CN120603956A - Variable viscosity inks for inkjet delivery of enzyme reagents - Google Patents

Abstract

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

Description

Variable viscosity inks for inkjet delivery of enzymatic reagents

Inkjet printing is a low cost, versatile technique for non-contact delivery of defined amounts of liquid to precise locations with minimal waste. This technique has been applied to oligonucleotide microarray synthesis using phosphoramidite chemistry and has been used to print enzymes directly onto a substrate in 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 rheological requirements of droplet formation, but also by changes in enzyme concentration and buffer conditions caused by evaporation losses when, for example, an enzyme-containing fluid is printed onto a microarray.

Recently, there has been interest in applying enzyme-based polynucleotide synthesis to problems not applicable to conventional chemical-based DNA synthesis, especially because of the mild aqueous reaction conditions of enzymatic processes. It has been observed that there is currently a tradeoff between the desired viscosity value of the ink for print drop formation and the desired viscosity value for effective enzymatic process performance following inkjet delivery. That is, ink viscosity optimized for drop formation is currently suboptimal for enzymatic activity, and vice versa.

Disclosure of Invention

The present invention relates to inks comprising variable viscosity modifiers for inkjet delivery of enzymes to reactive sites.

In one aspect, the invention relates to an inkjet-based method of enzymatically synthesizing a plurality of polynucleotides using such an ink, the method comprising the steps of (a) providing a substrate having an initiator (initiator) at a plurality of reaction sites, wherein each initiator has a free 3 '-hydroxyl group, and wherein each polynucleotide of the plurality of polynucleotides is assigned to (assigned to) a reaction site for synthesis, (b) dispensing at least one droplet of at least one synthetic reagent to each reaction site of the plurality of reaction sites by one or more inkjet pumps for a reaction cycle comprising the steps of (i) reacting, under extension conditions, an initiator or extension fragment having a free 3' -O-hydroxyl group with a3 '-O-protected nucleoside triphosphate and a template-free polymerase such that the initiator or extension fragment extends by incorporation of a 3' -O-protected nucleoside triphosphate to form an extension fragment, and (ii) deprotecting the extension fragment to form an extension fragment having a free 3 '-hydroxyl group, wherein the synthetic reagent comprises a 3' -O-protected nucleoside triphosphate, and the one or more template-free polymerase forms a viscosity modifying agent, wherein the one or more template-free polymerase has a viscosity modifying agent during the formation of the one or more than one viscosity modifying agent, and having a second viscosity of less than or equal to 2 mPa.s at each of the plurality of reaction sites during the reaction step of the reaction cycle, and (c) repeating step (b) until the plurality of polynucleotides are synthesized, wherein the first viscosity and the second viscosity are different. In some embodiments, the substrate is a planar substrate.

In another aspect, the invention relates to a template-free polymerase ink for inkjet printing comprising an aqueous solution comprising a template-free polymerase in a concentration of 1.0 to 30 μM, wherein each printed droplet has a volume of 0.1pL to 5nL of the aqueous solution per time the ink is printed onto a substrate, and wherein the ink comprises a variable viscosity modifier having a first viscosity of 2 to 20 mpa.s per time the temperature is 5 to 30 ℃ and a second viscosity of 1 to 2 mpa.s per time the temperature is 35 to 60 ℃, wherein the first viscosity and the second viscosity are different.

Brief description of the drawings

FIG. 1A contains a schematic representation of an enzymatic synthesis cycle in which 3' -O protected dNTPs are added to a nucleic acid strand, followed by deprotection.

Fig. 1B shows a droplet microarray with a hydrophobic-hydrophilic patterned surface that can be used as a synthetic support.

Fig. 2A-2C illustrate the synthesis cycle of four different embodiments of the present invention.

Fig. 3 shows an embodiment of the invention in which inkjet instruments are used to define reaction sites on an array to eliminate the problem of aligning inkjet delivery with preformed reaction sites.

Fig. 4A shows components of an exemplary inkjet system for use with the present invention, and fig. 4B shows an inkjet instrument for use in practicing several embodiments of the present invention.

FIG. 5 illustrates a technique for assessing the efficiency of inkjet-based enzymatic synthesis, which may be used to select variable viscosity modifiers.

Fig. 6 shows the change in viscosity of this example at different temperatures.

Fig. 7 shows the stability of extended inks stored at different temperatures.

Fig. 8 shows the results of 10 cycles poly (T) with manual EDS.

Detailed Description

While the invention is amenable to various modifications and alternative forms, specifics thereof have been shown by way of example in the drawings and will be described in detail. It should be understood, however, that the intention is not to limit the invention to the particular embodiments described. The intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention. Guidance for selecting materials and components to perform particular functions can be found in available papers and references concerning 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.

In one aspect, the invention relates to inks for delivering an enzyme-containing reagent in droplets formed by inkjet, and methods of using such inks for enzymatic synthesis of polynucleotides. Such inks comprise at least one variable viscosity modifier capable of exhibiting a first viscosity at the nozzles of the printhead during droplet formation and a second viscosity at the reaction sites for performing an enzymatic process after delivery. In general, variable viscosity modifiers exhibit different viscosity values in response to different physical conditions, such as temperature, at the nozzle during droplet formation and at the reaction site when an enzymatic process is performed after droplet delivery. In some embodiments, the nozzles of the printhead are maintained at a temperature lower than the temperature of the reaction sites, and a variable viscosity modifier is used that has a viscosity that decreases monotonically with increasing temperature. In other embodiments, the nozzles of the printhead are maintained at a temperature higher than the temperature of the reaction sites, and a variable viscosity modifier having a viscosity that monotonically increases with increasing temperature is used. In other embodiments, a variable viscosity modifier having shear thickening (or swelling (dilatant)) properties is used, such that the droplet formation conditions induce a viscosity of 2-20 mpa.s and the conditions at the reaction sites induce a viscosity of 1-2 mpa.s.

The variable modifier whose viscosity decreases with increasing temperature may include conventional viscosity modifiers at selected concentrations and 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) ₂ PEG, carboxymethyl cellulose, hydroxyethyl cellulose, and the like. Selected concentrations of the variable viscosity modifier in the inks of the present invention include, but are 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 15 ℃ or less, or 12 ℃ or less, or 10 ℃ or less of the at least one inkjet pump.

Variable viscosity modifiers whose viscosity decreases with increasing temperature may also include thermally reversible polymers, compositions incorporating poly (N-alkyl substituted acrylamides), preferably uncrosslinked, e.g., SASSI ET AL, electrophoreus, 17:1460-1469 (1996); schill, prog. Polymer Sci., 17:163-249 (1992), and the like. For example, the variable viscosity properties of poly (N-isopropylacrylamide) polymers can be readily modified by selecting the molecular weight, concentration, and appropriate comonomer (e.g. SigmaAldrich, Technical Bulletin, "Designing temperature and pH sensitive NIPAM based polymers;" Plate et al, Polymer Journal, 31(1): 21-27 (1999)).

In some embodiments, a template-free polymerase ink may be formulated comprising an uncrosslinked poly (N-alkyl substituted acrylamide) having a first viscosity of 2 to 20 mpa.s each time the temperature is 5 to 30 ℃ and a second viscosity of 1 to 2 mpa.s each time the temperature is 35 to 60 ℃.

Variable viscosity modifiers that increase in viscosity with increasing temperature may include poly (ethylene oxide) -poly (propylene oxide) -poly (ethylene oxide) triblock polymers such as F127, e.g., 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 readily modified by the choice of molecular weight, concentration and suitable comonomers. In some embodiments, a template-free polymerase ink may be formulated that includes an uncrosslinked poly (ethylene oxide) -poly (propylene oxide) -poly (ethylene oxide) triblock polymer having a first viscosity of 2 to 20 mpa.s each time the temperature is between 35 and 60 ℃ and a second viscosity of 1 to 2 mpa.s each time the temperature is between 5 and 30 ℃.

In further embodiments, the variable viscosity modifier may comprise a shear thickening or bulking agent (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 so that the ink viscosity is 3 mpa.s or more every time the shear force at the nozzle of the inkjet pump is 105 (s-1) or more.

In some embodiments, a template-free polymerase ink may be formulated that includes a polymer solution that gels upon cooling, the polymer solution having a first viscosity of 2-20 mpa.s each time the temperature is 5-30 ℃, and a second viscosity of 1-2 mpa.s each time the temperature is 35-60 ℃.

In some embodiments, the variable viscosity modifier may comprise a polymer solution that gels upon cooling. Such polymers are selected from at least one of hydrophobically modified polymers having UCST-type phase behavior, natural polymers, brush-grafted silica nanoparticles of a deblocking copolymer, 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 interactions.

In some embodiments, at least one of the synthesis reagent solutions is free of glycerol.

In some embodiments, there is more than one synthetic reagent solution, each solution having a different variable viscosity modifier.

In some embodiments, the ink with the variable viscosity modifier may also contain additional components as described below, including but not limited to conventional viscosity modifiers, surfactants, humectants, aldehyde scavengers, and the like, as described below.

In some embodiments, the invention relates to methods and compositions for inkjet-assisted synthesis of a plurality of polynucleotides at different sites on a substrate, respectively, using a template-free polymerase, such as a terminal deoxynucleotidyl transferase (TdT). Typically, such synthesis occurs on substrates comprising flat surfaces (e.g., surfaces of glass, silica, plastic, etc.), but may also occur on other surfaces, such as biological tissue or surface-immobilized cdnas extracted from tissue, for example. As used herein, "inkjet-assisted synthesis" means the delivery of one or more synthesis reagents to a reaction site in droplets produced by one or more inkjet pumps.

"Synthetic reagents" include any reagent used in the synthetic cycle to couple a monomer, particularly a 3' -O protected nucleoside triphosphate, to an initiator or extension fragment, such as a buffer comprising a template-free polymerase, a buffer comprising a 3' -O protected nucleotide monomer, a buffer comprising a mixture of a template-free polymerase and one or more 3' -O protected nucleoside triphosphates, a deprotected (or deblock) buffer, and the like. The term "deprotecting" agent, buffer, solution, etc. is used synonymously herein with the term "deblocking" agent, buffer, solution, etc., respectively. Likewise, the term "protected" with respect to a compound (e.g., dNTP) is used synonymously with the term "blocked" with respect to a compound. As used herein, the term "deprotection solution" (or its equivalent) means an agent that causes or facilitates removal of a protecting group (e.g., a 3' -O protecting group of a nucleotide). As described more fully below, the composition of the deprotection solution (and the deprotection reaction conditions) depends on the nature of the protecting group (or blocking group) to be removed. In various embodiments, the deprotection solution may contain specific reagents (e.g., reducing agents such as TCEP (tris (2-carboxyethyl) phosphine)), enzymes for enzymatic cleavage, scavengers, cofactors, and the like, which chemically react with the protecting groups and/or protecting moieties. In some embodiments, the deprotection solution may not comprise a particular reagent that reacts with the protecting group, but may comprise components that are compatible with the protecting group or facilitate physical cleavage of the protecting group, such as a pH buffer, for example, in the case of a photocleavable protecting group. Typically, in a reaction cycle of 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 1 minute to 30 minutes, or 3 minutes to 15 minutes. Typical extension reaction temperatures are Room Temperature (RT) to 80 ℃, or 20 ℃ to 60 ℃.

"Synthetic reagents" also include reagents for preparing substrates for polynucleotide synthesis, such as reagents for defining reaction sites, initiators, capping reagents, and the like. Typically, a "different reaction site" on a substrate is a discrete site because it is separate from other reaction sites, that is, for example, as illustrated in FIG. 1B, a discrete site does not have a boundary 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 "overwrite (overwriting)" embodiment described below for producing high-density bar codes on surfaces.

In some embodiments, the plurality of polynucleotides may be 2 to 500000, or 100 to 400000, or 100 to 200000, or 100 to 100000. 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 of a uniform deposition on an area comparable to that of a standard 25mm x 75mm 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.

The basic steps of template-free enzymatic synthesis of a polynucleotide are shown in FIG. 1A and described more fully below. Briefly, the synthetic method includes a step cycle that generally involves delivering at least one reagent to a reaction site that includes a buffer that is free of template polymerase, one or more buffers that each include one or more 3' -O protected dNTPs (i.e., monomers), a deprotection buffer, and a wash solution. In various embodiments of the invention, a template-free polymerase buffer, a buffer comprising 3' -O protected dNTP monomers, or a deprotection buffer may be delivered to the reaction site by droplets generated and delivered by an inkjet pump. For delivery by inkjet-generated droplets, these agents must be formulated to meet the rheological requirements of droplet formation. These formulations are referred to as "inks". Key rheology parameters affecting droplet formation are viscosity, density and surface tension, such as 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, Soft Matter, 4: 703-713 (2008) and similar references. Another key parameter related to drop volume is the nozzle diameter of the inkjet pump. In some embodiments, the nozzle diameter used in the present invention may be 10 μm to 100 μm. Thus, as described more fully below, one aspect of the invention includes reagent inks for template-free enzymatic synthesis of polynucleotides, and in particular inks comprising template-free polymerases, 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, in some cases all four monomer types for the purpose of synthesizing random sequence fragments of a polynucleotide, for example for the purpose of creating a polynucleotide tag or barcode.

According to the invention, inkjet-assisted enzymatic synthesis of polynucleotides may be carried out in various embodiments (wherein different reagents are delivered by inkjet pumps). Some of these embodiments are depicted in fig. 2A-2C. Fig. 2A shows two cycles in which a droplet (or droplet) comprising a template-free polymerase and a single species of monomer is delivered to a reaction site surrounded by a buffer droplet on a substrate. The surface of the reaction site (204) comprises a layer of initiator oligonucleotides (not shown) and is surrounded by a 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. Fig. 2A depicts the initiator as having a 3 '-terminal monomer "a" with a free 3' -hydroxyl group (not shown). In fig. 2A-2C, monomers A, B and C are each intended to represent any 3' -O protected dNTP monomer. The ink droplets (201) are dispensed by an inkjet pump (not shown) to a volume (202) at a reaction site (204) (or directly to the reaction site (204) if a drying step is performed). The ink droplets (201) contain 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 droplet-forming rheology requirements. The droplets (201) may also contain a wetting agent to minimize evaporation losses. In some embodiments, each time 3' -O-amino-NTP is used, the droplet (201) may further comprise an aldehyde scavenger. Droplets (201) are deposited on dried reaction sites (207) or coalesce with volumes (202) on undried reaction sites (221) to form a reaction mixture (206) in droplets (205), which are allowed to incubate (208) for a predetermined time to allow B monomers to couple to the 3' end of the initiator (or a previously extended or extended chain after an initial cycle). In some embodiments, this incubation occurs at a humidity above ambient to prevent drying during the incubation step.

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.

After the incubation time of the coupling reaction, the entire substrate surface is immersed or sprayed (209) in a deprotection buffer (210) for a predetermined time to allow removal of the protecting groups, which regenerates the free 3' -hydroxyl groups at the ends of the extended chains. After a predetermined deprotection time, the entire substrate surface is immersed in one or more wash buffers for one or more predetermined times to obtain reaction sites (211) with extended or extended chains or fragments (shown as "-AB") that are ready for the next coupling cycle.

In some embodiments, as described above, a drying step may be performed after the deprotection and washing (213) to minimize the likelihood of the droplets (211) diffusing or coalescing with adjacent droplets. Conventional drying techniques in inkjet printing, hot air or gas, radiation drying, etc., such as Hoynant et al, U.S. patent 8485096, may be used.

The next cycle is the same as the step of attaching the B monomer, except that the C monomer is coupled in the next cycle. Thus, droplets (212), which may have the same composition as droplets (201) except for the identity of the C monomer, are dispensed into the volume (211). As described above, the reaction mixture (214) in droplets (215) is incubated for a predetermined amount of time, after which the entire substrate is treated (218) with a deprotection buffer and wash solution (210) to give reaction sites (220) with extended chains, shown as "-ABC.

Depending on the nature of the substrate (203) in this and other embodiments, the coupling cycle may also include a drying step to prevent droplet spread and coalescence between adjacent reaction sites. This possibility of coalescence is minimized if the surface of the substrate (203) between the reaction sites (204) is sufficiently hydrophobic.

The embodiment of FIG. 2A can be performed by (a) providing a substrate having an initiator at a plurality of different reaction sites, wherein each initiator has a free 3' -hydroxyl group, and wherein each polynucleotide of the plurality of nucleotides is assigned to a different reaction site for synthesis, (b) dispensing at least one droplet of buffer solution comprising a mixture containing 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 type of 3' -O blocked dNTP dispensed to the reaction site depends on the predetermined sequence of the polynucleotide assigned to the reaction site, (c) incubating the template-free polymerase and 3' -O blocked dNTP at each reaction site such that the initiator or extension fragment at the reaction site is extended by incorporation of the 3' -O blocked dNTP to form a 3' -O blocked extension fragment, (d) extending the planar support by treatment of the planar support with a deblocking agent to form a free hydroxyl group, (c) repeating step (d) and (d) synthesizing the polynucleotide. 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, the plurality of droplets is 2 to 10, or 2 to 5, or 2 to 3. In other embodiments, the plurality of droplets may be 2 to 150, or 10 to 120. In some embodiments, a further step of cleaving the plurality of polynucleotides from the substrate is performed.

In some embodiments, the substrate is a planar substrate.

In some embodiments, a drying step may be included after step (d) or after step (d) and the washing step to minimize spreading or coalescence of the droplets when dispensing the next droplet.

Fig. 2B illustrates an embodiment in which monomer and template-free polymerase are delivered to a reaction site in droplets of different inkjet delivery. As described above, the surface of the reaction site (204) contains an initiator layer terminated with an A monomer shown as "-A". A droplet (252) comprising a template-free polymerase is dispensed (250) to the volume (202), after which a droplet (254) comprising B monomer is dispensed (256). In some embodiments, the order in which the template-free polymerase and monomers are dispensed may be reversed such that the monomers are dispensed prior to dispensing the template-free polymerase within the cycle. After incubating the reaction volume (265) for a predetermined time to allow the B monomer 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 then to one or more wash buffers to obtain a reaction site (268) having an extended chain "-AB". The next cycle proceeds in the same manner except that the C monomer is added, i.e., dispensing a droplet (270) containing the template-free polymerase, dispensing a droplet (272) containing the C monomer to form a reaction mixture (273), incubating the reaction mixture (273), deprotecting and washing (276) to give a reaction site (278) with extended chain "-ABC".

The embodiment of fig. 2B can be performed by (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 of the plurality of polynucleotides is assigned to a different reaction site for synthesis, (B) dispensing at least one droplet comprising a buffer solution containing a template-free polymerase to each reaction site by one or more inkjet pumps, (c) dispensing at least one droplet comprising a buffer solution comprising a 3' -O-blocked dATP, a 3' -O-blocked dCTP, a 3' -O-blocked dGTP, or a 3' -O-blocked dTTP to each reaction site by one or more inkjet pumps, wherein the kind of 3' -O-blocked dNTP dispensed to a reaction site depends on a predetermined sequence of the polynucleotide assigned to a reaction site, (d) incubating the template-free polymerase and the 3' -O-blocked dNTP at each reaction site such that the initiator or the extension fragment at the reaction site is extended by incorporation of the 3' -O-blocked dNTP to form a 3' -O-blocked dATP, (c) dispensing at each reaction site by one or more inkjet pumps, (d) forming a label by the extension of the polynucleotide at each reaction site, and (d) repeating steps (d) forming a free hydroxyl group at the reaction site. In some embodiments, a plurality of droplets are delivered to each reaction site during each cycle of steps (b) and (c). In some embodiments, such a plurality of droplets may comprise only a buffer comprising a template-free polymerase, or only a buffer comprising a 3' -O blocked nucleoside triphosphate, or a combination of both. In some embodiments, the plurality of droplets is 2 to 10, or 2 to 5, or 2 to 3. In other embodiments, the plurality of droplets may be 2 to 150, or 10 to 120. In some embodiments, a further step of cleaving the plurality of polynucleotides from the substrate is performed.

In some embodiments, the substrate is a planar substrate.

In some embodiments, before step (b), a step of drying each reaction site may be performed, and before step (c), a step of drying each reaction site may be performed.

FIG. 2C illustrates an embodiment in which multiple droplets of buffer containing template-free polymerase and monomer are delivered to a reaction site while a coupling reaction occurs in the droplets of the reaction site. An important problem associated with conducting reactions in droplets at the reaction site is the loss of fluid due to evaporation. This loss alters the concentration of enzymes, salts, and components such as viscosity modifiers, surfactants, etc., all of which may affect enzyme activity. Furthermore, in some embodiments, the concentration of template-free polymerase in the droplets delivered by inkjet may have to be lower than the optimal concentration for the coupling reaction, because at higher concentrations the polymerase increases the viscosity of the ink such that it adversely affects droplet formation, and thus, 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 sites such that during the coupling reaction, by such repeated addition, the concentration at the reaction sites increases while the buffer at the reaction sites continues to evaporate. Fig. 2C shows the step of a single reaction cycle, in which two droplets of buffer comprising template-free polymerase and monomer are delivered to the reaction site. In some embodiments, multiple droplets containing such reagents may be delivered to each reaction site during synthesis. The number of such droplet deliveries is a design choice depending 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 printheads are used for different reagents, and the like. In some embodiments, agents that alter the droplets on the substrate, such as surfactants, viscosity modifiers, detergents, wetting agents, etc., may be delivered in different droplets produced by different inkjet pumps in the inkjet printhead. The operation of this embodiment is similar to that of fig. 2A, except that two or more droplets are delivered to the reaction mixture. As described above, the surface of the reaction sites (204 a, containing either droplets (202), or 204b, not containing droplets) contains an initiator layer terminated with an a monomer shown as "-a". A first droplet of buffer comprising a template-free polymerase and B monomer is delivered to droplet (202) to form a droplet (282) of reaction mixture at reaction site (204).

Also, in some embodiments employing a drying step, droplet (283) will be delivered directly to reaction site (204 b) instead of droplet (202) at reaction site (204) to form reaction droplet (282).

The reaction droplet (282) continuously loses water by evaporation (285) during incubation (286), which reduces its volume and increases the concentration of all non-volatile components of the droplet, in particular the concentration of template-free polymerase, thereby improving the coupling activity in the reaction droplet (282). Coupling activity is further improved by delivering a second droplet of buffer comprising the template-free polymerase and B monomer separately or as a mixture. Although the concentration of polymerase decreases upon initial coalescence of delivery droplets (287) and reaction droplets (282), continued evaporation rapidly increases the concentration of polymerase in reaction droplets (282) such that it approaches the desired value. After delivery of a plurality of droplets comprising a buffer with polymerase and monomer, the entire surface of the substrate is immersed with a deprotection buffer (288) and one or more wash solutions to obtain reaction sites (294) with extended chains "-AB". The same procedure of delivering multiple droplets of reagent solution to address the evaporation-induced problems and the rheological constraints of droplet formation may also be applied to the embodiments of fig. 2B and 2C.

In some embodiments of fig. 2A-2C, the coupling cycle may further include a washing step after the deprotection step. In some such embodiments, the coupling cycle may further comprise a drying step after the washing step. As mentioned above, the drying step prior to successive coupling cycles will prevent the diffusion and possible coalescence of the reaction droplets at adjacent reaction sites. In some embodiments, washing and drying may be combined by using volatile wash solutions (e.g., acetonitrile, methanol, etc.) that readily evaporate between coupling cycles.

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

Although fig. 2A-2C illustrate a substrate having reaction sites continuously surrounded or occupied by droplets, this is not a requirement of all embodiments of the present invention. In some embodiments, the substrate with the reaction sites may be dried between cycles of steps such that, strictly speaking, the substrate is not always or discontinuously a droplet microarray throughout the synthesis process.

In some embodiments, including those described above, the plurality of polynucleotides (i.e., the number of reaction sites) enzymatically synthesized on the substrate with the inkjet delivery reagent is from 100 to 2 million, or from 100 to 1 million, or from 100 to 10 ten thousand, or from 100 to 50 ten thousand, or from 1000 to 1 million. in some embodiments, such a plurality of polynucleotides has a length of 1 cm ² to 500 cm ², Or 1 cm ² to 256 cm ²、1 cm² to 30cm ², or on a substrate having a surface area of 1 cm ² to 15 cm ², or on a substrate having a surface area of 1 cm ² to 7cm ², or on a substrate having a surface area of 7cm ² to 20 cm ². in some embodiments, the substrate may be prepared and surface treated before being cut or singulated (die) into smaller pieces for use. In some embodiments, polynucleotides synthesized according to the invention are 10 to 500 nucleotides, or 50 to 500 nucleotides, or 100 to 400 nucleotides, or 100 to 500 nucleotides in length. In some embodiments, the coupling efficiency per cycle in the synthesis of polynucleotides 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 polynucleotides in these length ranges is less than 15 minutes per cycle, or less than 10 minutes per cycle, or less than 7 minutes per cycle, or less than 5 minutes per cycle.

In some embodiments, the inkjet delivery of droplets may involve features on a substrate having dimensions directly related to its size or area, such as the width of square reaction sites or the diameter of circular reaction sites. Thus, in some embodiments, the reaction sites have a width or diameter of about 10 μm to about 1.0 cm. In some embodiments, droplets may be deposited to a reaction site having a width or diameter of about 1.0 μm to about 1.0mm, 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.

In some embodiments, the volume of reagent ink delivered to the reaction site is from 0.1 to 1000 pL, or from 0.5 to 500 pL, or from 1.0 to 250 pL, or from 1.0 to 100 pL, or from 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.

Device for inkjet synthesis

Delivery of fluids by inkjet printers is a well established technology that has been available for decades, so that a number of instructions can be found describing it and providing guidance for adapting it for new applications as in the present invention. Exemplary references ：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. patent 5474796; 10384189; 10669304; 6306; 6323043; 5847105, etc., provide guidance for constructing inkjet delivery systems. As described in Le (1999), inkjet pumps can be classified as "continuous" and "drop on demand" (DOD). In some embodiments, DOD inkjet pumps are used in the apparatus of the present invention, and in particular, piezoelectric inkjet pumps are of interest in various DOD inkjet printers. Drop formation in DOD inkjet printers is described, for example, in Dong et al Physics of Fluids, 18:072102 (2006). Such various inkjet pumps are available as banks or components (banks) of a large number of inkjet printers (e.g., tens to hundreds (10's to 100's)), which can be individually programmed to actuate and deliver droplets. Such inkjet printers and inkjet components (referred to herein as "inkjet heads") are commercially available from a number of manufacturers, including Epson, xaar, fujifilm and the like. As used herein, "inkjet pump" means a device capable of producing and ejecting droplets of a fluid. In some embodiments, an inkjet pump is a device capable of producing and ejecting fluid droplets at a predetermined rate and a predetermined uniform size. In some embodiments, the inkjet pump is capable of ejecting droplets each having about the same size of 0.1pL to 5.0nL, or 0.5pL to 1.0 nL. In some embodiments, the inkjet pump is capable of ejecting droplets at a rate of 1 to 100 kilohertz.

In some embodiments, the components of the ink jet device of the present invention may be arranged according to whether they are movable relative to each other or whether they are fixed, as shown in fig. 4A by component (602) and component (600), respectively. The computer and software (604) provide overall control of the system components, either directly or indirectly, through a controller. For example, the software may provide single pass reagent deposition, wherein the printhead (618) is stationary and the synthetic support holder (620) moves to deliver reagents to the reaction sites. Optionally, different software may provide one or more moving printheads (618) and/or moving a composite support holder (620) through various components, such as a print controller (606), a printhead driver (612), and a motion controller (610). Typically, the computer and software (604) controls the end-capping station (622), the rinsing station (624), the wiper (626), the inspection system (628), and the washing and possibly drying functions (630), if any.

The capping station (622) keeps the printhead wet and prevents drying of the ink. A rinse station (624) fills (prime) and flushes the printhead, which helps remove trapped air and debris and dried ink. The wiper (626) is used to remove excess ink and prevent cross-contamination. It may be part of a rinsing station. An inspection system (628) records the presence, absence, or size of a deposited reagent spot (spot) or an incorrectly placed reagent spot. The inspection system (628) may include a camera to capture an image of the composite support and image analysis software to extract processing information from the image. This information can be used in real time to optimize the synthesis or to implement corrective measures. The washing and drying functions (630) are performed by a fluid delivery system separate from the fluid delivery system for droplet delivery. The washing may comprise a deprotection step wherein the deprotection reagent is flowed through the synthetic substrate, optionally followed by a drying step. Drying may be achieved by blowing air or an inert gas (e.g. argon) onto the synthesis support or may be achieved by using a volatile solvent (e.g. methanol) in the washing step.

In some embodiments, a camera or microscope may be used to capture an image of the spot (i.e., the reaction site) and identify the missing spot, determine the spot size and spot location. Illumination for image capture may be from above, from the side, from below, or integrated into the substrate holder, whichever gives the best contrast in the absence or presence of dye in the ink. In the case of dyes (as described below), the dyes are selected so as not to interfere with the enzymatic reaction, not to react with the protecting groups of the nucleotides, and will be compatible with the enzyme and deprotection buffer. In some embodiments, each monomer will have a different distinguishable dye covering a different portion of the visible spectrum. In some embodiments, imaging of the array of reaction sites is performed during incubation of the extension reaction (30 seconds-10 minutes) and a sufficiently high magnification is used to observe individual spots, but not so high that scanning the array would take an undue amount of time. For standard microscope slides, the number of images taken in the imaging step may be 20 to 100. Images may be captured seamlessly in a video stream by scanning the substrate, or during a move-stop process. The captured images can be stitched using an algorithm with the aid of fiducial markers present on the slide. The fiducial markers also help determine if the slide has moved in the slide holder and help determine the spot location. In some embodiments, real-time image analysis that allows identification of missing or bad spot locations may be accompanied by automatic generation of new images and additional one or more prints.

An exemplary inkjet device for implementing various embodiments of the present invention (e.g., those of fig. 2A-2C) is shown schematically in fig. 4B. A plurality of DOD inkjet printers are housed in a printhead (680), the printhead (680) being capable of x-y and z movement relative to a drop microarray (657). In some embodiments, both the printhead (680) and the droplet microarray (657) are capable of x-y movement. In some embodiments, the printhead (680) is held in a fixed position and the array of drops (657) is moved x-y. In this example, "dATP reagent", "dCTP reagent", "dGTP reagent" and "dTTP reagent" (696) are each buffer formulations or inks 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, humectants, etc. required to meet the requirements of desired droplet formation and/or reduced evaporation losses. The printhead (680) includes a temperature regulator to maintain the ink at a temperature optimal for delivery and activity. In this embodiment, the reagents that flow or are delivered in bulk to the droplet microarray are a deprotection solution (or buffer) (695) and a wash solution (661). A droplet microarray (657) formed on a substrate (655) is located or mounted in a flow chamber (677), the flow chamber (677) comprising an inlet (652) and an outlet (653). The flow chamber (677) defines a flow path for reagents (not delivered by the printhead (680)) on the droplet microarray (657). Such reagents may flow continuously through the droplet microarray (657), or the reagents may be delivered to a flow chamber (677) where they remain for a predetermined incubation time and then removed or recycled. Such reagents may be moved over the reagent reservoir by conventional pumps or pressure heads. The flow chamber (677) includes a temperature control element (not shown) and a humidity control element (not shown) to maintain or optimize coupling reactivity. After exiting, the reagent is discarded into a waste container (656) or recycled. The timing of the inkjet discharge, positioning of the printhead (680), actuation of the valve (675) and valve (674) are controlled by a fluidics/inkjet controller (665), which fluidics/inkjet controller (665) may include imaging software that performs analysis of the array image obtained from the camera (697) and causes a change in reagent deposition, for example, when a coalescence reaction site is detected. In some embodiments, the printhead (680) may be driven by electronics available from Meteor (Meteor Inkjet Ltd, (Cambridge, UK)). For example, the print controller card (Print Controller Card, PCC) is synchronized with the encoder signals from the Thorlabs motion controller. A head driver card (HEAD DRIVER CARD, HDC) provides power and waveforms to the printheads. The drive electronics are controlled by the Meteor's digital printing front end, which includes MetDrop and MetWave software for optimizing the speckle parameters, and printing is initiated by the Thorlabs Kinesis software. The entire instrument control may be performed by instrument software (e.g., labView).

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

As described above, in some embodiments, the nozzle diameter used in the present invention may be 10 μm to 100 μm. In other embodiments, the inkjet nozzle size may be 20-30 μm for producing droplets of 10-20 pL size. In some embodiments, the nozzle diameter, synthetic reagent density, surface tension and viscosity are selected to dispense droplets to reaction sites having a volume of 2 pL to 5nL, or 2 pL to 1nL, or 2 pL to 500 pL, or 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 100 kHz.

In some embodiments, the inkjet-based synthesizer includes a drop detection assembly to monitor and record any anomalies in drop formation and delivery of the inkjet nozzles. In some embodiments, such drop monitoring may include a laser diode mounted perpendicular to the direction of printhead movement such that the stream of drops of each set of nozzles intersects the beam of light such that light scattering is caused if drops are present. Before each round of printing, the nozzles may be activated in turn by a light beam (fire), and the forward scattering of each drop is detected by a photodiode. The failed nozzle may be offline during synthesis. The apparatus of the present invention may also be equipped with a commercially available drop monitor, such as the Meteor drop monitor available from Meteor Inkjet Ltd (Cambridge, UK) and a camera for imaging the solid support and array of reaction sites. The latter allows monitoring of the array of reaction sites to detect the accuracy of droplet deposition, the size and geometry of the reaction sites, coalescence of the reaction sites, etc. In some embodiments, software may be provided to provide a complete image of an array on a slide or solid support by stitching together tiles (tiles) comprising smaller images, such as s. Preibisch, s. Saalfeld, p. Tomancak, bioinformatics, 2009, 25 (11), 1463-1465.

In certain embodiments, it may be desirable to prevent evaporation of the synthesis reagents and reaction mixture after deposition. Evaporation may be prevented in a number of different ways. In some embodiments, the synthesis cycle may be performed in a high humidity environment, such as a relative humidity of 75-85%. Alternatively or additionally, agents with evaporation retarders or wetting agents may be used, such as glycerol, polyethylene glycol, carboxymethyl cellulose, hydroxyethyl cellulose, and the like.

In some embodiments, a recirculating ink printhead is employed because this reduces problems of nozzle drying and/or clogging by enzymes. Recycled ink printheads are commercially available from, for example, fujifillm and are described in U.S. patents 8820899, 8534807, 8752946, 9144993, 9511598, 9457579, which are incorporated herein by reference.

Synthetic substrate

In some embodiments, the substrate 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 hydrophilic reaction sites, for example, after the flow of aqueous reagents or reactants across the surface. That is, in some embodiments, substrates for synthesis include so-called "drop microarrays," for example, as disclosed in the following exemplary references, which are incorporated by reference, U.S. patent 5474796; Chrisey et al, Nucleic Acids Research, 24(15): 3040-3047 (1996); Fixe et al, Materials Research Society Symposium Proceedings. Volume 723, 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 create a plurality of discrete hydrophilic regions, which can be used directly or further treated as reaction sites, for example, attachment initiators. In some embodiments, each of the plurality of discrete hydrophilic regions is surrounded by a hydrophobic region. The discrete hydrophilic regions may have various shapes, but are typically circular or rectangular or square for ease of manufacture. In some embodiments, the reaction sites have an area and capacity to accommodate an aqueous reaction mixture as described above. Although the synthetic substrates of some embodiments may include droplet microarrays, such arrays may undergo a drying step that removes liquid from the reaction sites during the synthesis process. That is, in some embodiments, a synthetic substrate comprising a droplet microarray may be free of droplets from time to time, e.g., after an extension cycle that ends in a drying step. The hydrophilic-hydrophobic configuration allows droplets to form on the surface of a droplet microarray after inkjet delivery of the synthetic reagent to the hydrophilic region or by flowing a "bulk" aqueous solution (e.g., a synthetic reagent or wash solution) over the substrate. As disclosed in the above references, the droplets retained by the hydrophilic region may be used as a reaction chamber or vessel. Such a process is shown in fig. 1B. The 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 submerged (156) by the aqueous solution (158), both the hydrophobic region (152) and the hydrophilic region (154) are submerged. As the aqueous solution (158) is discharged (160), some of the aqueous solution is retained by the hydrophilic region (154) to form droplets (162) of a droplet microarray (164). Individual droplets, such as (162), may be referred to as "microarray droplets" to distinguish them from droplets (e.g., (162)) formed by an inkjet pump prior to delivery to a reaction site.

The preparation of the substrate with discrete reaction sites can be accomplished by known methods. For example, such a method may involve creating hydrophilic reaction sites by first applying a protective agent or resist (e.g., silicon oxide or similar material) on selected areas of the substrate surface. The unprotected areas are then coated with a hydrophobic agent to create a non-reactive surface. For example, the hydrophobic coating may be produced by chemical vapor deposition of (trideceth) -triethoxysilane onto the exposed oxide surrounding the protected circle. Finally, the protectant or resist is removed using the high surface tension solvents described herein and procedures known in the art (e.g., those described by Maskos & Southern, nucleic Acids res. 20:1679-1684 (1992)) to expose the pore areas of the array for further modification and nucleoside synthesis. Alternatively, the entire surface of the glass sheet substrate may be coated with a hydrophobic material, such as 3- (1, 1-dihydroperfluorooctyloxy) propyltriethoxysilane, which is ablated at the desired site to expose the underlying silica glass. The substrate is then coated with glycidoxypropyl trimethoxysilane (glycidyloxypropyl trimethoxysilane), which reacts only with glass, and is then "treated" with hexaethylene glycol and sulfuric acid to form a linker with hydroxyl groups on which chemicals can be synthesized (Brennan, U.S. Pat. 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)).

In some embodiments, the reaction sites may be formed on the substrate following 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 set of hydrophilic molecules comprising an aminosilane are attached to the surface of a substrate to form reaction sites. Such hydrophilic molecules may include N- (3-triethoxysilylpropyl) -4-Hydroxybutyramide (HAPS), 11-acetoxyundecyltriethoxysilane, N-decyltriethoxysilane, (3-aminopropyl) trimethoxysilane, (3-aminopropyl) triethoxysilane, 3-glycidoxypropyl trimethoxysilane (GOPS), or 3-iodo-propyl trimethoxysilane. A set of hydrophobic molecules comprising fluorosilane are attached to the substrate surface in areas other than the reaction sites. Such hydrophobic molecules may include perfluorooctyltrichlorosilane, octylchlorosilane, octadecyltrichlorosilane, (tridecafluoro-1, 2-tetrahydrooctyl) trichlorosilane or (tridecafluoro-1, 2-tetrahydrooctyl) trimethoxysilane. Following this attachment, a substrate for polynucleotide synthesis is prepared by coupling an initiator to the aminosilane at the reaction site. This coupling can be accomplished using any number of available homobifunctional or heterobifunctional linkers to form a covalent bond between an amino group on the substrate and a5 '-mercapto group or 5' -amino group on the initiator. Such linkers are available, for example, from Sigma-Aldrich (St. Louis, MO) and are described in, for example, the paper by Hermanson, bioconjugate Techniques, 3 ^rd Edition (ACADEMIC PRESS, 2013). The synthesis of polynucleotides having 5 '-mercapto 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 4757141 and similar references.

In some embodiments, an array of reaction sites may be formed using click chemistry by depositing droplets of a 5' -DBCO (dibenzocyclooctyl) labeled initiator (e.g., GLEN RESEACH) under coupling conditions onto a substrate (preferably a planar substrate) comprising an azide layer (e.g., polyAn D azide slide). In some embodiments, such a reaction may be performed as a copper-free click reaction with less damage to DNA, such as Dommerholt et al, top. Curr. Chem. (Z) 374:16 (2016).

Various substrates can be used to create arrays of reaction sites for enzymatic synthesis of polynucleotides. The substrate may be a rigid material including, but not limited to, glass, fused silica, silicon, such as silicon dioxide or silicon nitride, metal, such as gold or platinum, plastic, such as polytetrafluoroethylene, polypropylene, polystyrene, polycarbonate, and any combination thereof. The rigid surface may be made of a material selected from the group consisting of silicon, polystyrene, agarose, dextran, cellulosic polymers, polyacrylamide, polydimethylsiloxane (PDMS), and glass. The substrate may also comprise a flexible material that can be bent, folded or the like 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 foils for printers, poly (methyl methacrylate) (PMMA), methacrylate copolymers, styrene polymers, high refractive index polymers, fluoropolymers, polyethersulfones, polyimides containing cycloaliphatic structures, rubber, fabrics, metal foils, and any combination thereof.

In some embodiments, patterned surfaces of superhydrophobic and superhydrophilic regions can be formed on a substrate. Guidance in forming a droplet microarray having such a patterned surface 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).

Achieving precise alignment of droplet delivery with the reaction sites of the pre-fabricated droplet microarray is an important aspect of inkjet-assisted synthesis of polynucleotides. In some embodiments, such alignment tasks may be minimized or avoided by creating an array of reaction sites by depositing droplets of synthesis reagents onto an initiator oligonucleotide layer on a substrate immediately prior to synthesis to define the location of the reaction sites. After such initial deposition of the droplets, the initiator layer outside the droplet-defining sites is treated to render them inert to subsequent elongation, or to render them inert to elongation and to render them hydrophobic. After such initial surface treatment to create reaction sites, inkjet delivery of further or subsequent droplets to the same reaction sites will be accurate, as the same inkjet head and pump used to define the reaction site locations will be used to deliver subsequent droplets during synthesis of the polynucleotide. In some embodiments, the synthesis reagent delivered to the initiator layer comprises a mixture of a template-free polymerase and 3' -O protected dntps. These reagents extend the initiator to define a reaction site or region on the oligonucleotide layer that is occupied by an extension fragment having a 3' -O protecting end. The areas outside these areas are then treated to render them inert to elongation. In some embodiments, after an initial coupling step to define the reaction site, the entire substrate is exposed to a template-free polymerase and a terminator, such as dideoxynucleoside triphosphate (ddNTP) or similar reagent. In some embodiments, such ddNTP may be, for example, a ddNTP conjugated to a hydrophobic moiety, thereby rendering the coating outside the reaction site hydrophobic. Such hydrophobic moieties may be, for example, dye or quencher molecules, such as Black Hole Quencher molecules. Various terminators may be employed for this purpose. In particular, the terminator includes nucleoside triphosphates without a 3' -hydroxy substituent and includes 2',3' -dideoxyribonucleoside, and 2',3' -dideoxy-3 ' -haloribonucleosides, such as 3' -deoxy-3 ' -fluororibonucleosides or 2',3' -dideoxy-3 ' -fluororibonucleosides. Alternatively, ribofuranosyl analogs may 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 ：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, nucleic acids & nucleic acids, 7:613-617 (1988) are disclosed in the following references. Nucleotide terminators also include reversible nucleotide terminators, e.g., metzker et al Nucleic Acids Res., 22 (20): 4259 (1994).

Thus, in such embodiments, the starting material for the synthesis operation is a surface coated with a layer of initiator oligonucleotides. An exemplary fabrication of reactive sites on such starting materials is shown in fig. 3. The substrate (400) (e.g., a slide) has a layer (402) of initiator oligonucleotides having free 3 '-hydroxyl groups and attached to the substrate by their 5' -ends. In some embodiments, the initiator density may be, for example, 10 ¹¹ to 10 ¹³ chains/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 may be unprotected, and the droplet may contain a template-free polymerase and an initial 3' -O protected nucleoside triphosphate, thereby producing a 3' -O protected extension fragment at each reaction site. After such deposition, the initiator layer (402) is immersed and incubated (409) in a buffer (408) comprising a template-free polymerase and a terminator (e.g., as described above) to produce a droplet microarray (414), the droplet microarray (414) having a surface (410) outside of the reaction sites (e.g., 412), the surface (410) being inert to elongation or inert to elongation and hydrophobic, depending on the terminator selected.

Embodiments of the invention for synthesizing a plurality of polynucleotides employing reactive site formation as depicted in FIG. 3 may be performed by (a) providing a substrate, preferably a planar substrate, to which an initiator layer has been attached, wherein each initiator has a free 3' -hydroxyl group, (b) dispensing one or more droplets by one or more inkjet pumps to each of a plurality of sites on the initiator layer to define an array of reactive sites, wherein each droplet comprises a buffer solution comprising a mixture of template-free polymerase and 3' -O blocked dATP, 3' -O blocked dGTP or 3' -O blocked dTTP, and wherein each polynucleotide of the plurality of polynucleotides is assigned to a different reactive site for synthesis, (c) capping the free 3' -hydroxyl group of the initiator outside the reactive site, (d) dispensing by one or more inkjet pumps a buffer solution comprising a mixture of template-free polymerase and 3' -O blocked dATP, 3' -O blocked dGTP or 3' -O blocked dTTP to each of the reactive sites depending on the type of the template-free polymerase and 3' -O blocked dNTP, extending the primer at each of the pre-assigned reactive sites by a pre-determined sequence of the template-extended dNTPs, to form 3'-O blocked extension fragments, (f) deblocking the extension fragments at each reaction site by treatment of the planar support with a deblocking agent to form extension fragments having free 3' -hydroxyl groups, (g) repeating steps (d), (e) and (f) until a plurality of polynucleotides are synthesized.

One aspect of the invention is a method of preparing a reaction site array for template-free enzymatic synthesis of a plurality of polynucleotides. In some embodiments, such an array preparation method can be performed by (a) providing a surface to which an initiator is attached, (b) delivering droplets to a plurality of different locations on the surface with one or more inkjet pumps to form a plurality of reaction sites, the droplets comprising a synthetic reagent in the reaction sites that reacts with the initiator to remove 3'-O protecting groups or extend such initiator by adding 3' -O protected nucleoside triphosphates, and (c) capping the initiator on the surface outside the reaction sites. 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 a template-free polymerase and a 3' -O protected nucleoside triphosphate, such that the template-free polymerase catalyzes the addition of the 3'-O protected nucleoside triphosphate to produce a 3' -O protected extension fragment within the reaction site. Thus, the initiator outside the reaction site may be capped by immersing the surface in a capping reagent (e.g., a mixture comprising dideoxynucleoside triphosphates and a template-free polymerase). In some embodiments, the initiator on the surface may have a 3'-O protecting group, and the synthetic reagent delivered by the droplet may comprise a deprotecting agent that removes the 3' -O protecting group from the initiator to form a reactive site. In the newly formed reaction site, a reagent containing a 3' -O protected nucleoside triphosphate and a template-free polymerase is delivered, wherein the protecting group of the delivered nucleoside triphosphate is orthogonal to the protecting group of the surface initiator. Exemplary orthogonal 3' -O protecting groups are described below. For example, such orthogonal protecting groups may be azidomethyl and amino.

One of ordinary skill will appreciate that similar reaction site formation may be implemented for other embodiments, such as those depicted in fig. 2B and 2C. The skilled artisan will also appreciate that the optional steps described for the embodiments of fig. 2A-2C (e.g., washing, drying, treatment with protease, etc.) may also be implemented in the embodiments described above (including those of fig. 3).

In another embodiment, the initiation layer of the initiator oligonucleotide has all 3 '-O-amino protected or 3' -O-azidomethyl protected ends. The method steps in this embodiment are similar to those of fig. 3, except that the deprotection buffer is ink-jet printed on the substrate to define the reaction sites as discrete regions of initiator with free 3' -hydroxyl groups. Following this selective deprotection, the surface is treated with an aqueous solution of an aldehyde or ketone to form a stable, non-extendable hydrophilic or hydrophobic 3' -oxime. The aldehyde or ketone may be water-soluble, such as acetone, or slightly water-soluble and hydrophobic (e.g., valeraldehyde, aldehyde-PEG-DBCO, etc.) or very hydrophobic and water-insoluble (e.g., heptanal).

In another embodiment, a buffer comprising a template-free polymerase/3 ' -O protected dNTP mixture is printed on an initiator oligonucleotide layer having a free 3' -hydroxyl group as described above to define a reaction site for an extended initiator having a terminal end with 3' -O protection. The surface outside these defined sites is then treated with a template-free polymerase and azide or alkyne-derived ddNTP to block further 3' extension. Hydrophobic molecules with complementary click chemistry groups (e.g., DBCO, benzyl azide) can then be reacted with the ddNTP terminator to render the surface outside of the reaction site hydrophobic. Exemplary click chemistry pairs are described in Feng et al, adv, mate, interfaces, 1400269 (2014).

In another embodiment, for a substrate surface without a layer of initiator oligonucleotides, a buffer comprising initiator oligonucleotides having 5 'linker groups is ink-jet printed on a surface derivatized with complementary reactive groups (e.g., epoxy, azide/alkyne) such that the initiators are attached to the surface through their 5' ends. For these attached initiators, a coupling reaction cycle may be performed according to the present invention. In addition, unreacted complementary reactive groups can be quenched by reacting them with inert groups (e.g., ethanolamine for epoxy resins), and inert groups with hydrophobic character can be selected.

Template-free enzymatic synthesis method

Typically, a template-free (or equivalently, "template-independent") enzymatic polynucleotide synthesis method comprises repeated cycles of steps, for example as shown in fig. 1A, wherein in each cycle a predetermined nucleotide is coupled to an initiator or growing chain. General elements of templeless enzymatic synthesis are described in Champion et al, U.S. Pat. No. 10752887, ybert et al, international patent publication WO/2015/15923, ybert et al, international patent publication WO/2017/216472, godron et al, international patent publication WO/2020/120442, hyman, U.S. Pat. No. 5436143, HIATT ET AL, U.S. Pat. No. 5763594; 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).

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 elongation activity of the template-free polymerase (in the case of template-free polymerase inks), and (ii) the need to meet the rheological requirements of droplet formation. Key solution parameters that affect drop formation by an inkjet printer are viscosity, surface tension, liquid density, and inkjet nozzle diameter. For certain embodiments of the present invention, synthetic reagents prepared for non-droplet delivery to the reaction mixture may be reconstituted by the addition of viscosity modifiers, surface tension modifiers, density modifiers, and the like, to form a "printable ink" that may be delivered in droplets produced by an inkjet pump. By "printable" with respect to a reagent ink is meant that repeatable droplets can be ejected from the nozzle at a uniform velocity and volume, and without satellite droplets.

As shown in fig. 1A, an initiator polynucleotide (100) having a free 3' -hydroxyl group (130), for example, attached to a synthetic support (120), is provided. 3' -O protected dNTPs and a template-free polymerase, such as a terminal deoxynucleotidyl transferase (TdT) or a variant thereof, are added to the initiator polynucleotide (100) (or extended initiator polynucleotide in a subsequent cycle) under conditions effective (140) for enzymatic incorporation of the 3' -O protected dNTPs to the 3' end of the initiator polynucleotide (100) (or extended initiator polynucleotide) (e.g., ybert et al, WO/2017/216472; champion et al, WO 2019/135007). The reaction produces an extended initiator polynucleotide (160) with the 3' -hydroxyl protected. If the extension sequence is incomplete, another addition cycle is performed (180). If the extended initiator polynucleotide contains competing sequences, 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 cleavable nucleotides at predetermined positions within the original initiator polynucleotide. An exemplary cleavable nucleotide may be a uracil nucleotide cleaved by uracil DNA glycosylase. According to some embodiments of the invention, the cleavage agent may be delivered to the reaction site by droplets produced by an inkjet pump. In such embodiments, a known incomplete or defective polynucleotide at a reaction site may be separated from a fully competing polynucleotide, or may 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.

If the extended initiator polynucleotide is not the complete sequence (i.e., the end product), the 3'-O protecting group is removed to expose the free 3' -hydroxyl group (130), and the extended initiator polynucleotide is subjected to another cycle of nucleotide addition and deprotection.

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

In some embodiments, the initiator may comprise a non-nucleic acid compound having a free hydroxyl group to which TdT may couple a 3' -O protected dNTP, e.g., baiga, U.S. patent publications US2019/0078065 and US2019/0078126.

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

Various cleavable linkages or cleavable nucleotides may be used for this purpose. In some embodiments, cleavage of the desired polynucleotide leaves a native free 5 '-hydroxyl group on the cleaved strand, however, in alternative embodiments, the cleavage step may leave a moiety, such as 5' -phosphate, that may be removed in a subsequent step, such as by phosphatase treatment. The cleavage step may be performed chemically, thermally, enzymatically or photochemically. In some embodiments, the cleavable nucleotide may be a nucleotide analog, such as deoxyuridine or 8-oxo-deoxyguanosine, which is recognized by a specific glycosylase (e.g., uracil deoxyglycosylase followed by endonuclease VIII, and 8-oxo-guanine DNA glycosylase, respectively). In some embodiments, cleavage may be achieved by providing the initiator with deoxyinosine (which may be cleaved at the 3' end of the initiator by endonuclease V, leaving a 5' -phosphate on the released polynucleotide) as the penultimate 3' nucleotide. In some embodiments, the initiator may contain a terminal uridine such that after synthesis, the desired polynucleotide may 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, incorporated by reference, U.S. patent nos. 5739386, 5700642, and 5830655 and U.S. patent disclosures No. 2003/0186226 and 2004/0106728, and in the Urdea and Horn, U.S. patent No. 5367066.

Returning to fig. 1A, in some embodiments, in each synthesis step, nucleotides of the ordered sequence are coupled to the initiator nucleic acid in the presence of 3' -O protected dntps using a template-free polymerase, such as TdT. In some embodiments, a method of synthesizing an oligonucleotide comprises the steps of (a) providing an initiator having a free 3' -hydroxyl group, (b) reacting the initiator or extended intermediate having a free 3' -hydroxyl group with a template-free polymerase under extended (or extended) conditions in the presence of a 3' -O protected nucleoside triphosphate to produce a 3' -O protected extended intermediate, (c) deprotecting the extended intermediate to produce an extended 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 fragment" 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 to catalyze an extension reaction in which a 3'-O protected nucleoside triphosphate monomer is coupled (by formation of a phosphodiester linkage) with the free 3' -hydroxyl of a nucleic acid fragment, which may be, for example, an initiator or an extension fragment. Exemplary extension conditions include the choice of reaction temperature, reaction duration, pH, concentration of various salts, scavengers of unwanted reaction components, reagents to reduce secondary structure of the nucleic acid, and the like. In some embodiments, the initiator is provided as an oligonucleotide attached to (e.g., via its 5' end) a solid support. The above method may further comprise a washing step after the reacting or extending step and after the deprotecting step. For example, the reaction step may comprise the sub-step of removing unincorporated nucleoside triphosphates after a predetermined incubation period or reaction time, e.g., by washing. In some embodiments, such predetermined incubation period or reaction time may be 30 seconds to 30 minutes, or 1 minute to 15 minutes, or 1 minute to 10 minutes, or 30 seconds to 5 minutes.

In some embodiments, after the synthesis cycle of fig. 1A is completed, further steps may be performed to cleave the completed polynucleotide from the solid support. Such further steps may be performed at the reaction sites of the array. Additionally, some cleavage methods may result in the released product still requiring modification to convert it into a usable product. For example, in "endonuclease V-inosine" cleavage (described below), 5' -phosphate remains that must be removed for some applications. Thus, a further step of phosphatase treatment may be required.

When the predetermined sequence of the polynucleotide on the synthetic support comprises a reverse complement sub-sequence, a secondary intramolecular or intermolecular structure may be generated by the formation of hydrogen bonds between the reverse complement regions. In some embodiments, the base protecting moiety for the exocyclic amine is selected such that the hydrogen of the protected nitrogen cannot participate in hydrogen bonding, thereby preventing the formation of such secondary structures. That is, base protecting moieties may be used to prevent the formation of hydrogen bonds, such as in normal base pairing, e.g., between nucleosides A and T and between nucleosides G and C. At the end of the synthesis, the base protecting moiety may be removed and the polynucleotide product may be cleaved from the solid support, for example by cleavage thereof from its initiator.

In addition to providing 3' -O blocked dNTP monomers with base protecting groups, extension reactions can be performed at higher temperatures using thermostable template-free polymerases. For example, a thermostable template-free polymerase that is active above 40 ℃, or in some embodiments, a thermostable template-free polymerase that is active at 40-85 ℃, or in some embodiments, a thermostable template-free polymerase that is active at 40-65 ℃, may be used.

In some embodiments, the extension conditions may include adding a solvent to the extension reaction mixture that inhibits hydrogen bonding or base stacking. Such solvents include water-miscible solvents having a low dielectric constant, such as dimethyl sulfoxide (DMSO), methanol, and the like. Also, in some embodiments, the extension conditions may include providing a chaotropic agent including, but not limited to, n-butanol, ethanol, guanidine chloride, lithium perchlorate, lithium acetate, magnesium chloride, phenol, 2-propanol, sodium dodecyl sulfate, thiourea, urea, and the like. 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 structures, wherein such proteins include, but are not limited to, single-chain binding proteins, helicases, DNA ethyleneglycol enzymes (DNA glycolase), and the like.

When base protected dntps are used, the above-described method of fig. 1A 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 ammonia.

The above method may further comprise a capping step and a washing step after the reacting or extending step and after the deprotecting step. As described above, in some embodiments, a capping step may be included in which the unextended free 3' -hydroxy group is reacted with a compound that prevents any further extension of the capping chain. In some embodiments, such a compound may be a dideoxynucleoside triphosphate. In other embodiments, the unextended strand with the free 3 '-hydroxy group can be degraded by treatment with 3' -exonuclease activity (e.g., exo I). See, for example, hysman, U.S. patent 5436143. Also, in some embodiments, chains that have not been deblocked may be treated to remove the chains or render them inert to further elongation. When using capping agents (e.g., ddNTP), buffers or synthetic reagents containing such agents may be delivered by flowing or spraying such agents onto a substrate containing the reaction sites.

In some embodiments, the reaction conditions for the extension step (sometimes also referred to as the extension step or coupling step) may include 2.0. Mu.M purified TdT, 125-600. Mu.M 3'-O blocked dNTPs (e.g., 3' -O-NH ₂ blocked dNTPs), about 10 to about 500mM potassium dimethylarsinate buffer (pH 6.5-7.5), and about 0.01 to about 10mM divalent cations (e.g., COCl ₂ or MnCl ₂), where the extension reaction may be performed at a temperature of RT to 45℃for 3 minutes. It will be appreciated that whenever the aforementioned coupling agent is delivered by ink jet generated droplets, its viscosity, density and surface tension must be adjusted to render it printable ink. In this regard, the present invention includes, in part, the recognition and understanding that inks for delivering TdT to a reaction site can have a viscosity modified for droplet formation and an activity preserved by selection of a viscosity modifier, for example, when carboxymethyl cellulose is selected as the viscosity modifier.

In embodiments where the 3'-O blocked dNTPs are 3' -O-NH ₂ -blocked dNTPs, the reaction conditions for the deblocking step may include 700mM NaNO ₂, 1M sodium acetate (adjusted to pH 4.8-6.5 with acetic acid), where the deblocking reaction may be carried out at a temperature of RT to 45℃for 30 seconds to several minutes. Washing may be performed with a cacodylate buffer that does not have components of the coupling reaction (e.g., enzyme, monomer, divalent cation). If the above reagent composition is delivered to the reaction site by inkjet delivery, it will be appreciated that the composition will be altered to meet the rheological requirements of droplet formation by the nozzles of the inkjet printheads used.

In some embodiments, RNA synthesis may be accomplished by similar steps as described above, but using template-free polymerases and monomers specifically selected for RNA synthesis, such as polyA polymerase (PAP), polyA polymerase (PUP), etc., for example international patent publication WO2020/077227. For example, the system of the present invention, The apparatus and kit may perform a method of synthesizing a polyribonucleotide having a predetermined sequence comprising the steps of a) providing an initiator having a 3' -terminal nucleotide with a free 3' -hydroxyl group, and b) repeating the following cycles until the polyribonucleotide is formed, (i) contacting the initiator or extension fragment 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 fragment extends by incorporation of the 3' -O-blocked nucleoside triphosphate to form a 3' -O-blocked extension fragment, and (ii) deblocking the extension fragment to form an extension fragment having a free 3' -hydroxyl group, wherein the template-free polymerase is a poly (A) polymerase (PAP) or a poly (U) polymerase. in further embodiments, the initiator may be attached to a support, which may be a solid support, via the 5' end, and the above-described method may include the step of cleaving the polynucleotide from the initiator. In some embodiments, the reaction conditions for the extension or elongation step using PAP or PUP may include reaction condition 1 (for primer +AM-rATP) of 250. Mu.M AM-rATP, 0.1. Mu.M ATTO488- (rA) 5,1. Mu.M PAP, 1 xATP buffer (20 mM Tris-HCl, 0.6mM MnCl ₂, 0.02mM EDTA, 0.1% BSA, 10% glycerol, 100mM imidazole, pH 7-8), 37 ℃,30 minutes. Reaction conditions 2 (for primer +AM-rGTP) 250. Mu. MrGTP, 0.1. Mu.M ATTO488- (rA) 5, 1. Mu.M PAP, 1 XGTP buffer (0.6 mM MnCl ₂, 0.1% BSA, 10mM imidazole, pH 6), 37℃for 30 min. In the above, "AM-rNTP" refers to 3' -azidomethyl-O-ribonucleoside triphosphates. Many of the 3' -O blocked rNTPs used 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. Pat. No. 7057026, international patent publications WO2004/005667, WO91/06678;Canard et al, gene (); Metzker et al, Nucleic Acids Research, 22: 4259-4267 (1994);Meng et al, J. Org. Chem., 14: 3248-3252 (3006); U.S. patent publication 2005/037991, zavgorodny et al, tetrahedron Letters, 32 (51): 7593-7596 (1991) cited above. In a further specific embodiment, the 3' -blocked nucleotide triphosphate is blocked with a 3' -O-propargyl, 3' -O-azidomethyl, 3' -O-NH ₂ or 3' -O-allyl group. In other embodiments, the 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-t-butoxyethoxy, 3' -O- (2-cyanoethyl) and 3' -O-propargyl. As described above, if the above reagent composition is delivered to the reaction site by inkjet delivery, it will be appreciated that the composition will be altered to meet the rheological requirements of droplet formation by the nozzles of the inkjet printheads used.

3' -O protected nucleoside triphosphates

Depending on the particular application, the deblocking and/or cleavage step may include various chemical or physical conditions, such as light, heat, pH, the presence of a particular agent, such as an enzyme, capable of cleaving a specified chemical bond. Guidance in the selection of 3' -O blocking groups and corresponding deblocking conditions can be found in the following references, incorporated by reference herein, benner, U.S. Pat. Nos. 7544794 and 8212020, U.S. Pat. No. 5808045, U.S. Pat. No. 8808988, international patent publication WO91/06678, and the references cited below. In some embodiments, the cleavage agent (also sometimes referred to as a deblocking agent or deblocking agent) is a chemical cleavage agent, such as, for example, dithiothreitol (DTT). In alternative embodiments, the cleavage agent may be an enzymatic cleavage agent, such as, for example, a phosphatase, which may cleave the 3' -phosphate blocking group. Those skilled in the art will appreciate that the choice of 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 solid support, etc. (which necessitates gentle treatment). For example, phosphines, such as tris (2-carboxyethyl) phosphine (TCEP), may be used to cleave 3' o-azidomethyl groups, palladium complexes may be used to cleave 3' o-allyl groups, or sodium nitrite may be used to cleave 3' o-amino groups. In particular embodiments, the cleavage reaction involves TCEP, palladium complex or sodium nitrite.

As described above, in some embodiments, it is desirable to employ two or more blocking groups that can be removed using orthogonal deblocking conditions. The following exemplary blocking group pairs may be used in parallel synthesis embodiments. It will be appreciated that other pairs or groups containing more than two blocking groups may be used in these embodiments of the invention.

In some embodiments, removal of a particular enzymatically removable blocking group requires a particular enzyme. For example, an esterase (e.g., acetyl esterase) or similar enzyme may be used to remove ester-or acyl-based blocking groups, and a 3' phosphatase (e.g., T4 polynucleotide kinase) may be used to remove phosphate blocking groups. For example, 3' -O-phosphate can be removed by treatment with a solution of 100mM Tris-HCl (pH 6.5), 10mM MgCl ₂, 5mM 2-mercaptoethanol, and one unit of T4 polynucleotide kinase. The reaction was carried out at a temperature of 37 ℃ for 1 minute. As described above, if the aforementioned composition is delivered to the reaction site by inkjet delivery, it will be appreciated that the composition will be altered to meet the rheological requirements of droplet formation by the nozzles of the inkjet printheads used.

Other examples of synthesis and enzymatic deprotection of 3 '-O-ester protected dNTPs or 3' -O-phosphate protected dNTPs are described in International patent publication WO1991/006678 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, .

In some embodiments, the modified nucleotide comprises a modified nucleotide or nucleoside molecule 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 is attached to a group of the structure:

-O-Z

wherein-Z is any one of-C (R ') ₂-O-R"、-C(R')₂-N(R")₂、-C(R')₂-N(H)R"、-C(R')₂ -S-R ' and-C (R ') ₂ -F, wherein each R ' is or is part of a removable protecting group; each R ' is independently a hydrogen atom, an alkyl group, a substituted alkyl group, an arylalkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, a heterocyclic group, an acyl group, a cyano group, an alkoxy group, an aryloxy group, a heteroaryloxy group, or an amido group, or a detectable label attached via a linking group, 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 '" may be the same or different and is selected from the group consisting of a hydrogen atom and a halogen atom, and an alkyl group, provided that in some embodiments the alkyl group of each R ' "has from 1 to 3 carbon atoms, and wherein the molecules may react to produce an intermediate, wherein 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 dissociate under aqueous conditions to provide a group having a free 3' -OH, and in the case where two R ' S are not R ' and R ' is H. In certain embodiments, R' of the modified nucleotide or nucleoside is alkyl or substituted alkyl, provided that such alkyl or substituted alkyl has from 1 to 10 carbon atoms and from 0 to 4 oxygen or nitrogen heteroatoms. In certain embodiments, the modified nucleotide or nucleoside has the formula-Z of-C (R') ₂ -N3. In certain embodiments, Z is an azidomethyl group.

In some embodiments, Z is a cleavable organic moiety with or without a heteroatom having a molecular weight of 200 or less. In other embodiments, Z is a cleavable organic moiety with or without a heteroatom having a molecular weight of 100 or less. In other embodiments, Z is a cleavable organic moiety with or without a heteroatom having a molecular weight of 50 or less. In some embodiments, Z is an enzymatically cleavable organic moiety with or without a heteroatom having a molecular weight of 200 or less. In other embodiments, Z is an enzymatically cleavable organic moiety with or without a heteroatom having a molecular weight of 100 or less. In other embodiments, Z is an enzymatically cleavable organic moiety with or without a heteroatom having a molecular weight of 50 or less. In other embodiments, Z is an enzymatically cleavable ester group having a molecular weight of 200 or less. In other embodiments, Z is a phosphate group that can be removed by a 3' -phosphatase. In some embodiments, one or more of the following 3' -phosphatases may be used according to manufacturer recommended protocols, T4 polynucleotide kinase, calf intestinal alkaline phosphatase, recombinant shrimp alkaline phosphatase (e.g., available from NEW ENGLAND Biolabs, beverly, mass.).

In some embodiments, the 3 '-blocked nucleotide triphosphate is blocked with a 3' -O-azidomethyl, 3'-O-NH ₂, or 3' -O-allyl group.

In some embodiments, the 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-t-butoxyethoxy, 3' -O- (2-cyanoethyl) and 3' -O-propargyl.

The 3'-O blocked dNTPs without base protection can be purchased from commercial suppliers or synthesized using published techniques, for example, U.S. Pat. No. 7057026;Guo et al, proc. Natl. Acad. Sci., 105 (27): 9145-9150 (2008); benner, U.S. Pat. Nos. 7544794 and 8212020; international patent publication WO2004/005667, WO91/06678;Canard et al, gene (cited herein as );Metzker et al, Nucleic Acids Research, 22: 4259-4267 (1994);Meng et al, J. Org. Chem., 14: 3248-3252 (2006); U.S. Pat. publication 2005/037991. 3' -O blocked dNTPs with base protection can be synthesized as follows).

Template-free polymerase for polynucleotide synthesis

A variety of different template-free polymerases can be used in the methods of the invention. Template-free polymerases include, but are not limited to, the polX family of polymerases (including DNA polymerases β, λ, and μ), poly (A) polymerase (PAP), poly (U) polymerase (PUP), DNA polymerase θ, and the like, as described, for example, in reference 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 variants thereof are useful in template-free DNA synthesis.

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 10435676 (incorporated by reference).

Template-free polymerase ink

As described above, the synthetic reagents delivered by the inkjet pump must be formulated to maintain the activity of the synthetic reagents and meet the rheological requirements of droplet formation, which can be done through the use of variable viscosity modifiers. Such formulations are referred to herein as "inks". For example, meeting the first constraint (activity) may require that the template-free polymerase be present in the reaction mixture at some minimum concentration. However, due to the high protein viscosity, the concentration of the desired activity may prevent the second constraint, i.e. the droplet formation ability, from being met. In such a case, embodiments of the invention may require delivery of multiple droplets each having a lower concentration of polymerase, which in combination with evaporation, allows for the production of a desired level of polymerase concentration for activity. Such an embodiment is shown in fig. 2C.

Also as described above, key solution parameters affecting drop formation by an inkjet printer are viscosity, surface tension, density and diameter of the inkjet nozzles, which are related by the formula z= [ (]a)^(0.5)]/WhereinIs the density of the fluid and,Is the surface tension force of the surface of the plastic film,Is viscosity, a is the radius of the inkjet pump nozzle, and Z is 1 to 10 to reliably form droplets, such as Derby, j. This relationship applies to any synthetic reagent delivered by inkjet-generated droplets comprising (i) a template-free polymerase in its coupling buffer, (ii) a mixture of 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 apply this relationship by adjusting reactant densities, viscosity modifiers, surface tension modifiers, etc., to determine ink compositions capable of forming the desired droplets for a particular embodiment.

Thus, for certain embodiments of the present invention, synthetic reagents prepared for non-droplet delivery to the reaction mixture may be reconstituted by the addition of viscosity modifiers, surface tension modifiers, density modifiers, and the like, so as to form a "printable ink" that may be delivered in droplets produced by an inkjet pump. By "printable" with respect to a reagent ink is meant that repeatable droplets can be ejected from the nozzle at a uniform velocity and volume, and without satellite droplets.

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, polymerase delivery may 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, e.g., 10-100 pL. In some embodiments, the plurality of droplets delivered is 2 to 10, or 2 to 5, or 2 to 3. In other embodiments, the plurality of droplets may be 2 to 150, or 10 to 120. In some embodiments, each time the template-free polymerase is TdT, the plurality of droplets is the number required to achieve a concentration of TdT in the reaction mixture at the reaction site of 1 μm to 30 μm or a value of 2 μm to 20 μm. In some embodiments, the concentration of TdT in the ink is a concentration that produces about a 1:1 stoichiometry between the TdT molecule and the polynucleotide at the reaction site. In other embodiments, the concentration of TdT in the ink is a concentration that produces 1:1 or greater stoichiometry between the TdT molecule and the polynucleotide at the reaction site.

In some embodiments, the 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 such TdT in a buffer suitable for coupling activity is from 1. Mu.M to 20. Mu.M/mg. In some embodiments, such buffers comprise about 10 to about 500mM potassium dimethylarsinate buffer (pH 6.5 to 7.5) and about 0.01 to about 10mM divalent cations (e.g., COCl ₂ or MnCl ₂). In some embodiments, the extension reaction buffer is an acetate buffer, e.g., 0.1M acetate, 0.5M nacl, ph4.5.

In some embodiments, an immutable viscosity modifier may be used with a variable viscosity modifier. Such non-variable viscosity modifiers may be selected from the group consisting of ethylene glycol, polyethylene glycols of varying molecular weights, polyethylene glycol methyl ether, polyethylene glycol dimethyl ether, poly (vinyl alcohol), carboxymethyl cellulose and hydroxyethyl cellulose.

In some embodiments, a printable template-free polymerase ink, such as TdT ink, includes a surface tension modifier in addition to the viscosity modifier. Such a surface tension modifier may be a detergent. The detergent may be selected from Tween 20, triton X-100, CHAPS, NP-40, octyl thioglucoside, 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).

In some embodiments, the 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 such TdT in a buffer suitable for coupling activity is 1. Mu.M-50. Mu.M/mg, or 1. Mu.M-20. Mu.M/mg. In some embodiments, such buffers comprise about 10 to about 500mM potassium dimethylarsinate buffer (pH 6.5 to 7.5) and about 0.01 to about 10mM divalent cations (e.g., COCl ₂ or MnCl ₂), the concentration of such 3' -O protected dNTPs being 125-600. Mu.M. In some embodiments, the variable viscosity modifier is selected from the group consisting of uncrosslinked poly (N-alkyl substituted-acrylamides) and poly (ethylene oxide) -poly (propylene oxide) -poly (ethylene oxide) triblock polymers. In some embodiments, the printable TdT ink further comprises a surface tension modifier in addition to the variable viscosity modifier. Such a surface tension modifier may be a detergent. The detergent may be selected from Triton X-100, CHAPS, NP-40, octyl thioglucoside, octyl glucoside or dodecyl maltoside. Of particular interest are Tween 20 and Triton X-100.

In some embodiments, the printable ink comprising a template-free polymerase (e.g., tdT variant) comprises a humectant for reducing droplet evaporation. Suitable humectants include, but are not limited to, glycerin, alcohol sugars, ethylhexyl glycerin, panthenol, sorbitol, xylitol, maltitol, propylene glycol, hexylene glycol, butylene glycol, sodium lactate, hyaluronic acid, and polydextrose.

In some embodiments, the TdT inks of the present invention are delivered as droplets of 1 pL-200 pL, or 1 pL-100 pL, or 1 pL-50 pL.

In some embodiments, the invention relates to a terminal deoxynucleotidyl transferase (TdT) composition comprising droplets of an aqueous solution having a volume of 2 pL-5nL and comprising (i) TdT or 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 10mM, and a variable viscosity modifier. In some embodiments, the divalent cation is cobalt or manganese, and such compositions further comprise a surface tension modifier.

In some embodiments, such variable viscosity modifiers are selected from uncrosslinked poly (N-alkyl substituted-acrylamides) and poly (ethylene oxide) -poly (propylene oxide) -poly (ethylene oxide) triblock polymers. In some embodiments, each time a printable template-free polymerase ink (e.g., a printable TdT ink) comprises a 3' o-amino nucleotide, 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 a 3'-O protected 2' -deoxynucleoside triphosphate monomer at a concentration of 100-1000. Mu.M or 125-600. Mu.M.

In some embodiments, the variable viscosity modifier may comprise a polymer solution that gels upon cooling. Such polymers are selected from at least one of hydrophobically modified polymers having UCST-type phase behavior, natural polymers, diblock copolymer brush grafted silica nanoparticles, 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 interactions.

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

In some embodiments of the foregoing compositions, the variable viscosity modifier is selected from the group consisting of uncrosslinked poly (N-alkyl substituted-acrylamides) and poly (ethylene oxide) -poly (propylene oxide) -poly (ethylene oxide) triblock polymers. In some embodiments, the 3'-O protected 2' -deoxynucleoside triphosphate of the foregoing composition 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 foregoing compositions, the 3' -O protecting group is selected from the group consisting of 3' -O-methyl, 3' -O- (2-nitrobenzyl), 3' -O-allyl, 3' -O-amine, 3' -O-azidomethyl, 3' -O-t-butoxyethoxy, 3' -O- (2-cyanoethyl), and 3' -O-propargyl.

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 pseudo-capping of the 3' -amine by reacting with extraneous (adventitious) aldehydes or ketones (e.g., formaldehyde) that are prevalent in the environment (spurious capping). This is a particular problem for inkjet synthesis because the droplets of ink have a very high surface area to volume ratio, which enhances the absorption of ambient aldehydes. Thus, in embodiments of the invention that use 3' -O-amino-dNTP monomers, the template-free polymerase ink as described above further comprises 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 pseudo-end-capped polynucleotide 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 reagent that reacts with a compound having a chemical group of formula R-C (=o) H or R ¹-C(=O)-R², wherein R, R ¹ and R ² are typically alkyl or aryl groups. More specifically, in some embodiments, the aldehyde scavenger is a reagent that reacts with the R-C (=o) H or R ¹-C(=O)-R² groups on the compound at a sufficiently high rate that such compound does not react (or reacts only negligibly) with the 3 '-amine groups of the 3' -O-amino-nucleotide. As used herein, the term "scavenger" means a chemical substance added to a mixture to remove or deactivate impurities or compounds that result in undesired reaction products. In various embodiments, the aldehyde scavenger may be in solution, immobilized on a material for storage or synthesis, or coupled with a reagent used in the methods of the invention, e.g., a template-free polymerase, such as TdT.

As described above, the enzymatic synthesis may be performed using various reagents (referred to herein as "synthesis reagents") that may contain or consist of reactants, wash solutions, deprotection buffers, enzymes, and the like. (the term "synthesis reagent" means any reagent used in the synthesis cycle to couple monomers, particularly 3 '-O-amino-nucleoside triphosphates, to an initiator or extension fragment, such as a buffer comprising a template-free polymerase, a buffer comprising 3' -O protected nucleotide monomers, a deprotected (or deblocked) buffer, etc.) 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, an aldehyde scavenger is added to the reaction mixture as a component of a synthesis reagent comprising a template-free polymerase.

In some embodiments, for example using Sudo et al, U.S. patent publication No. 2020/0061225 or listed in fig. 8A-8B, the effective amount is provided by a concentration of 1 to 500mM, or in other embodiments, by a concentration of 1 to 200mM, or in other embodiments, by a concentration of 1 to 100 mM.

In some embodiments, the aldehyde scavenger used in the present invention comprises an O-substituted hydroxylamine or a polyhydroxy amine. In some embodiments, the O-substituted hydroxylamine used in the present invention is defined by the formula:

R¹-ONH₂,

Such as those disclosed in Sudo et al, U.S. patent publication No. US2020/0061225 or KITASAKA ET AL, U.S. patent 7241625, which are incorporated herein by reference. In some embodiments, R ¹ is a C _1-18 straight chain, A branched or cyclic alkyl group which may be substituted with at least one substituent selected from the group consisting of a halogen atom; C _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 group, which may be substituted with halogen atom, A C _1-6 alkyl group, a C _1-6 alkoxy group, a C _1-6 haloalkyl group, a C _1-6 haloalkoxy group, A carboxyl group, a hydroxyl group, a mercapto group, a cyano group or a nitro group, a C _4-14 heteroaryl group which may be substituted by a halogen atom, a C _1-6 alkyl group, a C _1-6 alkoxy group, a C _1-6 haloalkyl group, C _1-6 haloalkoxy group, carboxyl group, hydroxyl group, mercapto group, cyano group or nitro group, alkoxycarbonyl group represented by the following formula:

-(C=O)-O-R²

and a carbamoyl group represented by the following formula:

-(C=O)-NR³(R³)

Wherein R ² is a C _1-18 linear, branched or cyclic alkyl group which may be substituted at a chemically acceptable optional position with at least one substituent selected from the group consisting of a carboxyl group, a hydroxyl group, a mercapto group, a halogen atom, a C _1-6 alkoxy group, a C _1-6 haloalkoxy group, a C _6-14 aryl group, and a C _4-14 heteroaryl group, and wherein each R ³ may be the same or different and is each independently a C _1-18 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, a C _1-6 alkoxy group, a C _1-6 haloalkoxy group, a C _6-14 aryl group, and a C _4-14 heteroaryl group, a C _6-14 aryl group, a C _4-14 heteroaryl group, or a hydrogen atom.

In particular, exemplary O-substituted hydroxylamines or polyhydroxy amines that may be used in the present invention are shown in fig. 3A and 3B as compounds (1) through (14), wherein compound (1) is also referred to herein as a "BOX" reagent.

In some embodiments, the aldehyde scavenger comprises a carbonyl compound disclosed by pacific, U.S. patent 5446195 or Burdeniuc et al, U.S. patent publication US20160369035, which are incorporated herein by reference and defined by the following formula:

Wherein R and R' are CH ₃ or H [ O (CH ₂)_m]_n O-, and wherein m and n are selected from the group consisting of m=1 and n=1, 3-19; m=2 and n=2-19; or m=3 and n=1-19; Y is- -CH ₂ - -or- -CH ₂-CO-CH₂ - -.

In some embodiments of the invention, the template-free polymerase ink as described above further comprises a dye to allow monitoring of the position, size, shape and possible overlap of the reaction sites, in particular monitoring of possible coalescence of the reaction mixture at adjacent sites, either upon initial dispensing of reagents to define the reaction sites or upon subsequent droplet dispensing during synthesis. For this purpose, there are a number of fluorescent and non-fluorescent dyes that are alternatives. The main criteria used are that the dye (i) does not adversely affect the properties of any of the reaction components, (ii) is sufficiently bright or sufficiently dense to make the droplets or reaction sites easily detectable, (iii) is spectrally different if more than one is used, and (iv) does not affect the rheological properties of the ink. In some embodiments, food dyes are used in the inks of the present invention. In other embodiments, pH indicator dyes are used in the inks of the present invention. In other embodiments, fluorescent dyes are used in the inks of the present invention. Exemplary dyes for template-free polymerase inks include Brilliant Blue FCF, FAST GREEN FCF, ponceau 4R and Sunset Yellow FCF. In some embodiments, the food dye is used at a concentration of 1 to 20mM or a concentration of 1 to 10 mM.

Examples

Method for evaluating reaction conditions in enzymatic inkjet synthesis

In this example, test beds for evaluating different inkjet reaction conditions were created. Because very small amounts of material at each reaction site are difficult to analyze, slides are prepared that allow 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 zones are created by depositing an equal volume of a base-cleavable or photo-cleavable initiator in each zone. An exemplary glass slide (900) is shown in fig. 5 (which may be PolyAn D-epoxy coated glass slide). It contains 24 circular areas (902), each of which contains approximately the same number of reaction sites (not shown). Regions (902) were created on the slide (900) by manually spotting 20 μm 5' -amino-derived photocleavable initiator to 24 positions (following the protocol recommended by the slide manufacturer). Briefly, after overnight incubation at 70% relative humidity, the slides were then heated to 80 ℃ for 5 minutes, washed for 1 hour in 1M ethanolamine pH8, 30 minutes in SSC 4X, 30 minutes in SSC 2X 0.1% SDS, 30 minutes in SSC 0.2X, and 30 minutes in MQ. Exemplary initiators may have the sequence 5 '-amino-C12-10T (PC) 4T (FAM-T) 18T-3', wherein C12 is a 12 carbon alkyl linker, T is thymidine, PC is a photocleavable linker (e.g., horgan, WO 2021/048142), and FAM-T is fluorescein-labeled thymidine. After performing the experiment, the sequences were photocleaved from a specific region by immersing the region in PBS (e.g., 40 μl/region) and irradiating the region with 365nm light (e.g., analytical Jena, 95-0252-02, UVLMS-38, 8W intensity at 3 "1500 μw/cm ² for 365 nm), after which the pooled sequences were loaded onto a gel as shown in fig. 5. In the example of fig. 5, the regions are divided into 8 groups of 3 regions each. In groups 1-4, 23 cycles of extension reactions were performed, after which the resulting products in 3 regions of each group were photocleaved, pooled and loaded (909) onto their respective lanes 1-4 (914). In groups 5-8, no synthesis reaction was performed. Non-extended initiator was processed exactly the same as in groups 1-4, but loaded into lanes 5-8 (916). The gel provides convenient and sensitive measurement of the effects of various reaction parameter variations, 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.

Evaluation of the influence of temperature on viscosity and enzymatic inkjet Synthesis

In this example, the effect of temperature on ink viscosity was evaluated. An extender ink was prepared as described in table 1 below.

Table 1 ink compositions

As shown in fig. 6, it can be seen that the viscosity decreases significantly with increasing temperature, which is expected.

Stability of the extension inks was also evaluated at different temperatures. As shown in fig. 7, the benefits of storing ink at low temperature can be seen, as well as the inability to synthesize with ink stored in the printhead at 37 ℃ because the ink precipitates within 24 hours.

In this example, a manual synthesis test was performed to evaluate the efficacy of the reaction at different temperatures.

The slide is completely covered with photo-cleavable initiator. An exemplary slide (900) is shown in fig. 5 (which may be PolyAn D-azide coated glass slide). Immobilization was performed by incubating 5 μm of the 5' dbco photocleavable initiator in NaOAc/NaCl buffer at ph=4.5 for 1 hour at 70% RH. The slides were then washed with a series of buffers, rinsed with MQ and dried.

Exemplary initiators may have the 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, WO 2021/048142) and FAM-T is fluorescein-labeled thymidine.

The manual synthesis was then performed in silicon washers at 22 ℃, 37 ℃ or 4 ℃ (one slide) i) incubated with extension ink (70% RH, 5 minutes at 4 ℃, 20 ℃ or 37 ℃), ii) incubated with deprotection buffer (70% RH, 3 minutes), iii) rinsed with H2O milli-Q, iv) dried with compressed air.

After experiments were performed, the gasket was filled with PBS and a specific region was irradiated with 365nm light (e.g., 8W intensity at Analytik Jena, 95-0252-02, UVLMS-38, 3 "1500 mW/cm2 for 365 nm) to photocleavage the sequence from that region.

The extended oligomers at 4 ℃,20 ℃ and 37 ℃ were gel-electrophoresed together with the non-extended oligomers used as reference.

The gel provides convenient and sensitive measurements of the effects of various reaction parameter variations, including but not limited to reactant concentration, presence, absence and concentration of rheological components, extension temperature, different template-free polymerases, secondary structure modifiers, and the like.

As can be seen from fig. 8, the synthesis at 37 ℃ is better than the synthesis at4 ℃, and the amount of impurities decreases with higher temperature. As can be seen from fig. 6 and 7, for better results, a high viscosity is required before synthesis and a lower viscosity is required during synthesis.

Definition of the definition

Unless specifically defined otherwise herein, terms and symbols of nucleic acid chemistry, biochemistry, genetics and molecular biology as used herein follow terms and symbols in standard papers and textbooks in the art, e.g. Kornberg and Baker, DNA Replication, Second Edition (W.H. 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).

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

"Polynucleotide" means a linear polymer of nucleotide monomers or analogs thereof. The monomers comprising the polynucleotide may be capable of specifically binding to the native polynucleotide by a regular pattern of monomer-monomer interactions, such as Watson-Crick base pairing, base stacking, hoogsteen or reverse Hoogsteen base pairing, and the like. Such monomers and their internucleoside linkages may be naturally occurring or may be analogues thereof, for example naturally occurring or non-naturally occurring analogues. Non-naturally occurring analogs can include PNAs, phosphorothioate internucleoside linkages, bases containing linking groups that allow for attachment of a label (e.g., a fluorophore or hapten), and the like. Whenever the use of a polynucleotide requires enzymatic processing, such as extension by a polymerase, ligation by a ligase, or the like, one of ordinary skill in the art will understand that in those instances the polynucleotide will not contain internucleoside linkages, sugar moieties, or certain analogs of bases at any or some positions. Polynucleotides are typically several monomer units in size, e.g., 5-40, to thousands of monomer units. Whenever a polynucleotide is represented by a letter sequence (uppercase or lowercase), for example, "ATGCCTG", it will be understood that the sequence of nucleotides is 5'→3' from left to right, and that "a" represents deoxyadenosine, "C" represents deoxycytidine, "G" represents deoxyguanosine, and "T" represents thymidine, "I" represents deoxyinosine, "U" represents uridine, unless otherwise indicated or apparent from context. Unless otherwise indicated, the terms and atom numbering conventions will follow those disclosed in STRACHAN AND READ, human Molecular Genetics 2 (Wiley-Lists, new York, 1999). Typically, polynucleotides comprise 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 comprise non-natural nucleotide analogs, including, for example, modified bases, sugars, or internucleoside linkages. Those skilled in the art will appreciate that when an enzyme has a particular requirement for polynucleotide substrate activity, e.g., single-stranded DNA, RNA/DNA duplex, etc., then the selection of the appropriate composition of the polynucleotide substrate is well within the knowledge of the ordinarily skilled artisan, particularly under the guidance of papers such as Sambrook et al Molecular Cloning, second Edition (Cold Spring Harbor Laboratory, new York, 1989), and similar references. Likewise, a polynucleotide may refer to a single-stranded form or a double-stranded form (i.e., a duplex of the polynucleotide and its corresponding complement). The ordinarily skilled artisan will recognize from the context of the use of the term which form is intended or whether two forms are intended.

Claims (20)

1. A method for enzymatically synthesizing a plurality of polynucleotides at a reaction site on a substrate for inkjet printing, 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 polynucleotide of the plurality of polynucleotides is assigned to a reaction site for synthesis;

(b) Reacting an initiator or 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 extension fragment extends 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 mixture of a 3' -O protected nucleoside triphosphate, a template-free polymerase, and a 3' -O protected nucleoside triphosphate, or a deprotected solution, wherein the at least one synthesis reagent comprises a variable viscosity modifier having a first viscosity during formation of the at least one droplet and a second viscosity at each of the plurality of reaction sites during a reaction step of the reaction cycle;

(c) Repeating step (b) until the plurality of polynucleotides is synthesized;

Wherein the first viscosity and the second viscosity are different.

2. The method of 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 of any of the preceding claims, wherein the first viscosity is from 2 to 20 mPas, inclusive, and the second viscosity is from 1 to 2 mPas, inclusive.

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

5. The method of any of the preceding claims, wherein the variable viscosity modifier comprises a thermally reversible polymer that decreases in viscosity with increasing temperature.

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

7. The method of any 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) ₂ PEG, carboxymethyl cellulose, hydroxyethyl cellulose, or a combination thereof.

8. The method of any one of the preceding claims, wherein step (b) further comprises washing the extension fragment after the deprotecting step.

9. The method of any one of the preceding claims, wherein each of the reaction sites is different and non-overlapping with the other reaction sites.

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

11. The method of any one of the preceding claims, comprising the step of capping the initiator or extended fragment having free 3' -O-hydroxy groups that failed to extend after the step of reacting.

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

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

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

15. The method of any of the preceding claims, wherein there is more than one solution of synthetic 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 μΜ to 30 μΜ, wherein each of the printed droplets has a volume of 0.1pL to 5nL of the aqueous solution each time the ink is printed as droplets onto a substrate, and wherein the ink comprises a variable viscosity modifier having a first viscosity of 2 mpa.s to 20 mpa.s each time the temperature is 5 ℃ to 30 ℃ and a second viscosity of 2 mpa.s to 3 mpa.s each time the temperature is 35 ℃ to 60 ℃, wherein the first viscosity and the second viscosity are different.

17. The templated 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) ₂ PEG, or a combination thereof.

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

19. The templated polymerase ink according to any 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.