Variable viscosity inks for inkjet delivery of enzymatic reagents

Abstract

The present invention relates to formulations of terminal deoxynucleotidyl transferase (TdT) inks using variable viscosity modifiers and their use for inkjet assisted synthesis of a plurality of polynucleotides at reaction sites on a substrate.

Description

[0001] Inkjet printing is a low-cost, versatile technology for contactlessly delivering defined amounts of liquid to precise locations with minimal waste. The technology has been applied to oligonucleotide microarray synthesis using phosphoramidite chemistry and has been used to print enzymes directly onto substrates for the production of enzyme-based biosensors. For the latter application of inkjet printing, it has been observed that enzyme activity is affected not only by shear forces and the rheological requirements of droplet formation, but also by changes in enzyme concentration and buffer conditions caused by evaporative losses when, for example, an enzyme-containing fluid is printed onto a microarray.

[0002] Recently, there has been interest in applying enzyme-based polynucleotide synthesis to problems that are not amenable to conventional chemical-based DNA synthesis, particularly due to the mild aqueous reaction conditions of enzymatic processes. It has been observed that there is currently a trade-off between the ink viscosity values ​​required for printing droplet formation and the viscosity values ​​required for efficient enzymatic processing after inkjet delivery. That is, ink viscosities optimized for droplet formation are currently suboptimal for enzymatic activity, and vice versa. Summary of the Invention

[0003] The present invention relates to inks containing variable viscosity modifiers for inkjet delivery of enzymes to reaction sites.

[0004] In one aspect, the present invention relates to an inkjet-based method for the enzymatic synthesis of multiple polynucleotides using such an ink, the method comprising the steps of: (a) providing a substrate having initiators at a plurality of reaction sites, wherein each initiator has a free 3'-hydroxyl group, and wherein each of the multiple polynucleotides is assigned to a reaction site for synthesis; (b) dispensing at least one droplet of at least one synthesis reagent to each of the plurality of reaction sites via one or more inkjet pumps for a reaction cycle, the reaction cycle comprising the steps of: (i) reacting an initiator or an extension fragment having a free 3'-O-hydroxyl group with a 3'-O-protected nucleoside triphosphate and a template-free polymerase under extension conditions such that the initiator or the extension fragment is extended by incorporation of the 3'-O-protected nucleoside triphosphate to form a 3'-O-protected extension fragment, and (ii) deprotecting the extension fragment to form an extension fragment having a free 3'-hydroxyl group, wherein the synthesis reagent comprises a template-free polymerase, a 3'-O-protected nucleoside triphosphate, a mixture of a template-free polymerase and a 3'-O-protected nucleoside triphosphate, or a deprotection solution; wherein the at least one synthesis reagent comprises a variable viscosity modifier having a first viscosity greater than 2 mPa·s during formation of the at least one droplet at the one or more inkjet pumps and a second viscosity less than or equal to 2 mPa·s at each of the plurality of reaction sites during the reaction steps of the reaction cycle; and (c) repeating step (b) until the multiple polynucleotides are synthesized; wherein the first viscosity and the second viscosity are different. In some embodiments, the substrate is a planar substrate.

[0005] In another aspect, the present invention relates to a template-free polymerase ink for inkjet printing, comprising: an aqueous solution comprising a template-free polymerase at a concentration of 1.0 μM to 30 μM; wherein each printed droplet has a volume of 0.1 pL to 5 nL of the aqueous solution whenever the ink is printed onto a substrate, and wherein the ink comprises a variable viscosity modifier having a first viscosity of 2 - 20 mPa·s whenever the temperature is 5 - 30°C and a second viscosity of 1 - 2 mPa·s whenever the temperature is 35 - 60°C; wherein the first viscosity and the second viscosity are different.

[0006] BRIEF DESCRIPTION OF THE DRAWINGS

[0007] Figure 1A Schematic of an enzymatic synthesis cycle where 3'-O-protected dNTP is added to a nucleic acid strand and then deprotected.

[0008] Figure 1BShown is a droplet microarray with a hydrophobic-hydrophilic patterned surface that can be used as a synthesis support.

[0009] Figures 2A to 2C Synthesis cycles for four different embodiments of the present invention are shown.

[0010] Figure 3 An embodiment of the invention is shown in which an inkjet instrument is used to define the reaction sites on the array, eliminating the problem of aligning inkjet delivery with preformed reaction sites.

[0011] Figure 4A shows components of an exemplary inkjet system for use with the present invention, Figure 4B An inkjet apparatus for practicing several embodiments of the present invention is shown.

[0012] Figure 5 A technique for evaluating the efficiency of inkjet-based enzymatic synthesis is presented, which can be used to select variable viscosity modifiers.

[0013] Figure 6 The change in viscosity of this example at different temperatures is shown.

[0014] Figure 7 The stability of the extended ink stored at different temperatures is shown.

[0015] Figure 8 Results for 10 cycles of poly(T) performed with manual EDS are shown. DETAILED DESCRIPTION

[0016] Although the present invention can be subjected to various modifications and alternative forms, its details have been illustrated by the examples in the accompanying drawings and will be described in detail. However, it should be understood that the purpose is not to limit the present invention to the specific embodiment described. The purpose is to cover all modifications, equivalents and substitutes that fall within the spirit and scope of the present invention. Guidance for selecting materials and components to perform specific functions can be found in available papers and references about scientific instruments, including but not limited to Moore et al, Building Scientific Apparatus, Third Edition (Perseus Books, Cambridge, MA); Hermanson, Bioconjugate Techniques, 3rd Edition (Academic Press, 2013) and similar references.

[0017] On the one hand, the present invention relates to an ink for delivering an enzyme-containing reagent in a droplet formed by inkjet, and a method for enzymatically synthesizing polynucleotides using this ink. This ink comprises at least one variable viscosity modifier, and the modifier can show a first viscosity at the nozzle of a printhead during droplet formation, and shows a second viscosity for carrying out an enzymatic process at the reaction site after delivery. Typically, the variable viscosity modifier responds to different physical conditions, such as temperature, at the reaction site during droplet formation and when implementing an enzymatic process after droplet delivery. In some embodiments, the nozzle of the printhead is maintained at a temperature lower than the temperature of the reaction site, and uses a variable viscosity modifier with a viscosity that monotonically decreases as the temperature rises. In other embodiments, the nozzle of the printhead is maintained at a temperature higher than the temperature of the reaction site, and uses a variable viscosity modifier with a viscosity that monotonically increases as the temperature rises. In other embodiments, a variable viscosity modifier with shear thickening (or dilatant) properties is used such that the droplet formation conditions induce a viscosity of 2-20 mPa.s and the conditions at the reaction site induce a viscosity of 1-2 mPa.s.

[0018] Variable viscosity modifiers whose viscosity decreases with increasing temperature may include conventional viscosity modifiers at selected concentrations and within selected temperature ranges. Such conventional viscosity modifiers include, but are not limited to, ethylene glycol, polyvinyl alcohol, dimethyl sulfoxide (DMSO), polyethylene glycol (PEG), polyethylene glycol methyl ether (OMe)PEG, polyethylene glycol dimethyl ether (OMe)2PEG, carboxymethyl cellulose, hydroxyethyl cellulose, and the like. The selected concentration of the variable viscosity modifier in the ink of the present invention includes, but is not limited to, 5-50% by volume of the solution, preferably 20-40% by volume of the solution. The selected temperature range includes, but is not limited to, a temperature of at least one inkjet pump of 15°C or less, or 12°C or less, or 10°C or less.

[0019] Variable viscosity modifiers whose viscosity decreases with increasing temperature can also include thermoreversible polymers, preferably uncrosslinked, compositions incorporating poly(N-alkyl substituted acrylamide), preferably uncrosslinked, such as Sassi et al, Electrophoresis, 17: 1460-1469 (1996); Schild, Prog. Polymer Sci., 17: 163-249 (1992), etc. For example, the variable viscosity properties of poly(N-isopropylacrylamide) polymers can be readily modified by selecting molecular weight, concentration, and appropriate comonomers (e.g., Sigma Aldrich, Technical Bulletin, "Designing temperature and pH sensitive NIPAM based polymers;" Plate et al, Polymer Journal, 31(1): 21-27 (1999)).

[0020] In some embodiments, a template-free polymerase ink can be formulated comprising an uncrosslinked poly(N-alkyl-substituted acrylamide) having a first viscosity of 2-20 mPa.s whenever the temperature is between 5-30°C and a second viscosity of 1-2 mPa.s whenever the temperature is between 35-60°C.

[0021] Variable viscosity modifiers whose viscosity increases with increasing temperature can include poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) triblock polymers, such as F127, for example, Wu et al, Electrophoresis, 19:231-241 (1998); Rill et al, Proc. Natl. Acad. Sci., 95: 1534-1539 (1998), etc. The variable viscosity properties of poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) triblock polymers can be easily modified by selecting molecular weight, concentration, and appropriate comonomers. In some embodiments, a template-free polymerase ink can be formulated comprising an uncrosslinked poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) triblock polymer having a first viscosity of 2-20 mPa.s whenever the temperature is between 35-60°C and a second viscosity of 1-2 mPa.s whenever the temperature is between 5-30°C.

[0022] In further embodiments, the variable viscosity modifier may comprise a shear thickener or dilatant, including but not limited to Rheovis PU 1331, Rheovis PE 1330 (BASF), and the like. Other such variable viscosity modifiers may include poly(N-isopropylacrylamide). The concentration and temperature are selected such that the ink viscosity is 3 mPa.s or greater whenever the shear force at the nozzle of the inkjet pump is 105 (s-1) or greater.

[0023] In some embodiments, a template-free polymerase ink can be formulated comprising a polymer solution that gels upon cooling and has a first viscosity of 2-20 mPa.s whenever the temperature is between 5-30°C and a second viscosity of 1-2 mPa.s whenever the temperature is between 35-60°C.

[0024] In some embodiments, the variable viscosity modifier may comprise a polymer solution that gels upon cooling. The polymer is selected from at least one of a hydrophobically modified polymer exhibiting UCST-type phase behavior, a natural polymer, silica nanoparticles grafted with a deblocked copolymer brush, poly(PEO-co-styrene), and combinations thereof. The polymer may be dissolved in an ionic liquid or in a solution of PNIPAM microgel and host-guest interaction.

[0025] In some embodiments, at least one synthetic reagent solution is free of glycerol.

[0026] In some embodiments, there is more than one synthesis reagent solution, each solution having a different variable viscosity modifier.

[0027] In some embodiments, the ink having a variable viscosity modifier may also include additional components as described below, including but not limited to conventional viscosity modifiers, surfactants, humectants, aldehyde scavengers, etc., as described below.

[0028] In some embodiments, the present invention relates to methods and compositions for inkjet-assisted synthesis of multiple polynucleotides, each at a different site on a substrate, using a template-free polymerase, such as terminal deoxynucleotidyl transferase (TdT). Typically, such synthesis occurs on a substrate comprising a flat surface (e.g., a surface of glass, silica, silicon oxide, plastic, etc.), but may also occur on other surfaces, such as biological tissue or surface-immobilized cDNA extracted from tissue. As used herein, "inkjet-assisted synthesis" means that one or more synthesis reagents are delivered to the reaction site in droplets generated by one or more inkjet pumps.

[0029] "Synthesis reagents" include any reagents for coupling monomers, particularly 3'-O protected nucleoside triphosphates to initiators or extension segments in a synthesis cycle, such as buffers comprising no template polymerase, buffers comprising 3'-O protected nucleotide monomers, buffers comprising a mixture of no template polymerase and one or more 3'-O protected nucleoside triphosphates, deprotection (or deblocking) buffers, etc. The terms "deprotection" agent, buffer, solution, etc. are used synonymously with the terms "deblocking" agent, buffer, solution, etc., respectively, in this article. Similarly, the term "protected" of a related compound (such as dNTP) is used synonymously with the term "blocked" of a related compound. As used herein, the term "deprotection solution" (or its equivalent) means a reagent that causes or promotes the removal of a protecting group (such as the 3'-O protecting group of a nucleotide). As described more fully below, the composition of the deprotection solution (and deprotection reaction conditions) depends on the property of the protecting group (or blocking group) to be removed. In various embodiments, the deprotection solution may contain specific reagents that chemically react with the protecting group and / or protecting portion (e.g., a reducing agent, such as TCEP (tris(2-carboxyethyl)phosphine)), enzymes for enzymatic cleavage, scavengers, cofactors, and the like. In some embodiments, the deprotection solution may not contain specific reagents that react with the protecting group, but, for example, in the case of a photocleavable protecting group, may contain components that are compatible with the protecting group or promote physical cleavage of the protecting group, such as a pH buffer. Typically, in a reaction cycle for extending a polynucleotide fragment, in the deprotection step, the deprotection solution is incubated with the 3'-O protected extension fragment for a predetermined incubation time. Typical incubation times (i.e., the duration of the incubation step) are from 1 minute to 30 minutes; or from 3 minutes to 30 minutes; or from 3 minutes to 15 minutes. Typical extension reaction temperatures are from room temperature (RT) to 80°C; or from 20°C to 80°C; or from 20°C to 60°C.

[0030] "Synthesis reagents" also include reagents used to prepare the substrate for polynucleotide synthesis, such as reagents for defining reaction sites, initiators, capping agents, etc. Typically, a "distinct reaction site" on a substrate is a discrete site because it is separated from other reaction sites; that is, for example Figure 1B As illustrated, discrete sites do not have boundaries with, or overlap with, another reaction site. In other words, discrete or distinct reaction sites do not abut or overlap with other reaction sites. Exceptions to this general arrangement include the "overwriting" embodiment described below for generating high-density barcodes on surfaces.

[0031] In some embodiments, the plurality of polynucleotides may be 2 to 500,000; or 100 to 400,000; or 100 to 200,000; or 100 to 100,000. The number of the plurality of polynucleotides may be the same as or different from the number of the plurality of reaction sites. In some embodiments, the number of the plurality of reaction sites may be greater than the number of the plurality of polynucleotides. In some embodiments, each of the plurality of reaction sites has a density comparable to that uniformly deposited on an area comparable to the area of ​​a standard 25 mm x 75 mm microscope slide. In some embodiments, the array of reaction sites formed by uniform deposition may be a linear array; and in other embodiments, the array of reaction sites formed by uniform deposition may be a hexagonal array.

[0032] The basic steps of template-free enzymatic synthesis of polynucleotides are as follows Figure 1AAs shown in, and more fully described below.In brief, synthetic method comprises the step cycle that is generally related to sending at least one following reagent to the reaction site: the buffer comprising no template polymerase, one or more buffers that each comprise the dNTP (i.e. monomer) of one or more 3'-O protections, deprotection buffer and washing solution.In various embodiments of the present invention, no template polymerase buffer, the buffer comprising the dNTP monomer of 3'-O protection, or deprotection buffer can be sent to the reaction site by the droplet produced and delivered by an inkjet pump.In order to deliver by the droplet produced by inkjet, these reagents must be formulated to meet the rheological requirements of droplet formation.These preparations are called "inks". The key rheological parameters that affect droplet formation are viscosity, density, and surface tension, for example, Derby, Annu. Rev. Mater. Sci., 40: 395-414 (2010); Derby, J. Mater. Chem., 18: 5717-57-21 (2008); Calvert, Chem. Mater., 13: 3299-3305 (2001); Tekin et al, SoftMatter, 4: 703-713 (2008) and similar references. Another key parameter related to droplet volume is the nozzle diameter of the inkjet pump. In some embodiments, the nozzle diameter used in the present invention can be 10 μm to 100 μm. Thus, as described more fully below, one aspect of the present invention includes reagent inks for template-free enzymatic synthesis of polynucleotides, and in particular, inks comprising a template-free polymerase, in particular inks comprising terminal deoxynucleotidyl transferase (TdT) or inks comprising TdT and one or more 3'-O-protected dNTP monomers. In some embodiments, the inks of the present invention may comprise more than one 3'-O-protected nucleoside triphosphate, and in some cases, for the purpose of synthesizing random sequence fragments of polynucleotides, for example, for the purpose of creating polynucleotide tags or barcodes, may comprise all four monomer types.

[0033] According to the present invention, inkjet-assisted enzymatic synthesis of polynucleotides can be implemented in various embodiments wherein different reagents are delivered by an inkjet pump. Figures 2A to 2C Some of these embodiments are described in . Figure 2A Two cycles are shown in which droplets (or microdroplets) containing a template-free polymerase and a single species of monomer are delivered to a reaction site surrounded by a buffer droplet on a substrate. The surface of the reaction site (204) contains a layer of initiator oligonucleotide (not shown) and is surrounded by the hydrophobic surface of the substrate (203), which allows the reaction site (204) to be surrounded by a volume (202) of aqueous liquid on the surface (203) without diffusing or coalescing with liquid from another reaction site. Figure 2A The initiator is depicted as having a 3'-terminal monomer "A" with a free 3'-hydroxyl group (not shown). Figures 2A to 2C In the figure, monomers A, B and C are each intended to represent any 3'-O protected dNTP monomer. The ink droplet (201) is dispensed into a volume (202) at a reaction site (204) by an inkjet pump (not shown) (or directly into the reaction site (204) if a drying step is performed). The ink droplet (201) comprises a predetermined concentration of template-free polymerase and a predetermined concentration of monomer B, as well as salt and buffer components for polymerase activity, and viscosity modifiers and surface tension modifiers required to meet the rheological requirements of droplet formation. The droplet (201) may also include a wetting agent to minimize evaporation losses. In some embodiments, whenever a 3'-O-amino-NTP is used, the droplet (201) may also include an aldehyde scavenger. The droplets (201) are deposited on dried reaction sites (207) or coalesced with the volume (202) on undried reaction sites (221) to form a reaction mixture (206) in the droplet (205), which is allowed to incubate (208) for a predetermined time to allow the B monomer to couple to the 3' end of the initiator (or the previously extended or extended chain after the initial cycle). In some embodiments, this incubation occurs at above ambient humidity to prevent drying during the incubation step.

[0034] In some embodiments, a separate step of drying the reaction sites may be performed to prevent fluid accumulation and / or coalescence with the reaction mixture at adjacent reaction sites.

[0035] After the incubation time for the coupling reaction, the entire substrate surface is immersed or sprayed (209) in a deprotection buffer (210) for a predetermined time to allow the removal of the protecting groups, which regenerates the free 3'-hydroxyl group at the end of the extended chain. After the predetermined deprotection time, the entire substrate surface is immersed in one or more wash buffers for a predetermined time one or more times to obtain a reaction site (211) with an extended or extended chain or fragment (shown as "-AB"), which is ready for the next coupling cycle.

[0036] In some embodiments, as described above, a drying step may be performed after deprotection and washing (213) to minimize the possibility of droplets (211) spreading or coalescing with adjacent droplets. Conventional drying techniques used in inkjet printing, such as hot air or gas, radiation drying, etc., may be used, for example, as described in Hoynant et al, U.S. Patent 8,485,096.

[0037] The steps of the next cycle are identical to those for attaching the B monomer, except that the C monomer is coupled in the next cycle. Thus, a droplet (212) having the same composition as the droplet (201), possibly except for the identity of the C monomer, is dispensed into the volume (211). As described above, the reaction mixture (214) in the droplet (215) is incubated for a predetermined amount of time, after which the entire substrate is treated (218) with a deprotection buffer and a wash solution (210) to yield reactive sites (220) with an extended chain, shown as "-ABC".

[0038] Depending on the nature of the substrate (203) in this and other embodiments, the coupling cycle may also include a drying step to prevent droplets from spreading and coalescing between adjacent reaction sites. If the surface of the substrate (203) between the reaction sites (204) is sufficiently hydrophobic, the possibility of such coalescence is minimized.

[0039] Figure 2A The embodiment of the present invention can be carried out by the following steps: (a) providing a substrate having initiators at a plurality of different reaction sites, wherein each initiator has a free 3'-hydroxyl group, and wherein each polynucleotide in a plurality of nucleotides is assigned to a different reaction site for synthesis; (b) dispensing at least one droplet of a buffer solution comprising a mixture of a template-free polymerase and 3'-O-blocked dATP, 3'-O-blocked dCTP, 3'-O-blocked dGTP or 3'-O-blocked dTTP to each reaction site by one or more inkjet pumps, wherein the 3'-O-blocked dCTP dispensed to the reaction site is free of dATP, 3'-O-blocked dGTP or 3'-O-blocked dTTP. The type of '-O blocked dNTP depends on the predetermined sequence of the polynucleotide assigned to the reaction site; (c) incubating a template-free polymerase and a 3'-O blocked dNTP at each reaction site so that the initiator or extension segment at the reaction site is extended by incorporating the 3'-O blocked dNTP to form a 3'-O blocked extension segment; (d) by treating the planar support with a deblocking agent, the extension segment is deblocked at each reaction site to form an extension segment with a free 3'-hydroxyl group; (e) repeating steps (b), (c) and (d) until a plurality of polynucleotides are synthesized. In some embodiments, a plurality of droplets are delivered to each reaction site during each cycle of steps (b), (c) and (d). In some embodiments, a plurality of droplets are 2 to 10, or 2 to 5, or 2 to 3. In other embodiments, a plurality of droplets can be 2 to 150, or 10 to 120. In some embodiments, a further step of cutting a plurality of polynucleotides from the substrate is implemented.

[0040] In some embodiments, the substrate is a planar substrate.

[0041] In some embodiments, a drying step may be included after step (d) or after step (d) and a washing step to minimize spreading or coalescence of the droplet when the next droplet is dispensed.

[0042] Figure 2B An embodiment is described in which monomers and template-free polymerases are delivered to a reaction site in droplets delivered by different inkjets. As described above, the surface of the reaction site (204) comprises an initiator layer terminated with an A monomer shown as "-A". Droplets (252) comprising template-free polymerases are dispensed (250) to the volume (202), after which droplets (254) comprising B monomers are dispensed (256). In some embodiments, the order in which template-free polymerases and monomers are dispensed can be reversed so that monomers are dispensed before template-free polymerases are dispensed within the cycle. After the reaction volume (265) is incubated for a predetermined time to allow the B monomers to couple with the initiator or extended chain at the reaction site (204), the entire surface of the substrate is exposed (268) to a deprotection buffer (267) and subsequently to one or more wash buffers to obtain a reaction site (268) with an extended chain "-AB". The next cycle is performed in the same manner except that the C monomer is added, i.e., droplets containing a template-free polymerase are dispensed (270), droplets containing a C monomer are dispensed (272) to form a reaction mixture (273), the reaction mixture is incubated (273), deprotected and washed (276) to obtain a reaction site with an extended chain "-ABC" (278).

[0043] Figure 2BThe embodiment of the present invention can be carried out by the following steps: (a) providing a substrate having initiators at a plurality of different reaction sites, wherein each initiator has a free 3'-hydroxyl group, and wherein each polynucleotide in a plurality of polynucleotides is assigned to a different reaction site for synthesis; (b) dispensing at least one droplet of a buffer solution containing a template-free polymerase to each reaction site by one or more inkjet pumps; (c) dispensing at least one droplet of a buffer solution containing 3'-O-blocked dATP, 3'-O-blocked dCTP, 3'-O-blocked dGTP or 3'-O-blocked dTTP to each reaction site by one or more inkjet pumps. Reaction site, wherein the type of 3'-O blocked dNTP assigned to the reaction site depends on the predetermined sequence of the polynucleotide assigned to the reaction site; (d) incubating the template-free polymerase and 3'-O blocked dNTP at each reaction site so that the initiator or extension segment at the reaction site is extended by incorporating the 3'-O blocked dNTP to form a 3'-O blocked extension segment; (e) by treating the planar support with a deblocking agent, the extension segment is deblocked at each reaction site to form an extension segment with a free 3'-hydroxyl group; (f) repeating steps (b), (c), (d) and (e) until a plurality of polynucleotides are synthesized. In some embodiments, multiple droplets are delivered to each reaction site during each cycle of steps (b) and (c). In some embodiments, such multiple droplets can include only a buffer containing a template-free polymerase, or only a buffer containing 3'-O blocked nucleoside triphosphates, or a combination of the two. In some embodiments, the multiple droplets are 2 to 10, or 2 to 5, or 2 to 3. In other embodiments, the plurality of droplets can be from 2 to 150, or from 10 to 120. In some embodiments, a further step of cleaving the plurality of polynucleotides from the substrate is performed.

[0044] In some embodiments, the substrate is a planar substrate.

[0045] In some embodiments, prior to step (b), a step of drying each reaction site may be performed, and prior to step (c), a step of drying each reaction site may be performed.

[0046] Figure 2CAn embodiment is described in which multiple droplets of a buffer containing a template-free polymerase and monomers are delivered to the reaction site while the coupled reaction occurs in the droplets at the reaction site. An important problem associated with conducting the reaction in droplets at the reaction site is fluid loss due to evaporation. This loss changes the concentration of enzymes, salts, and components such as viscosity modifiers, surfactants, etc., all of which may affect enzyme activity. In addition, in some embodiments, the concentration of the template-free polymerase in the droplets delivered by inkjet may have to be lower than the optimal concentration for the coupled reaction because at higher concentrations the polymerase increases the viscosity of the ink, causing it to adversely affect droplet formation; therefore, in order to be able to deliver a sufficient concentration of polymerase by inkjet, two or more droplets of low concentration polymerase are delivered to the reaction site so that during the coupled reaction, the concentration at the reaction site increases through this repeated addition while the buffer at the reaction site continues to evaporate. Figure 2C The steps of a single reaction cycle are shown, in which two droplets of a buffer solution containing a template-free polymerase and a monomer are delivered to the reaction site. In some embodiments, multiple droplets containing such reagents can be delivered to each reaction site during the synthesis. The number of such droplet deliveries is a design choice that depends on factors such as the reaction site droplet size, the delivery droplet size, the relative humidity, whether the wetting agent is a component of the ink, the duration of the coupling reaction, whether different print heads are used for different reagents, etc. In some embodiments, reagents that alter the droplets on the substrate, such as surfactants, viscosity modifiers, detergents, wetting agents, etc., can be delivered in different droplets generated by different inkjet pumps in the inkjet printhead. The operation of this embodiment is similar to that of the embodiment, except that two or more droplets are delivered to the reaction mixture. Figure 2A As described above, the surface of the reaction site (204a, containing a droplet (202), or 204b, not containing a droplet) comprises an initiator layer terminated with an A monomer shown as "-A". A first droplet of a buffer solution containing a template-free polymerase and a B monomer is delivered to the droplet (202) to form a droplet (282) of a reaction mixture at the reaction site (204).

[0047] Likewise, in some embodiments employing a drying step, droplet (283) will be delivered directly to reaction site (204b), rather than droplet (202) at reaction site (204), to form reaction droplet (282).

[0048] The reaction droplet (282) continues to lose water by evaporation (285) during the incubation (286), which reduces its volume and increases the concentration of all non-volatile components of the droplet, in particular the concentration of the template-free polymerase, thereby improving the coupling activity in the reaction droplet (282). The coupling activity is further improved by delivering a second droplet of a buffer solution containing the template-free polymerase and the B monomer separately or as a mixture. Although the concentration of the polymerase decreases during the initial coalescence of the delivery droplet (287) and the reaction droplet (282), continued evaporation rapidly increases the concentration of the polymerase in the reaction droplet (282) so that it approaches the desired value. After delivering multiple droplets containing the buffer solution with the polymerase and the monomer, the entire surface of the substrate is immersed in a deprotection buffer (288) and one or more washing solutions to obtain reaction sites (294) with extended chains "-AB". The same procedure of delivering multiple droplets of reagent solution to solve the problems caused by evaporation and the rheological constraints of droplet formation can also be applied Figure 2B and Figure 2C implementation plan.

[0049] exist Figures 2A to 2C In some embodiments, the coupling cycle may also include a washing step after the deprotection step. In some such embodiments, the coupling cycle may also include a drying step after the washing step. As mentioned above, the drying step before the continuous coupling cycle will prevent the diffusion and possible coalescence of the reaction droplets in adjacent reaction sites. In some embodiments, washing and drying can be combined by using a volatile washing solution (such as acetonitrile, methanol, etc.) that evaporates easily between coupling cycles.

[0050] As described above, embodiments of the methods of the present invention may include one or more washing steps in which a washing solution is flowed or sprayed onto a substrate comprising an array of reaction sites. The washing solution may comprise a variety of solvents, including but not limited to water, acetonitrile, methanol, PBS or other buffered saline solutions. In some embodiments, the washing solution may comprise one or more proteases, such as proteinase K, for the purpose of removing any polymerase that may adhere to the reaction sites. In other words, Figures 2A to 2C Embodiments may further comprise the step of treating the reaction site with one or more proteases to remove or inactivate polymerase that may have accumulated at the reaction site.

[0051] although Figures 2A to 2C A substrate having reaction sites continuously surrounded or occupied by droplets is shown, but this is not a requirement for all embodiments of the invention. In some embodiments, the substrate having reaction sites can be dried between cycles of steps so that, strictly speaking, the substrate is not always or continuously a droplet microarray throughout the synthesis process.

[0052] In some embodiments, including those described above, the plurality of polynucleotides enzymatically synthesized on a substrate having inkjet-delivered reagents (i.e., the number of reaction sites) is from 100 to 2 million, or from 100 to 1 million, or from 100 to 100,000, or from 100 to 500,000, or from 1,000 to 1 million. In some embodiments, such a plurality of polynucleotides is synthesized on a substrate having 1 cm 2 Up to 500 cm 2 , or 1 cm 2 Up to 256 cm 2 , 1 cm 2 Up to 30 cm 2 or synthesized on a substrate with a surface area of ​​1 cm 2 Up to 15 cm 2 or synthesized on a substrate with a surface area of ​​1 cm 2 Up to 7 cm 2 or on a substrate with a surface area of ​​7 cm 2 Up to 20 cm 2 In some embodiments, the polynucleotide synthesized according to the present invention has a length of 10 to 500 nucleotides, or 50 to 500 nucleotides, or 100 to 400 nucleotides, or 100 to 500 nucleotides. In some embodiments, the coupling efficiency of each cycle in the synthesis of the polynucleotide in these length ranges is at least 98%, or at least 99%, or at least 99.5%, or at least 99.8%, or at least 99.9%. In some embodiments, the coupling cycle time in the synthesis of the polynucleotide in these length ranges is that each cycle is less than 15 minutes, or each cycle is less than 10 minutes, or each cycle is less than 7 minutes, or each cycle is less than 5 minutes.

[0053] In some embodiments, inkjet delivery of droplets may be directed to features on a substrate having dimensions directly related to its size or area, such as the width of a square reaction site or the diameter of a circular reaction site. Thus, in some embodiments, the reaction site has a width or diameter of about 10 μm to about 1.0 cm. In some embodiments, droplets may be deposited onto reaction sites having a width or diameter of about 1.0 μm to about 1.0 mm, typically about 5.0 μm to 500 μm, more typically about 10 μm to 200 μm, and more typically about 20 μm to about 100 μm.

[0054] In some embodiments, the volume of reagent ink delivered to the reaction sites is 0.1 to 1000 pL, or 0.5 to 500 pL, or 1.0 to 250 pL, or 1.0 to 100 pL, or 2 to 50 pL. In some embodiments, the reagent ink is delivered to each reaction site in a predetermined number of droplets or "pulses" generated by the printhead, where, for example, each pulse has a volume of about 2.4 picoliters.

[0055] Equipment for inkjet synthesis

[0056] Delivery of fluids by inkjet printers is a mature technology that has been available for decades, so a large amount of information describing it and providing guidance for adapting it to new applications such as the present invention can be found. Exemplary references providing guidance on building inkjet delivery systems include: Lausted et al, Genome Biology, 5: R58 (2004); Le, Recent Progress in Ink Jet Technologies II, chapter 1, pgs. 1-14 (1999); Derby (2010, cited above); Zapka, editor, "Handbook of Industrial Inkjet Printing," (Wiley-VCH, Weinheim, Germany); U.S. Patents 5474796; 10384189; 10669304; 6306; 6323043; 5847105, etc. As described by Le (1999), inkjet pumps can be classified as "continuous" and "drop-on-demand" (DOD). In some embodiments, a DOD inkjet pump is used in the apparatus of the present invention, and in particular, in various DOD inkjet printers, a piezoelectric inkjet pump is of interest. For example, droplet formation in a DOD inkjet printer is described in Dong et al, Physics of Fluids, 18:072102 (2006). Various such inkjet pumps are available banks or assemblies of a large number of inkjet printers (e.g., tens to hundreds (10's to 100's)) that can be individually programmed to actuate and deliver droplets. Such inkjet printers and inkjet assemblies (referred to herein as "inkjet heads") are commercially available from many manufacturers, including Epson, Xaar, Fujifilm, etc. As used herein, "inkjet pump" means a device capable of generating and ejecting fluid droplets. In some embodiments, an inkjet pump is a device capable of generating and ejecting fluid droplets at a predetermined rate and a predetermined uniform size. In some embodiments, the inkjet pump is capable of ejecting droplets that are each approximately the same size of 0.1 pL to 5.0 nL, or the same size of 0.5 pL to 1.0 nL. In some embodiments, the inkjet pump is capable of ejecting droplets at a rate of 1 to 100 kilohertz.

[0057] In some embodiments, the components of the inkjet device of the present invention can be arranged according to whether they can move relative to each other or whether they are fixed, such as Figure 4A602 and 600, respectively. The computer and software (604) provide overall control of the system components directly or indirectly through the controller. For example, the software can provide a single-pass reagent deposition, wherein the print head (618) is fixed and the synthetic support holder (620) moves to deliver the reagent to the reaction site. Alternatively, different software can provide one or more mobile print heads (618) and / or mobile synthetic support holder (620) through various components, such as a print controller (606), a print head driver (612), and a motion controller (610). Typically, the computer and software (604) control the end-capping station (622), the rinse station (624), the wiper (626), the inspection system (628), and the washing and possible drying functions (630), if any.

[0058] The end-capping station (622) keeps the print head moist and prevents the ink from drying. The flushing station (624) primes and flushes the print head, which helps remove trapped air and debris as well as dried ink. The wiper (626) is used to remove excess ink and prevent cross-contamination. It can be part of the flushing station. The inspection system (628) records the presence, absence, or size of deposited reagent spots or incorrectly placed reagent spots. The inspection system (628) can include a camera that takes an image of the synthetic support and image analysis software that extracts processing information from the image. This information can be used in real time to optimize synthesis or implement corrective measures. The washing and drying function (630) is performed by a fluid delivery system separate from the fluid delivery system for droplet delivery. Washing can include a deprotection step in which the deprotection reagent flows over the synthetic substrate, optionally followed by a drying step. Drying can be achieved by blowing air or an inert gas (e.g., argon) onto the synthetic support, or can be achieved by using a volatile solvent (e.g., methanol) in the washing step.

[0059] In some embodiments, a camera or microscope can be used to capture the image of a spot (i.e., a reaction site) and identify the lost spot, determine spot size and spot position. The lighting for image capture can be from above, from the side, from below, or integrated into a substrate holder, and either one provides the best contrast in the absence or presence of a dye in the ink. When using a dye (as described below), the dye is selected so that it does not interfere with the enzymatic reaction, does not react with the protecting group of nucleotides, and will be compatible with enzyme and deprotection buffer. In some embodiments, each monomer will have different distinguishable dyes, covering different parts of the visible spectrum. In some embodiments, the imaging of the reaction site array is carried out during the incubation period of the extension reaction (30 seconds-10 minutes), and a sufficiently high magnification is used to observe a single spot, but not too high so that the scanning array will require excessive duration. For standard microscope slides, the number of images taken in the imaging step can be 20 to 100. The image can be seamlessly captured in a video stream by scanning the substrate, or captured during a move-stop process. The captured image can be stitched together with the help of an algorithm and the fiducial markers present on the slide. The fiducial marks also help determine if the slide has moved in the slide holder and help determine spot location.In some embodiments, real-time image analysis that allows identification of missing spots or poor spot locations can be accompanied by automatic generation of a new image and additional print(s).

[0060] Various embodiments for implementing the present invention (e.g., Figures 2A to 2C Exemplary inkjet devices of those Figure 4B. A plurality of DOD inkjet printers are housed in a print head (680) that is capable of xy and z movement relative to the droplet microarray (657). In some embodiments, both the print head (680) and the droplet microarray (657) are capable of xy movement. In some embodiments, the print head (680) remains in a fixed position and the droplet array (657) is subjected to xy movement. In this example, "dATP reagent," "dCTP reagent," "dGTP reagent," and "dTTP reagent" (696) are each a buffered formulation or ink comprising a template-free polymerase, the corresponding 3'-O protected dNTP, salts and cofactors necessary or useful for polymerase activity, and viscosity and surface tension modifiers, wetting agents, etc., required to meet the desired droplet formation and / or reduce evaporation loss requirements. The print head (680) includes a temperature regulator to maintain the ink at a temperature that is optimal for delivery and activity. In this embodiment, the reagents that flow to or are delivered to the droplet microarray in bulk are a deprotection solution (or buffer) (695) and a washing solution (661). The droplet microarray (657) formed on the substrate (655) is located or mounted in a flow chamber (677), which includes an inlet (652) and an outlet (653). The flow chamber (677) defines the flow path of the reagents (not delivered by the print head (680)) on the droplet microarray (657). Such reagents can flow continuously through the droplet microarray (657), or the reagents can be delivered to the flow chamber (677), where they are maintained for a predetermined incubation time and then removed or recycled. Such reagents can be moved on the reagent reservoir by a conventional pump or pressure head. The flow chamber (677) includes a temperature control element (not shown) and a humidity control element (not shown) to maintain or optimize the activity of the coupling reaction. After leaving, the reagents are discarded into a waste container (656) or recycled. The timing of inkjet discharge, the positioning of the print head (680), the actuation of valves (675) and valves (674) are controlled by a fluidics / inkjet controller (665), which may include imaging software that performs analysis of the array image obtained from the camera (697) and, for example, causes a change in reagent deposition when a coalescence reaction site is detected. In some embodiments, the print head (680) can be driven by electronics available from Meteor (Meteor Inkjet Ltd, (Cambridge, UK)). For example, a Print Controller Card (PCC) is synchronized with encoder signals from a Thorlabs motion controller. A Head Driver Card (HDC) provides power and waveforms to the print head.The drive electronics are controlled by Meteor's digital printing front-end, which includes MetDrop and MetWave software for optimizing spot parameters. Printing is initiated by Thorlabs' Kinesis software. Complete instrument control can be performed by the instrument software (e.g., LabView).

[0061] Typically, the distance between the inkjet nozzle and the substrate surface can be from about 10 μm to 10 mm, or from about 100 μm to 2 mm, or from about 200 μm to 1 mm, or from 500 μm to 3 mm. The droplet velocity can be from 1 to 10 meters per second. The printhead movement can be from 1 to 30 cm / second, or from 5 to 30 cm / second, or from 20 to 30 cm / second. As described more fully below, the printhead can have different droplet delivery modes, such as a single-pass mode, a multi-pass mode, and a move-stop mode.

[0062] As mentioned above, in some embodiments, the nozzle diameter for the present invention can be 10 μm to 100 μm. In other embodiments, the inkjet nozzle size can be 20-30 μm, for producing a droplet of 10-20 pL size. In some embodiments, nozzle diameter, synthesis reagent density, surface tension and viscosity are selected so that the droplet is distributed to a reaction site with 2 pL to 5nL or 2 pL to 1nL or 2 pL to 500 pL or at a volume of 2 pL to 100 pL. In some embodiments, the inkjet pump is a DOD inkjet pump and has a droplet generation rate of 1 to 100kHz.

[0063] In some embodiments, the synthesizer based on inkjet includes a droplet detection assembly to monitor and record any anomaly of the inkjet nozzle in droplet formation and delivery. In some embodiments, this droplet monitoring can include a laser diode installed perpendicular to the print head motion direction so that the droplet stream of each group of nozzles intersects with the light beam, so that if there is a droplet, light scattering is caused. Before each round of printing, the nozzle can be started (fired) in sequence by the light beam, and the forward scattering of each droplet is detected by a photodiode. The nozzle that failed to start can be offline during synthesis. The equipment of the present invention can also be equipped with a commercially available droplet monitor, such as the Meteor droplet monitor available from Meteor Inkjet Ltd (Cambridge, UK) and a camera that solid support and reaction site array are imaged. The latter allows monitoring the reaction site array to detect the accuracy of droplet deposition, the size and geometric structure of the reaction site, the coalescence of the reaction site, etc. In some embodiments, software can be provided to provide a complete image of an array on a slide or solid support by stitching together tiles comprising smaller images, e.g., S. Preibisch, S. Saalfeld, P. Tomancak, Bioinformatics, 2009, 25(11), 1463-1465.

[0064] In certain embodiments, it may be desirable to prevent synthesis reagents and reaction mixture from evaporating after deposition. Can prevent evaporation in a variety of different ways. In some embodiments, the synthesis cycle can be carried out in a high humidity environment, such as a relative humidity of 75-85%. Alternatively or additionally, a reagent with an evaporation delay agent or wetting agent can be used, such as glycerol, polyethylene glycol, carboxymethyl cellulose, hydroxyethyl cellulose etc.

[0065] In some embodiments, a recirculating ink printhead is employed because this reduces the problem of nozzle drying and / or clogging due to enzymes. Recirculating ink printheads are commercially available from, for example, Fujifilm and are described in U.S. Patents 8,820,899, 8,534,807, 8,752,946, 9,144,993, 9,511,598, and 9,457,579, which are incorporated herein by reference.

[0066] synthetic base

[0067] In some embodiments, the substrate used for synthesis comprises a surface that has been patterned with hydrophobic and hydrophilic regions, wherein discrete hydrophilic reaction sites are formed. These allow droplet formation on the hydrophilic reaction sites, for example, after flowing aqueous reagents or reactants across the surface. That is, in some embodiments, the substrate used for synthesis includes a so-called "droplet microarray", for example, as disclosed in the following exemplary references, which are incorporated by reference: Brennan, U.S. Patent 5474796; Chrisey et al, Nucleic Acids Research, 24(15): 3040-3047(1996); Fixe et al, Materials Research Society Symposium Proceedings. Volume723, Molecularly Imprinted Materials - Sensors and Other Devices. Symposia (San Francisco, California on April 2-5, 2002); Goldfarb, U.S. Patent Publication 2008 / 0166667; Gopinath et al, ACS Nano, 8(12): 12030-12040 (2014); Hong et al, Microfluid. Nanofluid., 10: 991-997 (2011); Kumar et al, Nucleic Acids Research, 28(14): e71 (2000); Peck et al, U.S. Patent 10384189; Indermuhle et al, U.S. Patent 10669304; Wu et al, Thin Solid Films, 515: 4203-4208 (2007); Zhang et al, J. Phys. Chem., 111: 14521-14529 (2007) and similar references. As used herein, the term "droplet microarray" refers to a substrate, preferably a planar substrate, the surface of which has been treated to produce a plurality of discrete hydrophilic regions that can be used directly or after further treatment as reaction sites, such as for attaching an initiator. In some embodiments, each of the plurality of discrete hydrophilic regions is surrounded by a hydrophobic region. The discrete hydrophilic regions can have a variety of shapes, but for ease of manufacture, are typically circular, rectangular, or square. In some embodiments, the reaction sites have an area and capacity to accommodate an aqueous reaction mixture as described above.Although the synthetic substrate of some embodiments may include a droplet microarray, during the synthesis process, such an array may undergo a drying step to remove liquid from the reaction site. That is, in some embodiments, a synthetic substrate comprising a droplet microarray may be free of droplets from time to time, for example, after an extension cycle that ends in a drying step. The hydrophilic-hydrophobic configuration allows droplets to be formed on the surface of the droplet microarray after inkjet delivery of synthetic reagents to the hydrophilic region or by flowing a "bulk" aqueous solution (e.g., synthetic reagents or washing solution) on the substrate. As disclosed in the above references, the droplets retained by the hydrophilic region can be used as a reaction chamber or container. Such a process is. Figure 1B . A substrate (150) (which is a planar substrate) has a surface with hydrophobic regions (152) and discrete hydrophilic regions (154) that can serve as reaction sites. When the substrate (150) is flooded (156) with an aqueous solution (158), both the hydrophobic regions (152) and the hydrophilic regions (154) are submerged. When the aqueous solution (158) is drained (160), some of the aqueous solution is retained by the hydrophilic regions (154) to form droplets (162) of a droplet microarray (164). Individual droplets, such as (162), can be referred to as "microarray droplets" to distinguish them from droplets (such as (162)) formed by an inkjet pump before being delivered to the reaction sites.

[0068] The preparation of the substrate with discrete reaction sites can be completed by known methods. For example, such a method can be related to creating hydrophilic reaction sites by first applying a protective agent or resist (for example, silicon oxide or similar materials) on the selected area of ​​the substrate surface. Then, the unprotected area is coated with a hydrophobic agent to produce a non-reactive surface. For example, a hydrophobic coating can be produced by chemical vapor deposition of (tridecafluorotetrahydrooctyl)-triethoxysilane onto the exposed oxide around the protected circular object. Finally, using high surface tension solvents as described herein and procedures known in the art (such as Maskos & Southern, Nucl. Acids Res. 20:1679-1684 (1992) described those) to remove the protective agent or resist, exposing the hole area of ​​the array for further modification and nucleoside synthesis. Alternatively, the entire surface of the glass plate substrate can be coated with a hydrophobic material, such as 3-(1,1-dihydroperfluorooctyloxy) propyltriethoxysilane, which is ablated to expose the silica glass below at the desired site. The substrate is then coated with glycidyloxypropyl trimethoxysilane, which reacts only with glass, and then "treated" with hexaethylene glycol and sulfuric acid to form a linker with hydroxyl groups on which chemicals can be synthesized (Brennan, U.S. Patent No. 5,474,796). Arrays produced in this way can localize small volumes of solvent within the reaction sites by virtue of surface tension effects (Lopez et al., Science 260:647-649 (1993)).

[0069] In some embodiments, the reaction sites can be formed on the substrate following the photolithographic methods of Brennan, U.S. Patent 5474796; Peck et al, U.S. Patent 10384189; Indermuhle et al, U.S. Patent 10669304; Fixe et al (cited above); or similar references cited above. According to these methods, a group of hydrophilic molecules comprising aminosilanes are attached to the surface of the substrate to form the reaction sites. Such hydrophilic molecules can include N-(3-triethoxysilylpropyl)-4-hydroxybutyramide (HAPS), 11-acetoxyundecyltriethoxysilane, n-decyltriethoxysilane, (3-aminopropyl)trimethoxysilane, (3-aminopropyl)triethoxysilane, 3-glycidoxypropyltrimethoxysilane (GOPS) or 3-iodo-propyltrimethoxysilane. A group of hydrophobic molecules comprising fluorosilanes are attached to the substrate surface in the area outside the reaction sites. Such hydrophobic molecules can include perfluorooctyltrichlorosilane, octylchlorosilane, octadecyltrichlorosilane, (tridecafluoro-1,1,2,2-tetrahydrooctyl)trichlorosilane or (tridecafluoro-1,1,2,2-tetrahydrooctyl)trimethoxysilane. After such attachment, the substrate for polynucleotide synthesis is prepared by coupling the initiator to the aminosilane at the reaction site. Such coupling can be accomplished using any number of available homobifunctional linkers or heterobifunctional linkers to form a covalent bond between the amino group on the substrate and the 5'-thiol group or 5'-amino group on the initiator. Such linkers are available, for example, from Sigma-Aldrich (St. Louis, MO) and are described, for example, in Hermanson, Bioconjugate Techniques, 3 rd Edition (Academic Press, 2013). The synthesis of polynucleotides having 5'-thiol groups or 5'-amino groups is well known and described in Kupihar et al, Nucleosides Nucleotides & Nucleic Acids, 22(5-8): 1297-1299 (2003); Fung et al, U.S. Patent No. 4,757,141 and similar references.

[0070] In some embodiments, arrays of reaction sites can be formed using click chemistry by depositing droplets of a 5'-DBCO (dibenzocyclooctyl)-labeled initiator (e.g., Glen Reseach) onto a substrate (preferably a planar substrate) containing an azide layer (e.g., a PolyAn 2D azide slide) under coupling conditions. In some embodiments, such reactions can be performed as copper-free click reactions that are less damaging to DNA, such as Dommerholt et al, Top. Curr. Chem. (Z) 374: 16 (2016).

[0071] Various substrates can be used to create reaction site arrays for enzymatic synthesis of polynucleotides. The substrate can be a rigid material, including but not limited to glass; fused quartz; 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. The rigid surface can be made of a material selected from silicon, polystyrene, agarose, dextran, cellulose polymers, polyacrylamide, polydimethylsiloxane (PDMS), and glass. The substrate can also comprise a flexible material that can be bent, folded, or similarly manipulated without breaking. Exemplary flexible materials include, but are not limited to, nylon (unmodified nylon, modified nylon, transparent nylon), nitrocellulose, polypropylene, polycarbonate, polyethylene, polyurethane, polystyrene, acetal, acrylic materials, acrylonitrile, butadiene styrene (ABS), polyester films such as polyethylene terephthalate, polymethyl methacrylate or other acrylic resins, polyvinyl chloride or other vinyl resins, transparent PVC foil, transparent foil for printers, poly(methyl methacrylate) (PMMA), methacrylate copolymers, styrene polymers, high refractive index polymers, fluoropolymers, polyethersulfone, polyimides containing alicyclic structures, rubber, fabrics, metal foils, and any combination thereof.

[0072] In some embodiments, a patterned surface of superhydrophobic and superhydrophilic regions can be formed on a substrate. Guidance for forming droplet microarrays having such patterned surfaces is described in the following references, which are incorporated by reference: Feng et al, Adv. Mater. Interfaces, 1400269 (2014); Zhan et al, Trends Anal. Chem., 108: 183-194 (2018); Neto et al, Adv. Functional Mater., 201400503 (2014).

[0073] The accurate alignment of droplet delivery and the reaction site of prefabricated droplet microarray is an important aspect of the inkjet-assisted synthesis of polynucleotide. In some embodiments, by creating a reaction site array by immediately before the synthesis by depositing the droplet of synthesis reagent onto the initiator oligonucleotide layer on the substrate to define the position of the reaction site, such alignment tasks can be minimized or avoided. After this initial deposition of droplet, the initiator layer outside the droplet definition site is processed, so that they are inert to subsequent extension, or make them inert and make them hydrophobic to extension. After such initial surface treatment to create the reaction site, further or subsequent droplet delivery to the inkjet of the same reaction site will be accurate, because the identical inkjet head and pump for defining the reaction site position will be used to deliver subsequent droplet during the synthesis of polynucleotide. In some embodiments, the synthesis reagent delivered to the initiator layer comprises a mixture of template-free polymerase and 3'-O protected dNTPs. These reagents extend initiator to define the reaction site or the zone on the oligonucleotide layer occupied by the extension fragment with 3'-O protected end. Then, the region outside these regions is processed, so that they are inert to extension. In some embodiments, after the initial coupling step of limiting the reaction site, the entire substrate is exposed to a template-free polymerase and terminator, such as a dideoxynucleoside triphosphate (ddNTP) or similar reagent. In some embodiments, this ddNTP can be, for example, a ddNTP conjugated to a hydrophobic moiety, thereby making the coating outside the reaction site hydrophobic. Such a hydrophobic moiety can be, for example, a dye or a quencher molecule, such as a Black Hole Quencher® molecule. Various terminators can be used for this purpose. In particular, terminator includes nucleoside triphosphates without a 3'-hydroxyl substituent, and includes 2', 3'-dideoxyribonucleoside, 2', 3'-didehydroribonucleoside and 2', 3'-dideoxy-3'-halogenated ribonucleoside, such as 3'-deoxy-3'-fluororibonucleoside or 2', 3'-dideoxy-3'-fluororibonucleoside. Alternatively, ribofuranose analogs can be used as terminators, such as 2',3'-dideoxy-β-D-ribofuranosyl, β-D-arabinofuranosyl, 3'-deoxy-β-D-arabinofuranosyl, and the like.Other terminators are disclosed in the following references: Chidgeavadze et al., Nucleic Acids Res., 12: 1671-1686 (1984); Chidgeavadze et al., FEBS Lett., 183: 275-278 (1985); Izuta et al, Nucleosides & Nucleotides, 15: 683-692 (1996); and Krayevsky et al, Nucleosides & Nucleotides, 7: 613-617 (1988). Nucleotide terminators also include reversible nucleotide terminators, such as Metzker et al. Nucleic Acids Res., 22(20):4259 (1994).

[0074] Thus, in such an embodiment, the starting material for the synthesis operation is a surface coated with a layer of initiator oligonucleotides. An exemplary fabrication of reaction sites on such a starting material is described in Figure 3 The substrate (400) (e.g., a glass slide) has a layer of initiator oligonucleotides (402) that have free 3'-hydroxyl groups and are attached to the substrate via their 5'-ends. In some embodiments, the initiator density can be, for example, 10 11 to 10 13 Chain / cm 2 . An inkjet pump in an inkjet head (404) is used to deposit droplets (407) in a regular and repeatable pattern on a layer (402) defining reaction sites (e.g., 406). For example, the 3'-hydroxyl group of such an initiator can be unprotected, and the droplets can contain a template-free polymerase and an initial 3'-O-protected nucleoside triphosphate, thereby producing a 3'-O-protected extension segment at each reaction site. After such deposition, the initiator layer (402) is immersed and incubated (409) in a buffer (408) containing a template-free polymerase and a terminator (e.g., as described above) to produce a droplet microarray (414) having a surface (410) outside the reaction sites (e.g., 412) that is inert to extension or inert to extension and hydrophobic, depending on the terminator selected.

[0075] Use Figure 3An embodiment of the present invention for synthesizing a plurality of polynucleotides formed by the reaction sites described in the invention can be performed by the following steps: (a) providing a substrate, preferably a planar substrate, to which an initiator layer is attached, wherein each initiator has a free 3'-hydroxyl group; (b) dispensing one or more droplets to each of a plurality of sites on the initiator layer by one or more inkjet pumps to define an array of reaction sites, wherein each droplet contains a buffer solution containing a mixture of a template-free polymerase and 3'-O-blocked dATP, 3'-O-blocked dCTP, 3'-O-blocked dGTP or 3'-O-blocked dTTP, and wherein each of the plurality of polynucleotides is assigned to a different reaction site for synthesis; (c) capping the free 3'-hydroxyl groups of the initiators outside the reaction sites; (d) dispensing one or more droplets containing a template-free polymerase to a substrate by one or more inkjet pumps. At least one droplet of a buffered solution of a mixture of a polymerase and 3'-O-blocked dATP, 3'-O-blocked dCTP, 3'-O-blocked dGTP, or 3'-O-blocked dTTP is dispensed to each reaction site, wherein the type of the 3'-O-blocked dNTP dispensed to the reaction site depends on the predetermined sequence of the polynucleotide assigned to the reaction site; (e) incubating a template-free polymerase and 3'-O-blocked dNTPs at each reaction site such that the initiator or extension segment at the reaction site is extended by incorporation of the 3'-O-blocked dNTP to form a 3'-O-blocked extension segment; (f) deblocking the extension segment at each reaction site by treating the planar support with a deblocking agent to form an extension segment having a free 3'-hydroxyl group; (g) repeating steps (d), (e), and (f) until a plurality of polynucleotides are synthesized.

[0076] An aspect of the present invention is to prepare a method for a reaction site array for the template-free enzymatic synthesis of multiple polynucleotides. In some embodiments, this array preparation method can be carried out by the following steps: (a) a surface to which an initiator is attached is provided, (b) droplets are delivered to a plurality of different positions on the surface to form a plurality of reaction sites with one or more inkjet pumps, the droplets comprising a synthesis reagent reacting with the initiator in the reaction site, to remove 3'-O protecting groups or to extend such initiator by adding 3'-O protected nucleoside triphosphates, and (c) the initiator on the surface outside the reaction site is blocked. In some embodiments, the initiator on the surface of step (a) has a free 3'-hydroxyl group, and the synthesis reagent delivered in step (b) comprises template-free polymerase and 3'-O protected nucleoside triphosphates, so that template-free polymerase catalyzes the addition of 3'-O protected nucleoside triphosphates to produce 3'-O protected extension fragments in the reaction site. Therefore, the initiator outside the reaction site can be blocked by immersing the surface in a blocking reagent (e.g., a mixture containing dideoxynucleoside triphosphates and template-free polymerase). In some embodiments, the initiator on the surface can have a 3'-O blocking group, and the synthetic reagent delivered by the droplet can include a deprotecting agent that removes the 3'-O blocking group from the initiator to form a reaction site. In the newly formed reaction site, deliver the reagent of the nucleoside triphosphate and template-free polymerase containing the 3'-O protection, wherein the blocking group of the nucleoside triphosphate delivered is orthogonal to the blocking group of the initiator on the surface. Exemplary orthogonal 3'-O blocking groups are as described below. For example, such orthogonal blocking groups can be azidomethyl and amino.

[0077] A person of ordinary skill will appreciate that for other embodiments, e.g. Figure 2B and Figure 2C Similar reaction site formation can be implemented as those described in Figures 2A to 2C The optional steps described in the embodiment of the present invention (such as washing, drying, treating with protease, etc.) can also be performed in the above embodiment (including Figure 3 those that are implemented in .

[0078] In another embodiment, the starting layer of initiator oligonucleotides all have 3'-O-amino protected or 3'-O-azidomethyl protected termini. The method steps in this embodiment are similar to Figure 3The invention relates to a method for selectively deprotecting an aldehyde or ketone by treating the surface with an aqueous solution of an aldehyde or ketone to form a stable, non-extendable hydrophilic or hydrophobic 3'-oxime. The aldehyde or ketone can be water-soluble, such as acetone, or slightly water-soluble and hydrophobic (such as valeraldehyde, aldehyde-PEG-DBCO, etc.) or very hydrophobic and water-insoluble (such as heptanal).

[0079] In another embodiment, a buffer comprising a mixture of dNTPs without template polymerase / 3'-O protection is printed on an initiator oligonucleotide layer with free 3'-hydroxyl as described above to limit the reaction site of the initiator with an extension of the end with 3'-O protection. The surface outside these restricted sites is then treated with a ddNTP derived from a template-free polymerase and an azide or alkyne to block further 3' extension. The hydrophobic molecules with complementary click chemistry groups (e.g., DBCO, benzyl azide) can then react with the ddNTP terminator to make the surface outside the reaction site hydrophobic. Exemplary click chemistry is described in Feng et al, Adv. Mater. Interfaces, 1400269 (2014).

[0080] In another embodiment, for the substrate surface without initiator oligonucleotide layer, the buffer solution comprising the initiator oligonucleotide with 5 ' linker group will be inkjet printed on the surface derived with complementary reaction groups (such as epoxy resin, azide / alkyne) so that initiator is attached to the surface by their 5 ' ends.For the initiator of these attachments, the coupled reaction cycle can be carried out according to the present invention.In addition, unreacted complementary reaction groups can be quenched by making them react with inert groups (such as, ethanolamine is used for epoxy resin), and the inert groups with hydrophobic characteristics can be selected.

[0081] Template-free enzymatic synthesis method

[0082] Typically, template-free (or equivalently, "non-template-dependent") enzymatic polynucleotide synthesis methods involve repeated cycles of steps, such as Figure 1AAs shown, a predetermined nucleotide is coupled to the initiator or the growing chain in each cycle. General elements of template-free enzymatic synthesis are described in the following references: Champion et al, U.S. Patent 10,752,887; Ybert et al, International Patent Publication WO / 2015 / 159023; Ybert et al, International Patent Publication WO / 2017 / 216472; Godron et al, International Patent Publication WO / 2020 / 120442; Hyman, U.S. Patent 5,436,143; Hiatt et al, U.S. Patent 5,763,594; Jensen et al, Biochemistry, 57: 1821-1832 (2018); Mathews et al, Organic & Biomolecular Chemistry, DOI: 0.1039 / c6ob01371f (2016); Schmitz et al, Organic Lett., 1(11): 1729-1731 (1999).

[0083] In the present invention, the synthetic reagents delivered by the inkjet pump must be formulated to meet at least two constraints: (i) the need to maintain the extension activity of the template-free polymerase (in the case of a template-free polymerase ink), and (ii) the need to meet the rheological requirements of droplet formation. The key solution parameters that affect the droplet formation by the inkjet printer are viscosity, surface tension, liquid density and the diameter of the inkjet nozzle. For specific embodiments of the present invention, synthetic reagents prepared for non-droplet delivery to the reaction mixture can be reformulated by adding viscosity modifiers, surface tension modifiers and density modifiers, etc., to form a "printable ink" that can be delivered in the droplets generated by the inkjet pump. "Printable" with respect to the reagent ink means that repeatable droplets can be ejected from the nozzle at a uniform speed and volume, and there are no satellite droplets.

[0084] like Figure 1AAs shown, an initiator polynucleotide (100) having a free 3'-hydroxyl group (130) is provided, for example, attached to a synthesis support (120). Under effective conditions (140) for enzymatic incorporation of the 3'-O protected dNTP into the 3' end of the initiator polynucleotide (100) (or extended initiator polynucleotide), a 3'-O protected dNTP and a template-free polymerase, such as terminal deoxynucleotidyl transferase (TdT) or a variant thereof (e.g., Ybert et al, WO / 2017 / 216472; Champion et al, WO2019 / 135007), are added to the initiator polynucleotide (100) (or extended initiator polynucleotide in subsequent cycles). The reaction produces an extended initiator polynucleotide (160) with a protected 3'-hydroxyl group. If the extended sequence is incomplete, another addition cycle (180) is performed. If the extended initiator polynucleotide contains a competing sequence, the 3'-O protecting group can be removed or deprotected and the desired sequence can be cleaved from the original initiator polynucleotide (182). Such cleavage can be performed using any of a variety of single-strand cleavage techniques, for example, by inserting a cleavable nucleotide at a predetermined position within the original initiator polynucleotide. An exemplary cleavable nucleotide can be a uracil nucleotide that is cleaved by uracil DNA glycosylase. According to some embodiments of the present invention, the cleavage reagent can be delivered to the reaction site by droplets generated by an inkjet pump. In such embodiments, the known incomplete or defective polynucleotide at the reaction site can be separated from the fully competing polynucleotide, or can be selectively resynthesized by cleaving and resynthesizing the entire polynucleotide, or by cleaving or otherwise removing the incorrect sequence and resynthesizing only the defective portion of the polynucleotide.

[0085] If the extended initiator polynucleotide is not a complete sequence (ie, the final product), the 3'-O protecting group is removed to expose the free 3'-hydroxyl group (130), and the extended initiator polynucleotide undergoes another cycle of nucleotide addition and deprotection.

[0086] As used herein, "initiator" (or equivalent terms, such as "starting fragment," "initiator nucleic acid," "initiator oligonucleotide," etc.) generally refers to a short oligonucleotide sequence having a free 3'-hydroxyl group at its end, which can be further extended by a template-free polymerase (e.g., TdT). In one embodiment, the starting fragment is a DNA starting fragment. In an alternative embodiment, the starting fragment is an RNA starting fragment. In some embodiments, the starting fragment has 3-100 nucleotides, particularly 3-20 nucleotides. In some embodiments, the starting fragment is single-stranded. In an alternative embodiment, the starting fragment can be double-stranded. In some embodiments, the initiator oligonucleotide can be attached to a synthetic support by its 5' end; in other embodiments, the initiator oligonucleotide can be indirectly attached to a synthetic support by forming a duplex (e.g., by a covalent bond) with a complementary oligonucleotide directly attached to the synthetic support. In some embodiments, the synthetic support is a solid support, which can be a discrete area of ​​a solid planar solid, or can be a bead.

[0087] In some embodiments, the initiator can comprise a non-nucleic acid compound having a free hydroxyl group to which TdT can couple a 3'-O protected dNTP, such as Baiga, US Patent Publications US2019 / 0078065 and US2019 / 0078126.

[0088] After synthesis is complete, the polynucleotide having the desired nucleotide sequence can be released from the initiator and synthesis support by cleavage.

[0089] Various cleavable linkages or cleavable nucleotides can be used for this purpose. In some embodiments, the polynucleotide required for cleavage leaves a natural free 5'-hydroxyl group on the cleaved chain; however, in alternative embodiments, the cleavage step can leave a portion that can be removed in a subsequent step, such as by phosphatase treatment, such as a 5'-phosphate. The cleavage step can be performed chemically, thermally, enzymatically, or photochemically. In some embodiments, the cleavable nucleotide can be a nucleotide analog, such as deoxyuridine or 8-oxo-deoxyguanosine, which are recognized by specific glycosylases (e.g., uracil deoxyglycosylase followed by endonuclease VIII, and 8-oxoguanine DNA glycosylase, respectively). In some embodiments, cleavage can be achieved by providing an initiator with deoxyinosine (which can be cleaved by endonuclease V at the 3' end of the initiator, leaving a 5'-phosphate on the released polynucleotide) as the penultimate 3' nucleotide. In some embodiments, the initiator may contain a terminal uridine so that after synthesis, the desired polynucleotide can be cleaved from the initiator by treatment with KOH or a similar base. Further methods for cleaving single-stranded polynucleotides are disclosed in the following references, which are incorporated by reference: U.S. Patent Nos. 5,739,386, 5,700,642, and 5,830,655 and U.S. Patent Publication Nos. 2003 / 0186226 and 2004 / 0106728; and Urdea and Horn, U.S. Patent No. 5,367,066.

[0090] Back to Figure 1AIn some embodiments, in each synthesis step, an ordered sequence of nucleotides is coupled to an initiator nucleic acid using a template-free polymerase, such as TdT, in the presence of a 3'-O protected dNTP. In some embodiments, the method of synthesizing an oligonucleotide comprises the following steps: (a) providing an initiator having a free 3'-hydroxyl group; (b) reacting an initiator or an extension intermediate having a free 3'-hydroxyl group with a template-free polymerase in the presence of a 3'-O protected nucleoside triphosphate under elongation (or extension) conditions to produce a 3'-O protected extension intermediate; (c) deprotecting the extension intermediate to produce an extension intermediate having a free 3'-hydroxyl group; and (d) repeating steps (b) and (c) until a polynucleotide is synthesized. (Sometimes the terms "extension intermediate" or "extension segment" or "growing chain" are used interchangeably). As used herein, the term "extension conditions" means the physical and chemical conditions of the reaction mixture required for the template-free polymerase-catalyzed extension reaction, wherein the 3'-O protected nucleoside triphosphate monomer is coupled to the free 3'-hydroxyl of the nucleic acid fragment (by forming a phosphodiester bond), which can be, for example, an initiator or an extension fragment. Exemplary extension conditions include the selection of reaction temperature, duration of reaction, pH, the concentration of various salts, a scavenger of unwanted reaction components, a reagent that reduces nucleic acid secondary structure, etc. In some embodiments, the initiator is provided as an oligonucleotide attached to a solid support (e.g., by its 5' end). The above method can also include a washing step after the reaction or extension step and after the deprotection step. For example, the reaction step can include a substep of removing unincorporated nucleoside triphosphates by washing after a predetermined incubation period or reaction time. In some embodiments, this predetermined incubation period or reaction time can be 30 seconds to 30 minutes, or 1 minute to 30 minutes, or 1 minute to 15 minutes, or 1 minute to 10 minutes, or 30 seconds to 5 minutes.

[0091] In some embodiments, Figure 1A After the synthesis cycle is completed, a further step can be performed to cut the completed polynucleotide from the solid support. This further step can be performed at the reaction site of the array. Additionally, some cutting methods may cause the released product to still need to be modified to convert it into an available product. For example, in " endonuclease V-inosine " cutting (as described below), a 5'-phosphate that must be removed for some applications is left. Therefore, a further step of phosphatase treatment may be required.

[0092] In some embodiments, the polynucleotide of the present invention can be used to synthesize the polynucleotide of the present invention.When the predetermined sequence of the polynucleotide on the synthetic support comprises the reverse complementary subsequence, may produce secondary intramolecular or intermolecular structure by the formation of hydrogen bond between the reverse complementary district.In some embodiments, the base protection part of the amine outside the ring is selected so that the hydrogen of the nitrogen protected can not participate in hydrogen bonding, thereby prevents the formation of this secondary structure.That is to say, the base protection part can be used to prevent the formation of hydrogen bond, for example, in normal base pairing, form, for example, between nucleosides A and T and between nucleosides G and C.When the synthesis is finished, the base protection part can be removed, and the polynucleotide product can be cut off from the solid support, for example, by it being cut off from its initiator.

[0093] In addition to providing 3'-O blocked dNTP monomers with base protecting groups, a thermostable template-free polymerase can be used to perform the extension reaction at a higher temperature. For example, a thermostable template-free polymerase that is active above 40°C can be used; or, in some embodiments, a thermostable template-free polymerase that is active at 40-85°C can be used; or, in some embodiments, a thermostable template-free polymerase that is active at 40-65°C can be used.

[0094] In some embodiments, the extension conditions may include adding a solvent that inhibits hydrogen bonding or base stacking to the extension reaction mixture. Such solvents include water-miscible solvents with low dielectric constants, such as dimethyl sulfoxide (DMSO), methanol, etc. Similarly, in some embodiments, the extension conditions may include providing a chaotropic agent, which includes but is not limited to n-butanol, ethanol, guanidine chloride, lithium perchlorate, lithium acetate, magnesium chloride, phenol, 2-propanol, sodium lauryl sulfate, thiourea, urea, etc. In some embodiments, the extension conditions include the presence of a secondary structure-inhibiting amount of DMSO. In some embodiments, the extension conditions may include providing a DNA-binding protein that inhibits the formation of secondary structure, wherein such protein includes but is not limited to single-stranded binding protein, helicase, DNA glycolase, etc.

[0095] When using base-protected dNTPs, Figure 1A The above process may further comprise a step (e) of removing the base protecting moiety, which in the case of an acyl or amidine protecting group may, for example, comprise treatment with concentrated aqueous ammonia.

[0096] Said method can also comprise end-blocking step and after reaction or extension step and after deprotection step washing step.As mentioned above, in some embodiments, end-blocking step can be comprised, wherein the free 3 '-hydroxyl of not extending reacts with the compound reaction of any further extension of preventing end-blocking chain.In some embodiments, such compound can be dideoxynucleoside triphosphate.In other embodiments, the unextended chain with free 3 '-hydroxyl can be degraded by processing with 3 '-nuclease activity (such as Exo I).For example, referring to Hyman, United States Patent (USP) 5436143.Equally, in some embodiments, can process and fail to remove the chain of sealing to remove the chain or make it be inert to further extension.When using end-blocking agent (such as ddNTP), the buffer containing such reagent or synthesis reagent can be delivered by flowing on the substrate containing reaction site or spraying such reagent.

[0097] In some embodiments, the reaction conditions for the extension step (sometimes also referred to as the elongation step or coupling step) can include the following: 2.0 μM purified TdT; 125-600 μM 3'-O blocked dNTPs (e.g., 3'-O-NH2 blocked dNTPs); about 10 to about 500 mM potassium cacodylate buffer (pH 6.5-7.5) and about 0.01 to about 10 mM divalent cations (e.g., COCl2 or MnCl2), wherein the extension reaction can be carried out at a temperature of RT to 45°C for 3 minutes. It should be understood that whenever the aforementioned coupling reagent is delivered by inkjet-generated droplets, its viscosity, density, and surface tension must be adjusted to make it a printable ink. In this regard, the present invention, in part, includes the recognition and understanding that the ink used to deliver TdT to the reaction site can have a viscosity modified for droplet formation and activity preserved by the choice of viscosity modifier, for example, when carboxymethyl cellulose is selected as the viscosity modifier.

[0098] In embodiments where the 3'-O blocked dNTP is a 3'-O-NH2 blocked dNTP, the reaction conditions for the deblocking step may include the following: 700 mM NaNO2; 1 M sodium acetate (adjusted to a pH of 4.8-6.5 with acetic acid), wherein the deblocking reaction may be carried out at a temperature of RT to 45°C for 30 seconds to several minutes. Washing may be performed with a cacodylate buffer that does not contain components of the coupling reaction (e.g., enzymes, monomers, divalent cations). If the above reagent composition is delivered to the reaction site by inkjet delivery, it will be understood that the composition will be modified to meet the rheological requirements for droplet formation through the nozzle of the inkjet print head used.

[0099] In some embodiments, RNA synthesis can be accomplished by steps similar to those described above, but using template-free polymerases and monomers specifically selected for RNA synthesis, such as polyA polymerase (PAP), polyU polymerase (PUP), etc., as disclosed in International Patent Publication WO2020 / 077227. For example, the systems, apparatus, and kits of the present invention can implement a method for synthesizing a polyribonucleotide having a predetermined sequence, the method comprising the following steps: a) providing an initiator having a 3'-terminal nucleotide with a free 3'-hydroxyl group; and b) repeating the following cycle until the polyribonucleotide is formed: (i) contacting the initiator or extension segment having a free 3'-hydroxyl group with a 3'-O-blocked nucleoside triphosphate and a template-free polymerase under extension conditions, such that the initiator or extension segment is extended by incorporation of the 3'-O-blocked nucleoside triphosphate to form a 3'-O-blocked extension segment, and (ii) deblocking the extension segment to form an extension segment having a free 3'-hydroxyl group; wherein the template-free polymerase is a poly(A) polymerase (PAP) or a poly(U) polymerase. In a further embodiment, the initiator can be attached to a support via the 5' end, which can be a solid support, and the method can include a step of cleaving the polynucleotide from the initiator. In some embodiments, the reaction conditions for the extension or extension step using PAP or PUP can include the following: Reaction Condition 1 (for primer + AM-rATP): 250 μM AM-rATP, 0.1 μM ATTO488-(rA)5, 1 μM PAP, 1× ATP buffer (20 mM Tris-HCl, 0.6 mM MnCl2, 0.02 mM EDTA, 0.1% BSA, 10% glycerol, 100 mM imidazole, pH 7-8), 37°C, 30 minutes. Reaction condition 2 (for primers + AM-rGTP): 250 μM rGTP, 0.1 μM ATTO488-(rA)5, 1 μM PAP, 1× GTP buffer (0.6 mM MnCl2, 0.1% BSA, 10 mM imidazole, pH 6), 37°C, 30 min. In the above, "AM-rNTP" refers to 3'-azidomethyl-O-ribonucleoside triphosphate.Many 3'-O blocked rNTPs for use in the present invention can be purchased from commercial suppliers (e.g., Jena Bioscience, MyChemLabs, etc.) or synthesized using published techniques, such as U.S. Patent 7057026; International Patent Publication Nos. WO2004 / 005667, WO91 / 06678; Canard et al, Gene (cited above); Metzker et al, Nucleic Acids Research, 22: 4259-4267 (1994); Meng et al, J. Org. Chem., 14: 3248-3252 (3006); U.S. Patent Publication No. 2005 / 037991; Zavgorodny et al, Tetrahedron Letters, 32(51): 7593-7596 (1991). In further specific embodiments, the nucleotide triphosphate of 3'-blocking is by 3'-O-propargyl, 3'-O-azidomethyl, 3'-O-NH2 or 3'-O-allyl group blocking.In other embodiments, 3'-O blocking groups of the present invention include 3'-O-methyl, 3'-O-(2-nitrobenzyl), 3'-O-allyl, 3'-O-amine, 3'-O-azidomethyl, 3'-O-tert-butoxyethoxy, 3'-O-(2-cyanoethyl) and 3'-O-propargyl.As mentioned above, if by inkjet delivery, above-mentioned reagent composition is delivered to reaction site, then it should be understood that composition will be changed to meet the rheological requirement of forming droplet by the nozzle of employed inkjet print head.

[0100] 3'-O-protected nucleoside triphosphates

[0101] Depending on the specific application, the step of deblocking and / or cutting can include various chemical or physical conditions, such as light, heat, pH, the presence of specific reagents such as enzymes that can cut a specified chemical bond. Guidance in selecting 3'-O blocking groups and corresponding deblocking conditions can be found in the following references, which are incorporated by reference: Benner, U.S. Patents 7544794 and 8212020; U.S. Patent 5808045; U.S. Patent 8808988; International Patent Publication WO91 / 06678; and the references cited below. In some embodiments, the cutting agent (sometimes also referred to as a deblocking agent or deblocking agent) is a chemical cutting agent, for example, such as dithiothreitol (DTT). In alternative embodiments, the cutting agent can be an enzymatic cutting agent, for example, such as a phosphatase, which can cut a 3'-phosphate blocking group. It will be understood by those skilled in the art that the selection of the deblocking agent depends on the type of 3'-nucleotide blocking group used, whether one or more blocking groups are used, whether the initiator is attached to a living cell or organism or a solid support, etc. (this makes gentle treatment necessary). For example, a phosphine, such as tris(2-carboxyethyl)phosphine (TCEP), can be used to cleave a 3'O-azidomethyl group, a palladium complex can be used to cleave a 3'O-allyl group, or sodium nitrite can be used to cleave a 3'O-amino group. In certain embodiments, the cleavage reaction involves TCEP, a palladium complex, or sodium nitrite.

[0102] As mentioned above, in some embodiments, it is desirable to adopt two or more blocking groups that can be removed using orthogonal blocking conditions. The following exemplary blocking groups can be used in parallel synthesis embodiments. Should be understood that containing more than two other blocking groups can be used in these embodiments of the present invention.

[0103]

[0104] In some embodiments, the removal of a specific enzymatically removable blocking group requires a specific enzyme. For example, esterases (e.g., acetylesterases) or similar enzymes can be used to remove blocking groups based on esters or acyl groups, and 3' phosphatases (e.g., T4 polynucleotide kinases) can be used to remove phosphate blocking groups. For example, 3'-O-phosphates can be removed by treating with a solution of 100mM Tris-HCl (pH 6.5), 10mM MgCl2, 5mM 2-mercaptoethanol, and one unit of T4 polynucleotide kinase. The reaction is carried out at a temperature of 37°C for 1 minute. As described above, if the aforementioned composition is delivered to the reaction site by inkjet delivery, it will be understood that the composition will be changed to meet the rheological requirements of forming droplets by the nozzle of the inkjet print head used.

[0105] Other examples of the synthesis and enzymatic deprotection of 3'-O-ester protected dNTPs or 3'-O-phosphate protected dNTPs are described in the following references: Canard et al, Proc. Natl. Acad. Sci., 92:10859-10863 (1995); Canard et al, Gene, 148: 1-6 (1994); Cameron et al, Biochemistry, 16(23): 5120-5126 (1977); Rasolonjatovo et al, Nucleosides & Nucleotides, 18(4&5): 1021-1022 (1999); Ferrero et al, Monatshefte fur Chemie, 131: 585-616 (2000); Taunton-Rigby et al, J. Org. Chem., 38(5): 977-985 (1973); Uemura et al, Tetrahedron Lett., 30(29): 3819-3820 (1989); Becker et al, J. Biol. Chem., 242(5): 936-950 (1967); Tsien, International Patent Publication WO1991 / 006678.

[0106] In some embodiments, modified nucleotides include modified nucleotides or nucleoside molecules comprising a purine or pyrimidine base and a ribose or deoxyribose sugar moiety having a removable 3'-OH blocking group covalently attached thereto such that the 3' carbon atom has attached thereto a group of the following structure: -OZ wherein -Z is any of -C(R')2-OR", -C(R')2-N(R")2, -C(R')2-N(H)R", -C(R')2-SR", and -C(R')2-F, wherein each R" is a removable protecting group or is part of a removable protecting group; each R' is independently a hydrogen atom, an alkyl, substituted alkyl, arylalkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, acyl, cyano, alkoxy, aryloxy, heteroaryloxy, or amido group, or a detectable label attached via a linker; provided that, in some embodiments, such substituents have up to 10 carbon atoms and / or up to 5 oxygen or nitrogen heteroatoms; or (R')2 represents a group of formula =C(R"')2, wherein each R"' can be the same or different and is selected from hydrogen atoms and halogen atoms and alkyl group, with the proviso that in some embodiments, each R'' alkyl group has 1 to 3 carbon atoms; and wherein the molecule can react to produce an intermediate in which each R'' is exchanged for H, or in the case where Z is -(R')2-F, F is exchanged for OH, SH or NH2, preferably OH, which intermediate dissociates under aqueous conditions to provide a molecule with a free 3'-OH; with the proviso that in the case where Z is -C(R')2-SR", both R' groups are not H. In certain embodiments, R' of the modified nucleotide or nucleoside is an alkyl or substituted alkyl group, with the proviso that such alkyl or substituted alkyl group has 1 to 10 carbon atoms and 0 to 4 oxygen or nitrogen heteroatoms. In certain embodiments, -Z of the modified nucleotide or nucleoside is of the formula -C(R')2-N3. In certain embodiments, Z is an azidomethyl group.

[0107] In some embodiments, Z is a cleavable organic moiety with or without heteroatoms having a molecular weight of 200 or less. In other embodiments, Z is a cleavable organic moiety with or without heteroatoms having a molecular weight of 100 or less. In other embodiments, Z is a cleavable organic moiety with or without heteroatoms having a molecular weight of 50 or less. In some embodiments, Z is an enzymatically cleavable organic moiety with or without heteroatoms having a molecular weight of 200 or less. In other embodiments, Z is an enzymatically cleavable organic moiety with or without heteroatoms having a molecular weight of 100 or less. In other embodiments, Z is an enzymatically cleavable organic moiety with or without heteroatoms having a molecular weight of 50 or less. In other embodiments, Z is an enzymatically cleavable ester group with a molecular weight of 200 or less. In other embodiments, Z is a phosphate group that can be removed by 3'-phosphatase. In some embodiments, one or more of the following 3'-phosphatases can be used according to the manufacturer's recommended protocol: T4 polynucleotide kinase, calf intestinal alkaline phosphatase, recombinant shrimp alkaline phosphatase (eg, available from New England Biolabs, Beverly, MA).

[0108] In some embodiments, the 3'-blocked nucleotide triphosphate is blocked with a 3'-O-azidomethyl, 3'-O-NH2, or 3'-O-allyl group.

[0109] In some embodiments, 3'-O blocking groups of the present invention include 3'-O-methyl, 3'-O-(2-nitrobenzyl), 3'-O-allyl, 3'-O-amine, 3'-O-azidomethyl, 3'-O-tert-butoxyethoxy, 3'-O-(2-cyanoethyl), and 3'-O-propargyl.

[0110] 3'-O blocked dNTPs without base protection can be purchased from commercial suppliers or synthesized using published techniques, such as U.S. Patent 7057026; Guo et al, Proc. Natl. Acad. Sci., 105(27): 9145-9150 (2008); Benner, U.S. Patents 7544794 and 8212020; International Patent Publications WO2004 / 005667, WO91 / 06678; Canard et al, Gene (cited herein); Metzker et al, Nucleic Acids Research, 22:4259-4267 (1994); Meng et al, J. Org. Chem., 14: 3248-3252 (2006); U.S. Patent Publication 2005 / 037991. 3'-O blocked dNTPs with base protection can be synthesized as follows.

[0111] Template-free polymerases for polynucleotide synthesis

[0112] A variety of different template-free polymerases can be used in the methods of the present invention. Template-free polymerases include, but are not limited to, polX family polymerases (including DNA polymerases β, λ, and μ), poly(A) polymerases (PAP), poly(U) polymerases (PUP), DNA polymerases θ, and the like, as described, for example, 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 WO2020 / 099451; Champion et al, International Patent Publication WO / 2021 / 116270; Heinisch et al, International Patent Publication WO2021 / 018919. In particular, terminal deoxynucleotidyl transferase (TdT) and its variants are useful in template-free DNA synthesis.

[0113] In some embodiments, TdT variants are used in the present invention that exhibit increased incorporation activity relative to 3'-O-aminonucleoside triphosphates. For example, such TdT variants can be prepared using the techniques described in Champion et al, U.S. Patent No. 10,435,676 (incorporated by reference).

[0114] Template-free polymerase ink

[0115] As described above, the synthesis reagents delivered by the inkjet pump must be formulated to maintain the activity of the synthesis reagents and meet the rheological requirements of droplet formation, which can be achieved by using variable viscosity modifiers. Such formulations are referred to as "inks" in this article. For example, meeting the first constraint (activity) may require that the template-free polymerase be present in the reaction mixture at a certain minimum concentration. However, due to high protein viscosity, the concentration of the required activity may hinder meeting the second constraint, namely droplet formation ability. In this case, embodiments of the present invention may require the delivery of multiple droplets each with a lower concentration of polymerase, which, combined with evaporation, allows the generation of polymerase concentrations at the required level of activity. Such embodiments are Figure 2C Shown in.

[0116] As also mentioned above, the key solution parameters that affect droplet formation by an inkjet printer are viscosity, surface tension, density, and diameter of the inkjet nozzle, which are related by the following equation: Z = [( a)^(0.5)] / ,in is the density of the fluid, is the surface tension, is the viscosity, a is the radius of the inkjet pump nozzle, and z is 1 to 10 to reliably form droplets, for example, Derby, J. Mater. Chem., 18: 5717-5721 (2008). This relationship applies to any synthetic reagent delivered by droplets generated by inkjet, wherein the droplets include (i) a template-free polymerase in its coupling buffer, (ii) a mixture of a template-free polymerase and 3'-O protected dNTPs in its coupling buffer, (iii) 3'-O protected dNTPs in the buffer, (iv) a deprotection buffer, and (v) a buffer containing a 5'-linker derived initiator. One of ordinary skill in the art can implement the application of this relationship by adjusting reactant density, viscosity modifiers, surface tension modifiers, etc. to determine an ink composition capable of forming the desired droplets for a particular embodiment.

[0117] Thus, for certain embodiments of the present invention, synthetic reagents prepared for non-droplet delivery to a reaction mixture can be reformulated by adding viscosity modifiers, surface tension modifiers, density modifiers, etc., to form a "printable ink" that can be delivered in droplets generated by an inkjet pump. "Printable" with respect to the reagent ink means that reproducible droplets can be ejected from the nozzle at a uniform velocity and volume, and without satellite droplets.

[0118] In some embodiments, if the specific activity of the template-free polymerase is relatively low, such that a relatively large amount of protein must be delivered to the reaction site to complete the coupling step, the polymerase delivery can be performed by dispensing multiple droplets in each coupling cycle while allowing a controlled amount of evaporation to maintain the reaction volume within a specified range, for example, 10-100 pL. In some embodiments, the multiple droplets delivered are 2 to 10, or 2 to 5, or 2 to 3. In other embodiments, the multiple droplets can be 2 to 150, or 10 to 120. In some embodiments, whenever the template-free polymerase is TdT, the multiple droplets are the number required to achieve a TdT concentration in the reaction mixture at the reaction site of 1 μM to 30 μM or 2 μM to 20 μM. In some embodiments, the concentration of TdT in the ink is a concentration that produces a stoichiometry of about 1:1 between the TdT molecules and the polynucleotide at the reaction site. In other embodiments, the concentration of TdT in the ink is that which produces a stoichiometry of 1:1 or greater between the TdT molecules and the polynucleotide at the reaction site.

[0119] In some embodiments, the present invention includes a printable ink comprising a TdT variant and a viscosity modifier, such as a variable viscosity modifier. In some embodiments, the concentration of the TdT in a buffer suitable for coupling activity is 1 μM to 20 μM / mg. In some embodiments, the buffer comprises about 10 to about 500 mM potassium cacodylate buffer (pH 6.5 to 7.5) and about 0.01 to about 10 mM divalent cations (e.g., COCl2 or MnCl2). In some embodiments, the extension reaction buffer is an acetate buffer, such as 0.1 M acetate, 0.5 M NaCl, pH 4.5.

[0120] In some embodiments, a fixed viscosity modifier can be used together with a variable viscosity modifier. Such fixed viscosity modifiers can be selected from ethylene glycol, polyethylene glycols of different molecular weights, polyethylene glycol methyl ethers, polyethylene glycol dimethyl ethers, poly(vinyl alcohol), carboxymethyl cellulose, and hydroxyethyl cellulose.

[0121] In some embodiments, in addition to the viscosity modifier, the printable template-free polymerase ink, such as TdT ink, further comprises a surface tension modifier. This surface tension modifier can be a detergent. This detergent can be selected from Tween 20, Triton X-100, CHAPS, NP-40, octylthioglucoside, octyl glucoside, or dodecyl maltoside. Of particular interest is Triton X-100. Also of particular interest is Tween 20. Additional surface tension modifiers (i.e., surfactants) are disclosed in Buret, LabChip, 12: 422-433 (2012).

[0122] In some embodiments, the present invention includes a printable ink comprising a TdT variant, a 3'-O protected dNTP, and a variable viscosity modifier. In some embodiments, the concentration of the TdT in a buffer suitable for coupling activity is 1 μM to 50 μM / mg, or 1 μM to 20 μM / mg. In some embodiments, the buffer comprises about 10 to about 500 mM potassium cacodylate buffer (pH 6.5 to 7.5) and about 0.01 to about 10 mM divalent cation (e.g., COCl2 or MnCl2); the concentration of the 3'-O protected dNTP is 125-600 μM. In some embodiments, the variable viscosity modifier is selected from an uncrosslinked poly(N-alkyl-substituted-acrylamide) and a poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) triblock polymer. In some embodiments, the printable TdT ink further comprises a surface tension modifier in addition to the variable viscosity modifier. The surface tension modifier can be a detergent. The detergent can be selected from Triton X-100, CHAPS, NP-40, octylthioglucoside, octyl glucoside or dodecyl maltoside. Of particular interest are Tween 20 and Triton X-100.

[0123] In some embodiments, the printable ink comprising a template-free polymerase (e.g., a TdT variant) comprises a wetting agent for reducing droplet evaporation. Suitable wetting agents include, but are not limited to, glycerol, alcohol sugars, ethylhexylglycerol, panthenol, sorbitol, xylitol, maltitol, propylene glycol, hexylene glycol, butylene glycol, sodium lactate, hyaluronic acid, and polydextrose.

[0124] In some embodiments, the TdT inks of the present invention are delivered in droplets ranging from 1 pL to 200 pL, or from 1 pL to 100 pL, or from 1 pL to 50 pL.

[0125] In some embodiments, the present invention relates to a terminal deoxynucleotidyl transferase (TdT) composition comprising a droplet having a volume of 2 pL to 5 nL of aqueous solution and comprising (i) TdT or a variant thereof at a concentration of 1.0 μM to 30 μM or 2.0 μM to 20 μM, a divalent cation at a concentration of 0.01 mM to 10 mM, and a variable viscosity modifier. In some embodiments, the divalent cation is cobalt or manganese, and the composition further comprises a surface tension modifier.

[0126] In some embodiments, the variable viscosity modifier is selected from uncrosslinked poly(N-alkyl-substituted-acrylamide) and poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) triblock polymers. In some embodiments, whenever the printable template-free polymerase ink (e.g., printable TdT ink) comprises 3'O-aminonucleotides, any of the above compositions may further comprise an aldehyde scavenger (described more fully below). In some embodiments, any of the above compositions may further comprise 3'-O-protected 2'-deoxynucleoside triphosphate monomers at a concentration of 100-1000 μM or 125-600 μM.

[0127] In some embodiments, the variable viscosity modifier may comprise a polymer solution that gels upon cooling. The polymer is selected from at least one of a hydrophobically modified polymer exhibiting UCST-type phase behavior, a natural polymer, a deblocked copolymer brush grafted silica nanoparticle, poly(PEO-co-styrene), and combinations thereof. The polymer may be dissolved in an ionic liquid or in a solution of PNIPAM microgel and host-guest interaction.

[0128] In some embodiments, the present invention relates to a 3'-O-protected 2'-deoxynucleoside triphosphate composition comprising a droplet of an aqueous solution having a volume of 2 pL to 5 nL, and comprising (i) a 3'-O-protected 2'-deoxynucleoside triphosphate at a concentration of 125 to 600 μM and a variable viscosity modifier. In some embodiments, the 3'-O-protected 2'-deoxynucleoside triphosphate composition further comprises a surface tension modifier.

[0129] In some embodiments of the aforementioned compositions, the variable viscosity modifier is selected from uncrosslinked poly(N-alkyl-substituted-acrylamide) and poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) triblock polymers. In some embodiments, the 3'-O-protected 2'-deoxynucleoside triphosphate of the aforementioned compositions is 3'-O-protected 2'-deoxyadenosine triphosphate, 3'-O-protected 2'-deoxyguanosine triphosphate, 3'-O-protected 2'-deoxycytidine triphosphate, or 3'-O-protected 2'-deoxythymidine triphosphate. In some embodiments of the aforementioned compositions, the 3'-O protecting group is selected from 3'-O-methyl, 3'-O-(2-nitrobenzyl), 3'-O-allyl, 3'-O-amine, 3'-O-azidomethyl, 3'-O-tert-butoxyethoxy, 3'-O-(2-cyanoethyl), and 3'-O-propargyl.

[0130] As described above, in some embodiments of the present invention using 3'-O-amino-dNTP monomers, the presence of an aldehyde scavenger in the template-free polymerase ink reduces spurious capping of the 3'-amine by reacting with adventitious aldehydes or ketones (e.g., formaldehyde) that are ubiquitous in the environment. This is a particular problem in inkjet synthesis because the ink droplets have a very high surface area-to-volume ratio, which enhances the absorption of environmental aldehydes. Therefore, in embodiments of the present invention using 3'-O-amino-dNTP monomers, the template-free polymerase ink described above also contains an effective amount of at least one aldehyde scavenger. As used herein, an "effective amount" with respect to an aldehyde scavenger means an amount (or concentration) sufficient to produce a measurable reduction in the spurious capped polynucleotides in the product. Such measurements can be readily accomplished using conventional techniques, such as DNA sequence analysis of product samples, gel electrophoresis, and the like. As used herein, the term "aldehyde scavenger" includes ketone scavengers. In some embodiments, the aldehyde scavenger is a ketone scavenger having the formula RC(=O)H or R 1 -C(=O)-R 2 The reagent for the reaction of a compound with a chemical group, wherein R, R 1 and R 2 Typically, an alkyl or aryl group. More specifically, in some embodiments, an aldehyde scavenger is a reagent that reacts with RC(=O)H or R on the compound at a sufficiently high rate. 1 -C(=O)-R 2 The aldehyde scavenger is a compound that reacts with an aldehyde group such that such a compound does not react (or reacts only negligibly) with the 3'-amine group of a 3'-O-amino-nucleotide. As used herein, the term "scavenger" means a chemical substance that is added to a mixture to remove or inactivate impurities or compounds that lead to undesirable reaction products. In various embodiments, the aldehyde scavenger can be in solution, immobilized on a material for storage or synthesis, or coupled to a reagent used in the method of the invention, for example, a template-free polymerase such as TdT.

[0131] As described above, enzymatic synthesis can be performed using various reagents (referred to herein as "synthesis reagents"), which may contain reactants, wash solutions, deprotection buffers, enzymes, and the like or consist of reactants, wash solutions, deprotection buffers, enzymes, and the like. (The term "synthesis reagent" means any reagent used in a synthesis cycle to couple a monomer, particularly a 3'-O-amino-nucleoside triphosphate, to an initiator or extension segment, such as a buffer comprising a template-free polymerase, a buffer comprising a 3'-O protected nucleotide monomer, a deprotection (or deblocking) buffer, and the like.) In various embodiments, the aldehyde scavenger may be a component of one or more of the synthesis reagents. In some embodiments, the aldehyde scavenger may be added to the reaction mixture as a separate synthesis reagent (without other reactants, wash buffers, or enzymes). In some embodiments, the aldehyde scavenger is added to the reaction mixture as a component of a synthesis reagent comprising a template-free polymerase.

[0132] In, for example, using Sudo et al, US Patent Publication No. US2020 / 0061225 or Figure 8 A to Figure 8 In some embodiments of the aldehyde scavengers listed in B, an effective amount is provided by a concentration of 1 to 500 mM, or in other embodiments, an effective amount is provided by a concentration of 1 to 200 mM, or in other embodiments, an effective amount is provided by a concentration of 1 to 100 mM.

[0133] In some embodiments, the aldehyde scavenger used in the present invention comprises an O-substituted hydroxylamine or polyhydroxylamine. In some embodiments, the O-substituted hydroxylamine used in the present invention is defined by the following formula: R 1 -ONH2, For example, as disclosed in Sudo et al, US Patent Publication No. US2020 / 0061225 or Kitasaka et al, US Patent No. 7,241,625, which are incorporated herein by reference. 1 C 1-18 A linear, branched or cyclic alkyl group, which may be substituted with at least one substituent selected from the group consisting of: a halogen atom; 1-6 Alkoxy group; C 1-6 Haloalkyl group; C 1-6 Haloalkoxy group; carboxyl group; hydroxyl group; mercapto group; cyano group; nitro group; C 6-14 Aryl groups, which may be substituted by halogen atoms, C 1-6 Alkyl group, C 1-6 Alkoxy group, C 1-6 Haloalkyl group, C 1-6C is substituted with a haloalkoxy group, a carboxyl group, a hydroxyl group, a mercapto group, a cyano group or a nitro group; 4-14 Heteroaryl groups, which may be substituted by halogen atoms, C 1-6 Alkyl group, C 1-6 Alkoxy group, C 1-6 Haloalkyl group, C 1-6 substituted with a haloalkoxy group, a carboxyl group, a hydroxyl group, a mercapto group, a cyano group or a nitro group; an alkoxycarbonyl group represented by the following formula: -(C=O)-OR 2 and a carbamoyl group represented by the following formula: -(C=O)-NR 3 (R 3 ) where R 2 It is C 1-18 A linear, branched or cyclic alkyl group, which may be substituted at a chemically acceptable optional position by at least one substituent selected from the following: a carboxyl group; a hydroxyl group; a mercapto group; a halogen atom; a C 1-6 Alkoxy group; C 1-6 Haloalkoxy group; C 6-14 Aryl group; and C 4-14 heteroaryl group; and wherein each R 3 may be the same or different and are each independently C 1-18 A linear, branched or cyclic alkyl group, which may be substituted with at least one substituent selected from the group consisting of a carboxyl group; a hydroxyl group; a mercapto group; a halogen atom; 1-6 Alkoxy group; C 1-6 Haloalkoxy group; C 6-14 Aryl group; and C 4-14 Heteroaryl group; C 6-14 Aryl group, C 4-14 a heteroaryl group or a hydrogen atom.

[0134] In particular, exemplary O-substituted hydroxylamines or polyhydroxylamines that can be used in the present invention are Figure 3 A and Figure 3 Shown in B are compounds (1) to (14), wherein compound (1) is also referred to herein as a "BOX" reagent.

[0135] In some embodiments, the aldehyde scavenger includes a carbonyl compound disclosed by Pacifici, U.S. Patent 5,446,195 or Burdeniucet al, U.S. Patent Publication No. US20160369035, which are incorporated herein by reference, and is defined by the formula:

[0136] Where R and R' are CH3 or H[O(CH2) m ] n O-, and wherein m and n are selected from the combination of m and n consisting of: m=1 and n=1, 3-19; m=2 and n=2-19; ​​or m=3 and n=1-19; Y is --CH2- or --CH2-CO-CH2--.

[0137] In some embodiments of the present invention, the template-free polymerase ink as described above also includes dyes, to allow when initially distributing reagents to define reaction sites or during synthesis when subsequent droplet distribution, monitoring the position, size, shape and possible overlap of the reaction sites, particularly monitoring the possible coalescence of the reaction mixture at adjacent sites. For this purpose, a large amount of fluorescent and non-fluorescent dyes are available for selection. The main criteria used are that the dye (i) does not adversely affect the performance of any reaction component, (ii) is bright enough or dense enough to make droplets or reaction sites easy to detect, (iii) if more than one is used, then spectrally different, and (iv) does not affect the rheological properties of the ink. In some embodiments, food dyes are used in the ink of the present invention. In other embodiments, pH indicator dyes are used in the ink of the present invention. In other embodiments, fluorescent dyes are used in the ink of the present invention. Exemplary dyes for template-free polymerase inks include BrilliantBlue FCF, Fast Green FCF, Ponceau 4R and Sunset Yellow FCF. In some embodiments, food dyes are used with a concentration of 1 to 20mM or a concentration of 1 to 10mM.

[0138] Example

[0139] Method for evaluating reaction conditions in enzymatic inkjet synthesis

[0140] In this example, a test bed was created to evaluate different inkjet reaction conditions. Because very small amounts of material at each reaction site are difficult to analyze, a slide was prepared that allowed areas containing a large number of reaction sites (e.g., 10 or more) to be processed together and pooled for analysis by gel electrophoresis. The areas were created by depositing an equal volume of base-cleavable or photo-cleavable initiator in each area. Figure 5An exemplary slide (900) is shown in FIG (which can be a PolyAn 3D-epoxy coated glass slide). It contains 24 circular areas (902), each of which contains approximately the same number of reaction sites (not shown). Areas (902) are created on the slide (900) by manually spotting 20 μM 5'-amino-derived photocleavable initiator to 24 locations (following the protocol recommended by the slide manufacturer). Briefly, after incubation overnight at 70% relative humidity, the slide is then heated to 80°C for 5 minutes, washed in 1 M ethanolamine pH 8 for 1 hour, washed in SSC 4X for 30 minutes, washed in SSC 2X 0.1% SDS for 30 minutes, washed in SSC 0.2X for 30 minutes, and washed in MQ for 30 minutes. An exemplary initiator may have the following sequence: 5'-amino-C12-10T(PC)4T(FAM-T)18T-3', where C12 is a 12-carbon alkyl linker, T is thymidine, "PC" is a photocleavable linker (e.g., Horgan, WO2021 / 048142), and "FAM-T" is fluorescein-labeled thymidine. After performing the experiment, the initiator was prepared by immersing a specific area in PBS (e.g., 40 μL / area) and illuminating the area with 365 nm light (e.g., Analytik Jena, 95-0252-02, UVLMS-38, 8 W intensity at 3" is 1500 μW / cm for 365 nm. 2 ) to illuminate the region to photocut the sequence from the region, after which, as Figure 5 The pooled sequences were loaded onto the gel as shown. Figure 5 In the example of , the regions are divided into 8 groups of 3 regions each. In Groups 1-4, 23 extension reaction cycles are performed, after which the resulting products in each group of 3 regions are photocleaved, pooled, and loaded (909) onto their respective lanes 1-4 (914). In Groups 5-8, no synthesis reaction is performed. The non-extended initiator is processed exactly the same as Groups 1-4, but is loaded onto lanes 5-8 (916). The gel provides a convenient and sensitive measurement of the effects of changes in various reaction parameters, including but not limited to reactant concentrations, the presence, absence, and concentration of rheological components (e.g., surfactants, viscosity modifiers, etc.), different template-free polymerases, secondary structure modifiers, etc.

[0141] Evaluation of the Effect of Temperature on Viscosity and Enzymatic Inkjet Synthesis

[0142] In this example, the effect of temperature on ink viscosity was evaluated. Extension inks were prepared as described in Table 1 below.

[0143]

[0144] Table 1 Ink composition

[0145] like Figure 6 As shown, it can be seen that the viscosity decreases significantly with increasing temperature, which is expected.

[0146] The stability of the extended ink was also evaluated at different temperatures. Figure 7 As shown, the benefit of storing the ink at low temperature can be seen, and a synthesis could not be performed with an ink stored in the printhead at 37°C because the ink precipitated within 24 hours.

[0147] In this example, manual synthesis tests were performed to evaluate the efficiency of the reaction at different temperatures.

[0148] The slide is completely covered with the photocleavable initiator. Figure 5 An exemplary slide (900) is shown in FIG (which may be a PolyAn 2D-azide coated glass slide). Immobilization is performed by incubating 5 μM 5'DBCO photocleavable initiator in NaOAc / NaCl buffer at pH 4.5 for 1 hour at 70% RH. The slide is then washed with a series of buffers, rinsed with MQ, and dried.

[0149] An exemplary initiator can have the following sequence: 5'-DBCO-TEG-10T(PC)4T(FAM-T)18T-3', where DBCO is dibenzocyclooctyne, TEG is triethylene glycol, T is thymidine, "PC" is a photocleavable linker (e.g., Horgan, WO2021 / 048142), and "FAM-T" is fluorescein-labeled thymidine.

[0150] Manual synthesis was then performed in silicon gaskets at 22°C, 37°C, or 4°C (one slide) as follows: i) incubation with extension ink (70% RH, 5 min at 4°C, 20°C, or 37°C), ii) incubation with deprotection buffer (70% RH, 3 min), iii) rinsing with H2O milli-Q, and iv) drying with compressed air.

[0151] After performing the experiment, fill the gasket with PBS and irradiate the specific area with 365 nm light (e.g. Analytik Jena, 95-0252-02, UVLMS-38, 8 W intensity at 3" is 1500 mW / cm2 for 365 nm) to photocleave the sequence from that area.

[0152] Extended oligomers at 4°C, 20°C and 37°C were subjected to gel electrophoresis along with non-extended oligomers used as reference.

[0153] Gels provide convenient and sensitive measurements of the effects of changes in various reaction parameters, including but not limited to reactant concentrations, the presence, absence, and concentration of rheological components, extension temperature, different template-free polymerases, secondary structure modifiers, etc.

[0154] from Figure 8 It can be seen that the synthesis at 37 °C is better than that at 4 °C, and the amount of impurities decreases with higher temperature. Figure 6 and Figure 7 It can be seen that in order to obtain better results, a high viscosity is required before synthesis and a lower viscosity is required during synthesis.

[0155] definition

[0156] Unless otherwise specifically defined herein, the terms and symbols of nucleic acid chemistry, biochemistry, genetics, and molecular biology used herein follow those in standard treatises and textbooks in the field, e.g., Kornberg and Baker, DNA Replication, Second Edition (WH Freeman, New York, 1992); Lehninger, Biochemistry, Second Edition (Worth Publishers, New York, 1975); Strachan and Read, Human Molecular Genetics, Second Edition (Wiley-Liss, New York, 1999); Le, Recent Progress in Ink Jet Technologies II, chapter 1, pgs. 1-14 (1999); Zapka, editor, “Handbook of Industrial Inkjet Printing,” (Wiley-VCH, Weinheim, Germany).

[0157] "Humectant" is any hygroscopic substance that attracts and retains moisture. Exemplary humectants include, but are not limited to, glycerin, sugar alcohols, ethylhexylglycerin, panthenol, sorbitol, xylitol, maltitol, propylene glycol, hexylene glycol, butylene glycol, sodium lactate, hyaluronic acid, polydextrose, and the like.

[0158] " polynucleotide " means the linear polymer of nucleotide monomer or its analog.The monomer constituting polynucleotide can be able to pass through the regular pattern of monomer-monomer interaction, such as Watson-Crick type base pairing, base stacking, Hoogsteen or reverse Hoogsteen type base pairing, etc., with natural polynucleotide specific binding.Such monomer and their internucleoside linkage bond can be naturally occurring or can be its analog, such as naturally occurring or non-naturally occurring analog.Non-naturally occurring analog can include PNA, thiophosphate internucleoside linkage bond, the base containing the linking group that allows label (such as fluorophore or hapten) to attach etc.Whenever the use of polynucleotide needs enzymatic processing, such as by polymerase extension, by ligase connection etc., those of ordinary skill in the art will understand that, in those cases, polynucleotide will not contain some analog of internucleoside linkage bond, sugar moiety or base at any or some positions.The size of polynucleotide is generally several monomeric units, such as 5-40, to several thousand monomeric units. Whenever a polynucleotide is represented by a sequence of letters (uppercase or lowercase), such as "ATGCCTG", it will be understood that the nucleotides are in 5'→3' order from left to right, and that "A" represents deoxyadenosine, "C" represents deoxycytidine, "G" represents deoxyguanosine, and "T" represents thymidine, "I" represents deoxyinosine, and "U" represents uridine, unless otherwise indicated or obvious from the context. Unless otherwise indicated, the terminology and atom numbering conventions will follow those disclosed in Strachan and Read, Human Molecular Genetics 2 (Wiley-Liss, New York, 1999). Typically, polynucleotides contain four natural nucleosides (e.g., deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine for DNA, or their ribose counterparts for RNA) linked by phosphodiester linkages; however, they may also contain non-natural nucleotide analogs, for example, including modified bases, sugars, or internucleoside linkages. Those skilled in the art will appreciate that when an enzyme has a specific polynucleotide substrate activity requirement, such as single-stranded DNA, RNA / DNA duplexes, etc., then selecting an appropriate composition for the polynucleotide substrate is well within the knowledge of those skilled in the art, particularly with the guidance of papers such as Sambrook et al, Molecular Cloning, Second Edition (Cold Spring Harbor Laboratory, New York, 1989) and similar references. Similarly, a polynucleotide can refer to either a single-stranded form or a double-stranded form (i.e., a duplex of a polynucleotide and its corresponding complement). Those skilled in the art will understand from the context in which the term is used which form is intended or whether both forms are intended.

Claims

1. A method for enzymatically synthesizing a plurality of polynucleotides at reaction sites on a substrate for inkjet printing, the method comprising the steps of: (a) providing a substrate having an initiator at a plurality of reaction sites, wherein each initiator has a free 3'-hydroxyl group, and wherein each polynucleotide in the plurality of polynucleotides is assigned to a reaction site for synthesis; (b) dispensing at least one droplet of at least one synthesis reagent to each reaction site in a plurality of reaction sites to perform a reaction cycle, the reaction cycle comprising the steps of: (i) reacting an initiator or extension segment having a free 3'-O-hydroxyl group with a 3'-O-protected nucleoside triphosphate and a template-free polymerase under extension conditions such that the initiator or extension segment is extended by incorporation of the 3'-O-protected nucleoside triphosphate to form a 3'-O-protected extension segment, and (ii) deprotecting the extension segment to form an extension segment having a free 3'-hydroxyl group, wherein the synthesis reagent comprises a template-free polymerase, a 3'-O-protected nucleoside triphosphate, a mixture of the template-free polymerase and the 3'-O-protected nucleoside triphosphate, or a deprotection solution; wherein the at least one synthesis reagent comprises a variable viscosity modifier, the variable viscosity modifier having a first viscosity during formation of the at least one droplet and having a second viscosity at each reaction site in the plurality of reaction sites during the reaction step of the reaction cycle; (c) repeating step (b) until the plurality of polynucleotides are synthesized; The first viscosity and the second viscosity are different. 2 . The method according to claim 1 , wherein the first viscosity is greater than 2 mPa·s and the second viscosity is less than or equal to 2 mPa·s.

3. The method according to any one of the preceding claims, wherein the first viscosity is from 2 mPa.s to 20 mPa.s, endpoints exclusive; and the second viscosity is from 1 mPa.s to 2 mPa.s, endpoints inclusive.

4. The method according to any one of the preceding claims, wherein the at least one synthesis reagent is a template-free polymerase ink.

5. The method of any one of the preceding claims, wherein the variable viscosity modifier comprises a thermoreversible polymer whose viscosity decreases with increasing temperature.

6. The method of claims 1 to 4, wherein the variable viscosity modifier comprises a polymer solution that gels upon cooling.

7. The method of any one of the preceding claims, wherein the variable viscosity modifier comprises at least one of polyvinyl alcohol, dimethyl sulfoxide (DMSO), polyethylene glycol (PEG), polyethylene glycol methyl ether (OMe)PEG, polyethylene glycol dimethyl ether (OMe)2PEG, carboxymethyl cellulose, hydroxyethyl cellulose, or a combination thereof.

8. The method according to any one of the preceding claims, wherein the step (b) further comprises washing the extension segment after the deprotection step.

9. The method of any one of the preceding claims, wherein each of the reaction sites is distinct and does not overlap with other of the reaction sites.

10. The method according to any one of the preceding claims, wherein the step of reacting comprises incubating the reaction mixture for a predetermined duration.

11. The method according to any one of the preceding claims, comprising, after the step of reacting, a step of capping the initiator or extension fragment having a free 3'-O-hydroxyl group that has not been extended.

12. The method of any one of the preceding claims, wherein each of the plurality of polynucleotides is assigned to a different reaction site for synthesis.

13. The method according to any one of the preceding claims, wherein the solution of at least one synthesis reagent is free of glycerol.

14. The method according to any one of the preceding claims, wherein the variable viscosity modifier comprises 5% to 50% by volume of the solution of the at least one synthesis reagent, preferably 20% to 40% by volume of the solution of the at least one synthesis reagent.

15. A method according to any preceding claim, wherein there is more than one solution of synthesis reagent, each solution having a different variable viscosity modifier.

16. A template-free polymerase ink for inkjet printing, comprising: an aqueous solution comprising a template-free polymerase at a concentration of 1.0 μM to 30 μM; wherein whenever the ink is printed as droplets onto a substrate, the printed droplets each have a volume of 0.1 pL to 5 nL of the aqueous solution, and wherein the ink comprises a variable viscosity modifier having a first viscosity of 2 mPa.s to 20 mPa.s whenever the temperature is 5°C to 30°C, and a second viscosity of 2 mPa.s to 3 mPa.s whenever the temperature is 35°C to 60°C; wherein the first viscosity and the second viscosity are different.

17. The template-free polymerase ink of the preceding claim, wherein the variable viscosity modifier comprises at least one of polyvinyl alcohol, carboxymethyl cellulose, hydroxyethyl cellulose, dimethyl sulfoxide, polyethylene glycol (PEG), polyethylene glycol methyl ether (OMe)PEG, polyethylene glycol dimethyl ether (OMe)2PEG, or a combination thereof.

18. The template-free polymerase ink according to any one of claims 16 to 17, wherein the variable viscosity modifier comprises 5% to 50% by volume of the solution of the at least one synthesis reagent, preferably 20% to 40% by volume of the solution of the at least one synthesis reagent.

19. The template-free polymerase ink of any one of claims 16 to 18, wherein the variable viscosity modifier comprises a polymer solution that gels upon cooling.

20. The template-free polymerase ink of any one of claims 16 to 19, wherein the ink is free of glycerol.

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