WO2021110079A1

WO2021110079A1 - Immersion synthesis of oligonucleotides

Info

Publication number: WO2021110079A1
Application number: PCT/CN2020/133509
Authority: WO
Inventors: Benjamin ALLRED; Handong Li; Yongwei Zhang
Original assignee: Bgi Shenzhen Co., Ltd.
Priority date: 2019-12-03
Filing date: 2020-12-03
Publication date: 2021-06-10
Also published as: CN114761116A

Abstract

Provided are compositions and methods directed to chemically synthesizing oligonucleotides using an array system that contains multiple subset arrays, in which each subset array comprises primers. Each subset array can be selectively immersed in coupling solutions to extend the primers.

Description

PCT PATENT APPLICATION IMMERSION SYNTHESIS OF OLIGONUCLEOTIDES

CROSS‐REFERENCES TO RELATED APPLICATIONS

This application claims the benefit of and priority to U.S. Provisional Application No. 62/943,149, filed on December 3, 2019, which is hereby incorporated by reference in its entirety for all purposes.

REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA EFS‐WEB

The sequence listing is submitted electronically via EFS‐Web as an ASCII formatted sequence listing with a file named seqlist_092171‐1219303‐5087WOCN. txt, created on November 18, 2020, and having a size of 4 kb and is filed concurrently with the specification. The sequence listing contained in this ASCII formatted document is part of the specification and is herein incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

Production of DNA molecules with user‐defined sequences is a fundamental enabler of biological research and applications. Short (e.g., <100 nucleotides) can be called oligonucleotides.

Oligonucleotide synthesis is typically done on a solid phase support using phosphoramidite chemistry. In this approach, a solid support is provided with a first nucleoside linked using a chemistry that is resistant to the phosphoramidite reagents, but cleavable when synthesis of the oligonucleotide is completed. Nucleosides are iteratively added to the 3’ end in in a user‐defined order, to produce an oligonucleotide of interest. Each cycle in the process comprises steps of deblocking (detritylation) , coupling, oxidation, and capping.

To initiate each cycle, a blocked first nucleoside (first cycle) or a growing oligonucleotide chain in which the 3’ terminal nucleoside is blocked (subsequent cycles) attached to the solid support is deblocked in a deblocking solution. to begin the next cycle of base addition. Where the blocking, or protecting, group is 5 prime DMT (4, 4’ dimethoxytrityl) , the group is removed using trichloroacetic acid (TCA) or dichloroacetic acid (DCA) , in a solvent such as dichloromethane or toluene. This produces a free 5’ OH on the oligonucleotide.

In the coupling step, a next nucleoside is added to the free 5’‐OH of the solid‐support‐linked nucleoside or olitonucleotide) . A protected nucleoside is combined with the solid support as a phosphoramidite monomer in a coupling solution comprising a solvent such as acetonitrile, and then activated by an acidic catalyst such as ETT [5 (ethylthio) 1H tetrazole] . Other common activators include 1H‐tetrazole, 5‐ethylthio‐1H‐tetrazole, [64] 2‐benzylthiotetrazole, [65] [66] 4, 5‐dicyanoimid‐azole or a number of other reagents. Thymine does not require a protecting group, but adenine, cytosine, and guanine do, because they contain primary amino groups. Protected forms include N (6) benzoyl A, N (4) benzoyl C, N (2) isobutyryl G, N (6) phenoxyacetyl A, N (2) acetyl C, and N (2) isopropylphenoxyacetyl G. The newly added nucleoside becomes linked to the growing oligonucleotide by a phosphite triester linkage, generating a free diisopropylamino group.

The phosphite triester formed during the coupling reaction is relatively unstable, and, in the oxidation step, is converted to a more stable phosphorus species in an oxidation solution. This is done using iodine in the presence of water and pyridine, producing a phosphate triester. The new linkage becomes a standard DNA backbone with a β cyanoethyl protecting group on the free oxygen.

At this point, there may be residual nucleosides on the solid support that have unreacted 5’ OH. These nucleosides are capped, to prevent them from reacting in the next cycle, which would result in an oligonucleotide having a missing base. The capping is done in a capping solution using acetic anhydride and N‐methylimidazole in a solvent such as tetrahydrofuran, which react to form an intermediate that contains a small amount of pyridine. As a result, the unreacted 5’ OH nucleosides are acylated, which effectively terminates further elongation of oligonucleotides that missed the last cycle of synthesis.

When the synthesis is completed, the newly synthesized oligonucleotide is detached from the solid support by cleaving the linker to the first nucleoside, for example, by ester hydrolysis. The released oligonucleotide has a terminal, free 3’ OH. The protecting groups are removed from the individual bases, for example, using concentrated aqueous ammonia. Finally, the β cyanoethyl group on the free oxygen of the phosphate is removed to convert the phosphate triester to a native phosphate diester backbone.

Variations of this chemistry are known. See, for example, Obika and Sekine, eds., “Synthesis of Therapeutic Oligonucleotides, ” Springer, 1st ed. 2018; and Hughes, 2017, “Synthetic DNA, ” Methods in Molecular Biology 1472. Synthesis of oligoribonucleotides is described at, e.g., Marshall and Kaiser, 2004, “Recent advances in the high‐speed solid phase synthesis of RNA, ” Curr. Opin. Chem. Bio. 8: 222‐229, each of which is incorporated herein by reference.

Current production of oligonucleotides uses a single operating principle: microfluidic devices deliver a specific reagent to the growing, surface‐attached oligonucleotide. Reliance on this single principle is limiting innovation and accessibility to more high‐throughput and efficient production.

Microfluidic delivery limits throughput to the number of valves and tubes that can fit within a single instrument. The current record for conventional microfluidics is the parallel synthesis of 768 unique sequences. More sequences, around 1 million, can be synthesized using ink‐jet printing systems to deliver the reagents. The throughput advantage of ink‐jet over conventional microfluidics comes with the operation and maintenance costs associated with a complex and large instrument.

For microfluidics systems to achieve high‐yields and low error rates, the fluidic system be washed thoroughly between delivery of each reagent. Washing, purging, and refilling the system requires excessive amounts of reagents, produces expensive waste, and thus decreases the efficiency of the production.

While microfluidic systems have been sufficient to fulfill the current demand of oligonucleotides, an increase in demand will highlight the limitations of the current technology. New methods for oligonucleotide production are needed.

SUMMARY OF THE INVENTION

In one aspect, the disclosure provides a method for reagent delivery to any subset of an array of 4‐10,000 reactive surfaces, irrespective of the number of elements within the subset (0 to total number of array) and the position of the subset elements within the array. The disclosure also provides devices for selecting any subset of an array of 4‐10,000 reactive surfaces, irrespective of the number of elements within the subset (0 to total number of array) and the position of the subset elements within the array. The disclosure also provides a method for delivering different reagents to two or more array subsets described above.

In one aspect, the disclosure provides methods for synthesizing oligonucleotides comprising: (a) providing an array system comprising: (i) a plurality of subset arrays, wherein each subset array comprises a plurality of primers; (ii) a plurality of solutions comprising (1) a plurality of coupling solutions each comprising unincorporated nucleotides, (2) a solution comprising capping agents, and (c) a solution comprising deblocking agents; and (3) a mechical device to selectively move the plurality of subset arrays; (b) immersing one or more subset arrays in the coupling solution to extend the primers in the one or more subset arrays by at least one nucleotide; (c) immersing the same subset arrays from step (b) in the solution comprising capping agents; (d) immersing the same subset arrays from step (c) in the solution comprising deblocking agents; (e) repeating steps (b) to (d) one or more times until the synthesis of the oligonucleotides is complete.

In one aspect the invention provides an apparatus for producing heteropolymers by exposing oligonucleotide to reagents, comprising: a support structure; a plurality of rods coupled to the support structure in an array comprising at least one row of rods, wherein each of the plurality of rods comprises an immersion end and an actuation end extending between a longitudinal axis of the rod, wherein each of the plurality of rods comprises a reactive surface proximate to the immersion end and configured for the oligonucleotides to be immobilized thereon, and wherein the plurality of rods are configured to be translatable along the longitudinal axes of the rods relative to the support structure; a controller; and a plurality of linear actuators electrically coupled to the controller, wherein each of the plurality of linear actuators is coupled to the support structure and the actuation end of one of the plurality of rods and configured to translate in a direction of the longitudinal axes, wherein the controller is configured to independently actuate each of the plurality of linear actuators in order to independently cause translation of each of the plurality of rods between a retracted position and an extended position, in order to cause the oligonucleotides immobilized on the reactive surfaces to be independently submerged into different reagents in reservoirs of a reagent tray positioned below the array.

This disclosure also provides an apparatus for producing a plurality of oligonucleotides with different user‐defined sequences. The apparatus generally includes a support structure arrayed with a plurality of rods, wherein each of the rods comprises an immersion end and an actuation end extending between a longitudinal axis of the rod. Each rod comprises a reactive surface at or proximate to the immersion end, which is configured for affixing or immobilizing a protected nucleoside derivative or an oligonucleotide comprising such derivative at its terminus. The rods are translatable along the longitudinal axes of the rods relative to the support structure. The apparatus also includes linear actuators, each of which is coupled to the support structure and to the actuation end of one of the plurality of rods. The actuators are configured to translate in a direction of the longitudinal axes.

The device further comprises a controller or microprocessor that is programmed or configured to independently actuate each of the plurality of linear actuators, causing translation of each of the plurality of rods between a retracted position and an extended position. This arrangement allows the rods to be moved by its respective actuator independently of other rods on the support structure or apparatus. In this way, a protected nucleoside derivative or oligonucleotide that has been immobilized on the reactive surface of each rod may be independently submerged into its own sequence of different reagent solutions in reservoirs of a reagent tray positioned below the array, in accordance with the sequence being synthesized on each rod.

In operation, the reactive surface on the rods comprises a protected nucleoside derivative affixed or immobilized thereon, or an oligonucleotide comprising a protected nucleoside derivative at its terminus affixed or immobilized thereon, optionally by way of a covalent bond or a linker. The protected nucleoside derivative may be a nucleoside phosphoramidite, a 3'‐O‐ (N, N‐diisopropyl phosphoramidite) derivative of a nucleoside, or a 2’‐deoxyribonucleoside derivative.

The apparatus may be one component of a system that comprises other equipment and/or reagents useful for producing oligonucleotides. The other equipment may include one or more reagent trays configured to contact individual rods of the plurality of rods with one or more reagent solutions contained therein.

Also provided are various combinations of reagent solutions that are suitable for producing oligonucleotides according to this disclosure. The reagent solutions may be provided as part of a system that also includes the aforesaid apparatus, or as an independent kit, useful for synthesizing a phosphoramidite‐based oligonucleotide on this or another apparatus. Such reagent solutions may include in any combination reagents that are selected from deblocking solutions, coupling solutions each comprising a phosphoramidite protected nucleoside, oxidation solutions, capping solutions, and wash solutions. Generally, at least one of said reagent solutions are free from acetonitrile, dichloromethane and water. Typically, all of the reagent solutions are anhydrous and comprise a nonvolatile solvent, such as propylene carbonate, acetic acid, trifluorotoluene, butyronitrile, or adiponitrile, or another nonvolatile solvent.

When a coupling solution is included, it may comprise propylene carbonate and either DNA phosphoramidites or RNA phosphoramidites, wherein the coupling solution is substantially free of acetonitrile. The combination of reagent solutions may include four or more coupling solutions, each of which comprises a nucleoside comprising a different nucleobase. Exemplary coupling solutions comprise the DNA phosphoramidites dA (Bz) CEP, dC (Bz) CEP, dG (iBu) CEP, and dT CEP. When a deblocking solution is included in the combination, it may comprise propylene carbonate and be substantially free from acetonitrile, or it may comprise trichloroacetic acid. When an oxidation solution is included, it may comprise propylene carbonate and be substantially free from water, or it may comprise anhydrous acetic acid or a combination of acetic acid, I ₂, and pyridine. When a capping solution is included, it may comprise propylene carbonate and be substantially free of acetonitrile, or it may comprise acetic anhydride, 1‐methylimidazole, and pyridine.

This disclosure provides a method for reagent delivery to any subset of an array of 4 to 10,000 reactive surfaces, irrespective of the number of elements within the subset (between 0 and the total number of elements in the array, includive) , and the position of the subset elements within the array.

This disclosure also provides a method of delivering reagents or chemical synthesis that comprises delivering different reagents to each of a plurality of reactive surfaces in a predetermined order. The user starts with an array having a set of independently actuatable reactive surfaces, wherein actuation of a reactive surface causes the reactive surface to be immersed into a reagent solution in a reservoir. A first cycle of chemical synthesis is carried out that comprises the following six steps:

i) immersing a first subset of the set of reactive surfaces into a first reservoir comprising a first reagent solution, and withdrawing the first subset of reactive surfaces from the first reservoir, wherein the first subset of reactive surfaces are coordinately actuated;

ii) immersing a second subset of the set of reactive surfaces into a second reservoir comprising a second reagent solution, and withdrawing the second subset of reactive surfaces from the second reservoir, wherein the second subset of reactive surfaces are coordinately actuated;

iii) immersing a third subset of the set of reactive surfaces into a third reservoir comprising a third reagent solution, and withdrawing the third subset of reactive surfaces from the third reservoir, wherein the third subset of reactive surfaces are coordinately actuated; and then

iv) immersing a fourth subset of the set of reactive surfaces into the first reservoir comprising the first reagent solution, and withdrawing the fourth subset of reactive surfaces from the first reservoir, wherein the fourth subset of reactive surfaces are coordinately actuated;

v) immersing a fifth subset of the set of reactive surfaces into the second reservoir comprising the second reagent solution, and withdrawing the fifth subset of reactive surfaces from the second reservoir, wherein the fifth subset of reactive surfaces are coordinately actuated; and

vi) immersing a sixth subset of the set of reactive surfaces into the third reservoir comprising the third reagent solution, and withdrawing the sixth subset of reactive surfaces from the third reservoir, wherein the sixth subset of reactive surfaces are coordinately actuated.

In each cycle of the synthesis, the fourth subset comprises at least some reactive surfaces from each of the first, second and third subsets, the fifth subset comprises at least some reactive surfaces from each of the first, second and third subsets, and the sixth subset comprises at least some reactive surfaces from the first, second and third subsets.

To continue the synthesis method, 4 to 100 additional synthesis cycles are carried out iteratively, each additional synthesis cycle comprising steps (i) to (vi) . At least four of the additional cycles the first, second, and third subsets in the cycle are not all the same as the first, second, and third subsets of the previous cycle. This results in a plurality of different reagents being delivered to each of the reactive surfaces in a user defined order.

This method may be used for oligonucleotide synthesis, whereby each of the reactive surfaces produces an oligonucleotide having a different sequence. Each of the reactive surfaces provided in step a) comprises a protected nucleoside derivative or an oligonucleotide comprising a protected nucleoside derivative at its terminus immobilized thereon, such as a nucleoside phosphoramidite.

Typically, the first, second and third reagent solutions are coupling solution with different phosphoramidite monomers, as described in more detail elsewhere in this disclosure. More specifically, the first, second and third reagent solutions may be three coupling solutions that each contain a phosphoramidite protected nucleoside that is not contained in either of the other two coupling solutions. By way of example, the three coupling solutions each comprise propylene carbonate and DNA phosphoramidites selected from dA (Bz) CEP, dC (Bz) CEP, dG (iBu) CEP, and dT CEP.

Each synthesis cycle of the method may further comprise sequentially immersing each of the reactive surfaces into a plurality of reservoirs to advance the synthesis, as described in more detail below. Typically, for the purposes of oligonucleotide synthesis, one reservoir contains an oxidation solution, a second contains a capping solution, a third contains a deblocking solution, and a fourth contains a wash solution.

In each cycle of the synhtesis, an oligonucleotide on the reactive surface is generally lengthened by addition of a single subunit. Optionally, for at least some reactive surfaces and at least some synthesis cycles, as described below, the subunit is a phosphoramidite trinucleotide.

Other aspeces of the invention are presented in the detailed description that follows, in the drawings, and in the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Parallelized reagent delivery to a completely flexible subset of an array system.

FIG. 2A and FIG 2B: Devices for selecting a subset of an array system for reagent delivery. FIG. 2A illustrates a modular subset selector that can be included in the array system to selectively choose which subset arrays to dip or immerse in a solution of reagents. A mechanical device raises and lowers rods according to the instructions of a programmable controller. The rods that are at the lowest level then push the top of the corresponding synthesis solids supports (subset arrays) . A friction plate allows the solid supports to slip while being pressed and the position to be maintained when the device is removed. Thus, the device imprints the subset within the column (s) that the device is in contact with. This process is repeated through all the columns of the array, creating a programmable subset of solid supports to receive reagent by delivery. FIG. 2B shows another option in which a direct subset selector is used. Each solid support is directly connected to a solenoid device. To select the subset of supports to be lowered, a programmable controller triggers the corresponding solenoids.

FIG. 3: Demonstration of 25 cycles of DNA synthesis using dipping synthesis method.

FIG. 4: Reverse phase HPLC analysis of the synthesis yield.

DETAILED DESCRIPTION OF THE INVENTION

Devices and methods disclosed herein relate to the mechanical delivery of surface‐attached oligonucleotides to synthesis reagents by dipping or immersing the oligonucleotides into synthesis reagent solutions. Examples of synthesis reagent solutions are N‐mer solutions, deblocking (detritylation) reagent solutions, coupling reagent solutions, oxidation reagent solutions, capping reagent solutions, wash solutions, and optionally other solutions. The mechanical delivery of the surface‐attached oligonucleotides may be used to produce defined nucleic acids (e.g., DNA strands) as well as other heteropolymers.

Methods for the synthesis of DNA and RNA oligomers of defined sequence are known. Most commonly, synthesis is based on solid phase phosphoramidite chemistry. In this approach, a nucleoside monomer is chemically combined with a solid‐support‐linked nucleoside (the terminal 3' base of the oligonucleotide) to produce a core oligonucleotide, and the core oligonucleotide is extended in a series of synthesis cycles, in which one nucleotide is added per cycle.

According to the present invention, a device (or equivalently, apparatus) is use for oligonucleotide synthesis in a process involving mechanical delivery of surface‐attached oligonucleotides to synthesis reagent solutions. The device comprises a plurality movable elements, or “synthesis solid supports, ” coupled to a modular subset selector. In one embodiment, each individual synthesis solid support comprises an elongated body with an actuation end and an immersion end. The movable elements may have any shape consistent with operation of the device. For example, a synthesis solid support may be shaped as a cylinder. In this disclosure movable elements are generally referred to as “rods” for ease of reference. However, the synthesis solid support, or “rods, ” may have a non‐cylindrical or non‐elongated shape. For example, the synthesis solid support, or “rod, ” may be shaped as a rectangular cuboid, a rectangular parallelepiped, a prism or a cone.

The synthesis solid supports are supported by a support structure so that the synthesis solid supports may translate along a longitudinal axis of the elongated body, herein referred to as translation in a z‐direction. The plurality of the synthesis solid supports may be arranged in an ordered array, such as a rectilinear array. In one approach, rods are arranged in a line in a direction perpendicular to the z‐direction. In one approach the plurality of the synthesis solid supports may be arranged in a 2‐dimensional rectangular array, for example a plurality of parallel rows of synthesis solid supports arranged in a plane perpendicular to the z‐direction. In some embodiments, an array may contain 100,000 or more different rods with reactive surfaces. As discussed below, the array may be coupled to an actuator for moving the array (including the component rods) in the X and/or Y directions.

In embodiments, synthesis solid supports comprise a reactive surface at or proximate to the immersion end. In some embodiments, only the portion of the synthesis solid support immediately proximate to the immersion end comprises the reactive surface. The reactive surface may be configured for oligonucleotide synthesis, such as phosphoramidite synthesis. At the start of the synthesis process the reactive surface of the synthesis solid support may have attached thereto a nucleoside (or a plurality of nucleosides) that can act as 3’ terminal nucleotide of the oligonucleotide. The nucleoside (s) may be covalently attached, and may be attached via a linker. It will be recognized that a reactive surface of a single rod may have a plurality (e.g., a large number) of nucleosides or oligonucleotides attached thereon. Generally the nucleoside is blocked by a 5’ protecting group, such as 4, 4'‐dimethoxytrityl, that prevents addition of N‐mers. In an initial “deblocking” step the protecting group is removed, for example by immersing the reactive surface into a solution containing trichloroacetic acid in propylene carbonate.

A rod may be formed from a variety of materials such as glass (e.g., borosilicate glass, controlled pore glass) , silicon, plastic (e.g., macroporous polystyrene) , or metal, provided the surface of the immersion end of the rod is compatible with the reagents and solvents with which it makes contact. See Guzaev et al., 2013, “Solid‐phase supports for oligonucleotide synthesis. In: Current protocols in nucleic acid chemistry” John Wiley & Sons, Inc. pp. 3.1.1‐3.1.60. The reactive surface can further be modified with silane compounds, phosphonate compounds, coupling agents (e.g., N‐ (3‐triethoxysilylpropoyl) ‐4‐hydroxybutyramide) , alkyl chains, amino groups, hydroxyl groups, or fluorinated groups. For example, the reactive surface may be amine‐modified glass to which a cleavable linker is attached. The immersed portion of a rod can be fabricated from materials or treated to minimize the liquid adsorption and avoid contamination from transfer from one reagent to another. The dimensions of a rod in a device may vary considerably based on the design of the device. In some embodiments, the immersion surface of the rods is defined by a cross‐sectional dimension (e.g. diameter or diagonal) in the range of 0.1 mm to 10 mm. For example, the rods may be cylindrical and have a cross sectional diameter in the range of 0.1 mm to 10 mm, e.g., 0.5 to 5 mm in diameter.

The rods in an array are independently movable. Alternatively, the rods may be grouped (e.g., groups of 2‐4 rods) and each individual group is independently movable. For purposes of clarity, discussion in this disclosure assumes rods in an array are independently movable.

In a given synthesis step or given synthesis cycle, subsets of rods are extended or retracted in concert. When rods are “extended” the immersions ends may be in contact with (e.g., immersed in) a synthesis reagent solution. As illustrated in FIG. 1, an array comprises a plurality of rods (three rods are shown in FIG. 1) that may be independently extended or retracted so that the reactive surface moves up and down, e.g., in the Z axis. For purposes of this description, the reactive surface of a rod can occupy either one of two positions: “extended” or “retracted. ” It will be understood that movement of the rod surface to intermediate positions is also contemplated.

Each of the three rods may be independently extended or retracted so that the reactive surfaces translate up and down relative to the support structure. For example, as shown in Fig. 1, in “subset 1” the left rod is extended, and the middle and right rods are retracted, and in “subset 2” the middle and right rods as extended and the left rod is retracted.

The apparatus may comprise a modular subset selector, as shown for example in FIG. 2A. The modular subset selector may comprise a controller connected to a plurality of linear actuators, for example but not limited to solenoids as shown in the example of FIG. 2B. Each linear actuator may be independently actuated by the controller. In some embodiments, each linear actuator is coupled to the support structure and a synthesis solid support, so that each synthesis solid support may be independently actuated to translate in the z‐direction relative to the support structure between an extended position and a retracted position, and in embodiments any position in between the extended position and a the retracted position. In some embodiments, for example as shown in FIG. 2B, the apparatus may comprise a friction plate, wherein each rod extends through the friction plate. The friction plate prevented the rods and linear actuators from translating unless a threshold force to overcome the friction is exceeded.

The independent actuation of the rods allows for the surface‐attached oligonucleotides on each rod to be independently exposed to different combinations of synthesis reagents by submerging and/or not submerging different combinations (subsets) of rods into synthesis reagents. Synthesis reagents are contained in containers (called a ‘reagent tray’ ) positioned close to the array. The reagent tray refers to a collection of reservoirs, vessels, or wells that contain synthesis reagent solutions. As used herein, ‘reagent tray’ may refer to a collection of independent vessels (which may be physically linked) or to a single structure with a plurality of distinct integral reservoirs or wells. In one embodiment the reagent tray is a multiwell plate (such as a 12, 24, 48, 96 or 384 well plate) . During operation the reservoirs are positioned so that at least some the rods in the array are aligned over one or more reservoirs, such that translating those rods to the extended position allows the immersion ends (including the reactive surfaces) to be submerged in the selected reservoir, while when the rods are retracted the immersion ends (including the reactive surfaces) are not submerged. This allows each reactive surface to be independently exposed to a specified and predetermined sequence of solutions and allows synthesis of different oligonucleotides to be carried out on different rods simultaneously. A feature of the reservoirs is that they are “open” to allow movement of rods into the solutions. That is, reagents are not confined to tubing or microfluidic channels. Reagent solutions may be contained in a well or reservoir throughout the synthesis process. Operation of the device does not require transport of reagents from storage containers to synthesis sites.

With the array over a reagent tray, the controller may perform a setting step by actuating a first set of one or more rods to transition from the retracted position to the extended position. The transition in the setting step may cause the reactive surface (s) with the surface‐attached oligonucleotides to be submerged in the reservoirs, and therefore the setting step may also be a submerging step. Alternatively, after translation in the setting step, the controller may perform a separate submerging step wherein the entire array may be translated in the Z‐direction to cause the rods in the extended position to be submerged, and the rod in the retracted position not to be submerged. After a submersion, the controller may perform a withdrawal step to cause the submerged rods to withdrawal out of the reservoirs. In embodiments, the controller may perform a withdrawal step by actuating the rods in the extended position to transition from the extended position back to the retracted position, and/or cause the entire array to translate in the z‐direction away from the reagent tray. In some embodiments, after a submersion, for example of the first set of rods, the controller may perform a realigning step wherein the reagent tray and array may be moved relative to each other so that the rods are aligned over a different set of wells of the reagent tray and/or a different reagent tray. The steps of the setting step, the submerging step, withdrawal step, and realigning step, may be carried out one or more times in each synthesis cycle. For example, after the first subset of rods is submerged, the controller may actuate a second subset of rods to transition from them from the retracted position to the extended position. In some embodiments, the first set and second set of rods may comprise one or more rods in common. In some embodiments, in any given synthesis cycle, some rods are not immersed in any solution. The process may continue for subsequent cycles with any combination of rods being submerged. In embodiments, the controller may perform any number of cycles, for example 5 to 100 cycles.

In some embodiments, the support structure for the array may be coupled to an actuator for moving the array in the X and/or Y directions in order to (re) position the rods of an array over one or more stationary reagent trays. In some embodiments, one or more reagent trays may additionally or alternatively be coupled to actuators coupled to the controller in order to move the reagent trays in the X and/or Y direction. In some embodiments, the entire array and/or reagent tray may be coupled to an actuator in order to translate in the Z‐direction in order for the extended rods to be submerged in the wells of the reagent trays.

In some embodiments, for example as shown in FIG. 1, multiple rods in the extended position may be submerged into a common well during a cycle. In some embodiments, adjacent rods in the array may be placed in the extended position and submerged into an adjacent well containing different reagents during the same cycle. In some embodiments, during a cycle some rods may simultaneously translate between the retracted and extended positions. For example all rods, or a portion of the rods, to be submerged may extend to be submerged at the same and then may be retracted at the same time. In some embodiments, during a cycle some rods may sequentially translate between the retracted and extended positions. For example, a first rod may be submerged and withdrawn, followed by a second rod being submerged and then withdrawn. After all rods to be submerged in a cycle are submerged and withdrawn, the cycle may progress to the next step in order for the process to advance to the next cycle.

As illustrated in FIG. 1, when a rod is in an “extended” position the reactive surface (including any attached oligonucleotides) is dipped or immersed into a solution contained in an underlying reservoir while rods in the same array that are in the “retracted position” the reactive surface is not immersed.

As illustrated in FIG. 1, at any given time all of the rods of an array may be retacted, all may be extended, or some may be retracted and others (a “subset” ) may be extended. As noted, each rod is independently controlled so that in a first instance an array may be positioned over a first reservoir such that the surfaces of any rods in the extended postion are immersed in the solution contained in the first reservoir and and in a second instance that array may be positioned over a second reservoir such that the surfaces of any rods in the extended postion are immersed in the solution contained in the second reservoir.

The synthesis of multiple unique sequences is parallelized using the methods described herein by making an array of solid, reactive surfaces that are individually and flexibly controlled. Raising and lowering individual solid supports selects the subset of elements (subst arrays) that will be immersed in a solution. The subset that receives a reagent is flexible as to the number (0 to complete array) and position of the elements within it. In some embodiments all elements in the subset receive the reagent at the same time. The subset of reactive surface elements that require a specific reagent are selectively lowered below the remainder of the array that do not require the reagent.

As noted, the device may include 4 to 10,000, or more, independently controllable rods. A programmable computer or cpu may be used to actuate various subsets of rods using an interface that allows the user to select the sequence, componants and synthesis chemistry of the desired oligonucleotides. The computer may also be used to move an array, a reagent tray, of individual reservoir of the reagent tray into a desired position.

It will be appreciated that a subset of rods are rods that move (are retracted or extended) in concert. Generally a ‘subset’ refers to rods that move are extended into or retracted from the same reagent solution in concert. The number of subsets will be adjusted to based on the chemistry used and the composition of the oligonucleotides being synthesized. Without limitation, typically 4‐100 subsets, e.g., 4‐50 subsets, are identified in the course of synthesis.

Thus, the methods described herein use a user‐controlled and completely flexible subset of the arrays, in number and position, that is immersed simultaneously in the reagent. The number of sequences synthesized in parallel is often limited by electronic device size, instead of microfluidic device size, and off‐the‐shelf components are readily available to control the synthesis of more than 100 sequences per standard microtiter plate size. Larger arrays (>1000 in the same plate size) are achievable with customized robotics/electronic control.

In addition to the high density and flexibility of the array system, delivery of reagents by dipping or immersing the oligonucleotides into the reagents provides advantages in production efficiency. With the currently used microfluidic devices, all reagents pass through a common delivery tube and become contaminated or diluted, preventing them from being reused. In embodiments of the methods described herein, the solutions containing reagents are modified (relative to conventional phosphoramidite synthesis chemistry) to allow them to remain open and in the same container throughout the production. Thus, reagent consumption is minimized because the reagents are reused and the containers do not need emptying, refilling, or washing during the process.

The methods for synthesizing oligonucleotides described herein comprise: (a) providing an array system comprising: (i) a plurality of subset arrays, wherein each subset array comprises a plurality of primers; (ii) a pluality of solutions comprising a pluality of coupling solutions each comprising unincorporated nucleotides, a solution comprising capping agents, and a solution comprising deblocking agents; and (iii) a mechical device to selectively move the pluality of subset arrays; (b) immersing one or more subset arrays in the coupling solution to extend the primers in the one or more subset arrays by at least one nucleotide; (c) immersing the same subset arrays from step (b) in the solution comprising capping agents; (d) immersing the same subset arrays from step (c) in the solution comprising deblocking agents; (e) repeating steps (b) to (d) one or more times until the synthesis of the oligonucleotides is complete.

It will be appreciated that by programing the actuation of rods and movement of arrays and reagent trays reactive surfaces of each rod can be exposed to defined reagent solutions in a defined order, and that oligonucleotides of defined sequence can be synthesized on each rod.

SYNTHESIS REAGENT SOLUTIONS

As noted above, in the immersion system disclosed herein wells or reserviour containing reagent solutions are generally open (exposed to the environment) during the immersion steps. According to an embodiment of the invention, synthesis reagent solutions are modified, relative to solutions used in conventional methods, to allow them to remain open and in the same container throughout the production. As one example, non‐volatile solvents are generally used in place of conventional solvents. Volatility is a material quality that describes how readily a substance vaporizes. A highly volatile substance will quickly evaporate, while a substance with low volatility will remain condensed. Boiling point can be used as a measure of volatility, with a lower boiling point corresponding to a higher volatility. In some embodiments a solvent is referred to as “non‐volatile” (or, equivalently, low‐volatile) if it has a boiling point higher than 101 degrees C (measured at 760 torr) or higher than 110 degrees C, or higher than 115 degrees C, or higher than 150 degrees C, or higher than 200 degrees C. Examples (for illustration and not limitation) of “non‐volatile” solvents include propylene carbonate, acetic acid, trifluorotoluene, butyronitrile, and adiponitrile.

TABLE 1 and TABLE 2 provide examples, for illustration and not limitation, of reagents adapted to immersion synthesis.

Table 1

Comparison of reagent characteristics for standard and described chemistries

COUPLING SOLUTION AND UNINCORPORATED NUCLEOTIDES

In embodiments, each coupling solution can contain a phosphoramidite precursor to a single nucleotide (sometimes referred to herein as ‘unincorporated nucleotides’ ) . In some cases the phosphoramidite precursor to a single nucleotide of a 5’‐blocked nucleoside. For example, a coupling solution can be a solution comprising an ATP precursor, a solution comprising a TTP precursor, a solution comprising a GTP precursor, and a solution comprising a CTP precursor. Depending on the desired synthesis product, the coupling solutions may contain DNA phosphoramidites, RNA phosphoramidites, 2’ modified phosphoramidites, dye‐labeled phosphoramidites, and the like. The chemical structure of phosphoramites can vary depending on the specific synthesis protocol used and the reagent supplier. In one example, the phosphoramites are dA (Bz) β‐cyanoethyl phosphoramidite (CEP) [C ₄₇H ₅₂N ₇O ₇P] , dC (Bz) CEP [C ₄₆H ₅₂N ₅O ₈P] , dG (iBu) CEP [C ₄₄H ₅₄N ₇O ₈P] , dT CEP [C ₄₀H ₄₉N ₄O ₈P] . Reagents are widely available from comercial sources including Thermo Fisher Scientific Logo. Phosphoramidite reagents include 5'‐> 3' synthesis phosphoramidites. depurination resistant base phosphoramidites, standard base protection phosphoramidites, trimer phosphoramidites, standard RNA phosphoramidites, 2'‐OMe RNA synthesis and 2'‐MOE RNA synthesis reagents. The unincorporated nucleotides can include monomers (corresponding the single nucleotides in the synthesized oligonucleotide (s) , having nucleobases such as adenine (A) , cytosine (C) , guanine (G) , thymine (T) , uracil (U) , noncanonical nucleobases (e.g., inosine, xanthine, hypoxanthine, 2, 6‐diaminopurine, and 6, 8‐diaminopurine) , purines and pyrimidine, modified bases and base analogs, non‐nucleoside phosphramidites (e.g., for incorporation of groups such as labels, biotin, terminal phosphate groups, and the like, provided the monomers have structures compatible with a solid phase synthesis. Monomers can include ribose, deoxyribose, sugars or may include precursors for oligonucleotides with nonstandard linkages (e.g., peptide nucleic acids) . In RNA synthesis, the 2'‐hydroxy group may be protected with a TBDMS (t‐butyldimethylsilyl) group or a TOM (tri‐iso‐propylsilyloxymethyl) group. These groups are removable by treatment with fluoride ion.

The unincorporated nucleotides can include single nucleotides such as dATP, dTTP, dGTP, and dCTP. The unincorporated nucleotides can also include a plurality of 3‐mer nucleotides. In this manner, when producing DNA synthetically, elongation is not limited to one base per cycle. Phosphoramidite reagents can be produced that include 2 or more bases, with the natural phosphodiester backbone between the additional bases. Three base phosphoramidites are particularly applicable to DNA synthesis for protein expression because the genetic code is based on 3 base “codons. ” The optimized codons for protein expression are known, and any user‐defined sequence will consist of a string of codons instead of individual bases (when synthesis is performed with single bases, the codon is made in three steps) . To improve the yield, increase the maximal length of the synthesized oligonucleotide, and simplify the synthesis procedure, the DNA synthesis can be carried out by dipping with phosphoramidite “codons, ” or 3‐mer nucleotides.

The unincorporated nucleotides can be 3’ protected nucleotides. The protective group of the 3' protected nucleotides can be selected from the group consisting of 3’ O allyl, 3’ O methoxymethyl, 3’ O nitrobenzyl, 3’ O azidomethylene, and 3’ O aminoalkoxyl.

In some embodiments, each solution in the plurality of coupling solutions comprises an activator and phosphoramidite. A coupling solution can be provided in which the activator and phosphoramidite are pre‐mixed, and the acetonitrile of the standard solution is replaced with a non‐volatile solvent. In some embodiments, the activator reagent comprises DCI and propylene carbonate. In one embodiment, the DCI has a concentration of 0.35 M.

A coupling solution can also contain a plurality of 3‐mer nucleotides. An array system can contain multiple coupling solutions, in which each solution contains a plurality of 3‐mer nucleotides. In this manner, when producing DNA synthetically, elongation is not limited to one base per cycle. Phosphoramidite reagents can be produced that include 2 or more bases, with the natural phosphodiester backbone between the additional bases. Three base phosphoramidites are particularly applicable to DNA synthesis for protein expression because the genetic code is based on 3 base “codons. ” The optimized codons for protein expression are known, and any user‐defined sequence will consist of a string of codons instead of individual bases (when synthesis is performed with single bases, the codon is made in three steps) . To improve the yield, increase the maximal length of the synthesized oligonucleotide, and simplify the synthesis procedure, the DNA synthesis can be carried out by dipping with phosphoramidite “codons, ” or 3‐mer nucleotides. Protocols for preparation of phosphoramidite trinucleotides have been described. See, e.g., Suchsland et al., 2018, J. Org. Chem. 14: 397–406. doi: 10.3762/bjoc. 14.28; Kayushin et al., Nucleic Acids Research, 1996, 24, 3748‐3755; Mauriala et al., J Pharm Biomed Anal, 2004, 34, 199‐206, each of which is incorporated herein by reference.

DEBLOCKING SOLUTION

In one embodiment a deblocking solution is used in which dichloromethane of the standard solution is replaced with a non‐volatile solvent. In some embodiments, the deblocking reagent comprises trichloroacetic acid and propylene carbonate. In one embodiment, the trichloroacetic acid has a concentration of 3%m/v (mass/volume) .

OXIDATION SOLUTION

In one embodiment an oxidation solution is used in which water of the standard solution is replaced acetic acid and other, higher boiling, carboxylic acids, and acetonitrile is replaced with non‐volatile solvent. In some embodiments, the oxidation reagent comprises I ₂, acetic acid, pyridine, and propylene carbonate. In one embodiment, the I ₂ has a concentration of 0.02 M. In one embodiment, the acetic acid has a concentration of 10%v/v (volume/volume) .

CAPPING SOLUTION

Capping solution in which acetonitrile of the standard solution is replaced with a non‐volatile solvent. In some embodiments, the cap A reagent comprises acetic anhydride and propylene carbonate. In one embodiment, the acetic anhydride has a concentration of 10%v/v. In some embodiments, the cap B reagent comprises 1‐methylimidazole, pyridine, and propylene carbonate. In one embodiment, 1‐methylimidazole has a concentration of 10%v/v. In one embodiment, pyridine has a concentration of 10%v/v.

WASHING SOLUTION

In some embodiments a washing solution is used in which acetonitrile of the standard solution is replaced with a high‐boiling solvent, such as propylene carbonate.

In some embodiments the reactive surface is immersed into the synthesis reagent solution more than one time, e.g., 2 or 3 times. Washing or buffer exchange steps, for example, often involve multiple brief (e.g., 1 second to 30 second) immersions ( “dips” ) . The duration of immersion into active reagents can vary but is generally in the range of 1 to 10 minutes. See Table 3.

VARIATIONS AND OTHER HETEROPOLYMERS

Although the discussion above is focused on certain phosphoramidite chemistries (e.g., phosphotriester synthesis) , it will be appreciated that the method can be adapted to a broad variety of oligonucleotide synthesis chemistries that link phosphoramidite building blocks derived from protected 2'‐deoxynucleosides (dA, dC, dG, and T) , ribonucleosides (A, C, G, and U) , or chemically modified nucleosides, e.g. LNA or BNA.

In addition, the strategy and devices disclosed herein may be used in complex approach heteropolymers that require sequential addition of individual subunits. For example, polypeptides (heteropolymers of amino acids) and polysaccharides (heteropolymers of sugars) can be synthesized using the device described herein, with appropriate changes in reagents.

KITS

Kits are provided for synthesizing a phosphoramidite‐based oligonucleotide on a solid support. The kits comprise synthesis reagents adapted for use in the devices described above. Generally the solutions are anhydrous and The synthesis reagents differ from conventional reagents in that the synthesis reagents (e.g., one or more of coupling reagent solutions, deblocking reagent solution, oxidation reagent solutions, capping reagent solutions, and wash solutions) contain nonvolatile solvents (e.g., propylene carbonate, butyronitrile, adiponitrile, trifluorotoluene) and optionally are anhydrous.

In one embodiment at least one, and optionally all, synthesis reagent solutions are free from acetonitrile, dichloromethane and water. Exemplary nonvolatile solvents include propylene carbonate, acetic acid, trifluorotoluene, butyronitrile, and adiponitrile. In one embodiment the nonvolatile solvent has a boiling point greater than 110 degrees C or has other properties described hereinabove.

A kit may include coupling solutions that contain propylene carbonate and which do not contain acetonitrile. The coupling solutions may include, for example, DNA phosphoramidites or RNA phosphoramidites. In one example, the coupling solution contains DNA phosphoramidites such as dA (Bz) CEP, dC (Bz) CEP, dG (iBu) CEP, dT CEP.

A kit may include a deblocking solution that contains propylene carbonate and is free from acetonitrile. In one embodiment, the deblocking solution contains trichloroacetic acid.

A kit may include an oxidation reagent solution that contains propylene carbonate and is free from water. An oxidation solution may contain a carboxylic acid, such as anhydrous acetic acid. In one embodiment the kit includes an oxidation solution that contains acetic acid, I ₂, and, optionally, pyridine.

A kit may include a capping solution that is free from acetonitrile. The capping solution may contain propylene carbonate. The capping solution may contain acetic anhydride and/or 1‐methylimidazole and/or pyridine.

EXAMPLE

Solid‐phase preparation

Borosilicate glass rods (1 mm diameter, Sutter Instruments) were cleaned and modified with the silane coupling agent N‐ (3‐triethoxysilylpropoyl) ‐4‐hydroxybutyramide (Gelest) . The hydroxylated surface was then coupled to the chemical phosphorylation reagent (Glen Research) , which serves as a cleavable linker. Alternative universal, cleavable linkers may be couple to an amine modified glass surface. The modified glass rods were cut into 30 mm long pieces.

Oligonucleotide synthesis

The glass rods were attached to the automated dipping hardware. A reagent tray was placed underneath the dipping hardware, and synthesis reagents were placed in predetermined wells of the reagent tray. The reagents and solutions are listed in Table 2.

The complete system was enclosed in a box (housing) that maintains a low humidity atmosphere.

Reagents used for oligonucleotide synthesis may include a deblocking reagent, a monomer reagent, an activator reagent, an oxidation reagent, a cap A reagent, a cap B reagent, and a wash reagent.

In some embodiments, the monomer reagent comprises phosphoramidite (e.g., 0.067M) and propylene carbonate. In some embodiments, the cap A reagent comprises acetic anhydride (e.g., 10%v/v) and propylene carbonate. In some embodiments, the cap B reagent comprises 1‐methylimidazole (e.g., 10%w/v) , pyridine, and propylene carbonate. In one embodiment, 1‐methylimidazole has a concentration of 10%v/v. In one embodiment, pyridine has a concentration of 10%v/v.

Table 2.

Composition of reagents for oligonucleotide synthesis

The program controlling the dipping hardware was started, and the synthesis was carried out. Following the program, the hardware immerses 5 mm of each rod into the reagents in the order required to carry out oligonucleotide synthesis with phosphoramidites. An example of the sequence and duration of dips in one cycle is shown in Table 3. The time and number of dips was varied when initial experiments were carried out to determine which values optimized yield.

TABLE 3.

Dipping steps in one synthesis cycle.

One base was added to the oligonucleotide per synthesis cycle. The number of cycles and the identity of the base in the monomer dip in each cycle (adenine, cytosine, thymine, guanine) determines the sequence of the synthesized nucleotide. In this example, the following sequence was synthesized: 5’‐AAT CAC GAG ATA TCC CGC GCC CTC ATA GTA GCC GCC CTG T‐3’ (SEQ ID NO: 1) . The synthesis was carried out 3’ to 5’, so the addition was performed in the following order: TGT; CCC; GCC; GAT; GAT; ACT; CCC; GCG; CCC; TAT; AGA; GCA; CTA; A.

Post‐synthesis processing

The glass rods were removed from the dipping hardware and washed with acetonitrile. The glass was immersed in concentrated ammonium hydroxide and incubated for at least 17 hours at room temperature. The ammonium hydroxide solution was collected and evaporated, leaving the oligonucleotide as a residue. The oligonucleotide was resuspended in water and used in downstream applications.

Synthesis yield and fidelity

The synthesis yield was analyzed by reverse phase HPLC (FIG. 3) . FIG. 4 shows comparison of 40 mers synthesized by dipping and on standard equipment: (i) chromatogram of 40 mer synthesized by dipping, with a stepwise yield >97%; and (ii) chromatogram of the same 40 mer sequence synthesized on a standard column‐based oligonucleotide synthesizer. The retention time matches that of the 40 mer synthesized by dipping.

***

While the foregoing invention has been described in some detail for purposes of clarity and understanding, it will be appreciated by those skilled in the relevant arts, once they have been made familiar with this disclosure, that various changes in form and detail can be made without departing from the true scope of the invention in the appended claims. The invention is therefore not to be limited to the exact components or details of methodology or construction set forth above. Except to the extent necessary or inherent in the processes themselves, no particular order to steps or stages of methods or processes described in this disclosure, including the Figures, is intended or implied. In many cases the order of process steps may be varied without changing the purpose, effect, or import of the methods described.

All publications and patent documents cited herein are incorporated herein by reference as if each such publication or document was specifically and individually indicated to be incorporated herein by reference. Citation of publications and patent documents (patents, published patent applications, and unpublished patent applications) is not intended as an admission that any such document is pertinent prior art, nor does it constitute any admission as to the contents or date of the same.

Claims

An apparatus for producing a plurality of oligonucleotides with different user‐defined sequences, comprising:

a support structure;

a rod array comprising a plurality of rods coupled to the support structure, wherein each of the plurality of rods comprises an immersion end and an actuation end extending between a longitudinal axis of the rod,

wherein each of the plurality of rods comprises a reactive surface at or proximate to the immersion end,

wherein the reactive surface is configured for immobilizing a protected nucleoside derivative or an oligonucleotide comprising a protected nucleoside derivative at its terminus,

wherein the plurality of rods are configured to be translatable along the longitudinal axes of the rods relative to the support structure;

a controller; and

a plurality of linear actuators electrically coupled to the controller, wherein each of the plurality of linear actuators is coupled to the support structure and the actuation end of one of the plurality of rods and configured to translate in a direction of the longitudinal axes,

wherein the controller is configured to independently actuate each of the plurality of linear actuators in order to independently cause translation of each of the plurality of rods between a retracted position and an extended position, in order to cause the protected nucleoside derivative or oligonucleotide immobilized on the reactive surfaces to be independently submerged into different reagent solutions in reservoirs of a reagent tray positioned below the array.
The apparatus of claim 1, wherein the reactive surface on the rods comprises a protected nucleoside derivative immobilized thereon, optionally immobilized by a covalent bond.
The apparatus of claim 1, wherein the reactive surface comprises immobilized thereon an oligonucleotide comprising a protected nucleoside derivative at its terminus, optionally immobilized by a covalent bond.
The apparatus of claim 2 or claim 3, wherein the protected nucleoside derivative or oligonucleotide is immobilized on the reactive surface via a linker.
The apparatus of any of claims 2 to 4, wherein the protected nucleoside derivative is a nucleoside phosphoramidite.
The apparatus of any of claims 2 to 4, wherein the protected nucleoside derivative is a 3'‐O‐ (N, N‐diisopropyl phosphoramidite) derivative of a nucleoside.
The apparatus of any of claims 2 to 4, wherein the protected nucleoside derivative is a 2’‐deoxyribonucleoside derivative.
A system that comprises the apparatus of any preceding claim, in combination with one or more reagent trays configured to contact individual rods of the plurality of rods with one or more reagent solutions contained therein.
The system of claim 8, further comprising one or more reagent solutions selected from a deblocking solution, a coupling solution comprising a phosphoramidite protected nucleoside, an oxidation solution, and a capping solution.
A kit for synthesizing a phosphoramidite‐based oligonucleotide on a solid support or on an apparatus according to any of claims 1 to 7,

wherein the kit comprises one or more reagent solutions selected from a deblocking solution, a coupling solution comprising a phosphoramidite protected nucleoside, an oxidation solution, and a capping solution.
The system of claim 9 or the kit of claim 10, wherein at least one, and optionally all of said reagent solutions are free from acetonitrile, dichloromethane and water.
The system of claim 8 or 9 or kit of claims 10 to 11, wherein all said reagent solutions are anhydrous and comprise a nonvolatile solvent.
The system or kit of claim 12, wherein the nonvolatile solvent is selected from propylene carbonate, acetic acid, trifluorotoluene, butyronitrile, and adiponitrile.
The system or kit of claim 12, wherein the nonvolatile solvent has a boiling point greater than 110 degrees C.
The system or kit of any of claims 9 to 14, wherein said reagent solutions include a plurality of coupling solutions.
The system or kit of claim 16, wherein individual coupling solutions of the plurality of coupling solutions comprise propylene carbonate and either DNA phosphoramidites or RNA phosphoramidites, wherein the coupling solution is substantially free of acetonitrile.
The system or kit of claim 15 or claim 16, wherein said reagent solutions include four or more coupling solutions, each of which comprises a nucleoside comprising a different nucleobase.
The system or kit of claim 17, wherein coupling solution comprises the DNA phosphoramidites dA (Bz) CEP, dC (Bz) CEP, dG (iBu) CEP, and dT CEP.
The system or kit of any of claims 9 to 18, wherein said reagent solutions include a deblocking solution.
The system or kit of claim 19, wherein the deblocking solution comprises propylene carbonate and is substantially free from acetonitrile.
The system or kit of claim 19, wherein the deblocking solution comprises trichloroacetic acid.
The system or kit of any of claims 9 to 21, wherein said reagent solutions include an oxidation solution.
The system or kit of claim 22, wherein the oxidation solution comprises propylene carbonate and is substantially free from water.
The system or kit of claim 22, wherein the oxidation solution comprises anhydrous acetic acid or a combination of acetic acid, I ₂, and pyridine.
The system or kit of any of claims 9 to 24, wherein said reagent solutions include a capping solution.
The system or kit of claim 25, wherein the capping solution comprises propylene carbonate and is substantially free of acetonitrile
The system or kit of claim 25, wherein the capping solution comprises acetic anhydride, 1‐methylimidazole, and pyridine.
An apparatus for producing a plurality of oligonucleotides with different user‐defined sequences, comprising:

a support structure;

a rod array comprising a plurality of rods coupled to the support structure, wherein each of the plurality of rods comprises an immersion end and an actuation end extending between a longitudinal axis of the rod,

wherein each of the plurality of rods comprises a reactive surface at or proximate to the immersion end,

wherein the reactive surface is configured for immobilizing a protected nucleoside derivative or an oligonucleotide comprising a protected nucleoside derivative at its terminus,

wherein the plurality of rods are configured to be translatable along the longitudinal axes of the rods relative to the support structure;

a controller; and

a plurality of linear actuators electrically coupled to the controller, wherein each of the plurality of linear actuators is coupled to the support structure and the actuation end of one of the plurality of rods and configured to translate in a direction of the longitudinal axes,

wherein the controller is configured to independently actuate each of the plurality of linear actuators in order to independently cause translation of each of the plurality of rods between a retracted position and an extended position, in order to cause the protected nucleoside derivative or oligonucleotide immobilized on the reactive surfaces to be independently submerged into different reagent solutions in reservoirs of a reagent tray positioned below the array.
A method for reagent delivery to any subset of an array of 4 to 10,000 reactive surfaces, irrespective of the number of elements within the subset (0 to total number of array) and the position of the subset elements within the array.
A method of chemical synthesis that comprises delivering a plurality of different reagents to each of a plurality of reactive surfaces, wherein the reagents are delivered to each individual reactive surface in a predetermined order, the method comprising:

a) providing an array comprising a set of independently actuatable reactive surfaces, wherein actuation of a reactive surface causes the reactive surface to be immersed into a reagent solution in a reservoir;

b) carrying out a cycle of chemical synthesis, the cycle comprising steps (i) to (vi) :

i) immersing a first subset of the set of reactive surfaces into a first reservoir comprising a first reagent solution, and withdrawing the first subset of reactive surfaces from the first reservoir, wherein the first subset of reactive surfaces are coordinately actuated;

ii) immersing a second subset of the set of reactive surfaces into a second reservoir comprising a second reagent solution, and withdrawing the second subset of reactive surfaces from the second reservoir, wherein the second subset of reactive surfaces are coordinately actuated;

iii) immersing a third subset of the set of reactive surfaces into a third reservoir comprising a third reagent solution, and withdrawing the third subset of reactive surfaces from the third reservoir, wherein the third subset of reactive surfaces are coordinately actuated; and then

iv) immersing a fourth subset of the set of reactive surfaces into the first reservoir comprising the first reagent solution, and withdrawing the fourth subset of reactive surfaces from the first reservoir, wherein the fourth subset of reactive surfaces are coordinately actuated;

v) immersing a fifth subset of the set of reactive surfaces into the second reservoir comprising the second reagent solution, and withdrawing the fifth subset of reactive surfaces from the second reservoir, wherein the fifth subset of reactive surfaces are coordinately actuated;

vi) immersing a sixth subset of the set of reactive surfaces into the third reservoir comprising the third reagent solution, and withdrawing the sixth subset of reactive surfaces from the third reservoir, wherein the sixth subset of reactive surfaces are coordinately actuated;

wherein the fourth subset comprises at least some reactive surfaces from each of the first, second and third subsets, the fifth subset comprises at least some reactive surfaces from each of the first, second and third subsets, and the sixth subset comprises at least some reactive surfaces from the first, second and third subsets; and

c) iteratively carrying out 4 to 100 additional synthesis cycles, each additional synthesis cycle comprising steps (i) to (vi) , wherein in at least four of the additional cycles the first, second, and third subsets in the cycle are not all the same as the first, second, and third subsets of the previous cycle;

thereby delivering a plurality of different reagents to each of the reactive surfaces, wherein the reagents are delivered to each individual reactive surface in a user defined order.
The method of claim 30, which is a method of oligonucleotide synthesis, whereby each of the reactive surfaces produces an oligonucleotide having a different sequence.
The method of claim 30 or claim 31, wherein each of the reactive surfaces provided in step a) comprises a protected nucleoside derivative or an oligonucleotide comprising a protected nucleoside derivative at its terminus immobilized thereon.
The method of claim 32, wherein the protected nucleoside derivative is a nucleoside phosphoramidite.
The method of any of claims 30 to 33, wherein the first, second and third reagent solutions are three coupling solutions that each contain a phosphoramidite protected nucleoside that is not contained in either of the other two coupling solutions.
The method of claim 34, wherein the three coupling solutions each comprise propylene carbonate and either DNA phosphoramidites or RNA phosphoramidites, wherein the coupling solutions are substantially free of acetonitrile.
The method of claim 34 or claim 35, wherein the phosphoramidites in each of the coupling solutions is selected from the DNA phosphoramidites dA (Bz) CEP, dC (Bz) CEP, dG (iBu) CEP, and dT CEP.
The method of any of claims 30 to 36, wherein each synthesis cycle further comprises sequentially immersing each of the reactive surfaces into a plurality of reservoirs, one containing an oxidation solution, a second containing a capping solution, a third containing a deblocking solution, and a fourth containing a wash solution.
The method of claim 37, wherein the oxidation solution comprises propylene carbonate and is substantially free from water, or wherein the oxidation solution comprises anhydrous acetic acid or a combination of acetic acid, I ₂, and pyridine.
The method of claim 37 or claim 38, wherein the capping solution comprises propylene carbonate and is substantially free of acetonitrile, or wherein the capping solution comprises acetic anhydride, 1‐methylimidazole, and pyridine.
The method of any of claims 37 to 39, wherein the deblocking solution comprises propylene carbonate and is substantially free from acetonitrile, or wherein the deblocking solution comprises trichloroacetic acid.
The method of any of claims 30 to 40, whereby in each synthesis cycle, an oligonucleotide on the reactive surface is lengthened by addition of a single subunit.
The method of calim 41, wherein for at least some reactive surfaces and at least some synthesis cycles, the single subunit is a phosphoramidite trinucleotide.