Method for synthesizing polynucleotide arrays using photoactivated active ingredients

Abstract

The present invention provides methods for the synthesis of DNA polynucleotides and polynucleotides, methods for their deprotection, and methods for the use of such compounds and compositions containing such compounds. [Solution] A method for synthesizing a polynucleotide having a predetermined sequence, comprising: providing a support containing a protected nucleic acid in a first characteristic portion; contacting the support with a photoresist solution containing a photoacid generator; and exposing the support to light of a certain wavelength, wherein the photoacid generator generates a weak acid via a cascade reaction when exposed to light of the said wavelength, and the weak acid deprotects the nucleic acid in the first characteristic portion; and binding the protected nucleic acid to the deprotected nucleic acid in the first characteristic portion.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefit of U.S. Provisional Application No. 62 / 668,964 filed 9 May 2018, the disclosure of which is incorporated herein by reference in its entirety. [Background technology]

[0002] background Microarray technology is readily used in biological research because it provides unprecedented information about nucleic acids for a wide range of applications, including gene expression and genotyping. Generally, there are challenges associated with polynucleotide synthesis, such as undesirable reactions like incomplete deprotection or depurination.

[0003] When constructing arrays by stepwise photodirective monomer-specific synthesis, using strong photoacids in the deprotection reaction tends to depurine nucleic acids, while using weak photoacids can increase the likelihood of incomplete deprotection. If the deprotection reaction is incomplete at any point, it reduces the yield of the correct sequence at that location in the array, resulting in a mixture of undesirable products. Since the synthesis cycle may need to be repeated many times at each location on the array, even a slight decrease in reaction efficiency can have a dramatic impact on the overall yield.

[0004] Therefore, what is needed is an improved nucleic acid synthesis method that improves reaction efficiency and, consequently, the yield of full-length synthetic polynucleotides. [Overview of the project]

[0005] This specification describes a method for synthesizing a polynucleotide having a predetermined sequence. In some embodiments, such a method includes providing a support comprising a protected nucleic acid in a first feature portion; contacting the support with a photoresist solution comprising a photoacid generator; exposing the support to light of a certain wavelength, wherein the photoacid generator generates a weak acid via a cascade reaction when exposed to light of the wavelength, and the weak acid deprotects the nucleic acid in the first feature portion; and binding the protected nucleic acid to the deprotected nucleic acid in the first feature portion.

[0006] In some embodiments, the weak acid includes acetic acid, carbonic acid, phosphoric acid, sulfonic acid, trifluoromethanesulfonic acid, or benzoic acid.

[0007] In some embodiments, the photoacid generator comprises 4-tert-butylphenyl acid.

[0008] In some embodiments, the photoacid generator comprises 4-tert-butylphenyl acetate and PGMEA.

[0009] In some embodiments, the photoacid generator comprises 4-tert-butylphenyl carbonate and a compound selected from the group consisting of propylene carbonate, methylphenyl carbonate, and PGMEA.

[0010] In some embodiments, the photoacid generator comprises 4-tert-butylphenyl phosphate and phenyl phosphate.

[0011] In some embodiments, the photoacid generator comprises 4-tert-butylphenyl sulfonate and a compound selected from the group consisting of phenyl sulfate, 4-methylphenyl sulfate, dimethyl sulfate, methyltrifluoromethanesulfonate, and methylfluorosulfonate.

[0012] In some embodiments, the photoacid generator includes 4-tert-butylphenyl triflate and phenyl trifluoromethanesulfonate.

[0013] In some embodiments, the photoacid generator includes phenyl 4-tert-butylbenzoate and phenyl benzoate.

[0014] In some embodiments, the protected nucleic acid includes a DMT group.

[0015] In some embodiments, the DMT group is attached to the nucleic acid at the 5'-carbon.

[0016] In some embodiments, the wavelength of the light is about 350 nm.

[0017] In some embodiments, the method further includes repeating the step to synthesize a polynucleotide of a desired length and sequence.

[0018] In some embodiments, the support includes at least 10, at least 100, at least 1,000, or at least 10,000 features including the protected nucleic acid.

[0019] Disclosed herein is a method for synthesizing an array of polynucleotides each having a predetermined sequence. In some embodiments, such a method includes providing a support including an array of protected nucleic acids attached to a surface, contacting the support with a solution including a photoacid generator, exposing a selected region of the support to light of a certain wavelength, wherein the photoacid generator generates a weak acid via a cascade reaction when exposed to light of that wavelength to deprotect the nucleic acid at each location exposed to light of that wavelength, the exposing, and contacting a selected input nucleotide with the wafer to bind to the deprotected nucleic acid.

[0020] In some embodiments, the nucleotide includes a DMT protecting group.

[0021] In some embodiments, the method further includes repeating the step a number of times sufficient to generate an array of polynucleotides having a predetermined sequence and intended length, respectively.

[0022] This specification describes a method for coupling a nucleotide monomer to a polynucleotide bound to a support, the method comprising: (a) providing a support comprising a terminal nucleotide bound to the surface of the support in a first feature portion; (b) carrying out in situ synthesis of an NPPOC protecting group on the terminal nucleotide; (c) exposing the support in the first feature portion to light of a certain wavelength to remove the NPPOC protecting group from the terminal nucleotide; and (d) contacting the support with a protected input nucleotide to bind the protected input nucleotide to the deprotected terminal nucleotide in the first feature portion.

[0023] In some embodiments, the method further includes (e) deprotecting the protected input nucleic acid bound to the terminal nucleotide, and then repeating steps (a) to (e) to bind a second protected input nucleic acid.

[0024] In some embodiments, the method further includes repeating all steps a number of times sufficient to synthesize polynucleotides of the intended length and sequence, which are bonded to the substrate.

[0025] In some embodiments, the method further includes the step of bringing the support into contact with a photoresist solution containing ITX before exposing the support to light of a certain wavelength.

[0026] In some embodiments, the wavelength of the light is approximately 365 nm.

[0027] In some embodiments, the NPPOC protecting group is bonded to the 5' carbon of the terminal nucleotide.

[0028] In some embodiments, providing the support containing the terminal nucleotides includes the step of deprotecting the terminal nucleotides entirely before carrying out the in situ NPPOC synthesis.

[0029] In some embodiments, the step of carrying out the in situ synthesis of NPPOC includes contacting the support with an NPPOC synthesis solution containing 2-(2-nitrophenyl)propyl chloroformate and pyridine.

[0030] In some embodiments, the NPPOC synthesis solution further comprises 1-methyl-2-pyrrolidinone.

[0031] This specification describes a method for synthesizing an array of polynucleotides, each having a predetermined sequence, the method comprising: (a) providing a support comprising an array of protected nucleotides bound to its surface; (b) performing in situ synthesis of an NPPOC protecting group on the array of nucleotides; (c) selectively exposing the support to light of a certain wavelength to remove the NPPOC protecting group from selected nucleotides from the array of nucleotides to which the addition of selected input nucleotides is desired; (d) contacting the array with the selected input nucleotides so as to bind to the deprotected nucleotides; and (e) repeating steps (c) to (e) a number of times sufficient to complete a desired nucleotide addition layer, thereby synthesizing an array of polynucleotides, each having a predetermined sequence.

[0032] In some embodiments, this method further includes (f) deprotecting the input nucleotides bound to the array of polynucleotides as a whole before in situ synthesis of the NPPOC protecting group.

[0033] In some embodiments, the method further includes repeating steps (b) to (f) a number of times sufficient to generate an array of polynucleotides, each having a predetermined sequence and intended length, and deprotecting the input nucleotides bound to the polynucleotide array as a whole before in situ synthesis of the NPPOC protecting group.

[0034] In some embodiments, the selected input nucleotide includes a protecting group.

[0035] In some embodiments, the protecting group is a DMT. [Invention 1001] A method for synthesizing a polynucleotide having a predetermined sequence, To provide a support containing a protected nucleic acid in a first characteristic part, The support is brought into contact with a photoresist solution containing a photoacid generator, The exposure involves exposing the support to light of a certain wavelength, wherein the photoacid generator generates a weak acid via a cascade reaction when exposed to light of the said wavelength, and the weak acid deprotects the nucleic acid in the first characteristic portion. The protected nucleic acid is bound to the deprotected nucleic acid in the first characteristic portion, The method, including the method described above. [Invention 1002] The method of the present invention 1001, wherein the weak acid comprises acetic acid, carbonic acid, phosphoric acid, sulfonic acid, trifluoromethanesulfonic acid, or benzoic acid. [Invention 1003] The method of the present invention 1001, wherein the photoacid generator contains 4-tert-butylphenyl acid. [Invention 1004] The method of the present invention 1001, wherein the photoacid generator comprises 4-tert-butylacetic acid phenyl and PGMEA. [Invention 1005] The method of the present invention 1001, wherein the photoacid generator comprises 4-tert-butylphenyl carbonate and a compound selected from the group consisting of propylene carbonate, methylphenyl carbonate, and PGMEA. [Invention 1006] The method of the present invention 1001, wherein the photoacid generator comprises 4-tert-butylphenyl phosphate and phenyl phosphate. [Invention 1007] The method of the present invention 1001, wherein the photoacid generator comprises 4-tert-butylphenyl sulfonate and a compound selected from the group consisting of phenyl sulfate, 4-methylphenyl sulfate, dimethyl sulfate, methyltrifluoromethanesulfonate, and methylfluorosulfonate. [Invention 1008] The method of the present invention 1001, wherein the photoacid generator comprises 4-tert-butylphenyl triflate and phenyltrifluoromethanesulfonate. [Invention 1009] The method of the present invention 1001, wherein the photoacid generator comprises 4-tert-butylbenzoate phenyl and benzoate phenyl. [Invention 1010] The method of the present invention 1001, wherein the protected nucleic acid contains a DMT group. [Invention 1011] The method of the present invention 1001, wherein the DMT group is bonded to the nucleic acid at the 5' carbon. [Invention 1012] The method of the present invention 1001, wherein the wavelength of the light is approximately 350 nm. [Invention 1013] The method of the present invention 1001 further comprises repeating the above steps to synthesize polynucleotides of an intended length and sequence. [Invention 1014] The method of the present invention 1001, wherein the support comprises at least 10, at least 100, at least 1,000, or at least 10,000 feature portions containing the protected nucleic acid. [Invention 1015] A method for synthesizing an array of polynucleotides, each having a predetermined sequence, To provide a support containing an array of protected nucleic acids bound to its surface, The support is brought into contact with a solution containing a photoacid generator, Exposing a selected region of the support to light of a certain wavelength, wherein the photoacid generator generates a weak acid via a cascade reaction upon exposure to light of the said wavelength in order to deprotect the nucleic acid at each location exposed to the said wavelength of light, The selected input nucleotide is brought into contact with the wafer so as to bind to the deprotected nucleic acid, The method, including the method described above. [Invention 1016] The method of the present invention 1015, wherein the input nucleotide contains a DMT protecting group. [Invention 1017] The method of the present invention 1015, further comprising repeating the step a number of times sufficient to generate an array of polynucleotides having a predetermined sequence and an intended length, respectively. [Invention 1018] A method for linking a nucleotide monomer to a polynucleotide conjugated to a support, a. To provide a support comprising terminal nucleotides attached to the surface of the support in the first characteristic portion, b. Performing in situ synthesis of the NPPOC protecting group on the terminal nucleotide, c. Exposing the support to light of a certain wavelength in the first characteristic portion to remove the NPPOC protecting group from the terminal nucleotide, d. The support is brought into contact with the protected input nucleotide, thereby binding the protected input nucleotide to the deprotected terminal nucleotide in the first characteristic portion. The method, including the method described above. [Invention 1019] (e) The method of the present invention 1018, further comprising deprotecting the protected input nucleic acid bound to the terminal nucleotide, and then repeating steps (a) to (e) to bind a second protected input nucleic acid. [Invention 1020] The method of the present invention 1019, further comprising repeating all steps a number of times sufficient to synthesize polynucleotides of the intended length and sequence bound to the substrate. [Invention 1021] The method of the present invention 1018, further comprising contacting the support with a photoresist solution containing ITX before exposing the support to light of the wavelength. [Invention 1022] The method of the present invention 1018, wherein the wavelength of the light is approximately 365 nm. [Invention 1023] The method of the present invention 1018, wherein the NPPOC protecting group is bonded to the 5' carbon of the terminal nucleotide. [Invention 1024] The method of the present invention 1018, which provides the support containing the terminal nucleotides, comprising deprotecting the terminal nucleotides whole before carrying out the in situNPPOC synthesis. [Invention 1025] The method of the present invention 1018, wherein the in situ synthesis of NPPOC comprises contacting the support with an NPPOC synthesis solution containing 2-(2-nitrophenyl)propyl chloroformate and pyridine. [Invention 1026] The method of the present invention 1025, wherein the NPPOC synthesis solution further comprises 1-methyl-2-pyrrolidinone. [Invention 1027] A method for synthesizing an array of polynucleotides, each having a predetermined sequence, a. To provide a support containing an array of protected nucleotides bound to its surface, b. Performing in situ synthesis of the NPPOC protecting group on the nucleotide array, c. Selectively exposing the support to light of a certain wavelength to remove the NPPOC protecting group from selected nucleotides from the array of nucleotides to which the addition of selected input nucleotides is desired. d. Contacting the selected input nucleotides with the array so as to bind them to the deprotected nucleotides, e. Repeat steps (c) to (e) a sufficient number of times to complete the desired nucleotide addition layer, thereby synthesizing an array of polynucleotides, each having a predetermined sequence. The method, including the method described above. [Invention 1028] (f) The method of the present invention 1027, further comprising deprotecting the input nucleotides conjugated to the polynucleotide array before in situ synthesis of the NPPOC protecting group. [Invention 1029] The method of the present invention 1027, further comprising repeating steps (b) to (f) a number of times sufficient to generate an array of polynucleotides having a predetermined sequence and an intended length, and further deprotecting the input nucleotides bound to the array of polynucleotides before in situ synthesis of the NPPOC protecting group. [Invention 1030] The method of the present invention 1027, wherein the selected input nucleotide contains a protecting group. [Invention 1031] The method of the present invention 1030, wherein the protecting group is DMT.

[0036] The aforementioned and other objectives, features, and advantages are evident from the following description of specific embodiments of the Invention, as shown in the accompanying drawings, and similar reference letters across different drawings refer to the same parts. The drawings are not necessarily to scale and instead focus on illustrating the principles of various embodiments of the Invention. These drawings illustrate various embodiments of the Invention for illustrative purposes only. Those skilled in the art will readily recognize from the following considerations that alternative embodiments of the structures and methods illustrated herein can be adopted without departing from the principles of the Invention as described herein. [Brief explanation of the drawing]

[0037] [Figure 1] This figure shows an example of a solvent-induced cascade photoacid system. [Figure 2] This figure illustrates an exemplary method for sequence-specific synthesis of polynucleotide microarrays, using a solvent-induced cascade photoacid system to deprotect protected terminal nucleotides bound to a wafer. [Figure 3] This figure shows an example of a reaction scheme for the in situ synthesis of terminal nucleotides protected with NPPOC((3'-nitrophenylpropyloxycarbonyl)). [Figure 4] This figure shows an exemplary synthesis of a polynucleotide microarray, according to an embodiment of the present invention, in which an NPPOC protecting group is added to the terminal nucleotides in each feature region using an in-situ reaction, followed by a two-step photoactivated NPPOC deprotection reaction and nucleotide addition reaction. [Figure 5] This figure shows an exemplary synthesis of a polynucleotide microarray, according to one embodiment of the present invention, in which an NPPOC protecting group is added to the terminal nucleotides in each feature region using an in-situ reaction, followed by a one-step photoactivated NPPOC deprotection and nucleotide addition reaction. [Modes for carrying out the invention]

[0038] Detailed explanation Details of various embodiments of the present invention are described below. Other features, purposes, and advantages of the present invention will become apparent from the description and drawings, as well as from the claims.

[0039] definition Unless otherwise specified, terms used in the claims and specification are defined as follows:

[0040] As used herein, the term "wafer" refers to a slice of semiconductor material, such as silicon or germanium crystal, commonly used in the manufacture of integrated circuits. Wafers can vary in size along one dimension, for example, from 25.4 mm (1 inch) to 300 mm (11.8 inches), and their thickness can be, for example, from 275 μm to 775 μm.

[0041] As used herein, the terms “photomask,” “reticle,” or “mask” refer to an opaque plate with a transparent pattern or holes through which light can pass. In a typical exposure process, the pattern on the photomask is transferred to a photoresist.

[0042] As used herein, the terms “photoresist,” “resist,” or “photoactive material” refer to a photosensitive material that undergoes chemical modification, for example, changes its solubility in a solution or generates photoacids when exposed to electromagnetic radiation, particularly ultraviolet or deep ultraviolet light. Photoresists may include organic or inorganic compounds.

[0043] As used herein, the term "photoresist formulation" refers to a formulation comprising a photoactive compound and a photoprotective compound.

[0044] As used herein, the term “photoactive compound” refers to a compound that is modified upon exposure to electromagnetic radiation. Examples of such compounds include cationic photoinitiators. For example, in a preferred embodiment, a cationic photoinitiator is a photoacid generator (PAG) that produces a corresponding photoacid upon exposure to electromagnetic radiation. Examples of photoactive compounds are disclosed in International Patent Application PCT / US2013 / 070207, filed November 14, 2013, which is incorporated herein by reference in whole for all purposes. A photoinitiator is a compound specifically added to a formulation to convert electromagnetic radiation into chemical energy in the form of an initiating species, such as a free radical or a cation. The acid or other product of the photoactive compound, upon exposure to electromagnetic radiation, may then react with another compound in a chain reaction to produce a desired chemical reaction. Thus, the spatial orientation in which these chemical reactions occur is defined according to the pattern of electromagnetic radiation to which the solution or surface containing the photoactive compound is exposed. This pattern can be defined, for example, by a photomask or reticle.

[0045] As used herein, the terms “coupling molecule” or “monomer molecule” include any natural or synthetic nucleic acid optionally protected by a protecting group such as DMT(4,4'-dimethoxytrityl) or NPPOC(3'-nitrophenylpropyloxycarbonyl).

[0046] As used herein, the terms “coupling,” “coupling process,” or “coupling step” refer to the process of forming a bond between two or more molecules, such as linking molecules or coupling molecules.

[0047] As used herein, the term "coupling efficiency" refers to the probability that a monomer is successfully attached to a reaction site available for binding to the monomer (e.g., the ends of a polymer). For example, during the growth of a polynucleotide, an input nucleotide binds to a deprotected terminal nucleotide attached to the surface of the wafer. This can be determined collectively, for example, by simultaneously monitoring the attachment of a single monomer to several specific reaction sites.

[0048] As used herein, the term “protecting group” includes groups introduced into a molecule by chemical modification of a functional group to obtain chemoselectivity in a subsequent chemical reaction. Chemoselectivity refers to the ability to guide a chemical reaction along a desired pathway and obtain a pre-selected product compared to another product. For example, the use of DMT as a protecting group enables chemoselectivity in polynucleotide synthesis using a photomask and photoacid generator, thereby selectively removing the protecting group and causing a predetermined nucleic acid coupling reaction at a site defined by the photomask.

[0049] As used herein, the terms “microarray,” “array,” or “chip” refer to a substrate on which multiple probe molecules of a particular polynucleotide binding sequence are synthesized in an orderly and separate manner at different locations, thus forming an array. A particular polynucleotide sequence may be bound to the chip substrate via one or more different types of linker molecules. A “chip array” refers to a plate having multiple chips, for example, 24, 96, or 384 chips.

[0050] As used herein, the terms “capping,” “capping process,” or “capping step” refer to the addition of a molecule that prevents further reactions of the bound molecule.

[0051] "Nucleotide" and "nucleotide moiety" refer to, but are not limited to, subunits of nucleic acids (e.g., RNA, DNA, or their analogues), including phosphate groups, sugar groups, and nitrogen-containing bases, as well as analogues of these subunits. Other groups (e.g., protecting groups) can be attached to sugar groups and nitrogen-containing base groups.

[0052] A “nucleoside” refers to a nucleic acid subunit, such as a sugar group and a nitrogen-containing base. While the term “nucleotide” is used herein to describe embodiments of this disclosure, it should be noted that those skilled in the art will understand that the terms “nucleoside” and “nucleotide” are almost always interchangeable. Those skilled in the art will also understand that additional modifications to nucleosides may be necessary.

[0053] As used herein, the terms “nucleoside” and “nucleotide” will be understood to include not only naturally occurring purine and pyrimidine bases (e.g., adenine (A), thymine (T), cytosine (C), guanine (G), or uracil (U)) but also modified purine and pyrimidine bases and other modified heterocyclic bases (these parts are sometimes collectively referred to as “purine and pyrimidine bases and their analogues”). Such modifications include, for example, diaminopurine and its derivatives, inosine and its derivatives, alkylated purines or pyrimidines, acylated purines or pyrimidines, thiolated purines or pyrimidines, or the addition of protecting groups such as levlinyl, acetyl, difluoroacetyl, trifluoroacetyl, isobutyryl, benzoyl, 9-fluorenylmethoxycarbonyl, phenoxyacetyl, dimethylformamidine, and N1N-diphenylcarbamate. Purine or pyrimidine bases may also be analogues as described above. Suitable analogues are known to those skilled in the art and are described in relevant texts and literature.Common analogues include, but are not limited to, 1-methyladenine, 2-methyladenine, N6-retyladenine, N6-isopentyladenine, 2-methylthio-N6-isopentyladenine, N,N-dimethyladenine, 8-bromoadenine, 2-thiocytosine, 3-methylcytosine, 5-methylcytosine, 5-ethylcytosine, 4-acetylcytosine, 1-methylguanine, 2-methylguanine, 7-methylguanine, 2,2-dimethylguanine, 8-bromoguanine, 8-chloroguanine, 8-aminoguanine, 8-methylguanine, 8-thioguanine, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, Examples include 5-ethyluracil, 5-propyluracil, 5-methoxyuracil, 5-hydroxymethyluracil, 5-(carboxyhydroxymethyl)uracil, 5-(methylaminomethyl)uracil, 5-(carboxymethylaminomethyl)uracil, 2-thiouracil, 5-methyl-2-thiouracil, 5-(2-bromovinyl)uracil, uracil-5-oxyacetic acid, uracil-5-oxyacetic acid methyl ester, pseudouracil, 1-methylpsoiduracil, keosin, inosine, 1-methylinosine, hypoxanthine, xanthine, 2-aminopurine, 6-hydroxyaminopurine, 6-thiopurine, and 2,6-diaminopurine.

[0054] A "nucleotide monomer" refers to a molecule that is not incorporated into a larger oligonucleotide or polynucleotide chain and corresponds to a single nucleotide subunit. Nucleotide monomers may also have activating or protecting groups, if such groups are necessary for the intended use of the nucleotide monomer.

[0055] A "polynucleotide" generally refers to a nucleotide polymer containing any number of nucleotides greater than one. The terms "oligonucleotide" and "polynucleotide" are often used synonymously, depending on the context of the sentence and paragraph in which they are used.

[0056] A "polynucleotide intermediate" is a molecule that arises between steps in the chemical synthesis of a polynucleotide. The polynucleotide intermediate is subjected to further reactions to obtain the desired final product, such as a protected polynucleotide, which is then deprotected.

[0057] "Nucleotide-linked linkages" refer to chemical linkages between two nucleoside moieties, such as phosphodiester linkages in naturally occurring nucleic acids, or linkages known from the synthesis techniques of nucleic acids and nucleic acid analogs. Nucleotide-linked linkages may include phospho or phosphite groups, and may include linkages in which one or more oxygen atoms of the phospho or phosphite group are modified by substituents or substituted by other atoms (e.g., sulfur or nitrogen atoms of a mono- or dialkylamino group), or by groups (e.g., a methyl group or other alkyl or functionalized alkyl groups).

[0058] The “group” includes both substituted and unsubstituted forms. Typical substituents include one or more lower alkyl, amino, imino, amide, alkylamino, arylamino, alkoxy, aryloxy, thioalkyl, alkylthio, arylthio, aryl, hydroxyl, amino, amide, sulfonyl, thio, mercapto, imino, halo, cyano, nitro, nitroso, azide, carboxy, sulfide, sulfone, sulfoxy, phosphoryl, silyl, silyloxy, and boronyl substituents, or optionally, substitution of one or more available carbon atoms with non-hydrocarbyl substituents such as cyano, nitro, halogen, hydroxyl, sulfonic acid, sulfate, phosphonic acid, phosphate, and phosphonate. Any substituent is typically selected so as not to substantially adversely affect the reaction yield (e.g., not to reduce the reaction yield by more than 20% (or 10%, or 5%, or 1%) of the yield obtained by other methods without a particular substituent or combination of substituents). Examples of "phospho" groups include phosphodiesters, phosphotriesters, and H-phosphonate groups. In the case of either a phospho group or a phosphite group, the chemical moiety other than the substituted five-membered furyl ring can be bonded to the oxygen atom of the phospho or phosphite group that connects the furyl ring to the P atom.

[0059] The term "protecting group" is used in the conventional chemical sense to refer to a group that reversibly dereactively makes a functional group inactive under specific conditions of a desired reaction. Several protecting groups are well known to those skilled in the art. Protection / deprotection processes and examples of various protecting groups are described in Wuts and Greene, 2006, Greene's Protective Groups in Organic Synthesis, Wiley-Interscience, New York, NY. Any suitable protecting group known to those skilled in the art may be used. After the desired reaction, the protecting group may be removed to deprotect the protected functional group. All protecting groups should be removable (and therefore unstable) under conditions that do not decompose a significant proportion of the synthesized molecule. In contrast to protecting groups, "capping groups" permanently bond to a segment of a molecule to prevent any further chemical transformation of that segment. Note that the functional group protected by a protecting group may or may not be part of what is called the protecting group.

[0060] overview This specification describes compositions and methods for synthesizing polynucleotides (e.g., DNA polynucleotides) in situ on a solid substrate. In particular, this specification describes novel compositions and methods for synthesizing arrays of polynucleotide probes in the form of DNA (micro)arrays or DNA chips.

[0061] The experimental procedures described below may, in part, utilize methods that are well known and commonly used in the art. For example, standard techniques may be used for the isolation, purification, amplification, and cloning of DNA and RNA. Enzymatic reactions involving DNA ligases, DNA polymerases, restriction endonucleases, etc., may be carried out according to the manufacturer's specifications. Such techniques and various other techniques may generally be carried out in accordance with Sambrook et al., 1989, Molecular Cloning - A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel et al., 1993, Current Protocols in Molecular Biology, Volumes 1-3, John Wiley & Sons, Inc., New York, NY; and Kriegler, 1990, Gene Transfer and Expression: A Laboratory Manual, Stockton Press, New York, NY. Each of these is incorporated herein in whole by reference.

[0062] This application describes a method for sequence-specific synthesis of polynucleotides on a microarray platform by high-throughput parallel synthesis using photolithography. Polynucleotide structures, including nucleotide monomers and nucleotide moieties, are also described herein, each containing various types of protecting groups. These nucleotide monomers and moieties can be used in combination with the methods, processes, and / or compositions of the present invention for the deprotection of polynucleotides, particularly for polynucleotide synthesis on an array. Embodiments of the present invention enable the quantitative or semi-quantitative and rapid synthesis of desired full-length polynucleotide products.

[0063] In various examples of the compositions and methods described herein, schemes illustrating the synthesis of DNA polynucleotides are provided below.

[0064] Scheme I - Photoacid Cascade System for Nucleic Acid Synthesis According to some embodiments, the herein provides a photoacid cascade system for facilitating efficient deprotection during nucleic acid synthesis. In standard nucleic acid synthesis, protecting groups such as DMT can be removed by exposure to acid. However, exposure to strong acids can lead to depurination, and exposure to weak acids can result in incomplete deprotection, both of which impair the yield of the nucleotide synthesis reaction.

[0065] This specification provides a novel nucleotide synthesis reaction that demonstrates improved yields of polynucleotides synthesized on a microarray.

[0066] Provided herein is a novel nucleotide synthesis reaction that uses a photoactivated cascade reaction to generate a weak acid sufficient to improve the yield of deprotection while avoiding depurination.

[0067] Optical Cascade System In some embodiments, the herein provides nucleotide synthesis reactions that use photoactivated cascade reactions to produce weak acids sufficient to improve the yield of deprotection while avoiding depurination, which is a characteristic of deprotection of strong acids. In some cases, the photoactivated cascade reaction involves the reaction of acetate with phenyl acetate.

[0068] Figure 1 shows an exemplary photoacid cascade system useful for nucleotide synthesis. For this reaction, a solution of propylene glycol methyl ether acetate (PGMEA) containing poly(methyl methacrylate) (PMMA), 4-tert-butylphenyl acetate, and isopropylthioxanthone (ITX) is prepared. In a particular embodiment, the reaction of 4-tert-butylphenyl acetate with the acetate derived from PGMEA is catalyzed by ITX when the solution is exposed to light at a wavelength of 356 nm, producing acetic anhydride and 4-tert-butylphenol as intermediate compounds. Next, acetic anhydride and 4-tert-butylphenol react to form the original reagents, 4-tert-butylphenyl acetate and acetic acid. In this cascade reaction, acetic acid is produced in the photoactivated solution when exposed to light at 365 nm. The resulting acetic acid then reacts at the site of the DMT-protected nucleotide, which can efficiently deprotect the nucleotide.

[0069] In some embodiments, the photoactive solution is found in concentrations of approximately 0.1% by weight, 0.2% by weight, 0.3% by weight, 0.4% by weight, 0.5% by weight, 0.6% by weight, 0.7% by weight, 0.8% by weight, 0.9% by weight, 1.0% by weight, 1.1% by weight, 1.2% by weight, 1.3% by weight, 1.4% by weight, 1.5% by weight, 1.6% by weight, 1.7% by weight, 1.8% by weight, 1.9% by weight, 2.0% by weight, 2.1% by weight, 2.2% by weight, 2.3% by weight, and 2. 4% by weight, 2.5% by weight, 2.6% by weight, 2.7% by weight, 2.8% by weight, 2.9% by weight, 3.0% by weight, 3.1% by weight, 3.2% by weight, 3.3% by weight, 3.4% by weight, 3.5% by weight, 3.6% by weight, 3. 7% by weight, 3.8% by weight, 3.9% by weight, 4.0% by weight, 4.1% by weight, 4.2% by weight, 4.3% by weight, 4.4% by weight, 4.5% by weight, 4.6% by weight, 4.7% by weight, 4.8% by weight, 4.9% by weight, 5.0 Weight%, 5.1% by weight, 5.2% by weight, 5.3% by weight, 5.4% by weight, 5.5% by weight, 5.6% by weight, 5.7% by weight, 5.8% by weight, 5.9% by weight, 6.0% by weight, 6.1% by weight, 6.2% by weight, 6.3 Weight%, 6.4% by weight, 6.5% by weight, 6.6% by weight, 6.7% by weight, 6.8% by weight, 6.9% by weight, 7.0% by weight, 7.1% by weight, 7.2% by weight, 7.3% by weight, 7.4% by weight, 7.5% by weight, 7.6% by weight The photoactive solution contains PMMA in amounts of 1% (1%), 7.7% (1%), 7.8% (1%), 7.9% (1%), 8.0% (1%), 8.1% (1%), 8.2% (1%), 8.3% (1%), 8.4% (1%), 8.5% (1%), 8.6% (1%), 8.7% (1%), 8.8% (1%), 8.9% (1%), 9.0% (1%), 9.1% (1%), 9.2% (1%), 9.3% (1%), 9.4% (1%), 9.5% (1%), 9.6% (1%), 9.7% (1%), 9.8% (1%), 9.9% (1%), or 10.0% (1%). In a preferred embodiment, the photoactive solution contains about 0.5 to 5% (1%) PMMA. In a more preferred embodiment, the photoactive solution contains about 1 to 3% (1%) PMMA. In one embodiment, the photoactive solution contains about 2% (1%) PMMA.

[0070] In some embodiments, the photoactive solution is found in concentrations of approximately 0.1% by weight, 0.2% by weight, 0.3% by weight, 0.4% by weight, 0.5% by weight, 0.6% by weight, 0.7% by weight, 0.8% by weight, 0.9% by weight, 1.0% by weight, 1.1% by weight, 1.2% by weight, 1.3% by weight, 1.4% by weight, 1.5% by weight, 1.6% by weight, 1.7% by weight, 1.8% by weight, 1.9% by weight, 2.0% by weight, 2.1% by weight, 2.2% by weight, 2.3% by weight, and 2.4% by weight. Amount %, 2.5 wt%, 2.6 wt%, 2.7 wt%, 2.8 wt%, 2.9 wt%, 3.0 wt%, 3.1 wt%, 3.2 wt%, 3.3 wt%, 3.4 wt%, 3.5 wt%, 3.6 wt%, 3.7 wt% , 3.8% by weight, 3.9% by weight, 4.0% by weight, 4.1% by weight, 4.2% by weight, 4.3% by weight, 4.4% by weight, 4.5% by weight, 4.6% by weight, 4.7% by weight, 4.8% by weight, 4.9% by weight, 5.0% by weight, 5 .1% by weight, 5.2% by weight, 5.3% by weight, 5.4% by weight, 5.5% by weight, 5.6% by weight, 5.7% by weight, 5.8% by weight, 5.9% by weight, 6.0% by weight, 6.1% by weight, 6.2% by weight, 6.3% by weight, 6.4 Weight%, 6.5% by weight, 6.6% by weight, 6.7% by weight, 6.8% by weight, 6.9% by weight, 7.0% by weight, 7.1% by weight, 7.2% by weight, 7.3% by weight, 7.4% by weight, 7.5% by weight, 7.6% by weight, 7.7% by weight The photoactive solution contains 4-tert-butylacetic acid in amounts of %, 7.8% by weight, 7.9% by weight, 8.0% by weight, 8.1% by weight, 8.2% by weight, 8.3% by weight, 8.4% by weight, 8.5% by weight, 8.6% by weight, 8.7% by weight, 8.8% by weight, 8.9% by weight, 9.0% by weight, 9.1% by weight, 9.2% by weight, 9.3% by weight, 9.4% by weight, 9.5% by weight, 9.6% by weight, 9.7% by weight, 9.8% by weight, 9.9% by weight, or 10.0% by weight. In a preferred embodiment, the photoactive solution contains about 2 to 7% by weight of 4-tert-butylacetic acid. In a more preferred embodiment, the photoactive solution contains about 3 to 6% by weight of 4-tert-butylacetic acid. In one embodiment, the photoactive solution contains about 5% by weight of 4-tert-butylacetic acid.

[0071] In some embodiments, the photoactive solution is found in concentrations of approximately 0.1% by weight, 0.2% by weight, 0.3% by weight, 0.4% by weight, 0.5% by weight, 0.6% by weight, 0.7% by weight, 0.8% by weight, 0.9% by weight, 1.0% by weight, 1.1% by weight, 1.2% by weight, 1.3% by weight, 1.4% by weight, 1.5% by weight, 1.6% by weight, 1.7% by weight, 1.8% by weight, 1.9% by weight, 2.0% by weight, 2.1% by weight, 2.2% by weight, 2.3% by weight, and 2% by weight. .4% by weight, 2.5% by weight, 2.6% by weight, 2.7% by weight, 2.8% by weight, 2.9% by weight, 3.0% by weight, 3.1% by weight, 3.2% by weight, 3.3% by weight, 3.4% by weight, 3.5% by weight, 3.6% by weight, 3 .7% by weight, 3.8% by weight, 3.9% by weight, 4.0% by weight, 4.1% by weight, 4.2% by weight, 4.3% by weight, 4.4% by weight, 4.5% by weight, 4.6% by weight, 4.7% by weight, 4.8% by weight, 4.9% by weight, 5. 0% by weight, 5.1% by weight, 5.2% by weight, 5.3% by weight, 5.4% by weight, 5.5% by weight, 5.6% by weight, 5.7% by weight, 5.8% by weight, 5.9% by weight, 6.0% by weight, 6.1% by weight, 6.2% by weight, 6. 3% by weight, 6.4% by weight, 6.5% by weight, 6.6% by weight, 6.7% by weight, 6.8% by weight, 6.9% by weight, 7.0% by weight, 7.1% by weight, 7.2% by weight, 7.3% by weight, 7.4% by weight, 7.5% by weight, 7.6 The photoactive solution contains ITX in amounts of 10% by weight, 7.7% by weight, 7.8% by weight, 7.9% by weight, 8.0% by weight, 8.1% by weight, 8.2% by weight, 8.3% by weight, 8.4% by weight, 8.5% by weight, 8.6% by weight, 8.7% by weight, 8.8% by weight, 8.9% by weight, 9.0% by weight, 9.1% by weight, 9.2% by weight, 9.3% by weight, 9.4% by weight, 9.5% by weight, 9.6% by weight, 9.7% by weight, 9.8% by weight, 9.9% by weight, or 10.0% by weight. In a preferred embodiment, the photoactive solution contains about 2 to 7% by weight of ITX. In a more preferred embodiment, the photoactive solution contains about 3 to 6% by weight of ITX. In one embodiment, the photoactive solution contains about 5% by weight of ITX.

[0072] In certain embodiments, the photoactive solution comprises about 0.5–5% by weight of PMMA, about 2–7% by weight of 4-tert-butylphenyl acetate, and about 5% by weight of ITX. In more specific embodiments, the photoactive solution comprises about 1–3% by weight of PMMA, about 3–6% by weight of 4-tert-butylphenyl acetate, and about 3–6% by weight of ITX. In one embodiment, the photoactive solution comprises about 2% by weight of PMMA, about 5% by weight of 4-tert-butylphenyl acetate, and about 5% by weight of ITX.

[0073] In certain embodiments of the photoactivation cascade, the 4-tert-butylphenyl acetate-PGMEA system is utilized, but other acid cascade systems can be used to generate weak acids via a cascade system for site-specific deprotection. In some embodiments, the 4-tert-butylphenyl carbonate-propylene carbonate photoactivation acid cascade system is used to generate carbonate / acetic acid. In some embodiments, the 4-tert-butylphenyl carbonate-methylphenyl carbonate photoactivation acid cascade system is used to generate carbonate / acetic acid. In some embodiments, the 4-tert-butylphenyl carbonate-PGMEA photoactivation acid cascade system is used to generate carbonate / acetic acid. In some embodiments, the 4-tert-butylphenyl phosphate-phenyl phosphate photoactivation acid cascade system is used to generate phosphoric acid. In some embodiments, the 4-tert-butylphenyl sulfonate-phenyl sulfate photoactivation acid cascade system is used to generate sulfonic acid. In some embodiments, the 4-tert-butylphenyl sulfonate-4-methylphenyl sulfate photoactivated acid cascade system is used to produce sulfonic acid. In some embodiments, the 4-tert-butylphenyl sulfonate-dimethyl sulfate photoactivated acid cascade system is used to produce sulfonic acid. In some embodiments, the 4-tert-butylphenyl sulfonate-methyltrifluoromethanesulfonate photoactivated acid cascade system is used to produce sulfonic acid. In some embodiments, the 4-tert-butylphenyl sulfonate-methylfluorosulfonate photoactivated acid cascade system is used to produce sulfonic acid. In some embodiments, the 4-tert-butylphenyl triflate-phenyltrifluoromethanesulfonate photoactivated acid cascade system is used to produce trifluoromethanesulfonic acid. In some embodiments, the 4-tert-butylbenzoate phenyl-benzoate phenyl photoactivated acid cascade system is used to produce benzoic acid.

[0074] DMT deprotection and monomer coupling using photoactivated acid cascades Polynucleotides can be efficiently synthesized on an array using the photoactivated acid cascade solution described herein. Figure 2 shows an exemplary method for array polynucleotide synthesis.

[0075] As shown in Figure 2, a wafer containing nucleotides protected with 4,4'-dimethoxytrityl (DMT) may be provided. A photoactivated acid cascade solution may be coated onto the surface of the wafer. Light can also be applied locally to selected regions requiring the addition of selected nucleotides for nucleotide synthesis. In some embodiments, light is applied by using a photomask. The wafer containing the photoactivated solution can be exposed to light of a certain wavelength to initiate the acid cascade reaction, thereby deprotecting the DMT-protected nucleotides at the locations where the wafer is exposed to light. In some embodiments, the wafer is exposed for 500 milliseconds per field. In some embodiments, after the exposure is complete, a post-exposure delay time of 10 minutes at room temperature is performed before washing. After deprotection, the photoactivated solution may be washed from the surface, leaving a wafer containing selected molecules having deprotected nucleotides. The desired nucleotides can then be added to the wafer and bound to the deprotected nucleotides. In a preferred embodiment, the input nucleotides are protected with DMT, thereby enabling subsequent cycles of nucleotide addition according to the provided reaction scheme. A capping solution can be applied to the wafer to prevent the synthesis of deprotected molecules that did not bind to the input nucleotide.

[0076] The above cycle can be repeated as many times as necessary to synthesize the desired polynucleotide sequence at each location on the array.

[0077] In some embodiments, the post-exposure delay time is approximately 0.5 minutes, 1 minute, 1.5 minutes, 2 minutes, 2.5 minutes, 3 minutes, 3.5 minutes, 4 minutes, 4.5 minutes, 5 minutes, 5.5 minutes, 6 minutes, 6.5 minutes, 7 minutes, 7.5 minutes, 8 minutes, 8.5 minutes, 9 minutes, 9.5 minutes, 10 minutes, 10.5 minutes, 11 minutes, 11.5 minutes, 12 minutes, 12.5 minutes, 13 minutes, 13.5 minutes, 14 minutes, 14.5 minutes, or 15 minutes. In preferred embodiments, the post-exposure delay time is approximately 3 to 6 minutes.

[0078] Synthesis of scheme 2-in situNPPOC In some embodiments, the polynucleotide synthesis scheme relies on the in situ synthesis of the 3'-nitrophenylpropyloxycarbonyl (NPPOC) protecting group of free nucleotides bound to an array. An example of the reaction scheme for the in situ synthesis of NPPOC is shown in Figure 3. As illustrated, nucleotides having an unprotected 5' hydroxyl group can be converted to NPPOC-protected nucleotides by adding an in situ NPPOC synthesis solution containing 1-methyl-2-pyrrolidinone and pyridine-soluble 2-(2-nitrophenyl)propyl chloroformate.

[0079] Therefore, for both one-step and two-step NPPOC-protected polynucleotide synthesis schemes, according to some embodiments, NPPOC-protected nucleotides can be prepared as follows: A wafer containing a DMT-protected nucleotide (DMT-R1) is provided, as shown in Figures 4 and 5. The DMT protecting group is then removed entirely using a strong acid such as TCA. Next, the NPPOC is linked to the deprotected nucleotide according to the reaction scheme shown in Figure 3. In some embodiments, the in situ synthesis of NPPOCs to each of the deprotected nucleotides bound to the wafer is performed by spin-coating an in situ NPPOC synthesis solution containing 0.3 M 2-(2-nitrophenyl)propyl chloroformate dissolved in a solvent of 30% 1-methyl-2-pyrrolidinone and 70% pyridine onto the wafer. Next, the wafer is baked at 95°C for 2 minutes to entirely link the NPPOC protecting group to the deprotected nucleotide bound to the wafer. Next, the wafer can be stripped with 1-methyl-2-pyrrolidone and isopropyl alcohol.

[0080] From the resulting array of NPPOC-protected nucleotides, the addition of selected nucleotides at each spot can proceed according to a one- or two-step NPPOC deprotection and coupling procedure, as described herein. In some embodiments, once the layer of protective nucleotides is added to the nucleotides bound to the array as needed, the overall deprotection and NPPOC synthesis can be repeated the required number of times to achieve the desired polynucleotide sequence length at each location on the wafer.

[0081] In some embodiments, the array coated with the NPPOC synthetic solution is fired at 65°C, 70°C, 75°C, 80°C, 85°C, 90°C, 95°C, 100°C, 105°C, or 110°C. In preferred embodiments, the array coated with the NPPOC synthetic solution is fired at a temperature of approximately 80–100°C. In some embodiments, the array coated with the NPPOC synthetic solution is calcined for approximately 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, or 360 seconds. In preferred embodiments, the array coated with the NPPOC synthetic solution is calcined for approximately 60 to 240 seconds. In specific embodiments, the array coated with the NPPOC synthetic solution is calcined for approximately 120 seconds. In certain embodiments, the array coated with the NPPOC synthesis solution is fired at a temperature of approximately 80-100°C for approximately 60-240 seconds. In one embodiment, the array coated with the NPPOC synthesis solution is fired at approximately 95°C for approximately 120 seconds.

[0082] In some embodiments, the concentrations of 2-(2-nitrophenyl)propyl chloroformate in the in situ NPPOC synthesis solution are approximately 0.01 M, 0.02 M, 0.03 M, 0.04 M, 0.05 M, 0.06 M, 0.07 M, 0.08 M, 0.09 M, 0.1 M, 0.11 M, 0.12 M, 0.13 M, 0.14 M, 0.15 M, 0.16 M, 0.17 M, 0.18 M, 0.19 M, and 0.2 M. , 0.21M, 0.22M, 0.23M, 0.24M, 0.25M, 0.26M, 0.27M, 0.28M, 0.29M, 0.3M, 0.31M, 0.32M, 0.33M, 0.34M, 0.35M, 0.36M, 0.37M, 0.38M, 0.39M, 0.4M, 0 .41M, 0.42M, 0.43M, 0.44M, 0.45M, 0.46M, 0.47M, 0.48M, 0.49M, 0.50M, 0 .51M, 0.52M, 0.53M, 0.54M, 0.55M, 0.56M, 0.57M, 0.58M, 0.59M, 0.6M0.6 The values ​​are 1M, 0.62M, 0.63M, 0.64M, 0.65M, 0.66M, 0.67M, 0.68M, 0.69M, 0.7M, 0.71M, 0.72M, 0.73M, 0.74M, 0.75M, 0.76M, 0.77M, 0.78M, 0.79M, 0.8M, 0.81M, 0.82M, 0.83M, 0.84M, 0.85M, 0.86M, 0.87M, 0.88M, 0.89M, 0.90M, 0.91M, 0.92M, 0.93M, 0.94M, 0.95M, 0.96M, 0.97M, 0.98M, 0.99M, or 1.0M. In preferred embodiments, the concentration of 2-(2-nitrophenyl)propyl chloroformate in the in situ NPPOC synthesis solution is about 0.05 to 0.8 M. In more preferred embodiments, the concentration of 2-(2-nitrophenyl)propyl chloroformate in the in situ NPPOC synthesis solution is about 0.2 to 0.5 M.

[0083] In some embodiments, 2-2-(2-nitrophenyl)propyl chloroformate is dissolved in a solution containing 1-methyl-2-pyrrolidinone and pyridine in the following percentages provided in Table 1 to form an NPPOC synthesis solution.

[0084] (Table 1) Solvent composition of in situ NPPOC synthesis solution TIFF2026123166000002.tif25128TIFF2026123166000003.tif22895TIFF2026123166000004.tif22895

[0085] In situ protection of NPPOCs, followed by two-step photoactivation deprotection and synthesis. In some embodiments, the in situ NPPOC-protected nucleotide is deprotected according to a two-step synthesis scheme, as shown in Figure 4, for example, and linked to an input nucleotide (e.g., DMTR2). Therefore, a photoresist solution containing ITX is coated onto the surface of the wafer to facilitate photoactivated deprotection at a selected site on the array. The wafer is selectively exposed to remove NPPOC from the protected nucleotides linked to the array. The photoactivated solution is then washed off the wafer, and a solution containing the desired nucleotide is added to the wafer to bind to the deprotected nucleotide. In some embodiments, the input nucleotide is protected, for example, having a 5' carbon linked to a DMT group. These steps can be repeated until the synthesis layer is complete (i.e., until further addition to the NPPOC-protected nucleotide is no longer needed). Once the layer is complete, another round of synthesis steps may be performed, starting with the overall deprotection of the protected nucleotides linked to the array, followed by in situ NPPOC protection.

[0086] In situ protection of NPPOCs, one-step photoactivation deprotection, and synthesis. In some embodiments, the in situ NPPOC-protected nucleotide is deprotected and ligated to an input nucleotide according to a one-step synthesis scheme, for example, as shown in Figure 5. Therefore, a photoresist solution containing ITX is coated onto the wafer surface to facilitate photoactivated deprotection at a selected site on the array and selectively exposed to deprotect the nucleotides ligated to the array. The photoresist solution may also contain a nucleotide of choice to bind to the deprotected nucleotide, thereby allowing deprotection and ligation of the input nucleotide to occur in a single step. In some embodiments, the input nucleotide is protected, for example, having a 5' carbon bonded to a DMT group. These steps can be repeated until the synthesis layer is complete (i.e., until further addition to the NPPOC-protected nucleotide is no longer needed). Once the layer is complete, another round of synthesis steps may be performed, starting with the overall deprotection of the array-ligated protected nucleotide, followed by in situ NPPOC protection.

[0087] Functionalization of wafers In some embodiments, wafers containing an array of protective nucleotides bonded to the surface of the wafer, preferably via a linker, are provided to facilitate the synthesis of polynucleotides. In some embodiments, what is provided herein is a wafer functionalized to facilitate polynucleotide synthesis.

[0088] Wafer functionalization scheme In some embodiments, the wafer is functionalized according to the following reaction scheme. 1. Functionalize the wafer with amine groups (for example, using organically functionalized alkoxysilanes). 2. A linker is bonded to the amine groups of the functionalized wafer. The linker may contain protected amine groups. The linker may further contain carboxyl groups. In some embodiments, the carboxyl groups of the linker are bonded to the amine groups of the functionalized wafer. In some embodiments, the bond is formed by bonding the linker to the functionalized wafer. 3. Deprotect the amine group of the bonded linker. 4. The amine group is attached to a DMT-protected nucleotide, such as a DMT-protected phosphoramidite. This can result in an array in which DMT-R1 is attached to a wafer.

[0089] In one embodiment, the functionalization reaction scheme is as follows: 1,3-aminopropyltriethoxysilane (APTES) is added to functionalize the wafer with amine groups. 2. Free amines are linked to FMOC-NH-PEG6-CH2CH2COOH, forming an Fmoc protective linker bonded to the wafer surface. 3,4-methylpiperidine removes Fmoc from the amine group. 4. Add DMT-phosphoramidite thymine CED-5'cyanoethyl (a DMT-protected phosphoramidite group that allows coupling of the following nucleic acids). 5. Perform capping to the uncoupled linker. 6. The photoacid cascade system is implemented as described in this specification.

[0090] Organic functional alkoxysilanes As shown, silicon dioxide wafers can be functionalized by coating them with compounds containing free amine groups for subsequent bonding. In some embodiments, the wafers are coated with organically functional alkoxysilane molecules. In some embodiments, the wafers are coated with APTES. In some embodiments, the amounts are about 0.1% by weight, 0.2% by weight, 0.3% by weight, 0.4% by weight, 0.5% by weight, 0.6% by weight, 0.7% by weight, 0.8% by weight, 0.9% by weight, 1.0% by weight, 1.1% by weight, 1.2% by weight, 1.3% by weight, 1.4% by weight, 1.5% by weight, 1.6% by weight, 1.7% by weight, 1.8% by weight, 1.9% by weight, 2.0% by weight, 2.1% by weight, 2.2% by weight, 2.3% by weight, 2.4% by weight, 2. 5% by weight, 2.6% by weight, 2.7% by weight, 2.8% by weight, 2.9% by weight, 3.0% by weight, 3.1% by weight, 3.2% by weight, 3.3% by weight, 3.4% by weight, 3.5% by weight, 3.6% by weight, 3.7% by weight, 3. 8% by weight, 3.9% by weight, 4.0% by weight, 4.1% by weight, 4.2% by weight, 4.3% by weight, 4.4% by weight, 4.5% by weight, 4.6% by weight, 4.7% by weight, 4.8% by weight, 4.9% by weight, 5.0% by weight, 5.1 Weight%, 5.2% by weight, 5.3% by weight, 5.4% by weight, 5.5% by weight, 5.6% by weight, 5.7% by weight, 5.8% by weight, 5.9% by weight, 6.0% by weight, 6.1% by weight, 6.2% by weight, 6.3% by weight, 6.4 Weight%, 6.5% by weight, 6.6% by weight, 6.7% by weight, 6.8% by weight, 6.9% by weight, 7.0% by weight, 7.1% by weight, 7.2% by weight, 7.3% by weight, 7.4% by weight, 7.5% by weight, 7.6% by weight, 7.7% by weight Solutions of APTES in concentrations of 10% (0%), 7.8% by weight, 7.9% by weight, 8.0% by weight, 8.1% by weight, 8.2% by weight, 8.3% by weight, 8.4% by weight, 8.5% by weight, 8.6% by weight, 8.7% by weight, 8.8% by weight, 8.9% by weight, 9.0% by weight, 9.1% by weight, 9.2% by weight, 9.3% by weight, 9.4% by weight, 9.5% by weight, 9.6% by weight, 9.7% by weight, 9.8% by weight, 9.9% by weight, or 10.0% by weight are used. In some embodiments, a solution of APTES in concentrations of about 0.1 to 5.0% by weight is used. In preferred embodiments, a solution of APTES in concentrations of about 0.5 to 4.0% by weight is used. In even more preferred embodiments, a solution of APTES in concentrations of about 1.0 to 3.0% by weight is used.In one embodiment, a solution of approximately 2.0% by weight of APTES is used.

[0091] Linker In some embodiments, the linker is bonded to a wafer coated with free amine groups. In some embodiments, the linker includes a carboxylic acid group for bonding to the free amine group. In some embodiments, the linker further includes a chain for facilitating separation between the surface and the synthesized polynucleotide. The chain may include one or more PEG groups linked together. In some embodiments, the chain may include an aliphatic carbon chain. In some embodiments, the linker further includes a group for facilitating the bonding of nucleotides (e.g., phosphoramidites). In some embodiments, the group for facilitating the bonding of nucleotides is an amine group. In some embodiments, the amine group for facilitating the bonding of nucleotides is a protected amine group, such as an Fmoc protected amine group. In some embodiments, the linker includes a carboxylic acid group at one end, an amine group for facilitating the bonding of nucleotides at the other end, and a spacer molecule such as one or more PEG groups or an aliphatic chain connecting the amine group to the carboxylic acid group. For example, the linker may contain Fmoc-NH-PEG6-CH2CH2COOH, where the carboxyl group is bound to a surface-bound amine on the wafer, and the Fmoc-protected amine group can be deprotected to allow another molecule to be subsequently attached, thereby facilitating the synthesis of polynucleotides. The protecting amine group can be deprotected with a suitable reagent, such as a reagent containing 4-methylpiperidine.

[0092] Phosphoramidite (DMT protection) In some embodiments, modified DMT-protected nucleosides are bound to free amines bonded to the surface of a wafer in order to generate a functionalized array in which a series of DMT-protected nucleotides are bound to a wafer. In some embodiments, the amine group needs to be deprotected first to facilitate the binding of the DMT-protected nucleotides. In some embodiments, the amine group is located at the end of a linker bonded to the surface of the array.

[0093] In some embodiments, thymine CED-5'-cyanoethyl is bonded to a free amine group on the wafer surface, but phosphoramidites having other bases may also be used.

[0094] formulation Disclosed herein are formulations such as photoresist formulations, substitution formulations, activating formulations, and linker formulations. These formulations may be useful, for example, in the manufacture and use of polynucleotide microarrays disclosed herein.

[0095] Photoresist formulations Disclosed herein are photoresist formulations comprising a photoactive compound and a photoprotective compound. In some embodiments, the photoactive compound is a photoacid generator. Exposure of the photoactive compound to electromagnetic radiation can induce a photochemical event that generates a compound that continues to induce a secondary reaction of material transformation within a diffusion-limited radius. The photoresist formulation may comprise a photoactive compound comprising a radiation-sensitive catalyst precursor, e.g., a photoacid generator (PAG), a plurality of chemical groups that can react by deletion, addition, or rearrangement in the presence of the catalyst, and optional additives (e.g., surfactants, photosensitizers, and etching resists) to improve performance or processability.

[0096] In some embodiments, the photoacid generator is a cationic photoinitiator. A photoinitiator can be added to a formulation to convert absorbed light energy, UV or visible light, into chemical energy in the form of an initiator, such as a free radical or a cation. The ability of some types of cationic photoinitiators to function as potential photochemical sources of very strong protic or Lewis acids is generally the basis for their use in photoimaging applications. In some embodiments, the photoacid generator is an iodonium salt, polonium salt, or sulfonium salt. In some embodiments, the photoacid generator is (4-methoxyphenyl)phenyliodonium or trifluoromethanesulfonate. In some embodiments, the photoacid generator is (2,4-dihydroxyphenyl)dimethylsulfonium triflate or (4-methoxyphenyl)dimethylsulfonium triflate, as shown below. TIFF2026123166000005.tif22128

[0097] In some embodiments, the photoacid generator is an iodonium and sulfonium salt of triflate, phosphate and / or antimonate. In some embodiments, the photoacid generator is present in amounts less than 0.1%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, and 2.1% of the total formulation. %, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3.0%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4.0%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8% 4.9%, 5.0%, 5.1%, 5.2%, 5.3%, 5.4%, 5.5%, 5.6%, 5.7%, 5.8%, 5.9%, 6.0%, 6.1%, 6.2%, 6.3%, 6.4%, 6.5%, 6.6%, 6.7%, 6.8%, 6.9%, 7.0%, 7.1%, 7.2%, 7.3%, 7.4%, 7.5% The concentrations are 7.6%, 7.7%, 7.8%, 7.9%, 8.0%, 8.1%, 8.2%, 8.3%, 8.4%, 8.5%, 8.6%, 8.7%, 8.8%, 8.9%, 9.0%, 9.1%, 9.2%, 9.3%, 9.4%, 9.5%, 9.6%, 9.7%, 9.8%, 9.9%, or 10.0%. In some embodiments, the photoacid generator is present at a concentration of approximately 0.5–5% by weight of the total photoresist formulation.

[0098] In some embodiments, the photoprotective compound is titanium dioxide, zinc sulfide, magnesium fluoride, etc.

[0099] In some embodiments, the photoresist formulation further comprises a polymer and a solvent.

[0100] In some embodiments, the polymers are poly(vinyl alcohol), dextran, sodium alginate, poly(aspartic acid), poly(ethylene glycol), poly(ethylene oxide), poly(vinylpyrrolidone), poly(acrylic acid), poly(acrylic acid)-sodium salt, poly(acrylamide), poly(N-isopropylacrylamide), poly(hydroxyethyl acrylate), poly(acrylic acid), poly(sodium styrenesulfonate), poly(acrylamide-2-methyl-1-propanesulfonic acid), polysaccharides, and cellulose derivatives.

[0101] In some embodiments, the polymer is a non-crosslinkable, inert polymer. In some embodiments, the polymer is polyvinylpyrrolidone. The general structure of polyvinylpyrrolidone is as follows: where n is any positive integer greater than 1. TIFF2026123166000006.tif28128

[0102] In some embodiments, the polymer is a polymer of vinylpyrrolidone. In some embodiments, the polymer is polyvinylpyrrolidone. Polyvinylpyrrolidone is soluble in water and other polar solvents. When dry, the polymer is a light, flaky powder that generally readily absorbs up to 40% of its weight in water in the air. In solution, it has excellent wettability and readily forms films. In some embodiments, the polymer is vinylpyrrolidone or vinyl alcohol. In some embodiments, the polymer is polymethyl methacrylate.

[0103] In some embodiments, the polymer is present in amounts less than about 0.1% by weight, 0.1% by weight, 0.2% by weight, 0.3% by weight, 0.4% by weight, 0.5% by weight, 0.6% by weight, 0.7% by weight, 0.8% by weight, 0.9% by weight, 1.0% by weight, 1.1% by weight, 1.2% by weight, 1.3% by weight, 1.4% by weight, 1.5% by weight, 1.6% by weight, 1.7% by weight, 1.8% by weight, 1.9% by weight, 2.0% by weight, 2.1% by weight, 2.2% by weight, 2.3% by weight, and 2.4% by weight of the total formulation. The concentrations are 2.5% by weight, 2.6% by weight, 2.7% by weight, 2.8% by weight, 2.9% by weight, 3.0% by weight, 3.1% by weight, 3.2% by weight, 3.3% by weight, 3.4% by weight, 3.5% by weight, 3.6% by weight, 3.7% by weight, 3.8% by weight, 3.9% by weight, 4.0% by weight, 4.1% by weight, 4.2% by weight, 4.3% by weight, 4.4% by weight, 4.5% by weight, 4.6% by weight, 4.7% by weight, 4.8% by weight, 4.9% by weight, 5.0% by weight, or greater than 5.0% by weight. In some embodiments, the polymer is concentrated at a concentration of about 0.5 to 5% by weight of the total formulation. In some embodiments, the polymer is concentrated at a concentration of 2.5 to 5% by weight of the total formulation.

[0104] In some embodiments, the photoresist formulation comprises a photoacid generator and a photosensitizer in a polymer matrix dispersed in a solvent. In some embodiments, the polymer in the photoresist composition is generally inert and non-crosslinkable, but the photoactive compound readily generates a sufficient amount of photoacid upon exposure to electromagnetic radiation, resulting in a desired reaction that produces the product in an acceptable yield.

[0105] In some embodiments, the solvent is concentrated at a concentration of about 80–90% by weight of the total formulation. In some embodiments, the solvent is concentrated at a concentration of about 70%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% of the total formulation.

[0106] In some embodiments, the solvent is water, ethyl lactate, n-methylpyrrolidone, or a combination thereof. In some embodiments, ethyl lactate is more than 50% soluble in water to form the solvent. In some embodiments, the solvent contains about 10% propylene glycol methyl ether acetate (PGMEA) and about 90% DI water. In some embodiments, the solvent contains up to about 20% PGMEA. In some embodiments, the solvent contains 50% ethyl lactate and 50% n-methylpyrrolidone. In some embodiments, the solvent is n-methylpyrrolidone. In some embodiments, the solvent is water, an organic solvent, or a combination thereof. In some embodiments, the organic solvent is n-methylpyrrolidone, dimethylformamide, or a combination thereof. In some embodiments, the solvent is concentrated at a concentration of about 70% by weight, 70% by weight, 71% by weight, 72% by weight, 73% by weight, 74% by weight, 75% by weight, 76% by weight, 77% by weight, 78% by weight, 79% by weight, 80% by weight, 81% by weight, 82% by weight, 83% by weight, 84% by weight, 85% by weight, 86% by weight, 87% by weight, 88% by weight, 89% by weight, 90% by weight, 91% by weight, 92% by weight, 93% by weight, 94% by weight, 95% by weight, 96% by weight, 97% by weight, 98% by weight, 99% by weight, or greater than 99% by weight of the total formulation. In some embodiments, the solvent is concentrated at a concentration of about 80-90% by weight of the total formulation.

[0107] In certain embodiments, the photoresist formulation comprises about 0.5 to 5% by weight of a photoacid generator, about 80 to 90% by weight of a solvent, and about 0.1 to 5% by weight of a polymer. In certain embodiments, the photoresist formulation comprises about 0.5 to 5% by weight of a photoacid generator, about 80 to 97% by weight of a solvent, and about 2.5 to 5% by weight of a polymer.

[0108] In certain embodiments, the photoresist formulation comprises a photosensitizer, a photoactive compound, a polymer, and a solvent.

[0109] In some embodiments, the photoresist formulation forms a photoresist layer on the surface of the microarray.

[0110] Activated preparation Disclosed herein are activating formulations for activating carboxylic acids so that they react with free amino groups. In some embodiments, the activating formulation comprises an activator (also called a coupling reagent). In some embodiments, the coupling reagent is a carbodiimide or a triazole. In some embodiments, the carbodiimide is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. In some embodiments, the carboxylic acid group activating compound is N-hydroxysuccinimide (NHS). In some embodiments, the activating formulation optionally comprises a solvent and / or a polymer.

[0111] In some embodiments, the activating agent further comprises R2-acetic acid, such as bromoacetic acid, chloroacetic acid, fluoroacetic acid, or iodoacetic acid. In some embodiments, the activating agent further comprises a coupling molecule, such as a DMT-protecting nucleotide.

[0112] In some embodiments, the coupling reagent is selected from 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide [EDC], N-hydroxysuccinimide [NHS], 1,3-diisopropylcarbodiimide [DIC], hydroxybenzotriazole (HOBt), (O-(7-azabenzotriazole-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate) [HATU], benzotriazole-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate [PyBOP], and N,N-diisopropylethylamine [DIEA]. In some embodiments, the solvent is water. In some embodiments, the solvent is N-methylpyrrolidone (NMP). In some embodiments, the coupling reagent converts the carboxylic acid to a carbonyl group (i.e., activates the carboxylic acid group). In some embodiments, the carboxylic acid group is activated for about 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 45 minutes, or 60 minutes after exposure to the substitution reaction formulation.

[0113] In some embodiments, the coupling reagent is less than about 0.1% by weight, 0.1% by weight, 0.2% by weight, 0.3% by weight, 0.4% by weight, 0.5% by weight, 0.6% by weight, 0.7% by weight, 0.8% by weight, 0.9% by weight, 1.0% by weight, 1.1% by weight, 1.2% by weight, 1.3% by weight, 1.4% by weight, 1.5% by weight, 1.6% by weight, 1.7% by weight, 1.8% by weight, 1.9% by weight, 2.0% by weight, 2.1% by weight, 2.2% by weight, 2.3% by weight. The concentrations are 2.4% by weight, 2.5% by weight, 2.6% by weight, 2.7% by weight, 2.8% by weight, 2.9% by weight, 3.0% by weight, 3.1% by weight, 3.2% by weight, 3.3% by weight, 3.4% by weight, 3.5% by weight, 3.6% by weight, 3.7% by weight, 3.8% by weight, 3.9% by weight, 4.0% by weight, 4.1% by weight, 4.2% by weight, 4.3% by weight, 4.4% by weight, 4.5% by weight, 4.6% by weight, 4.7% by weight, 4.8% by weight, 4.9% by weight, or 5.0% by weight. In some embodiments, the coupling reagent is concentrated at a concentration of about 0.5 to 5% by weight of the total formulation. In some embodiments, the coupling reagent is concentrated at a concentration of 2 to 4% by weight of the total formulation.

[0114] In any of the above combinations, the formulation may be completely removable with water. Therefore, in some embodiments, water can be used to wash away the formulation after exposure.

[0115] In some embodiments, the activating formulation comprises 4% by weight of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 2% by weight of N-hydroxysuccinimide (NHS) dissolved in deionized water. In some embodiments, the activating formulation comprises 4% by weight of 1,3-diisopropylcarbodiimide (DIC) and 2% by weight of hydroxybenzotriazole (HOBt) dissolved in NMP. In some embodiments, the activating formulation comprises 4% by weight of (O-(7-azabenzotriazole-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate) (HATU) and 2% by weight of N,N-diisopropylethylamine (DIEA) dissolved in NMP. In some embodiments, the activating formulation comprises 4% by weight of benzotriazole-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) and 2% by weight of DIEA dissolved in NMP.

[0116] In some embodiments, the solvent is water. In some embodiments, the solvent is concentrated at a concentration of about 80–90% by weight of the total formulation. In some embodiments, the solvent is concentrated at a concentration of about 70%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% by weight of the total formulation.

[0117] In some embodiments, the polymer is polyvinylpyrrolidone and / or polyvinyl alcohol. In some embodiments, the polymer is present in amounts less than about 0.1% by weight, 0.1% by weight, 0.2% by weight, 0.3% by weight, 0.4% by weight, 0.5% by weight, 0.6% by weight, 0.7% by weight, 0.8% by weight, 0.9% by weight, 1.0% by weight, 1.1% by weight, 1.2% by weight, 1.3% by weight, 1.4% by weight, 1.5% by weight, 1.6% by weight, 1.7% by weight, 1.8% by weight, 1.9% by weight, 2.0% by weight, 2.1% by weight, 2.2% by weight, 2.3% by weight, and 2. The concentrations are 4% by weight, 2.5% by weight, 2.6% by weight, 2.7% by weight, 2.8% by weight, 2.9% by weight, 3.0% by weight, 3.1% by weight, 3.2% by weight, 3.3% by weight, 3.4% by weight, 3.5% by weight, 3.6% by weight, 3.7% by weight, 3.8% by weight, 3.9% by weight, 4.0% by weight, 4.1% by weight, 4.2% by weight, 4.3% by weight, 4.4% by weight, 4.5% by weight, 4.6% by weight, 4.7% by weight, 4.8% by weight, 4.9% by weight, or 5.0% by weight. In preferred embodiments, the polymer is present at a concentration of about 0.5 to 5% by weight of the total formulation.

[0118] In some embodiments, the coupling reagent is a carbodiimide. In some embodiments, the coupling reagent is a triazole. In some embodiments, the coupling reagent is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. In some embodiments, the coupling reagent is less than about 0.1% by weight, 0.1% by weight, 0.2% by weight, 0.3% by weight, 0.4% by weight, 0.5% by weight, 0.6% by weight, 0.7% by weight, 0.8% by weight, 0.9% by weight, 1.0% by weight, 1.1% by weight, 1.2% by weight, 1.3% by weight, 1.4% by weight, 1.5% by weight, 1.6% by weight, 1.7% by weight, 1.8% by weight, 1.9% by weight, 2.0% by weight, 2.1% by weight, 2.2% by weight, 2.3% by weight. The concentrations are 2.4% by weight, 2.5% by weight, 2.6% by weight, 2.7% by weight, 2.8% by weight, 2.9% by weight, 3.0% by weight, 3.1% by weight, 3.2% by weight, 3.3% by weight, 3.4% by weight, 3.5% by weight, 3.6% by weight, 3.7% by weight, 3.8% by weight, 3.9% by weight, 4.0% by weight, 4.1% by weight, 4.2% by weight, 4.3% by weight, 4.4% by weight, 4.5% by weight, 4.6% by weight, 4.7% by weight, 4.8% by weight, 4.9% by weight, or 5.0% by weight. In preferred embodiments, the coupling reagent is present at a concentration of approximately 0.5 to 5% by weight of the total formulation.

[0119] Linker preparations Linker formulations are also disclosed herein. Linker formulations may include components such as a solvent, a polymer, a linker molecule, and a coupling reagent. In some embodiments, the polymer comprises 1% by weight of polyvinyl alcohol and 2.5% by weight of polyvinylpyrrolidone, the linker molecule is 1.25% by weight of polyethylene oxide, the coupling reagent is 1% by weight of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and the solvent is water. In some embodiments, the polymer comprises 0.5 to 5% by weight of polyvinyl alcohol and 0.5 to 5% by weight of polyvinylpyrrolidone, the linker molecule is 0.5 to 5% by weight of polyethylene oxide, the coupling reagent is 0.5 to 5% by weight of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and the solvent is water.

[0120] In some embodiments, the solvent is water, an organic solvent, or a combination thereof. In some embodiments, the organic solvent is N-methylpyrrolidone, dimethylformamide, dichloromethane, dimethyl sulfoxide, or a combination thereof. In some embodiments, the solvent is concentrated at about 70% by weight, 70% by weight, 71% by weight, 72% by weight, 73% by weight, 74% by weight, 75% by weight, 76% by weight, 77% by weight, 78% by weight, 79% by weight, 80% by weight, 81% by weight, 82% by weight, 83% by weight, 84% by weight, 85% by weight, 86% by weight, 87% by weight, 88% by weight, 89% by weight, 90% by weight, 91% by weight, 92% by weight, 93% by weight, 94% by weight, 95% by weight, 96% by weight, 97% by weight, 98% by weight, 99% by weight, or greater than 99% by weight of the total formulation. In preferred embodiments, the solvent is concentrated at about 80-90% by weight of the total formulation.

[0121] In some embodiments, the polymer is polyvinylpyrrolidone and / or polyvinyl alcohol. The general structure of polyvinyl alcohol is given by the following equation, where n is any positive integer greater than 1. TIFF2026123166000007.tif23128

[0122] In some embodiments, the polymer is present in amounts less than about 0.1% by weight, 0.1% by weight, 0.2% by weight, 0.3% by weight, 0.4% by weight, 0.5% by weight, 0.6% by weight, 0.7% by weight, 0.8% by weight, 0.9% by weight, 1.0% by weight, 1.1% by weight, 1.2% by weight, 1.3% by weight, 1.4% by weight, 1.5% by weight, 1.6% by weight, 1.7% by weight, 1.8% by weight, 1.9% by weight, 2.0% by weight, 2.1% by weight, 2.2% by weight, 2.3% by weight, and 2. The concentrations are 4% by weight, 2.5% by weight, 2.6% by weight, 2.7% by weight, 2.8% by weight, 2.9% by weight, 3.0% by weight, 3.1% by weight, 3.2% by weight, 3.3% by weight, 3.4% by weight, 3.5% by weight, 3.6% by weight, 3.7% by weight, 3.8% by weight, 3.9% by weight, 4.0% by weight, 4.1% by weight, 4.2% by weight, 4.3% by weight, 4.4% by weight, 4.5% by weight, 4.6% by weight, 4.7% by weight, 4.8% by weight, 4.9% by weight, or 5.0% by weight. In preferred embodiments, the polymer is present at a concentration of about 0.5 to 5% by weight of the total formulation.

[0123] A linker molecule may be a molecule inserted between a surface disclosed herein and a polynucleotide chain being synthesized via a coupling molecule. While the linker molecule does not necessarily impart functionality to the resulting polynucleotide chain, such as molecular recognition, it can instead increase the distance between the surface and the polynucleotide chain, thereby improving the exposure of the functional region(s) of the polynucleotide chain on the surface. In some embodiments, the linker may be about 4 to about 40 atoms long for exposure. The linker molecule may be, for example, an arylacetylene, an ethylene glycol oligomer containing 2 to 10 monomer units (PEG), a diamine, a diacid, an amino acid, or a combination thereof. Examples of diamines include ethylenediamine and diaminopropane. Alternatively, the linker may be the same molecular type as the synthesized one (e.g., a nascent polymer or various coupling molecules), such as a polymer of polypeptides and amino acid derivatives, e.g., aminohexanoic acid, or a nucleic acid polymer. In some embodiments, the linker molecule is a molecule having a carboxyl group at the first end and a protecting group at the second end. In some embodiments, the protecting group is a t-butoxycarbonyl (t-Boc) protecting group or a 9-fluorenylmethoxycarbonyl (Fmoc) protecting group. In some embodiments, the linker molecule is or comprises an arylacetylene, polyethylene glycol, a nascent polypeptide, a diamine, a dioic acid, a peptide, a nucleic acid monomer or polymer, or a combination thereof.In some embodiments, the linker molecule is present in amounts less than about 0.1% by weight of the total formulation, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, and 2%. The concentrations are 0.4% by weight, 2.5% by weight, 2.6% by weight, 2.7% by weight, 2.8% by weight, 2.9% by weight, 3.0% by weight, 3.1% by weight, 3.2% by weight, 3.3% by weight, 3.4% by weight, 3.5% by weight, 3.6% by weight, 3.7% by weight, 3.8% by weight, 3.9% by weight, 4.0% by weight, 4.1% by weight, 4.2% by weight, 4.3% by weight, 4.4% by weight, 4.5% by weight, 4.6% by weight, 4.7% by weight, 4.8% by weight, 4.9% by weight, or 5.0% by weight. In preferred embodiments, the linker molecule is present at a concentration of about 0.5 to 5% by weight of the total formulation.

[0124] The unbonded (or free end) portion of the linker molecule may have reactive functional groups that block, protect, or otherwise prevent them from being used in reactions by protecting groups that can be removed. Protecting groups can be bonded to the linker molecule to protect its reactive functional groups. Possible protecting groups include all protecting groups that are unstable in acids and bases. For example, a linkeramine group can be protected by t-butoxycarbonyl (t-BOC or BOC) or benzyloxycarbonyl (CBZ), both of which are unstable in acids, or by 9-fluorenylmethoxycarbonyl (FMOC), which is unstable in bases.

[0125] Additional protecting groups that can be used include acid-unstable groups to protect the amino moiety: tert-amyloxycarbonyl, adamantyloxycarbonyl, 1-methylcyclobutyloxycarbonyl, 2-(p-biphenyl)propyl(2)oxycarbonyl, 2-(p-phenylazophenylyl)propyl(2)oxycarbonyl, alpha, alpha-dimethyl-3,5-dimethyloxybenzyloxycarbonyl, 2-phenylpropyl(2)oxycarbonyl, 4-methyloxybenzyloxycarbonyl, furfuryloxycarbonyl, triphenylmethyl(trityl), p-toluenesulfenylaminocarbonyl, dimethylphosphinotioyl, diphenylphosphinotioyl, 2-benzoyl-1-methylvinyl, o-nitrophenylsulfenyl, and 1-naphthylidene; for protecting the amino moiety. Examples of base-unstable groups include 9-fluorenylmethyloxycarbonyl, methylsulfonylethyloxycarbonyl, and 5-benzoisoazoylmethyleneoxycarbonyl; groups for protecting amino moieties that are unstable when reduced include dithiasuccinoyl, p-toluenesulfonyl, and piperidino-oxycarbonyl; groups for protecting amino moieties that are unstable when oxidized include (ethylthio)carbonyl; groups for protecting amino moieties that are unstable to other reagents (appropriate agents are listed in parentheses after the group) include phthaloyl(hydrazine), trifluoroacetyl(piperidine), and chloroacetyl(2-aminothiophenol); acid-unstable groups for protecting carboxylic acids include tert-butyl esters; and acid-unstable groups for protecting hydroxyl groups include dimethyltrityl. See also Greene, TW, Protective Groups in Organic Synthesis, Wiley-Interscience, NY, (1981).

[0126] substrate Substrates are also disclosed herein. In some embodiments, the substrate surface is planar (i.e., two-dimensional). In some embodiments, the substrate surface is functionalized with free amine groups.

[0127] In some embodiments, the surface is a material or group of materials having rigidity or semi-rigidity. In some embodiments, the surface may be substantially flat, but in some embodiments, it may be desirable to physically separate synthetic regions of different molecules or features having, for example, wells, raised regions, pins, pillars, etched trenches, etc. In certain embodiments, the surface may be porous. Examples of surface materials include silicon, biocompatible polymers such as poly(methyl methacrylate) (PMMA) and polydimethylsiloxane (PDMS), glass, SiO2 (e.g., thermooxide silicon wafers used in the semiconductor industry), quartz, silicon nitride, functionalized glass, gold, platinum, and aluminum. Examples of functionalized surfaces include amino-functionalized glass, carboxy-functionalized glass, and hydroxy-functionalized glass. Furthermore, the surface may be optionally coated by one or more layers to provide a second surface for molecular bonding or functionalization, increased or decreased reactivity, bonding detection, or other specialized applications. The surface material and / or layers(s) may be porous or non-porous. For example, the surface may be composed of porous silicon. Furthermore, the surface may be a silicon wafer or chip, as used in the semiconductor device manufacturing industry. In the case of a wafer or chip, multiple arrays can be synthesized on the wafer.

[0128] In some embodiments, the substrate includes a porous layer (i.e., a three-dimensional layer) containing functional groups for binding first monomer components. In some embodiments, the substrate surface includes pillars for binding or synthesizing polynucleotides. In some embodiments, a porous layer is added on top of the pillars.

[0129] Porous layer substrate The porous layer that can be used may be a flat, permeable polymer material with a porous structure having amine groups (either inherent to the constituent polymer or introduced into the porous layer) for binding the first polynucleotide components. For example, the porous layer may be composed of porous silicon having functional groups for binding polymer components bonded to the surface of the porous silicon. In another example, the porous layer may include a crosslinked polymer material. In some embodiments, the porous layer may be polystyrene, saccharose, dextran, polyacryloylmorpholine, polyacrylate, polymethylacrylate, polyacrylamide, polyacrylopyrrolidone, polyvinyl acetate, polyethylene glycol, agarose, Sepharose, other conventional chromatography-type materials and derivatives and mixtures thereof. In some embodiments, the porous layer constituent material is selected from poly(vinyl alcohol), dextran, sodium alginate, poly(aspartic acid), poly(ethylene glycol), poly(ethylene oxide), poly(vinylpyrrolidone), poly(acrylic acid), poly(acrylic acid)-sodium salt, poly(acrylamide), poly(N-isopropylacrylamide), poly(hydroxyethyl acrylate), poly(acrylic acid), poly(sodium styrenesulfonate), poly(2-acrylamide-2-methyl-1-propanesulfonic acid), polysaccharides, and cellulose derivatives. Preferably, the porous layer has a porosity of 10 to 80%. In some embodiments, the thickness of the porous layer is in the range of about 0.01 μm to about 1,000 μm. In preferred embodiments, the thickness of the porous layer is in the range of about 0.1 μm to about 500 μm. The pore size contained in the porous layer may be in the range of 2 nm to about 100 μm. In a preferred embodiment, the pore size may be approximately 10 nm to approximately 50 μm.

[0130] According to another embodiment of the present invention, a substrate is provided comprising a porous polymer material having a porosity of 10-80%, wherein reactive groups are adapted in use to interact by chemically bonding to the pore surface and chemically bonding to reactive species, such as deprotected monomer components or polymer chains. In some embodiments, the reactive groups are free amine groups. The free amine groups are free to bond, for example, to activated carboxylic acid groups of coupling molecules or substituted acetic acid molecules.

[0131] In one embodiment, the porous layer is in contact with a support layer. The support layer includes, for example, a metal, plastic, silicon, silicon oxide, or silicon nitride. In another embodiment, the porous layer can be in contact with a patterned surface, such as the top of a pillar substrate as described below.

[0132] Pillar substrate In some embodiments, the substrate may include a top and bottom surface, as well as a planar layer having a plurality of pillars, the plurality of pillars being operably connected to the layer at positionally defined locations, each pillar having a plane extending from the layer, the distance between the face of each pillar and the top surface of the layer being approximately 1,000 to 5,000 angstroms, and the plurality of pillars being approximately 10,000 / cm 2 It exists at a density exceeding that.

[0133] In some embodiments, the planar layer comprises metal, plastic, silicon, silicon oxide, or silicon nitride. In some embodiments, the metal is chromium. In some embodiments, the metal is chromium, titanium, aluminum, tungsten, gold, silver, tin, lead, thallium, indium, or a combination thereof. In some embodiments, the planar layer is at least 98.5–99% (by weight) of metal, plastic, silicon, silicon oxide, or silicon nitride. In some embodiments, the planar layer is 100% metal, silicon, silicon oxide, or silicon nitride. In some embodiments, the planar layer is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, or 99% of metal, silicon, silicon oxide, or silicon nitride. In some embodiments, the layer is a homogeneous layer of metal, silicon, silicon oxide, or silicon nitride.

[0134] In some embodiments, the distance between the surface of each pillar and the top surface of the planar layer is less than approximately 1,000, 2,000, 3,000, 3,500, 4,500, 5,000, 5,500, 6,000, 6,500, 7,000, 7,500, 8,000, 8,500, 9,000, 9,500, or 10,000 angstroms (or any integer in between). In certain embodiments, the distance between the surface of each pillar and the top surface of the planar layer may be less than approximately 1,000 to 5,000 angstroms.

[0135] In some embodiments, the surface of each pillar is parallel to the top surface of the layer. In some embodiments, the surface of each pillar is substantially parallel to the top surface of the layer.

[0136] In some embodiments, multiple pillars may be 500, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 11,000, or 12,000 / cm 2 They exist at a density greater than (or any integer in between). In some embodiments, multiple pillars exist at a density of 10,000 / cm³. 2They exist at a density exceeding. In some embodiments, the plurality of pillars have a density of about 10,000 / cm 2 ~ about 2.5 million / cm 2 (or any integer therebetween). In some embodiments, the plurality of pillars exist at a density exceeding 2.5 million / cm 2 .

[0137] In some embodiments, the surface area of each pillar surface is at least 1μm 2 . In some embodiments, the surface area of each pillar surface can be at least 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50μm 2 (or any integer therebetween). In some embodiments, the surface area of each pillar surface has a total area less than 10,000μm 2 . In some embodiments, the surface area of each pillar surface has a total area less than 500, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 11,000, or 12,000μm 2 (or any integer therebetween).

[0138] In some embodiments, the distance between the surface of each pillar and the lower surface of the layer is 2,000 - 7,000 angstroms. In some embodiments, the distance between the surface of each pillar and the lower surface of the layer is less than about 500, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 11,000, or 12,000 angstroms (or any integer therebetween). In some embodiments, the distance between the surface of each pillar and the lower surface of the layer is 7,000, 3,000, 4,000, 5,000, 6,000, or 7,000 angstroms (or any integer therebetween).

[0139] In some embodiments, the layer is 1,000 to 2,000 angstroms thick. In some embodiments, the layer is less than approximately 500, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 11,000, or 12,000 angstroms (or any integer in between).

[0140] In some embodiments, the center of each pillar is at least 2,000 angstroms from the center of any other pillar. In some embodiments, the center of each pillar is at least about 500, 1,000, 2,000, 3,000, or 4,000 angstroms (or any integer in between) from the center of any other pillar. In some embodiments, the center of each pillar is at least about 2 μm to 200 μm from the center of any other pillar.

[0141] In some embodiments, at least one pillar or each pillar contains silicon. In some embodiments, at least one pillar or each pillar contains silicon dioxide or silicon nitride. In some embodiments, at least one or each pillar is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, or 99% (by weight) silicon dioxide.

[0142] In some embodiments, the substrate includes linker molecules having free amino termini bonded to the surface of each pillar. In some embodiments, the substrate includes linker molecules having free amino termini bonded to the surface of at least one pillar. In some embodiments, the substrate includes linker molecules having protecting groups bonded to the surface of each pillar. In some embodiments, the substrate includes linker molecules having protecting groups bonded to the surface of at least one pillar. In some embodiments, the substrate includes coupling molecules bonded to the surface of at least one pillar. In some embodiments, the substrate includes coupling molecules bonded to the surface of each pillar. In some embodiments, the substrate includes a polymer in contact with at least one surface of the pillars. In some embodiments, the substrate includes a polymer in a gelatinous form in contact with at least one surface of the pillars. In some embodiments, the substrate includes a polymer in a solid form in contact with at least one surface of the pillars.

[0143] In some embodiments, at least one surface of the pillars of the substrate is derivatized. In some embodiments, the substrate includes polymer chains bonded to at least one surface of the pillars. In some embodiments, the polymer chains include polynucleotide chains. In some embodiments, the bond to the surface of at least one pillar is via covalent bonds.

[0144] In some embodiments, the surface of each pillar is square or rectangular in shape. In some embodiments, the substrate can be bonded to a silicon dioxide layer. The thickness of the silicon dioxide layer is approximately 0.5 μm to 3 μm. In some embodiments, the substrate can be bonded to a wafer, for example, a silicon wafer. The thickness of the silicon dioxide layer is approximately 700 μm to 750 μm.

[0145] array Arrays are also disclosed herein. In some embodiments, the array is a two-dimensional array. In some embodiments, the array has a surface including a substrate, the substrate including a planar layer having an upper surface and a lower surface.

[0146] In some embodiments, the two-dimensional array includes feature regions bonded to the surface at positionally defined locations, each feature region including a set of polynucleotide chains of a determinable sequence and intended length, wherein within each feature region, the fraction of polynucleotides in the set having the intended length is characterized by an average coupling efficiency of about 98% at each coupling step. In some embodiments, the array includes a plurality of pillars operably bonded to a layer at positionally defined locations, each pillar having a plane extending from the layer, the distance between the surface of each pillar and the top surface of the layer being 1,000 to 5,000 angstroms, and the plurality of pillars being 10,000 / cm 2 It exists at a higher density.

[0147] In some embodiments, the surface of the array is functionalized with free amine groups. In some embodiments, the surface density of free amine groups on the array is 10 / cm³. 2 , 100 / cm 2 , 1,000 / cm 2 , 10,000 / cm 2 , 100,000 / cm 2 1,000,000 / cm 2 , or 10,000,000 / cm 2 It exceeds 10 / cm². In some embodiments, the surface density of the feature portion on the array is 10 / cm². 2 , 100 / cm 2 , 1,000 / cm 2 , 10,000 / cm 2 , 100,000 / cm 2 1,000,000 / cm 2 , or 10,000,000 / cm 2 It exceeds.

[0148] In some embodiments, the array is a three-dimensional array, for example, a porous array including feature portions bonded to the surface of a porous array. In some embodiments, the surface of the porous array includes an outer surface and a surface defining the pore volume within the porous array. In some embodiments, the three-dimensional array includes feature portions bonded to the surface at positionally defined locations, each of which includes a set of polynucleotide chains with a determinable sequence and intended length. In one embodiment, within each individual feature portion, the fraction of polynucleotide chains in a set having an intended length is characterized by an average coupling efficiency of more than 98% at each coupling step.

[0149] In some embodiments, during polynucleotide synthesis, the average coupling efficiency of each coupling step for joining an input nucleotide to a terminal nucleotide is at least 98.5%. In some embodiments, the average coupling efficiency of each coupling step is at least 99%. In some embodiments, the average coupling efficiency of each coupling step is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%.

[0150] In some embodiments, the purity of each feature portion in a fraction of a predetermined polynucleotide chain of the full length is F of the fraction of a predetermined polynucleotide chain of the full length having a predetermined sequence and a predetermined total sequence length N, where F = 10 (N+1)·log(E / 100%)Characterized by the following: In some embodiments, F is characterized by having an average coupling efficiency E for coupling each monomer in a given sequence of at least 98.5%. In some embodiments, F is characterized by having an average coupling efficiency E for coupling each monomer in a given sequence of at least 98.5%. In some embodiments, the average coupling efficiency E for each coupling step is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%.

[0151] In some embodiments, the distribution of sequence lengths on the array is based on the synthesis of polynucleotide sequences of a defined length (e.g., 64 mers). In each coupling step, the length of sequences that do not result in the desired coupling when the capping solution is used is fixed at that length. The length distribution, depending on the step yield for each sequence length shorter than the complete sequence, is given by the following equation: F(N)=10 (N+1)·log(E / 100%) -10 (N)·log(E / 100%) In the formula, F(N) is the ratio of sequences on the array at lengths N shorter than the full-length sequence, and E is the average coupling efficiency (percentage). The exact value of E at each length N can also be used to generate the exact number of oligomers at each length.

[0152] The ratio of the total length array is obtained by the following formula: F(N)=10 (N)·log(E / 100%) In the formula, F(N) is the ratio of sequences on the array of full-length sequences (without further coupling steps), and E is the mean coupling efficiency.

[0153] In some embodiments, the sequence length N is at least 64 monomers in length, and the proportion of a given polynucleotide chain less than the total length is equal to (1-F). In some embodiments, the sequence length N is at least 65 monomers in length.

[0154] In some embodiments, each polynucleotide chain has a length of 5 to 100 monomers. In some embodiments, each polynucleotide chain has a length of at least 64 monomers. In some embodiments, each polynucleotide chain has a length of at least 65 monomers. In some embodiments, each polynucleotide chain has a length of at least 100 monomers or more than 100 monomers. In some embodiments, each polynucleotide chain has a length of at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 monomers. In some embodiments, each polynucleotide chain has a length of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 5 The number of monomers is 2, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 monomers, or greater than 100 monomers. In some embodiments, each polynucleotide chain contains one or more modified nucleotides. In some embodiments, each polynucleotide contains one or more ribonucleotides.

[0155] An array may include at least 1,000 distinct feature segments bonded to its surface. An array may include at least 10,000 distinct feature segments bonded to its surface. An array may include at least 100, 500, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, or more than 10,000 (or any integer in between) distinct feature segments bonded to its surface.

[0156] In some embodiments, the array includes at least 1,000 different polynucleotide chains bound to the surface. In some embodiments, the array includes at least 10,000 different polynucleotide chains bound to the surface. In some embodiments, the array includes at least 100, 500, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, or more than 10,000 (or any integer in between) different polynucleotide chains bound to the surface.

[0157] In some embodiments, each feature comprises at least 500 identical full-length polynucleotide chains, each identical polynucleotide chain having a predetermined total length of at least 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, or more than 100 monomers. In some embodiments, each feature portion includes identical polynucleotide chains of 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 lengths, each identical polynucleotide chain having a predetermined total length of at least 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, or more than 100 monomers.

[0158] In some embodiments, each of the positionally defined locations is in a different known location that is physically separated from each of the other positionally defined locations. In some embodiments, each of the positionally defined locations is a positionally distinguishable location. In some embodiments, each determinable array is a known array. In some embodiments, each determinable array is a distinct array.

[0159] In some embodiments, each feature is bonded to the surface of the array at a different positionally defined location, the positionally defined location of each feature corresponds to a positionally defined location of a pillar, and the uppermost surface of each pillar is at least 1 μm thick. 2 It is the size of

[0160] In some embodiments, the feature is covalently bonded to the surface. In some embodiments, the polynucleotide chain is bonded to the surface via a linker molecule or coupling molecule.

[0161] In some embodiments, the feature region comprises a plurality of distinct, nested, and overlapping polynucleotide chains, each containing a subsequence derived from a source DNA or RNA sequence having a known sequence. In some embodiments, each of the plurality of polynucleotide chains is substantially the same length.

[0162] In some embodiments, each polynucleotide chain within the feature region is substantially the same length. In some embodiments, each polynucleotide chain within the feature region is the same length. In some embodiments, the feature region comprises a plurality of polynucleotide chains, each having a randomly determinable sequence of monomers.

[0163] method Substrate manufacturing method Methods for fabricating substrates are also disclosed herein. In some embodiments, a method for fabricating a substrate includes bundling a porous layer to a support layer. The support layer may include any metal or plastic or silicon or silicon oxide or silicon nitride. In one embodiment, the substrate includes a plurality of amino groups bonded to the substrate for binding nucleic acid monomers during polynucleotide synthesis. In some embodiments, a method for fabricating a substrate includes bundling a porous layer to a plurality of pillars, the porous layer containing functional groups for binding compounds to the substrate, the plurality of pillars being bonded to a planar layer at positionally defined locations, each pillar having a plane extending from the planar layer, the distance between the surface of each pillar and the top surface of the planar layer being about 1,000 to 5,000 angstroms, and the plurality of pillars being about 10,000 / cm 2 It exists at a density exceeding that.

[0164] In some embodiments, the surface of each pillar is parallel to the upper surface of the planar layer. In some embodiments, the surface of each pillar is substantially parallel to the upper surface of the planar layer.

[0165] surface derivatization The substrate can be surface-derivatized within a semiconductor module as described in U.S. Patent Publication 2010 / 0240555 (which is incorporated herein by reference in its entirety for all purposes). An exemplary substrate comprises an oxide pillar ready for surface-derivatization. Surface derivatization is a method of adding aminosilane groups to a substrate to make free amino groups available for coupling biomolecules. In some embodiments, the first molecule to be bonded to the surface-derivatized substrate is t-Boc protected glycine. This coupling procedure is analogous to the standard Merrifield solid-phase peptide synthesis procedure, which is generally known to those skilled in the art.

[0166] In some embodiments, a method for preparing a substrate surface includes obtaining a surface containing silicon dioxide, contacting the surface with a photoresist formulation containing a photoactive compound and a photoprotective compound, optionally a polymer, and a solvent, and applying ultraviolet light to a positionally defined location on the top of the surface that is in contact with the photoresist formulation. In some embodiments, this method may include the step of removing the photoresist formulation located outside the positionally defined location.

[0167] Array manufacturing method Methods for manufacturing arrays are also disclosed herein. In some embodiments, the arrays disclosed herein are synthesized in situ on a surface, for example, a substrate disclosed herein. In some cases, the arrays are fabricated using photolithography. In exemplary embodiments, the substrate is brought into contact with a photoresist formulation. A mask can be used to control the exposure of radiation or light to specific locations on the surface containing free linker molecules or free coupling molecules having a protecting group. At the exposed locations, the protecting group is removed, resulting in one or more newly exposed reactive moieties on the coupling molecule or linker molecule. The desired linker or coupling molecule is then linked to an unprotected bonded molecule, for example, at a free amine group. This process can be repeated to synthesize numerous feature elements at specific locations or positionally defined locations on a surface (see, for example, U.S. Patent No. 5,143,854, U.S. Patent Publications 2007 / 0154946, 2007 / 0122841, 2007 / 0122842, 2008 / 0108149, and 2010 / 0093554 by Pirrung et al., each of which is incorporated herein by reference).

[0168] In some embodiments, the feature portion is bonded to the surface using a photoresist formulation comprising a photoactive compound, a photoprotective compound, and optionally a polymer and a solvent. In some embodiments, the feature portion is bonded to the surface using a photoresist formulation disclosed herein. In some embodiments, the photoresist formulation is removed using water.

[0169] The process for manufacturing the array is also described herein. In exemplary embodiments, a surface is provided that includes bound free amine groups or protected terminal nucleotides. The surface is brought into contact with a photoresist formulation comprising a photoactive compound, a photoprotective compound, and optionally a polymer and a solvent. The surface is exposed to electromagnetic radiation, e.g., ultraviolet (UV) light, in a deep ultraviolet scanner tool according to a pattern defined by a photomask, and the areas exposed to radiation undergo photoacid generation because a photoacid generator is present in the photoresist formulation. In some embodiments, the exposure energy is about 1 mJ / cm². 2 ~about 5J / cm 2 In a preferred embodiment, the exposure energy is approximately 1 mJ / cm². 2 ~about 100mJ / cm 2 In some embodiments, the radiation includes UV light at 248 nm. In some embodiments, the radiation includes ultraviolet light at 365 nm. The radiation for activating the photoacid generator can be of various wavelengths and is not limited to those disclosed herein.

[0170] The surface may be post-sintered in a post-exposure sintering module during exposure. Post-exposure sintering can act as a chemical amplification step. In the sintering step, the photoacid initially produced can be amplified, accelerating its diffusion rate into the substrate. The post-sintering temperature can vary from 75°C to 115°C, depending on the thickness of the porous surface or the planar layer of the substrate. The surface may be post-sintered for approximately 60 seconds to approximately 120 seconds.

[0171] Next, the photoresist formulation can be removed. Described herein is a method for completely removing the photoresist with deionized (DI) water. In some embodiments, this process is achieved in a developing module. In one embodiment, the wafer is rotated on a vacuum chuck for, for example, 60 to 90 seconds, and deionized water is supplied through a nozzle for about 30 seconds.

[0172] The photoresist formulation can be coated onto a surface within a coupling spin module. The coupling spin module may typically have 20 or more nozzles for dispensing the photoactive coupling formulation. These nozzles may be fabricated to dispense the photoactive coupling formulation by pressurizing cylinders holding these solutions or by a pump dispensing the required amount. In some embodiments, a pump is used to dispense 5–8 cc of the photoactive coupling formulation onto the substrate. In some embodiments, the substrate is rotated on a vacuum chuck for about 10 seconds to about 10 minutes. In some embodiments, the substrate is rotated for about 10 seconds to about 3 minutes. In some embodiments, the substrate is rotated at about 500 rpm to about 10,000 rpm. The substrate may be rotated on a vacuum chuck for 15–30 seconds to dispense the photoactive coupling formulation. The rotation speed can be set to 2000–2500 rpm. In some cases, the substrate is rotated at about 2000–2500 rpm for about 15–30 seconds. In some cases, the substrate is rotated at about 500 rpm for about 180 seconds. In some cases, the circuit board is rotated at approximately 10,000 rpm for about 10 seconds.

[0173] Optionally, a capping film solution coating can be applied to the surface to prevent unreacted amino groups on the substrate from reacting with subsequent coupling molecules. A capping film coating solution of solvent, polymer, and coupling molecule can be prepared. Usable solvents may be organic solvents such as N-methylpyrrolidone, dimethylformamide, or combinations thereof. The capping molecule is typically acetic anhydride, and the polymer may be polyvinylpyrrolidone, polyvinyl alcohol, polymethyl methacrylate, poly(methylisopropenyl)ketone, or poly(2-methylpentene-1-sulfone). In some embodiments, the capping molecule is ethanolamine.

[0174] A capping spin module may be used in this process. The capping spin module may include a single nozzle that can be fabricated to supply a cap film coating solution onto the substrate. This solution can be supplied by pressurizing a cylinder containing the cap film coating solution or by a pump that dispenses the exact amount required. In some embodiments, a pump is used to supply about 5–8 cc of cap coating solution onto the substrate. In some embodiments, the substrate is rotated on a vacuum chuck for about 10 seconds to about 10 minutes. In some embodiments, the substrate is rotated for about 10 seconds to about 3 minutes. In some embodiments, the substrate is rotated at about 500 rpm to about 10,000 rpm. The substrate may be rotated on a vacuum chuck for 15–30 seconds to supply the coupling formulation. The rotation speed can be set to 2000–2500 rpm. In some cases, the substrate is rotated at about 2000–2500 rpm for about 15–30 seconds. In some cases, the substrate is rotated at about 500 rpm for about 180 seconds. In some cases, the circuit board is rotated at approximately 10,000 rpm for about 10 seconds.

[0175] A substrate containing a capping solution can be fired in a capping firing module. The capping firing module may be a hot plate specially configured to accept wafers immediately after the capping film coat has been applied. In some embodiments, provided herein is a method of significantly accelerating the capping reaction by firing a spin-coated capping coat solution on a hot plate. Hot plate firing can reduce the capping time to less than two minutes.

[0176] In one embodiment, byproducts of the capping reaction are removed within a stripper module. The stripper module may comprise multiple nozzles (typically up to 10) configured to supply organic solvents such as acetone, isopropyl alcohol, N-methylpyrrolidone, dimethylformamide, and DI water. In some embodiments, the nozzles are designated for acetone, followed by isopropyl alcohol being supplied onto the rotating wafer. The spin speed can be set to 2000–2500 rpm for about 20 seconds.

[0177] This entire cycle can be repeated with different coupling molecules each time as desired to obtain a polynucleotide chain with a determinable sequence and intended length.

[0178] How to use polynucleotide microarrays Methods of using substrates, formulations, and / or arrays are also disclosed herein. Uses of the arrays disclosed herein include, for example, research purposes, therapeutic purposes, medical diagnostics, and / or stratification of one or more patients.

[0179] Any array described herein can be used as a research tool or for research purposes. In one embodiment, the array can be used in a high-throughput screening assay. For example, a polynucleotide array can be tested by subjecting the array to a DNA or RNA molecule and identifying the presence or absence of a complementary DNA, RNA, or PNA molecule, for example, by detecting at least one change between feature regions of the array.

[0180] In some embodiments, the array is used to detect sequence variants in a sample, such as single nucleotide polymorphisms (SNPs). Detection of sequence variants can be performed by observing the sequence-specific hybridization of a labeled molecule to a probe on the array. Detection of sequence variants can also be performed by binding a sequence suspected of containing a sequence variant to a probe on the array, followed by a polymerase elongation reaction using the labeled nucleotide. In preferred embodiments, a polynucleotide probe is synthesized on the array and hybridized to a nucleotide sequence from a sample suspected of containing a sequence variant. The polynucleotide may be enzymatically active; that is, under preferred polymerization conditions, the polynucleotide can act as a substrate for incorporating complementary nucleotides into a growing chain using polymerase.

[0181] The array can also be used for ligand binding screening assays, substrate specificity determination, or identification of complementary polynucleotide molecules expressed in vivo or in vitro within a particular cell. Such screening assays may utilize, for example, labeling techniques, protease assays, or binding assays, as may be known to those skilled in the art.

[0182] In some embodiments, the array is used to represent a predefined polynucleotide sequence using overlapping polynucleotide sequences. For example, a polynucleotide sequence of a known gene is divided into overlapping sequence segments of any length and any preferred overlapping frame, and the polynucleotide chains corresponding to each sequence segment are synthesized in situ as disclosed herein.

[0183] In some embodiments, a sample is applied to an array having multiple random polynucleotide chains. The random polynucleotide chains can be screened and BLAST can be used to determine homologous domains having, for example, 90% or more identity to a given nucleotide sequence. In some embodiments, the entire polynucleotide sequence can then be synthesized and used to identify potential markers and / or causes of the disease of interest.

[0184] In some embodiments, the array is used for high-throughput screening of one or more genetic factors. The expression of DNA or RNA related to a gene can be investigated by polynucleoid hybridization, which can then be used to estimate the relationship between the gene and the disease.

[0185] In another example, arrays can be used to identify one or more biomarkers. Biomarkers can be used for the diagnosis, prognosis, treatment, and management of diseases. Biomarkers may be expressed, absent, or expressed at different levels in an individual depending on the disease state, stage, and response to treatment. Biomarkers may be, for example, DNA, RNA, PNA, proteins (e.g., enzymes such as kinases), sugars, salts, fats, lipids, or ions.

[0186] The array can also be used for therapeutic purposes, for example, to identify one or more bioactive agents. Methods for identifying bioactive agents may include applying multiple test compounds to the array and identifying at least one test compound as a bioactive agent. Test compounds may be small molecules, aptamers, polynucleotides, chemicals, natural extracts, peptides, proteins, antibody fragments, antibody-like molecules, or antibodies. In some embodiments, the test compounds are hybridized DNA, RNA, or PNA sequences. Bioactive agents may be modifiers of therapeutic agents or therapeutic targets. Examples of therapeutic targets include phosphatases, proteases, ligases, signaling molecules, transcription factors, protein transporters, protein sorters, cell surface receptors, secretory factors, and cytoskeletal proteins.

[0187] In one embodiment, arrays for use in medical diagnostics are also provided. These arrays can be used to determine a response to the administration of a drug or vaccine. For example, an individual's response to a vaccine can be determined by detecting the individual's gene expression level using an array having polynucleotide chains representing specific genes associated with the induced immune response. Another diagnostic use is to test an individual for the presence of biomarkers, where a sample is taken from the subject and tested for the presence of one or more biomarkers.

[0188] Arrays can also be used to stratify patient populations based on the presence or absence of biomarkers that indicate a potential response to therapeutic treatment. Arrays can be used to identify known biomarkers and determine the appropriate treatment group. For example, samples from subjects with a certain condition can be applied to an array. Binding to the array may indicate the presence of a biomarker for that condition. Previous studies may show that biomarkers are associated with positive outcomes after treatment, while the absence of biomarkers is associated with negative or neutral outcomes after treatment. Because patients possess biomarkers, healthcare professionals can stratify patients into groups that receive treatment.

[0189] In some embodiments, a method for detecting the presence or absence of an expressed gene of interest in a sample is disclosed herein and comprises determining whether the gene of interest is expressed in the sample by obtaining an array that has come into contact with a sample suspected to contain the DNA or RNA sequence of the gene of interest, and detecting the presence or absence of binding to one or more feature regions of the array. In some embodiments, the DNA or RNA sequence of the gene of interest can be obtained from bodily fluids such as amniotic fluid, aqueous body fluids, vitreous fluid, bile, serum, breast milk, cerebrospinal fluid, earwax, chyle, endolymph, perilymph, feces, vaginal fluid (female ejaculate), gastric acid, gastric juice, lymph, mucus, ascites, pleural fluid, pus, saliva, sebum, semen, sweat, synovial fluid, tears, vaginal secretions, vomit, or urine.

[0190] In some embodiments, a method for identifying a vaccine candidate involves obtaining an array disclosed herein by contacting a sample derived from a subject previously administered with the vaccine candidate, wherein the sample comprises multiple DNA or RNA sequences, and determining the binding specificity of the multiple DNA or RNA sequences to one or more feature regions of the array. In some embodiments, the feature region comprises multiple distinct, nested, overlapping polynucleotide chains, each comprising a subsequence derived from a known nucleotide sequence.

[0191] Equivalents and Scope Those skilled in the art will be able to recognize or confirm many equivalents to the specific embodiments of the present invention described herein by means of routine experiments. The scope of the present invention is not intended to be limited to the foregoing description, but rather to be as described in the appended claims.

[0192] In the claims, articles such as “a,” “an,” and “the” may mean one or more unless the opposite is indicated or is otherwise evident from the context. A claim or statement containing “or” between one or more elements of a group is deemed satisfied if one, two or more, or all members of the group are present, used, or otherwise related in a given product or process, unless the opposite is indicated or is otherwise evident from the context. The present invention includes embodiments in which exactly one member of the group is present, used, or otherwise related in a given product or process. The present invention includes embodiments in which two or more, or all, members of the group are present, used, or otherwise related in a given product or process.

[0193] Where a range is given, it includes the endpoint. Furthermore, unless otherwise indicated or otherwise evident from the context and the understanding of those skilled in the art, it should be understood that a value expressed as a range may be considered any specific value or subrange within the defined range of different embodiments of the invention, up to one-tenth of the lower limit unit of the range, unless the context otherwise explicitly indicates.

[0194] All cited sources, such as references, publications, databases, database entries, and technologies cited herein, are incorporated by reference to this application, even if not explicitly stated in the citation. In the event of any conflict between the cited sources and the descriptions in this application, the descriptions in this application shall prevail.

[0195] Section and table headings are not intended to be limiting. [Examples]

[0196] The following are examples of specific embodiments for carrying out the present invention. These examples are presented for illustrative purposes only and are not intended to limit the scope of the present invention.

[0197] Example 1: Synthesis of a functionalized array for polynucleotide synthesis Functionalized silicon dioxide wafers for subsequent polynucleotide synthesis were prepared as follows.

[0198] Patterned silicon dioxide wafers were obtained from SVMI. The wafers were then functionalized using (3-aminopropyl)triethoxysilane (APTES) as follows: APTES was added to ethanol to a final concentration of 2% by weight, and the mixture was spin-coated onto the wafers. After spin-coating, the wafers were baked at 100°C for 30 minutes.

[0199] Next, Fmoc-NH-PEG6-CH2CH2COOH was bonded to APTES attached to the wafer surface. A solution of Fmoc-NH-PEG6-CH2CH2COOH was prepared by dissolving Fmoc-NH-PEG6-CH2CH2COOH in 1-methyl-2-pyrrolidone solvent. 1-hydroxy-7-azabenzotriazole (HOAt) was added to the solution. This solution was stirred at room temperature for 15 minutes. Diisopropylcarbodiimide (DIC) was added to a 5 mL aliquot of the obtained solution. This solution was heated at 60°C for 15 minutes.

[0200] The final concentrations of each reagent in 1-methyl-2-pyrrolidone solvent are as follows: Fmoc-NH-PEG6-CH2CH2COOH(0.1M) HOAt(0.1M) DIC(0.1M)

[0201] Next, this solution was spin-coated onto an APTES-functionalized wafer to link Fmoc-NH-PEG6-CH2CH2COOH to the APTES on the wafer surface. Then, the wafer was stripped with a solution of 1-methyl-2-pyrrolidone and isopropyl alcohol. As a result, the resulting wafer was functionalized by Fmoc-protected amine groups at the ends of the long chains bonded to the wafer surface.

[0202] 4-methylpiperidine was added to 1-methyl-2-pyrrolidinone solvent at a concentration of 20% by weight. 5 mL of the mixture was spin-coated onto a wafer.

[0203] To prepare the array for subsequent polynucleotide synthesis, the initial ligation step of DMT-protected phosphoramidites to Fmoc-protected NH groups was performed. Thymine CED phosphoramidites were dissolved in acetonitrile solvent, and the mixture was stirred at room temperature for 15 minutes. 5-benzothiotetrazole was added to the mixture, and then stirred again at room temperature. The final concentration of thymine CED phosphoramidite was 0.2 M, and the final concentration of 5-benzothiotetrazole was 0.25 M. Next, the mixture was spin-coated onto a wafer to ligate the DMT-protected phosphoramidites to NH-PEG6-CH2CH2COOH bound to the array surface. The wafer was then stripped with acetonitrile and isopropyl alcohol.

[0204] Solutions of Cap A (anhydrous acetate / pyridine / THF) and Cap B (THF containing 16% N-methylimidazole) were mixed together and stirred at room temperature for 5 minutes. Cap A and Cap B solutions were obtained from ChemGenes Corporation. Next, the mixture was spin-coated onto a wafer to cap any deprotected amine groups that were not bonded to the thymine CED phosphoramidite. The wafer was then stripped using acetonitrile and isopropyl alcohol. The oxidizing solution was spin-coated onto the wafer. The oxidizing solution was obtained from ChemGenes Corporation. The wafer was then stripped using acetonitrile and isopropyl alcohol.

[0205] Fluorescence experiments using 5(6)-FAM were performed to confirm the presence of terminally DMT-protected thymine CED phosphoramidite. Briefly, the array was incubated with 5(6)-FAM. Since 5(6)-FAM only binds to unprotected thymidine, a low fluorescence signal intensity may indicate successful DMT protection. After the unbound FAM was washed away, the initial signal intensity was measured. After measurement, the DMT group was cleaved using TCA. A linker (MMT-C6 CED-phosphoramidite) was ligated to the thymine residue to convert the OH group of the deprotected thymine to NH2. Next, the array was re-incubated with 5(6)-FAM. After the unbound FAM was washed away, the second signal intensity was measured. In this experiment, the first signal intensity was 134 and the second signal intensity was 65535. This experiment confirmed that the resulting array contained multiple terminally DMT-protected thymine CED phosphoramidites for subsequent nucleotide synthesis reactions.

[0206] Example 2: Polynucleotide synthesis in a microarray using a photoactivated acid cascade system A photoactive solution for inducing a photoactivated acid cascade reaction on an array was prepared as follows: PMMA was added to PGMEA in small amounts until a final concentration of 2% by weight was reached, and the mixture was stirred at room temperature for 48 hours to dissolve the PMMA. 4-tert-butylphenyl acetate was added to the PGMEA-PMMA solution until a final concentration of 5% by weight was reached, and the mixture was stirred at room temperature for 15 minutes. Finally, ITX was added to the resulting solution at a final concentration of 5% by weight, and the solution was stirred at room temperature for 15 minutes.

[0207] An array containing multiple DMT-protecting nucleotides (DMT-R1) was prepared as described in Example 1. The photoactive solution was spin-coated onto the wafer. Next, the wafer was exposed at selected locations using a Nikon NSRi10-365nm photolithography stepper tool with a photomask. The exposure time was 500 ms per field for light at a wavelength of 365 nm. After exposure, a post-exposure delay was performed by leaving the wafer at room temperature for 10 minutes to complete the deprotection reaction. Next, the wafer was stripped with acetone and isopropyl alcohol.

[0208] A solution containing the desired nucleotide for attachment to the deprotected nucleotide was prepared by dissolving the DMT-protected nucleotide together with 5-benzothiotetrazole in acetonitrile and stirring for 5 minutes. The final concentration of the DMT-protected nucleotide was 0.1 M, and the final concentration of the 5-benzothiotetrazole was 0.25 M. Next, this solution was spin-coated onto a wafer to link the DMT-protected nucleotide to the deprotected nucleotide attached to the wafer. The wafer was then stripped using acetonitrile and isopropyl alcohol.

[0209] The capping solutions, consisting of 50% Cap A and 50% Cap B, were mixed together at room temperature and spin-coated onto the wafer to cap any unprotected nucleotides on the wafer surface. The wafer was then stripped using acetonitrile and isopropyl alcohol. The oxidizing solution was spin-coated onto the wafer. The wafer was then stripped using acetonitrile and isopropyl alcohol.

[0210] The deprotection, coupling, and capping steps were repeated to synthesize each desired site-specific nucleotide, generating the desired array of sequence-specifically synthesized polynucleotides.

[0211] Example 3: Two-step polynucleotide synthesis on an array using in situ-synthesized NPPOC protection In this example, sequence-specific polynucleotide synthesis on an array is described using in situ-synthesized NPPOC protecting groups to facilitate site-directed nucleotide addition. The process performed was a two-step reaction in which photoactivated deprotection and nucleotide coupling were carried out in separate steps.

[0212] An array containing multiple DMT-protecting nucleotides (DMT-R1) was prepared as described in Example 1. DMT was completely removed from the DMT-protecting nucleotides bound to the wafer by spin-coating a solution of trichloroacetate (TCA) and dichloromethane onto the wafer. The wafer was then stripped using acetonitrile and isopropyl alcohol.

[0213] In situ synthesis of NPPOCs onto each of the deprotected nucleotides bound to the wafer was performed by spin-coating the wafer with a solution containing 0.3 M 2-(2-nitrophenyl)propyl chloroformate dissolved in a solvent of 30% 1-methyl-2-pyrrolidinone and 70% pyridine. The wafer was baked at 95°C for 2 minutes to fully ligate the NPPOC protecting groups to the deprotected nucleotides bound to the wafer. The wafer was then stripped with 1-methyl-2-pyrrolidinone and isopropyl alcohol.

[0214] A photoresist solution to facilitate the deprotection of NPPOC-protected nucleotides was prepared by adding PMMA at a concentration of 2 wt% to NMP and stirring the solution for 24 hours. ITX was added to this solution to a final concentration of 5 wt% and stirred at room temperature for 1 hour. Next, the photoresist solution was spin-coated onto a wafer.

[0215] Next, using a photomask, the wafer was exposed at selected locations with a Nikon NSR i10-365nm photolithography stepper tool to remove the NPPOC protecting group from the selected nucleotides to which the selected input nucleotides were desired. The exposure time was 5,000 ms per field for light at a wavelength of 365 nm. After exposure was complete, the wafer was stripped with acetone and IPA.

[0216] A solution containing the desired nucleotide for attachment to the deprotected nucleotide was prepared by dissolving the DMT-protected nucleotide together with 5-benzothiotetrazole in acetonitrile and stirring for 5 minutes. The final concentration of the DMT-protected nucleotide was 0.1 M, and the final concentration of the 5-benzothiotetrazole was 0.25 M. Next, this solution was spin-coated onto a wafer to link the DMT-protected nucleotide to the deprotected nucleotide attached to the wafer. The wafer was then stripped using acetonitrile and isopropyl alcohol.

[0217] The capping solutions, consisting of 50% Cap A and 50% Cap B, were mixed together at room temperature and spin-coated onto the wafer to cap any unprotected nucleotides on the wafer surface. The wafer was then stripped using acetonitrile and isopropyl alcohol. The oxidizing solution was spin-coated onto the wafer. The wafer was then stripped using acetonitrile and isopropyl alcohol.

[0218] The deprotection, coupling, and capping steps were repeated to synthesize each desired site-specific nucleotide, generating the desired array of sequence-specifically synthesized polynucleotides.

[0219] Example 4: One-step polynucleotide synthesis on an array using in situ-synthesized NPPOC protection This example describes sequence-specific polynucleotide synthesis on an array using in situ-synthesized NPPOC protecting groups to facilitate site-directed nucleotide addition. The process performed was a one-step reaction in which photoactivated deprotection and nucleotide coupling were carried out in a single step.

[0220] An array containing multiple DMT-protecting nucleotides (DMT-R1) was prepared as described in Example 1. DMT was completely removed from the DMT-protecting nucleotides bound to the wafer by spin-coating a solution of trichloroacetate (TCA) and dichloromethane onto the wafer. The wafer was then stripped using acetonitrile and isopropyl alcohol.

[0221] In situ synthesis of NPPOCs onto each of the deprotected nucleotides bound to the wafer was performed by spin-coating the wafer with a solution containing 0.3 M 2-(2-nitrophenyl)propyl chloroformate dissolved in a solvent of 30% 1-methyl-2-pyrrolidinone and 70% pyridine. The wafer was baked at 95°C for 2 minutes to fully ligate the NPPOC protecting groups to the deprotected nucleotides bound to the wafer. The wafer was then stripped with 1-methyl-2-pyrrolidinone and isopropyl alcohol.

[0222] A photoresist solution was prepared by adding PMMA at a concentration of 2% by weight to NMP and stirring the solution for 24 hours. ITX was then added to this solution to a final concentration of 5% by weight and stirred at room temperature for 1 hour.

[0223] Next, DMT-protected nucleotides and 5-benzothiotetrazole were added to the photoresist solution at final concentrations of 0.1 M and 0.25 M, respectively, to facilitate single-step NPPOC deprotection and input nucleotide addition. The solution was stirred at room temperature for 5 minutes. Then, the photoresist solution was spin-coated onto the wafer.

[0224] Next, using a photomask, the wafer was exposed at the selected location using a Nikon NSRi10-365nm photolithography stepper tool. The exposure time was 5,000 ms per field for 365 nm wavelength light. After exposure was complete, the wafer was stripped with acetone and IPA.

[0225] The capping solutions, consisting of 50% Cap A and 50% Cap B, were mixed together at room temperature and spin-coated onto the wafer to cap any unprotected nucleotides on the wafer surface. The wafer was then stripped using acetonitrile and isopropyl alcohol. The oxidizing solution was spin-coated onto the wafer. The wafer was then stripped using acetonitrile and isopropyl alcohol.

[0226] The deprotection, coupling, and capping steps were repeated to synthesize each desired site-specific nucleotide, generating the desired array of sequence-specifically synthesized polynucleotides.

[0227] Example 5: Testing the synthesis efficiency of each reaction scheme: 15mer sequence TIFF2026123166000008.tif5128 was grown on three different wafers using each of the three reaction schemes from Examples 2-4. For control, the same 15-mer sequence was grown using a substituted 2-nitrobenzyltrichloroacetate that produced trichloroacetate upon photodegradation. In the grown sequence, the 8th mer (underlined) was the location of the SNP.

[0228] We obtained four biotin-labeled oligonucleotide probes that have a pair sequence nearly complementary to the 15-mer sequence, but with a different polynucleotide at the 8th position. The complementary sequences are as follows: TIFF2026123166000009.tif25128

[0229] To evaluate the synthesis efficiency, hybridization and biotin labeling experiments were performed as follows.

[0230] Step 1: 1 μM biotin-labeled oligonucleotides having sequence 1, sequence 2, sequence 3, or sequence 4 were separately added to four different wells of a 96-well plate containing 99 μl of PBS solution.

[0231] Step 2: The pillar plate containing the silicon chip was placed on a 96-well plate to allow hybridization of the oligonucleotide probe to the 15-mer sequence grown on the chip. Hybridization was performed at room temperature for 30 minutes.

[0232] Step 3: The chip was washed with PBS solution for 2 minutes. The washing step was repeated 3 times.

[0233] Step 4: 1 μl of Alexa488 streptavidin was dissolved in 500 μl of PBS solution. 100 μl of this solution was added to four wells of a 96-well plate for 30 minutes.

[0234] Step 5: The chip was washed with PBS solution for 2 minutes. The washing step was repeated 3 times.

[0235] Step 6: The chip was washed with DI water for 2 minutes and dried with nitrogen. The chip was scanned using a Nikon A1R array scanner at a wavelength of 488 nm to detect hybridization.

[0236] The results are shown in Tables 2 - 4.

[0237] (Table 2) Type I (Example 2) - Photoacid cascade synthesis: TIFF2026123166000010.tif14150

[0238] (Table 3) Type II (Example 3) - in situ NPPOC synthesis and two-step deprotection and coupling TIFF2026123166000011.tif14150

[0239] (Table 4) Type III (Example 4) - In situ NPPOC synthesis and one-step deprotection and coupling TIFF2026123166000012.tif14150

[0240] (Table 5) Control sequences using substituted 2-nitrobenzyl trichloroacetate: TIFF2026123166000013.tif14150

[0241] Type 1: The results in Tables 2 and 5 show that good hybridization efficiency was achieved using Type 1, which produces acetate (low pH), compared to the control photoacid that produces trichloroacetate. While we do not wish to be bound by theory, Type 1 has a lower risk of depurination (one of the significant issues in photoacid-based polynucleotide microarrays). Because acetate is produced using a cascade mechanism, complete deprotection of the DMT group can be achieved without depurinating the purines in the sequence. A 1-mer coupling cycle took 25 minutes. Growing 15-mer took 375 minutes.

[0242] Type 2: In Type 2, depurination was minimal or non-existent due to the use of a photocleavable protecting group (NPPOC). The use of ITX enabled complete deprotection of the NPPOC group at 365 nm UV. These experiments demonstrated that in situ synthesis of NPPOC protection facilitates lower costs compared to the synthesis of individual NPPOC-protected monomers. A 1-mer coupling cycle took 22 minutes. Growing 15-mer took 330 minutes.

[0243] Type 3: Compared to Types 1 and 2, Type 3 coupling is performed in a single step, increasing the turnaround time for each step while maintaining the same hybridization efficiency. A coupling cycle for 1 mer took 18 minutes. Growing 15 mers took 270 minutes.

[0244] As observed from the signals from each synthetic scheme tested, only matching complementary sequences hybridized into the sequences grown on the chip. Under control conditions using the conventional substituted 2-nitrobenzyltrichloroacetate method, the signal intensity was significantly lower—less than half—compared to the polynucleotide arrays produced by reaction schemes I, II, and III. These results suggest that the efficiency of the three synthetic schemes tested above increased by more than twofold.

[0245] Other Embodiments The words used are not limiting, but rather descriptive, and it is understood that modifications can be made within the scope of the appended claims without departing from the true scope and spirit of the broader aspects of the invention.

[0246] While the present invention has been described with some length and detail with respect to several described embodiments, these are not intended to limit the scope to any such details or embodiments or any particular embodiment. Rather, they should be interpreted with reference to the appended claims to provide the broadest possible interpretation of such claims in light of the prior art, and thus to effectively encompass the intended scope of the present invention.

[0247] All publications, patent applications, patents, and other references referenced herein are incorporated in their entirety by reference. In the event of any conflict, this specification shall prevail, including definitions. In addition, section headings, materials, methods, and examples are illustrative and not intended to limit the scope of this specification.

[0248] Sequence information SEQUENCE LISTING <110> VIBRANT HOLDINGS, LLC <120> METHODS OF SYNTHESIZING A POLYNUCLEOTIDE ARRAY USING PHOTOACTIVATED AGENTS <150> US 62 / 668,964 <151> 2018-05-09 <160> 5 <170> PatentIn version 3.5 <210> 1 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 1 tgaaggcacg agaaa 15 <210> 2 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 2 tgaaggctcg agaaa 15 <210> 3 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 3 tgaaggcgcg agaaa 15 <210> 4 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 4 tgaaggcccg agaaa 15 <210> 5 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 5 acttccgcgc tcttt 15

Claims

1. A method for synthesizing a polynucleotide having a predetermined sequence, To provide a support containing a protected nucleic acid in a first characteristic part, The support is brought into contact with a photoresist solution containing a photoacid generator, The exposure involves exposing the support to light of a certain wavelength, wherein the photoacid generator generates a weak acid via a cascade reaction when exposed to light of the said wavelength, and the weak acid deprotects the nucleic acid in the first characteristic portion. The protected nucleic acid is bound to the deprotected nucleic acid in the first characteristic portion, The method, including the method described above.

2. The method according to claim 1, wherein the weak acid comprises acetic acid, carbonic acid, phosphoric acid, sulfonic acid, trifluoromethanesulfonic acid, or benzoic acid.

3. The method according to claim 1, wherein the photoacid generator comprises 4-tert-butylphenylic acid.

4. The method according to claim 1, wherein the photoacid generator comprises 4-tert-butylacetic acid phenyl and PGMEA.

5. The method according to claim 1, wherein the photoacid generator comprises 4-tert-butylphenyl carbonate and a compound selected from the group consisting of propylene carbonate, methylphenyl carbonate, and PGMEA.

6. The method according to claim 1, wherein the photoacid generator comprises 4-tert-butylphenyl phosphate and phenyl phosphate.

7. The method according to claim 1, wherein the photoacid generator comprises 4-tert-butylphenyl sulfonate and a compound selected from the group consisting of phenyl sulfate, 4-methylphenyl sulfate, dimethyl sulfate, methyltrifluoromethanesulfonate, and methylfluorosulfonate.

8. The method according to claim 1, wherein the photoacid generator comprises 4-tert-butylphenyl triflate and phenyltrifluoromethanesulfonate.

9. The method according to claim 1, wherein the photoacid generator comprises phenyl 4-tert-butylbenzoate and phenyl benzoate.

10. The method according to claim 1, wherein the protected nucleic acid comprises a DMT group.

11. The method according to claim 1, wherein the DMT group is bonded to the nucleic acid at its 5' carbon.

12. The method according to claim 1, wherein the wavelength of the light is approximately 350 nm.

13. The method according to claim 1, further comprising repeating the steps to synthesize polynucleotides of an intended length and sequence.

14. The method according to claim 1, wherein the support comprises at least 10, at least 100, at least 1,000, or at least 10,000 feature portions containing the protected nucleic acid.

15. A method for synthesizing an array of polynucleotides, each having a predetermined sequence, To provide a support containing an array of protected nucleic acids bound to its surface, The support is brought into contact with a solution containing a photoacid generator, Exposing a selected region of the support to light of a certain wavelength, wherein the photoacid generator generates a weak acid via a cascade reaction upon exposure to light of the said wavelength in order to deprotect the nucleic acid at each location exposed to the said wavelength of light, The selected input nucleotide is brought into contact with the wafer so as to bind to the deprotected nucleic acid, The method, including the method described above.

16. The method according to claim 15, wherein the input nucleotide comprises a DMT protecting group.

17. The method according to claim 15, further comprising repeating the step a number of times sufficient to generate an array of polynucleotides having a predetermined sequence and an intended length, respectively.

18. A method for linking a nucleotide monomer to a polynucleotide conjugated to a support, a. To provide a support comprising terminal nucleotides bonded to the surface of the support in the first characteristic part, b. Performing in situ synthesis of the NPPOC protecting group on the terminal nucleotide, c. Exposing the support to light of a certain wavelength in the first characteristic portion to remove the NPPOC protecting group from the terminal nucleotide, d. The support is brought into contact with the protected input nucleotide, thereby binding the protected input nucleotide to the deprotected terminal nucleotide in the first characteristic portion. The method, including the method described above.

19. The method according to claim 18, further comprising (e) deprotecting the protected input nucleic acid bound to the terminal nucleotide, and then repeating steps (a) to (e) to bind a second protected input nucleic acid.

20. The method according to claim 19, further comprising repeating all steps a number of times sufficient to synthesize polynucleotides of the intended length and sequence bound to the substrate.

21. The method according to claim 18, further comprising contacting the support with a photoresist solution containing ITX before exposing the support to light of the wavelength.

22. The method according to claim 18, wherein the wavelength of the light is approximately 365 nm.

23. The method according to claim 18, wherein the NPPOC protecting group is bonded to the 5' carbon of the terminal nucleotide.

24. The method of claim 18, wherein providing the support containing the terminal nucleotides comprises deprotecting the terminal nucleotides whole before carrying out the in situNPPOC synthesis.

25. The method according to claim 18, wherein the in situ synthesis of NPPOC comprises contacting the support with an NPPOC synthesis solution containing 2-(2-nitrophenyl)propyl chloroformate and pyridine.

26. The method according to claim 25, wherein the NPPOC synthesis solution further comprises 1-methyl-2-pyrrolidinone.

27. A method for synthesizing an array of polynucleotides, each having a predetermined sequence, a. To provide a support containing an array of protected nucleotides bound to its surface, b. Performing in situ synthesis of the NPPOC protecting group on the nucleotide array, c. Selectively exposing the support to light of a certain wavelength to remove the NPPOC protecting group from selected nucleotides from the array of nucleotides to which the addition of selected input nucleotides is desired, d. Contacting the selected input nucleotides with the array so as to bind them to the deprotected nucleotides, e. Repeating steps (c) to (e) a sufficient number of times to complete the desired nucleotide addition layer, thereby synthesizing an array of polynucleotides, each having a predetermined sequence, The method, including the method described above.

28. (f) The method of claim 27, further comprising deprotecting the input nucleotides bound to the polynucleotide array before in situ synthesis of the NPPOC protecting group.

29. The method according to claim 27, further comprising repeating steps (b) to (f) a number of times sufficient to generate an array of polynucleotides having a predetermined sequence and an intended length, and deprotecting the input nucleotides bound to the array of polynucleotides as a whole before in situ synthesis of the NPPOC protecting group.

30. The method according to claim 27, wherein the selected input nucleotide comprises a protecting group.

31. The method according to claim 30, wherein the protecting group is DMT.

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