm above the synthesis surface . The reference electrodes are in some cases adjacent to the synthesis surfaces, such as adjacent to a well or a channel. In some cases, each locus has one corresponding reference electrode. In some cases, each locus shares a common reference electrode with one or more adjacent loci. The devices described herein may comprise any number of reference electrodes. In some cases, the reference electrode is a single homogeneous plate. In some cases, the devices contain a plurality of reference electrodes. The reference electrode may be associated with multiple loci, such as 2, 3, 4, 5, 10 or more loci.

This document describes devices, compositions, systems and methods for the synthesis and storage of nucleic acids on a solid support, while the solid support is characterized by various sizes. In some cases, the size of the solid substrate is from about 40 to 120 mm by about 25 to 100 mm. In some cases, the size of the solid substrate is about 80 mm by about 50 mm. In some cases, the width of the solid substrate is at least or about 10, 20, 40, 60, 80, 100, 150, 200, 300, 400, 500 or more than 500 mm. In some cases, the height of the solid substrate is at least or about 10, 20, 40, 60, 80, 100, 150, 200, 300, 400, 500 or more than 500 mm. In some cases, the solid substrate has a flat surface area of at least or about 100; 200; 500; 1000; 2000; 4500; 5000; 10000; 12000; 15000; 20000; 30000; 40000; 50000 ^mm2 or more. AT

In some cases, the thickness of the solid substrate is about 50 to about 2000 mm, about 50 to about 1000 mm, about 100 to about 1000 mm, about 200 to about 1000 mm, or about 250 to about 1000 mm. Non-limiting examples of solid substrate thicknesses include 275, 375, 525, 625, 675, 725, 775, and 925 mm. In some cases, the thickness of the solid substrate is at least or about 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, or greater than 4.0 mm.

This document describes devices where two or more solid substrates are assembled. In some cases, solid substrates are bonded together on a larger element. The connection may involve the exchange of fluids, electrical signals or other media between solid substrates. This element can interact with any number of servers, computers, or network devices. For example, a plurality of solid substrates are integrated into a rack space that can be conveniently inserted into or removed from a server rack. A rack space may contain any number of solid substrates. In some cases, a rack space 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 substrates. In some cases, two or more solid substrates are not connected to each other. The rack space may provide access to nucleic acids (and information stored therein) residing on solid substrates, see, for example, FIG. 14. Access includes extraction of polynucleotides from solid supports, direct analysis of polynucleotides on a solid support, or any other method that allows manipulation or identification of information stored in nucleic acids. In some cases, information is available from a plurality of racks, a single rack, one solid support from a rack, a portion of a solid support, or a single locus on a solid support. In various cases, access involves the interaction of nucleic acids with additional devices such as mass spectrometers, HPLC instruments, sequencing instruments, PCR amplifiers, or other nucleic acid manipulation device. In some cases, access to information stored in nucleic acids is provided by cleaving polynucleotides from all or part of the solid support. In some cases, splitting involves the action of reagents (ammonia or another reagent), electric potential, radiation, heat, light, sound, or other form of energy that can affect chemical bonds. In some cases, cleavage occurs as a result of charging one or more electrodes near the polynucleotides. In some cases, electromagnetic radiation in the form of UV light is used to cleave polynucleotides. In some cases, a lamp is used to cleave the polynucleotides, and the mask provides sites for exposure of the surface to UV light. In some cases, a laser is used to cleave polynucleotides, and the open/closed shutter mode provides control over the exposure of the surface to UV light. In some cases, access to information stored in nucleic acids (including the removal/addition of racks, solid supports, reagents, nucleic acids, or other component) is fully automated.

The solid substrates described herein comprise an active site. In some cases, the active site contains addressable regions or loci for nucleic acid synthesis. In some cases, the active site contains addressable regions or loci for storing nucleic acids.

The active site provides various sizes. For example, the size of the active site is from about 1 to about 50 mm by about 1 to about 50 mm. In some cases, the active region provides 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 site provides 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. An exemplary active site within a solid substrate is shown in FIG. 13. Block 1307 includes an active site 1305 within a solid substrate 1303. Block 1307 also includes a microfluidic interface 1301.

Devices, compositions, systems and methods for the synthesis and storage of nucleic acids on a solid support are described herein, wherein the solid support is characterized by a certain number of sites (eg, spots) or positions for synthesis or storage. In some cases, the solid substrate provides up to or about 10,000 per 10,000 positions in a given area. In some cases, the solid substrate provides from about 1,000 to 20,000 by about 1,000 to 20,000 positions in a given area. In some cases, the solid support provides at least or about 10, 30, 50, 75, 100, 200, 300, 400, 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 12000 , 14000, 16000, 18000, 20000 positions at least or about 10, 30, 50, 75, 100, 200, 300, 400, 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000 , 10000, 12000, 14000, 16000, 18000, 20000 positions in this section. In some cases, the area of the site is up to 0.25, 0.5, 0.75, 1.0, 1.25, 1.5 or 2.0 square inches. In some cases, the solid support contains addressable loci with a step size of 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. In some cases, the solid support contains addressable loci with a step size of about 5 μm. In some cases, the solid support contains addressable loci with a step size of about 2 μm. In some cases, the solid support contains addressable loci with a value

- 10 039806 steps about 1 µm. In some cases, the solid support contains addressable loci with a step size of about 0.2 μm. In some instances, the solid support contains addressable loci in increments of about 0.2 to about 10 µm, about 0.2 to about 8 µm, about 0.5 to about 10 µm, about 1 to about 10 µm, from about 2 to about 8 microns, about 3 to about 5 microns, about 1 to about 3 microns, or about 0.5 to about 3 microns. In some cases, the solid support contains addressable loci with a step size of about 0.1 to about 3 µm, see, for example, FIG. 15, 16A and 16B.

The solid support for nucleic acid synthesis or storage described herein has a high data storage capacity. For example, the capacitance of the solid support is at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 200, 300, 40θ, 500, 600, 700, 800 , 900, 1000 or more than 1000 petabytes. In some cases, the storage capacity of the solid substrate is from about 1 to about 10 petabytes, or from about 1 to about 100 petabytes. In some cases, the capacity of a solid substrate is about 100 petabytes. In some cases, data is stored as addressable arrays of data blocks in the form of blobs. In some cases, the data is stored as addressable arrays of data blocks in the form of drops on a spot. In some cases, data is stored as addressable arrays of data blocks in dried wells. In some cases, addressable arrays contain at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 200, or more than 200 GB of data. In some cases, addressable arrays contain 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 as part of the source data. For example, an information element encodes between about 10 and about 100 megabytes of data and is stored in 1 petabyte of raw data. In some cases, at least or about 1, 10, 20, 30, 40, 50, 6θ, 70, 80, 90, 100, 150, 2θ0, 300, 400, 500, or more than 500 MB of data are encoded in the information element, and it is stored in 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, or more than 500 PB of original data.

Devices, compositions, systems, and methods are provided herein for synthesizing and storing nucleic acids on a solid support, wherein, after synthesis, polynucleotides are assembled into data blocks in the form of one or more droplets. In some cases, the polynucleotides are assembled into data blocks in the form of one or more droplets and stored. In some cases, the number of drops is at least or about 1, 10, 20, 50, 100, 200, 300, 500, 1000, 2500, 5000, 75000, 10000, 25000, 50000, 75000, 100000, 1 million, 5 million , 10 million, 25 million, 50 million, 75 million, 100 million, 250 million, 500 million, 750 million or more than 750 million drops. In some cases, the droplet size is 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more than 100 microns in diameter. In some cases, the droplet size is 1-100 µm, 10-90 µm, 20-80 µm, 30-70 µm or 40-50 µm in diameter.

In some cases, the polynucleotides assembled into data blocks contain the same sequence. In some cases, the polynucleotides also contain a non-identical sequence used as a label or barcode. For example, a non-identical sequence is used to designate polynucleotides stored on a solid support and to subsequently search for specific polynucleotides by a different sequence. An exemplary label or barcode length includes barcode sequences that are, without limitation, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more bases in length. In some cases, the length of the label or barcode is at least or about 10, 50, 75, 100, 200, 300, 400, or more than 400 base pairs.

This document provides devices, compositions, systems and methods for the synthesis and storage of nucleic acids on a solid support, and polynucleotides are collected in blocks of data characterized by redundancy. For example, data blocks contain from about 100 to about 1000 copies of each polynucleotide. In some cases, data blocks contain 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, data blocks have about 1000X to about 5000X synthesis redundancy. In some cases, the synthesis redundancy is at least or about 500X, 1000X, 1500X, 2000X, 2500X, 3000X, 3500X, 4000X, 5000X, 6000X, 7000X, 8000X, or more than 8000X. Polynucleotides synthesized using solid support methods as described herein come in various lengths. In some cases, polynucleotides are synthesized and additionally stored on a solid support. In some cases, the length of the polynucleotide is in the range from about 100 to about 1000 bases. In some cases, the length of the polynucleotides is at least or about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350 , 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000 or more than 2000 bases.

Storage of information based on nucleic acids.

This document presents devices, compositions, systems and methods for storing information (data) based on nucleic acids. An exemplary workflow is shown in FIG. 1. At the first stage, 101 sequences are obtained in digital form, in which the element of information (ie, digital information in the form of a binary code for computer processing) is encoded. Encryption scheme 103 is used to convert sequentially

- 11 039806 digitally from a binary code to a nucleic acid sequence 105. Select 107 a surface material for nucleic acid extension, a design of loci for nucleic acid extension (otherwise arranged spots) and reagents for nucleic acid synthesis. Prepare 108 the surface of the structure for the synthesis of nucleic acids. Carry out the synthesis of 109 de novo polynucleotides. Synthesized polynucleotides are stored 111 and are available for subsequent release 113, in whole or in part. After release, the polynucleotides, in whole or in part, are amenable to sequencing 115, subjected to decoding 117 in order to reverse convert the nucleic acid sequence into a digital sequence. The digital sequence is then assembled 119 to produce an alignment product in which the original information element is encoded.

Information elements.

Optionally, the initial stage of the data storage process disclosed herein includes obtaining or receiving one or more pieces of information in source code form. Information elements include, without limitation, textual, audio, and visual information. Illustrative sources of information items include, without limitation, books, periodicals, electronic databases, medical records, letters, forms, speech recordings, animal records, biological profiles, broadcast recordings, films, short videos, emails, accounting, call logs, data logs obtained during the use of Internet information resources, drawings, paintings, prints, photographs, pixel art and program code. Exemplary sources of information elements in the case of a biological profile include, without limitation, gene libraries, genomes, gene expression data, and protein activity data. Exemplary information element formats include, without limitation, .txt, .PDF, .doc, .docx, .ppt, .pptx, .xls, .xlsx, .rtf, jpg, .gif, .psd, .bmp, .tiff, .png and .mpeg. The sizes of individual files in which the information item is encoded, or multiple files in which the information items are encoded in digital format, include, without limitation, sizes up to 1024 bytes (which equals 1 KB), 1024 KB (which equals 1 MB), 1024 MB (which equals 1 GB), 1024 GB (which equals 1 TB), 1024 TB (which equals 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, 60o, 700, 80o, 900, 1000 or more than 1000 GB. 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 TB. 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 PB.

Structures for the synthesis of polynucleotides.

This document presents rigid or flexible structures for the synthesis of polynucleotides. In the case of rigid structures, this document provides a device having a structure for creating a library of polynucleotides. In some cases, the structure is a tablet. An exemplary structure 200 is shown in FIG. 2, structure 200 having about the same dimensions as a standard 96 well plate: 140 mm by 90 mm. Structure 200 contains clusters grouped into 24 regions or subfields 205, with each subfield 205 containing an array of 256 clusters 210. An expanded view of an illustrative subfield 205 is shown in FIG. 3. In the expanded view of the four clusters (FIG. 3), a single cluster 210 has a Y-axis cluster pitch (center-to-center distance of adjacent clusters) of 1079.210 µm or 1142.694 µm and an X-axis cluster pitch of 1125 µm. An exemplary cluster 210 is shown in FIG. 4, where the loci pitch along the Y axis (distance from the center to the center of adjacent loci) is 63.483 μm, and the loci pitch along the X axis is 75 μm. The width of the locus in the longest part, for example, the diameter of the round locus, is 50 µm, and the distance between the loci is 24 µm. The number of loci 405 in the exemplary cluster in FIG. 4 is 121. Loci can be flat, wells, or channels. An exemplary channel arrangement is shown in FIG. 5A, 5B, which show that the plate 505 includes 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 the flow paths from the main channel 510 to each of a plurality of channels 515. The tablet 505 described herein may comprise a plurality of main channels 510. The plurality of channels 515 collectively form a cluster within the main channel 510.

In the case of flexible structures, this document presents devices where the flexible structure is a continuous loop 601 wound around one or more fixed structures, such as a pair of rollers 603, or an interrupted flexible structure 607 wound around individual fixed structures, such as a pair of coils 605, see Fig. 6A, 6B. In some cases, the structures contain multiple regions for polynucleotide synthesis. Illustrative Structure

- 12 039806 is shown in FIG. 6C, where the plate contains separate regions 609 for polynucleotide synthesis. Separate areas 609 can be separated 611 by breaking off or cutting. Each of the individual regions may also be released, sequenced, decrypted, and read 613 or stored 615. An alternative structure is shown in FIG. 6D, in which the ribbon contains separate regions 617 for polynucleotide synthesis. Separate areas 617 can be separated 619 by breaking off or cutting. Each of the individual regions can also be released, sequenced, decoded and read 621 or stored 623. Flexible structures are provided herein containing a multi-loci surface for polynucleotide extension. Figures 7A-7C show an enlarged view of a locus within a flexible structure. Each locus in the region of the flexible structure 701 may be a substantially flat spot 703 (e.g., a flat surface), a channel 705, or a dimple 707. In some cases, the width of each locus of the structure is about 10 microns, and the distance between the centers of each structure is about 21 microns. The loci may be, without limitation, round, rectangular, conical, or rounded. Alternatively, or in combination, the structures are rigid. In some cases, rigid structures contain loci for polynucleotide synthesis. In some cases, rigid structures contain substantially flat regions, channels, or wells for polynucleotide synthesis.

In some instances, a well as described herein has a width to depth (or height) ratio of 1 to 0.01, with width being a measure of the width at the narrowest segment of the well. In some instances, a well as described herein has a width to depth (or height) ratio of 0.5 to 0.01, with width being a measure of the width at the narrowest segment of the well. In some cases, the well described herein has a width to depth (or height) ratio of about 0.01, 0.05, 0.1, 0.15, 0.16, 0.2, 0.5, or 1. As used herein, This document presents structures for the synthesis of polynucleotides containing a plurality of discrete loci for the synthesis of polynucleotides. Exemplary structures for loci include, without limitation, substantially flat regions, channels, pits, or projections. The structures described herein may contain multiple clusters, with each cluster containing multiple wells, loci, or channels. Alternatively, the structures described herein may provide for a uniform arrangement of wells, loci, or channels. The structures provided herein may comprise wells with a height or depth of about 5 to about 500 µm, about 5 to about 400 µm, about 5 to about 300 µm, about 5 to about 200 µm, about 5 to about 100 µm, about 5 to about 50 µm, or about 10 to about 50 µm. In some cases, the well height is less than 100, less than 80, less than 60, less than 40, or less than 20 microns. In some cases, the well height is about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500 microns or more. In some cases, the height or depth of the well is at least 10, 25, 50, 75, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more than 1000 nm. In some instances, the height or depth of the well is in the range of about 10 to about 1000 nm, about 25 to about 900 nm, about 50 to about 800 nm, about 75 to about 700 nm, about 100 to about 600 nm, or from about 200 to about 500 nm. In some cases, the height or depth of the well is in the range from about 50 nm to about 1 μm. In some cases, the well height is about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 700, 800, 900 or about 1000 nm.

Presented in this document structures for the synthesis of polynucleotides may contain channels. Channels may have a width to depth (or height) ratio of 1 to 0.01, with width being a measure of the width at the narrowest segment of the microchannel. In some cases, the channel described herein has a width to depth (or height) ratio of 0.5 to 0.01, with the width being a measure of the width at the narrowest segment of the microchannel. In some cases, the channel described herein has a width to depth (or height) ratio of about 0.01, 0.05, 0.1, 0.15, 0.16, 0.2, 0.5, or 1.

The present document describes structures for the synthesis of polynucleotides containing many discrete loci. Structures, without limitation, contain essentially flat areas, channels, ledges or wells for the synthesis of polynucleotides. In some cases, it is contemplated that the structures described herein comprise a plurality of channels, wherein the height or depth of the channel is from about 5 to about 500 microns, from about 5 to about 400 microns, from about 5 to about 300 microns, from about 5 to about 200 µm, about 5 to about 100 µm, about 5 to about 50 µm, or about 10 to about 50 µm. In some cases, the channel height is less than 100, less than 80, less than 60, less than 40, or less than 20 microns. In some cases, the channel height is about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500 microns or more. In some cases, the channel height or depth is at least 10, 25, 50, 75, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more than 1000 nm. In some instances, the channel height or depth is in the range of about 10 to about 1000 nm, about 25 to about 900 nm, about 50 to about 800 nm, about 75 to about 700 nm, about 100 to about 600 nm, or from about 200 to about 500. The channels described herein may be located on the surface within clusters or as a uniform field.

Width of the locus on the surface of the structure for synthesis described herein

- 13 039806 polynucleotides can be from about 0.1 to about 500 µm, from about 0.5 to about 500 µm, from about 1 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, 80, 70, 60, 50, 40, 30, 20, 10, 5, 1, or 0.5 µm. In some instances, the locus width is less than about 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 microns. In some cases, the locus width is at least 10, 25, 50, 75, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more than 1000 nm. In some instances, the locus width is in the range of about 10 to about 1000 nm, about 25 to about 900 nm, about 50 to about 800 nm, about 75 to about 700 nm, about 100 to about 600 nm, or about 200 to about 500 nm. In some cases, the locus width is in the range of about 50 to about 1000 nm. In some cases, the distance between the centers of two adjacent loci is from about 0.1 to about 500 microns, from 0.5 to about 500 microns, from about 1 to about 200 microns, from about 1 to about 100 microns, from about 5 to about 200 µm, about 5 to about 100 µm, about 5 to about 50 µm, or about 5 to about 30 µm, for example, about 20 µm. In some cases, the total locus width is about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 µm. In some cases, the total locus width is about 1 to 100 µm, 30 to 100 µm, or 50 to 70 µm. In some cases, the distance between the centers of two adjacent loci is from about 0.5 to about 2 µm, from 0.5 to about 2 µm, from about 0.75 to about 2 µm, from about 1 to about 2 µm, from about 0 2 to about 1 µm, about 0.5 to about 1.5 µm, about 0.5 to about 0.8 µm, or about 0.5 to about 1 µm, such as about 1 µm. In some cases, the total locus width is about 50 nm, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1 .1, 1.2, 1.3, 1.4 or 1.5 µm. In some cases, the total locus width is about 0.5 to 2 µm, 0.75 to 1 µm, or 0.9 to 2 µm.

In some cases, each locus is the basis for the synthesis of a population of polynucleotides having a different sequence from that of a population of polynucleotides grown at a different locus. This document presents surfaces that contain 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. This document presents surfaces that contain more than 2000; 5000; 10000; 20000; 30000; 50000; 100000; 200000; 300000; 400000; 500000; 600000; 700000; 800000; 900000; 1000000; 5000000 or 10000000 or more individual loci. In some cases, each cluster includes 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 loci. In some cases, each cluster includes 50 to 500, 50 to 200, 50 to 150, or 100 to 150 loci. In some cases, each cluster includes from 100 to 150 loci. In some cases, each cluster includes 109, 121, 130, or 137 loci.

This document presents loci with a width in the longest segment from 5 to 100 µm. In some cases, the loci are about 30, 35, 40, 45, 50, 55, or 60 µm wide at their longest segment. In some cases, the loci are channels with several segments, with the distance between the centers of each segment being from 5 to 50 μm. In some cases, the distance between the centers of each segment is about 5, 10, 15, 20 or 25 microns.

In some cases, the number of individual polynucleotides synthesized on the surface of the structure described herein depends on the number of individual loci available on the substrate. In some cases, the density of loci within a substrate cluster is at least or about 1, 10, 25, 50, 65, 75, 100, 130, 150, 175, 200, 300, 400, 500, 1000, 10 ⁴ , 105 , 10 ⁶ loci/mm ² more. In from from from from about about about

100 some up to up to cases backing contains from about about about about about

400 loci/ ^mm2 ,

500 loci/ ^mm2 ,

250 loci/ ^mm2 , up to about 500 loci/ ^mm2 , from from from about about about

150 up to up to about

500 loci/mm ² , about 500 loci/mm ² , about 250 loci/mm ² , about 10 to about 200 loci/mm ² , or about 50 to about 200 loci/mm ² . In some cases, the substrate contains from about 10 ⁴ to about 10 ⁵ loci/mm ² . In some cases, the substrate contains from about 105 to about 10 ⁷ loci/mm ² . In some cases, the substrate contains at least 105 loci/mm ² . In some cases, the substrate contains at least 106 loci/mm ² . In some cases, the substrate contains at least 10 ⁷ loci/mm ² . In some cases, the substrate contains from about 10 ⁴ to about 105 loci/mm ² . In some cases, the density of loci within a substrate cluster is at least or about 1, 10, 25, 50, 65, 75, 100, 130, 150, 175, 200, 300, 400, 500, 1000 loci/µm ² or more . In some cases, the substrate contains from about 10 to about 500 loci/µm ² from about 25 to about 400 loci/µm ² from about 50 to about 500 loci/µm ² from about 100 to about 500 loci/µm ² from about 10 to about 250 loci/µm ² , about 150 to about 500 loci/µm ² , about 50 to about 250 loci/µm ² , about 10 to about 200 loci/µm ² , or about 50 to about 200 loci / µm ² .

In some instances, the distance between the centers of two adjacent loci within a cluster is about 10 to 500 µm, about 10 to 200 µm, or about 10 to 100 µm. In some cases, the distance between the centers of two adjacent loci exceeds about 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 microns. In some cases, the distance between the centers of two adjacent loci is less than about 200, 150, 100, 80, 70, 60, 50, 40, 30, 20, or 10 microns. In some cases, the distance between

- 14 039806 centers of two adjacent loci is less than about 10000, 8000, 6000, 4000, 2000 1000, 800,

600, 400, 200, 150, 100 nm, 80 µm, 70, 60, 50, 40, 30, 20 or 10 nm. In some cases, each square meter of the structure described herein provides at least 10 ⁷ , 10 ⁸ , 10 ⁹ , 10 ¹⁰ ,

10 ¹¹ loci, where each locus carries one polynucleotide. In some instances, 10 ⁹ polynucleotides are contained in less than about 6, 5, 4, 3, 2, or 1 m ² of the structure described herein.

In some cases, the structure described herein provides the basis for the synthesis of more than 2000; 5000; 10000; 20000; 30000; 50000; 100000; 200000; 300000; 400000; 500000; 600000; 700000; 800000; 900000; 1000000; 1200000; 1400000; 1600000; 1800000; 2000000; 2500000; 3000000; 3500000; 4000000; 4500000; 5000000; 10,000,000 or more non-identical polynucleotides. In some cases, the structure provides the basis for the synthesis of more than 2000; 5000; 10000; 20000; 50000; 100000; 200000; 300000; 400000; 500000; 600000; 700000; 800000; 900000; 1000000; 1200000; 1400000; 1600000; 1800000; 2000000; 2500000; 3000000; 3500000; 4000000; 4500000; 5000000; 10,000,000 or more polynucleotides encoding individual sequences. In some cases, at least a portion of the polynucleotides have an identical sequence or are designed to be synthesized with an identical sequence. In some cases, the structure provides surface conditions 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 or more bases. In some formats, the polynucleotide synthesis structures described herein comprise polynucleotide synthesis sites that are evenly spaced.

In some cases, polynucleotides are synthesized at separate loci of the structure, with a population of polynucleotides being synthesized at each locus. In some cases, a population of polynucleotides is synthesized at each locus with a sequence different from that of a population of polynucleotides grown at a different locus. In some cases, structure loci are located within a plurality of clusters. In some cases, the structure contains at least 10, 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000, 20000, 30000.0 more clusters. In some cases, the structure contains more than 2000; 5000; 10000; 100000; 200000; 300000; 400000; 500000; 600000; 700000; 800000; 900000; 1000000; 1100000; 1200000; 1300000; 1400000; 1500000; 1600000; 1700000; 1800000; 1900000; 2000000; 300000; 400000; 500000; 600000; 700000; 800000; 900000; 1000000; 1200000; 1400000; 1600000; 1800000; 2000000; 2500000; 3000000; 3500000; 4000000; 4500000; 5000000 or 10000000 or more individual loci. In some cases, each cluster includes 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 includes 50 to 500, 100 to 150, or 100 to 200 loci. In some cases, each cluster includes 109, 121, 130, or 137 loci. In some cases, each cluster includes 5, 6, 7, 8, 9, 10, 11, or 12 loci. In some cases, polynucleotides from separate loci within the same cluster have sequences that, when assembled, encode for a contiguous longer polynucleotide with a given sequence.

Structure size.

In some cases, the size of the structure described herein about corresponds to the size of the plate (eg, chip), for example, from about 40 to 120 mm by about 25 to 100 mm. In some cases, the structure described herein has a diameter less than or equal to about 1000, 500, 450, 400, 300 mm, 250 nm, 200, 150, 100, or 50 mm. In some cases, the diameter of the substrate is from about 25 to 1000 mm, from about 25 to about 800 mm, from about 25 to about 600 mm, from about 25 to about 500 mm, from about 25 to about 400 mm, from about 25 to about 300 mm, or about 25 to about 200. Non-limiting examples of substrate sizes include about 300, 200, 150, 130, 100, 84, 76, 54, 51, and 25 mm. In some cases, the substrate has a flat surface area of at least 100; 200; 500; 1000; 2000; 4500; 5000; 10000; 12000; 15000; 20000; 30000; 40000; 50000 ^mm2 or more. In some cases, the thickness is about 50 to about 2000 mm, about 50 to about 1000 mm, about 100 to about 1000 mm, about 200 to about 1000 mm, or about 250 to about 1000 mm. Non-limiting examples of thicknesses include 275, 375, 525, 625, 675, 725, 775, and 925 mm. In some cases, the thickness is at least or about 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0 mm, or greater than 4.0 mm. In some cases, the thickness varies depending on the diameter and depends on the composition of the substrate. For example, the thickness of a structure containing materials other than silicon may be different from the thickness of a silicon structure of the same diameter. The thickness of the structure may be determined by the mechanical strength of the material used, and the structure must be thick enough to support its own weight without cracking during handling. In some cases, the size of the structure is more than about 1, 2, 3, 4, 5, 10, 15, 30, 40, 50 feet in any direction.

Materials.

Disclosed herein are devices containing a surface where the surface is modified to allow polynucleotide synthesis at desired locations and resulting in low error rates, low dropout rates, high yields, and high oligonucleotide representation. In some cases, the surfaces of the devices for the synthesis of polynucleotides presented herein are made of various materials that can be modified to provide a de novo polynucleotide synthesis reaction. In some cases, the devices are sufficiently conductive, such as being able to generate uniform electric fields throughout the device or part of it. The devices described herein may include a flexible material. Exemplary flexible materials include, without limitation, modified nylon, unmodified nylon, nitrocellulose, and polypropylene. The devices described herein may contain rigid material. Exemplary rigid materials include, without limitation, glass, quartz glass, silicon, silicon dioxide, silicon nitride, plastics (eg, polytetrafluoroethylene, polypropylene, polystyrene, polycarbonate, and mixtures thereof), and metals (eg, gold, platinum). In this document, devices may be made from a material including silicon, polystyrene, agarose, dextran, cellulose-based polymers, polyacrylamides, polydimethylsiloxane (PDMS), glass, or any combination thereof.In some instances, the devices disclosed herein are made using a combination of the materials listed herein, or any other suitable material known in the art.

The devices described herein may comprise a material having a range of tensile strengths. Exemplary materials having a 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 solid substrates described herein may have a tensile strength of 1-300, 1-40, 1-10, 1-5, or 3-11 MPa. The solid substrates described herein may have tensile strengths of 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 more. In some instances, the device described herein comprises a solid polynucleotide synthesis support made of a flexible material that can be stored as a continuous loop or roll, such as a tape or flexible sheet material.

Young's modulus determines the ability of a material to resist elastic (reversible) deformation under load. Exemplary materials having a range of Young's modulus for 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 solid substrates described herein may have Young's modulus values of 1-500, 1-40, 1-10, 1-5, or 3-11 GPa. The solid substrates described herein may have Young's modulus values of 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 inversely proportional to each other, the flexible material has a low Young's modulus and changes its shape significantly under load. In some cases, the solid substrate described herein has a surface with the flexibility provided by at least nylon.

In some cases, the devices disclosed herein comprise a silica backbone and a silica surface layer. Alternatively, the devices may have a silicon oxide backbone. The surface of the device provided herein may be textured, resulting in an increase in the total surface area for polynucleotide synthesis. In some cases, devices disclosed herein may contain at least 5%, 10%, 25%, 50%, 80%, 90%, 95%, or 99% silicon. In some cases, the devices disclosed herein are made from silicon-on-insulator (SOI) wafer technology.

The structure can be made from a variety of materials suitable for the methods and compositions of the present invention described herein. In certain cases, the materials from which the substrates/solid substrates of the present invention are made are characterized by a low level of binding of polynucleotides. In some situations, it is possible to use a material that is transparent to visible and/or UV light. You can use materials that are sufficiently conductive, such as those capable of forming uniform electric fields over the entire area of the substrates/solid substrates described herein or part of it. In some cases, such materials may be connected to a grounding device. In some cases, the substrate or solid substrate may be thermally conductive or insulating. The materials may be chemically inert and thermally stable to allow chemical or biochemical reactions to take place, such as a series of polynucleotide synthesis reactions. In the case of flexible materials, materials of interest may include: nylon, both modified and unmodified, nitrocellulose, polypropylene, etc.

In the case of rigid materials, specific materials of interest include glass; quartz glass; silicon, plastics (eg polytetrafluoroethylene, polypropylene, polystyrene, polycarbonate and mixtures thereof, etc.); metals (eg gold, platinum, etc.). The structure may be made from a material selected from the group consisting of silicon, polystyrene, agarose, dextran, cellulose-based polymers, polyacrylamides, polydimethylsiloxane (PDMS), and glass. The substrates/solid substrates or microstructures, reactors therein, may be made from a combination of the materials listed herein or any other suitable material known in the art.

In some cases, the substrate disclosed herein contains a machine-readable object. Machine-readable objects include, without limitation, magnetic media, reel-to-reel tape, cassette tape, magnetic tape, floppy disk, paper media, film, microfiche, endless tape (eg, conveyor belt), and any media suitable for storing electronic instructions. In some cases, the substrate contains a magnetic reel tape or a magnetic stripe. In some cases, the substrate contains a flexible circuit board.

The structures described herein may be transparent to visible and/or UV light. In some cases, the structures described herein are conductive enough to generate uniform electric fields throughout the structure or part of it. In some cases, the structures described herein are thermally conductive or insulated. In some cases, the structures are chemically inert and thermally stable to allow a chemical reaction to proceed, such as the synthesis of polynucleotides. In some cases, the substrate is magnetic. In some cases, the structures contain a metal or metal alloy.

Polynucleotide synthesis structures may be greater than 1, 2, 5, 10, 30, 50 or more feet in length in any direction. In the case of a flexible structure, the flexible structure is optionally stored in a wound state, for example in the form of a roll. In the case of a large rigid structure, eg more than 1 foot long, the rigid structure can be stored vertically or horizontally.

Surface preparation.

Provided herein are methods of facilitating the immobilization of a biomolecule on a substrate, where the surface of the structure described herein contains a material and/or is coated with a material that promotes a coupling reaction with the biomolecule for attachment. To prepare the structure for the purpose of immobilizing the biomolecule, surface modifications can be used that chemically and/or physically change the surface of the substrate using an additive or subtractive process in order to change one or more chemical and/or physical properties of the surface of the substrate or a selected site or surface area. For example, surface modification includes (1) changing the wettability of the surface, (2) functionalizing the surface, i.e. introduction, modification or replacement of surface functional groups, (3) defunctionalization of the surface, i.e. removal of surface functional groups, (4) other alteration of the chemical composition of the surface, such as by etching, (5) increase or decrease in surface roughness, (6) coating the surface, for example, a coating that has a different wettability than that of the surface, and /or (7) deposition of particles on the surface. In some cases, the surface of a structure is selectively functionalized to produce two or more distinct regions on the structure, at least one region having a surface or chemical property that is different from that of another region within the same structure. Such properties include, without limitation, surface energy, chemical end groups, concentration of a particular chemical, etc.

In some instances, the surface of the structure disclosed herein is modified to include one or more active agent-functionalized surfaces configured to bind to both the surface of the substrate and the biomolecule to allow coupling to the surface. In some cases, the surface is also functionalized using a passive material that does not effectively bind the biomolecule, thereby preventing attachment of the biomolecule at sites where the passive functionalization agent is bound. In some cases, the surface contains an active layer that defines the boundaries of only individual loci, which will contain biomolecules.

In some cases, the surface is brought into contact with a mixture of groups for functionalization, which are in different ratios. In some cases, the mixture contains at least 2, 3, 4, 5 or more different types of functionalization agents. In some cases, the mixture ratio of at least two types of surface functionalizers 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 to achieve the desired representation of the surfaces 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 surface of the substrate with functionalization agents in the appropriate ratio. In some cases, the agents in the mixture are selected from suitable reactive and inert materials, thereby diluting the density of reactive groups on the surface to the required level for subsequent reactions to take place. In some cases, the mixture of functionalization reagents contains one or more reagents that

- 17 039806 bind to the biomolecule and one or more reagents that do not bind to the biomolecule.

Therefore, modulation in relation to the reactants allows you to control the degree of binding of the biomolecule, which occurs in a particular site of functionalization.

In some cases, the method of functionalization of the substrate involves the deposition of silane molecules on the surface of the substrate. Silane molecules can be deposited on areas of the substrate with high surface energy. In some cases, the high surface energy region includes a passive functionalization reagent. The methods described herein provide that the silane group binds to the surface while the rest of the molecule provides some 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 chlorodimethyloctadecylsilane, dichloromethyloctadecylsilane, trichlorooctadecylsilane and trimethyloctadecylsilane, triethyloctadecylsilane. In some cases, the silane is an aminosilane. Examples of aminosilanes include, without limitation, 11-acetoxyundecyltriethoxysilane, n-decyltriethoxysilane, (3-aminopropyl)trimethoxysilane, (3-aminopropyl)triethoxysilane, glycidyloxypropyl/trimethoxysilane, and N-(3-triethoxysilylpropyl)-4-hydroxybutyramide. In some cases, the silane includes 11-acetoxyundecyltriethoxysilane, n-decyltriethoxysilane, (3-aminopropyl)trimethoxysilane, (3-aminopropyl)triethoxysilane, glycidyloxypropyl/trimethoxysilane, N-(3-triethoxysilylpropyl)4-hydroxybutyramide, or any combination thereof. In some cases, the functionalizing active agent is 11-acetoxyundecyltriethoxysilane. In some instances, the active functionalization agent is n-decyltriethoxysilane. In some cases, the functionalizing active agent is glycidyloxypropyltriethoxysilane (GOPS). In some cases, the silane is a fluorosilane. In some cases, the silane is a hydrocarbon-containing silane. In some cases, the silane is 3-iodopropyltrimethoxysilane. In some cases, the silane is octylchlorosilane.

In some cases, silanization is carried out on the surface through self-assembly of organofunctional alkoxysilane molecules. Organofunctional alkoxysilanes are classified according to their functions attributable to organic compounds. Non-limiting examples of siloxane-based functionalization reagents include hydroxyalkylsiloxanes (surface silylation, diborane functionalization, and alcohol oxidation with hydrogen peroxide), (dihydroxyalkyl)-siloxanediols (surface silylation and hydrolysis to form a diol), aminoalkylsiloxanes (in the case of amines, no intermediate is required). functionalization step), glycidoxysilanes (3-glycidoxypropyl-dimethyl-ethoxysilane, glycidoxytrimethoxysilane), mercaptosilanes (3-mercaptopropyl-trimethoxysilane, 3-4-epoxycyclohexylethyltrimethoxysilane or 3-mercaptopropyl-methyl-dimethoxysilane), bicycloheptenyltrichlorosilane, butylaldehyde-trimethoxysilane or dimeric secondary aminoalkylsiloxanes. Exemplary hydroxyalkylsiloxanes include allyltrichlorosilane converting to 3-hydroxypropyl or 7-oct-1-enyltrichlorosilane converting to 8-hydroxyoctyl. (Dihydroxyalkyl)-siloxanediols include glycidyltrimethoxysilane derivative of (2,3-dihydroxypropyloxy)propyl (GOPS). Aminoalkylsiloxanes include 3-aminopropyltrimethoxysilane, which converts to 3-aminopropyl (3-aminopropyl-triethoxysilane, 3-aminopropyl-diethoxy-methylsilane, 3-aminopropyl-dimethylethoxysilane or 3-aminopropyl-trimethoxysilane). In some cases, the dimeric secondary aminoalkylsiloxane is bis-(3-trimethoxysilylpropyl)amine, which converts to bis-(silyloxypropyl)amine.

Active agent functionalization sites may contain one or more different types of silanes, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more types of silane. In some instances, one of one or more silane species is present in the functionalization composition in an amount greater than that of the other silane species. For example, a mixed solution of silanes containing two types of silanes provides a ratio of one type of silane and another type of silane 99:1, 98:2, 97:3, 96:4, 95:5, 94:6, 93:7, 92: 8, 91:9, 90:10, 89:11, 88:12, 87:13, 86:14, 85:15, 84:16, 83:17, 82:18, 81:19, 80:20, 75:25, 70:30, 65:35, 60:40, 55:45. In some cases, the functionalizing active agent comprises 11-acetoxyundecyltriethoxysilane and n-decyltriethoxysilane. In some instances, the functionalizing active 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.

In some cases, functionalization involves applying a functionalizing agent to a structure using any application technique, including, without limitation, chemical vapor deposition (CVD), atomic layer deposition (ALD), plasma CVD (PECVD), plasma ALD (PEALD), CVD for metal organic deposition (MOCVD), catalytic CVD (HWCVD), initiated CVD (iCVD), modified CVD (MCVD), axial vapor deposition (VAD), external vapor deposition (OVD), physical vapor deposition (eg sputter deposition, vapor deposition) and molecular layer deposition (MLD).

Any step or component in the following functionalization method may be omitted or modified according to the properties required for the final functionalized substrate. In some cases, additional components and/or method steps are added to the process sequences set forth herein. In some cases, the substrate is first cleaned, for example, using a solution of piranha. An example cleaning process includes soaking the substrate in a piranha solution (eg 90% H2SO ₄ , 10% H2O2) at elevated temperature (eg 120°C) and rinsing (eg water) and drying the substrate (eg using nitrogen gas). The process optionally includes post-treatment with a piranha solution, comprising soaking the piranha-treated substrate in a basic solution (eg, NH4OH) followed by washing with an aqueous medium (eg, water). In some cases, the surface of the structure is subjected to plasma-chemical cleaning, optionally followed by soaking in piranha solution and optionally post-cleaning with piranha solution. An example of a plasma-chemical cleaning process involves etching in oxygen plasma. In some cases, an active functionalization agent is applied to the surface, followed by evaporation. In some cases, the substrate is functionalized with active agents prior to purification, for example, by treatment with a piranha solution and/or plasma-chemical purification.

The surface functionalization method optionally includes resist coating and resist stripping. In some cases, after surface functionalization with active agents, the substrate is spin-coated with a resist, such as SPR™ 3612 positive photoresist. In various cases, the surface functionalization method involves lithography with surface functionalization in the form of a pattern. In some cases, photolithography is carried out after coating with a resist. In some cases, after lithography, the surface is visually inspected for lithographic defects. In some cases, the surface functionalization method includes a cleaning step, whereby residues are removed from the substrate, for example, by plasma chemical cleaning or etching. In some cases, the stage of plasma-chemical purification is carried out during one of the stages after the implementation of the stage of lithography.

In some cases, the resist-coated surface is treated to remove the resist, such as after functionalization and/or after lithography. In some cases, the resist is removed with a solvent, such as a stripping solution containing N-methyl-2-pyrrolidone. In some cases, the removal of the resist involves treatment with sonication or ultrasound. In some cases, the resist is applied and removed, followed by active functionalization of the exposed areas to create the desired differential functionalization pattern.

In some instances, the methods and compositions described herein involve the application of a photoresist to obtain modified surface properties in selected areas, the application of the photoresist being based on the fluid properties of the surface that determine the spatial distribution of the photoresist. Without being bound by theory, surface tension effects associated with the applied fluid can determine the flow characteristics of the photoresist. For example, surface tension and/or capillary effects can promote the penetration of photoresist into small structures in a controlled manner before the solvents of the resist evaporate. In some cases, the contact points of the resist are fixed when interacting with sharp edges, thereby providing control over the progress of the fluid. Base structures can be designed based on the desired flow patterns that are used to deposit the photoresist during fabrication and functionalization processes. The solid organic layer remaining after the solvents have evaporated can be used for subsequent steps in the manufacturing process. Structures can be designed to control fluid flow by promoting or inhibiting the effects of capillary wicking into adjacent fluid flow paths. For example, the structure is designed to avoid overlap between the top and bottom edges, which assists in retaining fluid in the top structures, which allows for a specific location of the resist. In an alternative example, the top and bottom edges overlap, causing the applied fluid to wick into the underlying structures. Appropriate designs can be appropriately selected depending on the desired application of the resist.

In some cases, the structure described herein has a surface that contains a material with a thickness of at least or about 0.1, 0.5, 1, 2, 5, 10, or 25 nm, which provides a reactive group capable of binding nucleosides. Examples include, without limitation, glass and silicon, such as silicon dioxide and silicon nitride. In some instances, exemplary surfaces include nylon and PMMA.

In some cases, electromagnetic radiation is used to apply a pattern to the surface.

- 19 039806 in the form of UV light. In some cases, a lamp is used to apply the pattern to the surface, and the mask provides points of exposure to UV light on the surface. In some cases, a laser is used to pattern the surface, and the open/closed shutter mode provides control over the UV light exposure to the surface. The location of the laser can be used in combination with a flexible structure that is able to move. In this format, the coordination of laser exposure and movement of the flexible structure is used to create patterns from one or more agents with differing nucleoside coupling abilities.

This document describes polynucleotide synthesis surfaces that can be reused. Following synthesis and/or cleavage of polynucleotides, the surface may be washed, washed, cleaned, fired, etched, or otherwise functionally restored to a condition suitable for subsequent polynucleotide synthesis. The number of times a surface is reused and how the surface is recycled/prepared for reuse varies depending on subsequent applications. Surfaces prepared for reuse are in some cases reused at least 1, 2, 3, 5, 10, 20, 50, 100, 1000 or more times. In some cases, the remaining life of a surface or the number of times a surface is suitable for reuse is determined or predicted.

Systems for making materials.

In some cases, synthesized polynucleotides are stored on a support, such as a solid support. The nucleic acid reagents can be applied to the surface of the support by the intermittent feed method or the demand feed method. Examples of such methods include the electromechanical transfer method, the electrothermal transfer method, and the electrostatic attraction method. In the case of the electromechanical transfer method, piezoelectric elements, deformed by electrical impulses, cause droplets to be ejected. In the case of the electrothermal transfer method, bubbles are formed in the chamber of the device, and the expansion forces of the bubbles cause the droplets to be pushed out. In the case of the method based on electrostatic attraction, an electrostatic attraction force is used to push droplets onto the substrate. In some cases, the droplet frequency is from about 5 to about 500 kHz; about 5 to about 100 kHz; about 10 to about 500 kHz; about 10 kHz to about 100 kHz, or about 50 kHz to about 500 kHz. In some cases, the frequency is less than about 500, 200, 100, or 50 kHz.

Droplet size depends on device resolution. In some cases, the devices provide droplets of reagents with a size of from about 0.01 to about 20 pl, from about 0.01 to about 10 pl, from about 0.01 to about 1 pl, from about 0.01 to about 0.5 pl , from about 0.01 to about 0.01 pl, or from about 0.05 to about 1 pl. In some cases, the droplet size is less than about 1, 0.5, 0.2, 0.1, or 0.05 pl.

In some formats, the polynucleotide synthesis system is configured to provide a continuous polynucleotide synthesis process that utilizes the flexibility of the support to move in a reel-to-reel type process. Such a synthesis process takes place in a continuous flow line, with the substrate being moved during the various steps of polynucleotide synthesis by means of one or more spools to change the position of the substrate. In an exemplary case, the polynucleotide synthesis reaction involves rolling the support through: through a solvent bath, under an adder to add amidophosphites, through an oxidizing agent bath, through an acetonitrile wash bath, and through a deblocking bath. Optionally, the tape also passes through the bath of capping reagents. The spool-to-spool process allows the end product to be easily collected from the substrate containing the synthesized polynucleotides to a take-up spool, from where it can be transported for further processing or storage.

In some formats, the synthesis of polynucleotides takes place in a continuous process, during which a continuous flexible belt moves along a conveyor system. Similar to a reel-to-reel type process, the synthesis of polynucleotides on a continuous belt occurs in a continuous production line, with the substrate moving during the various stages of polynucleotide synthesis in a conveying process. However, in the process with a conveyor system, the continuous tape is again included in the stage of polynucleotide synthesis without winding and unwinding the tape, as in a reel-to-reel type process. In some formats, the polynucleotide synthesis steps are divided into zones and a continuous belt is conveyed through each zone one or more times per cycle. For example, a polynucleotide synthesis reaction may involve (1) conveying the support through a solvent bath, under an amidophosphite adder, through an oxidizing agent bath, through an acetonitrile wash bath, and through a deblocking bath per cycle. ; and then (2) repeating cycles to obtain synthesized polynucleotides of a given length. Following polynucleotide synthesis, the flexible support is removed from the conveyor system and optionally wound up for storage. For storage, winding can be carried out on a reel. In some cases, a flexible substrate containing ter

- 20 039806 moplastic material, coated with a nucleoside coupling reagent. The coating is applied in a pattern to the loci so that each loci has a diameter of about 10 µm, with the distance between the centers of two adjacent loci being about 21 µm. In this case, the size of the locus is sufficient to accommodate a fixed droplet with a volume of 0.2 pl during the stage of polynucleotide synthesis with the introduction of reagents. In some cases, the density of loci is about 2.2 billion loci/m ² (1 locus/441 x 10' ¹² m ² ). In some cases, a 4.5 m ² substrate contains about 10 billion loci, each 10 µm in diameter.

In some formats, the device for applying one or more reactants to the substrate during the synthesis reaction is configured to introduce the reactants and/or nucleosides as monomers for synthesis based on the use of nucleoside amidophosphites. Polynucleotide synthesis reagents include polynucleotide extension reagents and wash buffers. As non-limiting examples, the apparatus provides for the application of cleaning agents, coupling agents, capping agents, oxidizing agents, deblockers, acetonitrile, gases such as nitrogen gas, and any combination thereof. In addition, the device optionally provides for the introduction of reagents to prepare and/or maintain the integrity of the substrate. In some instances, the polynucleotide synthesizer provides droplets less than about 200, 100, or 50 µm in diameter in a volume of less than about 1000, 500, 100, 50, or 20 pl. In some instances, the polynucleotide synthesizer provides an application of about 1 to 10,000, 1 to 5,000, 100 to 5,000, or 1,000 to 5,000 drops/sec.

Devices, methods, systems and compositions are described herein wherein polynucleotide synthesis reagents are recycled or reused. Recycling of reagents may involve the collection, storage and use of unused reagents, or the purification/conversion of used reagents. For example, the reagent bath is recycled and used to perform the polynucleotide synthesis step on the same or different surface. Reagents described herein can be recycled 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 times or more. Alternatively, or in combination, the reagent solution containing the reaction by-product is filtered to remove the by-product and the reagent solution is used for additional polynucleotide synthesis reactions.

Polynucleotide synthesis systems often use multiple integrated or non-integrated elements. In some cases, the polynucleotide synthesis system contains one or more elements suitable for subsequent processing of the synthesized polynucleotides. As an example, the system includes a temperature controlled element such as a thermal cycler. In some instances, a temperature controlled element is applied to a plurality of individual reactors for performing nucleic acid assembly, eg, by X-ray analysis and/or nucleic acid amplification, eg, by PCR.

Synthesis of polynucleotides de novo.

This document provides systems and methods for synthesizing polynucleotides on a high density support in short periods 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 bases are synthesized in one day.

In some cases, at least 10x108, 10x109, 10x10 ¹⁰ , 10x1011 or 10x10 ¹² polynucleotides are synthesized in one day. In some cases, each synthesized polynucleotide contains at least 20, 50, 100, 200, 300, 400, or 500 nitrogenous bases. In some cases, these bases are synthesized with an overall mean error rate of less than about 1 in 100; 200; 300; 400; 500; 1000; 2000; 5000; 10000; 15000; 20,000 bases. In some cases, such error rates are observed for at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.5% or more of the synthesized polynucleotides. In some instances, at least 90%, 95%, 98%, 99%, 99.5% or more of the synthesized polynucleotides do not differ from the target sequence they encode. In some cases, the error rate for polynucleotides synthesized on a support using the methods and systems described herein is less than about 1 in 200. In some cases, the error rate for polynucleotides synthesized on a support using the methods and systems described herein, is less than about 1 in 1000. In some cases, the error rate for polynucleotides synthesized on a support using the methods and systems described herein is less than about 1 in 2000. In some cases, the error rate for polynucleotides synthesized on a support using methods and systems described herein is less than about 1 in 3,000. In some cases, the error rate for polynucleotides synthesized on a support using the methods and systems described herein is less than about 1 in 5,000. mating, deletion twists and/or substitutions in polynucleotides synthesized on a support. The term error rate refers to the comparison of the total number of synthesized polynucleotides with a population of given polynucleotide sequences. In some instances, the synthesized polynucleotides disclosed herein contain a 12 to base binding. In some cases, the binding contains 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.

This document describes methods, systems, devices and compositions, where the control of chemical reactions used in the synthesis of polynucleotides, is carried out using electrochemical processes. In some cases, the control of electrochemical reactions is carried out using any source of energy, such as light, heat, radiation or electricity. For example, electrodes are used to monitor chemical reactions at all or part of discrete loci on a surface. In some cases, electrodes are charged by applying an electrical potential to the electrode in order to control one or more of the chemical reactions involved in the synthesis of polynucleotides. In some cases, such electrodes are addressable. In some cases, one or more electrodes control any number of chemical reaction steps described herein. Electrochemical reactions may include oxidation, reduction, acid-base reactions, or other reactions controlled by an electrode. In some cases, the electrodes generate electrons or protons, which are used as reagents for chemical transformations. In some cases, the electrodes directly generate a reactant such as acid. In some cases, the acid is a proton. In some cases, the electrodes directly generate a reactant such as a base. Acids or bases are often used to cleave protecting groups or influence the kinetics of various reactions in polynucleotide synthesis, for example by adjusting the pH of the reaction solution. In some cases, electrochemically controlled polynucleotide synthesis reactions involve redox active metals or other redox active organic substances. In some cases, metal or organic catalysts are used for such electrochemical reactions. In some cases, acids are generated from the oxidation of quinones.

The control of chemical reactions is not limited to the electrochemical generation of reagents; reactivity can be influenced indirectly by biophysical alterations of substrates or reactants via electrical fields (or gradients) generated by the electrodes. In some cases, substrates include, without limitation, nucleic acids. In some cases, electric fields are generated that repel certain reactants or substrates from the electrode or surface, or attract certain reactants or substrates to the electrode or surface. In some cases, such fields are generated by applying an electrical potential to one or more electrodes. For example, negatively charged nucleic acids are repelled by a negatively charged electrode surface. In some cases, such repulsions or attractions of polynucleotides or other reactants, due to the action of local electric fields, move the polynucleotides or other reactants into or out of the region of the device or structure for synthesis. In some cases, the electrodes generate electric fields that repel polynucleotides from a surface for synthesis, structure, or device. In some cases, the electrodes generate electric fields that attract polynucleotides to a synthesis surface, structure, or device. In some cases, protons are repelled from a positively charged surface with limited contact of protons with substrates or their parts. In some cases, repulsive or attractive forces are used to ensure or block the flow of reagents or substrates to certain areas of the surface for synthesis. In some cases, the nucleosides as monomers are prevented from contacting the polynucleotide chain by applying an electric field in the vicinity of one or both components. Such schemes provide for the introduction of certain reagents, which may eliminate the need for protecting groups, since the concentration or degree of contact between the reagents and/or substrates is controlled. In some cases, nucleosides are used for the synthesis of polynucleotides as monomers without protective groups. Alternatively, the application of an electric field in the vicinity of one or both components stimulates the contact of nucleosides as monomers with the polynucleotide chain. In addition, the application of electric fields to the substrate may change the reactivity or conformation of the substrates. In an exemplary application, electric fields generated by electrodes are used to prevent interaction between polynucleotides from adjacent loci. In some cases, the substrate is a polynucleotide, optionally attached to a surface. In some cases, the application of an electric field provides a change in the three-dimensional structure of the polynucleotide. Such changes involve the folding or unfolding of various structures such as helices, hairpins, loops, or other 3-dimensional nucleic acid structures. Such modifications are useful for manipulation of nucleic acids within wells, channels, or other structures. In some cases, electric fields are applied to nucleic acids as a substrate to prevent the formation of secondary structures. In some cases, electric fields eliminate the need for linkers or attachment to a solid support during polynucleotide synthesis.

- 22 039806

A suitable method for synthesizing supported polynucleotides according to the present disclosure is the amidophosphite method, comprising the controlled addition of amidophosphite as a building block, i.e. nucleoside amidophosphite, for extending the polynucleotide chain during the coupling step, which results in the formation of a phosphite triester bond between the amidophosphite as a structural unit and the nucleoside bound to the substrate. In some cases, the nucleoside amidophosphite is applied to the activated support. In some cases, the nucleoside amidophosphite is supported on a support with an activator. In some cases, nucleoside amidophosphites are added to the substrate at 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-fold excess or more compared to the amount of nucleosides bound to the substrate. In some cases, the addition of the nucleoside amidophosphite is carried out in an anhydrous medium, such as anhydrous acetonitrile. After the addition and attachment of the nucleoside amidophosphite in the coupling step, the support is optionally washed. In some cases, the coupling step is repeated one or more additional times, optionally with a washing step in between additions to the nucleoside amidophosphite support. In some instances, the method for synthesizing polynucleotides used herein involves 1, 2, 3 or more consecutive coupling steps. In many cases, prior to coupling, the support-bound nucleoside is deprotected by deprotection, the function of the protecting group being to prevent polymerization. Protecting groups can be any chemical group that prevents the elongation of the polynucleotide chain. In some cases, the protecting group is cleaved off (or removed) in the presence of an acid. In some cases, the protecting group is cleaved off in the presence of a base. In some cases, the protecting group is removed by exposure to electromagnetic radiation, such as by exposure to light, heat, or other source of energy. In some cases, the protecting group is removed by an oxidation or reduction reaction. In some cases, the protecting group is a triarylmethyl group. In some cases, the protecting group is an aryl ether. In some cases, the protecting group is a disulfide. In some cases, the protecting group is an acid-labile silane. In some cases, the protecting group is an acetal. In some cases, the protecting group is a ketal. In some cases, the protecting group is an enol ether. In some cases, the protecting group is a methoxybenzyl group. In some cases, the protecting group is 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 a tert-butyl ester. In some cases, the protecting group is a base-labile group.

Following the coupling, amidophosphite methods for synthesizing polynucleotides optionally include a capping step. In the capping step, the growing polynucleotide is treated with a capping agent. The capping step is generally designed to block unreacted support-binding 5'-OH groups after coupling from further chain extension, thereby preventing the formation of polynucleotides with internal base deletions. In addition, amidophosphites activated with 1H-tetrazole often react to a small extent at the O6 position of guanosine. Without going into theory, when 1 ₂ /water is oxidized, this by-product, probably through O ₆ -K ⁷ migration, undergoes depurination. The apurine sites may eventually be cleaved during the final deprotection of the polynucleotide, thereby causing a reduction in the yield of the full length product. The O ₆ modifications can be removed by treatment with a capping agent prior to oxidation with 1 ₂ /water. In some cases, the inclusion of a capping step during polynucleotide synthesis provides a reduction in the error rate compared to synthesis without capping. By way of example, the capping step includes treating the supported polynucleotide with a mixture of acetic anhydride and 1-methylimidazole. After the capping step, the support is optionally washed.

After addition of the nucleoside amidophosphite, and optionally after capping and one or more washing steps, the support described herein contains bound nascent nucleic acid that can be oxidized. The oxidation step comprises the oxidation of a phosphite triester to a four-coordinate phosphate triester, a protected precursor to form a naturally occurring phosphodiester internucleoside bond. In some cases, triesters of phosphite acid are oxidized electrochemically. In some cases, oxidation of the growing polynucleotide is achieved by treatment with iodine and water, optionally in the presence of a weak base such as pyridine, lutidine, or collidine. Sometimes the oxidation is carried out under anhydrous conditions using tert-butyl hydroperoxide or (1S)-(+)-(10-camphorsulfonyl)oxaziridine (CSO). In some methods, the capping step is carried out after the oxidation. The second capping step allows the substrate to dry out, since residual water after oxidation, which may remain, may interfere with subsequent coupling. After oxidation, the support and the growing polynucleotide are optionally washed. In some

- 23 039806 cases, the oxidation step is replaced by a sulfurization step in order to obtain polynucleotide thiophosphates, and any capping steps can be carried out after sulfurization. Many reagents are capable of efficiently transferring sulfur, including, without limitation,

-(dimethylaminomethylidene)amino)-3H-1,2,4-dithiazole-3-thione, DDTT, 3H-1,2-benzodithiol3-one 1,1-dioxide, also known as Bocage's reagent, and N,N disulfide, NΓN'-tetraethylthiuram (TETD).

In order for the subsequent nucleoside incorporation cycle to occur via coupling, the protected 5' end (or 3' end if the synthesis is carried out in the 5' to 3' direction) of the support-bound nascent polynucleotide is removed so that the primary hydroxyl group can react with following nucleoside amidophosphite. In some cases, the protecting group is DMT and deprotection occurs using trichloroacetic acid in dichloromethane. In some cases, the protecting group is DMT and the deprotection occurs under the action of electrochemically generated protons. Conducting detritylation for longer periods of time, or using stronger than recommended acid solutions, may result in increased depurination of the solid support bound polynucleotide, and thus reduce the yield of the desired full length product. The methods and compositions described herein provide controlled deblocking conditions that limit unwanted depurination reactions. In some cases, the supported polynucleotide is washed after release. In some cases, efficient washing after deblocking results in the synthesized polynucleotides having a low error rate.

The methods described herein for synthesizing supported polynucleotides may include a repeating sequence of the following steps: depositing a protected monomer on the surface of the support element to bind to either the surface, a linker, or a previously deprotected monomer; deprotection of the introduced monomer so that it can react with the subsequently introduced protected monomer; and introducing another protected monomer for binding. One or more intermediate steps include oxidation and/or sulfurization. In some cases, one or more washing steps are performed before or after one or all of the steps.

The methods described herein for synthesizing supported polynucleotides may include an oxidation step. For example, the methods include a repeating sequence of the following steps: depositing a protected monomer on the surface of a support element to bind either to the surface, or to a linker, or to a deprotected monomer; deprotection of the introduced monomer so that it can react with the subsequently introduced protected monomer; introducing another protected monomer for binding and oxidation and/or sulfurization. In some cases, one or more washing steps are performed before or after one or all of the steps.

The methods described herein for synthesizing supported polynucleotides may further comprise a repeating sequence of the following steps: depositing a protected monomer on the surface of the support element to bind to either the surface, a linker, or a previously deprotected monomer; deprotection of the introduced monomer so that it can react with the subsequently introduced protected monomer; and oxidation and/or sulfurization. In some cases, one or more washing steps are performed before or after one or all of the steps.

The methods described herein for synthesizing supported polynucleotides may further comprise a repeating sequence of the following steps: depositing a protected monomer on the surface of the support element to bind to either the surface, a linker, or a previously deprotected monomer; and oxidation and/or sulfurization. In some cases, one or more washing steps are performed before or after one or all of the steps.

The methods described herein for synthesizing supported polynucleotides may further comprise a repeating sequence of the following steps: depositing a protected monomer on the surface of the support element to bind to either the surface, a linker, or a previously deprotected monomer; deprotection of the introduced monomer so that it can react with the subsequently introduced protected monomer; and oxidation and/or sulfurization. In some cases, one or more washing steps are performed before or after one or all of the steps.

In some cases, polynucleotides are synthesized using photolabile protecting groups, while the hydroxyl groups formed on the surface are blocked using photolabile protecting groups. By exposing the surface to UV light, for example through a photolithographic mask, a pattern of free hydroxyl groups can be obtained on the surface. Such hydroxyl groups can react with photoprotected nucleoside amidophosphites, in accordance with amidophosphite chemistry. A second photolithographic mask may be applied and the surface may be exposed to UV light to form a second pattern of hydroxyl groups, followed by coupling with the 5'-photoprotected nucleoside amidophosphite. Similarly, oligomeric chains can be patterned and extended. Without being bound by theory, the lability of the photocleavable group depends on the wavelength and polarity of the solvent used, and the rate of photocleavage may depend on the duration of exposure and light intensity. This method can take into account a number of factors, such as the accuracy of mask alignment, the efficiency of removing photoprotective groups, and the yield at the amidophosphite coupling step. In addition, unanticipated light penetration into neighboring sites can be minimized. The density of the synthesized oligomers on the spot can be controlled by adjusting the loading of the leader nucleoside onto the synthesis surface.

The surface of the support described herein, which provides the basis for the synthesis of polynucleotides, can be chemically modified to allow the synthesized polynucleotide chain to be cleaved from the surface. In some cases, the polynucleotide chain is cleaved off simultaneously with the deprotection of the polynucleotide. In some cases, the polynucleotide chain is cleaved off after deprotection of the polynucleotide. In an illustrative scheme, a trialkoxysilylamine, such as (CH3CH ₂ O)3Si-(CH ₂ ) ₂ -NH ₂ , reacts with the surface SiOH groups of the support, followed by the reaction of succinic anhydride with an amine to form an amide bond and a free -OH on which the nucleic acid chain grows. Cleavage includes gas-assisted cleavage using ammonia or methylamine. In some cases, cleavage includes cleavage of the linker with electrically generated reagents such as acids or bases. In some cases, after being released from the surface, the polynucleotides are assembled into larger nucleic acids, which are sequenced and decoded to extract the stored information.

The surfaces described herein, after polynucleotide cleavage, can be reused to carry out additional rounds of polynucleotide synthesis. For example, the linker can be reused without further processing/chemical modifications. In some cases, the linker is non-covalently bonded to the surface of the support or polynucleotide. In some embodiments, the linker remains attached to the polynucleotide after it has been cleaved from the surface. In some embodiments, linkers contain reversible covalent bonds, such as esters, amides, ketals, beta-substituted ketones, heterocycles, or other groups that can be reversibly cleaved off. In some cases, such reversible cleavage reactions are controlled by the addition or removal of reagents, or by electrode-controlled electrochemical processes. Optionally, chemical linkers or surface-bound chemical groups are regenerated after a certain number of cycles to restore reactivity and eliminate the formation of unwanted by-products on such linkers or surface-bound chemical groups.

Assembly.

Polynucleotides can be designed so that together they cover a large region of a given sequence in which information is encoded. In some cases, larger polynucleotides are obtained through ligation reactions with the connection of the synthesized polynucleotides. One example of a ligation reaction is polymerase cyclic assembly (PCA). In some instances, at least a portion of the polynucleotides are designed to include an additional region that is a substrate for binding universal primers. In the case of PCA reactions, pre-synthesized polynucleotides include areas of overlap with each other (eg, the overlapping sequence is 4, 20, 40 or more bases). During polymerase reaction cycles, polynucleotides are annealed with complementary fragments, and the gaps are then filled with polymerase. Thus, each cycle provides an increase in the length of different fragments in a random way, depending on which polynucleotides find each other. Complementarity between fragments ensures the formation of full-length double-stranded DNA. In some cases, after completion of the X-ray reaction, an error correction step is carried out using enzymes involved in the detection and repair of mismatched nucleotides in order to remove the mismatched nucleotides from the sequence. After obtaining larger fragments of the target sequence, they can be amplified. In some cases, for example, a target sequence containing 5' and 3' adapter sequences is amplified by a polymerase chain reaction (PCR) that includes modified primers that hybridize to the adapter sequences. In some cases, the modified primers contain one or more uracil bases. The use of modified primers allows the removal of primers by enzymatic reactions based on targeting the modified base and/or gaps left by enzymes that cleave the modified base pair from the fragment. The result is a double-stranded amplification product that lacks adapter sequence residues. Thus, many amplification products can be obtained

- 25 039806 in parallel using the same set of primers to obtain different fragments of double-stranded DNA.

Error correction can be performed on synthesized polynucleotides and/or post-assembly products. An exemplary error correction strategy includes site-directed mutagenesis by PCR with overlapping error correction primers, which is optionally combined with two or more rounds of cloning and sequencing. In certain instances, double-stranded nucleic acids with nucleotide mismatches, loops and small loops, chemically altered bases, and/or other heteroduplexes are selectively removed from correctly synthesized nucleic acid populations. In some cases, error correction is performed using proteins/enzymes that recognize and bind to or near mismatched or mismatched bases within double stranded nucleic acids to create a single or double strand break or to initiate a strand transfer event. Non-limiting examples of error correction proteins/enzymes include endonucleases (T7 endonuclease I, E. coli endonuclease V, T4 endonuclease VII, mung bean nuclease, Celi, E. coli endonuclease IV, UVDE), restriction enzymes, glycosylases, ribonucleases, enzymes involved in the repair of mismatched nucleotides, resolvases, helicases, ligases, antibodies specific for mismatched nucleotides and their variants. Examples of specific error correction enzymes include T4 endonuclease 7, T7 endonuclease 1, S1, mung bean nuclease, MutY, MutS, MutH, MutL, Cleavase, CELI, and HINF1. In some cases, mismatched nucleotide binding protein MutS (Thermus aquaticus) is used to remove unsuccessful products from the population of synthesized products. In some cases, error correction is carried out using the Correctase enzyme. In some cases, error correction is performed using the SURVEYOR endonuclease (Transgenomic), a mismatch-specific DNA endonuclease that scans the DNA heteroduplex for known and unknown mutations and polymorphisms.

Sequencing.

After extraction and/or amplification of polynucleotides from the surface of the structure, a suitable sequencing technology can be used to sequence the polynucleotides. In some cases, the DNA sequence is read on a support or within a structural element. In some cases, the polynucleotides stored on the support are extracted, optionally assembled into larger nucleic acids, and then sequenced.

The polynucleotides synthesized and stored on the structures described herein encode data that can be deciphered by reading the sequence of the synthesized polynucleotides and converting the sequence into a machine-readable binary code. In some cases, sequences require assembly, and it may be necessary for the assembly step to be performed at the nucleic acid sequence level or at the digital sequence level.

This document provides discovery systems containing a device capable of sequencing stored polynucleotides either directly on the structure and/or after removal from the main structure. In cases where the structure is a reel tape of flexible material, the detection system includes a device for holding and advancing the structure through the detection area and a detector located adjacent to the detection area to detect a signal emanating from a piece of tape when the specified piece of tape is in detection areas. In some cases, the signal indicates the presence of a polynucleotide. In some cases, a signal indicates the presence of a polynucleotide sequence (eg, a fluorescent signal). In some cases, the information encoded in the polynucleotides on the continuous tape is read by the computer as the tape is conveyed through a detector operatively connected to the computer. In some cases, the detection system comprises a computer system containing a polynucleotide sequencing device, a database for storing and retrieving data related to the polynucleotide sequence, software for converting the DNA code of the polynucleotide sequence into binary code, a computer for reading the binary code, or any combination of the above. .

This document presents sequencing systems that can be integrated into the devices described herein. Various sequencing methods are well known in the art and include nitrogenous base recognition, which determines the identity of the base in the target polynucleotide. In some instances, polynucleotides synthesized using the methods, devices, compositions, and systems described herein are sequenced after cleavage from a synthesis surface. In some cases, sequencing occurs during or simultaneously with the synthesis of polynucleotides, with recognition of nitrogenous bases occurring immediately after or immediately before the addition of the nucleoside as a monomer to the growing polynucleotide chain. Methods for recognizing nitrogenous bases include measuring the electric current generated as a result of polymerase-catalyzed addition

- 26039806 bases to the thread of the matrix. In some cases, surfaces for synthesis contain enzymes such as polymerases. In some cases, such enzymes are associated with electrodes or surfaces for synthesis.

Computer systems.

In various aspects, any of the systems described herein are operatively linked to a computer and optionally automated by a computer, either locally or remotely. In various instances, the methods and systems of the present invention may further provide for computer system software programs and their use. Accordingly, computerized control for synchronizing dosing/vacuum/replenishment functions such as controlling and synchronizing applicator movements, performing dosing and activating vacuum are within the scope of the present invention. In some instances, computer systems are programmed to match between a user-specified nucleotide sequence and the position of an applicator to deliver the appropriate reagents to specified areas of the substrate.

Computer system 800 illustrated in FIG. 8 may be understood as a logical device that can read instructions from storage media 811 and/or network port 805, which may optionally be connected to a server 809 containing non-removable media 812. The system may include a CPU 801, a disk drive 803, an optional input device, such as a keyboard 815 and/or a computer mouse 816, and an optional monitor 807. Data transmission may be provided through the specified communication channel to a server at a local or remote location. The communication channel may include any means of transmitting and/or receiving data. For example, the communication channel may be a network connection, a wireless connection, or an Internet connection. Such a connection can provide communication through the World Wide Web. It is contemplated that data related to the present disclosure may be transmitted over such networks or connections for reception and/or viewing by user 822.

Fig. 9 is a block diagram illustrating a first example of a computer system architecture that can be used with examples of the present invention. As shown in FIG. 5, an exemplary computer system may include a processor 902 for processing instructions. Non-limiting examples of processors include: Intel Xeon™ Processor, AMD Opteron™ Processor, Samsung ARM 1176JZ(F)-S v1.0™ 32-bit RISC Processor, Samsung S5PC100™ ARM Cortex-A8 Processor, Apple ARM Cortex-A8 Processor A4™, Marvell PXA 930™ processor, or functionally equivalent processor. Multiple threads of execution can be used for parallel processing. In some cases, multiple processors or processors with multiple cores may also be used, whether in a single computer system, in a cluster, or distributed across systems within a network of multiple computers, mobile phones, and/or PDAs.

As illustrated in FIG. 9, cache 904 may be coupled to or included with processor 902 to provide high-speed memory for instructions or data that have been recently or frequently used by processor 902. Processor 902 is coupled to northbridge 906 via processor bus 908. Northbridge 906 connected to random access memory (RAM) 910 via memory bus 912 and controls access to RAM 910 of processor 902. Northbridge 906 is also connected to southbridge 914 via internal bus 916. Southbridge 914, in turn, is connected to the peripheral bus 918. The peripheral bus may be, for example, PCI, PCI-X, PCI Express, or another peripheral bus. The northbridge and southbridge are often referred to as the chipset for the processor, and they control the communication between the processor, RAM, and peripheral components on the 918 peripheral bus. In some alternative architectures, the northbridge functionality may be included in the processor instead of using a separate northbridge.

In some cases, system 900 may include an accelerator 922 coupled to a peripheral bus 918. The accelerator may include field programmable gate arrays (FPGAs) or other hardware to speed up certain processing. For example, the accelerator can be used for adaptive data restructuring or for evaluating algebraic expressions used in processing extended sets.

Software and data is stored on external storage 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, as well as operating system-based application software for data storage management and optimization, in accordance with exemplary embodiments. of the present invention.

According to this example, system 900 also includes network cards (NICs) 920 and 921 connected

- 27 039806 with a peripheral bus, to provide network interfaces for external storage, such as a network attached storage device (NAS), and other computer systems that can be used for distributed parallel processing.

Fig. 10 is a diagram showing a network 1000 with multiple computer systems 1002a and 1002b, multiple mobile phones and personal digital assistants 1002c, and a network attached storage (NAS) 1004a and 1004b. According to exemplary embodiments, systems 1002a, 1002b, and 1002c can provide data storage management and data access optimization for data stored on a network-attached storage device (NAS) 1004a and 1004b. The data may be mathematically modeled and computed using distributed parallel processing within computer systems 1002a and 1002b and systems using mobile phones and personal digital assistants 1002c. Computer systems 1002a and 1002b and systems using mobile phones and personal digital assistants 1002c can also provide parallel processing for adaptive data restructuring for data stored on network attached storage (NAS) 1004a and 1004b. In FIG. 10 is only an example, and a wide variety of other computer architectures and systems can be applied to various embodiments of the present invention. For example, a blade server can be used to provide parallel processing. Blade servers with processors can be connected via a backplane to provide parallel processing. The storage device can also be connected to the backplane or, as a Network Attached Storage (NAS) device, through a separate network interface.

According to some exemplary embodiments, the processors may contain separate memory spaces and communicate data through network interfaces, a backplane, or other connectors for parallel processing by other processors. In other embodiments, some or all of the processors may use a shared virtual address memory space.

Fig. 11 is a block diagram of a multiprocessor computer system 1100 utilizing shared virtual address memory space in accordance with an exemplary embodiment. The system includes a plurality of processors 1102a-f that may have access to a common memory subsystem 1104. The system includes a plurality of programmable processors for algorithmic control of hardware memory (MAP) 1106a-f in the memory subsystem 1104. Each MAP 1106a-f may contain memory 1108a -f and one or more Field Programmable Gate Arrays (FPGA) 1110a-f. The MAP provides a configurable functional node and specific algorithms, or portions of the algorithms, can be provided to the FPGA 1110a-f for processing in close coordination with the respective processor. For example, MAP can be used to evaluate algebraic expressions against a data model and to perform adaptive data restructuring in exemplary embodiments. According to this example, each MAP is available to all processors for these purposes. In one configuration, each MAP may use direct memory access (DMA) to access associative memory 1108a-f, allowing it to perform tasks independently and asynchronously from the associated microprocessor 1102a-f. In this configuration, a MAP can pass results directly to another MAP for pipelining and parallel execution of algorithms.

The above computer architectures and systems are exemplary only, and a wide variety of other architectures and systems using computers, mobile phones, and personal digital assistants, including systems using any combination of general purpose processors, coprocessors, FPGAs, and other programmable logic devices, systems on a chip (SOC), 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. With respect to the exemplary embodiments, any of a variety of storage media can be used, including random access storage, hard drives, flash memory, tape drives, disk array, network attached storage (NAS), and other local or distributed storage devices and systems. .

In exemplary embodiments, the computer system may be implemented using software modules operating within any of the above or other computer architectures and systems. In other embodiments, the functions of the system may be implemented partially or entirely in firmware, programmable logic devices such as field programmable gate arrays (FPGAs), systems on a chip (SOCs), application specific integrated circuits (ASICs), and other processing and logic devices. elements. For example, the processor group and optimizer can be implemented with hardware acceleration through the use of a hardware accelerator.

Embodiments.

This document presents methods for storing information, including a) providing

- 28 039806 structure containing the surface; b) introducing at least one nucleotide onto the surface, wherein at least one nucleotide reacts with a polynucleotide attached to the surface; and c) repeating step b) to synthesize a plurality of polynucleotides on the surface, wherein the density of unique polynucleotides on the surface is at least 100 x 10 ⁶ polynucleotides/cm ² . Also provided herein are methods, said method further comprising cleaving at least one polynucleotide from a surface, the polynucleotide being dissolved in the droplet. Also provided herein are methods, said method further comprising sequencing at least one polynucleotide from a surface. In addition, methods are presented herein, said method further comprising drying the surface. In addition, methods are provided herein, said method further comprising washing nucleotides from a surface. In addition, methods are presented herein in which the surface is a solid substrate. In addition, methods are presented herein in which the surface contains glass, quartz glass, silicon, silicon dioxide, silicon nitride, plastics, metals, or combinations of the above.

This document presents methods for storing information, including a) providing a structure containing a surface; b) applying to the surface of a droplet containing at least one nucleotide, wherein at least one nucleotide reacts with a polynucleotide attached to the surface; and c) repeating step b) to effect the synthesis of a plurality of polynucleotides on the surface, wherein the droplet volume is less than about 100 femtoliters.

This document presents methods for storing information, including a) providing a structure containing a surface; b) introducing at least one nucleotide onto the surface, wherein at least one nucleotide reacts with a polynucleotide attached to the surface; and c) repeating step b) to synthesize a plurality of surface polynucleotides, wherein the time to repeat step b) using four different nucleotides is less than about 100 ms. Also provided herein are methods, said method further comprising one or more washing steps. In addition, methods are provided herein, said method further comprising deblocking, oxidizing, washing, capping, or any combination thereof.

Provided herein are information storage devices comprising a chip containing an array of addressable loci, wherein one or more addressable loci comprise at least one electrode, a synthesis surface, and at least one fluid port, with the synthesis surface in each of addressable loci contains at least one polynucleotide extending from the surface, and the density of addressable loci on the chip is at least 100x10 ⁶ polynucleotide/cm ² . Also provided herein are devices wherein the length of at least one polynucleotide is from about 150 to about 500 bases. Also provided herein are devices where at least one polynucleotide is about 200 bases in length. In addition, devices are presented herein, said device further comprising a reagent reservoir. In addition, devices are presented herein, said device further comprising a heating or cooling element. Also provided herein are devices where at least one addressable locus contains a droplet. In addition, this document presents devices where the droplet diameter is less than 50 microns.

Provided herein are methods for storing information, the method comprising: a) converting at least one piece of information in the form of at least one sequence in digital form into at least one nucleic acid sequence; b) synthesizing a plurality of polynucleotides with given sequences co-coding for at least one nucleic acid sequence; c) applying to the surface of a droplet containing at least one nucleotide, wherein at least one nucleotide reacts with a polynucleotide attached to the surface; d) repeating step c) to effect synthesis of a plurality of polynucleotides on the surface; and e) storing a plurality of polynucleotides with a droplet volume of less than about 100 femtoliters.

Provided herein are methods for storing information, the method comprising: a) converting at least one piece of information in the form of at least one sequence in digital form into at least one nucleic acid sequence; b) synthesizing a plurality of polynucleotides with given sequences co-coding for at least one nucleic acid sequence; c) applying to the surface of a droplet containing at least one nucleotide, wherein at least one nucleotide reacts with a polynucleotide attached to the surface; d) repeating step c) to effect synthesis of a plurality of polynucleotides on the surface; and e) storing a plurality of polynucleotides, wherein the time to repeat step c) using four different nucleotides is

- 29 039806 less than about 100 ms.

Provided herein are methods for synthesizing polynucleotides comprising a) providing a structure containing a surface, the surface containing a plurality of polynucleotide extension loci; and b) synthesizing a plurality of surface-extending polynucleotides, the synthesizing comprising introducing one or more reagents by applying a potential to the surface. Methods are also presented in which the potential is an electrical potential. In addition, the document presents methods in which the surface is a solid substrate.

This document provides information storage devices using any of the methods described herein. This document provides systems for storing information using any of the methods described herein.

The following examples are set forth to more clearly illustrate the principle and practice of the embodiments disclosed herein for those skilled in the art, and should not be construed as limiting the scope of any of the claimed embodiments. Unless otherwise noted, all parts and percentages are by weight.

Examples

Example 1. Functionalization of the surface of the device.

The device was functionalized to allow the attachment and synthesis of a library of polynucleotides. The surface of the device was first wet cleaned using a piranha solution containing 90% H2SO4 and 10% H2O2 for 20 minutes. The device was washed in several beakers with DI-water, kept under a tap with a stream of DI-water for 5 min and dried with N2. The device was then soaked in NH4OH (1:100; 3 ml:300 ml) for 5 min, rinsed with DI-water using a manual dispenser gun, soaked successively in three beakers with DI-water for 1 min each time, and then washed again with DI-water using a manual dispenser gun. Then the device was subjected to plasma-chemical cleaning by exposing the surface of the device to O ₂ . A SAMCO PC-300 instrument was used for etching with O ₂ plasma at 250 W for 1 min in a downward mode.

The cleaned surface of the device was functionalized with active agents using a solution containing N-(3-triethoxysilylpropyl)-4-hydroxybutyramide using a YES-1224P system for vapor deposition using the following parameters: 0.5-1 torr, 60 min, 70°C, evaporator temperature 135°C. The surface of the device was coated with resist using a Brewer Science 200X spin coater. The device was spin coated with SPRx 3612 photoresist at 2500 rpm for 40 seconds. The device was pre-fired for 30 min at 90° C. on a Brewer heating plate. The device was photolithographed using a Karl Suss MA6 mask photolithograph. The device was exposed for 2.2 seconds and developed for 1 minute in MSF 26A. The remaining developer was washed out with a manual dispenser gun and the device was soaked in water for 5 minutes. The device was fired for 30 min at 100° C. in an oven followed by a visual check for lithographic defects using a Nikon L200. A cleaning process was used to remove residual resist using a SAMCO PC-300 O ₂ plasma etcher at 250 W for 1 minute.

The surface of the device was functionalized by passive means using 100 μl of a solution of perfluorooctyltrichlorosilane mixed with 10 μl of light mineral oil. The device was placed in the chamber, pumped for 10 minutes, and then the valve leading to the pump was closed and the device was left for 10 minutes. The chamber was ventilated with air. The device was subjected to a stripping procedure by performing two soaks for 5 min in 500 ml NMP at 70° C. with ultrasonication at maximum power (9 in the Crest system). Then the device was soaked for 5 min in 500 ml of isopropanol at room temperature with ultrasonication at maximum power. The device was immersed in 300 ml of 200 proof ethanol and purged with N ₂ . The functionalized surface was activated to function as a scaffold for polynucleotide synthesis.

Example 2. High-precision storage and assembly of information based on DNA.

Digital information, selected in the form of binary data with a total volume of about 0.2 GB, included the contents of the Universal Declaration of Human Rights in more than 100 languages, the top 100 books from Project Gutenberg, and a seed database. The digital information was encrypted into a nucleic acid sequence and divided into lines. More than 10 million non-identical polynucleotides have been synthesized on a rigid silicon surface, each corresponding to a specific string. Each non-identical polynucleotide was 200 bases or less in length. The synthesized polynucleotides were collected and sequenced and decoded back into a digital code, and when compared with the original at least one sequence in digital form, the accuracy of the original digital information was 100%.

- 30 039806

Example 3 High Density Storage System.

Polynucleotides are synthesized de novo using the methods described herein. After synthesis, the polynucleotides are collected as a single droplet and transferred to a solid silicon support for storage. The solid substrate measures 86x54 mm and is 1-2 mm thick. The capacity of a solid substrate is 1-10 petabytes (PB), represented as an addressable array of 10 GB data blocks. The physical division into data blocks is redundantly encoded as a leading sequence, with each polynucleotide in the data block having a common start sequence and any other sequence information for designation or retrieval. Data blocks are presented as water drops with dissolved polynucleotides, with a physical redundancy of 100-1000 copies of each polynucleotide. The length of polynucleotides is in the range of 100-1000 bases. The volume of a drop is equivalent to spheres with a diameter of 40–50 µm. The solid substrate also allows for up to 10000x10000 positions in less than 1 sq. inches.

An exemplary solid support is shown in FIG. 12A, 12V. In FIG. 12A shows the front side of a solid substrate made of glass and containing a transparent window for the array and fluid holes. In FIG. 12B shows the rear side of the solid substrate, which is an electronic circuit containing electrical contacts (LGA with 1 mm pitch) and a thermal contact surface under the solid substrate region.

After droplets are added to the solid support, the solid support can be dried and later resolvated for subsequent applications. Alternatively, the solid support is dried and stored. Since the blobs within each data block contain sequence information for designation or retrieval, certain data blocks are extracted from the plurality of data blocks based on the sequence information.

Example 4 Access High Density Storage System.

Polynucleotides were synthesized de novo using the methods described herein. After synthesis, the polynucleotides were collected as a single drop and transferred to a solid paper support for storage. The solid backing measures 3.5x2.5 inches. The capacity of a solid substrate is 1 petabyte, represented as a 32x32 addressable array containing 1024 spots. Each spot contains a 1 terabyte pool, see, for example, FIG. 17 and 18.

At least one 10-100 MB piece of information is encoded in DNA and stored in 1 PB of data. Next, 10-100 MB of encoded DNA is extracted by random accessing the encoded DNA and extracting the encoded DNA from a 1 TB pool.

Example 5 Local control of polynucleotide synthesis on a solid support.

Polynucleotides of 240 bases are synthesized on a solid support using the methods described herein. The dsDNA polynucleotides are approximately 80 nm long and the ssDNA polynucleotides are approximately 160 nm long. The solid support contains an array of wells with parameters of 500 nm (depth) x 400 nm (diameter) (the volume is approximately 0.628 femtoliter). Each of the wells contains an addressable locus, addressable electrodes within the side walls of each well (area is 50,000 nm ² /electrode) and a 250 nm (diameter) polynucleotide synthesis/attachment surface in an addressable message with a 250 nm (diameter) addressable bottom electrode. The electrodes are independently addressable.

The polynucleotides are present on the synthesis surface at a density of 1 polynucleotide per 50 nm ² . The wells are separated in 1.0 µm increments. 10 nm thick side electrodes (located about 100 nm above the polynucleotide surface) are charged to create an H ⁺ ion concentration gradient that removes the protecting groups (where the polynucleotide is capped with an acid cleavable blocking group) from the 5'-OH -groups on certain polynucleotides at loci on the surface for synthesis. The H ⁺ ions are then removed. (See Fig. 16B). The nucleoside amidophosphite is added as a monomer and the polynucleotides will be available for coupling with the nucleosides at the unblocked sites. For the synthesis of polynucleotides, the cycles of deprotection and combination are repeated. By applying a series of electrode-controlled masks prior to addition to the surface of each monomer type, the desired polynucleotides are synthesized at precise locations on the surface in a controlled sequence.

Example 6. Local control of polynucleotide synthesis on a solid support using electric fields.

Polynucleotides of 240 bases are synthesized on a solid support using the methods described herein. The solid support contains an array of wells with parameters of 500 nm (depth) x 500 nm (diameter). Each of the wells contains an addressable locus, addressable electrodes within the side walls of each well (area is 50,000 nm ² /electrode) and a 250 nm (diameter) polynucleotide synthesis/attachment surface in an addressable message with a 250 nm (diameter) addressable bottom electrode. The electrodes are independently addressable.

- 31 039806

The polynucleotides are present on the synthesis surface at a density of 1 polynucleotide per 50 nm ² . The wells are separated in 1.0 µm increments. The nucleoside amidophosphite is added as a monomer and the polynucleotides will be available for coupling with the nucleosides at the unblocked sites. For the synthesis of polynucleotides, the cycles of deprotection and combination are repeated. By applying a series of electrode-controlled masks prior to addition to the surface of each monomer type, the desired polynucleotides are synthesized at precise locations on the surface in a controlled sequence. The lower electrodes at the bottom of the well are activated, resulting in the release of polynucleotides attached to it. The side electrodes are charged to generate an electric field that moves the polynucleotides out of the well. Nucleoside amidophosphites are then added as monomers, which grow onto reusable linkers attached to the surface, and the synthesis is repeated.

Example 7 Local control of polynucleotide synthesis on a solid support.

Polynucleotides of 240 bases are synthesized on a solid support using the methods described herein. The dsDNA polynucleotides are approximately 80 nm long and the ssDNA polynucleotides are approximately 160 nm long. The solid support contains an array of addressable electrodes with a diameter of 100 nm on the surface for the synthesis/attachment of polynucleotides (see Fig. 19).

The polynucleotides are present on the synthesis surface at a density of 1 polynucleotide per 39.27 nm ² . The wells are separated in 0.25 µm increments. The electrodes are charged to create an H ⁺ ion concentration gradient that removes protecting groups (where the polynucleotide is blocked by an acid-cleavable blocking group) from the 5'-OH groups on certain polynucleotides at the synthesis surface loci. The H ⁺ ions are then removed. The nucleoside amidophosphite is added as a monomer and the polynucleotides will be available for coupling with the nucleosides at the unblocked sites. For the synthesis of polynucleotides, the cycles of deprotection and combination are repeated. By applying a series of electrode-controlled masks prior to addition to the surface of each monomer type, the desired polynucleotides are synthesized at precise locations on the surface in a controlled sequence.

While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, modifications and substitutions will occur to those skilled in the art without departing from the present invention. It should be understood that in the practice of the present invention, various alternatives to the embodiments of the present invention described herein may be used. It is intended that the following claims define the scope of the present invention, and that methods and structures within the scope of this claim and their equivalents are also covered by it.

CLAIM

Claims (19)

CLAIM 1. An information storage device comprising a solid support, the solid support comprising a plurality of wells, each of the wells containing an addressable locus containing a surface for polynucleotide synthesis located in the lower region of each of the wells; a bottom electrode in addressable communication with the surface for polynucleotide synthesis; and at least one side electrode located on the side wall of each of the wells, with at least one side electrode located at a distance of 50 to 200 nm from the lower region. 2. Device according to claim 1, wherein the solid support contains addressable loci with a density of at least 100x106 addressable loci/cm ² . 3. The device according to claim 1, where the wells are separated with a step of not more than 250 nm. 4. The device according to claim 1, where each of the wells contains a depth of from 100 to 1000 nm. 5. The device according to claim 1, where each of the wells contains the largest cross-sectional diameter from 100 to 800 nm. 6. The device according to claim 1, where the lower electrode provides the largest cross-sectional area from 10 ⁴ to 105 nm ² . 7. The device according to claim 1, wherein at least one side electrode is located at a distance of 75 to 125 nm from the lower region. 8. The device according to claim 1, wherein at least one side electrode provides a height of 5 to 25 nm. 9. The device according to claim 1, containing at least two side electrodes. - 32 039806 10. The apparatus of claim 1, wherein at least one side electrode and the bottom electrode are independently addressable. 11. A method of storing information, including: a) providing the device according to claim 1; b) providing instructions for the synthesis of polynucleotides; c) introducing at least one nucleoside onto the synthesis surface, the at least one nucleoside being coupled to the polynucleotide attached to the synthesis surface; and d) repeating step c) to synthesize a plurality of polynucleotides on a synthesis surface, the instructions providing for at least one sequence encoding the plurality of polynucleotides. 12. The method of claim 11, wherein the method further comprises cleaving at least one polynucleotide from the surface, the polynucleotide being dissolved in the droplet. 13. The method of claim 11, wherein the method further comprises sequencing at least one polynucleotide from the surface. 14. The method of claim 11 wherein the nucleoside comprises a nucleoside amidophosphite. 15. The method of claim 11, wherein the method further comprises drying the surface. 16. The method of claim 11, wherein the method further comprises a cleavage step, the cleavage step comprising applying an electrical potential to the bottom electrode to generate a cleavage reagent. 17. The method of claim 11, wherein the method further comprises the step of capping. 18. The method of claim 11, wherein the method further comprises an oxidation step. 19. The method of claim 11, wherein the method further comprises a deblocking step, wherein the deblocking step includes applying an electrical potential to at least one side electrode to generate a deprotectant.