Devices and methods for synthesis

Abstract

Compositions, devices, systems, and methods for DNA oligomer synthesis are provided herein.Furthermore, devices are provided that include addressable electrodes to control polynucleotide synthesis (such as deprotection, extension, or cleavage).The compositions, devices, systems, and methods described herein provide improved synthesis, storage, density, and retrieval of biomolecule-based information.

Description

[Technical field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 63 / 274,397, filed November 1, 2021, which is incorporated by reference in its entirety. [Background technology]

[0002] Biomolecules (e.g., nucleic acids) have applications in research, medicine, and information storage. However, there is a need for high-density, scalable, automated, precise, and efficient systems for producing biomolecules. Incorporation by Reference

[0003] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. Summary of the Invention [Means for solving the problem]

[0004] Provided herein is a method of synthesizing a polynucleotide, the method comprising: (a) contacting at least one nucleoside bound to a solid support with a protected nucleoside, the protected nucleoside comprising a protecting group, the protecting group comprising an alkene, and the solid support comprising an addressable array; (b) contacting an amount of a reagent with the protected nucleoside, resulting in deprotection of a terminal nucleoside of the protected nucleoside; and (c) repeating steps (a) and (b) to synthesize a polynucleotide. Provided herein further is a method in which the amount of the reagent is less than about 15 mol % of the protected nucleoside. Provided herein further is a method in which the amount of the reagent is less than about 5 mol % of the protected nucleoside. Provided herein further is a method in which the protected nucleoside comprises a 5' or 3' protecting group. Provided herein further is a method in which the protecting group comprises an allyl group. Provided herein further is a method in which the protecting group comprises an O-allyl group. Further provided herein is a method in which the reagent is a transition metal catalyst. Further provided herein is a method in which the transition metal catalyst is in the oxidation state zero. Further provided herein is a method in which the transition metal catalyst comprises a Pd(0) catalyst. Further provided herein is a method in which the transition metal catalyst comprises one or more phosphine ligands. Further provided herein is a method in which the transition metal catalyst comprises Pd(Ph3)4. Further provided herein is a method further comprising contacting the protected nucleoside with a nucleophile. Further provided herein is a method in which the nucleophile is a C nucleophile. Further provided herein is a method in which the nucleophile is an N,N-dimethylbarbiturate. Further provided herein is a method in which the reagent comprises a heteroaromatic group. Further provided herein is a method in which the reagent is a tetrazine. Further provided herein is a method in which the nucleophile comprises a phosphine. Further provided herein is a method in which the nucleophile is PPh3. Further provided herein is a method in which contacting the reagent with the protected nucleoside is less than about 10 minutes. Further provided herein are methods wherein contacting the reagent with the protected nucleoside takes less than about 5 minutes.Further provided herein is a method further comprising capping. Further provided herein is a method wherein the capping comprises treatment with acetyl chloride or acetic anhydride. Further provided herein is a method wherein the protected nucleoside comprises at least two alkenyl groups. Further provided herein is a method wherein the deprotection comprises removal of one or more alkenyl groups. Further provided herein is a method comprising an oxidation step after step (c). Further provided herein is a method comprising an oxidation step after step (b). Further provided herein is a method wherein the polynucleotide is 50-300 bases in length. Further provided herein is a method wherein the polynucleotide is 50-300 bases in length. Further provided herein is a method wherein the protected nucleoside comprises the formula:

[0005] [ka] or

[0006] [ka] wherein R 1 is a linear or branched alkenylene; R 2 is alkylene or alkenylene, each of which is independently unsubstituted or substituted; R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , or R 9 are each independently H, OH, halogen, O-alkyl, N-alkyl, O-alkyl-O-alkyl, N3, or R 3 and R 6 come together to form a ring, B is a monocyclic or bicyclic C 4-6 Further provided herein is a method in which R is a heterocyclic ring. 1Further provided herein are methods in which R 1 is linear C 2-6 Further provided herein is a method in which R is an alkenylene. 1 Further provided herein is a method in which R 1 Further provided herein are methods in which R is a branched alkenylene. 1 is branched chain C 3-8 Further provided herein is a method in which R is an alkenylene. 2 Further provided herein are methods in which R is a substituted alkylene. 2 C is substituted 2-4 Further provided herein is a method in which R 2 Further provided herein is a process where R 2 Further provided herein are methods in which R is unsubstituted alkenylene. 2 is unsubstituted C 2-4 Further provided herein is a method in which R 2 Further provided herein is a method where B is an allyl. Further provided herein is a method where B is a monocyclic C4 heterocyclic ring. Further provided herein is a method where B is a bicyclic C5 heterocyclic ring. Further provided herein is a method where B is a nucleobase. Further provided herein is a method where R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , and R 9 and R 3 is H and R 4 Further provided herein are methods in which R 3 and R 4 Further provided herein is a method in which one or both of R 3 and R 4 wherein one or both of is -OCH3 or -OCH2CH2OCH3. Additionally, a protected nucleoside may be provided herein having the structure

[0007] [ka] Further provided herein is a method for preparing a protected nucleoside having the structure

[0008] [ka] Provided herein is a method comprising:

[0009] Provided herein is a device configured to perform the methods described herein.Further provided herein is a device in which the addressable array comprises at least 1000 addressable loci for synthesis.Further provided herein is a device in which the addressable array comprises a pitch distance of 10-200 nm.

[0010] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which: [Brief description of the drawings]

[0011] [Figure 1] 1 illustrates an exemplary device for nucleic acid-based data storage configured to deblock polynucleotides with electrochemically generated acid, according to some embodiments. [Diagram 2] 1 illustrates a silicon-based prior art device having a porous growth layer over an electrode according to some embodiments. [Figure 3A] 1 illustrates a silicon-based polynucleotide synthesis surface comprising patterned conductive anodes, according to some embodiments. [Figure 3B]1 illustrates a silicon-based polynucleotide synthesis surface comprising a patterned conductive anode and a recessed shield electrode, according to some embodiments. [Figure 4] 1 illustrates a silicon-based polynucleotide synthesis surface including oxide islands on a conductive layer according to some embodiments. The oxide islands are patterned as exemplary arrangements only, and in some cases are randomly arranged. [Figure 5A] 1 illustrates a silicon-based polynucleotide synthesis surface including patterned conductive anodes and a thermal oxide layer on top of an optional p-type or n-type silicon layer according to some embodiments. Polynucleotide growth occurs in the pores between the anodes. Vertical interconnect access (VIA) is not shown for clarity. [Figure 5B] 1 illustrates a silicon-based polynucleotide synthesis surface including patterned oxide islands on a conductive anode layer and an optional thermal oxide layer on a p-type or n-type silicon layer according to some embodiments. Polynucleotide growth occurs on the oxide islands. Vertical interconnect accesses (VIAs) are not shown for clarity. [Figure 5C] 1 illustrates a silicon-based polynucleotide synthesis surface including patterned oxide islands on a conductive anode layer and a thermal oxide layer on an optional p-type or n-type silicon layer according to some embodiments. The conductive layers are sandwiched on both sides by additional bonding layers. A single bonding layer is shown for clarity only, and in some cases the surface includes multiple bonding layers. Polynucleotide growth occurs on the oxide islands. Vertical interconnect accesses (VIAs) are not shown for clarity. [Figure 6A] 1 illustrates a device including a "sandwiched" anode above the surface of a cathode, according to some embodiments. [Figure 6B]1 illustrates a device including an anode that is located substantially in the same plane as the cathode ("in-plane"), according to some embodiments. [Figure 7A] FIG. 1 illustrates a cross-sectional view of a high-density device for polynucleotide synthesis according to some embodiments. Two exemplary addressable device arrays are shown for clarity only. [Figure 7B] 1 illustrates a top view of a high density device for polynucleotide synthesis according to some embodiments. Nine exemplary addressable device arrays are shown for clarity only. [Figure 7C] FIG. 1 illustrates a top view of a high-density device for polynucleotide synthesis. Four exemplary addressable device arrays are shown for clarity only. [Figure 7D] FIG. 1 illustrates a cross-sectional view of a high-density device for polynucleotide synthesis according to some embodiments. Two exemplary device arrays are shown for clarity only. [Figure 7E] FIG. 1 illustrates a top view of a high-density device for polynucleotide synthesis according to some embodiments. An exemplary array of 16 addressable devices is shown for clarity only. [Figure 8] 1 shows a schematic diagram of a CMOS integrated device array according to some embodiments. [Figure 9] 1 is an example of a rack-type instrument. Such an instrument, according to some embodiments, may contain hundreds or thousands of solid support arrays. [Figure 10A] FIG. 1 is a front view of an example solid support array, according to some embodiments. Such an array may, in some cases, contain thousands or millions of the polynucleotide synthesis devices described herein. [Figure 10B] FIG. 2 is a rear view of an example solid support array, according to some embodiments. [Figure 11] FIG. 1 is a schematic diagram of a solid support including an active area and a fluidic interface, according to some embodiments. [Figure 12A] 1 illustrates a continuous loop arrangement of a flexible structure according to some embodiments. [Figure 12B] 1 illustrates a reel-to-reel arrangement of a flexible structure according to some embodiments. [Figure 12C] 1 shows a scheme for the release and extraction of synthesized polynucleotides according to some embodiments. [Figure 12D] 1 shows a scheme for the release and extraction of synthesized polynucleotides according to some embodiments. [Figure 13] Illustrates chemical synthesis of DNA using 5'-alloc DNA phosphoramidite monomers according to some embodiments. Steps are labeled as follows: top right: phosphoramidite coupling, bottom: oxidation and capping, 1) I2 / pyr / H2O, 2) Ac2O, top left: Pd-catalyzed deprotection of Alloc, Pd(PPh3)4, PPh3, N,N' dimethylbarbituric acid. [Figure 14A] HPLC chromatograms of 15-mer DNA molecules synthesized using 5'-alloc DNA phosphoramidite monomers and subsequent deprotection conditions according to some embodiments. The window is labeled Chromatographic Results. The top plot is labeled Samples from ESI TIC Scan Frag=200.0V 202201210 DSP_000DD 20201207.d, with the y-axis representing counts from 0 to 7x108 in unit intervals of 1x108. The bottom plot is labeled Samples from DAD1-A:Sig260.0,4,0 Ref=360.0 0.100.0 20201210 DSP_000DD 20201207.d, with the y-axis representing counts from -0.1x103 to 1x103 in unit intervals of 0.1x103. Both plots have a common x-axis labeled response versus acquisition time (min) from 0 to 19 in unit intervals of 0.5. [Figure 14B]1 shows MS analysis of a 15-mer DNA molecule synthesized using 5'-alloc DNA phosphoramidite monomers and subsequent deprotection conditions according to some embodiments. The window is titled Deconvolution Results, with display choices of delta mass, monoisotopic, match tolerance 5 ppm, and peak-to-peak profile. The plot is labeled Sample from ESI scan (rt: 9.825-10.106 min, 18 scans), Frag=200.0V 20201210 DSP_000DD 20201207.d, Deconvolution. The y-axis represents counts from 0 to 2.4x106 at intervals of 0.2x106. The x-axis is from 0 to 1250 at unit intervals of 500 and is labeled Counts vs. Mass-to-Charge (m / z). The peaks labeled (from left to right) are: 357.98, 1500.25 (second largest peak), 2146.31, 2755.41, 3363.49, 3971.58, 4500.73 (largest peak), 6016.39, 9000.45, 10321.88, and 11914.75. [Figure 15] Figure 1 shows chemical synthesis of DNA using PO-alloc protecting groups according to some embodiments. Steps are labeled as follows: right: phosphoramidite coupling, left: 1) Pd-catalyzed Alloc deprotection and capping, Pd(PPh3)4, PPh3, N,N' dimethylbarbituric acid, 2) Ac2O, top left: oxidation and cleavage, 1) I2 / pyr / H2O, 2) NHMe2. [Figure 16] FIG. 1 illustrates DNA oligomer synthesis on an electrochemical platform with Pd(0)-catalyzed deprotection of 5′-O-alloc protected DNA phosphoramidite moieties with nucleophiles according to some embodiments. [Figure 17] 1 illustrates the use of an electrochemical platform for electrophoretic applications, according to some embodiments. Charged species can be selectively attracted to or repelled from reaction sites. [Figure 18] 1 illustrates an example computer system according to some embodiments. [Figure 19] 1 is a block diagram illustrating the architecture of a computer system according to some embodiments. Pd0(R3P)4 4- is shown for illustrative purposes only. [Figure 20] FIG. 1 illustrates a network configured to incorporate multiple computer systems, multiple mobile phones and personal data assistants, and network attached storage (NAS), according to some embodiments. [Figure 21] FIG. 1 is a block diagram of a multi-processor computer system using a shared virtual address memory space according to some embodiments. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] There is a need for higher density synthesis systems, for example, those capable of synthesizing large and diverse libraries of biomolecules (e.g., nucleic acids). In some cases, nucleic acid libraries are useful for gene assembly, antibody design, next generation sequencing capture / enrichment, and data storage. In the case of data storage, the amount of information generated and stored is exponentially increasing, so there is a need for higher capacity storage systems. Provided herein is a method for increasing the throughput of biomolecule synthesis using efficient and simplified deprotection and oxidation chemistry. definition

[0013] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0014] Throughout this disclosure, numerical characteristics are presented in range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of any embodiment. Thus, the description of a range shall be considered to have specifically disclosed all possible subranges, as well as individual numerical values ​​within that range, to the tenth of the unit of the lower limit, unless otherwise expressly indicated by the context. For example, the description of a range such as 1-6 shall be considered to have specifically disclosed subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, as well as individual values ​​within the range, e.g., 1.1, 2, 2.3, 5, and 5.9. This applies regardless of the breadth of the range. The upper and lower limits of these intervening ranges may independently be included in the smaller ranges, and are also encompassed by the invention, subject to any specifically excluded limits in the described range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention, unless the context clearly indicates otherwise.

[0015] The terms used herein are for the purpose of describing particular embodiments only and are not intended to limit any embodiment. As used herein, the singular forms "a", "an" and "the" are intended to include the plural unless the context clearly indicates otherwise. Furthermore, it will be understood that the terms "comprises" and / or "comprising" as used herein specify the presence of stated features, integers, steps, operations, elements, and / or components, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0016] Unless specifically indicated or clear from the context, as used herein, the term "about" in reference to a numerical value or range of numerical values ​​is understood to mean the numerical value set forth, as well as values ​​±10% thereof, or, for values ​​set forth as a range, values ​​10% below the stated lower limit and 10% above the stated upper limit.

[0017] As used herein, the terms "preselected sequence," "predefined sequence," or "predetermined sequence" are used interchangeably. These terms mean that the sequence of the polymer is known and is selected prior to the synthesis or assembly of the polymer. In particular, various embodiments are described herein primarily with respect to the preparation of nucleic acid molecules, where the sequence of the polynucleotide is known and selected prior to the synthesis or assembly of the nucleic acid molecule.

[0018] As used herein, the term "symbol" generally refers to an index of a unit of digital information. Digital information may be divided or converted into one or more symbols. In one example, a symbol may be a bit, which may have a numerical value. In some examples, a symbol may have a value of "0" or "1." In some examples, digital information may be represented as a sequence of symbols or a string of symbols. In some examples, a sequence of symbols or a string of symbols may constitute binary data.

[0019] Methods and compositions are provided herein for the production of synthetic (e.g., de novo synthesis or chemically synthesised) polynucleotides.Polynucleotides may also be referred to as oligonucleotides or oligos.The polynucleotide sequences described herein may include DNA or RNA, unless otherwise indicated.

[0020] "Amino" refers to the -NH2 radical.

[0021] "Cyano" refers to the -CN radical.

[0022] "Nitro" refers to the -NO2 radical.

[0023] "Oxa" refers to the --O- radical.

[0024] "Oxo" refers to the =O radical.

[0025] "Thioxo" refers to the =S radical.

[0026] "Imino" refers to the =NH radical.

[0027] "Oximo" refers to the =N-OH radical.

[0028] "Hydrazino" refers to the =N-NH2 radical.

[0029] "Alkyl" refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, containing no unsaturation, and having from 1 to 15 carbon atoms (e.g., C1-C 15 In certain embodiments, alkyl comprises 1 to 13 carbon atoms (e.g., C1-C 13 In certain embodiments, the alkyl comprises 1-8 carbon atoms (e.g., C1-C8 alkyl). In other embodiments, the alkyl comprises 1-5 carbon atoms (e.g., C1-C5 alkyl). In other embodiments, the alkyl comprises 1-4 carbon atoms (e.g., C1-C4 alkyl). In other embodiments, the alkyl comprises 1-3 carbon atoms (e.g., C1-C3 alkyl). In other embodiments, the alkyl comprises 1-2 carbon atoms (e.g., C1-C2 alkyl). In other embodiments, the alkyl comprises 1 carbon atom (e.g., C1 alkyl). In other embodiments, the alkyl comprises 5-15 carbon atoms (e.g., C5-C6 alkyl). 15In other embodiments, the alkyl group comprises 5 to 8 carbon atoms (e.g., C5-C8 alkyl). In other embodiments, the alkyl group comprises 2 to 5 carbon atoms (e.g., C2-C5 alkyl). In other embodiments, the alkyl group comprises 3 to 5 carbon atoms (e.g., C3-C5 alkyl). In other embodiments, the alkyl group is selected from methyl, ethyl, 1-propyl (n-propyl), 1-methylethyl (iso-propyl), 1-butyl (n-butyl), 1-methylpropyl (sec-butyl), 2-methylpropyl (iso-butyl), 1,1-dimethylethyl (tert-butyl), 1-pentyl (n-pentyl). The alkyl is attached to the remainder of the molecule by a single bond. Unless otherwise specifically stated herein, the alkyl group may be selected from the following substituents: halo, cyano, nitro, oxo, thioxo, imino, oximo, trimethylsilanyl, -OR, -O- ... a , -SR a , -OC(O)-R a , -N(R a )2, -C(O)R a , -C(O)OR a , -C(O)N(R a )2, -N(R a )C(O)OR a , -OC(O)-N(R a )2, -N(R a )C(O)R a , -N(R a )S(O) t R a (t is 1 or 2), -S(O) t OR a (t is 1 or 2), -S(O) t R a (t is 1 or 2), and -S(O) t N(R a )2 (t is 1 or 2), where each R aare independently hydrogen, alkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), fluoroalkyl, carbocyclyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), carbocyclylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), aryl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), aralkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heterocyclyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heterocyclylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heteroaryl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), or heteroarylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl).

[0030] "Alkoxy" refers to a radical attached through an oxygen atom of the formula --O-alkyl, where alkyl is an alkyl chain as defined above.

[0031] "Alkenyl" refers to a straight or branched hydrocarbon chain radical group consisting solely of carbon and hydrogen atoms, containing at least one carbon-carbon double bond, and having from 2 to 12 carbon atoms. In certain embodiments, an alkenyl contains from 2 to 8 carbon atoms. In other embodiments, an alkenyl contains from 2 to 4 carbon atoms. An alkenyl is attached to the remainder of the molecule by a single bond, for example, ethenyl (i.e., vinyl), prop-1-enyl (i.e., allyl), but-1-enyl, pent-1-enyl, penta-1,4-dienyl, and the like. Unless otherwise specifically stated in the specification, an alkenyl group may contain the following substituents: halo, cyano, nitro, oxo, thioxo, imino, oximo, trimethylsilanyl, -OR, -O- ... a , -SR a , -OC(O)-Ra , -N(R a )2, -C(O)R a , -C(O)OR a , -C(O)N(R a )2, -N(R a )C(O)OR a , -OC(O)-N(R a )2, -N(R a )C(O)R a , -N(R a )S(O) t R a (t is 1 or 2), -S(O) t OR a (t is 1 or 2), -S(O) t R a (t is 1 or 2), and -S(O) t N(R a )2 (t is 1 or 2), where each R a are independently hydrogen, alkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), fluoroalkyl, carbocyclyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), carbocyclylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), aryl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), aralkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heterocyclyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heterocyclylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heteroaryl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), or heteroarylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl).

[0032] "Alkynyl" refers to a straight or branched hydrocarbon chain radical group consisting solely of carbon and hydrogen atoms, containing at least one carbon-carbon triple bond, and having from 2 to 12 carbon atoms. In certain embodiments, an alkynyl contains from 2 to 8 carbon atoms. In other embodiments, an alkynyl contains from 2 to 6 carbon atoms. In other embodiments, an alkynyl contains from 2 to 4 carbon atoms. An alkynyl is attached to the remainder of the molecule by a single bond, and is, for example, ethynyl, propynyl, butynyl, pentynyl, hexynyl, and the like. Unless otherwise specifically stated in the specification, an alkynyl group may contain the following substituents: halo, cyano, nitro, oxo, thioxo, imino, oximo, trimethylsilanyl, -OR, -O- ... a , -SR a , -OC(O)-R a , -N(R a )2, -C(O)R a , -C(O)OR a , -C(O)N(R a )2, -N(R a )C(O)OR a , -OC(O)-N(R a )2, -N(R a )C(O)R a , -N(R a )S(O) t R a (t is 1 or 2), -S(O) t OR a (t is 1 or 2), -S(O) t R a (t is 1 or 2), and -S(O) t N(R a )2 (t is 1 or 2), where each R aare independently hydrogen, alkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), fluoroalkyl, carbocyclyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), carbocyclylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), aryl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), aralkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heterocyclyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heterocyclylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heteroaryl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), or heteroarylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl).

[0033] "Alkylene" or "alkylene chain" refers to a straight or branched divalent hydrocarbon chain, consisting solely of carbon and hydrogen, containing no unsaturation, having 1 to 12 carbon atoms, that links the remainder of the molecule to a radical group, such as methylene, ethylene, propylene, n-butylene, and the like. The alkylene chain is attached to the remainder of the molecule through a single bond and to the radical group through a single bond. The points of attachment of the alkylene chain to the remainder of the molecule and to the radical group are through one carbon in the alkylene chain, or through any two carbons in the chain. In certain embodiments, the alkylene comprises 1 to 8 carbon atoms (e.g., C1-C8 alkylene). In other embodiments, the alkylene comprises 1 to 5 carbon atoms (e.g., C1-C5 alkylene). In other embodiments, the alkylene comprises 1 to 4 carbon atoms (e.g., C1-C4 alkylene). In other embodiments, the alkylene comprises 1 to 3 carbon atoms (e.g., C1-C3 alkylene). In other embodiments, an alkylene comprises 1 to 2 carbon atoms (e.g., a C1-C2 alkylene). In other embodiments, an alkylene comprises 1 carbon atom (e.g., a C1 alkylene). In other embodiments, an alkylene comprises 5 to 8 carbon atoms (e.g., a C5-C8 alkylene). In other embodiments, an alkylene comprises 2 to 5 carbon atoms (e.g., a C2-C5 alkylene). In other embodiments, an alkylene comprises 3 to 5 carbon atoms (e.g., a C3-C5 alkylene). Unless otherwise specifically stated in the specification, an alkylene chain may include the following substituents: halo, cyano, nitro, oxo, thioxo, imino, oximo, trimethylsilanyl, -OR. a , -SR a , -OC(O)-R a , -N(R a )2, -C(O)R a , -C(O)OR a , -C(O)N(R a )2, -N(R a )C(O)OR a , -OC(O)-N(R a )2, -N(R a )C(O)R a , -N(R a)S(O) t R a (t is 1 or 2), -S(O) t OR a (t is 1 or 2), -S(O) t R a (t is 1 or 2), and -S(O) t N(R a )2 (t is 1 or 2), where each R a are independently hydrogen, alkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), fluoroalkyl, carbocyclyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), carbocyclylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), aryl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), aralkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heterocyclyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heterocyclylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heteroaryl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), or heteroarylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl).

[0034] "Aryl" refers to a radical derived from an aromatic monocyclic or polycyclic hydrocarbon ring system by removing one hydrogen atom from a ring carbon atom. The aromatic monocyclic or polycyclic hydrocarbon ring system contains only hydrogen and 5 to 18 carbon atoms, where at least one of the rings in the ring system is fully unsaturated, i.e., contains a cyclic delocalized (4n+2) pi-electron system according to the Hückel theory. Ring systems from which aryl groups are derived include, but are not limited to, groups such as benzene, fluorene, indane, indene, tetralin, and naphthalene. Unless otherwise specifically stated in the specification, the term “aryl” or the prefix “ar-” (e.g., in “aralkyl”) means any of the following: alkyl, alkenyl, alkynyl, halo, fluoroalkyl, cyano, nitro, optionally substituted aryl, optionally substituted aralkyl, optionally substituted aralkenyl, optionally substituted aralkynyl, optionally substituted carbocyclyl, optionally substituted carbocyclylalkyl, optionally substituted heterocyclyl, optionally substituted heterocyclylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, -R b -OR a , -R b -OC(O)-R a , -R b -OC(O)-OR a , -R b -OC(O)-N(R a )2, -R b -N(R a )2, -R b -C(O)R a , -R b -C(O)OR a , -R b -C(O)N(R a )2, -R b -OR c -C(O)N(R a )2, -R b -N(R a )C(O)OR a , -R b -N(R a )C(O)R a , -Rb -N(R a )S(O) t R a (t is 1 or 2), -R b -S(O) t R a (t is 1 or 2), -R b -S(O) t OR a (t is 1 or 2), and -R b -S(O) t N(R a t is 1 or 2, where each R a are independently hydrogen, alkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), fluoroalkyl, cycloalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), cycloalkylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), aryl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), aralkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heterocyclyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heterocyclylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heteroaryl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), or heteroarylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl); and each R b is independently a direct bond or a straight or branched alkylene or alkenylene chain; R c is a straight or branched alkylene or alkenylene chain and each of the above substituents is unsubstituted unless otherwise indicated.

[0035] "Aralkyl" is a group of the formula -R c -aryl radical, where R c is an alkylene chain as defined above, e.g., methylene, ethylene, etc. The alkylene chain part of the aralkyl radical is optionally substituted as described above for an alkylene chain. The aryl part of the aralkyl radical is optionally substituted as described above for an aryl group.

[0036] "Carbocyclyl" or "cycloalkyl" refers to a stable non-aromatic monocyclic or polycyclic hydrocarbon radical, consisting solely of carbon and hydrogen atoms, including fused or bridged ring systems, having 3 to 15 carbon atoms. In certain embodiments, a carbocyclyl contains 3 to 10 carbon atoms. In other embodiments, a carbocyclyl contains 5 to 7 carbon atoms. A carbocyclyl is attached to the remainder of the molecule by a single bond. A carbocyclyl is saturated (i.e., contains only a single C-C bond) or unsaturated (i.e., contains one or more double or triple bonds). A fully saturated carbocyclyl radical is also referred to as a "cycloalkyl". Examples of monocyclic cycloalkyls include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Unsaturated carbocyclyls are also referred to as "cycloalkenyls". Examples of monocyclic cycloalkenyls include, for example, cyclopentenyl, cyclohexenyl, cycloheptenyl, and cyclooctenyl. Polycyclic carbocyclyl radicals include, for example, adamantyl, norbornyl (i.e., bicyclo[2.2.1]heptanyl), norbornenyl, decalinyl, 7,7-dimethyl-bicyclo[2.2.1]heptanyl, and the like. Unless otherwise specifically stated in the specification, the term “carbocyclyl” includes any of the following radicals: alkyl, alkenyl, alkynyl, halo, fluoroalkyl, oxo, thioxo, cyano, nitro, optionally substituted aryl, optionally substituted aralkyl, optionally substituted aralkenyl, optionally substituted aralkynyl, optionally substituted carbocyclyl, optionally substituted carbocyclylalkyl, optionally substituted heterocyclyl, optionally substituted heterocyclylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, -R b -OR a , -R b -OC(O)-R a , -R b -OC(O)-OR a , -R b -OC(O)-N(R a)2, -R b -N(R a )2, -R b -C(O)R a , -R b -C(O)OR a , -R b -C(O)N(R a )2, -R b -OR c -C(O)N(R a )2, -R b -N(R a )C(O)OR a , -R b -N(R a )C(O)R a , -R b -N(R a )S(O) t R a (t is 1 or 2), -R b -S(O) t R a (t is 1 or 2), -R b -S(O) t OR a (t is 1 or 2), and -R b -S(O) t N(R a )2, where t is 1 or 2, where each R aare independently hydrogen, alkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), fluoroalkyl, cycloalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), cycloalkylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), aryl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), aralkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heterocyclyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heterocyclylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heteroaryl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), or heteroarylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl); and each R b is independently a direct bond or a straight or branched alkylene or alkenylene chain; R c is a straight or branched alkylene or alkenylene chain and each of the above substituents is unsubstituted unless otherwise indicated.

[0037] A "carbocyclylalkyl" is a group of the formula -R c -carbocyclyl radical, where R c is an alkylene chain as defined above. The alkylene chain and the carbocyclyl radical are optionally substituted as defined above.

[0038] "Halo" or "halogen" refers to a bromo, chloro, fluoro, or iodo substituent.

[0039] "Fluoroalkyl" refers to an alkyl radical, as defined above, substituted by one or more fluoro radicals, as defined above, such as trifluoromethyl, difluoromethyl, fluoromethyl, 2,2,2-trifluoroethyl, 1-fluoromethyl-2-fluoroethyl, etc. In some embodiments, the alkyl portion of the fluoroalkyl radical is optionally substituted as defined above for an alkyl group.

[0040] "Heterocyclyl" or "heterocycloalkyl" refers to a stable 3- to 18-membered non-aromatic ring radical containing 2 to 12 carbon atoms and 1 to 6 heteroatoms selected from nitrogen, oxygen, and sulfur. Unless otherwise specifically stated herein, a heterocyclyl radical is a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, which optionally includes fused or bridged ring systems. The heteroatoms in a heterocyclyl radical are optionally oxidized. If one or more nitrogen atoms are present, they are optionally quaternized. The heterocyclyl radical is partially or fully saturated. The heterocyclyl is attached to the remainder of the molecule through any atom of the ring. Examples of such heterocyclyl radicals include, but are not limited to, dioxolanyl, thienyl[1,3]dithianyl, decahydroisoquinolyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-oxo-thiomorpholinyl, and 1,1-dioxo-thiomorpholinyl. Unless otherwise specifically stated in the specification, the term “heterocyclyl” includes any of the following radicals: alkyl, alkenyl, alkynyl, halo, fluoroalkyl, thioxo, cyano, nitro, optionally substituted aryl, optionally substituted aralkyl, optionally substituted aralkenyl, optionally substituted aralkynyl, optionally substituted carbocyclyl, optionally substituted carbocyclylalkyl, optionally substituted heterocyclyl, optionally substituted heterocyclylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, -R b -OR a , -R b -OC(O)-R a , -R b-OC(O)-OR a , -R b -OC(O)-N(R a )2, -R b -N(R a )2, -R b -C(O)R a , -R b -C(O)OR a , -R b -C(O)N(R a )2, -R b -OR c -C(O)N(R a )2, -R b -N(R a )C(O)OR a , -R b -N(R a )C(O)R a , -R b -N(R a )S(O) t R a (t is 1 or 2), -R b -S(O) t R a (t is 1 or 2), -R b -S(O) t OR a (t is 1 or 2), and -R b -S(O) t N(R a )2, where t is 1 or 2, wherein each R aare independently hydrogen, alkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), fluoroalkyl, cycloalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), cycloalkylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), aryl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), aralkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heterocyclyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heterocyclylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heteroaryl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), or heteroarylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl); and each R b is independently a direct bond or a straight or branched alkylene or alkenylene chain; R c is a straight or branched alkylene or alkenylene chain and each of the above substituents is unsubstituted unless otherwise indicated.

[0041] "N-heterocyclyl" or "N-linked heterocyclyl" refers to a heterocyclyl radical as defined above that contains at least one nitrogen and the point of attachment of the heterocyclyl radical to the remainder of the molecule is through a nitrogen atom in the heterocyclyl radical. The N-heterocyclyl radical is optionally substituted as described above for heterocyclyl radicals. Examples of such N-heterocyclyl radicals include, but are not limited to, 1-morpholinyl, 1-piperidinyl, 1-piperazinyl, 1-pyrrolidinyl, pyrazolidinyl, imidazolinyl, and imidazolidinyl.

[0042] "C-heterocyclyl" or "C-linked heterocyclyl" refers to a heterocyclyl radical as defined above that contains at least one heteroatom and the point of attachment of the heterocyclyl radical to the remainder of the molecule is through a carbon atom in the heterocyclyl radical. The C-heterocyclyl radical is optionally substituted as described above for heterocyclyl radicals. Examples of such C-heterocyclyl radicals include, but are not limited to, 2-morpholinyl, 2- or 3- or 4-piperidinyl, 2-piperazinyl, 2- or 3-pyrrolidinyl, and the like.

[0043] "Heteroaryl" refers to a radical derived from a 3-18 membered aromatic ring radical containing 2-17 carbon atoms and 1-6 heteroatoms selected from nitrogen, oxygen, and sulfur. As used herein, a heteroaryl radical is a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, where at least one of the rings in the ring system is fully unsaturated, i.e., contains a cyclic delocalized (4n+2) pi-electron system according to the Hückel theory. Heteroaryl includes fused or bridged ring systems. The heteroatoms in the heteroaryl radical are optionally oxidized. If one or more nitrogen atoms are present, they are optionally quaternized. Heteroaryl is bonded to the remainder of the molecule through any atom of the ring. Examples of heteroaryl include azepinyl, acridinyl, benzimidazolyl, benzindolyl, 1,3-benzodioxolyl, benzofuranyl, benzoxazolyl, benzo[d]thiazolyl, benzothiadiazolyl, benzo[b][1,4]dioxepinyl, benzo[b][1,4]oxazinyl, 1,4-benzodioxanyl, benzonaphthofuranyl, benzoxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl, benzopyranonyl, benzofuranyl, benzofuranoyl, benzothienyl (benzothiophenyl), benzothieno[3,2-d]pyrimidinyl, benzotriazolyl, benzo[4,6]imidazo[ 1,2-a]pyridinyl, carbazolyl, cinnolinyl, cyclopenta[d]pyrimidinyl, 6,7-dihydro-5H-cyclopenta[4,5]thieno[2,3-d]pyrimidinyl, 5,6-dihydrobenzo[h]quinazolinyl, 5,6-dihydrobenzo[h]cinnolinyl, 6,7-dihydro-5H-benzo[6,7]cyclohepta[1,2-c]pyridazinyl, dibenzofuranyl, dibenzothiophenyl, furanyl, furanonyl, furo[3,2-c]pyridinyl, 5,6,7,8,9,10-hexahydrocycloocta[d]pyrimidinyl, 5,6,7,8,9,10-hexahydrocycloocta[d]pyridazinyl, 5,6,7,8,9,10-Hexahydrocycloocta[d]pyridinyl, isothiazolyl, imidazolyl, indazolyl, indolyl, indazolyl, isoindolyl, indolinyl, isoindolinyl, isoquinolyl, indolizinyl, isoxazolyl, 5,8-methano-5,6,7,8-tetrahydroquinazolinyl, naphthyridinyl, 1,6-naphthyridinonyl, oxadiazolyl, 2-oxoazolyl Zepinyl, oxazolyl, oxiranyl, 5,6,6a,7,8,9,10,10a-octahydrobenzo[h]quinazolinyl, 1-phenyl-1H-pyrrolyl, phenazinyl, phenothiazinyl, phenoxazinyl, phthalazinyl, pteridinyl, purinyl, pyrrolyl, pyrazolyl, pyrazolo[3,4-d]pyrimidinyl, pyridinyl, pyrido[3,2-d]pyrimidinyl, pyri do[3,4-d]pyrimidinyl, pyrazinyl, pyrimidinyl, pyridazinyl, pyrrolyl, quinazolinyl, quinoxalinyl, quinolinyl, isoquinolinyl, tetrahydroquinolinyl, 5,6,7,8-tetrahydroquinazolinyl, 5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidinyl, 6,7,8,9-tetrahydro-5H-cyclohepta[4,5]thieno[2,3-d]pyrimidinyl, These include, but are not limited to, eno[2,3-d]pyrimidinyl, 5,6,7,8-tetrahydropyrido[4,5-c]pyridazinyl, thiazolyl, thiadiazolyl, triazolyl, tetrazolyl, triazinyl, thieno[2,3-d]pyrimidinyl, thieno[3,2-d]pyrimidinyl, thieno[2,3-c]pyridinyl, and thiophenyl (i.e., thienyl). Unless otherwise specifically stated in the specification, the term "heteroaryl" includes any of the following radicals: alkyl, alkenyl, alkynyl, halo, fluoroalkyl, haloalkenyl, haloalkynyl, oxo, thioxo, cyano, nitro, optionally substituted aryl, optionally substituted aralkyl, optionally substituted aralkenyl, optionally substituted aralkynyl, optionally substituted carbocyclyl, optionally substituted carbocyclylalkyl, optionally substituted heterocyclyl, optionally substituted heterocyclylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, -R, b -OR a, -R b -OC(O)-R a , -R b -OC(O)-OR a , -R b -OC(O)-N(R a )2, -R b -N(R a )2, -R b -C(O)R a , -R b -C(O)OR a , -R b -C(O)N(R a )2, -R b -OR c -C(O)N(R a )2, -R b -N(R a )C(O)OR a , -R b -N(R a )C(O)R a , -R b -N(R a )S(O) t R a (t is 1 or 2), -R b -S(O) t R a (t is 1 or 2), -R b -S(O) t OR a (t is 1 or 2), and -R b -S(O) t N(R a t is 1 or 2, wherein each R aare independently hydrogen, alkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), fluoroalkyl, cycloalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), cycloalkylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), aryl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), aralkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heterocyclyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heterocyclylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), heteroaryl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl), or heteroarylalkyl (optionally substituted with halogen, hydroxy, methoxy, or trifluoromethyl); and each R b is independently a direct bond or a straight or branched alkylene or alkenylene chain; R c is a straight or branched alkylene or alkenylene chain and each of the above substituents is unsubstituted unless otherwise indicated.

[0044] "N-heteroaryl" refers to a heteroaryl radical as defined above that contains at least one nitrogen and the point of attachment of the heteroaryl radical to the remainder of the molecule is through a nitrogen atom in the heteroaryl radical. The N-heteroaryl radical is optionally substituted as described above for heteroaryl radicals.

[0045] "C-heteroaryl" refers to a heteroaryl radical as defined above, where the point of attachment of the heteroaryl radical to the remainder of the molecule is through a carbon atom in the heteroaryl radical. The C-heteroaryl radical is optionally substituted as described above for heteroaryl radicals.

[0046] The compounds disclosed herein, in some embodiments, contain one or more asymmetric centers, thus resulting in enantiomers, diastereomers, and other stereoisomeric forms defined in terms of absolute stereochemistry as (R)- or (S)-. It is intended that all stereoisomeric forms of the compounds disclosed herein are contemplated by the present disclosure, unless otherwise indicated. When the compounds described herein contain an alkene double bond, it is intended that the present disclosure includes both E and Z geometric isomers (e.g., cis or trans), unless otherwise specified. Similarly, all possible isomers, as well as their racemic and optically pure forms, and all tautomeric forms are also intended to be included. The term "geometric isomer" refers to the E or Z geometric isomers (e.g., cis or trans) of the alkene double bond. The term "positional isomer" refers to structural isomers around a central ring, such as ortho, meta, and para isomers around a benzene ring.

[0047] Methods and compositions for the production of synthetic (e.g., de novo synthesized, enzymatically synthesized, chemically synthesized) biomolecules are provided herein. In some cases, the biomolecules are synthesized in a template-independent manner. In some cases, the biomolecules include polynucleotides. Polynucleotides may also be referred to as oligonucleotides or oligos. The polynucleotide sequences described herein may include DNA or RNA unless otherwise indicated. In some cases, the biomolecules include polymers including two or more monomers. Biomolecules refer in some cases to polymers such as nucleic acids (e.g., DNA, RNA), carbohydrates (e.g., sugars), peptides / proteins, lipids, fatty acids, terpenes, peptoids, or mixtures thereof. In some cases, the biomolecules may be synthesized in an iterative manner using methods well known in the art (with or without protecting groups). In some cases, the biomolecules may be synthesized in an iterative manner from monomers, dimers, trimers, or other suitable building blocks. Biomolecule Synthesis

[0048] Provided herein are systems and methods for synthesizing high density biomolecules (e.g., polynucleotides) on a substrate in a short time and with low error rates. In some cases, the methods include the use of electrochemical deprotection. In some cases, the electrochemical deprotection is facilitated by the use of a transition metal catalyst. In some cases, site-specific deprotection is achieved using an addressable array.

[0049] A method for synthesizing a polynucleotide is provided herein. The polynucleotide can be synthesized by contacting at least one nucleoside bound to a solid support with a protected nucleoside. In some cases, the protected nucleoside includes a protecting group. In some cases, the protecting group includes an alkene. In some cases, the solid support includes an addressable array. In some cases, the method can include contacting an amount of a reagent with the protected nucleoside. In some cases, the contacting results in deprotection of the terminal nucleoside of the protected nucleoside. In some cases, one or more steps provided herein can be repeated to synthesize a polynucleotide. In some cases, the method provided herein includes one or more steps of (a) contacting at least one nucleoside bound to a solid support with a protected nucleoside, (b) contacting an amount of a reagent with the protected nucleoside, and (c) repeating steps (a) and (b) to synthesize a polynucleotide. In some cases, the reagent is used to deprotect nucleotides, oxidize the phosphate backbone, perform a capping reaction, facilitate the coupling of nucleotide monomers, or perform other reactions. In some cases, the reagent comprises an enzyme. In some cases, the enzyme comprises a polymerase.

[0050] Described herein are methods, systems, devices, and compositions in which chemical reactions used in polynucleotide synthesis are controlled using electrochemistry. Electrochemical reactions are controlled in some cases by an energy source, such as light, heat, irradiation, electricity, or any other energy source. For example, electrodes are used to control one or more chemical reactions at all or a portion of individual locations on a surface. In some cases, electrodes are charged by applying a potential to the electrodes to control one or more steps (e.g., chemical steps) in polynucleotide synthesis. In some cases, the electrodes are addressable. Any number of steps described herein, e.g., chemical steps, are controlled in some cases by one or more electrodes. The electrochemical reactions in the chemical steps can include oxidation, reduction, acid / base chemical reactions, or other reactions controlled by electrodes. In some cases, the electrodes generate electrons or protons that are used as reagents for chemical transformations in the reactions. In some cases, the electrodes directly generate reagents, e.g., acids. In some cases, the acids are protons. The electrodes, in some cases, generate reagents, such as bases, directly. Acids or bases are often used to cleave protecting groups or to affect the kinetics of various polynucleotide synthesis reactions, for example, by adjusting the pH of the reaction solution. Electrochemically controlled polynucleotide synthesis reactions, in some cases, include redox-active metals or other redox-active organic materials. In some cases, metal or organic catalysts are used in these electrochemical reactions. In some cases, exemplary methods are shown in FIG. 13 or FIG. 15. In some cases, the protected nucleosides include 5' or 3' protecting groups. In some cases, the protecting groups include allyl groups. In some cases, the protecting groups include O-allyl. In some cases, the synthesis proceeds in the 5' to 3' direction. In some cases, the synthesis proceeds in the 3' to 5' direction.

[0051] Control of chemical reactions may not be limited to electrochemical generation of reagents. In some cases, chemical reactivity is indirectly affected through biophysical changes of substrates or reagents via electric fields (or gradients) generated by electrodes. In some cases, substrates include, but are not limited to, nucleic acids. In some cases, electric fields are generated that repel or attract certain reagents or substrates toward or away from electrodes or surfaces. Such electric fields are generated in some cases by application of a potential to one or more electrodes. For example, negatively charged nucleic acids are repelled from negatively charged electrode surfaces. Such repulsion or attraction of polynucleotides or other reagents caused by local electric fields, in some cases, results in the movement of polynucleotides or other reagents into or out of regions of a synthesis device or structure. In some cases, electrodes generate electric fields that repel polynucleotides away from a synthesis surface, structure, or device. In some cases, electrodes generate electric fields that attract polynucleotides toward a synthesis surface, structure, or device. In some cases, protons are repelled away from a positively charged surface to limit contact of the protons with the substrate or one or more portions thereof. In some cases, repulsive or attractive forces are used to allow or block entry of reagents or substrates to certain areas of the synthesis surface. In some cases, nucleoside monomers are prevented from contacting polynucleotide chains by applying an electric field near one or both components. Such configurations allow gating of certain reagents, which may avoid the need for protecting groups if the concentration of reagents and / or substrates or the rate of contact between them are controlled. In some cases, unprotected nucleoside monomers are used for polynucleotide synthesis. Alternatively, applying an electric field near one or both components promotes contact of nucleoside monomers with polynucleotide chains. In addition, application of an electric field to a substrate may change the reactivity or composition of the substrate. In an exemplary application, an electric field generated by electrodes is used to prevent polynucleotides at adjacent locations from interacting.In some cases, the substrate is a polynucleotide, optionally attached to a surface. The application of an electric field in some cases changes the three-dimensional structure of the polynucleotide. Such changes include folding or unfolding of various structures, such as helices, hairpins, loops, or other three-dimensional nucleic acid structures. Such changes are useful for manipulating nucleic acids in wells, channels, or other structures. In some cases, the electric field is applied to the nucleic acid substrate to prevent secondary structures. In some cases, the electric field avoids the need for linkers or attachment to solid supports during polynucleotide synthesis.

[0052] A suitable method for polynucleotide synthesis on a substrate of the present disclosure is the phosphoramidite method, which involves the controlled addition of phosphoramidite building blocks, e.g., nucleoside phosphoramidites, to a growing polynucleotide chain in a coupling step that forms a phosphorous triester bond between the phosphoramidite building block and the nucleoside bound to the substrate. In some cases, the nucleoside phosphoramidite is provided to an activated substrate. In some cases, the nucleoside phosphoramidite is provided to the substrate together with an activator. In some cases, the nucleoside phosphoramidite is provided to the substrate in 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 of the nucleoside bound to the substrate. In some cases, the addition of the nucleoside phosphoramidite is carried out in an anhydrous environment, for example, anhydrous acetonitrile. After the addition and linkage of the nucleoside phosphoramidite in the coupling step, the substrate is optionally washed. In some cases, the coupling step is repeated one or more further times, optionally with a washing step between the addition of the nucleoside phosphoramidite to the substrate. In some cases, the polynucleotide synthesis method used herein includes one, two, three, or more consecutive coupling steps. In many cases, prior to coupling, the nucleoside bound to the substrate is deprotected by removing a protecting group, where the protecting group functions to prevent polymerization. The protecting group may include any chemical group that prevents the extension of the polynucleotide chain. In some cases, the protecting group is cleaved (or removed) in the presence of an acid. In some cases, the protecting group is cleaved (or removed) in the presence of a base. In some cases, the protecting group is removed using electromagnetic radiation, such as light, heat, or other energy sources. In some cases, the protecting group is removed by an oxidation or reduction reaction. In some cases, the protecting group comprises a triarylmethyl group.In some cases, the protecting group comprises an aryl ether. In some cases, the protecting group comprises a disulfide. In some cases, the protecting group comprises an acid labile silane. In some cases, the protecting group comprises an acetal. In some cases, the protecting group comprises a ketal. In some cases, the protecting group comprises an enol ether. In some cases, the protecting group comprises a methoxybenzyl group. In some cases, the protecting group comprises 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 comprises a base labile group.

[0053] After coupling, the phosphoramidite polynucleotide synthesis method optionally includes a capping step, in which the growing polynucleotide is treated with a capping agent. The capping step generally serves to block the 5'-OH group attached to the unreacted substrate after coupling from further chain extension, preventing the formation of polynucleotides with internal base deletions.

[0054] Furthermore, phosphoramidites activated with 1H-tetrazole often react to a small extent with the O6 position of guanosine. Upon oxidation with I2 / water, this by-product undergoes depurination, possibly via O6-N7 migration. The apurinic site may become cleaved during the final deprotection of the polynucleotide, thus reducing the yield of the full-length product. The O6 modification may be removed by treatment with a capping reagent prior to oxidation with I2 / water. In some cases, including a capping step during polynucleotide synthesis reduces the error rate compared to synthesis without capping. In some cases, the compounds and methods disclosed herein reduce depurination during polynucleotide synthesis. In some cases, the compounds and methods disclosed herein eliminate depurination during polynucleotide synthesis. In some embodiments, the compounds and methods disclosed herein do not require oxidation with an oxidizing agent, e.g., I2 / water. By way of example, the capping step includes treating the substrate-bound polynucleotide with a mixture of acetic anhydride and 1-methylimidazole. After the capping step, the substrate is optionally washed.

[0055] After addition of nucleoside phosphoramidites, and optionally after capping and one or more washing steps, the substrate described herein contains a bound growing nucleic acid that can be oxidized. The oxidation step involves oxidizing the phosphite triester to a tetracoordinate phosphate triester, which is a protected precursor of the naturally occurring phosphodiester internucleoside linkage. In some cases, the phosphite triester is 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. Oxidation may be performed under anhydrous conditions using tert-butyl hydroperoxide or (1S)-(+)-(10-camphorsulfonyl)-oxaziridine (CSO). In some methods, a capping step is performed after oxidation. A second capping step allows the substrate to dry, since residual water that may remain from oxidation can inhibit subsequent coupling. After oxidation, the substrate and growing polynucleotide are optionally washed. In some cases, the oxidation step is replaced by a sulfurization step to obtain polynucleotide phosphorothioates, in which case an optional capping step can be performed after sulfurization. Many reagents are capable of efficient sulfur transfer, including, but not limited to, 3-(dimethylaminomethylidene)amino)-3H-1,2,4-dithiazole-3-thione, DDTT, 3H-1,2-benzodithiol-3-one 1,1-dioxide (also known as Beaucage reagent), and N,N,N'N'-tetraethylthiuram disulfide (TETD).

[0056] For the subsequent nucleoside incorporation cycle to occur by coupling, the protected 5' end (or 3' end, if synthesis is performed in the 5' to 3' direction) of the growing polynucleotide bound to the substrate is removed so that the primary hydroxyl group can react with the next nucleoside phosphoramidite. In some embodiments, the protecting group comprises a straight or branched chain alkenylene. In some embodiments, the protecting group comprises an allyl group. In some embodiments, the protecting group comprises a straight or branched chain alkenylene carbonate. In some embodiments, the phosphoramidite comprises a dialkylamine. In some embodiments, the phosphoramidite comprises a dialkylamino variant of methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butylamine, or any combination thereof. In some embodiments, the formula:

[0057] [ka] or

[0058] [ka] wherein R 1 is a linear or branched alkenylene; R 2 is alkylene or alkenylene, each of which is independently substituted or unsubstituted; B is a monocyclic or bicyclic C 4-6 Disclosed herein are compounds that are heterocyclic rings. In some cases, B comprises a base corresponding to an A, T, C, G, or U nucleotide. In some embodiments, R 1 is a straight chain alkenylene. In some embodiments, R 1 is a linear C 2-6 In some embodiments, R is alkenylene. 1 is allyl. In some embodiments, R 1 is branched alkenylene. In some embodiments, R 1is a branched chain C 3-8 In some embodiments, R is alkenylene. 2 is a substituted alkylene. In some embodiments, R 2 is a substitution C 2-4 In some embodiments, R 2 is cyanoethyl. In some embodiments, R 2 is unsubstituted alkenylene. In some embodiments, R 2 is unsubstituted C 2-4 In some embodiments, R 2 is allyl. In some embodiments, R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , or R 9 Each of R is independently H, OH, halogen, O-alkyl, N-alkyl, O-alkyl-O-alkyl, N, or R and R taken together form a ring and B is a heterocyclic ring. 3 , R 4 , R 5 , R 6 , R 7 , R 8 , and R 9 At least three of R are H. In some cases, 3 , R 4 , R 5 , R 6 , R 7 , R 8 , and R 9 At least five of R are H. In some cases, 3 , R 4 , R 5 , R 6 , R 7 , R 8 , and R 9 is H. In some cases, R 3 , R 4 , R 5 , R 6 , R 7, R 8 , and R 9 At least one of R is F. In some cases, R 3 is H and R 4 is OH. In some cases, R 3 and R 4 One or both of R is F. In some cases, 3 and R 4 In some cases, one or both of R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , and R 9 At least one of R is -OCH3. 3 , R 4 , R 5 , R 6 , R 7 , R 8 , and R 9 At least one of is -OCH2CH2OCH3. In some cases, R3 and R6 taken together form a ring. In some cases, R3 and R6 taken together form a ring, where the ring comprises -CH2-O-. In some embodiments, the formula:

[0059] [ka] wherein R 1 is a linear or branched alkenylene; R 2 is alkylene or alkenylene, each of which is independently substituted or unsubstituted; B is a monocyclic or bicyclic C 4-6 Disclosed herein are compounds in which R is a heterocyclic ring. 1 is a straight chain alkenylene. In some embodiments, R 1 is a linear C 2-6 In some embodiments, R is alkenylene.1 is allyl. In some embodiments, R 1 is branched alkenylene. In some embodiments, R 1 is a branched chain C 3-8 In some embodiments, R is alkenylene. 2 is a substituted alkylene. In some embodiments, R 2 is a substitution C 2-4 In some embodiments, R 2 is cyanoethyl. In some embodiments, R 2 is unsubstituted alkenylene. In some embodiments, R 2 is unsubstituted C 2-4 In some embodiments, R 2 is an allyl.

[0060] In some embodiments, B is a monocyclic C4 heterocyclic ring. In some embodiments, B is a bicyclic C5 heterocyclic ring. In some embodiments, B is a nucleobase. In some embodiments, B is a modified nucleobase. In some embodiments, B is a group represented by the formula:

[0061] [ka] ,

[0062] [ka] ,

[0063] [ka] ,

[0064] [ka] ,or

[0065] [ka] In some embodiments, B has the formula:

[0066] [ka] ,

[0067] [ka] ,

[0068] [ka] ,or

[0069] [ka] each of which is independently substituted or unsubstituted.

[0070] In some embodiments, the compound is

[0071] [ka] In some embodiments, the compound is

[0072] [ka] It is.

[0073] The methods and compositions described herein provide controlled deblocking conditions that limit undesired depurination reactions. In some embodiments, the deblocking step includes contacting a quantity of a reagent with the protected nucleoside, which results in deprotection of the terminal nucleoside of the protected nucleoside. In some embodiments, the reagent is a transition metal catalyst. In some embodiments, the reagent is a zero-valent transition metal catalyst. In some embodiments, the O-alloc protecting group can be removed by site-selective reduction of a Pd(II) precursor (FIGS. 16-17). In some embodiments, the O-alloc protecting group can be removed without the use of an acid. In some embodiments, the reagent includes a Pd(0) catalyst. In some embodiments, the Pd(0) catalyst has a nitrogen-based ligand. In some embodiments, the Pd(0) catalyst includes an arylphosphine ligand. In some cases, the ligand includes 1,2-bis(diphenylphosphino)ethane. In some embodiments, the Pd(0) catalyst has a nitrogen-based ligand. In some embodiments, the Pd(0) catalyst comprises an alkylamino ligand. In some cases, the alkylamine ligand comprises TMEDA, TPPTS, diethylamine, DIPEA, and in some cases, the alkylamine ligand comprises C1-C 16 In some embodiments, the Pd(0) catalyst comprises a heteroaryl phosphine ligand. In some cases, the heteroaryl group comprises a substituted or unsubstituted pyridine, pyrazole, pyrazine, furan, thiophene, thiazole, isoxazole, isothiazole, oxazole, pyridazine, or triazine. In some embodiments, the reagent comprises Pd(Ph3)4, PdCl2(Ph3P)2, Pd(dba)2, or Pd(OAc)2. In some cases, the catalyst comprises a metal in oxidation state 2 (e.g., Pd 2+ Or Ni 2+In some embodiments, the reagent comprises a Ni catalyst. In some embodiments, the Ni catalyst comprises a CO ligand. In some embodiments, the Ni catalyst comprises an alkylamine ligand. In some embodiments, the alkylamino ligand comprises TMEDA. In some embodiments, the reagent comprises a Ni, Pd, or Pt catalyst. In some embodiments, the reagent comprises a Pt catalyst. In some embodiments, the reagent comprises a tetrazine. In some embodiments, the reagent comprises a modified tetrazine. In some embodiments, the amount of catalyst is about 0.001 mol % to about 25 mol % of the protected nucleoside. In some embodiments, the amount of catalyst is about 0.001 mol%, 0.005 mol%, 0.01 mol%, 0.05 mol%, 0.1 mol%, 0.5 mol%, 1 mol%, 3 mol%, 5 mol%, 8 mol%, 10 mol%, 12 mol%, 15 mol%, 20 mol%, or 25 mol% of the protected nucleoside. In some embodiments, the amount of catalyst is at least about 0.001 mol%, 0.005 mol%, 0.01 mol%, 0.05 mol%, 0.1 mol%, 0.5 mol%, 1 mol%, 3 mol%, 5 mol%, 8 mol%, 10 mol%, 12 mol%, 15 mol%, 20 mol%, or 25 mol% of the protected nucleoside. In some embodiments, the amount of catalyst is at most about 0.001 mol%, 0.005 mol%, 0.01 mol%, 0.05 mol%, 0.1 mol%, 0.5 mol%, 1 mol%, 3 mol%, 5 mol%, 8 mol%, 10 mol%, 12 mol%, 15 mol%, 20 mol%, or 25 mol% of the protected nucleoside. In some embodiments, the amount of catalyst is 0.001-30%, 0.001-10%, 0.001-5%, 0.001-1%, 0.001-0.1%, 0.001-0.01%, 0.01-10%, 0.01-5%, 0.01-1%, 0.01-1%, 0.1-50%, 0.1-25%, 0.1-15%, 0.1-10%, 0.1-5%, 0.1-2%, 0.1-1%, 1-50%, 1-75%, 1-25%, or 1-10% (mol %).In some embodiments, the amount of catalyst is less than about 15 mol% of the protected nucleoside. In some embodiments, the amount of catalyst is less than about 5 mol% of the protected nucleoside. In some embodiments, the amount of catalyst is less than 25 mol%, less than 20 mol%, less than 15 mol%, less than 12 mol%, less than 10 mol%, less than 8 mol%, less than 5 mol%, less than 3 mol%, less than 1 mol%, less than 0.01 mol%, or less than 0.001 mol% of the protected nucleoside. In some embodiments, the deblocking step further comprises contacting the protected nucleoside with a nucleophile, e.g., a C nucleophile. In some embodiments, the C nucleophile is N,N-dimethylbarbiturate. In some embodiments, the nucleophile is acetylacetonate. In some embodiments, the nucleophile comprises a phosphine. In some embodiments, the nucleophile is PPh3. In some embodiments, the deblocking step is performed in a chlorinated solvent. In some embodiments, the deblocking step is carried out in dichloromethane or chloroform.

[0074] The reagent can be contacted with the protected nucleoside for about 1 minute to about 20 minutes. In some embodiments, the contact between the reagent and the protected nucleoside is about 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, or 20 minutes. In some embodiments, the contact between the reagent and the protected nucleoside is at least about 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, or 20 minutes. In some embodiments, the contact between the reagent and the protected nucleoside is less than about 20 minutes, less than about 15 minutes, less than about 10 minutes, less than about 9 minutes, less than about 8 minutes, less than about 7 minutes, less than about 6 minutes, less than about 5 minutes, less than about 4 minutes, less than about 3 minutes, less than about 2 minutes, or less than about 1 minute. In some embodiments, the contact between the reagent and the protected nucleoside is less than about 10 minutes. In some embodiments, the contact between the reagent and the protected nucleoside is less than about 5 minutes. In some embodiments, the contact between the reagent and the protected nucleoside is 0.1-10, 0.1-8, 0.1-5, 0.1-4, 0.1-3, 0.1-2, 0.1-1, 0.1-0.5, 0.5-10, 0.5-5, 0.5-3, 0.5-2, 1-10, 1-7, 1-5, 1-4, 1-3, 1-2, 2-10, 2-8, 2-5, or 5-10 minutes. In some cases, the polynucleotide bound to the substrate is washed after deblocking. In some cases, efficient washing after deblocking contributes to the synthesis of polynucleotides with low error rates.

[0075] The method for synthesis of polynucleotides on a substrate described herein may include one or more repeated sequences of steps of applying a protected monomer to the surface of a substrate feature for linkage with either the surface, a linker, or a previously deprotected monomer, deprotecting the applied monomer so that it can react with a subsequently applied protected monomer, and applying another protected monomer for linkage. 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 steps.

[0076] The method for synthesis of polynucleotides on substrates described herein may include an oxidation step.For example, the method may include one or more repeating sequences of the steps of applying a protected monomer to the surface of substrate features for linking with either the surface, linker, or previously deprotected monomer, deprotecting the applied monomer so that it can react with the subsequently applied protected monomer, applying another protected monomer for linking, and oxidizing and / or sulfurizing.In some cases, one or more washing steps are performed before or after one or all steps.

[0077] The method for synthesis of polynucleotides on a substrate described herein may further include a repeating sequence of steps of applying a protected monomer to the surface of a substrate feature for linkage with either the surface, a linker, or a previously deprotected monomer, deprotecting the applied monomer so that it can react with a subsequently applied protected monomer, and oxidizing and / or sulfurizing.In some cases, one or more washing steps are performed before or after one or all steps.In some embodiments, an oxidation step is performed at the end of the synthesis.

[0078] The method for synthesis of polynucleotides on a substrate described herein may further comprise one or more repeated sequences of steps of applying a protected monomer to the surface of a substrate feature for linking with either the surface, a linker, or a previously deprotected monomer, oxidizing and / or sulfurizing.In some cases, one or more washing steps are performed before or after one or all steps.In some embodiments, one oxidation step is performed at the end of synthesis.

[0079] The methods for synthesis of polynucleotides on a substrate described herein may further include a repeating sequence of one or more of the steps of applying a protected monomer to the surface of a substrate feature for linkage with either the surface, a linker, or a previously deprotected monomer, deprotecting the applied monomer so that it can react with a subsequently applied protected monomer, and oxidizing and / or sulfurizing. In some cases, one or more washing steps are performed before or after one or all of the steps.

[0080] In some cases, polynucleotides are synthesized with photolabile protecting groups, where the hydroxyl groups generated on the surface are blocked by the photolabile protecting groups. When the surface is exposed to UV light, for example through a photolithographic mask, a pattern of free hydroxyl groups can be generated on the surface. These hydroxyl groups can react with the photoprotected nucleoside phosphoramidites by phosphoramidite chemistry. A second photolithographic mask can be applied and the surface exposed to UV light to generate a second pattern of hydroxyl groups, which can then be coupled with 5'-photoprotected nucleoside phosphoramidites. Similarly, patterns can be generated and the oligomer chains can be extended. Without being bound by theory, the instability of the photocleavable groups depends on the wavelength and polarity of the solvent used, and the rate of photocleavage can be affected by the duration of exposure and the intensity of the light. This method can take advantage of several factors, such as the accuracy of the mask alignment, the efficiency of removal of the photoprotecting groups, and the yield of the phosphoramidite coupling step. Furthermore, unintended light leakage to neighboring sites can be minimized. The density of synthesized oligomers per spot can be monitored by modulating the loading of leader nucleosides on the synthesis surface.

[0081] The surface of the substrate described herein that provides support for polynucleotide synthesis may be chemically modified to allow the synthesized polynucleotide chain to be cleaved from the surface. In some cases, the polynucleotide chain is cleaved at the same time that the polynucleotide is deprotected. In some cases, the polynucleotide chain is cleaved after the polynucleotide is deprotected. In an exemplary scheme, a trialkoxysilylamine, such as (CH3CHO)3Si-(CH2)2-NH2, is reacted with the surface SiOH group of the substrate, followed by reaction of the amine with succinic anhydride to create an amide linkage and a free OH, on which nucleic acid chain growth is supported. Cleavage includes gas cleavage with ammonia or methylamine. In some cases, cleavage includes cleavage of the linker with an electrogenerated reagent, such as acid or base. In some cases, the polynucleotide, once released from the surface, is assembled (either in vitro, in vivo, or in silico) into a larger nucleic acid that is sequenced and decoded to extract the stored information. In some cases, the polynucleotides are assembled using one or more indexes.

[0082] The surfaces described herein can be reused after polynucleotide cleavage to support additional cycles of polynucleotide synthesis. For example, the linker can be reused without additional treatment / chemical modification. In some cases, the linker is non-covalently attached to the substrate surface or polynucleotide. In some embodiments, the linker remains attached to the polynucleotide after cleavage from the surface. The linker, in some embodiments, comprises a reversible covalent bond, such as an ester, amide, ketal, beta-substituted ketone, heterocycle, or other group that can be reversibly cleaved. Such reversible cleavage reactions are controlled in some cases by the addition or removal of reagents or by electrochemical processes controlled by electrodes. Optionally, the chemical linker or surface-bound chemical group is regenerated after several cycles to restore reactivity and eliminate undesired by-product formation on such linker or surface-bound chemical group.

[0083] In some cases, the substrate is a flexible substrate. In some cases, the substrate is about 10 10 pieces, 10 11 pieces, 10 12 pieces, 10 13 pieces, 10 14 Pieces or 10 15 bases are synthesized per day. In some cases, at least about 10 10 pieces, 10 11 pieces, 10 12 pieces, 10 13 pieces, 10 14 Pieces or 10 15 bases are synthesized per day. In some cases, at most about 10 10 pieces, 10 11 pieces, 10 12 pieces, 10 13 pieces, 10 14 Pieces or 10 15 bases are synthesized per day. In some cases, about 10×10 8 pieces, 10×10 9 pieces, 10×10 10 pieces, 10×1011 Pieces, or 10x10 12 polynucleotides are synthesized per day. In some cases, at least about 10×10 8 pieces, 10×10 9 pieces, 10×10 10 pieces, 10×10 11 Pieces, or 10x10 12 polynucleotides are synthesized per day. In some cases, at most about 10×10 8 pieces, 10×10 9 pieces, 10×10 10 pieces, 10×10 11 Pieces, or 10x10 12polynucleotides are synthesized per day. In some cases, each polynucleotide synthesized comprises about 20, 50, 100, 200, 300, 400, or 500 nucleic acid bases. In some cases, each polynucleotide synthesized comprises at least about 20, 50, 100, 200, 300, 400, or 500 nucleic acid bases. In some cases, each polynucleotide synthesized comprises at most about 20, 50, 100, 200, 300, 400, or 500 nucleic acid bases. In some cases, these bases are synthesized with a total average error rate of about 1 per 100, 200, 300, 400, 500, 1000, 2000, 5000, 10000, 15000, 20000 bases. In some cases, these bases are synthesized with a total average error rate of less than about 1 error per 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 12000, 15000, 18000, 20000 bases. In some cases, these error rates are for about 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.5% or more of the polynucleotides synthesized. In some cases, these error rates are for at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.5% or more of the polynucleotides synthesized. In some cases, at least 90%, 95%, 98%, 99%, 99.5% or more of these synthesized polynucleotides do not differ from the predetermined sequence they encode. In some cases, the error rate of polynucleotides synthesized on substrates using the methods and systems described herein is less than about 1 in 200. In some cases, the error rate of polynucleotides synthesized on substrates using the methods and systems described herein is less than about 1 in 1,000.In some cases, the error rate of polynucleotides synthesized on a substrate using the methods and systems described herein is less than about 1 in 2,000. In some cases, the error rate of polynucleotides synthesized on a substrate using the methods and systems described herein is less than about 1 in 3,000. In some cases, the error rate of polynucleotides synthesized on a substrate using the methods and systems described herein is less than about 1 in 5,000. Individual types of error rate include mismatches, deletions, insertions, and / or substitutions of polynucleotides synthesized on a substrate. The term "error rate" refers to a comparison of a collection of polynucleotides synthesized to a collection of predetermined polynucleotide sequences. In some cases, each of the polynucleotides includes a plurality of different nucleotide bases (e.g., A, T, C, G, etc.).

[0084] In some cases, the synthesized polynucleotides disclosed herein include a tether of 12 to 25 bases. In some cases, the tether includes about 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. In some cases, the synthesized polynucleotides disclosed herein comprise a tether of about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, or about 250 bases.

[0085] Electrochemistry

[0086] Provided herein is a method of applying a voltage to the device described herein. Such a voltage can result in any number of different effects, such as electrochemical reactions with a solvent or solute. In some cases, the application of a voltage can modify molecules attached to the synthetic surface. In some cases, the molecules are polysaccharides, polynucleotides, polypeptides, or other polymers. Additionally, provided herein is a method of providing a voltage to the device described herein. In some cases, the voltage is used to chemically modify oligonucleotides attached to a solid support or surface. Such modifications can occur through direct electrochemical reactions of polynucleotides or through the generation of reagents.

[0087] The methods described herein may include providing a voltage to the device (applying a voltage) for a period of time. The applied voltage, in some cases, forms a circuit between the cathode and the anode, causing current to flow through the device, the solvent, and / or other components. In some cases, a layer of the device is configured as an anode or a cathode. In some cases, the device includes an anode positioned above a cathode face ("sandwich type"). In some cases, the device includes a cathode positioned above an anode face ("opposing cathode"). In some cases, a conductive layer is in electrical contact with the cathode layer. In some cases, the device includes an anode positioned on substantially the same face as the cathode. The application of the voltage is, in some cases, configured to perform a step of polynucleotide synthesis. In some cases, the device includes a conductive layer in fluid communication with a solvent. In some cases, the solvent includes a reagent.

[0088] The methods described herein may include applying a voltage to one or more devices described herein. In some cases, such a voltage results in deprotection of molecules (polynucleotides, polypeptides, polysaccharides, or other polymers) in one or more devices or regions. In some cases, application of a voltage in one or more devices results in deprotection of polynucleotides in one or more devices or regions within one or more devices. In some cases, a device is described as "inactive" if no reagents are produced in or near the device or region of the device. In some cases, a device is described as "active" if reagents are produced in or near the device or region of the device. In some cases, chemical reactions of polynucleotides occur in or near one or more active devices, or regions of one or more active devices. In some cases, voltages are provided to both active and inactive devices. In some cases, voltages are applied to inactive devices at levels that are insufficient to produce chemical reagents. In some cases, one or more voltages (or voltage levels) may be applied for a period of time. In some cases, a single voltage level is used (FIGS. 17A-17C). In some cases, the cathode voltage is held constant at 0V while the anode voltage is increased from 0V to 2V during a "pulse". In some cases, the cathode voltage is held constant at a negative voltage (e.g., -1V or other negative voltage) while the anode voltage is increased from 0V to 2V during a "pulse". In some cases, during a "pulse", the cathode voltage is decreased from 0V to a negative voltage (such as -1V) and the anode voltage is increased from 0V to 1V. In some cases, such voltages are synchronized. In some cases, the decrease in voltage at the cathode and the increase in voltage at the anode occur at approximately the same time. In some cases, such voltages are synchronized.In some cases, the voltage decrease at the cathode and the voltage increase at the anode occur within 1 second, 0.5 seconds, 0.1 seconds, 0.05 seconds, 0.01 seconds, 0.005 seconds, 0.001 seconds, or 0.0005 seconds of each other. In some cases, two voltage levels are used during the deprotection step. In some cases, two voltage levels are used, for example, a positive voltage and a neutral voltage. In some cases, three voltage levels are used, for example, a positive voltage, a neutral voltage (or zero / about zero), and a negative voltage. The two or three voltage levels used can be a positive voltage, a neutral voltage, a negative voltage, or any combination thereof.

[0089] In some cases, a voltage is applied to multiple electrodes in fluid communication with the same surface, for example, between a reaction electrode and a shield electrode. In some cases, the voltage between the reaction electrode and the shield electrode is synchronized. In some cases, the reagent is generated when the difference between the cathode voltage and the anode voltage exceeds a threshold. In some cases, synchronizing the positive anode voltage and the negative cathode voltage provides the advantage of reducing the magnitude of the voltage required to operate the device.

[0090] Different voltage configurations may be used for device activation. In a first configuration, the in-plane cathode, the counter-cathode, and other devices (e.g., proximal or distal devices) are grounded while the device is energized. In a second configuration, the counter-cathode is grounded and the in-plane cathode and other devices remain unconnected while the device is energized. In a third configuration, the in-plane cathode and other devices are grounded and the counter-cathode remains unconnected while the device is energized. In a fourth configuration, the in-plane cathode is grounded and the counter-cathode and other devices remain unconnected while the device is energized. In some cases, when the device is activated, the grounded in-plane cathode produces a deprotection-neutralizing product. In some cases, the grounded component (e.g., cathode) has a negative potential and the activated device (e.g., anode) is connected to a positive potential. In some cases, this configuration reduces the effective voltage required to drive the device.

[0091] A device may be described as a circuit between an anode and a cathode. In some cases, such a circuit is described as being in a device state such as "on", "off", "alternate resistance", etc. In some cases, the alternate resistance is a high resistance state, or a "disconnected" state. In some cases, the high resistance state is a higher resistance state than the off state (e.g., low / no voltage in the off state, but still grounded). In some cases, the high resistance state provides an effective amount of resistance to reduce current flow to one or more inactive devices. Without being bound by theory, the disconnected state reduces undesired deprotection in areas adjacent to the on devices in some cases. In some cases, the high resistance state provides an effective amount of resistance to reduce current flow to near zero to one or more inactive devices. In some cases, the off state is created by zero (or near zero) voltage between the inactive devices and the common cathode. In some cases, the off state exists even when a minimum voltage is applied between the inactive devices and the common cathode, which is less than the amount required for deprotection. In some cases, the high resistance state is created when the voltage between the inactive devices and the cathode is zero, resulting in a high resistance between the inactive devices and the common cathode. In some cases, the off state indicates that the voltage between the anode and the active device (cathode) is zero or negative. In some cases, the on state indicates that the voltage between the anode and the active device (cathode) is positive, sufficient for deprotection. In some cases, the inactive devices are in an off or alternate resistance state. In some cases, the active devices (for which deprotection is desired) are cycled (e.g., pulsed) between one or more on and off states for a period of time.In some cases, an active device (for which deprotection is desired) is cycled between one or more on and off states for a period of time, while an adjacent inactive device is maintained in an alternate resistance state.

[0092] In some cases, the methods described herein include one or more of: (a) providing a surface having (i) one or more electrodes proximate the surface and (ii) one or more in-plane cathodes proximate the surface and including a first plurality of protected biomolecules bound to the surface; (b) passing current through at least one electrode proximate a first region of the surface to electrochemically generate a reagent, where the reagent reacts with at least some of the first plurality of biomolecules in the first region; (c) coupling at least one protected biomolecule monomer to at least one deprotected biomolecule in the first region; and (d) repeating steps (a)-(c) to synthesize a biomolecule. In some cases, the biomolecule comprises a polynucleotide. In some cases, the biomolecule monomer comprises a nucleotide. In some cases, a negative voltage is applied to the cathode and a positive voltage is applied to the anode. In some cases, the absolute difference in voltage is about 2, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, or 0.2 volts. In some cases, the absolute difference in voltage is at least about 2, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, or 0.2 volts. In some cases, the absolute difference in voltage is about 2 volts or less, 1.9 volts or less, 1.8 volts or less, 1.7 volts or less, 1.6 volts or less, 1.5 volts or less, 1.4 volts or less, 1.3 volts or less, 1.2 volts or less, 1.1 volts or less, 1 volt or less, 0.9 volts or less, 0.8 volts or less, 0.7 volts or less, 0.6 volts or less, 0.5 volts or less, 0.4 volts or less, 0.3 volts or less, or 0.2 volts or less. In some cases, the absolute difference in voltage is about 0.2-2 volts or less, 0.5-2 volts or less, 0.8-2 volts or less, 1-2 volts or less, 1.5-2 volts or less, 0.2-1 volts or less, 0.2-0.5 volts or less, 0.5-1 volts or less, or 0.5-1.5 volts or less.In some cases, the absolute difference in voltage is 0.2 to 2 volts or less, 0.5 to 2 volts or less, 0.8 to 2 volts or less, 1 to 2 volts or less, 1.5 to 2 volts or less, 0.2 to 1 volt or less, 0.2 to 0.5 volts or less, 0.5 to 1 volt or less, or 0.5 to 1.5 volts or less.

[0093] A voltage may be applied to the cathode in addition to the anode. In some cases, the cathode is biased at a negative voltage with respect to ground. In some cases, biasing the voltage of the cathode (bias voltage) reduces the maximum anode voltage required for the electrochemical reaction (e.g., the voltage difference between the anode and cathode is equal to the anode voltage plus the magnitude of the negative bias voltage of the cathode). In some cases, the device includes a contact bias on the cathode. In some cases, the bias voltage of the cathode is switched (e.g., synchronized) every time the anode voltage is switched. In some cases, the cathode controls the electrochemistry of a single device. In some cases, the cathode controls the electrochemistry of multiple devices (a "common" cathode). In some cases, the use of a common cathode reduces the number of transistors required per device. In some cases, the bias voltage is −0.1 volts or less, −0.2 volts or less, −0.3 volts or less, −0.5 volts or less, −0.7 volts or less, −0.9 volts or less, −1.0 volts or less, −1.1 volts or less, −1.2 volts or less, −1.5 volts or less, −1.8 volts or less, −2.0 volts or less, −2.1 volts or less, −2.2 volts or less, or −2.5 volts or less. In some cases, the bias voltage is at least −0.1, −0.2, −0.3, −0.5, −0.7, −0.9, −1.0, −1.1, −1.2, −1.5, −1.8, −2.0, −2.1, −2.2, or at least −2.5 volts. In some cases, the bias voltage is about -0.1, -0.2, -0.3, -0.5, -0.7, -0.9, -1.0, -1.1, -1.2, -1.5, -1.8, -2.0, -2.1, -2.2, or about -2.5 volts. In some cases, the bias voltage is about -0.1 to -2.5 volts, -0.2 to -2.5 volts, -0.5 to -2.5 volts, -1.0 to -2.5 volts, -1.5 to -2.5 volts, -1.0 to -2.0 volts, -0.5 to -1.0 volts, -0.2 to -1.5 volts, or -2.0 to -2.5 volts.

[0094] The voltage between the two layers of the device or surface can vary. In some cases, the voltage is between the anode and the cathode. In some cases, the voltage is about 0.5-3, 1-3, 1.5-2.5, 1-2.5, or 1.5-2 volts. In some cases, the voltage is at least about 0.5, 0.75, 1, 1.2, 1.5, 1.7, 1.9, 2, 2.2, 2.4 volts, or greater than 2.4 volts. In some cases, the voltage is about 0.5, 0.75, 1, 1.2, 1.5, 1.7, 1.9, 2, 2.2, 2.4, or about 2.4 volts. In some cases, the voltage is about -0.1 to -2.5 volts, -0.2 to -2.5 volts, -0.5 to -2.5 volts, -1.0 to -2.5 volts, -1.5 to -2.5 volts, -1.0 to -2.0 volts, -0.5 to -1.0 volts, -0.2 to -1.5 volts, or -2.0 to -2.5 volts. In some cases, a conductive layer of the device is charged with a positive voltage. In some cases, a conductive layer of the device is charged with a negative voltage. In some cases, a first layer of the device is charged with a positive voltage while a second layer is charged with a negative voltage.

[0095] The duration for which the voltage is applied can vary for each synthesis cycle (e.g., deblocking, coupling, etc.). In some cases, the duration is the total duration for which the voltage is applied during a synthesis cycle. The voltage is applied for about 0.1 seconds or less, 0.2 seconds or less, 0.5 seconds or less, 0.8 seconds or less, 1 second or less, 2 seconds or less, 5 seconds or less, or 10 seconds or less in some cases. The voltage is applied for about 0.1-10, 0.5-10, 0.5-5, 0.1-5, 2-5, 2-10, 3-10, or 0.1-2 seconds in some cases. The voltage is applied for about 0.1, 0.2, 0.5, 0.8, 1, 2, 5, or about 10 seconds in some cases. The voltage is applied for about 0.1 milliseconds (ms) or less, 0.2 milliseconds or less, 0.5 milliseconds or less, 0.8 milliseconds or less, 1 millisecond or less, 2 milliseconds or less, 5 milliseconds or less, 10 milliseconds or less, 20 milliseconds or less, 50 milliseconds or less, 100 milliseconds or less, 200 milliseconds or less, 500 milliseconds or less, 800 milliseconds or less, or 1000 milliseconds or less in some cases. The voltage is applied for about 0.1, 0.2, 0.5, 0.8, 1, 2, 5, 10, 20, 50, 100, 200, 500, 800, or about 1000 milliseconds in some cases. The voltage is applied for about 0.1-1000, 0.5-500, 0.5-50, 0.1-5, 2-50, 2-100, 3-200, 0.1-10, 1-100, 1-50, or 0.1-2 milliseconds in some cases.

[0096] The voltage may be applied to an active device as a single "on" / "off" cycle or as a series of alternating "on" and "off" cycles. In some cases, the "on" state is a positive or negative voltage. The application of a voltage in an "on" state followed by an "off" state is defined as a "pulse" in some cases. In some cases, the voltage is applied in a series of pulses, e.g., 1-1000, 1-500, 1-300, 10-500, 10-100, 50-500, 50-200, 100-1000, 2-10, 2-8, 20-200, or 300-750 pulses. The number of pulses may be about 1, 2, 5, 10, 20, 50, 100, 200, 300, 500, 750, or 1000 pulses. The duration of voltage application can be divided by the number of pulses to define the pulse duration (or pulse width, or time per pulse). The pulse duration is, in some cases, about 0.1-1000, 0.5-500, 0.5-50, 0.1-5, 2-50, 2-100, 3-200, 0.1-10, 1-100, 1-50, or 0.1-2 milliseconds. In some cases, the polynucleotide synthesis surface is washed with solvent between pulses. In some cases, the polynucleotide synthesis surface is not washed with solvent between pulses. In some cases, a series of pulses is used to deliver voltage to the surface, followed by a washing step, which in some cases is followed by another series of pulses. In some cases, the pulses are the same voltage. In some cases, the pulses are not the same voltage. In some cases, the first pulse is positive and the second pulse is negative. In some cases, the time between the positive and negative voltages is substantially instantaneous. In some cases, the first pulse is about 2 volts and the second pulse is about −0.6 volts. In some cases, the first pulse is between 0.5 and 3 volts and the second pulse is between −0.1 and −1.0 volts.

[0097] The period between pulses can vary. In some cases, the period between pulses can vary to allow for dissipation of electrochemically generated reagents, without being bound by theory. In some cases, the time between pulses is about 0.1-10, 0.5-10, 0.5-5, 0.1-5, 2-5, 2-10, 3-10, or 0.1-2 seconds. In some cases, the ratio of time between on and off of a series of pulses is described as the duty cycle. In some cases, the duty cycle is about 1:100, 1:50, 1:20, 1:10, 1:5, 1:2, 1:1.5, 1:1.05, 1.05:1, 1.5:1, 2:1, or about 3:1.

[0098] The electrochemical reaction may result in reduced transfer of reagents to adjacent (or nearby) addressable devices (or solid supports) where other polynucleotides are synthesized. In some cases, the addressable device may be pulsed more than once. In some cases, the active device includes a first plurality of protected biomolecules. In some cases, the active device includes a second plurality of protected biomolecules (close to) an adjacent device. In some cases, the reduced transfer of reagents to the adjacent device results in improved synthesis fidelity on the device. For example, a high percentage of the first plurality of protected biomolecules in the active device are chemically modified, while a minimal percentage of the second plurality of protected biomolecules in the adjacent device are chemically modified. In some cases, the electrochemical deprotection chemically modifies the protected nucleosides. In some cases, about 90%, 95%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.97%, 99.99%, or 99.995% of the polynucleotides on the addressable device are chemically modified. In some cases, at least 90%, 95%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.97%, 99.99%, or at least 99.995% of the polynucleotides on the addressable device are chemically modified. In some cases, about 10%, 5%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.05%, 0.02%, 0.01%, 0.005%, 0.004%, 0.001%, or 0.0005% of the polynucleotides on adjacent addressable devices are chemically modified.In some cases, 10% or less, 5% or less, 3% or less, 2% or less, 1% or less, 0.5% or less, 0.4% or less, 0.3% or less, 0.2% or less, 0.1% or less, 0.05% or less, 0.02% or less, 0.01% or less, 0.005% or less, 0.004% or less, 0.001% or less, or 0.0005% or less of the polynucleotides on adjacent addressable devices are chemically modified. In some cases, about 90%, 95%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.97%, 99.99%, or 99.995% of the protected polynucleotides on an addressable device are chemically modified, and the pitch distance between the addressable devices is 1 micron or less. In some cases, at least 90%, 95%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.97%, 99.99%, or at least 99.995% of the protected polynucleotides on an addressable device are chemically modified and the pitch distance between the addressable devices is 1 micron or less. In some cases, about 10%, 5%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.05%, 0.02%, 0.01%, 0.005%, 0.004%, 0.001%, or 0.0005% of the protected polynucleotides on adjacent addressable devices are chemically modified and the pitch distance between the addressable devices is 1 micron or less. In some cases, 10% or less, 5% or less, 3% or less, 2% or less, 1% or less, 0.5% or less, 0.4% or less, 0.3% or less, 0.2% or less, 0.1% or less, 0.05% or less, 0.02% or less, 0.01% or less, 0.005% or less, 0.004% or less, 0.001% or less, or 0.0005% or less of the protected polynucleotides on adjacent addressable devices are chemically modified, and the pitch distance between the addressable devices is 1 micron or less.

[0099] The method described herein can result in the reduction of the synthesis time of biomolecules.In some cases, the biomolecule comprises a polynucleotide.In some cases, the polynucleotide is synthesized at a rate of at least 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 45, 50, 55, 60, 75, or at least 100 nt / hour.In some cases, the polynucleotide is synthesized at a rate of about 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 45, 50, 55, 60, 75, or at least 100 nt / hour. In some cases, polynucleotides are synthesized at a rate of 10-100, 10-75, 10-50, 10-25, 7, 15-25, 15-50, 15-75, 20-80, 20-50, 30-80, 30-50, 50-100, or 75-125 nt / hour.

[0100] Methods for producing devices and surfaces for polynucleotide synthesis are provided herein. A layer integrated into a solid support is described herein. In some cases, the layer includes an electrode or is configured to be used as an electrode. In some cases, the electrode is configured as a cathode or an anode. In some cases, the anode includes a metal oxide. In some cases, the nucleic acid is synthesized on the anode. In some cases, the nucleic acid is synthesized on a metal oxide layer. In some cases, the nucleic acid is synthesized on a porous metal oxide layer that includes a continuous metal layer underneath. The electrode includes at least one conductor in some cases and is made from materials well known in the art. In some cases, the electrode includes at least one conductor and one or more insulators or semiconductors. The material may include metals, nonmetals, mixed metal oxides, nitrides, carbides, silicon-based materials, or other materials. In some cases, the metal oxides include TiO2, Ta2O5, IrO2, RuO2, RhO2, Nb2O5, Al2O3, BaO, Y2O3, HfO2, SrO, or other metal oxides known in the art. In some cases, the metal carbides include TiC, WC, ThC2, ThC, VC, W2C, ZrC, HfC, NbC, TaC, Ta2C, or other metal carbides known in the art. In some cases, the metal nitrides include GaN, InN, BN, Be3N2, Cr2N, MoN, Si3N4, TaN, Th2N2, VN, ZrN, TiN, HfN, NbC, WN, TaN, or other metal nitrides known in the art. In some cases, the devices disclosed herein are manufactured using a combination of materials listed herein or any other suitable materials known in the art.

[0101] The solid support including the layer may be coated with additional materials, such as semiconductors or insulators. In some cases, a layer is configured for use as an electrode. In some cases, the electrodes are coated with materials for biomolecule binding and synthesis. In some cases, the electrodes are coated with materials for polynucleotide binding and synthesis. Each electrode can control one or more different sites for synthesis. In some cases, each site for synthesis has a polynucleotide density. In some cases, the density is greater than 10 nm 2 , 20nm 2 , 50 nm 2 , 100 nm 2 , 200 nm 2 , 500 nm 2 , 1,000 nm 2 , 2,000 nm 2 , 5,000 nm 2 At least one biomolecule per 10,000 nm 2 In some cases, the density is at least 1 oligo per 10 nm 2 Approximately 1 biomolecule per ~5,000 nm 2 Approximately 1 biomolecule per 50 nm 2 Approximately 1 biomolecule per ~500 nm 2 Approximately 1 biomolecule per 25 nm 2 Approximately 1 biomolecule per ~75 nm 2 In some cases, the density of polynucleotides is approximately 1 biomolecule per 25 nm 2 Approximately 1 biomolecule per ~75 nm 2 In some cases, the density is about 1 biomolecule per 10 nm 2 , 20, 50, 100, 200, 500, 1,000, 2,000, 5,000, or at least 1 biomolecule per 10,000 nm 2 In some cases, the density is at least 1 oligo per 10 nm 2 Approximately 1 oligo per ~5,000 nm 2 Approximately 1 oligo per 50 nm2 Approximately 1 oligo per ~500 nm 2 Approximately 1 oligo per 1000 nm, or 25 nm 2 Approximately 1 oligo per ~75 nm 2 In some cases, the density of the polynucleotides is about 1 oligo per 25 nm 2 Approximately 1 oligo per ~75 nm 2 Approximately one oligo per

[0102] Described herein is a device in which two or more solid supports are assembled. In some cases, the solid supports are interfaced with each other on a larger unit. The interface connection can include the exchange of fluid, electrical signals, or other exchange media between the solid supports. The unit can be interfaced with any number of servers, computers, or networked devices. For example, multiple solid supports are integrated into or attached to a rack unit, which can be easily inserted or removed from a server rack. A rack unit can include any number of solid supports. In some cases, a rack unit includes about 1, 2, 5, 10, 20, 50, 100, 200, 500, 1000, 2000, 5000, 10,000, 20,000, 50,000, 100,000, or 100,000 solid supports. In some cases, a rack unit contains at least 1, 2, 5, 10, 20, 50, 100, 200, 500, 1000, 2000, 5000, 10,000, 20,000, 50,000, 100,000, or 100,000 solid supports. In some cases, a rack unit contains at most 1, 2, 5, 10, 20, 50, 100, 200, 500, 1000, 2000, 5000, 10,000, 20,000, 50,000, 100,000, or 100,000 solid supports. In some cases, all or a portion of the solid supports of a rack unit are in fluid communication, electronic communication, or both. In some cases, the server rack includes about 10, 20, 50, 80, 100, 200, 500, 800, or 1000 rack units. In some cases, the server rack includes at least about 10, 20, 50, 80, 100, 200, 500, 800, or 1000 rack units. In some cases, the server rack includes at most about 10, 20, 50, 80, 100, 200, 500, 800, or 1000 rack units. In some cases, all or a portion of the rack units of the rack server are in fluid communication, electronic communication, or both.In some cases, two or more solid supports are not interfaced with each other. In some cases, two or more rack units including solid supports, such as those described herein, are stacked vertically. Fluid communication, electronic communication, or both can be formed using, as non-limiting examples, one or more tubes (e.g., microfluidic tubes), valves, actuators, robots, etc.

[0103] The nucleic acids present on the solid support (and the information stored therein) can be accessed from a rack unit. See, for example, FIG. 9. In some cases, the solid support is present on a solid support, e.g., a chip (FIGS. 10A-10B and FIG. 11). Accessing includes removal of the polynucleotides from the solid support, direct analysis of the polynucleotides on the solid support, or any other method that allows for manipulating or identifying the information stored in the nucleic acid. The information is accessed in some cases from multiple racks, a single rack, a single solid support in a rack, a portion of a solid support, or a single location on a solid support. In various cases, accessing includes interfacing the nucleic acid with an additional device, e.g., a mass spectrometer, HPLC, sequencing equipment, PCR thermocycler, or other device for manipulating nucleic acids. Accessing the nucleic acid information is achieved in some cases by cleaving the polynucleotides from all or a portion of the solid support.

[0104] In some cases, the rack units or rack servers are located in a data center. In some cases, the data center utilizes mechanical structures used to mount traditional computing and data storage resources to the rack units, such as openings adapted to support disk drives, processing blades, or other computer equipment. In some cases, a computer system, such as those provided herein, is used to retrieve polynucleotides from one or more rack units on one or more rack servers. In some cases, the retrieval of one or more rack units on one or more rack servers is directed by a user (e.g., a technician, researcher, customer, etc.), a computer system, or both. In some cases, the rack units can be retrieved from the rack servers using a robotic system, such as a robotic arm. In some cases, the robotic system communicates with the computer system. The robotic system can be used to interface any component of the data storage system with another component of the data storage system. In some cases, the interfacing includes transferring, storing, moving, processing, or retrieving. In some cases, the robotic system moves solid supports between components (e.g., units or chambers) of the data storage system. The components may include, by way of non-limiting examples, a synthesis unit, a storage unit, an amplification unit, and the like.

[0105] Cleavage, in some cases, involves exposure to chemical reagents (ammonia or other reagents), electrical potential, irradiation, heat, light, sound, or other forms of energy that can manipulate chemical bonds. In some cases, cleavage occurs by charging one or more electrodes in the vicinity of the polynucleotide. In some cases, electromagnetic radiation in the form of UV light is used to cleave the polynucleotide. In some cases, a lamp is used to cleave the polynucleotide, and a mask controls the location of the UV light exposure to the surface. In some cases, a laser is used to cleave the polynucleotide, and the open / closed state of a shutter controls the exposure of the UV light to the surface. In some cases, a computer system, such as those provided herein, directs the open / closed state of the shutter. In some cases, access to the nucleic acid information (including removal / addition of racks, solid supports, reagents, nucleic acids, or other components) is fully automated (e.g., using a computer system provided herein). In some cases, the chip has one or more contacts. In some cases, the chip includes at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 75, 100, or more than 200 contacts.

[0106] The solid support described herein comprises an active area. In some cases, the active area comprises an addressable solid support, region, or location for nucleic acid synthesis. In some cases, the active area comprises an addressable region or location for nucleic acid storage. In some cases, the active area is in fluid communication with a solvent or other reagent. The active area comprises a variety of dimensions. For example, the active area has a dimension of about 1 mm to about 50 mm by about 1 mm to about 50 mm. In some cases, the active area has a width of at least or about 0.5, 1, 1.5, 2, 2.5, 3, 5, 5, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, or more than 80 mm. In some cases, the active area comprises 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 greater than 80 mm. Exemplary active areas within a solid support are found in Figure 11. Package 2407 comprises active area 2405 within solid support 2403. Package 2407 also comprises fluidic interface 2401.

[0107] Described herein are devices, compositions, systems, and methods for solid support-based nucleic acid synthesis and storage. In some cases, the solid support has several sites (e.g., spots) or locations for synthesis or storage. In some cases, the solid support includes up to or about 10,000 x 10,000 locations within an area. In some cases, the solid support includes about 1000-20,000 x about 1000-20,000 locations within an area. In some cases, the solid support includes at least or about 10, 30, 50, 75, 100, 200, 300, 400, 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, 12,000, 14,000, 16,000, 18,000, 20,000, 22,000, 24,000, 28,000, 30,000, 32,000, 36,000, 38,000, 40,000, 40,000, 50,000, 50,000, 60,000, 70,000, 80,000, 90,000, 10,000, 12,000, 14,000, 16,000, 18,000, 20,000, 22,000, 24,000, 28,000, 30,000, 32,000, 36,000, 38,000, 40,000, 40 0, 20,000 by at least or about 10, 30, 50, 75, 100, 200, 300, 400, 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, 12,000, 14,000, 16,000, 18,000, 20,000 locations. In some cases, the area is at most 0.25, 0.5, 0.75, 1.0, 1.25, 1.5, or 2.0 square inches. In some cases, the solid support comprises addressable locations having a pitch 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 um, or greater than 10 um. In some cases, the solid support comprises addressable locations having a pitch of about 5 um. In some cases, the solid support comprises addressable locations having a pitch of about 2 um. In some cases, the solid support comprises addressable locations having a pitch of about 1 um. In some cases, the solid support comprises addressable locations having a pitch of about 0.2 um.In some cases, the solid support comprises addressable locations having a pitch of about 0.2 μm to about 10 um, about 0.2 to about 8 um, about 0.5 to about 10 um, about 1 μm to about 10 um, about 2 μm to about 8 um, about 3 μm to about 5 um, about 1 μm to about 3 μm, or about 0.5 μm to about 3 um. In some cases, the solid support comprises addressable locations having a pitch of about 0.1 μm to about 3 um. In some cases, the solid support comprises addressable locations having a pitch of at least or about 0.01, 0.02, 0.025, 0.03, 0.04, 0.05, 0.1, 0.15, .02, 0.25, 0.30, 0.35, 0.4, 0.45, 0.5, 0.6, 0.7, 0.8, 0.9, 1 um, or greater than 1 um. In some cases, the solid support includes addressable locations having a pitch of about 0.5 um. In some cases, the solid support includes addressable locations having a pitch of about 0.2 um. In some cases, the solid support includes addressable locations having a pitch of about 0.1 um. In some cases, the solid support includes addressable locations having a pitch of about 0.02 um. In some cases, the solid support includes addressable locations having a pitch of about 0.02 μm to about 1 um, about 0.02 to about 0.8 um, about 0.05 to about 0.1 um, about 0.1 μm to about 1 um, about 0.2 μm to about 0.8 um, about 0.3 μm to about 0.5 um, about 0.1 μm to about 0.3 μm, or about 0.05 μm to about 0.3 um. In some cases, the solid support includes addressable locations having a pitch of about 0.01 μm to about 0.3 um. See, for example, Figures 7B-7C.

[0108] The devices described herein can include high density addressable arrays for synthesis. In some cases, the device arrays can be up to 1 mm 2 In some cases, the device array comprises about 1, 10, 100, 500, 1000, 2000, 5000, 10,000, 20,000, 50,000, 1000,000, or 200,000 addressable locations per mm. 2In some cases, the device array comprises at least about 1, 10, 100, 500, 1000, 2000, 5000, 10,000, 20,000, 50,000, 1000,000, or at least 200,000 addressable locations per mm. 2 In some cases, the device array comprises at most about 1, 10, 100, 500, 1000, 2000, 5000, 10,000, 20,000, 50,000, 1000,000, or at least 200,000 addressable locations per mm. 2 Approximately 1-50,000, 1-10,000, 10-100,000, 50-100,000, 100-100,000, 100-50,000, 100-5000, 100-1000, 500-50,000, 500-10,000, 500-25,000, 1000-200,000, 1000-100,000, 1000-50,000, 1000-25,000, 1000-10,000, 5000-100,000, 5000-200,000, or 5000-50,000 locations per unit. Structures for Biomolecular Synthesis

[0109] Provided herein are rigid or flexible structures for biomolecule synthesis (e.g., polynucleotide synthesis). In the case of rigid structures, provided herein are devices having structures for the generation of libraries of polynucleotides. In some cases, the structures include plates.

[0110] In the case of a flexible structure, devices are provided herein in which the flexible structure includes one or more fixed structures, e.g., a continuous loop 2501 wrapped around a pair of rollers 2503, or a discontinuous flexible structure 2507 wrapped around a separate fixed structure, e.g., a pair of reels 2505. See Figures 12A-12B. In some cases, the structure includes multiple regions for polynucleotide synthesis. An exemplary structure is illustrated in Figure 12C, where a plate includes separate regions 2509 for polynucleotide synthesis. The separate regions 2509 may be separated by breaking or cutting (2511). Each of the separate regions may be further released, sequenced, decoded, read (2513), or stored (2515). An alternative structure is illustrated in Figure 12D, where a tape includes separate regions 2517 for polynucleotide synthesis. The distinct regions 2517 may be separated by breaking or cutting (2519). Each of the distinct regions may be further released, sequenced, decoded, read (2521), or stored (2523). Provided herein is a flexible structure having a surface with multiple locations for polynucleotide extension. In some cases, each location of the structure is about 10 μm wide and the distance between the centers of each structure is about 21 um. In some cases, each location of the structure is about 1 μm wide and the distance between the centers of each structure is about 2 um. In some cases, each location of the structure is about 0.1 μm wide and the distance between the centers of each structure is about 0.2 um. The locations may include, without limitation, circular, rectangular, tapered, or rounded shapes. Alternatively, or in combination, the structure is rigid. In some cases, the rigid structure includes locations for polynucleotide synthesis. In some cases, the rigid structures include substantially planar regions, channels, or wells for polynucleotide synthesis, hi some cases, the structures are patterned into one or more layers of the device.

[0111] The wells described herein may include any size or dimension. In some cases, the wells described herein have a width to depth (or height) ratio of 1 to 0.01, where the width is a measurement of the width at the narrowest segment of the well. In some cases, the wells described herein have a width to depth (or height) ratio of 0.5 to 0.01, where the width is a measurement of the width at the narrowest segment of the well. In some cases, the wells described herein have 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. Provided herein are structures for polynucleotide synthesis that include multiple separate locations for polynucleotide synthesis. Exemplary structures of locations include, without limitation, substantially planar regions, channels, wells, or protrusions. The structures described herein may include multiple clusters, each cluster including multiple wells, locations, or channels. Alternatively, the structures described herein may include a uniform arrangement of wells, sites, or channels. The structures provided herein may include wells having a height or depth of about 5 μm to about 500 um, about 5 μm to about 400 um, about 5 μm to about 300 um, about 5 μm to about 200 um, about 5 μm to about 100 um, about 5 μm to about 50 um, or about 10 μm to about 50 um. In some cases, the height of the wells is less than 100 um, less than 80 um, less than 60 um, less than 40 μm, or less than 20 um. In some cases, the height of the wells is about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500 μm, or more. In some cases, the height or depth of the wells is at least 10, 25, 50, 75, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 nm, or greater than 1000 nm. In some cases, the height or depth of the wells is in the range of about 10 nm to about 1000 nm, about 25 nm to about 900 nm, about 50 nm to about 800 nm, about 75 nm to about 700 nm, about 100 nm to about 600 nm, or about 200 nm to about 500 nm.In some cases, the height or depth of the wells ranges from about 50 nm to about 1 um, hi some cases, the height of the wells is about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 700, 800, 900, or about 1000 nm.

[0112] The structures for polynucleotide synthesis provided herein may include a channel. The ratio of width to depth (or height) of the channel may be 1 to 0.01, where the width is a measurement of the width at the narrowest segment of the microchannel. In some cases, the channels described herein have a ratio of width to depth (or height) of 0.5 to 0.01, where the width is a measurement of the width at the narrowest segment of the microchannel. In some cases, the channels described herein have a ratio of width to depth (or height) of about 0.01, 0.05, 0.1, 0.15, 0.16, 0.2, 0.5, or 1.

[0113] Described herein are structures for polynucleotide synthesis that include a plurality of distinct locations. The structures include, without limitation, substantially planar regions, channels, protrusions, or wells for polynucleotide synthesis. In some cases, structures described herein are provided that include a plurality of channels, where the height or depth of the channels is about 5 μm to about 500 μm, about 5 μm to about 400 μm, about 5 μm to about 300 μm, about 5 μm to about 200 μm, about 5 μm to about 100 μm, about 5 μm to about 50 μm, or about 10 μm to about 50 μm. In some cases, the height of the channels is less than 100 μm, less than 80 μm, less than 60 μm, less than 40 μm, or less than 20 μm. In some cases, the height of the channel is about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500 μm, or more. In some cases, the height or depth of the channel is at least 10, 25, 50, 75, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 nm, or more than 1000 nm. In some cases, the height or depth of the channel is in the range of about 10 nm to about 1000 nm, about 25 nm to about 900 nm, about 50 nm to about 800 nm, about 75 nm to about 700 nm, about 100 nm to about 600 nm, or about 200 nm to about 500. The channels described herein may be arranged on a surface in clusters or as a uniform field.

[0114] The width of a location on a surface of a structure for polynucleotide synthesis described herein can be about 0.1 μm to about 500 μm, about 0.5 μm to about 500 μm, about 1 μm to about 200 μm, about 1 μm to about 100 μm, about 5 μm to about 100 μm, or about 0.1 μm to about 100 μm, such as about 90 μm, 80 μm, 70 μm, 60 μm, 50 μm, 40 μm, 30 μm, 20 μm, 10 μm, 5 μm, 1 μm, or 0.5 μm. In some cases, the width of the location is less than about 100 μm, less than 90 μm, less than 80 μm, less than 70 μm, less than 60 μm, less than 50 μm, less than 40 μm, less than 30 μm, less than 20 μm, or less than 10 μm. In some cases, the width of the location is at least 10, 25, 50, 75, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1000 nm, or greater than 1000 nm. In some cases, the width of the location is in the range of about 10 nm to about 1000 nm, about 25 nm to about 900 nm, about 50 nm to about 800 nm, about 75 nm to about 700 nm, about 100 nm to about 600 nm, or about 200 nm to about 500. In some cases, the width of the location is in the range of about 50 nm to about 1000 nm. In some cases, the distance between the centers of two adjacent locations is about 0.1 μm to about 500 μm, 0.5 μm to about 500 μm, about 1 μm to about 200 μm, about 1 μm to about 100 μm, about 5 μm to about 200 μm, about 5 μm to about 100 μm, about 5 μm to about 50 μm, or about 5 μm to about 30 μm, such as about 20 μm. In some cases, the total width of the locations is about 5 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, or 100 μm. In some cases, the total width of the locations is about 1 μm to 100 μm, 30 μm to 100 μm, or 50 μm to 70 μm. In some cases, the distance between the centers of two adjacent locations is about 0.5 μm to about 2 μm, 0.5 μm to about 2 μm, about 0.75 μm to about 2 μm, about 1 μm to about 2 μm, about 0.2 μm to about 1 μm, about 0.5 μm to about 1.5 μm, about 0.5 μm to about 0.8 μm, or about 0.5 μm to about 1 μm, e.g., about 1 μm.In some cases, the total width of the location is about 50 nm, 0.1 μm, 0.2 μm, 0.3 μm, 0.4 μm, 0.5 μm, 0.6 μm, 0.7 μm, 0.8 μm, 0.9 μm, 1 μm, 1.1 μm, 1.2 μm, 1.3 μm, 1.4 μm, or 1.5 μm. In some cases, the total width of the location is about 0.5 μm to 2 μm, 0.75 μm to 1 μm, or 0.9 μm to 2 μm. In some cases, the location is substantially planar.

[0115] In some cases, each location supports synthesis of a population of polynucleotides having a different sequence than a population of polynucleotides grown at another location. Provided herein are surfaces that include at least 10, 100, 256, 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000, 20000, 30000, 40000, 50000, or more clusters. Provided herein are surfaces that include greater than 2,000, greater than 5,000, greater than 10,000, greater than 20,000, greater than 30,000, greater than 50,000, greater than 100,000, greater than 200,000, greater than 300,000, greater than 400,000, greater than 500,000, greater than 600,000, greater than 700,000, greater than 800,000, greater than 900,000, greater than 1,000,000, greater than 5,000,000, or greater than 10,000,000 or more distinct locations. In some cases, each cluster includes about 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 locations. In some cases, each cluster includes about 50-500, 50-200, 50-150, or 100-150 locations. In some cases, each cluster includes 100-150 locations. In some cases, each cluster includes 109, 121, 130, or 137 locations.

[0116] Provided herein are locations where the width at the longest segment is about 5-100 μm. In some cases, the location is about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 μm wide at the longest segment. In some cases, the location is a channel having multiple segments, where the distance between the centers of each segment is 5-50 μm. In some cases, the distance between the centers of each segment is about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 μm.

[0117] Provided herein are locations where the width of the longest segment is between 5 and 500 nm. In some cases, the location is where the width of the longest segment is about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nm. In some cases, the location is a channel having multiple segments, where the distance between the centers of each segment is between 5 and 50 nm. In some cases, the distance between the centers of each segment is about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 150, 170, 180, 190, or 200 nm.

[0118] In some cases, the number of distinct polynucleotides synthesized on the surface of the structures described herein depends on the number of distinct locations available on the substrate. In some cases, the density of locations within a cluster of the substrate is at least or about 1 mm 2 1 place per piece, 1mm 2 10 locations per mm 2 25 locations per 1mm 2 50 locations per mm 2 65 locations per 1mm 2 75 locations per 1mm 2 100 locations per mm2 130 locations per 1mm 2 150 locations per 1mm 2 175 locations per 1mm 2 200 locations per 1mm 2 300 locations per 1mm 2 400 locations per 1mm 2 500 locations per 1mm 2 1,000 locations per mm 2 10 per 4 Location of each piece, 1mm 2 10 per 5 Location of each piece, 1mm 2 10 per 6 In some cases, the substrate may be 1 mm 2 Approximately 10 pieces to 1mm per 2 Approximately 500 locations per 1mm 2 Approximately 25 pieces ~ 1mm per 2 Approximately 400 locations per 1mm 2 Approximately 50 pieces ~ 1mm 2 Approximately 500 locations per 1mm 2 Approximately 100 pieces ~ 1mm 2 Approximately 500 locations per 1mm 2 Approximately 150 pieces ~ 1mm 2 Approximately 500 locations per 1mm 2 Approximately 10 pieces to 1mm per 2 Approximately 250 locations per 1mm 2 Approximately 50 pieces ~ 1mm 2 Approximately 250 locations per 1mm 2 Approximately 10 pieces to 1mm per 2 Approximately 200 locations per mm 2 Approximately 50 pieces ~ 1mm 2 In some cases, the substrate contains approximately 200 locations per mm 2 About 10 per 4 pieces~1mm 2 About 10 per 5 In some cases, the substrate includes a 2 About 10 per 5 pieces~1mm 2About 10 per 7 In some cases, the substrate includes a 2 At least 10 per 5 In some cases, the substrate includes a 2 At least 10 per 6 In some cases, the substrate includes a 2 At least 10 per 7 In some cases, the substrate includes a 2 About 10 per 4 pieces~1mm 2 About 10 per 5 In some cases, the density of the locations within the cluster of the substrate is at least or about 1 um 2 1 piece per location, 1um 2 10 spots per 1um 2 25 spots per 1um 2 50 spots per 1um 2 65 spots per 1um 2 75 spots per 1um 2 100 spots per 1um 2 130 spots per 1um 2 150 spots per 1um 2 175 spots per 1um 2 200 spots per 1um 2 300 spots per 1um 2 400 spots per 1um 2 500 spots per 1um 2 In some cases, the substrate is 1 um thick or more. 2 Approximately 10 pieces ~ 1um per 2 Approximately 500 spots per 1um 2 Approximately 25 pieces per 1um 2 Approximately 400 spots per 1um 2 Approximately 50 pieces per 1um 2 Approximately 500 spots per 1um 2 Approximately 100 pieces per 1um 2 Approximately 500 spots per 1um 2Approximately 150 pieces per 1um 2 Approximately 500 spots per 1um 2 Approximately 10 pieces ~ 1um per 2 Approximately 250 spots per 1um 2 Approximately 50 pieces per 1um 2 Approximately 250 spots per 1um 2 Approximately 10 pieces ~ 1um per 2 Approximately 200 spots per micron, or 1 um 2 Approximately 50 pieces per 1um 2 Contains approximately 200 locations per

[0119] In some cases, the distance between the centers of two adjacent locations in a cluster is about 10 μm to about 500 um, about 10 μm to about 200 um, or about 10 μm to about 100 um. In some cases, the distance between the centers of two adjacent locations is greater than about 10 um, greater than 20 um, greater than 30 um, greater than 40 um, greater than 50 um, greater than 60 um, greater than 70 um, greater than 80 um, greater than 90 μm, or greater than 100 um. In some cases, the distance between the centers of two adjacent locations is less than about 200 um, less than 150 um, less than 100 um, less than 80 um, less than 70 um, less than 60 um, less than 50 um, less than 40 um, less than 30 um, less than 20 μm, or less than 10 um. In some cases, the distance between the centers of two adjacent locations is less than about 10,000 nm, 8,000 nm, 6,000 nm, 4,000 nm, 2,000 nm, 1,000 nm, 800 nm, 600 nm, 400 nm, 200 nm, 150 nm, 100 nm, 80 um, 70 nm, 60 nm, 50 nm, 40 nm, 30 nm, 20 nm, or 10 nm. In some cases, at least 10 7 pieces, 10 8 pieces, 10 9 pieces, 10 10 pieces, 10 11 There are possible locations, each location supporting one polynucleotide. In some cases, 9Each polynucleotide is about 6 m long. 2 Less than 5m 2 Less than 4m 2 Less than 3m 2 Less than 2m 2 Less than or equal to 1m 2 Less than one embodiment of the present invention is supported on the structures described herein.

[0120] In some cases, the structures described herein may have more than 2,000, more than 5,000, more than 10,000, more than 20,000, more than 30,000, more than 50,000, more than 100,000, more than 200,000, more than 300,000, more than 400,000, more than 500,000, more than 600,000, more than 700,000, more than 800,000, more than 900,000, more than 1,000,000, or more than 2,000,000. In one embodiment, the present invention provides support for the synthesis of greater than 0, greater than 1,200,000, greater than 1,400,000, greater than 1,600,000, greater than 1,800,000, greater than 2,000,000, greater than 2,500,000, greater than 3,000,000, greater than 3,500,000, greater than 4,000,000, greater than 4,500,000, greater than 5,000,000, greater than 10,000,000 or more non-identical polynucleotides. In some cases, the structure may have more than 2,000, more than 5,000, more than 10,000, more than 20,000, more than 50,000, more than 100,000, more than 200,000, more than 300,000, more than 400,000, more than 500,000, more than 600,000, more than 700,000, more than 800,000, more than 900,000, more than 1,000,000, more than 1,200,000, or more than 2,000,000. In some embodiments, the present invention provides support for the synthesis of polynucleotides encoding more than 1,000, more than 1,400,000, more than 1,600,000, more than 1,800,000, more than 2,000,000, more than 2,500,000, more than 3,000,000, more than 3,500,000, more than 4,000,000, more than 4,500,000, more than 5,000,000, more than 10,000,000, or more distinct sequences. In some cases, at least a portion of the polynucleotides have the same sequence or are configured to be synthesized with the same sequence.In some cases, the structures provide a surface environment for the growth of polynucleotides having at least 50, 60, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 bases, or more. In some configurations, the structures for polynucleotide synthesis described herein include sites for polynucleotide synthesis in a uniform arrangement.

[0121] In some cases, polynucleotides are synthesized at separate locations of the structure, where each location supports the synthesis of a population of polynucleotides. In some cases, each location supports the synthesis of a population of polynucleotides having a different sequence than the population of polynucleotides grown at another location. In some cases, the locations of the structure are located within multiple clusters. In some cases, the structure includes at least 10, 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000, 20000, 30000, 40000, 50000, or more clusters. In some cases, the structure may include more than 2,000, more than 5,000, more than 10,000, more than 100,000, more than 200,000, more than 300,000, more than 400,000, more than 500,000, more than 600,000, more than 700,000, more than 800,000, more than 900,000, more than 1,000,000, more than 1,100,000, more than 1,200,000, more than 1,300,000, more than 1,400,000, more than 1,500,000, more than 1,600,000, more than 1,700,000, more than 1,800,000, more than 1,900,000, This includes more than 00,000, more than 300,000, more than 400,000, more than 500,000, more than 600,000, more than 700,000, more than 800,000, more than 900,000, more than 1,000,000, more than 1,200,000, more than 1,400,000, more than 1,600,000, more than 1,800,000, more than 2,000,000, more than 2,500,000, more than 3,000,000, more than 3,500,000, more than 4,000,000, more than 4,500,000, more than 5,000,000, or more than 10,000,000 or more separate locations.In some cases, each cluster contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 130, 150 or more locations. In some cases, each cluster contains 50-500, 100-150, or 100-200 locations. In some cases, each cluster contains 109, 121, 130, or 137 locations. In some cases, each cluster contains 5, 6, 7, 8, 9, 10, 11, or 12 locations. In some cases, polynucleotides from separate locations within a cluster have a sequence that, when assembled, encodes a contiguous longer polynucleotide of a given sequence.

[0122] Size of the structure

[0123] In some cases, the structures described herein are approximately the size of a plate (e.g., chip or wafer), e.g., about 40-120 mm by about 25-100 mm. In some cases, the structures described herein have a diameter of about 1000 mm, 500 mm, 450 mm, 400 mm, 300 mm, 250 mm, 200 mm, 150 mm, 100 mm, or 50 mm or less. In some cases, the diameter of the substrate is about 25 mm to 1000 mm, about 25 mm to about 800 mm, about 25 mm to about 600 mm, about 25 mm to about 500 mm, about 25 mm to about 400 mm, about 25 mm to about 300 mm, or about 25 mm to about 200 mm. Non-limiting examples of substrate sizes include about 300 mm, 200 mm, 150 mm, 130 mm, 100 mm, 84 mm, 76 mm, 54 mm, 51 mm, and 25 mm. In some cases, the substrate is at least 100 mm. 2 , 200mm 2 , 500mm 2 , 1,000mm 2 , 2,000mm 2 , 4,500mm 2 , 5,000mm 2 , 10,000mm 2 , 12,000mm2 , 15,000mm 2 , 20,000mm 2 , 30,000mm 2 , 40,000mm 2 , 50,000mm 2 , or more. In some cases, the thickness is about 50 mm to about 2000 mm, about 50 mm to about 1000 mm, about 100 mm to about 1000 mm, about 200 mm to about 1000 mm, or about 250 mm to about 1000 mm. Non-limiting examples of thickness include 275 mm, 375 mm, 525 mm, 625 mm, 675 mm, 725 mm, 775 mm, and 925 mm. In some cases, the thickness is at least or about 0.5 mm, 1.0 mm, 1.5 mm, 2.0 mm, 2.5 mm, 3.0 mm, 3.5 mm, 4.0 mm, or more than 4.0 mm. In some cases, the thickness varies with diameter and depends on the composition of the substrate. For example, a structure including a material other than silicon may have a different thickness than 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 structure is greater than about 1 foot, greater than 2 feet, greater than 3 feet, greater than 4 feet, greater than 5 feet, greater than 10 feet, greater than 15 feet, greater than 30 feet, greater than 40 feet, greater than 50 feet in any one dimension. In some cases, the structure includes an array of polynucleotide synthesis devices. In some cases, the structure is integrated into CMOS.

[0124] material

[0125] A device comprising a surface, the surface being modified to support polynucleotide synthesis at a predetermined location, resulting in low error rate, low dropout rate, high yield, and high oligo occurrence. In some cases, the surface of the device for polynucleotide synthesis provided herein is manufactured from various materials that can be modified to support de novo polynucleotide synthesis reactions. In some cases, the device is sufficiently conductive, e.g., capable of forming a uniform electric field over the entirety or a portion of the device. In some cases, the device includes one or more conductive layers. The device described herein may include a flexible material. Exemplary flexible materials include, without limitation, modified nylon, unmodified nylon, nitrocellulose, and polypropylene. The device described herein may include a rigid material. Exemplary rigid materials include, without limitation, glass, fused silica, silicon, silicon dioxide, silicon nitride, plastics (e.g., polytetrafluoroethylene, polypropylene, polystyrene, polycarbonate, and blends thereof), and metals (e.g., gold, platinum). The devices disclosed herein may be manufactured from materials including silicon, polystyrene, agarose, dextran, cellulose polymers, polyacrylamide, polydimethylsiloxane (PDMS), glass, or any combination thereof. In some cases, the devices disclosed herein are manufactured using combinations of the materials listed herein or any other suitable materials known in the art.

[0126] The devices described herein may include materials with various tensile strengths. Exemplary materials with various 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 supports described herein may have a tensile strength of 1-300, 1-40, 1-10, 1-5, or 3-11 MPa. The solid supports described herein may have a tensile strength 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 cases, the devices described herein include a solid support for polynucleotide synthesis in the form of a flexible material, such as a tape or flexible sheet, that can be stored in a continuous loop or reel.

[0127] Young's modulus is a measure of a material's resistance to elastic (recoverable) deformation under load. Exemplary materials with various Young's modulus stiffnesses 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 supports described herein may have a Young's modulus of 1-500, 1-40, 1-10, 1-5, or 3-11 GPa. The solid supports described herein may have a Young's modulus 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 flexibility and stiffness are inversely related to each other, flexible materials have low Young's modulus and undergo large changes in shape when subjected to load. In some cases, the solid supports described herein have a surface that has at least the flexibility of nylon.

[0128] In some cases, the devices disclosed herein include a silicon dioxide base and a surface layer of silicon oxide. Alternatively, the devices may have a silicon oxide base. The surfaces of the devices provided herein may be textured, providing an increased overall surface area for polynucleotide synthesis. The devices disclosed herein, in some cases, include at least 5%, 10%, 25%, 50%, 80%, 90%, 95%, or 99% silicon. The devices disclosed herein, in some cases, are fabricated from silicon-on-insulator (SOI) wafers.

[0129] The structure can be fabricated from a variety of materials suitable for the methods and compositions of the device described herein. In some cases, the material from which the substrate / solid support of the device is fabricated exhibits low levels of polynucleotide binding. In some circumstances, materials that are transparent to visible and / or UV light can be used. Sufficiently conductive materials can be utilized, such as those that can form a uniform electric field across all or a portion of the substrate / solid support described herein. In some cases, such materials can be connected to electrical ground. In some cases, the substrate or solid support can be thermally conductive or thermally insulating. These materials can be chemically and heat resistant to support chemical or biochemical reactions, such as a series of polynucleotide synthesis reactions. For flexible materials, materials of interest can include nylon, nitrocellulose, polypropylene, and the like, both modified and unmodified.

[0130] For rigid materials, specific materials of interest include glass, fused silica, silicon, plastics (e.g., polytetrafluoroethylene, polypropylene, polystyrene, polycarbonate, and blends thereof, etc.), metals (e.g., gold, platinum, etc.). The structures can be made from materials selected from the group consisting of silicon, polystyrene, agarose, dextran, cellulose polymers, polyacrylamide, polydimethylsiloxane (PDMS), and glass. The substrate / solid support or microstructure thereof, the reactor, can be made using a combination of materials listed herein or any other suitable materials known in the art.

[0131] In some cases, the substrate disclosed herein comprises a computer-readable material. Computer-readable materials include, without limitation, magnetic media, reel-to-reel tape, cartridge tape, cassette tape, flexible disk, paper media, film, microfiche, continuous tape (e.g., belt), and any medium suitable for storing electronic instructions. In some cases, the substrate comprises a magnetic reel-to-reel tape or a magnetic belt. In some cases, the substrate comprises a flexible printed circuit board.

[0132] The structures described herein may be transparent to visible and / or UV light. In some cases, the structures described herein are sufficiently conductive to form a uniform electric field across all or a portion of the structure. In some cases, the structures described herein are thermally conductive or thermally insulating. In some cases, the structures are chemically and thermally resistant to support chemical reactions, e.g., polynucleotide synthesis reactions. In some cases, the substrate is magnetic. In some cases, the structures include a metal or metal alloy. The structures described herein may be integrated into the racks described herein, e.g., rack units in rack servers.

[0133] Structures for polynucleotide synthesis may be 1, 2, 5, 10, 30, 50 feet or more in length in any dimension. In the case of flexible structures, the flexible structures are optionally stored wound, for example, on a reel. In the case of large rigid structures, for example, over a foot in length, the rigid structures may be stored vertically or horizontally.

[0134] surface preparation

[0135] A method for supporting the immobilization of biomolecules on a substrate is provided herein, in which the surface of the structure described herein comprises and / or is coated with a material that promotes a coupling reaction with biomolecules for binding. To prepare the structure for immobilization of biomolecules, surface modification can be utilized to chemically and / or physically modify the substrate surface by additive or subtractive processes to change one or more chemical and / or physical properties of the substrate surface or selected sites or regions of the surface. For example, surface modification includes one or more of: (1) changing the wettability of the surface; (2) functionalizing the surface, e.g., providing, modifying, or replacing surface functional groups; (3) defunctionalizing the surface, e.g., removing surface functional groups; (4) changing the chemical composition of the surface by other methods, e.g., etching; (5) increasing or decreasing the roughness of the surface; (6) providing a coating on the surface, e.g., a coating that exhibits a wettability different from the wettability of the surface; and / or (7) depositing particulates on the surface. In some cases, the surface of a structure is selectively functionalized to provide two or more distinct areas on the structure, where at least one area has different surface or chemical properties than another area of ​​the same structure, such properties including, without limitation, surface energy, chemical termination, surface concentration of chemical moieties, etc.

[0136] In some cases, the surface of the structures disclosed herein is modified to include one or more actively functionalized surfaces configured to bind both the surface of the substrate and the biomolecule, thereby supporting the coupling reaction to the surface. In some cases, the surface is also functionalized with a passive material that does not efficiently bind to the biomolecule, thereby preventing the binding of the biomolecule at the site where the passive functionalization agent is attached. In some cases, the surface includes an active layer that only defines separate locations for the support of the biomolecule.

[0137] In some cases, the surface is contacted with a mixture of functional groups in any different ratio. In some cases, the mixture includes at least 2, 3, 4, 5, or more different types of functionalizing agents. In some cases, the ratio of the at least two types of surface functionalizing agents in the mixture is about 1:1, 1:2, 1:5, 1:10, 2:10, 3:10, 4:10, 5:10, 6:10, 7:10, 8:10, 9:10, or any other ratio that achieves the desired surface appearance of the two groups. In some cases, by providing a suitable ratio of functionalizing agents to the substrate surface, the desired surface tension, wettability, water contact angle, and / or other suitable solvent contact angle is achieved. In some cases, the agents in the mixture are selected from suitable reactive and inactive moieties, and thus the surface density of reactive groups is diluted to a desired level for downstream reactions. In some cases, the mixture of functionalizing reagents includes one or more reagents that bind to biomolecules and one or more reagents that do not bind to biomolecules. Thus, by tailoring the reagents it is possible to control the amount of biomolecule binding that occurs at the distinct functionalized areas.

[0138] In some cases, the method for functionalization of a substrate includes deposition of a silane molecule on the surface of the substrate. The silane molecule may be deposited on a high energy surface of the substrate. In some cases, the high surface energy region includes a passive functionalization reagent. The methods described herein provide a silane group that binds to the surface, and the remainder of the molecule provides a distance from the surface and a terminal free hydroxyl group to which a biomolecule is attached. In some cases, the silane is an organofunctional alkoxysilane molecule. Non-limiting examples of organofunctional alkoxysilane molecules include dimethylchloro-octodecyl-silane, methyldichloro-octodecyl-silane, trichloro-octodecyl-silane, trimethyl-octodecyl-silane, triethyl-octodecyl-silane. 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 comprises 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 active functionalization agent comprises 11-acetoxyundecyltriethoxysilane. In some cases, the active functionalization agent comprises n-decyltriethoxysilane. In some cases, the active functionalizing agent includes glycidyloxypropyltriethoxysilane (GOPS). In some cases, the silane is a fluorosilane. In some cases, the silane is a hydrocarbon silane. In some cases, the silane is 3-iodopropyltrimethoxysilane.In some cases, the silane is octylchlorosilane.

[0139] In some cases, silanization is carried out on surfaces through self-assembly with organofunctional alkoxysilane molecules, which are classified according to their organofunctional group. Non-limiting examples of siloxane functionalizing reagents include hydroxyalkylsiloxanes (surface silylated, functionalized with diborane, and alcohols oxidized with hydrogen peroxide), diol (dihydroxyalkyl)siloxanes (surface silylated and hydrolyzed to diols), aminoalkylsiloxanes (amines that do not require an intermediate functionalization step), glycidoxysilanes (3-glycidoxypropyl-dimethyl-ethoxysilane, glycidoxy-trimethoxysilane), mercaptosilanes (3-mercaptopropyl-trimethoxysilane, 3-4 epoxycyclohexyl-ethyltrimethoxysilane, or 3-mercaptopropyl-methyl-dimethoxysilane), bicycloheptenyl-trichlorosilane, butyl-aldehyde-trimethoxysilane, or dimeric secondary aminoalkylsiloxanes. Exemplary hydroxyalkylsiloxanes include allyltrichlorosilane, which is converted to 3-hydroxypropyl, or 7-oct-1-enyltrichlorosilane, which is converted to 8-hydroxyoctyl. Diol(dihydroxyalkyl)siloxanes include (2,3-dihydroxypropyloxy)propyl (GOPS) from glycidyltrimethoxysilane. Aminoalkylsiloxanes include 3-aminopropyltrimethoxysilane, which is converted to 3-aminopropyl (3-aminopropyl-triethoxysilane, 3-aminopropyl-diethoxy-methylsilane, 3-aminopropyl-dimethyl-ethoxysilane, or 3-aminopropyl-trimethoxysilane). In some cases, the dimeric secondary aminoalkylsiloxane is bis(3-trimethoxysilylpropyl)amine, which is converted to bis(silyloxypropyl)amine.

[0140] The active functionalized area may include one or more different types of silanes, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more types of silanes. In some cases, one of the one or more types of silanes is present in the functionalized composition in a greater amount than another silane. For example, a mixed silane solution having two types of silanes has a ratio of one silane to the other silane of 99:1, 98:2, 97:3, 96:4, 95:5, 94:6, 93:7, 92:8, 91:9, 90:10, 89:11, 88:12, 87:13, 86:14, 85:15, 84:16, 83:17, 82:18, 81:19, 80:20, 75:25, 70:30, 65:35, 60:40, 55:45. In some cases, the active functionalizing agent includes 11-acetoxyundecyltriethoxysilane and n-decyltriethoxysilane. In some cases, the active functionalizing agent includes 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.

[0141] In some cases, the functionalization includes depositing a functionalizing agent onto the structure by any deposition technique, including, but not limited to, chemical vapor deposition (CVD), atomic layer deposition (ALD), plasma enhanced CVD (PECVD), plasma enhanced ALD (PEALD), metal organic CVD (MOCVD), hot wire CVD (HWCVD), initiated CVD (iCVD), modified CVD (MCVD), vapor axial deposition (VAD), outside vapor deposition (OVD), physical vapor deposition (e.g., sputter deposition, evaporative deposition), and molecular layer deposition (MLD).

[0142] Any step or component in the following functionalization process may be omitted or modified depending on the properties desired for the final functionalized substrate. In some cases, additional components and / or process steps are added to the process workflow embodied herein. In some cases, the substrate is first cleaned, for example, using piranha solution. An example of a cleaning process includes immersing the substrate in piranha solution (e.g., 90% H2SO4, 10% H2O2) at high temperature (e.g., 120°C) and rinsing (e.g., water) and drying (e.g., nitrogen gas) the substrate. This process optionally includes a piranha post-treatment that includes immersing the piranha-treated substrate in a basic solution (e.g., NH4OH) followed by an aqueous rinse (e.g., water). In some cases, the surface of the structure is optionally plasma cleaned after the piranha immersion and optional piranha post-treatment. An example of a plasma cleaning process includes oxygen plasma etching. In some cases, an active functionalizing agent is deposited on the surface, followed by evaporation. In some cases, the substrate is actively functionalized prior to cleaning, for example, by piranha treatment and / or plasma cleaning.

[0143] The process for surface functionalization optionally includes resist coating and resist stripping. In some cases, after active surface functionalization, the substrate is spin-coated with resist, e.g., SPR™ 3612 positive photoresist. The process for surface functionalization includes lithography with patterned functionalization in various cases. In some cases, photolithography is performed after resist coating. In some cases, after lithography, the surface is visually inspected for lithographic defects. The process for surface functionalization includes a cleaning step in some cases, whereby substrate residues are removed, for example, by plasma cleaning or etching. In some cases, the plasma cleaning step is performed at any step after the lithography step.

[0144] In some cases, the resist-coated surface is treated to remove the resist, for example, after functionalization and / or after lithography. In some cases, the resist is removed with a stripping solution that includes a solvent, for example, N-methyl-2-pyrrolidone. In some cases, resist stripping includes sonication or ultrasonic treatment. In some cases, the resist is coated and stripped, followed by active functionalization of the exposed areas to create the desired differential functionalization pattern.

[0145] In some cases, the methods and compositions described herein relate to the application of photoresist for the production of modified surface properties in selective areas, where the application of photoresist relies on surface fluid properties that define the spatial distribution of the photoresist. Without being bound by theory, the flow of the photoresist may be defined by surface tension effects associated with the applied fluid. For example, surface tension and / or capillary action effects may facilitate drawing the photoresist into small features in a controlled manner before the resist solvent evaporates. In some cases, the resist contacts are pinned by sharp edges, thereby controlling the advancement of the fluid. The underlying structure may be designed based on the desired flow pattern used to apply the photoresist during the fabrication and functionalization process. The solid organic layer that remains after the solvent evaporates may be used to continue the subsequent steps of the fabrication process. The structure may be designed to control the flow of the fluid by promoting or suppressing the wicking effect into adjacent fluid paths. For example, the structure may be designed to avoid overlap between the top and bottom ends, which facilitates the retention of fluid within the upper structure and allows for specific placement of the resist. In an alternative example, the top and bottom edges overlap, resulting in wicking of the applied fluid to the bottom structure. Depending on the desired application of the resist, an appropriate design may be selected accordingly.

[0146] In some cases, the structure described herein has a surface that comprises a material having a thickness of at least 0.1 nm, 0.5 nm, 1 nm, 2 nm, 5 nm, 10 nm, or 25 nm, which comprises a reactive group that can be bound to nucleosides. Examples include, without limitation, glass and silicon, such as silicon dioxide and silicon nitride. In some cases, exemplary surfaces include nylon and PMMA.

[0147] In some cases, electromagnetic radiation in the form of UV light is used for surface patterning. In some cases, a lamp is used for surface patterning, and a mask controls the location of the UV light exposure on the surface. In some cases, a laser is used for surface patterning, and the opening and closing of a shutter controls the exposure of the UV light to the surface. The laser arrangement can be used in combination with a flexible structure that can move. In such an arrangement, the coordination of laser exposure and the movement of the flexible structure is used to create a pattern of one or more agents with different nucleoside coupling capabilities.

[0148] A reusable surface for polynucleotide synthesis is described herein. After polynucleotide synthesis and / or cleavage, the surface can be soaked, washed, cleaned, baked, etched, or otherwise functionally restored to a state suitable for subsequent polynucleotide synthesis. The number of times the surface is reused and the method of recycling / preparing the surface for reuse vary depending on the subsequent application. In some cases, the surface prepared for reuse is reused about 1, 2, 3, 5, 10, 20, 50, 100, 1,000 times or more. In some cases, the surface prepared for reuse is reused at least 1, 2, 3, 5, 10, 20, 50, 100, 1,000 times or more. In some cases, the remaining "lifetime", i.e., the number of times the surface is suitable for reuse, is measured or predicted.

[0149] Material Deposition Systems

[0150] In some cases, the synthesized polynucleotides are stored on a substrate, such as a solid support. The nucleic acid reagents can be deposited on the substrate surface in a non-continuous or drop-on-demand manner. Examples of such methods include electromechanical transport, electrothermal transport, and electrostatic attraction. In electromechanical transport, droplets are ejected by a piezoelectric element deformed by an electric pulse. In electrothermal transport, bubbles are generated in the chamber of the device, and droplets are ejected by the expansion force of the bubbles. In electrostatic attraction, droplets are ejected onto the substrate using electrostatic attraction. In some cases, the drop frequency is about 5KHz to about 500KHz, about 5KHz to about 100KHz, about 10KHz to about 500KHz, about 10KHz to about 100KHz, or about 50KHz to about 500KHz. In some cases, the frequency is less than about 500KHz, less than 200KHz, less than 100KHz, or less than 50KHz.

[0151] The size of the dispensed droplets correlates with the resolution of the device. In some cases, the device deposits droplets of reagent in sizes of about 0.01 pl to about 20 pl, about 0.01 pl to about 10 pl, about 0.01 pl to about 1 pl, about 0.01 pl to about 0.5 pl, about 0.01 pl to about 0.01 pl, or about 0.05 pl to about 1 pl. In some cases, the droplet size is less than about 1 pl, less than 0.5 pl, less than 0.2 pl, less than 0.1 pl, or less than 0.05 pl.

[0152] In some arrangements, the configuration of the polynucleotide synthesis system allows for a continuous polynucleotide synthesis process that utilizes the flexibility of the substrate for movement in a reel-to-reel type process. The synthesis process operates in a continuous production line manner, where the substrate passes through various stages of polynucleotide synthesis by rotating the position of the substrate using one or more reels. In an exemplary case, the polynucleotide synthesis reaction includes advancing the substrate through a solvent bath, under a deposition device for phosphoramidite deposition, through an oxidizer bath, through an acetonitrile wash bath, and through a deblocking bath. Optionally, the tape moves through a capping bath. The reel-to-reel type process allows the final product of the substrate, including the synthesized polynucleotide, to be easily collected on a take-up reel, where it can be sent for further processing or storage.

[0153] In some arrangements, polynucleotide synthesis proceeds in a continuous process as a continuous flexible tape is transported along a conveyor belt system. Similar to reel-to-reel type processes, polynucleotide synthesis on continuous tape operates in a production line manner, with the substrate passing through various stages of polynucleotide synthesis during transport. However, in conveyor belt processes, the continuous tape returns to the polynucleotide synthesis step without winding and unwinding the tape as in reel-to-reel processes. In some arrangements, the polynucleotide synthesis step is divided into zones, and the continuous tape is transported through each zone one or more times in one cycle. For example, a polynucleotide synthesis reaction may include (1) passing a substrate through a solvent bath, transporting it under a deposition device for phosphoramidite deposition, through an oxidizer bath, through an acetonitrile wash bath, and through a block bath in one cycle, and then (2) repeating this cycle to achieve the synthesis of a polynucleotide of a predetermined length. After polynucleotide synthesis, the flexible substrate is removed from the conveyor belt system and optionally wound up for storage. The winding may be wound onto a reel for storage. In some cases, a flexible substrate comprising a thermoplastic material is coated with a nucleoside coupling reagent. The coating is patterned into locations such that each location has a diameter of about 10 um and the center-to-center distance between two adjacent locations is about 21 μm. In this case, the location size is sufficient to accommodate a 0.2 pl sessile drop volume during the polynucleotide synthesis deposition step. In some cases, the density of locations is greater than 1 m. 2 Approximately 2.2 billion locations per location (1 location / 441 x 10 -12 m 2 ) In some cases, 4.5m 2 The substrate contains approximately 10 billion loci, each with a diameter of 10 μm.

[0154] In some arrangements, polynucleotide synthesis proceeds on a rack unit, for example, as described herein. In some cases, one or more fluids containing reagents for polynucleotide synthesis are delivered to at least one rack unit through one or more tubes. In some cases, a rack unit containing at least one solid support is continuously exposed to reagents to achieve polynucleotide synthesis. In some cases, a UV spectrometer or other suitable device is used to measure the concentration of polynucleotides on the solid support. In some cases, reagents are delivered as gas using a vacuum (e.g., to cleave polynucleotides on the solid support). The rack unit may further include one or more electrodes. The one or more electrodes may be connected to an electrical system in a rack server to deliver a voltage to one or more locations on the solid support in the rack unit.

[0155] In some arrangements, the device for applying one or more reagents to the substrate during the synthesis reaction is configured to deposit reagents for nucleoside phosphoramidite-based synthesis and / or nucleoside monomers. Reagents for polynucleotide synthesis include reagents for polynucleotide extension and wash buffers. As non-limiting examples, the device deposits cleaning reagents, coupling reagents, capping reagents, oxidizing agents, deblocking agents, gases such as acetonitrile, nitrogen gas, and any combination thereof. In addition, the device optionally deposits reagents for preparation and / or maintenance of substrate integrity. In some cases, the polynucleotide synthesis apparatus deposits droplets having a diameter of less than about 200 μm, less than 100 μm, or less than 50 μm, with a volume of less than about 1000 pl, less than 500 pl, less than 100 pl, less than 50 pl, or less than 20 pl. In some cases, the polynucleotide synthesizer deposits about 1-10,000, 1-5,000, 100-5,000, or 1,000-5,000 droplets per second.

[0156] Described herein are devices, methods, systems, and compositions in which reagents for polynucleotide synthesis are recycled or reused. Recycling of reagents can include collection, storage, and use of unused reagents, or purification / conversion of used reagents. For example, a reagent bath is recycled and used for a polynucleotide synthesis step on the same or a different surface. Reagents described herein can be recycled 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times. Alternatively, or in combination, a reagent solution containing reaction by-products is filtered to remove the by-products, and the reagent solution is used for additional polynucleotide synthesis reactions.

[0157] Many integrated or non-integrated elements are often used in polynucleotide synthesis systems. In some cases, the polynucleotide synthesis system includes one or more elements useful for downstream processing of the synthesized polynucleotide. As an example, the system includes a temperature control element, such as a thermal cycling device. In some cases, the temperature control element is used with multiple disassembled reactors to perform nucleic acid assembly, such as PCA, and / or nucleic acid amplification, such as PCR. In some cases, the synthesized polynucleotide is transferred to an amplification unit, a storage unit, a sequencing unit, or any combination thereof. In some cases, the synthesized polynucleotide may be transferred using a robotic system (e.g., a robotic arm or picker). In some cases, the synthesized polynucleotide may be transferred onto a solid support as described herein. In some cases, the synthesized polynucleotide may not be transferred onto a solid support as described herein. For example, the synthesized polynucleotide may be transferred in a capsule, a tube, or any other suitable structure. The synthesized polynucleotide may be transferred in a liquid, gas, or solid state.

[0158] High-density synthesis device

[0159] Provided herein are devices, methods, compositions, and systems for biomolecule synthesis on solid supports. In some cases, electrochemistry is used to control the synthesis of biomolecules through steps such as deprotection, coupling, or cleavage. Further provided herein are devices, methods, compositions, and systems for storage and synthesis of nucleic acids on solid supports. In some cases, the solid support comprises a surface. In some cases, the surface comprises one or more features. In some cases, the features comprise one or more locations for biomolecule synthesis. Further provided herein are multiple devices that are combined to form a larger array or chip. Further provided herein are devices that comprise one or more addressable solid supports. Further provided herein are devices and methods configured for electrochemical deprotection or deblocking during biomolecules (e.g., polynucleotide synthesis). Further provided herein are devices that comprise one or more addressable solid supports for polynucleotide synthesis. In some cases, a voltage is applied to the device to perform one or more steps for polynucleotide synthesis, such as electrophoretic applications. Such devices, in some cases, include "active" or "inactive" states, each state including one or more voltage states and / or one or more resistance states (e.g., "on", "off", or "disconnected"). The states of the devices can be controlled using a computer system provided herein. The computer system may be integrated into a data storage system provided herein. The device or array of addressable supports, in some cases, provides addressable control of high density nucleic acid synthesis and / or storage. Additionally, devices are provided herein that include an in-plane cathode that reduces migration of reagents to nearby or adjacent devices (e.g., diffusion control).

[0160] Devices for polynucleotide synthesis are provided herein (e.g., FIG. 1). Such devices include a solid support 100 that, in some cases, includes a plurality of features 106 for polynucleotide synthesis. Such devices may include conductive elements or electrodes 102. Such electrodes may function as anodes or cathodes. The polynucleotide 104 includes a protecting or blocking group 105 attached to the terminal base during at least one synthesis cycle. Applying a voltage through the electrodes 102 during the synthesis step 107 produces, in some cases, reagents that can be used to process the polynucleotide 104. The length of time the voltage is applied, the number of times it is applied, and other variables have a large effect on the extent of the desired reaction and the reduction of undesired side reactions. The geometry of the device surface and electrodes may also affect the efficiency of the chemical reaction.

[0161] Provided herein is a device for polynucleotide synthesis comprising layers of material. Such devices may comprise any number of layers of material, including conductive, semiconductive, or insulating materials. A conventional device 200 comprises a base layer 201, conductive materials 202a / 202b, 205 (one or more conductive layers configured for use as electrodes, which may be embedded in the base layer, such as 202a, or may be on top of the base layer, such as 202b), and a porous growth layer surface 203 (FIG. 2). In some cases, conductive layer 202a is in electrical contact with layer 202b. Each of such layers may be individually patterned to generate features for polynucleotide synthesis, such as pores, holes, wells, channels, or other shapes (e.g., FIG. 7B and FIG. 7C). Various layers of such devices are combined in some cases to form an addressable solid support. The layers or surfaces of such devices may be in fluid communication with solvents, solutes, or other reagents used during polynucleotide synthesis.

[0162] Further described herein are devices with multiple surfaces. In some cases, the surfaces include features for polynucleotide synthesis in proximity to the conductive material. In some cases, the devices described herein include 1, 2, 5, 10, 50, 100, or even thousands of surfaces per device. In some cases, a voltage is applied to one or more layers of the devices described herein to facilitate polynucleotide synthesis. In some cases, a voltage is applied to one or more layers of the devices described herein to facilitate a step in polynucleotide synthesis, such as deblocking. Different layers on different surfaces of different devices are often supplied with voltages at different times or different voltages. For example, a positive voltage is applied to a first layer and a negative voltage is applied to a second layer of the same or different device. In some cases, one or more layers on different devices are supplied with voltages while other layers are disconnected from ground. In some cases, the base layer includes additional circuitry, such as a complementary metal oxide semiconductor (CMOS) device. In some cases, the various layers of one or more devices are connected horizontally via routing and / or vertically using vias. In some cases, the various layers of one or more devices are connected to a CMOS layer horizontally via routing and / or vertically using vias. In some cases, the various layers of one or more devices are connected to a CMOS device through wire bonds, pogo pin contacts, or Si vias (TSVs). In some cases, an array of devices is independently addressable. In some cases, a layer or component of a device that includes a conductive material functions as a cathode or anode when a voltage is applied.

[0163] A first device 300A provided herein comprises a base layer 301 and a patterned top layer 305 (FIG. 3A). In some cases, the top layers 305 and 302b comprise a conductive material. In some cases, the device comprises a conductive layer 302a present in the base layer. In some cases, a polynucleotide synthesis surface 306 is formed on a solvent-exposed surface of the base layer 301. Such a device provides fluid communication between the polynucleotide synthesis surface 306 and the top layer 305. In some cases, the patterned top layer comprises a plurality of voids that facilitate fluid communication between the polynucleotide synthesis surface 306 and the top layer 305. In some cases, the voids comprise any size or shape, including but not limited to wells, channels, or other shapes.

[0164] A second device 300B provided herein comprises a base layer 301, a recessed shield electrode 308, and a patterned top layer 305 (FIG. 3B). In some cases, the top layers 305 and 302b comprise conductive materials. In some cases, the device comprises a conductive layer 302a present in the base layer. In some cases, the polynucleotide synthesis surface 306 is formed by pores in the top layer 305. Such a device provides fluid communication between the polynucleotide synthesis surface 306 and the top layer. In some cases, the recessed shield electrode 308 does not contact either the synthesis surface 306 or the top layer 305. In some cases, a voltage passes through the shield electrode 308 to affect the flow of ions in a solvent in contact with the synthesis surface 306. In some cases, a different voltage is applied to the shield electrode 308 compared to the voltage applied to the top layer 305. In some cases, the voltage applied to the shield electrode 308 is synchronized with an adjacent or nearby conductive layer (e.g., 302b). In some cases, the time for which the voltage is applied between the shield electrode and the proximal anode is 0.1 microseconds or less, 0.2 microseconds or less, 0.5 microseconds or less, 0.8 microseconds or less, 1.0 microseconds or less, 1.2 microseconds or less, 1.5 microseconds or less, 1.8 microseconds or less, 2 microseconds or less, 5 microseconds or less, 8 microseconds or less, 10 microseconds or less, 12 microseconds or less, 15 microseconds or less, 20 microseconds or less, 50 microseconds or less, 80 microseconds or less, or 100 microseconds or less. In some cases, the time for which the voltage is applied between the shield electrode and the proximal anode is about 0.1 microseconds, 0.2, 0.5, 0.8, 1.0, 1.2, 1.5, 1.8, 2, 5, 8, 10, 12, 15, 20, 50, 80, or about 100 microseconds. In some cases, the time that the voltage is applied between the shield electrode and the proximal anode is 0.1-1, 0.1-5, 0.1-10, 0.1-100, 0.5-10, 0.5-100, 1-10, 1-50, 1-100, 5-50, 10-100, or 50-100 microseconds.

[0165] A third device 400 provided herein comprises a base layer 401, an intermediate layer 405, and a top layer 406 (FIG. 4). In some cases, the intermediate layer 405 and layer 402a comprise a conductive material. In some cases, the top layer comprises a polynucleotide synthesis surface 406. Such a device provides fluid communication between the polynucleotide synthesis surface 406 and the intermediate layer 405. The polynucleotide synthesis surface 406 is patterned, in some cases, as a cylinder, a substantially rectangular shape, a channel, or other shape. In some cases, the polynucleotide synthesis surface 406 is randomly distributed. In some cases, the intermediate layer 405 comprises a thermal oxide. The device comprises one or more additional bonding layers between the synthesis surface 406 and the bottom layer 401 in some cases. In some cases, the intermediate layer is 1-100, 1-50, 1-25, 1-10, 1-5, 2-25, 2-50, 5-50, 5-25, 5-75, 10-100, 10-50, or 50-100 nm thick. In some cases, the intermediate layer is 1 nm or less, 2 nm or less, 5 nm or less, 10 nm or less, 15 nm or less, 20 nm or less, 25 nm or less, 30 nm or less, 50 nm or less, 75 nm or less, 100 nm or less, or 150 nm or less thick. In some cases, the intermediate layer is about 1, 2, 5, 10, 15, 20, 25, 30, 50, 75, 100, or about 150 nm thick. In some cases, the top layer is 1-100, 1-50, 1-25, 1-10, 1-5, 2-25, 2-50, 5-50, 5-25, 5-75, 10-100, 10-50, or 50-100 nm thick. In some cases, the top layer is 1 nm or less, 2 nm or less, 5 nm or less, 10 nm or less, 15 nm or less, 20 nm or less, 25 nm or less, 30 nm or less, 50 nm or less, 75 nm or less, 100 nm or less, or 150 nm or less thick. In some cases, the top layer is about 1, 2, 5, 10, 15, 20, 25, 30, 50, 75, 100, or about 150 nm thick.

[0166] A fourth device 500A provided herein comprises a base layer 501, a first intermediate layer 511, and a top layer 505 (FIG. 5A). In some cases, the first intermediate layer comprises a polynucleotide synthesis surface 506. In some cases, the smallest feature dimension is 512. In some cases, the device comprises one or more of a) a base layer comprising silicon, b) an intermediate layer comprising an oxide, and c) a top layer comprising a conductive material. In some cases, the top layer is configured to produce an electrochemically generated reagent when a voltage is applied. In some cases, the intermediate layer and the top layer are in fluid communication with a solvent. In some cases, the intermediate layer is positioned between the base layer and the top layer. In some cases, the intermediate layer is configured for binding of molecules. In some cases, the solid support comprises a plurality of features. In some cases, the top layer comprises a plurality of voids configured to allow fluid communication between a solvent and the intermediate layer. In some cases, at least some of the voids are centered above one or more features. In some cases, the void comprises a well or channel. In some cases, the well or channel is 1-5, 1-10, 1-15, 1-20, 1-25, 1-50, 1-75, 1-100, 1-150, 1-200, or 1-500 nm deep. In some cases, the well or channel is 1 nm or less, 2 nm or less, 3 nm or less, 5 nm or less, 10 nm or less, 15 nm or less, 20 nm or less, 25 nm or less, 30 nm or less, 40 nm or less, 50 nm or less, 75 nm or less, 100 nm or less, 150 nm or less, 200 nm or less, or 500 nm or less deep. In some cases, a plurality of features are located on the intermediate layer. In some cases, the smallest feature dimension is proportional to the diffusion distance of a reagent generated proximate the conductive layer. In some cases, the intermediate layer has a thickness of 1-100, 1-50, 1-25, 1-10, 1-5, 2-25, 2-50, 5-50, 5-25, 5-75, 10-100, 10-50, or 50-100 nm.In some cases, the intermediate layer is 1 nm or less, 2 nm or less, 5 nm or less, 10 nm or less, 15 nm or less, 20 nm or less, 25 nm or less, 30 nm or less, 50 nm or less, 75 nm or less, 100 nm or less, or 150 nm or less in thickness. In some cases, the intermediate layer is about 1, 2, 5, 10, 15, 20, 25, 30, 50, 75, 100, or about 150 nm in thickness. In some cases, the top layer is 1-100, 1-50, 1-25, 1-10, 1-5, 2-25, 2-50, 5-50, 5-25, 5-75, 10-100, 10-50, or 50-100 nm in thickness. In some cases, the top layer is 1 nm or less, 2 nm or less, 5 nm or less, 10 nm or less, 15 nm or less, 20 nm or less, 25 nm or less, 30 nm or less, 50 nm or less, 75 nm or less, 100 nm or less, or 150 nm or less in thickness. In some cases, the top layer is about 1, 2, 5, 10, 15, 20, 25, 30, 50, 75, 100, or about 150 nm in thickness. In some cases, the device comprises one or more conductive layers configured for use as a cathode. In some cases, the device comprises one or more in-plane cathodes.

[0167] A fifth device 500B provided herein comprises a base layer 501, a first intermediate layer 511, a second intermediate layer 505, and a top layer 506 (FIG. 5B). In some cases, polynucleotides are synthesized on the top layer 506. The polynucleotide synthesis surface 506 is patterned in some cases as a cylinder, a substantially rectangular shape, a channel, or other shape. In some cases, the polynucleotide synthesis surface 506 is randomly patterned. In some cases, the smallest feature dimension is 512. In some cases, the device comprises additional binding layers 515 and 516, as shown in device 500C (FIG. 5C). In some cases, the smallest feature dimension is proportional to the diffusion distance of the reagents generated proximal to the conductive layer.

[0168] In some cases, device 600A includes a conductive layer 611 configured for use as a cathode above a surface of one or more conductive layers 605 / 602b (attached to lower conductive layer 602a) configured for use as an anode (FIG. 6A). In some cases, the anode is in fluid communication with one or more locations for polynucleotide synthesis 606. In another configuration, as in device 600B of FIG. 6B, conductive layer 611 is configured for use as a cathode and positioned on the same surface as one or more conductive layers 605 / 602b (attached to lower conductive layer 602a) configured for use as an anode. In some cases, the device includes one or more of a) a base layer including silicon, b) a middle layer including a conductive material (the middle layer is configured to produce an electrochemically generated reagent when a voltage is applied), and c) a top layer including an oxide. In some cases, the middle layer and the top layer are in fluid communication with a solvent. In some cases, the middle layer is positioned between the base layer and the top layer. In some cases, the top layer is configured for molecular attachment. In some cases, the solid support includes multiple features. In some cases, an in-plane cathode in close proximity to the active anode layer reduces excess migration of electrochemically generated reagents into adjacent devices.

[0169] A sixth device described herein comprises multiple device arrays (or addressable solid supports), as shown in Figures 7A-7E. Although Figure 7A shows two such device arrays for clarity, such devices may comprise any number of device arrays. Nine such device arrays are shown in Figure 7B with routing connections that allow for individual or group addressable control of the device arrays. In some cases, device array 1 is individually addressable from device arrays 2 and 3. Although four such device arrays are shown in Figure 7C for clarity only, any number of devices may be arranged in this manner. Four devices are addressable in groups 1, 2, and 4, as shown for clarity only, although in some cases the number of addressable groups will be equal to or less than the total number of device arrays. The conductive layer 802 in some cases generates a reagent (e.g., acid) for electrochemical deprotection of biomolecules, e.g., polynucleotides. In some cases, 802 is configured for use as an anode. In some cases, the conductive layer is also configured for use as a cathode, as shown in FIG. 7C. A cross-sectional view of the device of FIG. 7C or 8E is shown in FIG. 7D. In some cases, polynucleotides are synthesized on the oxide layer 805. In some cases, polynucleotides are synthesized on the conductive layer 802. Such devices are addressable in some cases by a first routing layer 801a and a second routing layer 801b. Such devices may include any number of routing layers, for example, 1, 2, 3, 4, 5, 10, 20, 50, 100, or more than 100 routing layers. Routing in different horizontal planes is connected in some cases by one or more vertical interconnect accesses (VIAs) 803 and 804. Such devices may include any number, for example, 1, 2, 3, 4, 5, 10, 20, 50, 100, 1000, or more than 1000 vias per square micron.The number and size of the routing and vias are in some cases proportional to the number of addressable solid supports on the device. A device (similar to FIG. 7C) with a 16 device array is shown in FIG. 7E. Routing 801b is in the upper layer relative to routing 801a in the device. The 16 device array of FIG. 7E is in some cases addressable in groups of seven, although other configurations are also consistent with the devices and methods described herein. In some cases, the device is integrated with additional circuitry, e.g., CMOS (FIG. 8). In some cases, the location and surrounding anode are circular, rectangular, square, or other shapes. In some cases, the device is fabricated with the following approximate dimensions: p=device pitch; d=device size=2 / 5p; s=oxide growth feature=1 / 5p; n=cathode neck=1 / 5p; g=device-cathode gap=1 / 5p; and t=platinum thickness=1 / 50p. In some cases, the device pitch is 5 nm or less, 10 nm or less, 15 nm or less, 20 nm or less, 25 nm or less, 30 nm or less, 35 nm or less, 40 nm or less, 45 nm or less, 50 nm or less, 60 nm or less, 75 nm or less, 85 nm or less, 100 nm or less, 125 nm or less, 150 nm or less, 175 nm or less, 200 nm or less, 250 nm or less, 500 nm or less, or 1000 nm or less. In some cases, the device pitch is about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 75, 85, 100, 125, 150, 175, 200, 250, 500, or about 1000 nm. In some cases, the device pitch is 5-500, 5-100, 10-200, 10-500, 25-500, 50-500, 100-500, 100-1000, 250-1000, 500-1000, or 100-1000 nm. In some cases, the minimum distance between the cathode and anode (g, the gap between the device and the cathode) is 5-50%, 5-25%, 5-30%, 10-30%, 10-50%, 10-20%, 20-50%, or 30-75% of the pitch distance.In some cases, the minimum width (n) of the cathode is 5-50%, 5-25%, 5-30%, 10-30%, 10-50%, 10-20%, 20-50%, or 30-75% of the pitch distance. In some cases, the minimum width (n) of the cathode is 5-50%, 5-25%, 5-30%, 10-30%, 10-50%, 10-20%, 20-50%, or 30-75% of the pitch distance. In some cases, the maximum dimension (d, linear distance or diameter) of the anode is 5-50%, 5-25%, 5-30%, 10-30%, 10-50%, 10-20%, 20-50%, or 30-75% of the pitch distance. In some cases, the via closest to the anode has a maximum dimension (linear distance or diameter) that is 5-50%, 5-25%, 5-30%, 10-30%, 10-50%, 10-20%, 20-50%, or 30-75% of the pitch distance. In some cases, the feature size (s, linear distance or diameter) is 5-50%, 5-25%, 5-30%, 10-30%, 10-50%, 10-20%, 20-50%, or 30-75% of the pitch distance. In some cases, the thickness of one or more of the anode or cathode is 0.55-5% or less, 0.5-2.5% or less, 0.5-3% or less, 1-3% or less, 1-5% or less, 1-2% or less, 2-5% or less, or 3-7.5% or less of the pitch distance.

[0170] The device may include any number of device arrays. In some cases, the device includes at least 10, 50, 100, 1000, 10,000, 100,000, or more than 100,000 device arrays in a single device. In some cases, the device includes about 10, 50, 100, 1000, 10,000, 100,000, or about 100,000 device arrays in a single device. In some cases, the device includes 10-50, 10-5000, 10-10,000, 100-1000, 100-10,000, 1000-100,000, 1000-10,000, or 1000-100,000 device arrays in a single device.

[0171] In some cases, the device comprises one or more base layers. In some cases, the base layer comprises a first layer comprising an oxide, a second layer comprising a carbide, and a third layer comprising a nitride. In some cases, the device comprises at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more than 75 base layers. In some cases, the device comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, or more than 30 vias. In some cases, the device comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, or more than 30 routing elements. In some cases, the first layer (of the base layer) comprises an oxide, e.g., SiO2. In some cases, the second layer (of the base layer) comprises a carbide, e.g., silicon carbide. In some cases, the third layer (of the base layer) comprises a nitride, e.g., silicon nitride. Additional materials may also be used for these layers. In some cases, the device comprises a top layer. In some cases, the top layer comprises one or more device layers and one or more in-plane cathodes. In some cases, the in-plane cathode comprises a first layer comprising an oxide, a second layer comprising a metal-doped nitride, and a third layer comprising a metal. In some cases, the first layer (of the cathode) comprises silicon oxide. In some cases, the second layer (of the cathode) comprises titanium nitride doped with chromium. In some cases, the third layer (of the cathode) comprises platinum. In some cases, the device layer comprises a first layer comprising an oxide, a second layer comprising a metal-doped nitride, a third layer comprising a metal, a fourth layer comprising a metal, and a fifth layer comprising an oxide. In some cases, the first layer (of the device layer) comprises silicon oxide. In some cases, the second layer (of the device layers) comprises chromium doped titanium nitride. In some cases, the third layer (of the device layers) comprises platinum. In some cases, the fourth layer (of the device layers) comprises ruthenium. In some cases, the fifth layer (of the device layers) comprises titanium.In some cases, the sixth layer (of the device layers) comprises silicon oxide. In some cases, polynucleotides are synthesized on the fifth layer of the device layers. Additional materials may also be used in these layers.

[0172] The device array can be scaled to any size or dimension. In some cases, the device array is about 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 0.8, 1, 2, 5, 8, or about 10 microns in width. In some cases, the device array is 0.01 microns or less, 0.02 microns or less, 0.05 microns or less, 0.1 microns or less, 0.2 microns or less, 0.5 microns or less, 0.8 microns or less, 1 micron or less, 2 microns or less, 5 microns or less, 8 microns or less, or 10 microns or less in width. In some cases, the device array is 0.01-10, 0.1-10, 0.1-1, 0.5-1, 1-10, or 5-30 microns in width. In some cases, the device arrays are spaced apart by about 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 0.8, 1, 2, 5, 8, or about 10 microns. In some cases, the device arrays are spaced apart by 0.01 microns or less, 0.02 microns or less, 0.05 microns or less, 0.1 microns or less, 0.2 microns or less, 0.5 microns or less, 0.8 microns or less, 1 micron or less, 2 microns or less, 5 microns or less, 8 microns or less, or 10 microns or less. In some cases, the device arrays are spaced apart by 0.01-10, 0.1-10, 0.1-1, 0.5-1, 1-10, or 5-30 microns.

[0173] Devices with addressable device arrays can be addressed in various patterns or configurations. In some cases, only certain groups (or clusters) of devices in the array are activated at the same time. In some cases, the device array is addressed according to Figures 13A-13G. Any number of device arrays can be activated at the same time. In some cases, about 1%, 2%, 3%, 5%, 7%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 75%, 95%, or about 100% of the device arrays in the devices described herein are activated at the same time. In some cases, 1% or less, 2% or less, 3% or less, 5% or less, 7% or less, 10% or less, 15% or less, 20% or less, 30% or less, 40% or less, 50% or less, 60% or less, 75% or less, 95% or less, or 99% or less of the device arrays in the devices described herein are activated at the same time. In some cases, at least 1%, 2%, 3%, 5%, 7%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 75%, 95%, or at least 99% of the device arrays in a device described herein are activated simultaneously. In some cases, 1-2%, 1-5%, 1-10%, 1-20%, 1-50%, 2-10%, 2-50%, 5-50%, 5-90%, 10-25%, 10-95%, or 15-95% of the device arrays in a device described herein are activated simultaneously.

[0174] The devices can be controlled (activated, deactivated, disconnected) collectively. In some cases, the devices in the array include clusters of smaller devices. In some cases, the device cluster includes at least 25, 50, 60, 70, 80, 90, 100, 125, 150, 200, or more than 500 devices in the cluster. In some cases, the devices in the cluster are independently addressable. In some cases, the device cluster includes at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 addressable devices. In some cases, the device cluster includes at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 cathodes.

[0175] The devices described herein can be manufactured using a number of methods, such as masking methods. In some cases, lift-off manufacturing methods are used. The lift-off method, in some cases, includes adding a sacrificial layer (e.g., photoresist or "PR") to a base layer coated with an oxide layer, adding a conductive layer, and removing the sacrificial layer. In some cases, dry etching methods are used. The dry etching method, in some cases, includes adding one or more layers, such as an oxide layer, a first intermediate layer (e.g., TiN or other material), a conductive layer (e.g., platinum), a second intermediate layer (e.g., TiN or other material), and a sacrificial layer (e.g., photoresist) to a base layer, partially removing the second intermediate layer to expose the conductive layer, partially removing the conductive layer to expose the first intermediate layer, partially removing the first conductive layer to expose the first intermediate layer, and partially removing the first intermediate layer to expose the oxide layer.

[0176] The device can be configured such that a minimum dimension of a plurality of features is equal to or less than the diffusion length of an electrochemically generated reagent, in some cases the diffusion length is 1000 nm or less, 750 nm or less, 500 nm or less, 400 nm or less, 300 nm or less, 250 nm or less, 225 nm or less, 200 nm or less, 150 nm or less, 125 nm or less, 100 nm or less, 75 nm or less, 50 nm or less, 25 nm or less, 10 nm or less, or 5 nm or less.

[0177] The device may be configured with one or more vias or routing components. In some cases, the vias and routing are 10 nm or less, 20 nm or less, 50 nm or less, 100 nm or less, 150 nm or less, 200 nm or less, 250 nm or less, 300 nm or less, 350 nm or less, or 500 nm or less in length. In some cases, the vias and routing are about 10, 20, 50, 100, 150, 200, 250, 300, 350, or about 500 nm in length. In some cases, the vias and routing are between 10-500, 10-350, 10-200, 10-100, 10-50, 50-500, 50-300, 50-250, 50-200, 50-100, 100-300, 100-500, 100-200, 200-500, or 300-500. In some cases, the vias comprise a conductive material. In some cases, the vias comprise a metal as described herein. In some cases, the vias comprise copper. In some cases, the vias comprise substantially copper.

[0178] assembly

[0179] Polynucleotides can be designed to collectively span large regions of a given sequence that code for information. In some cases, a ligation reaction joins synthesized polynucleotides to generate a larger polynucleotide. One example of a ligation reaction is polymerase chain assembly (PCA). In some cases, at least a portion of the polynucleotide is designed to include an additional region that is a substrate for universal primer binding. For PCA reactions, pre-synthesized polynucleotides include portions that overlap each other (e.g., 4, 20, 40, or more bases with overlapping sequences). During polymerase cycles, polynucleotides anneal to complementary fragments and are then filled in by polymerase. Thus, with each cycle, the length of the various fragments randomly increases depending on which polynucleotides find each other. Complementarity between the fragments allows the formation of a complete long double-stranded DNA. In some cases, after the PCA reaction is completed, an error correction step is performed using a mismatch repair detection enzyme to remove mismatches in the sequence. Once a larger fragment of the target sequence is generated, it can be amplified. For example, in some cases, a target sequence containing 5' and 3' terminal adapter sequences is amplified in a polymerase chain reaction (PCR) containing 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 for removal of the primers by an enzymatic reaction centered on targeting the modified bases and / or by a gap left by an enzyme that cleaves the modified base pair from the fragment. This leaves a double-stranded amplification product that is free of remnants of the adapter sequence. In this method, multiple amplification products can be generated in parallel using the same primer set to generate different fragments of double-stranded DNA.

[0180] Error correction may be performed on the synthesized polynucleotide and / or assembled product. An exemplary strategy for error correction includes site-specific mutagenesis by overlap extension PCR to correct errors, which is optionally combined with two or more rounds of cloning and sequencing. In certain cases, double-stranded nucleic acids with mismatches, bulges, small loops, chemically modified bases, and / or other heteroduplexes are selectively removed from the population of correctly synthesized nucleic acids. In some cases, error correction is performed using proteins / enzymes that recognize and bind to or next to mismatches or unpaired bases in double-stranded nucleic acids to create single-strand or double-strand breaks or initiate strand transfer translocation events. Non-limiting examples of proteins / enzymes for error correction include endonucleases (T7 endonuclease I, E. coli endonuclease V, T4 endonuclease VII, mung bean nuclease, Cell, E. coli endonuclease IV, UVDE), restriction enzymes, glycosylases, ribonucleases, mismatch repair enzymes, resolvases, helicases, ligases, mismatch-specific antibodies, and variants thereof. Examples of specific error correction enzymes include T4 endonuclease 7, T7 endonuclease 1, S1, mung bean endonuclease, MutY, MutS, MutH, MutL, cleavase, CELI, and HINF1. In some cases, the DNA mismatch binding protein MutS (Thermus aquaticus) is used to remove failed products from a population of synthesized products. In some cases, error correction is performed using a correctase enzyme. In some cases, error correction is performed using SURVEYOR endonuclease (Transgenomic), a mismatch-specific DNA endonuclease that scans heteroduplex DNA for known and unknown mutations and polymorphisms.

[0181] Nucleic acid-based information storage

[0182] Devices, compositions, systems, and methods for nucleic acid-based information (data) storage are provided herein. Biological molecules, such as DNA molecules, provide a suitable host for information storage due to their stability over time and enhanced information coding capacity, in contrast to traditional binary information coding. In the first step, a digital sequence (e.g., digital information in binary code for processing by a computer) that codes for an item of information is received. An encryption scheme is applied to convert the digital sequence from one or more symbols (e.g., binary code) to a nucleic acid sequence. The surface material for nucleic acid extension, the design of the location (e.g., placement spot) for nucleic acid extension, and the reagent for nucleic acid synthesis are selected. The surface of the structure is prepared for nucleic acid synthesis. De novo polynucleotide synthesis is then performed. The synthesized polynucleotides are stored and available in whole or in part for subsequent release. Once released, the polynucleotides are sequenced in whole or in part and subjected to decoding to convert the nucleic acid sequence back to a digital sequence. The digital sequence is then assembled to obtain an alignment that codes for the original item of information.

[0183] The nucleic acid encoding the digital information may include an error correction component. In some cases, the error correction component includes an error correction code, such as a Reed-Solomon (RS) code, an LDPC code, a polar code, or a turbo code. In some cases, the error correction code distributes the digital data to be stored across multiple polynucleotides. In some cases, distributing the data across multiple polynucleotides builds in redundancy to correct losses (e.g., losses of oligos). In some cases, the digital information can be restored in the presence of errors. In some cases, the error correction component includes a parity base. In some cases, the error correction component includes an index sequence. In some cases, the index sequence defines the location or address of the digital information encoded in the nucleic acid. In some cases, the index sequence defines the source of the digital information. The nucleic acid encoding the digital information includes overlaps with one or more nucleic acids in the same library or set in some cases. In some cases, the error correction component includes overlapping or redundant regions. In some cases, an algorithm is applied to the sequenced nucleic acid to reduce errors. In some cases, the error correction algorithm includes a consensus sequence, Hash Encoded, Decoded by Greedy Exhaustive Search (HEDGES), or other methods.

[0184] Nucleic acids encoding digital information can be stored in different media. In some cases, the nucleic acids are stored as essentially dry or lyophilized powders. In some cases, the nucleic acids are stored in a buffer. In some cases, the nucleic acids are stored on chips, wafers, or other silicon solid supports. In some cases, the nucleic acids are stored inside an organism (or a population of organisms), such as a plasmid or genome.

[0185] The solid support for nucleic acid synthesis or storage described herein has a large capacity for data storage. For example, the capacity of the solid support is at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 petabytes, or more than 1000 petabytes. In some cases, the capacity of the solid support is about 1 petabyte to about 10 petabytes, or about 1 petabyte to about 100 petabytes. In some cases, the capacity of the solid support is about 100 petabytes. In some cases, the data is stored as a packet array addressable as droplets. In some cases, the data is stored as a packet array addressable as droplets on spots. In some cases, the data is stored as a packet array addressable as dry wells. In some cases, the addressable array contains at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 200 terabytes, or more than 200 terabytes of data. In some cases, the addressable array contains at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 200 gigabytes, or more than 200 gigabytes of data. In some cases, the addressable array contains at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 200 terabytes, or more than 200 terabytes of data. In some cases, the item of information is stored in the background of the data. For example, the item of information encodes about 10 to about 100 megabytes of data and is stored in the background of 1 petabyte of data.In some cases, an item of information encodes at least or greater than about 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, or 500 megabytes of data and is stored with greater than 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, or 500 petabytes of background data. In some cases, storage capacity is based on device pitch (FIG. 26), with smaller device pitches allowing for more storage.

[0186] Information item

[0187] Optionally, an initial step of the data storage process disclosed herein includes obtaining or receiving one or more items of information in the form of an initial code. Items of information include, without limitation, text, audio, and visual information. Exemplary sources of items of information include, without limitation, books, periodicals, electronic databases, medical records, letters, forms, voice recordings, animal recordings, biological profiles, broadcasts, movies, short videos, emails, bookkeeping phone logs, Internet activity logs, drawings, paintings, printouts, photographs, pixelated graphics, and software code. Exemplary sources of biological profiles of items of information include, without limitation, gene libraries, genomes, gene expression data, and protein activity data. Exemplary formats of items of information include, without limitation, .txt, .PDF, .doc, .docx, .ppt, .pptx, .xls, .xlsx, .rtf, .jpg, .gif, .psd, .bmp, .tiff, .png, and .mpeg. The size of an individual file encoding an item of information in digital form, or the amount of files encoding an item of information, can include, without limitation, up to 1024 bytes (equivalent to 1 KB), 1024 KB (equivalent to 1 MB), 1024 MB (equivalent to 1 GB), 1024 GB (equivalent to 1 TB), 1024 TB (equivalent to 1 PB), 1 exabyte, 1 zettabyte, 1 yottabyte, 1 xenottabyte, or more. In some cases, the amount of digital information is at least 1 gigabyte (GB). In some cases, the amount of digital information is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 gigabytes, or more than 1000 gigabytes. In some cases, the amount of digital information is at least 1 terabyte (TB). In some cases, the amount of digital information is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 terabytes, or greater than 1000 terabytes.In some cases, the amount of digital information is at least 1 petabyte (PB). In some cases, the amount of digital information is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 petabytes, or more than 1000 petabytes. In some cases, the digital information does not include genomic data obtained from an organism. The item of information is encoded in some cases. Non-limiting examples of coding methods include 1 bit / base, 2 bits / base, 4 bits / base, or other encoding methods.

[0188] Sequencing

[0189] After extracting and / or amplifying polynucleotides from the surface of the structure, the polynucleotides may be sequenced using a suitable sequencing technology. In some cases, DNA sequences are read on the substrate or within the features of the structure. In some cases, the polynucleotides stored on the substrate are extracted, optionally assembled into longer nucleic acids, and then sequenced. In some cases, the polynucleotides stored on the substrate are not extracted and assembled into longer nucleic acids. In some cases, the sequence of the polynucleotides of the sequence can be assembled in silico.

[0190] The polynucleotides synthesized and stored on the structures described herein encode data that can be decoded by reading the sequence of the synthesized polynucleotide and converting the sequence into a set of computer-readable symbols (e.g., binary code). In some cases, the sequence requires assembly, and the assembly step may need to be at the nucleic acid sequence stage or the digital sequence stage. Assembly may be accomplished using one or more indexes of the sequence. One or more indexes may be used to group or align one or more sequences to decode the information encoded in the polynucleotide.

[0191] Provided herein is a detection system that includes a device that can sequence the stored polynucleotides either directly in the structure and / or after removal from the main structure. When the structure is a reel-to-reel tape of flexible material, the detection system includes a device for holding and advancing the structure through the detection position, and a detector that is located in close proximity to the detection position for detecting a signal generated from a section of the tape when the section is at the detection position. In some cases, the signal indicates the presence of the polynucleotide. In some cases, the signal indicates the sequence of the polynucleotide (e.g., a fluorescent signal). In some cases, the information encoded in the polynucleotide on the continuous tape is read by a computer as the tape is continuously conveyed through a detector operably connected to the computer. In some cases, the detection system includes a computer system that includes a polynucleotide sequencing device, a database for storage and retrieval of 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 thereof.

[0192] Provided herein is a sequencing system that can be integrated into the device described herein. In some cases, the sequencing system is a parallel sequencing system. Various methods of sequencing are well known in the art, including "base calling," in which the identity of a base in a target polynucleotide is identified. In some cases, polynucleotides synthesized using the methods, devices, compositions, and systems described herein are sequenced after cleavage from the synthesis surface. In some cases, sequencing is performed during or simultaneously with polynucleotide synthesis, and base calling is performed immediately or immediately before the extension of nucleoside monomers into a growing polynucleotide chain. Methods of base calling include measuring the current / voltage generated by the polymerase-catalyzed addition of bases to a template strand. In some cases, the synthesis surface includes an enzyme, e.g., a polymerase. In some cases, such an enzyme is bound to an electrode or a synthesis surface. In some cases, the enzyme includes terminal deoxynucleotidyl transferase or a variant thereof.

[0193] Computer Systems

[0194] In various embodiments, any of the systems described herein are operably linked to a computer and are optionally automated through a computer, either locally or remotely. In various cases, the methods and systems described herein further include a software program on a computer system and its use. Thus, computer control for synchronization of dispense / vacuum / replenish functions, such as coordination and synchronization of material deposition device movement, dispense operations, and vacuum operation, is within the scope of the disclosure provided herein. In some cases, the computer system is programmed to interface between a user-specified base sequence and a material deposition device position to deliver the correct reagent to a designated area of ​​the substrate. As an example, a computer system, such as the system shown in FIG. 18 or FIG. 19, can be used to encode data represented as a set of symbols into another set of symbols. For example, data can be represented as numeric symbols, such as binary values ​​of "0" and "1," and the computer system can execute a program that includes an error correction code (e.g., RS code, LDPC code, turbo code, etc.). In some cases, the computer system executes a program that converts data into a plurality of nucleic acid sequences, a program that converts a plurality of nucleic acid sequences into data, or both. In some examples, the program can be a machine learning algorithm. In some examples, the machine learning algorithm can determine the nucleotide base based on a signal (e.g., an electrical signal, such as a current or voltage).

[0195] The program may be executed on the computer system provided herein. In some cases, the program includes a statistical algorithm or a machine learning algorithm. In some cases, an algorithm including machine learning (ML) is used to associate a signal (e.g., current / voltage) with a nucleoside monomer added to a polynucleotide. In some cases, an algorithm including ML may be trained using training data to associate a signal (e.g., current / voltage) with a nucleoside monomer added to a polynucleotide. In some cases, the algorithm includes a classical ML algorithm for classification and / or clustering (e.g., K-means clustering, mean shift clustering, density-based spatial clustering of applications with noise (DBSCAN), expectation maximization (EM) clustering, agglomerative hierarchical clustering, logistic regression, naive Bayes, K-nearest neighbors, random forest or decision tree, gradient boosting, support vector machine (SVM), or a combination thereof).

[0196] In some cases, the algorithm includes a learning algorithm that includes layers, such as one or more neural networks. A neural network may include connected nodes in a network, which may perform functions such as transforming or translating input data. In some examples, an output from a given node may be sent as an input to another node. In some embodiments, a node in a network may include an input unit, a hidden unit, an output unit, or a combination thereof. In some cases, an input node may be connected to one or more hidden units. In some cases, one or more hidden units may be connected to an output unit. A node may receive an input and generate an output based on an activation function. In some embodiments, the input or output may be a tensor, a matrix, a vector, an array, or a scalar. In some embodiments, the activation function may be a rectified linear unit (ReLU) activation function, a sigmoid activation function, or a hyperbolic tangent activation function. In some embodiments, the activation function may be a softmax activation function. The connections between the nodes may further include weights for adjusting the input data to a given node (e.g., weights for activating the input data or deactivating the input data). In some embodiments, the weights may be learned by the neural network. In some embodiments, the neural network may be trained using gradient-based optimization. In some cases, the gradient-based optimization may include one or more loss functions. In some examples, the gradient-based optimization may be conjugate gradient descent, stochastic gradient descent, or variations thereof (e.g., adaptive moment estimation (Adam)). In further examples, the gradients in the gradient-based optimization may be calculated using backpropagation. In some embodiments, the nodes may be organized into a graph to generate a network (e.g., a graph neural network).In some embodiments, the nodes may be organized into one or more layers to generate a network (e.g., a feedforward neural network, a convolutional neural network (CNN), a recurrent neural network (RNN), etc.). In some cases, the neural network may be a deep neural network composed of more than one layer.

[0197] In some cases, the neural network may include one or more recurrent layers. In some examples, the one or more recurrent layers may be one or more long short-term memory (LSTM) layers or gated recurrent units (GRUs) that can perform sequential data classification and clustering. In some embodiments, the neural network may include one or more convolutional layers. The inputs and outputs may be tensors that represent variables or attributes (e.g., features) in the dataset, which may be referred to as feature maps (or activation maps). In some cases, the convolutions may be one-dimensional (1D) convolutions, two-dimensional (2D) convolutions, three-dimensional (3D) convolutions, or any combination thereof. In further cases, the convolutions may be 1D transposed convolutions, 2D transposed convolutions, 3D transposed convolutions, or any combination thereof. In some examples, the 1D convolutional layers may be suitable for time series data because they can classify time series through parallel convolutions. In some instances, the convolution layer can be used to analyze signals (e.g., current / voltage) to nucleoside monomers added to a polynucleotide.

[0198] The layers in the neural network may further include one or more pooling layers before or after the convolutional layers. The one or more pooling layers may reduce the dimensionality of the feature maps using a filter that summarises regions of the matrix. This may downsample the number of outputs, thus reducing the parameters and computational resources required for the neural network. In some embodiments, the one or more pooling layers may be max pooling, min pooling, average pooling, global pooling, norm pooling, or a combination thereof. Max pooling may reduce the dimensionality of the data by taking only the maximum value in a region of the matrix, which helps capture important features. In some embodiments, the one or more pooling layers may be one-dimensional (1D), two-dimensional (2D), three-dimensional (3D), or any combination thereof. The neural network may further include one or more flattening layers that may flatten the input and send it to the next layer. In some cases, the input may be flattened by reducing it to a one-dimensional array. The flattened input may be used to output a classification of the object (e.g., a classification of the signal (e.g., current / voltage) for the nucleoside monomers added to the polynucleotide). The neural network may further include one or more dropout layers. The dropout layers may be used during training of the neural network (e.g., to perform binary or multi-class classification). The one or more dropout layers may randomly set certain weights to 0, which may set corresponding elements in the feature map to 0, allowing the neural network to avoid overfitting. The neural network may further include one or more dense layers that constitute a fully connected network. In the dense layers, information may be passed through the fully connected network, a predicted classification of the object may be generated, and an error may be calculated. In some embodiments, the error may be backpropagated to improve the prediction. The one or more dense layers may include a softmax activation function that may convert a vector of numerical values ​​into a vector of probabilities.These probabilities can then be used to classify, for example, signals (eg, currents and / or voltages) into nucleoside monomers added to a polynucleotide.

[0199] The computer system 3200 illustrated in FIG. 18 can be understood as a logical device capable of reading instructions from a medium 3211 and / or a network port 3205, which may optionally be connected to a server 3209 having a fixed medium 3212. The system may include a CPU 3201, a disk drive 3203, optional input devices, such as a keyboard 3215 and / or a mouse 3216, and an optional monitor 3207. Data communication may be accomplished through a designated communication medium to a server at a local or remote location. The communication medium may include any means of transmitting and / or receiving data. For example, the communication medium may be a network connection, a wireless connection, or an Internet connection. Such a connection may provide communication via the World Wide Web. It is envisioned that data related to the present disclosure may be transmitted over such a network or connection for receipt and / or review by a party 3222.

[0200] FIG. 19 is a block diagram illustrating a first exemplary architecture of a computer system that can be used in connection with an exemplary example of the present disclosure. As shown in FIG. 18, the exemplary computer system may include a processor 3302 for processing instructions. Non-limiting examples of processors include Intel Xeon™ processors, AMD Opteron™ processors, Samsung 32-bit RISC ARM 1176JZ(F)-S v1.0™ processors, ARM Cortex-A8 Samsung S5PC100™ processors, ARM Cortex-A8 Apple A4™ processors, Marvell PXA 930™ processors, or functionally equivalent processors. Multiple execution threads may be used for parallel processing. In some cases, multiple processors or processors with multiple cores may be used, either in a single computer system, in a cluster, or distributed across a system over a network, including multiple computers, mobile phones, and / or personal data assistant devices.

[0201] As illustrated in FIG. 19, a high speed cache 3304 may be connected to or incorporated into the processor 3302 to provide high speed memory for recently or frequently used instructions or data by the processor 3302. The processor 3302 is connected to a north bridge 3306 by a processor bus 3308. The north bridge 3306 is connected to a random access memory (RAM) 3310 by a memory bus 3312 and manages access to the RAM 3310 by the processor 3302. The north bridge 3306 is also connected to a south bridge 3314 by a chipset bus 3316. The south bridge 3314 is connected to a peripheral bus 3318. The peripheral bus may be, for example, a PCI, PCI-X, PCI Express, or other peripheral bus. The north bridge and south bridge are often referred to as the processor chipset and manage data transfers between the processor, RAM, and peripheral components on the peripheral bus 3318. In some alternative architectures, the functionality of the north bridge may be incorporated into the processor instead of using a separate north bridge chip.

[0202] In some cases, the system 3300 may include an accelerator card 3322 attached to the peripheral bus 3318. The accelerator may include a field programmable gate array (FPGA) or other hardware for accelerating certain operations. For example, an accelerator may be used for adaptive data restructuring, or evaluation of algebraic expressions used in extended set processing.

[0203] Software and data may be stored on external storage 3324 and loaded into RAM 3310 and / or cache 3304 for use by the processor. The system 3300 includes an operating system for managing system resources, non-limiting examples of which include Linux, Windows™, MACOS™, BlackBerry OS™, iOS™, and other functionally equivalent operating systems, as well as application software that runs on the operating system to manage data storage and optimization in accordance with exemplary embodiments of the present disclosure.

[0204] In this example, system 3300 also includes network interface cards (NICs) 3320 and 3321 connected to the peripheral bus to provide a network interface to external storage, such as network attached storage (NAS) and other computer systems that can be used for distributed parallel processing.

[0205] 20 illustrates a network 3400 having multiple computer systems 3402a and 3402b, multiple mobile phones and personal data assistants 3402c, and network attached storage (NAS) 3404a and 3404b. In an exemplary embodiment, systems 3402a, 3402b, and 3402c can manage data storage and optimize data access for data stored in network attached storage (NAS) 3404a and 3404b. Mathematical models can be used on the data and evaluated using distributed parallel processing across computer systems 3402a and 3402b and mobile phone and personal data assistant systems 3402c. Computer systems 3402a and 3402b and mobile phone and personal data assistant systems 3402c can also provide parallel processing for adaptive data reconstruction of data stored in network attached storage (NAS) 3404a and 3404b. 20 illustrates only one example, and a wide variety of other computer architectures and systems may be used in conjunction with various embodiments of the present disclosure. For example, blade servers may be used to provide parallel processing. Processor blades may be connected through a backplane to provide parallel processing. Storage may also be connected to the backplane or through a separate network interface as network attached storage (NAS).

[0206] In some exemplary embodiments, the processors may maintain separate memory spaces and transmit data over a network interface, backplane, or other connector for parallel processing by other processors, hi other embodiments, some or all of the processors may use a shared virtual address memory space.

[0207] FIG. 21 is a block diagram of a multiprocessor computer system 3500 using a shared virtual address memory space, according to an exemplary embodiment. The system includes multiple processors 3502a-f that can access a shared memory subsystem 3504. The system incorporates multiple programmable hardware memory algorithm processors (MAPs) 806a-f within the memory subsystem 3504. Each MAP 3506a-f may include a memory 3508a-f and one or more field programmable gate arrays (FPGAs) 3510a-f. The MAPs provide configurable functional units, whereby a particular algorithm, or a portion of an algorithm, may be provided to the FPGAs 3510a-f for processing in close coordination with the respective processor. For example, the MAPs may be used to evaluate algebraic expressions on a data model, and in an exemplary embodiment, to perform adaptive data restructuring. In this example, each MAP is globally accessible by all of the processors for these purposes. In one configuration, each MAP can use direct memory access (DMA) to access an associated memory 3508a-f and perform tasks independently and asynchronously from the respective microprocessor 3502a-f. In this configuration, a MAP can send results directly to another MAP for pipelined and parallel execution of algorithms.

[0208] The above computer architectures and systems are merely examples, and a wide variety of other computer, cell phone, and personal data assistant architectures and systems can be used in connection with the exemplary embodiments, including systems using general purpose processors, co-processors, FPGAs, and other programmable logic devices, systems on chips (SOCs), application specific integrated circuits (ASICs), and any combination of other processing and logic elements. In some embodiments, all or part of the computer system can be implemented in software or hardware. Any of a wide variety of data storage media can be used in connection with the exemplary embodiments, including random access memory, hard drives, flash memory, tape drives, disk arrays, network attached storage (NAS), and other local or distributed data storage devices and systems.

[0209] In an exemplary embodiment, the computer system may be implemented using software modules executing on any of the above or other computer architectures and systems. In other embodiments, the system's functionality may be implemented partially or fully in firmware, programmable logic devices such as field programmable gate arrays (FPGAs), systems on chips (SOCs), application specific integrated circuits (ASICs), or other processing and logic elements. For example, the set processor and optimizer may be implemented with hardware acceleration through the use of a hardware accelerator card.

[0210] The following examples are presented to more clearly illustrate to one skilled in the art the principles and practice of the embodiments disclosed herein, and should not be construed as limiting the scope of any claimed embodiments. Unless otherwise stated, all parts and percentages are by weight. EXAMPLES

[0211] Example 1: Chemical synthesis of DNA using 5'-O-alloc DNA phosphoramidite monomers. dT 15-mer DNA oligomers were synthesized using the synthesis cycle illustrated in Figure 13. 5'-O-alloc deprotection conditions were as follows: 800 μM Pd(Ph3)4, 10 mM PPh3, N,N-dimethylbarbituric acid in THF, reaction time 3 min. The integrity of the 15-mer DNA oligomers was confirmed using high performance liquid chromatography (HPLC) and mass spectrometry (MS) analysis.

[0212] Figure 13 illustrates the chemical synthesis of DNA using 5'-alloc DNA phosphoramidite monomers. First, phosphoramidite coupling was achieved using 5'-O-alloc protected phosphoramidite monomers. Second, the coupled phosphoramidite moieties were subjected to oxidation and capping. Third, the coupled phosphoramidite moieties were subjected to Pd-catalyzed alloc deprotection.

[0213] Figure 14A shows an HPLC analysis of a 15-mer DNA oligomer synthesized using 5'-O-alloc deprotection. Figure 14B shows an MS analysis of a 15-mer DNA oligomer synthesized using 5'-O-alloc deprotection.

[0214] Example 2: Chemical synthesis of DNA using PO-alloc DNA phosphoramidite monomers. dT 15-mer DNA oligomers were synthesized by replacing the PO-cyanoethyl group with a PO-alloc protecting group. Stable H-phosphonates are generated with each cycle (simultaneous with deprotection) and oxidized as a single step at the end of the synthesis.

[0215] Figure 15 shows the chemical synthesis of DNA using PO-alloc protecting group. First, phosphoramidite coupling is achieved using 5'-O-alloc protected and PO-alloc protected phosphoramidite monomers. Second, the coupled phosphoramidite moieties are subjected to Pd-catalyzed alloc deprotection and capping. After repeating all coupling steps, the final intermediate is subjected to oxidation and cleavage to obtain the desired oligomeric molecule.

[0216] Example 3: DNA oligomer synthesis on an electrochemical platform The synthesis of DNA oligomers is carried out on an electrochemical platform by conjugating base pairs to a solid support. 5'-O-alloc protected DMTr DNA phosphoramidite monomers are coupled and subjected to deprotection with nucleophiles catalyzed by Pd(0). This species can be generated by site-selective reduction of a Pd(II) precursor.

[0217] FIG. 16 illustrates DNA oligomer synthesis on an electrochemical platform in which 5′-O-alloc protected DNA phosphoramidite moieties are deprotected with nucleophiles catalyzed by Pd(0).

[0218] Example 4: Electrophoresis Applications Figure 17 illustrates the use of electrochemical platforms in electrophoretic applications where charged species are selectively attracted to or repelled from reaction sites. In one example, negatively charged arylsulfonate-containing phosphine ligands are attracted to (to facilitate deprotection) or repelled from (to prevent deprotection) reaction sites. Alternatively, positively charged ligands are used.

[0219] Example 5: High-density array device A library of at least 10,000 polynucleotides having a length of 150 bases is synthesized utilizing a solid support containing an array of addressable locations for polynucleotide synthesis according to the general methods of Examples 1 or 2. The locations have a pitch distance of 10-200 nm, and the array contains at least 1000 addressable locations.

[0220] 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 alternatives will occur to those skilled in the art without departing from the present invention. It should be understood that various alternatives to the embodiments of the present invention described herein may be used to practice the present invention. The following claims define the scope of the present invention, and it is intended that methods and structures within the scope of these claims, and their equivalents, be covered thereby.

Claims

1. 1. A method for synthesizing a polynucleotide, comprising: (a) contacting at least one nucleoside bound to a solid support with a protected nucleoside, wherein the protected nucleoside comprises a protecting group, the protecting group comprises an alkene, and the solid support comprises an addressable array; (b) contacting an amount of a reagent with the protected nucleoside, wherein the contacting results in deprotection of a terminal nucleoside of the protected nucleoside; (c) repeating steps (a) and (b) to synthesize the polynucleotide; and A method comprising:

2. 10. The method of claim 1, wherein the amount of the reagent is less than about 15 mole % of the protected nucleoside.

3. The method of claim 1, wherein the protected nucleoside comprises a 5' or 3' protecting group.

4. The method of claim 3, wherein the protecting group comprises an allyl group.

5. The method of claim 1, wherein the reagent comprises a heteroaromatic group, a tetrazine, and / or a transition metal catalyst.

6. The method of claim 5, wherein the reagent comprises a transition metal catalyst, the transition metal catalyst having an oxidation state of zero, comprising a Pd(0) catalyst, comprising one or more phosphine ligands, and / or comprising Pd(Ph 3 ) 4 .

7. The method of claim 1, further comprising contacting the protected nucleoside with a C nucleophile, a phosphine, PPh 3 , and / or N,N-dimethylbarbiturate.

8. The method of claim 1, wherein the protected nucleoside comprises at least two alkenyl groups.

9. The method of claim 1, wherein the polynucleotide is 50 to 300 bases in length.

10. The method of claim 1, further comprising applying a voltage to a solvent in fluid communication with the protected polynucleotide.

11. The protected nucleoside is represented by the formula 【Chemistry 1】 or 【Chemistry 2】 wherein R 1 is a linear or branched alkenylene; R 2 is alkylene or alkenylene, each of which is independently unsubstituted or substituted; R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , or R 9 are each independently H, OH, halogen, O-alkyl, N-alkyl, O-alkyl-O-alkyl, azide, or R 3 and R 6 together form a ring; B is a monocyclic or bicyclic C 4-6 heterocyclic ring; The method of claim 1.

12. R 1 is straight chain alkenylene, straight chain C 2-6 alkenylene, allyl, branched chain alkenylene, or branched chain C 3-8 alkenylene; 12. The method of claim 11, wherein R 2 is substituted alkylene, substituted C 2-4 alkylene, cyanoethyl, unsubstituted alkenylene, unsubstituted C 2-4 alkylene, or allyl.

13. The method of claim 11, wherein B is a monocyclic C 4 heterocyclic ring, a bicyclic C 5 heterocyclic ring, or a nucleobase.

14. The method of claim 11, wherein one or both of R 3 and R 4 is F, OCH 3 , or OCH 2 CH 2 OCH 3 .

15. The protected nucleoside has the structure 【Transformation 3】 or 【Chemistry 4】 12. The method of claim 11, comprising:

16. A first capping step after contacting the at least one nucleoside bound to the solid support with the protected nucleoside; an oxidation step; a second capping step after the oxidation step, allowing the substrate to dry; The method of claim 1 further comprising:

17. The method of claim 16, wherein the first capping step and / or the second capping step includes treatment with acetyl chloride or acetic anhydride.

18. The method of claim 1, wherein contacting the reagent with the protected nucleoside is for less than about 10 minutes.

19. The method described in claim 10, wherein the voltage is between about 0.5 volts and about 3 volts, and the voltage is applied for between about 0.1 seconds and about 10 seconds.

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