WO2024163450A2

WO2024163450A2 - Methods and systems for polymer synthesis by contacting synthesis surfaces with compartmentalized liquid reagents

Info

Publication number: WO2024163450A2
Application number: PCT/US2024/013513
Authority: WO
Inventors: Tuan Tran; Evan Foster; Joseph KOSCINSKI, Jr.; Christopher HOFF; Margarita KHARITON; Julia Swavola; Sebastian PALLUK; Urmi BHAUMIK
Original assignee: Ansa Biotechnologies, Inc.
Priority date: 2023-01-30
Filing date: 2024-01-30
Publication date: 2024-08-08
Also published as: WO2024163450A3

Abstract

Disclosed herein are methods and devices for improved de novo cyclic enzymatic polymer synthesis using a protected monomer. Protected monomers include those with a protecting group that prevents addition of more than one monomer during a synthesis step, including the catalytic enzyme attached to the monomer. In specific embodiments, the polymer synthesis method is an enzymatic de novo polynucleotide synthesis method using protected nucleotides, such as nucleotides comprising a reversible terminator or nucleotides bound to a catalytic polymerase.

Description

METHODS AND SYSTEMS FOR POLYMER SYNTHESIS BY CONTACTING

SYNTHESIS SURFACES WITH COMPARTMENTALIZED LIQUID REAGENTS

CROSS-REFERENCE TO RELATED APPLIC TION

[0001] This application claims priority to and the benefit of U.S. Provisional Application No. 63/441,969, filed on January 30, 2023, the entire disclosure of which is incorporated by reference herein for all purposes.

BACKGROUND

[0002] Polymer synthesis may be performed in a number of different ways to yield different varieties of polymers of a specified length and monomer sequence. One type of polymer synthesis that is vital to the life sciences industry is de novo polynucleotide synthesis. De novo DNA synthesis has been used in several applications such as biosynthesis, genetic engineering, genomics, and synthetic biology. In biosynthesis, de novo DNA synthesis is used to produce enzymes, antibodies, and other biomolecules. In genetic engineering, de novo DNA synthesis is used to create genetically modified organisms. And in genomics, de novo DNA synthesis is used to construct genetic maps, as well as to sequence and analyze genomes.

[0003] A prominent conventional approach to de novo polynucleotide synthesis is phosphoramidite-based synthesis. Technologies around phospharamidite-based oligonucleotide synthesis have made remarkable progress in the past decades, including updates to microfluidics and microchip-based oligo synthesis using inkjet printing to deliver reagents, enabling more efficient large-scale oligonucleotide synthesis. However, due to natural limitations of chemical reactions, further improvement to accuracy and length of oligonucleotides synthesized by phosphoramidite chemistry-based methods have been difficult to obtain. Recently emerged enzymatic methods for de novo DNA synthesis show great potential for improved synthesis accuracy and length, as well as faster coupling times. [0004] However, both enzymatic and phosphoramidite-based DNA synthesis methods are performed on an oligonucleotide attached to a solid support, and current methods deliver and remove several different reagents, such as by microfluidics, to and from the synthesized oligonucleotide to perform the steps required each cycle. The time required for fluidic management limits oligo speed synthesis gains achieved by improved coupling time during enzymatic synthesis. Small differences (i.e., increases) in cycle time lead to a much longer time for oligonucleotide synthesis, as they are multiplied by the number of cycles performed. This becomes increasingly important if enzymatic synthesis is to fulfill its promise for longer oligonucleotide synthesis.

[0005] Furthermore, any issues with reagent delivery during microfluidic or inkjet-based delivery of the reagents, such as bubble formation or incomplete delivery of reagents, may decrease the accuracy of synthesis, resulting in a failed extension step. Very small increases in error rate lead to large differences in the final number of perfect sequences synthesized according to a Poisson distribution. For example, synthesis of a 500 mer oligonucleotide with a 0.5% error rate at each cycle lead to 8.2% correct sequences in the final product. However, a 1% error rate leads to just 0.7% correct sequencees. A 2% error rate results in fewer than 0.01% correct sequences.

[0006] What is needed therefore, are improved methods and systems for de novo polynucleotide synthesis. In particular, systems that build on emerging advantages from enzymatic -based methods to further reduce the total cycle time and the error rate of nucleotide addition during de novo polynucleotide synthesis can significantly impact the field of enzymatic de novo polynucleotide synthesis.

SUMMARY OF THE INVENTION

[0007] In accordance with a first inventive facet, a system for multiplex polymer synthesis includes a plurality of elements. Each of the elements has a synthesis surface on which a base molecule for polymer synthesis is bound. The system further includes a plate having resolved loci where each locus is adapted for containing a respective liquid reagent volume. The system further includes at least one actuator for actuating the plurality of elements and/or the plate between i) first relative positions wherein the synthesis surfaces are separate from the resolved loci, and ii) second relative positions wherein the synthesis surfaces are within sufficient proximity to the resolved loci to contact the respective liquid reagent volumes when present at the resolved loci on the plate surface.

[0008] The resolved loci may be wells or locations on a patterned surface. The plate may be a patterned surface separating the resolved loci in some embodiments. The patterned surface may have a pattern of hydrophobic regions and hydrophilic regions. The resolved loci may be separated by hydrophobic regions on the patterned surface. The resolved loci may be hydrophilic regions on the patterned surface. At least a subset of the resolved loci may be spatially separated to be capable of alignment with the plurality of elements. [0009] The elements may be rods, plates, or filaments. The synthesis surface may be functionalized to bind to the base molecule. The elements may be sized and shaped to fit into the wells. The system may have an elements holder for holding the plurality of elements so that the plurality of elements may be actuated together and an actuatable component secured to the elements holder. The actuatable component may be actuatable by the at least one actuator.

[0010] The base molecule may be a synthesis initiator comprising a reactive group for coupling to a first monomer in a cycled polymer synthesis reaction. The base molecule may be capable of binding to a synthesis initiator having a reactive group for coupling to a first monomer in a cycled polymer synthesis reaction. The binding of the base molecule to said synthesis initiator may be via one or more linking molecules. The base molecule may be a first oligonucleotide.

[0011] The first oligonucleotide may include a free 3' hydroxyl group. The first oligonucleotide may have a single-stranded region at the 3' end. The first oligonucleotide may be capable of hybridizing specifically to a second oligonucleotide, wherein the second oligonucleotide has a free 3' hydroxyl end at the distal end from the functionalized surface when hybridized to the first oligonucleotide. The second oligonucleotide may comprise a single- stranded region at the 3' hydroxyl end when hybridized to the first oligonucleotide. [0012] The at least one actuator may be operationally linked to and configured to align the plurality of elements and at least a subset of the resolved loci across a first plane. The at least one actuator may be operationally linked to and configured to move the aligned plurality of elements and at least a subset of the resolved loci between said first and second relative positions along an axis orthogonal to the first plane. The at least one actuator may include a first actuator operationally linked to and configured to align the plurality of elements and at least a subset of the resolved loci across a first plane and a second actuator operationally linked to and configured to move the aligned plurality of elements and the at least a subset of the resolved loci between said first and second relative positions along an axis orthogonal to the first plane.

[0013] The system may include a dispenser for dispensing liquid reagents to the resolved loci. The dispenser may have one or more nozzles each capable of dispensing a liquid reagent to the resolved loci. The system may be configured to simultaneously i) actuate the plurality of elements or a first plate between said first relative positions and said second relative positions, and ii) dispense liquid reagents to the resolved loci on a second plate using said dispenser.

[0014] The dispenser may be connected via a fluidic pathway to a reagent container for holding a polymer extension solution. The polymer extension solution may comprise a monomer and an enzyme capable of catalyzing addition of the monomer to a polymer comprising said base molecule and bound to the synthesis surface. The enzyme may be a polymerase. The polymerase may be a template-independent polymerase. The enzyme may be bound to the monomer via a linker to form a protected monomer. The monomer may comprise a protecting group. The monomer may be a nucleotide.

[0015] The dispenser may be connected via a fluidic pathway to a reagent container for holding a deprotection solution. The deprotection solution may be capable of removing a protecting group from a monomer. The deprotection solution may comprise a linker cleavage reagent capable of separating an enzyme bound to a monomer via a cleavable linker. The dispenser may be connected via a fluidic pathway to a reagent container for holding a reaction quenching solution. The dispenser may be connected via a fluidic pathway to a reagent container for holding a wash solution.

[0016] In accordance with another inventive facet, a system for multiplex polymer synthesis includes a plurality of elements. Each of the elements has a synthesis surface on which a base molecule for polymer synthesis is bound. The system further includes a plate comprising resolved loci containing a liquid reagent. The system additionally includes at least one actuator for actuating the plurality of elements or the plate to cause the synthesis surfaces to be at least partially immersed into corresponding ones of the resolved loci containing the liquid reagent to perform one or more polymer synthesis steps on the synthesis surfaces.

[0017] In accordance with an additional inventive facet, a system for polymer synthesis, includes elements, wherein each of the elements has a synthesis surface on which a base molecule for synthesizing a polymer is bound. The system also includes at least one actuator for actuating the elements for the synthesis surfaces of the elements to be at least partially immersed in a liquid reagent volume to cause one or more steps of polymer synthesis. Further, the system includes a processor configured for executing computer programming instructions to control actuation by the actuator.

[0018] Each of the elements may be a rod that is suspended by an element holder and has a length that extends along a longitudinal axis from a proximal end to a distal end. Each of the elements may float in the element holder such that it may be displaced longitudinally relative to the element holder in response to contacting an object with the distal end of the element. The elements may comprise at least one of metal, plastic, or glass. The processor may be configured to cause the at least one actuator to at least partially immerse the elements in the liquid reagent volume multiple times.

[0019] The system may include a dispenser for dispensing liquid reagent volumes into compartments. The compartments may be wells of a reaction plate or loci on a patterned surface. The processor may be configured to cause the dispenser to dispense a liquid reagent volume comprising a polymerase and a nucleotide into at least one of the compartments. The processor may be configured to cause the dispenser to dispense different nucleotide identities in distinct ones of the compartments. The processor may be configured to cause the at least one actuator to at least partially immerse one of the elements in each of the distinct ones of the compartments in a sequence. The dispenser may be fluidically connected to a reservoir holding a reaction quenching solution. The processor may be configured to cause the dispenser to dispense the reaction quenching solution from the reservoir in one of the compartments, and the processor may be configured to cause the at least one actuator to at least partially immerse one of the elements in the compartment in which the reaction quenching solution has been dispensed. The processor may be configured to cause the dispenser to dispense a wash solution in one of the compartments, and the processor may be configured to cause the at least one actuator to at least partially immerse one of the elements in the compartment in which the wash solution has been dispensed. The processor may be configured to cause the dispenser to dispense a deprotection solution in one of the compartments, and the processor may be configured to cause the at least one actuator to at least partially immerse one of the elements in the compartment in which the enzymatic linker cleaver solution has been dispensed. The deprotection solution may contain a blocker removal reagent. The deprotection solution may contain an enzymatic linker cleaver.

[0020] In accordance with a further inventive facet, a system for polynucleotide synthesis includes an element having a functionalized surface bound to a polynucleotide synthesis primer comprising a free 3' hydroxyl end. The system has at least one actuator for actuating the element for the functionalized surface of the element to be inserted in a liquid reagent comprising a polymerase and a nucleotide to cause polynucleotide synthesis. The system also has a processor configured for controlling the at least one actuator to contact the functionalized surface of the element into the liquid reagent under conditions suitable for polymerase catalyzed extension of said polynucleotide at said free 3' hydroxyl end.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The foregoing and other objects, features and advantages will be apparent from the following description of particular embodiments, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead placed upon illustrating the principles of various embodiments.

[0022] Figure 1 depicts a block diagram of a synthesis system that is suitable for exemplary embodiments.

[0023] Figure 2 depicts a first illustrative configuration of the synthesis system.

[0024] Figure 3 depicts a second illustrative configuration of the synthesis system.

[0025] Figure 4 depicts an illustrative element and an illustrative well that are suitable for the exemplary embodiments.

[0026] Figure 5 depicts illustrative cross-sections of element designs of exemplary embodiments.

[0027] Figure 6 depicts a longitudinal view of illustrative element designs of exemplary embodiments.

[0028] Figures 7 A and 7B depict a floating elements design of an exemplary embodiment. [0029] Figure 8 depicts an illustrative reaction plate for use in exemplary embodiments. [0030] Figure 9 depicts a portion of an illustrative patterned surface for use in exemplary embodiments.

[0031] Figure 10 depicts a flowchart of illustrative steps that may be performed in exemplary embodiments to synthesize polymers.

[0032] Figure 11 depicts illustrative operations that may be performed in exemplary embodiments to perform hybridization of elements of an element array.

[0033] Figure 12 depicts a flowchart of illustrative steps that may be performed in exemplary embodiments as part of the synthesis process.

[0034] Figure 13 depicts a flowchart of illustrative steps that may be performed in exemplary embodiments to perform a cycle of the synthesis process.

[0035] Figure 14 depicts an illustrative pattern of loading polymer extension solutions in wells of a reaction plate in exemplary embodiments. DETAILED DESCRIPTION

[0036] The details of various embodiments are set forth in the description below. Other features, objects, and advantages will be apparent from the description and the drawings, and from the claims.

Polymer Synthesis

[0037] Provided herein is a system for performing steps in a method of polymer synthesis to generate polymers of desired length and sequence according to the embodiments described herein. In some embodiments, the steps are performed by dipping a reaction surface comprising a bound synthesis initiator (which may also include previously added monomers) into a contained solution comprising the desired reagents. The method is amenable to multiplexed polymer synthesis, such that a plurality of elements having an end with the reaction surface and bound synthesis initiator may be used and simultaneously dipped into a plurality of contained liquid reagents (such as in wells or droplets on a surface) aligned with the reaction surfaces. In some embodiments, the contained liquid reagent comprise a polymer extension solution with a monomer of a specific identity and and enzyme capable of adding the monomer to the synthesis initiator.

[0038] In some embodiments, synthesis of a polymer comprises adding protected monomers stepwise to a synthesis initiator (e.g., an initial oligonucleotide) via the cycled steps of: addition of monomer comprising a protecting group (i.e., a protected monomer) to a synthesis initiator or extended polymer comprising previously added monomers, binding of the monomer to the end of the synthesis initiator or extended polymer catalyzed by the enzyme, and removal of the protecting group from the monomer to allow addition of a subsequent monomer to the extended polymer. These steps can be repeated until a desired polymer sequence and length is synthesized.

[0039] As used herein, a “protecting group” bound to the monomer (e.g., a nucleotide) is a group capable of preventing addition of another monomer once the monomer has been added to the synthesis initiator or extended polymer. After addition of the desired protected monomer and removal of excess monomer during an extension cycle, the extended polymer is immersed in a monomer deprotection solution capable of removing the protecting group from the monomer.

[0040] In some embodiments, the protecting group can be the enzyme that catalyzes addition of the monomer to the synthesis initiator or extended polymer, wherein the enzyme is linked to the monomer (i.e., a monomer-enzyme conjugate). In this embodiment, the enzyme can sterically hinder addition of a subsequent monomer after addition of the protected monomer to the polymer. A monomer deprotection solution that removes the enzyme from the monomer can then be used, such as a linker cleavage solution.

[0041] Both the monomer addition and protecting group removal steps may be quenched by immersing the extended polymer in an appropriate reaction quenching solution, such as EDTA. In addition, washing steps may be used between steps by immersing the extended polymer in a wash buffer.

Enzymatic DNA Synthesis

[0042] In some embodiments, provided herein is a system for performing steps in a method of polynucleotide synthesis to generate polynucleotides of desired length and sequence according to the embodiments described herein. In some embodiments, the steps are performed by dipping a reaction surface comprising a bound synthesis initiator (which may also include previously added nucleotides) into a contained solution comprising the desired reagents. The method is amenable to multiplexed polynucleotide synthesis, such that a plurality of elements having an end with the reaction surface and bound synthesis initiator may be used and simultaneously dipped into a plurality of contained liquid reagents (such as in wells or droplets on a surface) aligned with the reaction surfaces. In some embodiments, the contained liquid reagent comprise a polymer extension solution with a nucleotide of a specific identity and and polymerase capable of adding the nucleotide to the synthesis initiator.

[0043] In some embodiments, synthesis of a polynucleotide comprises adding protected nucleotides stepwise to an oligonucleotide bound to the reaction surface on the element via the cycled steps of: addition of nucleotide comprising a protecting group (i.e., a protected nucleotide) to a synthesis initiator or extended polynucleotide comprising previously added nucleotides, binding of the nucleotide to the end of the synthesis initiator or extended polynucleotide catalyzed by the polymerase, and removal of the protecting group from the nucleotide to allow addition of a subsequent nucleotide to the extended polynucleotide. These steps can be repeated until a desired polynucleotide sequence and length is synthesized.

[0044] The protecting group bound to the nucleotide (e.g., a nucleotide) is a group capable of preventing addition of another nucleotide once the nucleotide has been added to the synthesis initiator or extended polynucleotide. After addition of the desired protected nucleotide and removal of excess nucleotide during an extension cycle, the extended polynucleotide is immersed in a nucleotide deprotection solution capable of removing the protecting group from the nucleotide.

[0045] In some embodiments, the protecting group is the polymerase that catalyzes addition of the nucleotide to the surface-bound polynucleotide, wherein the polymerase is linked to the nucleotide (i.e., a nucleotide-polymerase conjugate). In this embodiment, the polymerase can sterically hinder addition of a subsequent nucleotide after addition of the protected nucleotide to the polynucleotide. A monomer deprotection solution that removes the polymerase from the nucleotide can then be used to remove the protecting group, such as a linker cleavage solution.

[0046] In some embodiments, the protecting group is a reversible terminator bound to the nucleotide. A monomer deprotection solution that removes the reversible terminator from the nucleotide can then be used to remove the protecting group, such as a linker cleavage solution. In some embodiments, both a reversible terminator and a polymerase bound to the nucleotide may be used.

[0047] Both the nucleotide addition and protecting group removal steps may be quenched by immersing the extended polynucleotide in an appropriate reaction quenching solution, such as EDTA. In addition, washing steps may be used between steps by immersing the extended polynucleotide in a wash buffer.

[0048] Exemplary embodiments may provide methods and systems for polymer synthesis, such as de novo enzymatic polynucleotide synthesis. In the exemplary embodiments, synthesis surfaces on elements may have a base molecule bound to the synthesis surfaces. The synthesis surfaces may be placed in contact with liquid reagent volumes, such as a polymer extension solution comprising a monomer and an enzyme capable of catalyzing addition of the monomer to a synthesis initiator or polymer, to synthesize polymers. In some exemplary embodiments, the system may include an array of elements that have the functionalized surfaces at least on their distal portions, and the elements may be moved by one or more actuators to be contacted with (i.e., at least partially immersed) into a liquid reagent in a resolved loci on a surface, such as a plate of wells or a plate with a patterned surface holding a polymer extension solution, to perform the synthesis.

[0049] Resolved loci are fluidically disconnected from each other and capable of containing a liquid reagent on a surface separate from other liquid reagents, e.g., a substrate or plate can have a first resolved locus capable of containing a first polymer extension solution (such as a contained droplet or well) for a first oligonucleotide and a second resolved locus capable of containing a second polymer extension solution (such as a contained droplet or well) for a second oligonucleotide, the first and second polymer extension solutions being fluidically isolated from each other (and other polymer extension solutions) on the surface or plate. Such fluidic isolation is maintained when a corresponding synthesis surface is dipped into the polymer extension solution.

[0050] In some embodiments, a plate comprising resolved loci is a droplet microarray comprising a substrate having a hydrophobic -hydrophilic patterned surface on which a plurality of resolved loci correspond to hydrophilic locations each of which is capable of hosting a liquid reagent. Preparation of substrates with discrete resolved loci for containing liquid reagent can be accomplished by known methods. For example, such methods can involve the creation of hydrophilic reaction sites by first applying a protectant, or resist, over selected areas over the surface of a substrate, such as a silicon oxide, or like material. The unprotected areas are then coated with a hydrophobic agent to yield an unreactive surface. For example, a hydrophobic coating can be created by chemical vapor deposition of (tridecafluorotetrahydrooctyl)-triethoxysilane onto the exposed oxide surrounding the protected circles. Finally, the protectant, or resist, is removed exposing the well regions of the array for further modification and nucleoside synthesis using the high surface tension solvents described herein and procedures known in the art such as those described by Maskos & Southern, Nucl. Acids Res. 20:1679-1684 (1992). Alternatively, the entire surface of a glass plate substrate can be coated with hydrophobic material, such as 3-(l,l- dihydroperfluoroctyloxy)propyltriethoxysilane, which is ablated at desired loci to expose the underlying silicon dioxide glass. The substrate is then coated with glycidyloxypropyl trimethoxysilane, which reacts only with the glass, and which is subsequently “treated” with hexaethylene glycol and sulfuric acid to form an hydroxyl group-bearing linker upon which chemical species can be synthesized (Brennan, U.S. Pat. No. 5,474,796). Arrays produced in such a manner can localize small volumes of solvent within the reaction site by virtue of surface tension effects (Lopez et ah, Science 260:647-649 (1993)).

[0051] The number of elements may be matched with the number and positioning of wells on a plate or locations on a patterned surface plate holding a polymer extension solution or other liquid reagent volume. [0052] The systems of the exemplary embodiments may also perform operations, such as hybridization of the functionalized surfaces, heating or cooling of the liquid reagent volumes, agitation of the liquid reagent volumes and washing of the dipping elements (i.e., synthesis elements) and/or plates in order to properly realize the synthesis.

[0053] The exemplary embodiments may enable reduced polymer synthesis times by reducing the time required for each cycle of synthesis. The approach of these exemplary embodiments separates the loading operations of loading wells on a plate or resolved loci on a patterned surface with the liquid reagent volumes from the synthesis operations involving contacting synthesis surfaces with the liquid reagent volumes. This separation of the fluidic steps from the synthesis steps helps to reduce cycle time as the fluidic steps are performed beforehand and need not be performed as part of the synthesis steps. The liquid reagent volumes may be pre-mixed in the wells or the resolved loci on the patterned surface. The synthesis may be quicker than conventional polymer synthesis systems.

[0054] Furthermore, the delivery of reagent to the polymer by dipping the polymer into a reagent will also mitigate errors in DNA synthesis caused by conventional methods of reagent delivery to the reaction site, such as bubble formation or incomplete reagent delivery. With a single dispense of a reagent to the polymer, any issues with reagent delivery could result in inhibition of a synthesis or deprotection reaction. However, re-dipping the synthesis surface comprising the attached polymer or synthesis initiator can lead to increased robustness as the reaction (e.g., monomer addition or deprotection) is initiated more than once, mitigating any issues that may arise during a single dip or a single reagent delivery. Therefore, in some embodiments, a polymer may be dipped (e.g., immersing the polymer / synthesis surface into a liquid reagent compartmentalized in at a resolved locus) and redipped into the same liquid reagent (e.g., a polymer extension solution) multiple times to agitate the solution and/or to reduce the impact liquid reagent delivery problems, such as bubble formation or incomplete reagent delivery. For example, although a desired reaction from one instance of dipping the polymer into the liquid reagent may be inhibited as described above, repeated dipping into the liquid reagent allows for additional attempts to perform the reaction in a different configuration, e.g., a bubble may be moved so that it does not inhibit the reaction in at least some of the dips.

[0055] The exemplary embodiments are applicable to polymer synthesis and are particularly applicable to enzymatic polynucleotide synthesis as described below. The discussion below generally focuses on polymer synthesis but, in some instances, focuses on polynucleotide synthesis.

[0056] Figure 1 depicts a block diagram of components of an illustrative synthesis system 100 for performing polymer synthesis, such as enzymatic polynucleotide synthesis, in exemplary embodiments. Illustrative configurations of these components are discussed below. The synthesis system 100 may include an element array 102. The element array 102 may include a number of elements organized in an array. As is explained below, each element may include a synthesis surface or surfaces on at least a portion of the element to which a base molecule is bound or will be bound.

[0057] In some embodients, the synthesis surface comprises a functionalized surface capable of binding to the base molecule. For example, a polynucleotide modified with a disulfide group can be covalently bound to a surface that has been modified with a thiol group (-SH). Other functional groups that are useful for binding oligonucleotides include amino groups (-NH2) and carboxyl groups (-COOH). In some embodiments, surfaces such as glass or silicon oxide can be functionalized with silanes such as aminopropyltrimethoxy silane (APTMS) and these can be used to covalently bind amine- modified oligonucleotides. In some embodiments, gold, silver, and titanium surfaces can be functionalized with thiol groups and can be used for immobilizing thiolated oligonucleotides to form a “self-assembled monolayers (SAM)”. In some embodiments, a functionalized surface is chemically or naturally charged like glass or silicon, where oligonucleotides can be adsorbed via electrostatic interactions. In some embodiments, surfaces such as glass or silicon oxide can be functionalized with silanes such as aminopropyltrimethoxy silane (APTMS) and these can be used to covalently bind amine-modified oligonucleotides.

[0058] In some embodiments, a base molecule is a synthesis initiator comprising a reactive group for coupling to a monomer in a cycled polymer synthesis reaction. For example, a polynucleotide sequence bound to a synthesis surface and having a free 3' hydroxyl at its end may be a synthesis initiator. In some embodiments, a synthesis initiator comprises a non- nucleic acid compound having a free hydroxyl to which a TdT may couple a nucleotide, e.g. as described in U.S. patent publications US2019/0078065 and US2019/0078126.

[0059] In some embodiments, a base molecule is a compound bound to the reaction surface and is capable of binding to the reaction initiator. For example, a reaction initiator oligonucleotide having a sufficiently complementary sequence to hybridize to a base molecule oligonucleotide bound to the surface at its 3' end may be used. In this embodiment, the synthesis reaction does not occur off of the covalently bound base molecule, but occurs at the end of the hybridized oligonucleotide. In this embodiment, an additional step of hybridization of the reaction initator to the base molecule bound to the reaction surface may be bound. Further, in this embodiment, the system may only comprise a base molecule bound to the surface without a bound reaction initiator before use in the processese described herein. Thus, a system comprising a base molecule bound to the reaction surface of an element can include a reaction initiator (when the base molecule is the reaction initiator) or is primed to bind to a reaction initiator (i.e., comprises a base molecule capable of binding to the reaction initiator).

[0060] The number of elements in an array and the arrangements of the elements may match that of a group of wells in a plate of wells or of other resolved loci, such as a group of hydrophilic locations in a patterned surface plate that includes a pattern of hydrophilic locations and hydrophobic locations. The elements of the element array 102 may be held by an element array holder 104.

[0061] A wide variety of substrates may be employed for creating synthesis surfaces or elements for enzymatic synthesis of polynucleotides. Substrates may be a rigid material including, without limitation, glass; fused silica; silicon such as silicon dioxide or silicon nitride; metals such as gold or platinum; plastics such as polytetrafluoroethylene, polypropylene, polystyrene, polycarbonate, and any combination thereof. A rigid surface can be fabricated from a material selected from the group consisting of silicon, polystyrene, agarose, dextran, cellulosic polymers, polyacrylamides, polydimethylsiloxane (PDMS), and glass. Substrates may also comprise flexible materials, which is capable of being bent, folded or similarly manipulated without breakage. Exemplary flexible materials include, without limitation, nylon (unmodified nylon, modified nylon, clear nylon), nitrocellulose, polypropylene, polycarbonate, polyethylene, polyurethane, polystyrene, acetal, acrylic, acrylonitrile, butadiene styrene (ABS), polyester films such as polyethylene terephthalate, polymethyl methacrylate or other acrylics, polyvinyl chloride or other vinyl resin, transparent PVC foil, transparent foil for printers, Poly (methyl methacrylate) (PMMA), methacrylate copolymers, styrenic polymers, high refractive index polymers, fluorine-containing polymers, polyethersulfone, polyimides containing an alicyclic structure, rubber, fabric, metal foils, and any combination thereof.

[0062] The element array holder 104 may be actuatable by actuator(s) 106, which may actuate one or more actuatable components 109, such as a robotic arm, that may hold or contain the element array holder 104. The one or more actuator(s) 106 may include driver(s)

108, such as electric motors, for driving motion of the one or more actuatable components

109. In other exemplary embodiments, magnetic elements and electrical elements may serve as the driver(s) 108. The driver(s) 108 may drive motion of the one or more actuatable components 109 to move up or down, move laterally and/or even rotate. The driver 108 may cause linear actuation in the X and Y dimensions of a plane to position the elements of the element array 102 to be in alignment above resolved loci of the plate (e.g., the wells of a plate or hydrophilic locations on a patterned surface plate) 128 and to move the elements into contact with the liquid reagent volumes in the resolved loci of the plate 128 as part of the polymer synthesis. In some exemplary embodiments, multiple drivers 108 may be provided. [0063] The actuation by the one or more actuator(s) 106 may be under the control of a processor 110 that executes computer programming instructions of control application 112 stored in a storage 114. The control application 112 may include computer programming instructions for controlling operations of the synthesis system 100. The control application 112 may invoke operations and direct activities of the components of the synthesis system 100 described herein 100. The processor 110 may be a central processing unit (CPU), a graphics processing unit (GPU), an application specific integrated circuit (ASIC), a field programmable gate array (FPGA), or other type of processor. In some exemplary embodiments, more than one processor may be provided. The storage 114 may be a non- transitory computer-readable storage medium, such as one or more of random access memory (RAM), read only memory (ROM), solid state memory, magnetic disk storage, optical disk storage or the like.

[0064] The synthesis system 100 may include a display 116 for displaying, graphical textual and video content. The display 116 may display a user interface (UI) 118 under the control of the control application 112. The UI 118 may display useful information to a user of the synthesis system 100 and may be used to enter commands or invoke operations in the synthesis system. For example, a user may cause actuation of the one or more actuators 106 by entering commands via input devices 120, such as a mouse, a keyboard, a thumbpad or the like. In some embodiments, the display 116 may be a touchscreen that is capable of receiving input by the user touching the screen of the display 116. In some embodiments, the input devices 120 may also include knobs, levers and buttons that are used to control operation of the synthesis system 100. In some exemplary embodiments, the processor 110, storage 114, the display 116 and the input devices 120 may be part of a computer system, such as a workstation, personal computer, laptop computer, tablet computer, smartphone or other computing device, that is coupled to the other components of the synthesis system 100. The coupling may be a hardwired connection or a network connection, such as via a wired or wireless network. Alternatively, these components 110, 114, 116 and 120 may be integrated as part of the synthesis system 100, such as in a common housing or in one of multiple housings of the synthesis system 100.

[0065] The synthesis system 100 may also include a plate washer 122 for washing reaction plates. In some embodiments, the washing reaction plates can include a uniform bath for immersing multiple reaction surfaces into. In some embodiments, a wash solution can be contained in wells or patterned surfaces 128 that are used in the synthesis system 100. The synthesis system 100 may include a washing fountain 124. Dispenser(s) 126 may be provided for dispensing solution to the resolved loci on plates or patterned surfaces 128. The dispenser(s) 126 may have, for example, fluidic connections with a reagent holder 127A containing a wash solution, a reagent container 127B containing a quenching solution for quenching a reaction, a reagent container 127C containing a deprotection solution and a reagent container 127D, containing a reagent solution, such as a polymer extension solution. A thermal controller 130 may be provided for heating and/or cooling the reaction plates or patterned surfaces 128 or the liquid reagent volumes held therein. This may ensure that the reagents involved in the synthesis are at a suitable temperature for reactions that occur during the polymer synthesis. A shaker 132 may be provided for shaking the reaction plates or patterned surfaces 128 as needed to properly complete the polymer synthesis.

[0066] Figure 2 depicts a first illustrative configuration 200 of the synthesis system. In this synthesis system 200, an element array 202 is held in an element array holder 204. The element array holder 204 is secured in an arm 205 that is actuatable by an actuator (not shown), like an electric motor. The element array holder 204 may be moved up and down (i.e., along the Z axis) relative to the plane of the top surface of the reaction plate 206, where at least some of the resolved loci hold a liquid reagent volume. The element array holder 204 is positioned on a platform 208. The platform 208 is actuatable in the X direction. The platform 208 may be actuated to slide along the rail 210 to be positioned under the element array 202 so that at least some of the elements are in alignment with at least some of the resolved loci (e.g., wells) of the plate 206. The elements may be placed in contact with the liquid reagent volumes in the resolved loci as will be described below as part of the synthesis. Subsequently, the platform 208 may be actuated to slide on the rail 210 back to the position shown in Figure 2.

[0067] Plates 212 are positioned on a platform 218. The platform 218 may slide along rail 214 in the X direction and/or rail 216 in the Y direction. The plates 212 may include a plate where at least some of the resolved loci contain quenching solution, and a plate where at least some of the resolved loci contain cleaving solution. One or more of the reaction plates 212 may contain wells holding a wash solution. The platform 218 may be actuated by one or more of the actuators to move individual one of the plates 212 under the element array 202 so that elements may be placed in contact with contents in at least some of the resolved loci of the individual plates. This facilitates performing quenching, cleaving, and washing steps as described below.

[0068] Figure 3 depicts a second illustrative configuration 300 of the synthesis system. In this illustrative configuration 300, plates 301 ride on levitating carriers 304. The levitating carriers 304 are levitated and propelled over a platform 302 by magnets and electrical components found in magnetic levitation systems. In some embodiments, the levitating carriers are controlled using two sets of magnets: one set that provides the lifting force and another set that provides the guidance and stability.

[0069] In some embodiments, superconducting magnets are used. The levitating carrier can be made of a magnetic material, such as iron, and has a magnetization that is parallel to the direction of the lifting force. To move the plate, the direction of the magnetic field can be changed. This can be done by adjusting the current in the superconducting magnets or by using additional magnets that can generate a secondary magnetic field. This change in the direction of the magnetic field will cause the plate to move, following the direction of the magnetic field. In some embodiments, magnetic levitation is performed using permanent magnets. In this case, a combination of repulsive and attractive forces between the magnets are used to create the levitation. To move the levitated plate, the magnetic field and position of the permanent magnets are adjusted.

[0070] In this illustrative configuration 300, the levitating carriers 304 are actuated under computer programming control to move between stations during the synthesis process. The levitating carriers 304 may be actuated to the dispensing subsystem 306. The dispensing subsystem station 306 contains dispensers for dispensing liquid reagent volumes, such as a polymer extension solution, in at least some of the wells of the plates 301. The bath fill, drain subsystem station 308 may contain dispensers for dispensing quenching solution, cleaving solution and wash solution to wells of reaction plates 301. The levitating carriers 304 may also be actuated to be positioned at a thermal subsystem station 312. The thermal subsystem station 312 may contain heating elements and/or cooling elements that may be placed in contact with the reaction plates 301 or the liquid reagent volumes of at least some of the resolved loci of the plates 301 to adjust the temperature of the liquid reagent volumes. The levitating carriers 304 may be actuated to be positioned at the dipper subsystem station 310. The dipper subsystem station 310 may include an element array that is actuatable up and down relative to the planar upper face of the platform 302. The actuation of the element array may bring at least some of the elements of the element array into contact with liquid reagent volumes in at least some of the wells of reaction plates 301 as part of the synthesis process. The element array may also be actuated to bring at least some of the elements into contact with a quenching solution, a deprotection solution and/or a wash solution in wells of the plates 301 as part of the synthesis process.

[0071] Figure 4 depicts an illustrative element 400 and an illustrative well that are suitable for the exemplary embodiments. In this example, the element 400 is a cylindrical rod. The element 400 may be made of suitable materials, such as stainless steel, glass, plastic or the like. The element 400 may have one or more synthesis surfaces 404 on which a base molecule, like a polynucleotide, is bound. In the example shown in Figure 4, the distal end of the element 400 is where the synthesis surface 404 is located. In this example, the synthesis surface may extend about the circumference of the cylinder of the element 400. The remainder of the length of the element extending from the synthesis surface 404 to the proximal end of the element 400 may be covered with a hydrophobic coating, such as polytetrafluoroethylene (PTFE), perylene, polyimide or another coating. In some embodiments, where the synthesis surface is functionalized, the hydrophobic coating 406 limits the amount of unintended functionalization and extension beyond the functionalized surface.

[0072] A well 402 is formed in the plate 408. The well 402 holds a liquid reagent volume, such as a polymer extension solution (e.g., a polymerase-nucleotide conjugate solution), 410 that has been dispensed to the well 402. The shape and size of the well and the dipping element 400 are chosen so that the dipping element may be actuated downward into the well 402 to contact the polymer extension solution 410 as part of the synthesis. Specifically, the actuation at least partially immerses the distal portion of the functionalized surface 404 into the polymer extension solution 410. The distal portion of the functionalized surface 404 may remain in the polymer extension solution 410 for a predetermined period of time (e.g., 30 to 90 seconds) and then is removed by actuating the dipping element 400 out of the well 402. The downward and upward actuation are indicated in Figure 4 by the arrow 412.

[0073] The elements may assume a number of different configurations. The examples depicted thus far have been cylindrical rods with a circular cross-sections 502, such as shown in Figure 5. The cross-section of an element need not be circular. Instead, the cross-section of an element may be, for example, oval 504 or triangular 506. Further, the cross-section of an element may be square 508 or rectangular 510. These cross-section shapes shown in Figure 5 are intended to be illustrative and not limiting. The elements may have other cross- sectional shapes that are not shown.

[0074] An element does not have to be a rod. Figure 6 depicts a longitudinal side view of various alternate element designs. An element may be a rectangular plate 602 with a rectangular face of small thickness. An element may be a small a straight filament 604. The element may be an oval plate 606 with a wide oval face and a small thickness. Still further, an element may be a diamond- shaped plate 608 with a wide face and a small thickness.

More generally, the element may assume many different forms. The forms depicted herein are intended to be illustrative and not limiting.

[0075] The elements may be held by the element array so as to “float.” As shown in Figure 7A, an element 700 is held by the element array holder 702. An opening 704 is provided in the element array holder. The opening is sized and shaped to provide a passage through which the proximal end of the element 700 may pass. A top portion 706 is larger than the diameter of the opening 704. Thus, the element 700 is suspended the top portion 706 and passes through the opening 704. The diameter of the opening 704 may be large enough for the element to pass but to limit the degree to which the element may move laterally and angulate. The element “floats” in that it may move upward in direction 708 freely. Gravity pulls the element downward until the top portion 506 rests on the top surface of the element array holder.

[0076] The floating is demonstrated in Figure 7B. The element 700 has been actuated downward to contact the bottom surface 712 of the interior of the well 710. Since, the element 700 floats, the element 700 moves upward in direction 708 as shown so that the top portion of the element 700 no longer is resting of the top surface of the element array holder 702. The floating prevents the element from being damage and also prevents the well 710 from being damaged when the distal end of the element 500 contacts the bottom surface 712 of the well 710. Each element of the element array may be configured to float in this fashion. [0077] In some embodiments, other mechanisms, such as attachment of elements to a spring to allow some movement of the element when contacting a hard surface, while returning it to its original position after removal, may also be used.

[0078] Figure 8 depicts an example of a reaction plate 800 that has an array of wells 802 organized in a grid. In this example, the reaction plate 800 has an 8 x 12 grid of wells 802, for a total of 96 wells 802. Reaction plates with fewer or additional wells may be used in the exemplary embodiments. Each well 802 may be conical or cylindrical in shape with a circular opening 804. As mentioned above, at least some of the wells 802 may hold liquid reagent volumes, wash solution, deprotection solution, or quenching solution in some exemplary embodiments.

[0079] Figure 9 depicts a portion of a patterned surface plate 900 that may be used in exemplary embodiments. The patterned surface plate 900 may, for example, have hydrophilic regions like the circular regions 902 shown in Figure 9. The patterned surface plate 900 also may include hydrophobic regions 904 that surround the hydrophilic regions 902. As such, when a liquid reagent volume is applied to the hydrophilic regions by a dispenser, there is a propensity for the liquid reagent volume, which contains some water, to remain in the hydrophilic region and to not migrate to the hydrophobic regions 904. A wash solution, a deprotection solution, or a quenching solution also may be applied to such a patterned surface and stay resident on the hydrophilic regions 902. It should be appreciated that in other embodiments, a solution that has an affinity for hydrophobic regions may be dispensed on to the surface of the patterned surface. In that instance, the pattern on the patterned surface 900 may be reversed so that the regions that are hydrophobic and the regions that are hydrophilic may be reversed (i.e., regions 902 would be hydrophobic and regions 904 would be hydrophilic).

[0080] Figure 10 depicts a flowchart 1000 of illustrative steps that may be performed by the synthesis system 100 in exemplary embodiments to synthesize a polymer. At 1002, one or more surfaces on at least some of the elements in the element array 102 are processed so that the one or more surfaces of the elements become synthesis surfaces that are configured to act as sites where synthesis of portions of the polymer may take place. In the case where the polymer is an oligonucleotide, portions of the polymer are added iteratively by dipping the elements into different polymer extension solutions comprising a pretermined monomer and a polymerase in a predefined sequence as described below. Where the polymer is an oligonucleotide, the synthesis surface may be processed to have the one or more synthesis surfaces functionalized with a surface-bound DNA tag. At 1004, in some exemplary embodiments, hybridization is performed so that a starter sequence having a sufficiently complementary sequence is hybridized to a surface bound DNA tag. Figure 11 depicts operations 1100, 1102, and 1104 that may be performed in some exemplary embodiments as part of the hybridization 1004. Initially, at 1000, the dipping elements array may be washed by a washer. At 1102, the washed dipping elements may be immersed into a hybridization buffer so that the starter sequence is hybridized to the surface-bound DNA tag. At 1104, the dipping elements array is washed again by the washer.

[0081] In some exemplary embodiments, the hybridization 1004 need not be performed. Hence, the depiction of this operation in Figure 10 is in phantom form. For example, in one alternative, the starter sequence may be directly bound to the surface through a 5' amine functional group. This surface bound starter can be hybridized for quality control purposes or to prevent base-pairing between the starter sequence and the growing nucleic acid oligo on the surface, but hybridization is not necessary for the extension of the tag with a nucleotide since the surface-bound starter has an exposed 3' hydroxyl.

[0082] At 1006, the synthesis processing is performed to cause the desired polymer to be secured to the elements of the element array and ultimately cleaved from the elements. Figure 12 depicts a flowchart of illustrative steps that may be performed in exemplary embodiments in synthesis processing. The synthesis processing is iterative and has numerous cycles. A cycle represents the portion of the processing where a portion of the polymer is attached to the elements of the element array 102. In synthesizing an oligonucleotide, each cycle attaches a protected nucleotide to the elements. Thus, at 1202, the next cycle is initiated. At 1204, processing for the cycle is performed as described below. At 1206, after the cycle processing is completed, a check is made whether the last cycle has been processed. If not, the next cycle of processing continues with a new polymer extension solution or portion of the polymer being attached beginning again at 1202. When the last cycle has been completed, the full polymer has been attached to the elements of the element array. The polymer must then be detached from the elements. Where hybridization has been used, de-hybridization must be performed at 1208. At 1210, the synthesized polymers, such as oligonucleotides, may be harvested. The polymers may, for example be eluted from the surfaces of the wells of a reaction plate or of the hydrophilic locations on a patterned surface. [0083] Figure 13 depicts a flowchart 1300 of illustrative steps that may be performed in exemplary embodiments during processing for a single cycle. At 1302, polymer extension solution(s) is/are dispensed into the wells of a plate or locations on a patterned surface by dispenser(s) 126. A single liquid polymer extension solution may be dispensed to all of the wells or locations or different respective polymer extension solutions may be dispensed among corresponding subsets of the wells or locations. For example, as shown in Figure 14, wells in region 1402 (i.e., columns 1-6) may be filled with a first polymer extension solution comprising a first monomer; wells in region 1404 (i.e., columns 7-12) may be filled with a second polymer extension solution comprising a second monomer; wells in region 1406 (i.e., columns 13-18) may be filled with a third polymer extension solution comprising a third monomer; and wells in region 1408 (i.e., columns 19-24) may be filled with a fourth polymer extension solution comprising a fourth monomer. The wells of the plates or the hydrophilic locations on a patterned surface plate are filled with the desired polymer extension solution depending on the desired sequence of the product on each dipping element. During successive cycles, the elements may be dipped into the wells of the next successive region 1402, 1404, 1406 or 1408. The wells or locations on the patterned surface can either be prefilled or filled in-line by a high throughput dispenser (such as the Formulatrix Tempest). [0084] At 1304, in a coupling step, the synthesis surfaces of the elements are placed in contact with the liquid reagent volumes in the wells or locations. In some exemplary embodiments, the synthesis surfaces are at least partially immersed in the liquid reagent volumes for a specified period of time. The contact may be achieved by the actuator(s) 106 actuating the actuatable components 109 to move the element array holder 104, the reaction plate or patterned surface 128 or both of those items. The resulting movement caused by the actuation may be in the X, Y and/or Z direction, such as in the configurations 200 and 300 of Figures 2 and 3, or may include rotational or angular movement. The synthesis system 100 may contact the elements with the liquid reagent volumes (e.g., polymer extension solution) at the resolved loci for a sufficient time for monomer addition. During this reaction time, all of the elements may be immersed in the liquid reagent volumes simultaneously. The element array 102 can be formatted to match a subset of the wells or hydrophilic locations. For example, a 1536 well reaction plate may be filled with four monomer coupling steps, and used with an array of 384 dipping elements with a 4.5 mm pitch. Similarly, a lower throughput system using 8, 16, 24 or any subset of 1536 dipping elements may be aligned in an array to access wells of a 96, 384, or 1536 reaction well plate or locations on a patterned surface.

[0085] In order to achieve short reaction times during this coupling step, the plate or patterned surface may be heated by a thermal controller 130. The reaction plates or patterned surfaces 128 also may be chilled by the thermal controller 130 as a means of increasing stability before they are needed for a coupling step. In some embodiments, a Peltier may be used to rapidly control plate temperature. Alternatively, cold and warm stations may serve as the thermal controller 130 to change the temperature of the reagents in the reaction plates or patterned surfaces. Preformatted reaction plates or patterned surfaces also may be stored in a cold cabinet for several days at a time.

[0086] If necessary, agitation within the coupling well or location can be achieved by plunging the element into the well or location multiple times. Agitation may also be achieved by using a shaker 132, such as an orbital shaker or orbital stage motion, to move the reaction plate or patterned surface with respect to the dipping elements.

[0087] At 1304, the elements that were placed in contact with the polymer extension solution(s) may be placed in contact with a quenching solution for quenching the reaction. At 1306, the same elements may be placed in contact with a deprotection solution, such as proteinase K (ProK). These stations can be recirculating, continuously flowing, or static baths. The ProK cleavage step may be heated in order to increase the enzymatic reaction rate. At 1308, the elements are washed. The washing of the elements can be carried out in dedicated stations of the synthesis system 100, such as described above. Between coupling cycles, the reaction plate or patterned surface may be washed with the plate washer 122 and reloaded with new polymer extension solution in order to save plasticware space and cost. For example, if using an array of 384 elements with a 1536 reaction well plate, the wash cycle may be every four coupling steps.

[0088] While exemplary embodiments have been described herein, various changes in form and detail may be made with departing from the scope of the attached claims.

Enzymatic Synthesis using Protected Nucleotides and Conjugates

[0089] In some embodiments, synthesis of a polynucleotide comprises adding nucleotides stepwise to a synthesis initiator (e.g., an initial oligonucleotide) via the cycled steps of: addition of polymerase-nucleotide conjugate to an oligonucleotide, binding of the nucleotide to the 3' end of the oligonucleotide catalyzed by the polymerase, and cleavage of the polymerase from the added nucleotide. These steps can be repeated until a desired polynucleotide is synthesized.

[0090] In some embodiments, synthesis of a polynucleotide comprises adding nucleotides stepwise to a starter molecule (e.g., an initial oligonucleotide) via the cycled steps of: addition of polymerase-nucleotide conjugate to an oligonucleotide, binding of the nucleotide to the 3' end of the oligonucleotide catalyzed by the polymerase, and cleavage of the polymerase from the added nucleotide. These steps can be repeated until a desired polynucleotide is synthesized.

[0091] In some embodiments, the present disclosure includes a method of synthesizing a polynucleotide comprising contacting a precursor polynucleotide with a nucleotide and a polymerase, wherein said nucleotide comprises comprising a protecting group bound to a base pairing oxygen or nitrogen on the nucleobase. In some embodiments, the method of synthesizing a polynucleotide comprises removing a blocking group, such as a conjugated polymerase or a reversible terminator, after addition of a nucleotide to a precursor polynucleotide. In some embodiments, the method of synthesizing a polynucleotide comprises repeating contacting, adding, and optionally removing a blocking group described herein one or more times. In some embodiments, removal of one or more protecting groups described herein comprises exposing said polynucleotide to a chemical or photolytic condition capable of removing said one or more protecting groups from said protected nucleobases.)

[0092] In some embodiments, the nucleotides described herein comprise a protecting group that is a reversible terminator group, such as such as an O- azidomethyl or O-NH2 group on the 3' position of the sugar or an (alpha-tertbutyl-2- nitrobcnzyljoxymcthl group on the 5 position of pyrimidines or the 7 position of 7- deazapurines (for an overview see, e.g. Chen et al., Genomics, Proteomics & Bioinformatics 2013 11: 34-40). In these embodiments, the nucleotide analog prevents or hinders further elongation once incorporated into a nucleic acid to achieve controlled termination of synthesis. In some embodiments, when used as part of a conjugate, the RTdNTP- polymerase conjugates do not rely on the shielding effect to achieve termination, e.g. when a 3' modified RTdNTP is tethered to the polymerase, the linker used may exceed 100 A or 200 A in length. Conjugates

[0093] In some embodiments, the nucleotides described herein comprise a protecting group that is the polymerase linked to the nucleotide. Accordingly, described herein are methods of nucleic acid synthesis using conjugates comprising a polymerase and a nucleotide, wherein the polymerase and the nucleotide are linked via a linker that comprises a cleavable linkage. The polymerase moiety of a conjugate can elongate a nucleic acid using its linked nucleotide (i.e., the polymerase can catalyze the attachment of a nucleotide to which it is joined onto a nucleic acid) and remains attached to the elongated nucleic acid via the linker until the linker is cleaved.

[0094] When a conjugate comprising a polymerase and a nucleoside polyphosphate is incubated with a nucleic acid, it preferentially elongates the nucleic acid using its tethered nucleotide (as opposed to using the nucleotide of another conjugate molecule). As described above, the polymerase then remains attached to the nucleic acid via its tether to the added nucleotide until exposed to some stimulus that causes cleavage of the linkage to the added nucleotide. In this situation, further extensions by polymerase-nucleotide conjugates are hindered (i.e., the nucleotide is “shielded” or “protected”) when: 1) the attached polymerase molecule hinders other conjugates from accessing the 3' OH of the extended DNA molecule and 2), other nucleoside polyphosphates in the system are hindered from accessing the catalytic site of the polymerase that remains attached to the 3' end of the extended nucleic acid. (The extent of shielding may be described as the extent to which both of these interactions are hindered.) To enable subsequent extensions, the linker tethering the incorporated nucleotide to the polymerase can be cleaved, releasing the polymerase from the nucleic acid and therefore re-exposing its 3' OH group for subsequent elongation.

[0095] Methods for nucleic acid synthesis provided herein that employ the shielding effect to achieve termination comprise an extension step wherein a nucleic acid is exposed to conjugates preferentially in the absence of free (i.e. untethered) nucleoside polyphosphates, because the termination mechanism of shielding may not prevent their incorporation into the nucleic acid.

[0096] In some embodiments, termination of further elongation may be "complete," meaning that after a nucleic acid molecule has been elongated by a conjugate, further elongations cannot occur during the reaction. In other embodiments, termination of further elongation may be "incomplete," meaning that further elongations can occur during the reaction but at a substantially decreased rate compared to the initial elongation, e.g., 100 times slower, or 1000 times slower, or 10,000 times slower, or more. Conjugates that achieve incomplete termination may still be used to extend a nucleic acid by predominantly a single nucleotide (e.g., in methods for nucleic acid synthesis and sequencing) when the reaction is stopped after an appropriate amount of time. In some embodiments, the reagent containing the conjugate may additionally contain polymerases without tethered nucleoside polyphosphates.

[0097] Reagents based on conjugates employing the shielding effect to achieve termination preferentially only contain polymerase-nucleotide conjugates in which all polymerases remain folded in the active conformation. In some cases, if the polymerase moiety of a conjugate is unfolded, its tethered nucleoside polyphosphate may become more accessible to the polymerase moieties of other conjugate molecules. In these cases, the unshielded nucleotides may be more readily incorporated by other conjugate molecules, circumventing the termination mechanism.

[0098] Polymerase-nucleotide conjugates employing the shielding effect to achieve termination are preferentially only labeled with a single nucleoside polyphosphate moiety. Polymerase-nucleotide conjugates labeled with multiple nucleoside polyphosphates that can access the catalytic site can, in some cases, incorporate multiple nucleoside polyphosphates into the same nucleic acid. Additional tethered nucleotides may therefore lead to additional, undesired nucleotide incorporations into a nucleic acid during a reaction. Furthermore, only one tethered nucleoside polyphosphates can occupy the (buried) catalytic site of its polymerase at a time so the other tethered nucleoside polyphosphate(s) may have an increasing accessibility to the polymerase moieties of other conjugate molecules, as discussed below.

[0099] Polymerase-nucleotide conjugates employing the shielding effect to achieve termination preferentially comprise as short of a linker as possible that still enables the nucleoside polyphosphate to frequently access the catalytic site of its tethered polymerase molecule in a productive conformation, in order to enable fast incorporation of the nucleotide into a nucleic acid. Such conjugates may also preferentially employ an attachment position of the linker to the polymerase as close to the catalytic site as possible, enabling use of a shorter linker. The length of the linker will determine the maximum distance from the attachment point a tethered nucleoside polyphosphate or a tethered nucleic acid can reach. A smaller distance may lead to a reduced accessibility of the tethered moiety to other polymerase- nucleotide molecules, as discussed below. In some embodiments, linkers are approximately 24 and 28 A long. Shorter linkers, e.g. with lengths of 8-15 A may increase shielding; longer linkers, e.g. linkers longer than 50 A, 70 A or 100 A, may reduce shielding. The shielding effect may be influenced by a combination of factors including, but not limited to, the structure of the polymerase, the length of the linker, the structure of the linker, the attachment position of the linker to the polymerase, the binding affinity of the nucleoside polyphosphate to the catalytic site of the polymerase, the binding affinity of the nucleic acid to the polymerase, the preferred conformation of the polymerase, and/or the preferred conformation of the linker.

[00100] One contribution to shielding can be steric effects that block the 3' OH of a nucleic acid that has been elongated by a conjugate from reaching into the catalytic site of another conjugate's polymerase moiety. Steric effects may also hinder a tethered nucleoside polyphosphate from reaching into the catalytic site of another polymerase-nucleotide conjugate molecule due to clashes between the conjugates that would occur during such approaches. These steric effects may result in complete termination if they completely block productive interactions between the tethered nucleoside polyphosphate (or elongated nucleic acid) of one conjugate molecule with another conjugate molecule, or may result in incomplete termination if they only hinder such intermolecular interactions.

[00101] Another contribution to shielding arises from the binding affinity of the tethered nucleoside polyphosphate to the catalytic site of the polymerase. The tethered nucleoside polyphosphate of a conjugate will have a high effective concentration with respect to the catalytic site of its tethered polymerase so it may remain bound to that site much of the time. When the nucleoside polyphosphate is bound to the catalytic site of its tethered polymerase molecule, it is unavailable for incorporation by other polymerase molecules. Thus, tethering reduces the effective concentration of nucleoside polyphosphates available for intermolecular incorporation (i.e. incorporation catalyzed by a polymerase molecule to which the nucleotide is not tethered). This shielding effect can enhance termination by reducing the rate by which a nucleic acid is elongated using the nucleoside polyphosphate moiety of one conjugate molecule by the polymerase moiety of another conjugate molecule.

[00102] Another contribution to shielding arises from the binding affinity of the 3' region of a nucleic acid molecule to the catalytic site of a polymerase molecule. After elongation by a conjugate, the nucleic acid is tethered to the conjugate via its 3' terminal nucleotide and will have a high effective concentration with respect to the catalytic site of its tethered polymerase so it may remain bound to that site much of the time. When the nucleic acid is bound to the catalytic site of its tethered polymerase molecule, it is unavailable for elongation by other conjugate molecules. This effect can enhance termination by reducing the rate by which a nucleic acid that has been elongated by a first conjugate is further elongated by other conjugate molecules.

[00103] In some embodiments, the polymerase-nucleotide conjugates comprise additional moieties that sterically hinder the tethered nucleoside polyphosphate (or a tethered nucleic acid post-elongation) from approaching the catalytic sites of another conjugate molecule. Such moieties include polypeptides or protein domains that can be inserted into a loop of the polymerase, and those and other bulky molecules such as polymers that can be site- specifically ligated e.g. to an inserted unnatural amino acid or specific polypeptide tag. [00104] In some embodiments, the linker is attached to the 5 position of pyrimidines or the 7 position of 7-deazapurines. In other embodiments, the linker may be attached to an exocyclic amine of a nucleobase, e.g. by N-alkylating the exocyclic amine of cytosine with a nitrobenzyl moiety as discussed herein. In other embodiments, the linker may be attached to any suitable atom of the nucleotide to form a conjugate, such as the phosphate, sugar, or base of the nucleotide, as will be apparent to those skilled in the art. In some embodiments, the linker is attached to the alpha-phosphate, sugar, or base of the nucleotide so that the polymerase remains attached to the nucleotide after addition to the 3’ end of an oligonucleotide. In some embodiments, the linker is attached to the P-phosphate, %- phosphate, 5-phosphate, s-phosphate, (^-phosphate, or y -phosphate of a nucleotide. In some embodiments, the linker is attached to the terminal phosphate of a nucleotide.

[00105] Certain polymerases have a high tolerance for modification of certain parts of a nucleotide, e.g. modifications of the 5-position of pyrimidines and the 7-position of purines are well-tolerated by some polymerases (He and Seela., Nucleic Acids Research 30.24 (2002): 5485-5496.; or Hottin et al., Chemistry. 2017 Feb 10;23(9):2109-2118). In some embodiments, the linker is attached to these positions.

[00106] In some embodiments, a polymerase-nucleotide conjugate is prepared by first synthesizing an intermediate compound comprising a linker and a nucleotide (referred to herein as a "linker-nucleotide"), and then the intermediate compound is attached to the polymerase. By way of non-limiting examples, in some embodiments, nucleosides with substitutions compared to natural nucleosides, e.g. pyrimidines with 5 -hydroxymethyl or 5- propargylamino substituents, or 7- deazapurines with 7-hydroxymethyl or 7-propargylamino substituents may be useful starting materials for preparing linker- nucleotides. An exemplary 1 set of nucleosides with 5- and 7- hydroxymethyl substituents that may be useful for preparing linker-nucleotides is shown below:

[00107] An exemplary set of nucleosides with 5- and 7-deaza-7-propargylamino substituents that may be useful for preparing linker-nucleotides is shown below:

[00108] These nucleosides are also commercially available as deoxyribonucleoside polyphosphates.

[00109] In some embodiments a method of preparation (e.g., comprising an intermediate compound), the conjugate comprises a linker-nucleotide.

[00110] In some embodiments, the linker-nucleotide comprises a nucleotide. In some embodiments, the linker-nucleotide comprises a nucleotide polyphosphate or a modified nucleotide polyphosphate. In some embodiments, the linker-nucleotide comprises a nucleotide triphosphate or a modified nucleotide triphosphate. Any suitable nucleotide may be used. It is understood that a nucleotide comprises a nucleobase (e.g. adenine, guanine, cytosine thymine or uracil), a sugar (e.g. a ribose or a deoxyribose), and a polyphosphate. It is understood that a nucleoside comprises a nucleobase (e.g. adenine, guanine, cytosine thymine or uracil) and a sugar (e.g. a ribose or a deoxyribose).

[00111] In some embodiments, the linker-nucleotide comprises a nucleotide polyphosphate. In some embodiments, the linker-nucleotide comprises a modified nucleotide polyphosphate. It is understood that the polyphosphate portion of a nucleotide can be a monophosphate, a diphosphate, a triphosphate, a tetraphosphate, a heptaphosphate, or a pentaphosphate. In some embodiments, the nucleotide polyphosphate comprises a nucleoside triphosphate or a modified nucleoside triphosphate. In some embodiments, the linker-nucleotide comprises a nucleotide tetraphosphate or a modified nucleotide tetraphosphate. In some embodiments, the linker-nucleotide comprises a nucleotide pentaphosphate or a modified nucleotide pentaphosphate. In some embodiments, the linker-nucleotide comprises a nucleotide hexaphosphate or a modified nucleotide hexaphosphate.

[00112] In some embodiments, the linker-nucleotide comprises a modified nucleobase. In some embodiments, the linker-nucleotide comprises a modified nucleobase. In some embodiments, the modified nucleobase comprises an O- or N-linked modification. In some embodiments, the O- or N-linked modification is removable following incorporation of the nucleotide portion of the linker-nucleotide into a polynucleotide. In some embodiments, the O- or N-linked modification is removable by a photolytic process. In some embodiments, the photolytic process comprises exposure to UV light, wherein the UV light comprises wavelengths at 365 nm and/or 405 nm. In some embodiments, the O- or N-linked modification is removable by a chemical process. In some embodiments, the chemical process is selected from a beta-elimination reaction, a Pd-catalyzed deallylation, and a reduction reaction. In some embodiments, the O- or N-linked modification is removable by an enzymatic process. In some embodiments, the enzymatic process comprises removal by an alkyltransferase or methyltransferase.

[00113] In some embodiments, the O- or N-linked modification reduces or eliminates Watson-Crick base pairing in a polynucleotide comprising the modified nucleobase. In some embodiments, the O- or N-linked modification reduces or eliminates secondary structure in a polynucleotide comprising the modified nucleobase. In some embodiments of the method, following removal of the O- or N-linked modification the modified nucleobase comprises a natural nucleobase. In some embodiments, the natural nucleobase is guanine, cytosine, adenine, thymine, or uracil.

[00114] The conjugates provided herein comprise a polymerase tethered to a nucleotide via a linker.

[00115] Any suitable linker for tethering a nucleoside polyphosphate to a polymerase is contemplated for use in the methods described herein. In some embodiments, the linker is specifically attached to a cysteine residue of the polymerase using a sulfhydryl- specific attachment chemistry. Illustrative sulfhydryl specific attachment chemistries include, without limitation, ortho-pyridyl disulfide (OPSS), maleimide functionalities, 3- arylpropiolonitrile functionalities, allenamide functionalities, haloacetyl functionalities such as iodoacetyl or bromoacetyl, alkyl halides or perfluroaryl groups that can favorably react with sulfhydryls surrounded by a specific amino acid sequence (Zhang, Chi, et al. Nature chemistry 8, (2015) 120-128.). Other attachment chemistries for specific labeling of cysteine residues will be apparent to those skilled in the art or are described in the pertinent literature and texts (e.g., Kim, Younggyu, et al, Bioconjugate chemistry 19.3 (2008): 786-791.).

[00116] In some embodiments, the linker is attached to a lysine residue via an amine - reactive functionality (e.g. NHS esters, Sulfo-NHS esters, tetra- or pentafluorophenyl esters, isothiocyanates, sulfonyl chlorides, etc.). In some embodiments, the linker is attached to the polymerase via attachment to a genetically inserted unnatural amino acid, e.g. p- propargyloxyphenylalanine or p- azidophenylalanine that could undergo azide-alkyne Huisgen cycloaddition, though many suitable unnatural amino acids suitable for site-specific labeling exist and can be found in the literature (e.g. as described in Lang and Chin., Chemical reviews 114.9 (2014): 4764-4806.).

[00117] In some embodiments, the linker may be specifically attached to the polymerase N- terminus. In some embodiments, the polymerase is mutated to have an N-terminal serine or threonine residue, which may be specifically oxidized to generate an N-terminal aldehyde for subsequent coupling to e.g. a hydrazide. In some embodiments, the polymerase is mutated to have an N-terminal cysteine residue that can be specifically labeled with an aldehyde to form a thiazolidine. In some embodiments, an N-terminal cysteine residue can be labeled with a peptide linker via Native Chemical Ligation.

[00118] In some embodiments, a peptide tag sequence may be inserted into the polymerase that can be specifically labeled with a synthetic group by an enzyme, e.g. as demonstrated in the literature using biotin ligase, transglutaminase, lipoic acid ligase, bacterial sortase and phosphopantetheinyl transferase (e.g. as described in refs. 74-78 of Stephanopoulos & Francis Nat. Chem. Biol. 7, (2011) 876-884).

[00119] In some embodiments, the linker is attached to a labeling domain fused to the polymerase. For example, a linker with a corresponding reactive moiety may be used to covalently label SNAP tags, CLIP tags, HaloTags and acyl carrier protein domains (e.g. as described in refs. 79-82 of Stephanopoulos & Francis Nat. Chem. Biol. 7, (2011) 876-884). [00120] In some embodiments, the linker is attached to an aldehyde specifically generated within the polymerase, as described in Carrico et al. (Nat. Chem. Biol. 3, (2007) 321 - 322). For example, after insertion of an amino acid sequence that is recognized by the enzyme formylglycine-generating enzyme (FGE) into the polymerase, it may be exposed to FGE, which will specifically convert a cysteine residue in the recognition sequence to formylglycine (i.e. producing an aldehyde). This aldehyde may then be specifically labeled with e.g. a hydrazide or aminooxy moiety of a linker. [00121] In some embodiments, a linker may be attached to the polymerase via non-covalent binding of a moiety of the linker to a moiety fused to the polymerase. Examples of such attachment strategies include fusing a polymerase to streptavidin that can bind a biotin moiety of a linker, or fusing a polymerase to anti-digoxigenin that can bind a digoxigenin moiety of a linker. In some embodiments, site-specific labeling may lead to an attachment of the linker to the polymerase that may readily be reversed (e.g. an ortho-pyridyl disulfide (OPSS) group that forms a disulfide bond with a cysteine that can be cleaved using reducing agents, e.g. using TCEP), other attachment chemistries will produce permanent attachments. [00122] In some embodiments, the polymerase is mutated to ensure specific attachment of the tethered nucleotide to a particular location of the polymerase, as will be apparent to those skilled in the art. For example, with sulfhydryl-specific attachment chemistries such as maleimides or ortho-pyridyl disulfides, accessible cysteine residues in the wild-type polymerase may be mutated to a non-cysteine residue to prevent labeling at those positions. On this "reactive cysteine-free" background, a cysteine residue may be introduced by mutation at the desired attachment position. These mutations preferentially do not interfere with the activity of the polymerase.

[00123] Other strategies for site-specific attachment of synthetic groups to proteins will be apparent to those skilled in the art and are reviewed in literature, (e.g., Stephanopoulos & Francis Nat. Chem. Biol. 7, (2011) 876-884).

[00124] As described above, when a conjugate comprising a polymerase (e.g., a templateindependent polymerase) and a nucleotide is incubated with a nucleic acid or a polynucleotide, it preferentially elongates (i.e., extends) the nucleic acid or polynucleotide by incorporating the tethered nucleotide or modified nucleotide (as opposed to using the nucleotide or modified nucleotide of another conjugate molecule) into the nucleic acid or polynucleotide. In some embodiments, a polymerase in a polymerase-nucleotide conjugate is folded in an active conformation. In other embodiments, a polymerase in a polymerasenucleotide conjugate is unfolded.

[00125] Any polymerase capable of extending a polynucleotide, incorporating a nucleotide into a polynucleotide, or incorporating a nucleotide analog into a polynucleotide is envisaged for use in the methods described herein. In some embodiments, the polynucleotide is single stranded. In some embodiments, the polynucleotide is double stranded. In some embodiments, the polynucleotide is immobilized on a solid support. [00126] For DNA synthesis applications, in particular template-independent polymerases, e.g., a terminal deoxynucleotidyl transferase (TdT) or DNA nucleotidylexotransferase, which terms are used interchangeably to refer to an enzyme having activity as described for E.C. class 2.7.7.31 may be used.

[00127] In some embodiments, methods of the present disclosure use conjugates comprising template-independent polymerases. In some embodiments, conjugates comprise a Pol-X family polymerase. In some embodiments, conjugates comprise a polymerase Terminal deoxynucleotidyl Transferase (TdT), or a variant thereof (e.g., a non-wild-type TdT, e.g., a modified TdT). In some embodiments of the method, the template-independent polymerase is a TdT or a variant thereof (i.e., a modified TdT).

[00128] A variety of different template-free polymerases are available for use in methods of embodiments. Template-free polymerases include, but are not limited to, polX family polymerases (including DNA polymerases b, 1 and m), poly(A) polymerases (PAPs), poly(U) polymerases (PUPs), DNA polymerase 0, and the like, for example, described in the following references: Ybert et al, International patent publication WO2017/216472;

Champion et al, U.S. patent 10435676; Champion et al, International patent publication W02020/099451; Heinisch et al, International patent publication W02021/018919. In particular, terminal deoxynucleotidyltransferases (TdTs) and variants thereof are useful in template-free DNA synthesis.

[00129] In some embodiments of the method, the polymerase is a fusion protein. In some embodiments of the method, the fusion protein comprises maltose binding protein (MBP). [00130] In some embodiments of the method, the TdT or variant thereof may be operably linked to a linker moiety including a covalent or non-covalent bond; amino acid tag (e.g., poly-amino acid tag, poly-His tag, 6His-tag; chemical compound (e.g., polyethylene glycol); protein-protein binding pair (e.g., biotin-avidin); affinity coupling; capture probes; or any combination of these. The linker moiety can be separate from or part of a TdT variant.

[00131] Illustrative examples of polymerases with the ability to extend single stranded nucleic acids include, but are not limited to, Polymerase Theta (Kent et al., eLife 5 (2016): el3740.), polymerase mu (Juarez et al., Nucleic acids research 34.16 (2006): 4572-4582.; or McElhinny et all., Molecular cell 19.3 (2005): 357-366.) or polymerases where templateindependent activity is induced, e.g. by the insertion of elements of a template-independent polymerase (Juarez et al., Nucleic acids research 34.16 (2006): 4572-4582). In other DNA synthesis applications, the polymerase can be a template-dependent polymerase i.e., a DNA- directed DNA polymerase (which terms are used interchangeably to refer to an enzyme having activity 2.7.7.7 using the IUBMB nomenclature).

[00132] In some embodiments, such as RNA synthesis applications, tethered ribonucleotides (e.g., ribonucleostide polyphosphates) may be used. In some such embodiments, a RNA specific nucleotidyl transferase, such as E. coli Poly(A) Polymerase (IUBMB EC 2.7.7.19) or Poly(U) Polymerase, among others, may be employed. The RNA nucleotidyl transferases can contain modifications, e.g., single point mutations, that influence the substrate specificity towards a specific rNTP (Lunde et al., Nucleic acids research 40.19 (2012): 9815-9824.). In some embodiments, a very short tether between an RNA nucleotidyl transferase and a ribonucleotide (e.g., ribonucleoside triphosphate) may be used to induce a high effective concentration of the ribonucleotide (e.g., ribonucleoside polyphosphate), thereby forcing incorporation of an rNTP that might not be the natural substrate of the nucleotidyl transferase.

[00133] In some embodiments, a conjugate of the present disclosure comprises a linker. In some such embodiments, the linker comprises at least the atoms that connect the nucleotide to the polymerase. The linker can attach to the base, the sugar, or the aphosphate of the nucleotide or modified nucleotide to the polymerase. In some embodiments, the polymerase and the nucleotide are attached with a linker. In some such embodiments, the polymerase and the nucleotide are covalently linked (via the linker) and the distance between the linked atom of the nucleotide and the polymerase to which it is attached can be, for example, in the range of about 4-100 A, about 15-40 A or about 20-30 A, or a distance appropriate for the position on the polymerase to which the nucleotide (e.g., nucleoside polyphosphate) is tethered. Any suitable linker for tethering the nucleotide or modified nucleotide to the polymerase is contemplated in the methods described herein. In some embodiments, the linker comprises a poly ether or a polyethylene glycol (PEG). In some embodiments, the linker comprises one or more peptide bonds. In some embodiments, the linker comprises one or more sarcosines. In some embodiments, the linker comprises one or more glycines. In some embodiments, the linker comprises one or more prolines. In some embodiments, the linker comprises a carbamate. In some embodiments, the linker joins to the nucleotide at an atom of the nucleobase that is not involved in base pairing. In such embodiments, the linker is considered to be at least the atoms that connect the polymerase to any atom in the monocyclic or polycyclic ring system bonded to the T position of the sugar (e.g. pyrimidine or purine or 7- deazapurine or 8-aza-7-deazapurine). In some embodiments, the linker joins to the nucleotide at an atom of the nucleobase that is involved in base pairing. In some embodiments, the linker is joined to the sugar or to the a-phosphate of the nucleotide. In some embodiments, the linker is sufficiently long to allow the nucleotide (e.g., nucleoside polyphosphate) to access the active site of the polymerase to which it is tethered. As described in greater detail herein, the polymerase of a conjugate is capable of catalyzing the addition of the nucleotide to which it is linked onto the 3' end of a nucleic acid.

[00134] As described herein, a the linker may be attached to various positions on a nucleotide (e.g., of a conjugate of the present disclosure), and a variety of cleavage strategies may be used. It is understood that the cleavage strategy will be determined by the type of linker joining the nucleotide or modified nucleotide and the polymerase. Any suitable method for cleaving a linker is contemplated in the methods described herein.

[00135] In some embodiments, the linker is cleaved, wherein following cleavage of the linker, a nucleotide comprising a chemical group from the retained portion of the linker (i.e. a scar) is formed, e.g. illustrative, non-limiting, chemical groups (i.e. scars) following linker cleavage are shown below. In some embodiments, the chemical group e.g.is removed by a chemical, photolytic, or enzymatic process.

[00136] In some embodiments, the linker may be cleaved by exposure to any suitable reducing agent such as dithiothreitol (DTT), P-mercaptoethanol, or tris(2- carboxyethyl)phosphine (TCEP). For example, a linker comprising a 4- (disulfaneyl)butanoyloxy-methyl group attached to the 5 position of a pyrimidine or the 7 position of a 7-deazapurine may be cleaved by reducing agents (e.g. DTT) to produce a 4- mercaptobutanoyloxymethyl scar on the nucleobase. This scar may undergo intramolecular thiolactonization to eliminate a 2-oxothiolane, leaving a smaller hydroxymethyl scar on the nucleobase. An example of such a linker attached to the 5-position of cytosine is depicted below, but the strategy is applicable to any suitable nucleobase:

[00137] In other embodiments, the linker may be cleaved by exposure to light. For example a linker comprising a (2-nitrobenzyl)oxymethyl group may be cleaved with 365 nm light, leaving a hydroxymethyl scar, e.g. as depicted for cytosine below, but the strategy is applicable to any suitable nucleobase:

(where, e.g., R' ' =H or R' '=CH3 or R' =i-Bu.)

[00138] In other embodiments, the linker may comprise a 3-(((2- nitrobenzyl)oxy)carbonyl)aminopropynyl group that may be cleaved with 365 nm light to release a nucleobase with a propargylamino scar. This strategy is applicable to any suitable nucleobase:

_£

[00139] In other embodiments, the linker may comprise an acyloxymethyl group that may be cleaved with a suitable esterase to release a nucleobase with a hydroxymethyl scar, e.g. as depicted for cytosine below, but the strategy is applicable to any suitable nucleobase:

[00140] In such embodiments, the linker may comprise additional atoms (included in R' above) adjacent to the ester that increase the activity of the esterase towards the ester bond. In other embodiments, the linker may comprise an N-acyl-aminopropynyl group that may be cleaved with a peptidase to release a nucleobase with propargylamino scar, e.g. as depicted for 5 -propargylamino cytosine below, but the strategy is applicable to any suitable nucleobase:

[00141] In such embodiments, the linker may comprise additional atoms (included in R' above) adjacent to the amide that increase the activity of the peptidase towards the amide

bond.

[00142] Equivalents and Scope

[00143] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments in accordance with the embodiments described herein. The scope of the claims is not intended to be limited to the above Description, but rather is as set forth in the appended claims.

[00144] In the claims, articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. Embodiments may have exactly one member of the group is present, employed in, or otherwise relevant to a given product or process. Embodiments may have more than one, or all of the group members present in, employed in, or otherwise relevant to a given product or process.

[00145] It is also noted that the term “comprising” is intended to be open and permits but does not require the inclusion of additional elements or steps. When the term “comprising” is used herein, the term “consisting of’ is thus also encompassed and disclosed.

[00146] Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. [00147] All cited sources, for example, references, publications, databases, database entries, and art cited herein, are incorporated into this application by reference, even if not expressly stated in the citation. In case of conflicting statements of a cited source and the instant application, the statement in the instant application shall control.

[00148] Section and table headings are not intended to be limiting.

OTHER EMBODIMENTS

[00149] It is to be understood that the words which have been used are words of description rather than limitation, and that changes may be made without departing from the true scope and spirit of the claims.

[00150] While several embodiments are described herein, it is not intended that the claims should be limited to any such particulars or embodiments or to any particular embodiment, but rather the claims should be construed to provide the broadest possible interpretation of such claims in view of the prior art.

[00151] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, section headings, the materials, methods, and examples are illustrative only and not intended to be limiting.

Claims

1. A system for multiplex polymer synthesis, comprising; a plurality of elements, each of the elements comprising a synthesis surface on which a base molecule for polymer synthesis is bound; a plate comprising resolved loci each adapted for containing a respective liquid reagent volume; and at least one actuator for actuating the plurality of elements and/or the plate between i) first relative positions wherein the synthesis surfaces are separate from the resolved loci, and ii) second relative positions wherein the synthesis surfaces are within sufficient proximity to the resolved loci to contact the respective liquid reagent volumes when present at the resolved loci on the plate surface.

2. The system of claim 1, wherein the resolved loci are wells.

3. The system of claim 1, wherein the plate comprises a patterned surface separating said resolved loci.

4. The system of claim 3, wherein the patterned surface has a pattern of hydrophobic regions and hydrophilic regions.

5. The system of claim 3, wherein the resolved loci are separated by hydrophobic regions on the patterned surface.

6. The system of claim 3 or 5, wherein the resolved loci are hydrophilic regions on the patterned surface.

7. The system of claim 1, wherein at least a subset of the resolved loci are spatially separated to be capable of alignment with the plurality of elements.

8. The system of claim 1, wherein the elements are one of rods, plates, or filaments.

9. The system of claim 1, wherein the synthesis surface is functionalized to bind to said base molecule.

10. The system of claim 2, wherein the elements are sized and shaped to fit into the wells.

11. The system of claim 1, further comprising: an elements holder for holding the plurality of elements so that the plurality of elements may be actuated together; and an actuatable component secured to the elements holder, the actuatable component being actuatable by the at least one actuator.

12. The system of claim 1, wherein the base molecule is a synthesis initiator comprising a reactive group for coupling to a first monomer in a cycled polymer synthesis reaction.

13. The system of claim 1, wherein the base molecule is capable of binding to a synthesis initiator comprising a reactive group for coupling to a first monomer in a cycled polymer synthesis reaction.

14. The system of claim 13, wherein said binding of the base molecule to said synthesis initiator is via one or more linking molecules.

15. The system of claim 1, wherein the base molecule is a first oligonucleotide.

16. The system of claim 15, wherein the first oligonucleotide comprises a free 3' hydroxyl group.

17. The system of claim 16, wherein the first oligonucleotide comprises a single- stranded region at the 3' end.

18. The system of claim 15, wherein the first oligonucleotide is capable of hybridizing specifically to a second oligonucleotide, wherein the second oligonucleotide comprises a free 3' hydroxyl end when hybridized to the first oligonucleotide.

19. The system of claim 18, wherein the second oligonucleotide comprises a singlestranded region at the 3' hydroxyl end when hybridized to the first oligonucleotide.

20. The system of claim 1, wherein the at least one actuator is: operationally linked to and configured to align the plurality of elements and at least a subset of the resolved loci across a first plane; and operationally linked to and configured to move the aligned plurality of elements and the at least a subset of the resolved loci between said first and second relative positions along an axis orthogonal to the first plane.

21. The system of claim 1, wherein the at least one actuator comprises: a first actuator operationally linked to and configured to align the plurality of elements and at least a subset of the resolved loci across a first plane; and

22. a second actuator operationally linked to and configured to move the aligned plurality of elements and the at least a subset of the resolved loci between said first and second relative positions along an axis orthogonal to the first plane. The system of claim 1, wherein said system comprises a dispenser for dispensing liquid reagents to the resolved loci.

23. The system of claim 22, wherein said dispenser comprises one or more nozzles each capable of dispensing a liquid reagent to the resolved loci.

24. The system of claim 22, wherein said system is configured to simultaneously i) actuate the plurality of elements or a first plate between said first relative positions and said second relative positions, and ii) dispense liquid reagents to the resolved loci on a second plate using said dispenser.

25. The system of claim 22, wherein the dispenser is connected via a fluidic pathway to a reagent container for holding a polymer extension solution , wherein said polymer extension solution comprises a monomer and an enzyme capable of catalyzing addition of the monomer to a synthesis initiator or a polymer comprising said base molecule.

26. The system of claim 25, wherein the enzyme is a polymerase.

27. The system of claim 26, wherein the polymerase is a template-independent polymerase.

28. The system of any one of claims 25-27, wherein the enzyme is bound to the monomer via a linker, forming a protected monomer.

29. The system of any one of claims 25-28, wherein the monomer comprises a protecting group.

30. The system of claim 28 or 29, wherein the monomer is a nucleotide.

31. The system of claim 22, wherein the dispenser is connected via a fluidic pathway to a reagent container for holding a deprotection solution.

32. The system of claim 31, wherein said deprotection solution is capable of removing a protecting group from a monomer.

33. The system of claim 31, wherein said deprotection solution comprises a linker cleavage reagent capable of separating an enzyme bound to a monomer via a cleavable linker.

34. The system of claim 22, wherein the dispenser is connected via a fluidic pathway to a reagent container for holding a reaction quenching solution.

35. The system of claim 22, wherein the dispenser is connected via a fluidic pathway to a reagent container for holding a wash solution.

36. A system for multiplex polymer synthesis, comprising; a plurality of elements, each of the elements comprising a synthesis surface on which a base molecule for polymer synthesis is bound; a plate comprising resolved loci containing a liquid reagent; and at least one actuator for actuating the plurality of elements or the plate to cause the synthesis surfaces to be at least partially immersed into corresponding ones of the resolved loci containing the liquid reagent to perform one or more polymer synthesis steps on the synthesis surfaces.

37. The system of claim 36, wherein the resolved loci are wells.

38. The system of claim 36, wherein the plate comprises a patterned surface separating said resolved loci

39. The system of claim 38, wherein the patterned surface has a pattern of hydrophobic regions and hydrophilic regions.

40. The system of claim 38, wherein the resolved loci are separated by hydrophobic regions on the patterned surface.

41. The system of claim 38 or 40, wherein the resolved loci are hydrophilic regions on the patterned surface.

42. The system of claim 36, wherein at least a subset of the resolved loci are spatially separated to be capable of alignment with the plurality of elements.

43. The system of claim 36, wherein the elements are one of rods, plates, or filaments.

44. The system of claim 36, wherein the synthesis surface is functionalized to bind to said base molecule.

45. The system of claim 37, wherein the elements are sized and shaped to fit into the wells.

46. The system of claim 36, further comprising: an elements holder for holding the plurality of elements so that the plurality of elements may be actuated together; and an actuatable component secured to the elements holder, the actuatable component being actuatable by the at least one actuator.

47. The system of claim 36, wherein the base molecule is a synthesis initiator comprising a reactive group for coupling to a first monomer in a cycled polymer synthesis reaction.

48. The system of claim 36, wherein the base molecule is capable of binding to a synthesis intiator comprising a reactive group for coupling to a first monomer in a cycled polymer synthesis reaction.

49. The system of claim 48, wherein said binding of the base molecule to said synthesis initiator is via one or more linking molecules.

50. The system of claim 36, wherein the base molecule is a first oligonucleotide.

51. The system of claim 50, wherein the first oligonucleotide comprises a free 3' hydroxyl group.

52. The system of claim 51, wherein the first oligonucleotide comprises a single- stranded region at the 3' end.

53. The system of claim 50, wherein the first oligonucleotide is capable of hybridizing specifically to a second oligonucleotide, wherein the second oligonucleotide comprises a free 3' hydroxyl end when hybridized to the first oligonucleotide.

54. The system of claim 53, wherein the second polynucleotide comprises a singlestranded region at the 3' hydroxyl end when hybridized to the first polynucleotide.

55. The system of claim 36, wherein the at least one actuator is: operationally linked to and configured to align the plurality of elements and at least a subset of the resolved loci across a first plane; and operationally linked to and configured to move the aligned plurality of elements and the liquid reagent of the at least a subset of the resolved loci into contact with each other along an axis orthogonal to the first plane.

56. The system of claim 36, wherein the at least one actuator comprises: a first actuator operationally linked to and configured to align the plurality of elements and at least a subset of the resolved loci across a first plane; and a second actuator operationally linked to and configured to move the aligned plurality of elements and the liquid reagent of the at least a subset of the resolved loci into contact with each other along an axis orthogonal to the first plane.

57. The system of claim 36, wherein said system comprises a dispenser for dispensing said liquid reagent to the resolved loci.

58. The system of claim 57, wherein said dispenser comprises one or more nozzles each capable of dispensing a liquid reagent to the resolved loci.

59. The system of claim 36, wherein said system is configured to simultaneously i) actuate the plurality of elements or a first plate to cause the synthesis surfaces to be at least partially immersed into corresponding ones of the resolved loci containing the liquid reagent, and ii) dispense liquid reagents to the resolved loci on a second plate using said dispenser.

60. The system of claim 36, wherein the liquid reagent comprises a polymer extension solution , wherein said polymer extension solution comprises a monomer and an enzyme capable of catalyzing addition of the monomer to a polymer comprising said base molecule bound to the synthesis surface .

61. The system of claim 60, wherein the enzyme is a polymerase.

62. The system of claim 61, wherein the polymerase is a template-independent polymerase.

63. The system of any one of claims 60-62, wherein the enzyme is bound to the monomer via a linker, forming a protected monomer.

64. The system of any one of claims 60-63, wherein the monomer comprises a protecting group.

65. The system of claim 63 or 64, wherein the monomer is a nucleotide.

66. The system of any one of claims 60-65, wherein the liquid reagents are distributed on said resolved loci on said plate in an ordered manner by monomer identity.

67. The system of claim 60, wherein the liquid reagent comprises a deprotection solution.

68. The system of claim 67, wherein said deprotection solution is capable of removing a protecting group from a monomer.

69. The system of claim 67, wherein said deprotection solution comprises a linker cleavage reagent capable of separating an enzyme bound to a monomer via a cleavable linker.

70. The system of claim 60, wherein the liquid reagent comprises reaction quenching solution.

71. The system of claim 60, wherein the liquid reagent comprises a wash solution.

72. A system for polymer synthesis, comprising: elements, wherein each of the elements has a synthesis surface on which a base molecule for synthesizing a polymer is bound; at least one actuator for actuating the elements for the synthesis surfaces of the elements to be at least partially immersed in a liquid reagent volume to cause one or more steps of polymer synthesis; and a processor configured for executing computer programming instructions to control actuation by the actuator.

73. The system of claim 72, wherein the liquid reagent volume is contained in wells on a plate or is positioned on hydrophilic regions of a patterned surface.

74. The system of claim 73, wherein the elements are sized and shaped to fit into the wells on the plate or into the liquid reagent volume on the hydrophilic regions.

75. The system of claim 72, wherein the liquid reagent comprises a polymer extension solution, wherein said polymer extension solution comprises a monomer and an enzyme capable of catalyzing addition of the monomer to a polymer comprising said base molecule bound to the synthesis surface.

76. The system of claim 75, wherein the enzyme is a polymerase.

77. The system of claim 76, wherein the polymerase is a template-independent polymerase.

78. The system of any one of claims 75-77, wherein the enzyme is bound to the monomer via a linker, forming a protected monomer.

79. The system of any one of claims 75-77, wherein the monomer comprises a protecting group.

80. The system of claim 78 or 79, wherein the monomer is a nucleotide.

81. The system of any one of claims 76-80, wherein the liquid reagents are distributed on said resolved loci on said plate in an ordered manner by monomer identity.

82. The system of claim 72, wherein the liquid reagent comprises a deprotection solution.

83. The system of claim 82, wherein said deprotection solution is capable of removing a protecting group from a monomer.

84. The system of claim 82, wherein said deprotection solution comprises a linker cleavage reagent capable of separating an enzyme bound to a monomer via a cleavable linker.

85. The system of claim 72, wherein the liquid reagent comprises reaction quenching solution.

86. The system of claim 72, wherein the liquid reagent comprises a wash solution.

87. The system of claim 72, wherein each of the elements is a rod that is suspended by an element holder and has a length that extends along a longitudinal axis from a proximal end to a distal end.

88. The system of claim 87, wherein each of the elements may be displaced longitudinally relative to the element holder in response to contacting an object with the distal end of the element.

89. The system of claim 72, wherein the elements comprise at least one of metal, plastic, or glass.

90. The system of claim 72, wherein the processor is configured to cause the at least one actuator to at least partially immerse the elements in the liquid reagent volume multiple times.

91. The system of claim 72, further comprising a dispenser for dispensing liquid reagent volumes into compartments.

92. The system of claim 91, wherein the compartments are wells of a reaction plate or loci on a patterned surface.

93. The system of claim 91, wherein the processor is configured to cause the dispenser to dispense a liquid reagent volume comprising a polymerase and a nucleotide into at least one of the compartments.

94. The system of claim 91, wherein the processor is configured to cause the dispenser to dispense different nucleotide identities in distinct ones of the compartments and wherein the processor is configured to cause the at least one actuator to at least partially immerse one of the elements in each of the distinct ones of the compartments in a sequence.

95. The system of claim 91, wherein the dispenser is fluidically connected to a reservoir holding a reaction quenching solution, wherein the processor is configured to cause the dispenser to dispense the reaction quenching solution from the reservoir in one of the compartments and wherein the processor is configured to cause the at least one actuator to at least partially immerse one of the elements in the compartment in which the reaction quenching solution has been dispensed.

96. The system of claim 91, wherein the processor is configured to cause the dispenser to dispense a wash solution in one of the compartments and wherein the processor is configured to cause the at least one actuator to at least partially immerse one of the elements in the compartment in which the wash solution has been dispensed.

97. The system of claim 91, wherein the processor is configured to cause the dispenser to dispense a deprotection solution solutionin one of the compartments and wherein the processor is configured to cause the at least one actuator to at least partially immerse one of the elements in the compartment in which the enzymatic linker cleaver solution has been dispensed.

98. The system of claim 97, wherein the deprotection solution contains a blocker removal reagent.

99. The system of claim 97, wherein the deprotection solution contains an enzymatic linker cleaver.

100. A system for polynucleotide synthesis, comprising: an element having a functionalized surface bound to a polynucleotide synthesis primer comprising a free 3' hydroxyl end; at least one actuator for actuating the element for the functionalized surface of the element to be inserted in a liquid reagent comprising a polymerase and a nucleotide to cause polynucleotide synthesis; and a processor configured for controlling the at least one actuator to: contact the functionalized surface of the element into the liquid reagent under conditions suitable for polymerase catalyzed extension of said polynucleotide at said free 3' hydroxyl end.

101. The system for polynucleotide synthesis of claim 100, wherein the nucleotide is protected.

102. A method of polymer synthesis, comprising: providing an element comprising a synthesis surface on which a base molecule for polymer synthesis is bound; immersing the synthesis surface into a liquid reagent; and removing said synthesis surface from said liquid reagent.

103. The method of claim 102, wherein said liquid reagent is contained in a resolved locus on the surface of a plate.

104. The method of claim 102 or 103, wherein said liquid reagent comprises a polymer extension solution comprising a monomer and an enzyme capable of catalyzing addition of the monomer to a synthesis initiator or a polymer comprising said base molecule.

105. A method of multiplexed polymer synthesis, comprising: providing a plurality of elements, each of the elements comprising a synthesis surface on which a base molecule for polymer synthesis is bound; immersing the plurality of synthesis surfaces into a polymer extension solution contained in resolved loci on the surface of a plate, wherein said polymer extension solution comprises a monomer and an enzyme capable of catalyzing addition of said monomer to a synthesis initiator or polymer comprising said base molecule; and removing said synthesis surface from said polymer extension solution.

106. The method of claim 105, wherein the monomer comprises a protecting group configured to prevent addition of more than one monomer to said synthesis initiator or polymer during said immersion into the polymer extension solution.

107. The method of claim 106, wherein the protecting group is the enzyme bound to the monomer via a linker.

108. The method of claim 106, wherein the protecting group is a reversible terminator,

109. The method of any one of claims 105-108, wherein the enzyme is a polymerase, and the monomer is a nucleotide.

110. The method of claim 109, wherein the polymerase is a template-independent polymerase.

111. The method of any one of claims 105-110, further comprising immersing said synthesis surfaces in a monomer deprotection solution capable of removing the protecting group from the monomer.

112. The method of claim 111, wherein said monomer deprotection solution comprises a linker cleavage reagent capable of separating said enzyme bound to said monomer via a linker.

113. The method of any one of claims 105-112, further comprising immersing said synthesis surfaces in a reaction quenching solution.

114. The method of any one of claims 105-113, further comprising immersing said synthesis surfaces in a wash buffer.

115. The method of any one of claims 105-114, further comprising repeating the immersing steps recited in any one of claims 105-114 one or more times to synthesize a polymer.

116. The method of any one of claims 105-115, wherein any of said immersing steps comprises immersing said synthesis surfaces into said polymer extension solution, deprotection solution, quenching solution, or wash buffer and removing said synthesis surface a plurality of times.

117. The method of any one of claims 105-116, further comprising dispensing said polymer extension solution into said resolved loci prior to said immersion of said synthesis surface into said polymer extension solution.

118. The method of claim 117, wherein immersion of the plurality of synthesis surfaces into polymer extension solutions on a first plate occurs concurrently with said dispensing said polymer extension solution into said resolved loci on a second plate.

119. The method of claim 105 or 117, wherein the polymer extension solutions are dispensed into said resolved loci on said plate in an ordered manner by monomer identity.

120. The method of claim 105, wherein the resolved loci are wells.

121. The method of claim 105, wherein the plate comprises a patterned surface separating said resolved loci.

122. The method of claim 121, wherein the patterned surface has a pattern of hydrophobic regions and hydrophilic regions.

123. The method of claim 121, wherein the resolved loci are separated by hydrophobic regions on the patterned surface.

124. The method of claim 121 or 123, wherein the resolved loci are hydrophilic regions on the patterned surface.

125. The method of claim 105, wherein at least a subset of the resolved loci are spatially separated to be capable of alignment with the plurality of elements.

126. The method of claim 105, wherein the elements are one of rods, plates, or filaments.

127. The method of claim 105, wherein the synthesis surface is functionalized to bind to said base molecule.

128. The system of claim 120, wherein the elements are sized and shaped to fit into the wells.

129. The method of claim 105, wherein the elements are held in place by an elements holder so that the elements may be actuated together.

130. The method of claim 105, wherein the base molecule is a synthesis initiator comprising a reactive group for coupling to a first monomer in a cycled polymer synthesis reaction.

131. The method of claim 105, wherein the base molecule is capable of binding to a synthesis intiator comprising a reactive group for coupling to a first monomer in a cycled polymer synthesis reaction.

132. The method of claim 105, wherein said binding of the base molecule to said synthesis initiator is via one or more linking molecules.

133. The method of claim 105, wherein said immersing the plurality of synthesis surfaces into the polymer extension solutions comprises: aligning the plurality of elements and at least a subset of the resolved loci across a first plane; and moving the aligned plurality of elements and the polymer extension solution of the at least a subset of the resolved loci into contact with each other along an axis orthogonal to the first plane.

134. A method of multiplexed polynucleotide synthesis, comprising: providing a plurality of elements, each of the elements comprising a synthesis surface on which an oligonucleotide is bound; immersing the plurality of synthesis surfaces into a polymer extension solution contained in resolved loci on the surface of a plate, wherein said polymer extension solution comprises a nucleotide comprising a protecting group and a polymerase capable of catalyzing addition of said nucleotide to said oligonucleotide; and removing said synthesis surface from said polymer extension solution.

135. The method of claim 134, further comprising immersing said synthesis surfaces in a monomer deprotection solution capable of removing the protecting group from the nucleotide.

136. The method of claim 134 or 135, wherein the protecting group is the polymerase bound to the nucleotide via a linker.

137. The method of claim 136, wherein the monomer deprotection solution comprises comprises a linker cleavage reagent capable of separating the polymerase from the nucleotide.

138. The method of claim 134 or 135, wherein the protecting group is a reversible terminator.

139. The method of any one of claims 135-138, further comprising repeating said immersing said synthesis surfaces in said polymer extension solution and said monomer deprotection solution to generate a polynucleotide of a desired sequence and length.