Substrates, systems, and methods for nucleic acid array synthesis

Abstract

To provide formulations, substrates, and arrays for the synthesis of PNA chains and PNA-DNA chimera on microarrays.SOLUTION: A photo-protective compound that shields any PNA monomers, PNA polymers, or PNA-DNA chimera already attached to a microarray from radiation exposure during the synthesis of the PNA or PNA-DNA chains is comprised. In some embodiments, substrates and arrays comprise a porous or a planar layer for the synthesis and attachment of PNA or DNA monomers, or PNA or PNA-DNA polymers. In some embodiments, disclosed herein are formulations and methods for high-efficiency coupling of PNA monomers or PNA polymers to a microarray substrate.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 272,057, filed December 28, 2015, the disclosure of which is incorporated herein by reference. [Background technology]

[0002] background A typical microarray system generally consists of biomolecular probes, e.g., DNA, or RNA, formatted on a solid flat surface such as a glass, plastic, or silicon chip, and the equipment required to handle the sample (automated robotics), read the reporter molecules (scanners), and analyze the data (bioinformatics tools). Microarray technology facilitates monitoring many probes per square centimeter. Advantages of using multiple probes include, but are not limited to, speed, adaptability, comprehensiveness, and relatively low mass production costs. Applications of such arrays include, but are not limited to, diagnostic microbiology, including pathogen detection and identification, antimicrobial resistance studies, epidemiological strain classification, cancer genetic studies, analysis of microbial infections using host genome expression, and polymorphism profiling.

[0003] Peptide nucleic acids (PNAs) are DNA analogs with a peptide-like backbone, with each subunit containing a natural or unnatural base. One such backbone is composed of repeating N-(2-aminoethyl)glycine units linked by amide bonds, as described in PE Nielsen et al., Science, 254, 1497-1500 (1991). Furthermore, U.S. Patent No. 6,395,474 discloses a new class of compounds (PNAs) that bind to both DNA and RNA, forming PNA / DNA or PNA / RNA duplexes. PNA / oligonucleotide hybrids have higher thermal stability and higher biological stability than the corresponding DNA (or RNA) hybrids. This specificity of binding to DNA and RNA has paved the way for PNA applications in biotechnology, including the identification of single nucleotide polymorphisms in PCR-based assays. PNAs have the potential to be a highly sensitive method for directly detecting binding to RNA or DNA. However, for robust analyses, the reaction volume can be significantly reduced and the detection process simplified by using a relatively large number of high quality oligomers arranged in a microarray format.

[0004] Two methods of synthesizing PNA microarrays are well known to those skilled in the art, including (1) synthesizing different single probes, respectively, based on solid-phase synthesis techniques, and then attaching these probes to different locations on a microarray support by spot synthesis or adsorption; and (2) using in situ ultraviolet-controlled photolithographic synthesis using a light mask.

[0005] An example of the first method is disclosed in US Patent Application Publication No. 2006 / 0147949 (Patent Document 2), which involves the production of a PNA chip in which a probe PNA containing a desired DNA sequence is immobilized on a plastic support coated with an epoxy-containing polymer by an epoxy-containing polymer layer in an efficient and cost-effective manner. However, this method has drawbacks including, but not limited to, low spatial resolution and a time-consuming process with high synthesis costs of single probes to form a PNA array.

[0006] An example of the second method is disclosed in US Patent No. 6,359,125 (Patent Document 3). US Patent No. 6,359,125 (Patent Document 3) includes a process for preparing a PNA probe array immobilized on a solid matrix by using a polymeric photoacid generator. The speed of this process is faster than that of the first method by using parallel processing when synthesizing the probes on the microarray. However, the process limiting factor is the high synthesis cost of the PNA monomers required for parallel synthesis.

[0007] Thus, both prior art methods suffer from various deficiencies, including, inter alia, low or inconsistent coupling efficiency over multiple coupling cycles, synthesis cost, and difficulty in spatially controlling the synthesis of different PNA probes.Furthermore, photolithography methods are compromised by the effect of nucleic acids becoming unstable (i.e., being destroyed) when exposed to short wavelength radiation (e.g., 248 nm ultraviolet light) that is required to obtain high PNA probe density on microarrays.

[0008] PNA-DNA chimeras are oligomeric molecules that contain separate PNA and nucleotide portions. PNA-DNA chimeras can be synthesized by covalently linking PNA monomer sequences together with nucleotide sequences in virtually any combination or sequence.

[0009] Egholm et al. (US Pat. No. 6,316,230) provide a method and kit for primer extension of PNA-DNA chimeras from template nucleic acids using polymerase, nucleotide 5'-triphosphates, and primer extension reagents. The invention is based on the discovery that PNA-DNA chimeras can perform primer extension under a wide range of experimental conditions and variables. DNA sequencing methods can benefit from the use of PNA-DNA chimeras, where the high affinity and specificity imparted by the PNA moiety in the PNA-DNA chimera provides additional specificity. This method suffers from the disadvantages of low spatial resolution and high cost of synthesis of single probes to form PNA-DNA chimera arrays, as well as being a time-consuming process.

[0010] Therefore, what is needed are methods and compositions that address these and other shortcomings to PNA synthesis and PNA-DNA chimeric probe synthesis on microarrays, but still allow the arrays to be successfully used, for example, for SNP detection from samples. [Prior art documents] [Patent documents]

[0011] [Patent Document 1] U.S. Patent No. 6,395,474 [Patent Document 2] US Patent Application Publication No. 2006 / 0147949 [Patent Document 3] U.S. Patent No. 6,359,125 [Patent Document 4] U.S. Patent No. 6,316,230 [Non-patent literature]

[0012] [Non-Patent Document 1] PE Nielsen et al., Science, 254, 1497-1500(1991) Summary of the Invention

[0013] overview Provided herein are methods and compositions for improving the synthesis of PNA and PNA-DNA chimeras on arrays, and methods for using these arrays.The present invention provides novel supports, systems and methods for nucleic acid microarray synthesis, as detailed below.In particular, the present invention includes a novel method for synthesizing UV-directed PNA monomers and PNA polymers from submonomers at addressable locations on microarrays, which can economically prepare PNA microarrays with high spot density of PNA probes.

[0014] In some embodiments, the present specification discloses an array with high spot density of PNA and PNA-DNA chimeric probes and economically prepares PNA and PNA-DNA chimeric probes, and the method of manufacturing the array.The present specification shows a novel method of synthesizing UV-controlled PNA monomers and PNA polymers at addressable locations, which are derived from submonomers, and then UV-controlled DNA oligomers and DNA polymers.This combines the advantages of PNA (high specificity and coupling yield) and DNA oligomers (can be detected on chip using PCR reaction), thereby enabling better detection accuracy.

[0015] Aspects of the invention include formulations, supports, and arrays. Aspects also include methods for making and using the formulations, supports, and arrays.

[0016] In some embodiments, the present application provides an array of features attached to a surface at a defined location, each of said features comprises a plurality of PNA polymers with determinable sequence and intended length, said plurality of PNA polymers comprises a length distribution characterized by coupling efficiency of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99% or 99.5%.In some embodiments, the length distribution of said plurality of PNA polymers shorter than intended length is represented by the formula: F(N)=10 (N+1)·log(E / 100%) -10 (N)·log(E / 100%) where N=actual length of the PNA polymer, E=coupling efficiency, and E is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, or 99.5%. In some embodiments, the proportion of PNA polymers of intended length is characterized by the formula: F(N)=10 (N)·log(E / 100%) where N=the intended length of the PNA polymer, E=the average coupling efficiency, and E is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, or 99.5%.

[0017] In some embodiments, the array comprises at least 10,000 features, hi some embodiments, the array comprises at least 100,000 features.

[0018] In some embodiments, the intended length of the PNA polymers on the array is at least 30. In some embodiments, the intended length of the PNA polymers on the array is at least 40. In some embodiments, the intended length of the PNA polymers on the array is at least 50. In some embodiments, the intended length of the PNA polymers on the array is at least 75.

[0019] In some embodiments, the PNA polymers on the array are PNA / nucleic acid chimeras, and the PNA polymers further comprise one or more nucleic acid residues. In some embodiments, the nucleic acid is a deoxyribonucleic acid.

[0020] In some embodiments, the array comprises at least 10,000 features per square centimeter. In some embodiments, the array comprises at least 20,000, at least 40,000, at least 100,000, at least 200,000, at least 500,000, at least 1 million, at least 2 million, at least 5 million, at least 10 million, at least 20 million, or at least 50 million features per square centimeter. In some embodiments, the array comprises pillars and the surface is the top surface of the pillars. In some embodiments, the area of ​​the top surface of the pillars is at least 1 μm 2 It is.

[0021] Also provided herein is a method for making a PNA or PNA-DNA chimera array, comprising the steps of creating a pattern with a light mask, exposing a photoresist to ultraviolet light through the light mask, and generating an acid or base in the pattern of the array as a result of the exposure to the ultraviolet light.

[0022] In some embodiments, a base is generated from the photobase generator by UV exposure. In some embodiments, the photobase generator is 1,3-Bis[(2-nitrobenzyl)oxycarbonyl-4-piperidyl]propane, 1,3-Bis[1-(9-fluorenylmethoxycarbonyl)-4-piperidyl]propane, 1,5,7-triazabicyclo[4.4.0]dec-5-enyl-phenyl glyoxylate, 1,5,7-triazabicyclo[4.4.0]dec-5-enyl-4-nitrophenyl glyoxylate, 1,5,7 -triazabicyclo[4.4.0]dec-5-enyl-tetraphenylborate, 1,8-diazabicyclo[5.4.0]undec-7-enyl-tetraphenylborate, 1-phenacyl-(1-azonia-4-azabicyclo[2,2,2]octane)-tetraphenylborate, and 1-naphthoylmethyl-(1-azonia-4-azabicyclo[2,2,2]octane)-tetraphenylborate or similar. In some embodiments, the photobase generator is 1,3-Bis[(2-nitrobenzyl)oxycarbonyl-4-piperidyl]propane. In some embodiments, the base from the photobase generator cleaves the protecting group from the amine group.

[0023] In some embodiments, the method of making a PNA or PNA-DNA chimeric array comprises coupling PNA monomers to deprotected amine groups.

[0024] In some embodiments, an acid is generated from the photoacid generator upon exposure to UV light. In some embodiments, the photoacid generator is selected from the group consisting of iodonium salts, polonium salts, and sulfonium salts. In some embodiments, the photoacid generator is selected from the group consisting of Bis(4-tert-butylphenyl)iodonium perfluoro-1-butanesulfonate, Bis(4-tert-butylphenyl)iodonium p-toluenesulfonate, Bis(4-tert-butylphenyl)iodonium triflate, Boc-methoxyphenyldiphenylsulfonium triflate, (tert-butoxycarbonylmethoxynaphthyl)-diphenylsulfonium triflate, (4-tert-butylphenyl)diphenylsulfonium triflate, diphenyliodonium hexafluorophosphate, diphenyliodonium perfluoro-1-butanesulfonate, diphenyliodon ... triflate, (4-tert-butylphenyl)diphenylsulfonium triflate, diphenyliodonium trif iodophenyl)diphenylsulfonium triflate, (4-methoxyphenyl)diphenylsulfonium triflate, (4-methylphenyl)diphenylsulfonium triflate, (4-methylthiophenyl)methylphenylsulfonium triflate, Tris(4-tert-butylphenyl)sulfonium triflate, (4-methoxyphenyl)phenylsulfonium triflate, (4-methoxyphenyl)phenyliodonium triflate, 4-methoxyphenyl)phenyliodonium trifluoromethanesulfonate, (4-methoxyphenyl)dimethylsulfonium triflate, (2,4-dihydroxyphenyl)dimethylsulfonium triflate or similar. In some embodiments, the photoacid generator is an iodonium or sulfonium salt of a triflate, phosphate, or antimonate. In some embodiments, the photoacid generator is (4-iodophenyl)diphenylsulfonium triflate. In some embodiments, the acid generated by the photoacid generator cleaves the protecting group from the carboxylic acid group.

[0025] In some embodiments, the method for creating a PNA-DNA chimeric array further comprises coupling DNA monomers to the deprotected carboxylic acid groups.In some embodiments, coupling PNA monomers comprises activating the substituted acetic acid with an activating agent, and coupling the activated acetic acid to the unprotected amine groups in the selectively exposed portions, and the substitution of acetic acid comprises a leaving group.In some embodiments, coupling PNA monomers is performed simultaneously at multiple sites on the array.

[0026] In some embodiments, the leaving group is halo. In some embodiments, the coupling further comprises replacing the leaving group of the acetic acid with a diamino-alkane, wherein one amine of the diamino-alkane is protected. In some embodiments, the diamino-alkane is ethylenediamine.

[0027] In some embodiments, the step of coupling further comprises activating the PNA monomer acetate with an activating agent, and coupling the activated PNA monomer acetate to the unprotected amine of the diamino-alkane. In some embodiments, the PNA monomer acetate is R-thymine-1-acetate, R-(cytosin-1-yl)-acetate, R-adenine-9-yl-acetate, R-guanine-9-acetate, or R-uracil-1-acetate, where R is H or a protecting group.

[0028] Also provided herein is a method for analyzing a sample containing nucleic acid obtained from a subject, the method comprising the steps of: contacting the sample with a PNA or PNA-DNA array described herein under conditions that promote hybridization between the sample and the array; detecting a signal from the array, the signal indicating the presence, absence, or amount of sample hybridized to the array at one or more of the characteristic locations; and analyzing the signal, thereby analyzing the sample.

[0029] In some embodiments, analyzing the sample comprises determining a nucleic acid sequence present in the sample based on the signal. In some embodiments, analyzing the sample comprises determining the presence or absence of SNPs present in the sample based on the signal. In some embodiments, the array comprises PNA / DNA chimeras, and the method further comprises performing a primer extension reaction after the hybridization between the sample and the array.

[0030] In some embodiments, the photoactive compound is about 0.5-5% by weight of the total formulation. In some embodiments, the photoprotective compound protects any compounds or coupling molecules attached to the microarray from electromagnetic radiation. Examples of coupling molecules include, but are not limited to, peptide nucleic acids ("PNAs"), and the like. In some embodiments, the coupling molecule is 1-2% by weight of the total formulation. In some embodiments, the coupling molecule includes a protected group. In some embodiments, the group is protected by Fmoc.

[0031] In some embodiments, each PNA strand or PNA-DNA strand is at least 6 monomers in length. In some embodiments, each PNA strand or PNA-DNA strand is at least 6 monomers, 10 monomers, 15 monomers, 20 monomers, 25 monomers, 30 monomers, 35 monomers, 40 monomers, 45 monomers, 50 monomers, 55 monomers, or 60 monomers in length. In some embodiments, each PNA strand or PNA-DNA strand comprises one or more L-chiral PNA monomers. In some embodiments, each PNA strand or PNA-DNA strand comprises one or more D-chiral PNA monomers. In some embodiments, each PNA strand or PNA-DNA strand comprises one or more synthetic PNA or DNA monomers. In some embodiments, the array comprises at least 1,000 different PNA strands or PNA-DNA strands attached to a surface. In some embodiments, the array comprises at least 10,000 different PNA strands or PNA-DNA strands attached to a surface.

[0032] In some embodiments, each of the defined locations is at a different known location that is physically separated from each of the other defined locations. In some embodiments, each of the defined locations comprises a plurality of identical PNA sequences or PNA-DNA sequences. In some embodiments, each of the defined locations comprises a plurality of identical PNA sequences that are different from the other defined locations. In some embodiments, each of the defined locations is a distinct location. In certain embodiments, each determinable PNA sequence or PNA-DNA sequence corresponds to a known nucleotide sequence. In certain embodiments, each determinable PNA-DNA sequence is a distinct sequence. In some embodiments, the features are covalently attached to the surface. In some embodiments, the PNA strand or PNA-DNA strand is attached to the surface via a linker or coupling molecule. [The present invention 1001] An array of features attached to a surface at defined locations, each of the features comprising a plurality of PNA polymers of determinable sequence and intended length, the plurality of PNA polymers comprising a distribution of lengths characterized by a coupling efficiency of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, or 99.5%. [The present invention 1002] The distribution of lengths of a plurality of PNA polymers shorter than the intended length is determined by the formula: F(N)=10 (N+1)·log(E / 100%) -10 (N)·log(E / 100%) Arrays of the present invention 1001, characterized by: where N=actual length of the PNA polymer, E=coupling efficiency, and E is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, or 99.5%. [The present invention 1003] The ratio of PNA polymers of intended length is determined according to the formula: F(N)=10 (N)·log(E / 100%) Arrays of the present invention 1001, characterized by: where N=intended length of the PNA polymer, E=average coupling efficiency, and E is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, or 99.5%. [The present invention 1004] Any of the foregoing arrays of the invention comprising at least 10,000 features. [The present invention 1005] The array of the invention 1004 comprising at least 100,000 features. [The present invention 1006] Any of the preceding arrays of the invention, wherein the intended length of the PNA polymer is at least 30. [The present invention 1007] Any of the preceding arrays of the invention, wherein the intended length of the PNA polymer is at least 40. [The present invention 1008] Any of the preceding arrays of the invention, wherein the intended length of the PNA polymer is at least 50. [The present invention 1009] Any of the preceding arrays of the invention, wherein the intended length of the PNA polymer is at least 75. [The present invention 1010] Any of the aforementioned arrays of the invention, wherein the PNA polymer is a PNA / nucleic acid chimera, said PNA polymer further comprising one or more nucleic acid residues. [The present invention 1011] The array of the invention 1010, wherein the nucleic acid is deoxyribonucleic acid. [The present invention 1012] Any of the aforementioned arrays of the invention comprising at least 10,000 features per square centimeter. [The present invention 1013] Arrays of the invention 1012 comprising at least 20,000, at least 40,000, at least 100,000, at least 200,000, at least 500,000, at least 1 million, at least 2 million, at least 5 million, at least 10 million, at least 20 million, or at least 50 million features per square centimeter. [The present invention 1014] Any of the aforementioned arrays of the invention, wherein the array comprises pillars and the surface is a top surface of the pillars. [The present invention 1015] The top surface of the pillar is at least 1 μm 2 Array of the present invention 1014 having an area of ​​1014. [The present invention 1016] Creating a pattern using a photomask; exposing the photoresist to ultraviolet light through the photomask; and generating an acid or base in said pattern on said array as a result of said exposure to said ultraviolet light. A method for making any one of the arrays 1001 to 1015 of the present invention, comprising the steps of: [The present invention 1017] The method of the present invention 1016, wherein a base is generated from a photobase generator upon exposure to light. [The present invention 1018] The photobase generator is 1,3-Bis[(2-nitrobenzyl)oxycarbonyl-4-piperidyl]propane, 1,3-Bis[1-(9-fluorenylmethoxycarbonyl)-4-piperidyl]propane, 1,5,7-triazabicyclo[4.4.0]dec-5-enyl-phenyl glyoxylate, 1,5,7-triazabicyclo[4.4.0]dec-5-enyl-4-nitrophenyl glyoxylate, 1,5,7-triazabicyclo[4 1.4.0]dec-5-enyl-tetraphenylborate, 1,8-diazabicyclo[5.4.0]undec-7-enyl-tetraphenylborate, 1-phenacyl-(1-azonia-4-azabicyclo[2,2,2]octane)-tetraphenylborate, and 1-naphthoylmethyl-(1-azonia-4-azabicyclo[2,2,2]octane)-tetraphenylborate or the like. [The present invention 1019] The method of claim 1018, wherein the photobase generator is 1,3-Bis[(2-nitrobenzyl)oxycarbonyl-4-piperidyl]propane. [The present invention 1020] The method of any of claims 1017 to 1019, wherein the protecting group is cleaved from the amine group by a base. [The present invention 1021] The method of claim 1020, further comprising the step of coupling a PNA monomer to the deprotected amine group. [The present invention 1022] The method of the present invention 1016, wherein an acid is generated from a photoacid generator upon exposure to light. [The present invention 1023] The method of claim 1022, wherein the photoacid generator is selected from the group consisting of iodonium salts, polonium salts, and sulfonium salts. [The present invention 1024] The photoacid generator is Bis(4-tert-butylphenyl)iodonium perfluoro-1-butanesulfonate, Bis(4-tert-butylphenyl)iodonium p-toluenesulfonate, Bis(4-tert-butylphenyl)iodonium triflate, Boc-methoxyphenyldiphenylsulfonium triflate, (tert-butoxycarbonylmethoxynaphthyl)-diphenylsulfonium triflate, (4-tert-butylphenyl)diphenylsulfonium triflate, diphenyliodonium hexafluorophosphate, diphenyliodonium perfluoro-1-butanesulfonate, diphenyliodonium triflate, (4-iodophenyl)diphenylsulfonium triflate, (4-methoxyphenyl)diphenylsulfonium triflate, (4-methylphenyl)diphenylsulfonium triflate, 1023. The method of claim 1022, wherein the arylphenyl sulfonium triflate is selected from the group consisting of (4-methylthiophenyl)diphenylsulfonium triflate, (4-methylthiophenyl)methylphenylsulfonium triflate, Tris(4-tert-butylphenyl)sulfonium triflate, (4-methoxyphenyl)phenylsulfonium triflate, (4-methoxyphenyl)phenyliodonium triflate, 4-methoxyphenyl)phenyliodonium trifluoromethanesulfonate, (4-methoxyphenyl)dimethylsulfonium triflate, and (2,4-dihydroxyphenyl)dimethylsulfonium triflate, or the like. [The present invention 1025] The method of claim 1022, wherein the photoacid generator is selected from the group consisting of iodonium and sulfonium salts of triflates, phosphates, and antimonates. [The present invention 1026] The method of claim 1022, wherein the photoacid generator is (4-iodophenyl)diphenylsulfonium triflate. [The present invention 1027] The method of any one of claims 1022 to 1026, wherein the protecting group is cleaved from the carboxylic acid group by acid. [The present invention 1028] The method of claim 1027, further comprising the step of coupling a DNA monomer to the deprotected carboxylic acid group. [The present invention 1029] The step of coupling the PNA monomers comprises: activating the substituted acetic acid with an activating agent; and Coupling activated acetic acid to unprotected amine groups in selectively exposed areas. wherein the replacement of the acetic acid includes a leaving group. [The present invention 1030] 1029. The method of claim 1029, wherein the step of coupling PNA monomers is performed simultaneously at multiple sites on an array. [The present invention 1031] The process of claim 1029, wherein the leaving group is halo. [The present invention 1032] The coupling step comprises: Replacing the acetic acid leaving group with a diamino-alkane 1029. The method of claim 1029, further comprising: [The present invention 1033] The process of claim 1032, wherein the diamino-alkane is ethylenediamine. [The present invention 1034] The coupling step comprises: activating the PNA monomer acetate with an activating agent; and Coupling the activated PNA monomer acetate to the unprotected amine of a diamino-alkane The method of any one of claims 1029 to 1030, further comprising: [The present invention 1035] 1035. The method of claim 1034, wherein the PNA monomer acetate is R-thymine-1-acetate, R-(cytosin-1-yl)-acetate, R-adenine-9-yl-acetate, R-guanine-9-acetate, or R-uracil-1-acetate, where R is H or a protecting group. [The present invention 1036] 1. A method for analyzing a sample containing nucleic acid obtained from a subject, the method comprising the steps of: contacting the sample with any of the arrays of the present invention 1001 to 1015 under conditions that promote hybridization between the sample and the array; detecting a signal from the array, the signal being indicative of the presence, absence, or amount of a sample hybridized to the array at one or more of the characteristic locations; and Analysing the signal, thereby analyzing the sample. [The present invention 1037] The method of claim 1036, wherein the analyzing step comprises determining a nucleic acid sequence present in the sample based on the signal. [The present invention 1038] The method of claim 1036, wherein the analyzing step comprises determining the presence or absence of a SNP present in the sample based on the signal. [The present invention 1039] The method of claim 1038, wherein the array comprises PNA / DNA chimeras, and further comprises the step of performing a primer extension reaction after hybridization between the sample and the array. [Brief description of the drawings]

[0033] A brief description of some drawings These and other features, aspects, and advantages of the present invention will become better understood with regard to the following description and accompanying drawings.

[0034] [Figure 1] 1 illustrates a method for producing a PNA array, according to some embodiments. [Figure 2A] 1 shows a method for producing a PNA-DNA chimeric array and reaction schemes for selected steps, according to some embodiments. [Figure 2B] 1 shows a method for producing a PNA-DNA chimeric array and reaction schemes for selected steps, according to some embodiments. [Diagram 3] 1 illustrates an exemplary synthetic scheme for the reverse phosphoramidite approach. [Figure 4]1 illustrates a general synthetic scheme for attaching custom or preselected oligonucleotides to a PNA oligomer. [Diagram 5] 1 illustrates phosphoramidite chemistry for accessing sequences from the 3' position of a donor nucleotide to the 3' position of an acceptor nucleotide. [Figure 6] 4 shows fluorescein intensity for measuring hybridization of correct and mismatched sequences attached to PNA molecules bound to an array, according to some embodiments. [Figure 7] 1 illustrates an exemplary single-base primer extension reaction for detecting sequence variants. The figure discloses SEQ ID NOs: 18 and 19, respectively, in order of appearance. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0035] Detailed Description Terms used in the claims and specification, unless otherwise specified, are defined as set forth below.

[0036] The term "wafer" as used herein refers to a thin slice of semiconductor material, such as silicon or germanium crystal, commonly used in integrated circuit fabrication. Wafers may come in a variety of sizes, e.g., from 25.4 mm (1 inch) to 300 mm (11.8 inches) along one dimension, and may be, for example, from 275 μm to 775 μm in thickness.

[0037] As used herein, the term "optical mask" or "reticle" or "mask" refers to a light-opaque plate with optically transparent patterns or holes that allow light to pass through. In a typical exposure process, the pattern of the optical mask is transferred onto a photoresist.

[0038] As used herein, the term "photoresist" or "resist" or "photoactive material" refers to a photosensitive material that undergoes chemical modification, e.g., changes its solubility in solution or generates photoacid, when exposed to electromagnetic radiation, particularly ultraviolet or deep ultraviolet radiation. Photoresists include organic or inorganic compounds.

[0039] As used herein, the term "photoresist formulation" refers to a formulation that includes a photoactive compound and a photoprotective compound.

[0040] The term "photoactive compound" as used herein refers to a compound that is modified when exposed to electromagnetic radiation. These compounds include, for example, cationic photoinitiators, such as photoacid generators (PAGs) that generate a corresponding photoacid or photobase generators (PBGs) that generate a corresponding photobase when exposed to electromagnetic radiation. Examples of photoactive compounds are disclosed in International Patent Application No. PCT / US2013 / 070207, filed November 14, 2013, which is incorporated herein in its entirety for all purposes. Photoinitiators are compounds that are specifically added to formulations to convert electromagnetic radiation into chemical energy in the form of initiating species, e.g., free radicals or cations. The acid, base, or other product of the photoactive compound exposed to electromagnetic radiation can 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 electromagnetic radiation pattern used when the solution or surface containing the photoactive compound is exposed. This pattern may be defined, for example, by a light mask or reticle.

[0041] The term "photoprotective" compound, as used herein, refers to an organic or inorganic compound that blocks electromagnetic radiation by absorbing or scattering the radiation such that the energy of the radiation is reduced beyond the photoprotective compound along the direction of the radiation. Examples of inorganic photoprotective compounds include, but are not limited to, titanium dioxide, zinc sulfide, and magnesium fluoride.

[0042] The term "coupling molecule" or "monomer molecule" as used herein includes any peptide nucleic acid (PNA) or PNA monomer acetate, natural or synthetic, in which the nitrogen is optionally protected with a fluorenylmethyloxycarbonyl (Fmoc or F-Moc) group or a t-butoxycarbonyl (tboc or Boc) group. Alternatively, these terms include substituted acetates, where the substitution is a leaving group. Examples of coupling molecules include R-thymine-1-acetate, R-(cytosin-1-yl)-acetate, R-adenine-9-yl-acetate, R-guanine-9-acetate, and R-uracil-1-acetate, where R is H or a protecting group of a nucleic acid monomer. Other examples are described below.

[0043] As used herein, the term "coupling" or "coupling process" or "coupling step" refers to a process of forming a bond between two or more molecules, such as linker molecules or coupling molecules. The bond may be a covalent bond, such as a peptide bond. A peptide bond is formed when a carboxyl group of one coupling molecule reacts with an amino group of another coupling molecule to form a water molecule (H 2 A -C(=O)NH- bond is a chemical bond formed between two molecules when the two molecules release an -C(=O)NH- bond. This is a dehydration synthesis reaction (also known as a condensation reaction) and usually occurs between amino acids. The resulting -C(=O)NH- bond is called a peptide bond and the resulting molecule is an amide. As used herein, coupling a PNA monomer includes coupling a portion of a PNA or a PNA precursor and then fabricating the PNA.

[0044] The term "coupling efficiency" as used herein refers to the likelihood of a monomer successfully adding to a reactive site (e.g., a reactive site at the end of a polymer) that can bind to a monomer. For example, during the growth of a PNA polymer (also called a PNA sequence) in the C→N direction, a PNA monomer with a free carboxyl group will bind to another PNA monomer with a free amine group under appropriate conditions. Coupling efficiency indicates the likelihood of a free carboxylic acid adding to a free amino group under certain conditions. Coupling efficiency can be ascertained collectively, for example, by simultaneously monitoring the addition of one type of monomer to several individual reactive sites.

[0045] As used herein, the term "biomarker" includes, but is not limited to, PNA, DNA, RNA, proteins (e.g., enzymes such as kinases), peptides, sugars, salts, fats, lipids, ions, and the like.

[0046] As used herein, the term "linker molecule" or "spacer molecule" includes any molecule that does not add any functionality to the resulting PNA monomer, or PNA polymer, or PNA-DNA chimeric polymer, but that spaces the monomer or polymer apart, extending it from the support, thus increasing the distance between the support surface and the growing PNA or PNA-DNA sequence. This generally reduces steric hindrance with the support for reactions involving PNA or PNA-DNA polymers (including unimolecular folding and multimolecular binding reactions), thereby improving the performance of assays that measure one or more aspects of PNA or PNA-DNA function.

[0047] The term "developer" as used herein refers to a solution that can selectively dissolve exposed or unexposed material. Typically, developers are water-based solutions with trace amounts of base added. Examples include tetramethylammonium hydroxide dissolved in a water-based developer. Developers are used to define the initial pattern where a commercially available photoresist is used.

[0048] The term "protecting group" as used herein includes groups introduced into molecules by chemical modification of functional groups to obtain chemoselectivity in subsequent chemical reactions. "Chemical selectivity" refers to directing a chemical reaction to a desired path to obtain a preselected product compared to another product. For example, when tboc is used as a protecting group, the protecting group is selectively removed by the chemoselectivity of PNA synthesis using a photomask and a photoacid generator, allowing a predetermined direct PNA coupling reaction to occur at the location defined by the photomask.

[0049] As used herein, the term "microarray", "array" or "chip" refers to a support on which a plurality of probe molecules of specific PNA, RNA or DNA binding sequences are attached in an ordered manner at separate locations, thus forming a microarray. The specific PNA, RNA or DNA binding sequences may be linked to the chip support via one or more different types of linker molecules. A "chip array" refers to a plate having a plurality of chips, for example, 24, 96 or 384 chips.

[0050] As used herein, the term "probe molecule" refers to, but is not limited to, peptide nucleic acid ("PNA"), DNA binding sequences, oligonucleotides, nucleic acids, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), nucleotide mimics, chelators, side chain modified peptide sequences, biomarkers, and the like. As used herein, the term "feature" refers to a particular probe molecule that is attached to a microarray. As used herein, the term "ligand" refers to a molecule, agent, analyte, or compound of interest that can bind to one or more features.

[0051] As used herein, the term "microarray system" or "chip array system" refers to a system that typically consists of biomolecular probes formatted on a solid flat surface such as a glass, plastic, or silicon chip, and the equipment needed to handle the samples (automated robotics), read the reporter molecules (scanners), and analyze the data (bioinformatics tools).

[0052] As used herein, the terms "patterned area" or "pattern" or "location" refer to the areas on a substrate where various features are grown. These patterns can be defined using a photomask.

[0053] The term "derivatization" as used herein refers to the process of chemically modifying a surface to make it suitable for biomolecule synthesis. Typically, derivatization involves the following steps: rendering the support hydrophilic, adding aminosilane groups, and attaching linker molecules.

[0054] As used herein, the term "capping" or "capping process" or "capping step" refers to the addition of a molecule that prevents further reaction of the attached molecule. For example, amino groups are typically capped with an acetic anhydride molecule to prevent further formation of peptide bonds. In another embodiment, ethanolamine is used.

[0055] As used herein, the term "diffusion" refers to the spread of, for example, a photoacid or photobase, via random motion from an area of ​​high concentration to an area of ​​low concentration.

[0056] The term "dye molecule" as used herein refers to a dye, which is typically a colored substance that can be bound to a support. Dye molecules can be useful in detecting binding between features on an array and a molecule of interest.

[0057] As used herein, the terms "immunological binding" and "immunological binding properties" refer to the type of non-covalent interactions that occur between an immunoglobulin molecule and an antigen for which the immunoglobulin shows specificity.

[0058] The term "biological sample" as used herein refers to a sample derived from biological tissue or fluid that can be assayed for an analyte of interest. Such samples include, but are not limited to, sputum, amniotic fluid, blood, blood cells (e.g., white blood cells), tissue or fine needle biopsy samples, urine, peritoneal fluid, and pleural fluid, or cells derived therefrom. Biological samples may also include tissue sections, such as frozen sections taken for histological purposes. Although samples are typically taken from human patients, the assays can be used to detect analytes of interest in samples derived from any organism (e.g., mammals, bacteria, viruses, algae, or yeast) or mammals, such as dogs, cats, sheep, cows, and pigs. Samples may be prepared, if necessary, by dilution with an appropriate buffer solution, and may be concentrated if desired.

[0059] As used herein, the term "assay" refers to a type of biochemical test that measures the presence or concentration of a substance of interest in a solution that may contain a complex mixture of substances.

[0060] The term percent "identity" in the context of two or more nucleic acid sequences or PNA sequences refers to two or more sequences or subsequences that have a certain percentage of nucleic acid bases that are identical when compared and aligned for maximum correspondence, as determined using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to those of skill in the art) or by visual inspection. Depending on the application, the percent "identity" may exist over a region of the sequences being compared, for example, over a functional domain, or over the full length of the two sequences being compared.

[0061] In sequence comparison, typically, a sequence serves as a reference sequence, and the reference sequence and test sequence are compared.When using sequence comparison algorithm, test sequence and reference sequence are input into computer, and if necessary, the coordinate of subsequence is designated, and sequence algorithm program parameters are designated.Then, sequence comparison algorithm calculates the percent sequence identity of test sequence compared with reference sequence based on designated program parameters.

[0062] Optimal sequence alignment for comparison can be performed, for example, using the local homology algorithm of Smith & Waterman Adv. Appl. Math. 2:482 (1981), the homology alignment algorithm of Needleman & Wunsch J. Mol. Biol. 48:443 (1970), the search for similarity method of Pearson & Lipman Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally, Ausubel et al., infra).

[0063] One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm described in Altschul et al., J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available at the website of the National Center for Biotechnology Information.

[0064] Unless otherwise indicated, as used herein, whether used alone or as part of a substituent group, refers to a saturated, branched, or straight-chain monovalent hydrocarbon group obtained by removing one hydrogen atom from a carbon atom of a parent alkane. Representative alkyl groups include, but are not limited to, methyl; ethyl; propyl, e.g., propan-1-yl, propan-2-yl; butyl, e.g., butan-1-yl, butan-2-yl, 2-methyl-propan-1-yl, 2-methyl-propan-2-yl, and the like. In a preferred embodiment, the alkyl group is C 1-6 Alkyl, C 1-3 Alkyl is especially preferred. "Alkoxyl" groups are oxygen ethers formed from the straight or branched chain alkyl groups previously described.

[0065] As used herein, "halo" or "halogen" is intended to mean chlorine, bromine, fluorine, and iodine. "Halo-substituted" is intended to mean a group substituted with at least one halogen atom, preferably at least one fluorine atom. Suitable examples include -CF 3 These include, but are not limited to:

[0066] The term "cycloalkyl" as used herein refers to a stable saturated or partially saturated monocyclic or bicyclic ring structure containing 3 to 8 ring carbons, preferably 5 to 7 ring carbons. Examples of such cyclic alkyl rings include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or cycloheptyl.

[0067] The term "alkenyl" refers to an unsaturated branched, straight-chain, or cyclic monovalent hydrocarbon group having at least one carbon-carbon double bond derived by the removal of a hydrogen atom from a single carbon atom of a parent alkene. The group may be in the cis or trans conformation about the double bond. Representative alkenyl groups include, but are not limited to, ethenyl; propenyl, e.g., prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl, prop-2-en-2-yl, cycloprop-1-en-1-yl; cycloprop-2-en-1-yl; butenyl, e.g., but-1-en-1-yl, but-1-en-2-yl, 2-methyl-prop-1-en-1-yl, but-2-en-1-yl, but-2-en-1-yl, but-2-en-2-yl, buta-1,3-dien-1-yl, buta-1,3-dien-2-yl, cyclobut-1-en-1-yl, cyclobut-1-en-3-yl, cyclobuta-1,3-dien-1-yl, and the like.

[0068] The term "heteroaryl" refers to a monovalent heteroaromatic group derived by removing a hydrogen atom from an atom of a parent heteroaromatic ring structure. Representative heteroaryl groups include monocyclic and bicyclic structures in which one or both rings are heteroaromatic. The heteroaromatic ring may contain 1 to 4 heteroatoms selected from O, N, and S. Examples include, but are not limited to, groups derived from carbazole, furan, imidazole, indazole, indole, indolizine, isoindole, isoquinoline, isothiazole, isoxazole, naphthyridine, oxadiazole, oxazole, purine, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pyrrole, pyrrolidine, quinazoline, quinoline, quinolizine, quinoxaline, tetrazole, thiadiazole, thiazole, thiophene, triazole, xanthene, and the like.

[0069] The term "aryl" as used herein refers to an aromatic group containing a stable 6-membered monocyclic, 10-membered bicyclic, or 14-membered tricyclic aromatic ring structure composed of carbon atoms. Examples of aryl groups include, but are not limited to, phenyl or naphthalenyl.

[0070] The term "heterocyclyl" refers to a 3- to 12-membered, saturated or partially saturated monocyclic (monocyclic), bicyclic, or fused ring system consisting of carbon atoms and one to six heteroatoms selected from N, O, and S. The heterocyclyl group can be attached at any heteroatom or carbon atom to create a stable structure. Bicyclic heterocyclyl groups include systems in which one or both rings contain heteroatoms. Examples of heterocyclyl groups include, but are not limited to, 2-imidazoline, imidazolidine; morpholine, oxazoline, oxazolidine, 2-pyrroline, 3-pyrroline, pyrrolidine, pyridone, pyrimidone, piperazine, piperidine, indoline, tetrahydrofuran, 2-pyrroline, 3-pyrroline, 2-imidazoline, 2-pyrazoline, indolinone, thiomorpholine, tetrahydropyran, tetrahydroquinoline, tetrahydroquinazoline, [1,2,5]thiadiazolidine 1,1-dioxide, [1,2,3]oxathiazolidine 2,2-dioxide, and the like.

[0071] The term "cis-trans isomers" refers to stereoisomeric olefins or cycloalkanes (or heteroanalogs) that differ in the position of their atoms (or groups) relative to a reference plane. In cis isomers, the atoms with highest priority are on the same side; in trans isomers, the atoms with highest priority are on opposite sides.

[0072] The term "substituted" refers to a group in which one or more hydrogen atoms are each independently replaced with the same or different substituents.

[0073] With reference to substituents, the term "independently" means that when more than one of such substituents is possible, such substituents may be the same or different from one another.

[0074] The term "oxo," whether used alone or as part of a substituent, refers to an O= attached to a carbon or sulfur atom. For example, phthalimide and saccharin are examples of compounds that have oxo substituents.

[0075] The term "PNA-DNA chimera" refers to an oligomer composed of (i) a contiguous stretch of PNA monomer units and (ii) a contiguous stretch of nucleotide monomer units having an enzymatically extendable terminus.

[0076] The term "primer extension" refers to the enzymatic addition of monomeric nucleotide units to a primer while the primer is hybridized (annealed) to a template nucleic acid, i.e., polymerization.

[0077] It must be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0078] Compositions and synthesis schemes PNA synthesis on microarrays The present application provides a method for making peptide nucleic acid (PNA) compounds, including PNA monomers and PNA polymers, and PNA-DNA chimeras on a microarray platform by high-throughput parallel synthesis using photolithography. In some embodiments, 248 nm ultraviolet light is used to make PNA or PNA-DNA chimera microarrays.

[0079] Figure 1 and scheme 1 illustrate suggested synthetic routes according to some embodiments for PNA oligomer synthesis. Using this scheme, the following guidelines, and examples, one skilled in the art can develop similar or similar methods for obtaining a specific PNA compound or a microarray with multiple PNA compounds within the scope of the present invention. In some embodiments, the PNA compound is coupled to a microarray at a defined location. These methods are representative of synthetic schemes, but should not be interpreted as limiting the scope of the present invention.

[0080] In some embodiments, the method includes providing a microarray wafer having a surface that includes protected amine groups attached thereto, as illustrated in FIG. 1 (FIG. 1, part no. 1). The protecting groups for the amines are R 1 and R 1 In some embodiments, the aryloxycarbonyl group is selected from the group consisting of carbobenzyloxy (Cbz), p-methoxybenzylcarbonyl (Moz), tert-butyloxycarbonyl (Boc), carbamate group, methoxytrityl (Mmt), dimethoxytrityl (DMT), trityl (Trt), tosyl (Ts), tert-amyloxycarbonyl, adamantyloxycarbonyl, 1-methylcyclobutyloxycarbonyl, 2-(p-biphenyl)propyl(2)oxycarbonyl, 2-(p-phenylazophenylyl)oxycarbonyl, and the like. )propyl(2)oxycarbonyl, α,α-dimethyl-3,5-dimethyloxybenzyloxy-carbonyl, 2-phenylpropyl(2)oxycarbonyl, 4-methyloxybenzyloxycarbonyl, furfuryloxycarbonyl, p-toluenesulfenylaminocarbonyl, dimethylphosphinothioyl, diphenylphosphinothioyl, 2-benzoyl-1-methylvinyl, o-nitrophenylsulfenyl, and 1-naphthylidene, all of which are acid labile. In some embodiments, the amine protecting groups are acetyl (Ac), benzoyl (Bz), 9-fluorenylmethyloxycarbonyl (Fmoc), methylsulfonylethyloxycarbonyl, and 5-benzisoazolylmethyleneoxycarbonyl, which are generally base labile.

[0081] In some embodiments, the microarray wafer is spin-coated with a photoresist formulation (Figure 1, part number 2). In some embodiments, the photoresist formulation comprises a photoactive compound and a photoprotective compound. The photoactive compound is also called a photocleavable reagent. In some embodiments, the photoactive compound is a photoacid generator (PAG) or a photobase generator (PBG). In some embodiments, the polymer is 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(-acrylamido-2-methyl-1-propanesulfonic acid), polysaccharides, and cellulose derivatives. In some embodiments, the photoprotective compound is titanium dioxide, zinc sulfide, and magnesium fluoride.

[0082] In some embodiments, the method comprises forming a photoresist layer on the wafer surface by applying a photoresist formulation. The photoresist layer protects the groups or compounds, including PNA compounds, attached to the microarray from radiation, and the wafer is exposed to radiation. In some embodiments, the protected groups or compounds are included in a layer disposed between the wafer surface and the photoresist layer (Figure 1, part number 2).

[0083] In some embodiments, the photoresist formulation further comprises a polymer and a solvent.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, dimethylsulfoxide, propylene glycol methyl ether acetate (PGMEA), ethyl lactate, ethoxyethyl acetate, or a combination thereof.

[0084] In some embodiments, the wafer is spin-coated with the photoresist formulation at a speed ranging from 2000 rpm to 4000 rpm, preferably from 2500 rpm to 3000 rpm, for 10 to 180 seconds, preferably from 60 to 120 seconds. After the photoresist formulation is spin-coated, the wafer is exposed to radiation according to a pattern defined by a photomask, and since a photoacid generator or a photobase generator is present in the photoresist formulation, an acid or base is generated at the locations exposed to the radiation. In some embodiments, the radiation comprises 248 nm ultraviolet light with a deep UV scanner tool. In some embodiments, the radiation comprises 365 nm ultraviolet light. The radiation that activates the photoacid generator or photobase generator may be at a range of wavelengths and is not limited to those disclosed herein. In some embodiments, the exposure energy is 1 mJ / cm 2 ~100mJ / cm 2 , preferably 30 to 60 mJ / cm 2 The range is.

[0085] In some embodiments, the surface of the wafer is post-baked after radiation exposure in a bake module, hi some embodiments, the post-bake temperature is between 75° C. and 115° C. for about 60 seconds, typically not more than 180 seconds.

[0086] In some embodiments, the photoacid or photobase generator generates a photoacid or photobase upon exposure to radiation, and then the photoacid or photobase removes the amino group protection of the exposed areas on the microarray wafer surface (step 1 of Scheme 1), followed by stripping off the photoresist layer (Figure 1, part no. 3). In some embodiments, the generated photoacid or photobase (also called reacted photocleavable reagent) diffuses out of the photoresist layer into a layer containing a protected group or compound, e.g., a PNA compound, attached to the microarray surface.

[0087] In some embodiments, the unprotected (free) amino group is R 2 - Activated R by spin-coating with an activation formulation containing acetic acid and an activator.2 -acetic acid and reacted in a bake module at a temperature of 55° C. to 115° C. for 60 to 240 seconds (step 2 of Scheme 1; Figure, part no. 4). 2 is a leaving group. In some embodiments, the leaving group is bromo, chloro, fluoro, iodo, etc. In some embodiments, R 2 -Acetic acid includes bromoacetic acid, chloroacetic acid, fluoroacetic acid, iodoacetic acid, and the like.

[0088] In some embodiments, the activating agent is 1-ethyl-3-(3-dimethyl-aminopropyl)-carbodiimide (EDC), N-hydroxysuccinimide (NHS), 1,3-diisopropyl-carbodiimide (DIC), hydroxybenzotriazole (HOBt), O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (HPT), benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (B ... pyridoxafluorophosphate (PyBOP), N,N-diisopropylethylamine (DIEA), N-hydroxysuccinimide (HOSu), N-hydroxy-5-norbornene-2,3-dicarboximide (HONB), 6-chloro-1-hydroxybenzotriazole (6-Cl-HOBt), 3-hydroxy-4-oxo-3,4-dihydro-1,2,3-benzotriazine (HODhbt) and its aza derivative (HODhat), or any combination thereof.

[0089] In some embodiments, R 2- The concentration of acetic acid is less than 0.1% by weight of the total formulation concentration or is 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%, 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%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.8%, 3.9%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.5%, 3.6%, 3.7 ... 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 more than 5.0% by weight.

[0090] In some embodiments, the concentration of the activator is less than 0.1% by weight of the total formulation concentration, or is 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%, 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%, 6.5%, 6.6%, 6.7%, 6.8%, 6.9%, 7.0%, 7.1%, 7.2%, 7.3%, 7.4%, 7.5%, 7.5%, 7.6%, 7.7%, 7.8%, 7.9%, 8.0%, 8.1%, 8.1%, 8.2%, 8.3%, 8.4%, 8.5%, 8.5%, 8.6%, 8.7%, 8.8%, 8.9%, 9.0%, 9.0%, 9.0%, 9 %, 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 wt%, 3.9 wt%, 4.0 wt%, 4.1 wt%, 4.2 wt%, 4.3 wt%, 4.4 wt%, 4.5 wt%, 4.6 wt%, 4.7 wt%, 4.8 wt%, 4.9 wt%, 5.0 wt%, or more than 5.0 wt%.

[0091] In some embodiments, activated R 2 After coupling of -acetic acid, monoamino-protected ethylenediamine (monoR 1 (also called protected ethylenediamine) and a protecting group R 1This is followed by a substitution reaction with (step 3 of Scheme 1; Figure 1, part no. 5). In some embodiments, the substitution reaction involves a monoamino-protected diamino-alkane. "Monoamino-protected" refers to only one of the two amino groups being protected. In some embodiments, a monoR 1 The replacement formulation containing the protected ethylenediamine is spin-coated onto the wafer and reacted in a bake module at a temperature of 55° C. to 115° C. for 30 to 300 seconds, preferably 120 seconds.

[0092] In some embodiments, the protecting group R 1 is any amino protecting group as described above. In some embodiments, the concentration of monoamino protected ethylenediamine is less than 0.1% by weight of the total formulation concentration, or is 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%, 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 ...8%, 3.9%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.8%, 3.9%, 3.1%, 3.2%, 3.3%, %, 2.4 wt%, 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 wt%, 3.9 wt%, 4.0 wt%, 4.1 wt%, 4.2 wt%, 4.3 wt%, 4.4 wt%, 4.5 wt%, 4.6 wt%, 4.7 wt%, 4.8 wt%, 4.9 wt%, 5.0 wt%, or more than 5.0 wt%.

[0093] In some embodiments, the substitution reaction is followed by a coupling reaction with a PNA monomer acetate (step 4 of Scheme 1; Figure 1, part no. 6). In some embodiments, the PNA monomer acetate includes, but is not limited to, R-thymine-1-acetate, R-(cytosin-1-yl)-acetate, R-adenine-9-yl-acetate, R-guanine-9-acetate, and R-uracil-1-acetate. 3-acetic acid, where R is H or a protecting group of the nucleic acid monomer, in some embodiments Boc, Bis-Boc, Alloc, benzoyl, acetyl, Fmoc, trityl, any of the amino protecting groups described above. In some embodiments, the PNA monomer acetate is activated with an activating agent before coupling to the substituted amine. In some embodiments, the wafer is spin-coated with an activation formulation that includes the PNA monomer acetate and an activating agent and reacted for 90-300 seconds.

[0094] In some embodiments, the activating agent is 1-ethyl-3-(3-dimethyl-aminopropyl)-carbodiimide (EDC), N-hydroxysuccinimide (NHS), 1,3-diisopropyl-carbodiimide (DIC), hydroxybenzotriazole (HOBt), O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (HPT), benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (B ... pyridoxafluorophosphate (PyBOP), N,N-diisopropylethylamine (DIEA), N-hydroxysuccinimide (HOSu), N-hydroxy-5-norbornene-2,3-dicarboximide (HONB), 6-chloro-1-hydroxybenzotriazole (6-Cl-HOBt), 3-hydroxy-4-oxo-3,4-dihydro-1,2,3-benzotriazine (HODhbt) and its aza derivative (HODhat), or any combination thereof.

[0095] In some embodiments, the wafer surface is optionally modified so that unreacted amino groups on the wafer are coupled to the next coupling molecule, e.g., activated R 2-The capping solution is spin-coated on the wafer to prevent it from reacting with acetic acid. In some embodiments, the capping solution includes a capping molecule, a solvent, a polymer, and a coupling molecule. In some embodiments, the solvent is an organic solvent such as N-methylpyrrolidone, dimethylformamide, or a combination thereof. In some embodiments, the capping molecule is acetic anhydride, and the polymer is polyvinylpyrrolidone, polyvinyl alcohol, polymethylmethacrylate, poly-(methyl-isopropenyl)-ketone, or poly-(2-methyl-pentene-1-sulfone). In some embodiments, the capping solution is spin-coated on the wafer at 1500-3500 rpm for at least 30 seconds, and reacted in a bake module at a temperature of 55°C-95°C for 30 seconds-90 seconds, preferably 60 seconds, to complete one cycle.

[0096] In some embodiments, the entire synthesis cycle (FIG. 1, parts 1-6) is repeated with a specific PNA monomer acetate in each cycle to generate a specific PNA sequence of the desired length.

[0097] Examples of the described synthetic cycles are further illustrated in Scheme 1 and Examples 1-8. Compounds similar to the target compounds of these examples can be made following similar synthetic routes. The disclosed compounds are useful in microarray fabrication as described herein. The wavy lines in the disclosed schemes TIFF2025013596000002.tif4128 shows the coupling of specific compounds to the microarray surface. Scheme 1 TIFF2025013596000003.tif95139

[0098] When the compounds according to the invention have at least one chiral center, they may exist as corresponding enantiomers. When the compounds have two or more chiral centers, they may further exist as diastereomers. When the process for preparing the compounds according to the invention results in a mixture of stereoisomers, these isomers can be separated by techniques such as preparative chromatography. The compounds may be prepared in racemic form or as individual enantiomers or diastereomers by stereospecific synthesis or resolution. The compounds may be resolved into their component enantiomers or diastereomers by techniques such as, for example, forming a stereoisomeric pair by salt formation with an optically active base, followed by fractional crystallization and free acid regeneration. The compounds may also be resolved by forming stereoisomeric esters or amides, followed by chromatographic separation and removal of the chiral auxiliary. Alternatively, the compounds may be resolved by chiral HPLC columns. It should be understood that all of the stereoisomers, racemic mixtures, diastereomers, geometric isomers, and enantiomers are encompassed within the scope of the present invention.

[0099] Additionally, some of the crystalline forms of the compounds may exist as polymorphs and, as such, are intended to be included in the present invention. Additionally, some of the compounds may form solvates with water (i.e., hydrates) or common organic solvents and, as such, are intended to be encompassed within the scope of the present invention.

[0100] Coupling of DNA monomers to PNA on a microarray Figure 1 and Figure 2A and Figure 2B illustrate suggested synthetic routes according to some embodiments for PNA-DNA chimera oligomer synthesis. Using this scheme, the following guidelines, and examples, one skilled in the art can develop similar or similar methods for obtaining a particular PNA-DNA chimera or a microarray with multiple PNA-DNA chimeras within the scope of the present invention. In some embodiments, the PNA-DNA chimera is coupled to a microarray at a defined location. These methods are representative of synthetic schemes, but should not be construed as limiting the scope of the present invention.

[0101] In some embodiments, PNA-DNA chimeras are synthesized through three distinct types of covalent bonds (i.e., covalent attachments): (i) covalent bonds between a PNA monomer and another PNA monomer, PNA oligomer, or free amine attached to the surface of the array; (ii) covalent bonds between a DNA monomer and the PNA monomer at the end of a PNA oligomer attached to the surface of the array; or (iii) covalent bonds between a DNA monomer and the DNA monomer at the end of a PNA-DNA chimera oligomer. The covalent bonds of PNA monomers are as described above.

[0102] 2A and 2B illustrate suggested synthetic routes, according to some embodiments, for covalently attaching a DNA monomer to a PNA oligomer (steps 7-10) and for covalently attaching a DNA monomer to the 5' end of a PNA-DNA chimeric oligomer (steps 11-13).

[0103] In some embodiments, as illustrated in Figure 2A, the method comprises preparing a microarray wafer having a surface that comprises protected amine groups attached to the termini of PNA oligomers (Figure 2A, part number 7). Examples of suitable protecting groups are provided herein. In some embodiments, PNA oligomers are synthesized on the array according to the methods provided herein.

[0104] In some embodiments, the microarray wafer is spin-coated with a photoresist formulation (FIG. 2A, part no. 8). In some embodiments, the photoresist formulation comprises a photoactive compound and a photoprotective compound. The photoactive compound is also called a photocleavable reagent. In some embodiments, the photoactive compound is a photoacid generator (PAG) or a photobase generator (PBG). In some embodiments, the polymer is 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(-acrylamido-2-methyl-1-propanesulfonic acid), polysaccharides, and cellulose derivatives. In some embodiments, the photoprotective compound is titanium dioxide, zinc sulfide, and magnesium fluoride.

[0105] In some embodiments, the method comprises forming a photoresist layer on the wafer surface by applying a photoresist formulation. The photoresist layer protects the groups or compounds, including PNA compounds, attached to the microarray from radiation, and the wafer is exposed to radiation. In some embodiments, the protected groups or compounds are included in a layer disposed between the wafer surface and the photoresist layer (Figure 1, part number 2).

[0106] In some embodiments, the photoresist formulation further comprises a polymer and a solvent.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, dimethylsulfoxide, propylene glycol methyl ether acetate (PGMEA), ethyl lactate, ethoxyethyl acetate, or a combination thereof.

[0107] In some embodiments, the wafer is spin-coated with the photoresist formulation at a speed ranging from 2000 rpm to 4000 rpm, preferably from 2500 rpm to 3000 rpm, for 10 to 180 seconds, preferably from 60 to 120 seconds. After the photoresist formulation is spin-coated, the wafer is exposed to radiation according to a pattern defined by a photomask, and since a photoacid generator or a photobase generator is present in the photoresist formulation, an acid or base is generated at the locations exposed to the radiation. In some embodiments, the radiation comprises 248 nm ultraviolet light with a deep UV scanner tool. In some embodiments, the radiation comprises 365 nm ultraviolet light. The radiation that activates the photoacid generator or photobase generator may be at a range of wavelengths and is not limited to those disclosed herein. In some embodiments, the exposure energy is 1 mJ / cm 2 ~100mJ / cm 2 , preferably 30 to 60 mJ / cm 2 The range is.

[0108] In some embodiments, the surface of the wafer is post-baked after radiation exposure in a bake module, hi some embodiments, the post-bake temperature is between 75° C. and 115° C. for about 60 seconds, typically not more than 180 seconds.

[0109] In some embodiments, the photoacid or photobase generator generates a photoacid or photobase upon exposure to radiation, and then the photoacid or photobase removes the amino group protection of the exposed areas on the microarray wafer surface, followed by stripping off the photoresist layer (FIG. 2A, part no. 9). In some embodiments, the generated photoacid or photobase (also called reacted photocleavable reagent) diffuses out of the photoresist layer into a layer containing a protected group or compound, e.g., a PNA compound, attached to the microarray surface.

[0110] In some embodiments, the unprotected (free) amino group is coupled to an activated reverse phosphoramidite by spin-coating an activation formulation containing the reverse phosphoramidite and an activator, and reacted in a bake module at a temperature of 75°C to 115°C for 60 to 180 seconds (Figures 2A and 2B, part number 10). In some embodiments, the reverse phosphoramidite contains a dimethoxytrityl ("DMT") or similar protecting group on the 3'-hydroxyl of the deoxynucleoside. In some embodiments, the reverse amidite is a 3'-R4-R5 phosphoramidite. In some embodiments, the activator is a tetrazole catalyst.

[0111] The exemplary synthetic scheme shown in FIG. 3 and described below illustrates the "reverse" (ie, coupling to the 5' position of a growing oligomer) phosphoramidite approach described above.

[0112] In some embodiments, the linker or peptide / nucleotide can be covalently attached to the nucleotide via a phosphoester route (including the use of phosphodiester, phosphotriester, phosphite triester, phosphorothioate activated groups). In these examples, dimethoxytrityl (DMT) is used, but other protecting groups are well known to those skilled in the art. For example, the phosphoramidite chemistry and 5'-O-(α-methyl-6-nitropiperonyloxycarbonyl) (MeNPOC) protecting group described above could be used at the 5' or 3' position of any nucleotide. Such changes and modifications using common protecting or "blocking" groups are within the ability of those skilled in the art to use.

[0113] In some embodiments, the concentration of reverse amidite is less than 0.1% by weight of the total formulation concentration or is 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%, 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%, 7.5%, 7.6%, 7.7%, 7.8%, 7.9%, 8.0%, 8.1%, 8.1%. 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 or more than 5.0% by weight.

[0114] In some embodiments, the concentration of the activator is less than 0.1% by weight of the total formulation concentration, or is 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%, 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%, 6.5%, 6.6%, 6.7%, 6.8%, 6.9%, 7.0%, 7.1%, 7.2%, 7.3%, 7.4%, 7.5%, 7.5%, 7.6%, 7.7%, 7.8%, 7.9%, 8.0%, 8.1%, 8.1%, 8.2%, 8.3%, 8.4%, 8.5%, 8.5%, 8.6%, 8.7%, 8.8%, 8.9%, 9.0%, 9.0%, 9.0%, 9 %, 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 wt%, 3.9 wt%, 4.0 wt%, 4.1 wt%, 4.2 wt%, 4.3 wt%, 4.4 wt%, 4.5 wt%, 4.6 wt%, 4.7 wt%, 4.8 wt%, 4.9 wt%, 5.0 wt%, or more than 5.0 wt%.

[0115] In some embodiments, the wafer surface is optionally spin-coated with a capping solution to prevent unreacted amino groups on the support from reacting with the next coupling molecule. The capping solution can be prepared as follows: solvent, polymer, and coupling molecule. The capping solution is spin-coated on the wafer at 1500-3500 rpm for at least 30 seconds and reacted in a bake module at a temperature of 55° C.-95° C. for 30 seconds-90 seconds, preferably 60 seconds, to complete one cycle. The phosphite triester formed in the coupling step is then converted to a stable form, which is accomplished by iodine oxidation in the presence of water and pyridine.

[0116] In some embodiments, a general synthetic scheme (otherwise known in the art as oligonucleotide synthesis) for adding custom or preselected oligonucleotides to a sequence is described in the exemplary synthesis as shown in Figure 4. As one skilled in the art will appreciate, this synthesis adds a nucleotide through the 5' position of the donor nucleotide to the 3' position of the acceptor nucleotide. In some embodiments, the same phosphoramidite chemistry can be used to access a sequence through the 3' position of the donor nucleotide to the 3' position of the acceptor nucleotide, as shown in Figure 5.

[0117] Thus, in some embodiments, one skilled in the art can mix and match DMT, otherwise known protecting groups, and phosphoramidate activating groups at the 5' and 3' positions of the acceptor and donor nucleotides to couple donors and acceptors together as desired, i.e., to produce any combination via the 5' position. Thus, the amino acids of the PNAs disclosed herein can be coupled to nucleic acids according to some embodiments of the methods described herein.

[0118] Coupling of DNA monomers to PNA-DNA chimeric oligos on a microarray In some embodiments, as illustrated in Figure 2A and Figure 2B, the method includes preparing a microarray wafer having a surface that includes a protected carboxylic acid group attached to the 5' end of a DNA monomer on a PNA-DNA chimeric oligomer (Figure 2A and Figure 2B, part number 10 or 13). Examples of suitable protecting groups are provided herein. In some embodiments, the PNA-DNA chimeric oligomer is synthesized on the array according to the method provided herein.

[0119] In some embodiments, the microarray wafer is spin-coated with a photoresist formulation (FIG. 2A, part number 11). In some embodiments, the photoresist formulation comprises a photoactive compound and a photoprotective compound. The photoactive compound is also called a photocleavable reagent. In some embodiments, the photoactive compound is a photoacid generator (PAG) or a photobase generator (PBG). In some embodiments, the polymer is 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(-acrylamido-2-methyl-1-propanesulfonic acid), polysaccharides, and cellulose derivatives. In some embodiments, the photoprotective compound is titanium dioxide, zinc sulfide, and magnesium fluoride.

[0120] In some embodiments, the method comprises forming a photoresist layer on the wafer surface by applying a photoresist formulation. The photoresist layer protects the groups or compounds, including PNA compounds, attached to the microarray from radiation, and the wafer is exposed to radiation. In some embodiments, the protected groups or compounds are included in a layer disposed between the wafer surface and the photoresist layer (Figure 1, part number 11).

[0121] In some embodiments, the photoresist formulation further comprises a polymer and a solvent.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, dimethylsulfoxide, propylene glycol methyl ether acetate (PGMEA), ethyl lactate, ethoxyethyl acetate, or a combination thereof.

[0122] In some embodiments, the wafer is spin-coated with the photoresist formulation at a speed ranging from 2000 rpm to 4000 rpm, preferably from 2500 rpm to 3000 rpm, for 10 to 180 seconds, preferably from 60 to 120 seconds. After the photoresist formulation is spin-coated, the wafer is exposed to radiation according to a pattern defined by a photomask, and since a photoacid generator or a photobase generator is present in the photoresist formulation, an acid or base is generated at the locations exposed to the radiation. In some embodiments, the radiation comprises 248 nm ultraviolet light with a deep UV scanner tool. In some embodiments, the radiation comprises 365 nm ultraviolet light. The radiation that activates the photoacid generator or photobase generator may be at a range of wavelengths and is not limited to those disclosed herein. In some embodiments, the exposure energy is 1 mJ / cm 2 ~100mJ / cm 2 , preferably 30 to 60 mJ / cm 2 The range is.

[0123] In some embodiments, the surface of the wafer is post-baked after radiation exposure in a bake module, hi some embodiments, the post-bake temperature is between 75° C. and 115° C. for about 60 seconds, typically not more than 180 seconds.

[0124] In some embodiments, the photoacid or photobase generator generates a photoacid or photobase upon exposure to radiation, and then the photoacid or photobase removes the amino group protection of the exposed areas on the microarray wafer surface, followed by stripping off the photoresist layer (FIG. 2A, part no. 12). In some embodiments, the generated photoacid or photobase (also called reacted photocleavable reagent) diffuses out of the photoresist layer into a layer containing a protected group or compound, such as a PNA-DNA chimeric oligo compound, attached to the microarray surface.

[0125] In some embodiments, the unprotected (free) carboxylic acid group is coupled to the activated reverse phosphoramidite by spin-coating an activation formulation containing the reverse phosphoramidite and an activator, and reacted in a bake module at a temperature of 75°C to 115°C for 60 to 180 seconds (Figure 2A and Figure 2B, part number 13). In some embodiments, the reverse amidite is a 3'-R4-R6 phosphoramidite. In some embodiments, the activator is a tetrazole catalyst.

[0126] In some embodiments, the concentration of the 3'-R4-R6 phosphoramidite is less than 0.1% by weight of the total formulation concentration, or is less than 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%, 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%, 7.5%, 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%, 9.5%, 9.6%, 9.7%, 9.8%, 9.9%, 10.0%, 10.1%, 10 %, 2.4 wt%, 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 wt%, 3.9 wt%, 4.0 wt%, 4.1 wt%, 4.2 wt%, 4.3 wt%, 4.4 wt%, 4.5 wt%, 4.6 wt%, 4.7 wt%, 4.8 wt%, 4.9 wt%, 5.0 wt%, or more than 5.0 wt%.

[0127] In some embodiments, the concentration of the activator is 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%, 2.4%, 2.5%, 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%, 4.5% by weight of the total formulation concentration. amount %, 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 wt%, 3.9 wt%, 4.0 wt%, 4.1 wt%, 4.2 wt%, 4.3 wt%, 4.4 wt%, 4.5 wt%, 4.6 wt%, 4.7 wt%, 4.8 wt%, 4.9 wt%, less than 5.0 wt% or more than 5.0 wt%.

[0128] In some embodiments, the wafer surface is optionally spin-coated with a capping solution to prevent unreacted amino groups on the support from reacting with the next coupling molecule. The capping solution can be prepared as follows: solvent, polymer, and coupling molecule. The capping solution is spin-coated on the wafer at 1500-3500 rpm for at least 30 seconds and reacted in a bake module at a temperature of 55° C.-95° C. for 30 seconds-90 seconds, preferably 60 seconds, to complete one cycle. The phosphite triester formed in the coupling step is then converted to a stable form, which is accomplished by iodine oxidation in the presence of water and pyridine.

[0129] In some embodiments, the entire DNA synthesis cycle (Figures 2A and 2B, parts 11-13) is repeated using a specific 3'-R4-R6 phosphoramidite in each cycle to generate a specific DNA sequence of desired length at the end of the PNA-DNA chimeric oligomer.

[0130] formulation Disclosed herein are formulations such as photoresist formulations, replacement formulations, activation formulations and linker formulations.These formulations may be useful in manufacturing and / or using, for example, in manufacturing and / or using the PNA microarrays disclosed herein.Generally, the components of each formulation disclosed herein are soluble in water at room temperature (about 25°C).

[0131] Photoresist Formulation Photoresist formulations are disclosed herein.In some embodiments, the photoresist formulations include components such as photoactive compounds (also called photocleavable reagents) and photoprotective compounds.In some embodiments, the photoactive compounds are photoacid generators or photobase generators.In some embodiments, the photoresist formulations further include polymers and solvents.

[0132] In some embodiments, the polymer is 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(-acrylamido-2-methyl-1-propanesulfonic acid), polysaccharides, and cellulose derivatives. In some embodiments, the photoprotective compound is titanium dioxide, zinc sulfide, magnesium fluoride, and the like.

[0133] In one aspect, the photoresist formulation comprises a photoactive compound. The photoactive compound may comprise a photobase generator or a photoacid generator. Exposure of the photoactive compound to electromagnetic radiation is a primary photochemical event that generates a compound that continues to induce secondary reactions of material transformation within a diffusion-limited range. The photoresist formulation may comprise a photoactive compound that includes a radiation-sensitive catalyst precursor, e.g., a photoacid generator (PAG); a plurality of chemical groups that can react by elimination, addition, or rearrangement in the presence of the catalyst; and optional additives that improve performance or processability, e.g., surfactants, photosensitizers, and etch resistors.

[0134] In some embodiments, the photoresist formulation comprises a photobase 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, while the photoactive compound readily generates a sufficient amount of photobase upon exposure to electromagnetic radiation to cause the desired reaction to occur and produce the product in acceptable yield.

[0135] In some embodiments, the photoresist formulation may include various components such as a photosensitizer, a photoactive compound, a polymer, and a solvent.

[0136] In some embodiments, the photoactive compound may be a photoacid generator (PAG) or a photobase generator (PBG). The photoacid generator (or PAG) is a cationic photoinitiator. A photoinitiator is a compound that is specifically added to a formulation to convert absorbed light energy, UV, or visible light, into chemical energy in the form of an initiating species, e.g., free radicals or cations. Cationic photoinitiators are widely used in photolithography. In general, the ability of some types of cationic photoinitiators to act as a hidden photochemical source of very strong protonic or Lewis acids is the basis for their use in the photoimaging field. In some embodiments, the photoacid generator is an iodonium salt, a polonium salt, or a 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. TIFF2025013596000004.tif23128

[0137] In some embodiments, the photoacid generator is an iodonium and sulfonium salt of a triflate, phosphate, and / or antimonate. In some embodiments, the photoacid generator is about 0.5-5% by weight of the total formulation. In some embodiments, the photoacid generator is present in an amount of about 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%, 2.1%, 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%, 4.1%, 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%, 7.5%, 7.6%, 7.7%, 7.8%, 7.9%, 8.0%, 8.1%, 8.2%, 8.3%, 8.4%, 8.5%, 8.5%, 8.6%, 8.7%, 8.8%, 8.9%, 9.0%, 9.1%, 9.1%, 9.2%, 9.3%, 9.4%, 0.5% 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, less than 7.0% by weight or more than 7.0% by weight.

[0138] In some embodiments, the photobase generator is 1,3-Bis[(2-nitrobenzyl)oxycarbonyl-4-piperidyl]propane or 1,3-Bis[(1-(9-fluorenylmethoxycarbonyl)-4-piperidyl]propane. The photobase generator must be present in the composition of the invention in an amount sufficient to deprotect the monomer so that it can be attached to the support. In some embodiments, the photobase generator is About 0.5-5% by weight of the total formulation concentration. In some embodiments, the photobase generator is about 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%, 2.1%, 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 ... 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 Amount%, 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, less than 7.0% by weight or more than 7.0% by weight.

[0139] In some embodiments, the photoresist formulation forms a photoresist layer on the surface of the microarray, and the concentration of the photoresist layer is less than 0.1% by weight of the total formulation concentration, or is 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%, 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 ...%. %, 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 or more.

[0140] In some embodiments, the solvent is about 80-90% by weight of the total formulation concentration. In some embodiments, the solvent is less than 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 more by weight of the total formulation concentration.

[0141] In some embodiments, the polymer is a non-crosslinked 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: TIFF2025013596000005.tif30128

[0142] 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, polyvinylpyrrolidone is a light, flaky powder that typically readily absorbs up to 40% of its weight in atmospheric water vapor. In the dissolved state, polyvinylpyrrolidone has excellent wettability and readily forms a thin film. In some embodiments, the polymer is vinylpyrrolidone or vinyl alcohol. In some embodiments, the polymer is polymethylmethacrylate.

[0143] In some embodiments, the polymer is present at 2.5-5% by weight of the total formulation. In some embodiments, the polymer is present at about 0.5-5% by weight of the total formulation. In some embodiments, the polymer is present at about 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%, 2.1%, 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 ...8%, 3.9%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 3.0%, 3.1%, 3.2 0.5% 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, less than 5.0% by weight or more than 5.0% by weight.

[0144] In some embodiments, the solvent is water, ethyl lactate, n-methylpyrrolidone, or a combination thereof. In some embodiments, the ethyl lactate can be dissolved in water to greater than 50% to form a solvent. In some embodiments, the solvent may be about 10% propylene glycol methyl ether acetate (PGMEA) and about 90% DI water. In some embodiments, the solvent may include up to about 20% PGMEA. In some embodiments, the solvent may include 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.

[0145] In some embodiments, the solvent is about 80-90% by weight of the total formulation concentration. In some embodiments, the solvent is less than 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 more by weight of the total formulation concentration.

[0146] Replacement preparations In some embodiments, the replacement formulation comprises 1-2% by weight of the total formulation concentration of monoR 1 In some embodiments, monoR 1 The protected ethylenediamine is present at about 0.5 to 5% by weight of the total formulation concentration. 1The protected ethylenediamine is present in an amount of about 0.1%, 0.1% by weight, 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%, 2.4%, 2.5% by weight of the total formulation concentration. %, 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% or more by weight. 1 contains a mono-protected amino group, e.g., a group protected via t-Boc or F-Moc chemistry. In most cases, mono R 1 Increasing the concentration of protected ethylenediamine gives the best performance.

[0147] Activated Preparation Disclosed herein is an activation formulation for activating carboxylic acid so that free amino group and carboxylic acid react.In some embodiments, the activation formulation comprises an activating agent (also called 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 activation compound is N-hydroxysuccinimide (NHS).In some embodiments, the activation formulation optionally comprises a solvent and / or a polymer.

[0148] In some embodiments, the activating formulation is 2-acetic acid, such as bromoacetic acid, chloroacetic acid, fluoroacetic acid, iodoacetic acid. In some embodiments, the activation formulation further comprises a coupling molecule, such as PNA monomer acetic acid. In some embodiments, the PNA monomer acetic acid is R-thymine-1-acetic acid, R-(cytosin-1-yl)-acetic acid, R-adenine-9-yl-acetic acid, R-guanine-9-acetic acid, and R-uracil-1-acetic acid, where R is H or a protecting group of nucleic acid monomer, in some embodiments, Boc, Bis-Boc, Alloc, benzoyl, acetyl, Fmoc, trityl, any of the above amino protecting groups.

[0149] 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-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate) [HATU], benzotriazol-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 a carboxylic acid to a carbonyl group (i.e., activation of the carboxylic acid group). In some embodiments, the carboxylic acid groups are activated for 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 45 minutes, or 60 minutes after exposure to the displacement reaction formulation.

[0150] In some embodiments, the coupling reagent is 2-4% by weight of the total formulation. In some embodiments, the coupling reagent is about 0.5-5% by weight of the total formulation. In some embodiments, the coupling reagent is about 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%, 2.1%, 2.2%, 2.3%, 2.4% by weight of the total formulation. , 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 wt%, 3.9 wt%, 4.0 wt%, 4.1 wt%, 4.2 wt%, 4.3 wt%, 4.4 wt%, 4.5 wt%, 4.6 wt%, 4.7 wt%, 4.8 wt%, 4.9 wt%, less than 5.0 wt% or more than 5.0 wt%.

[0151] In any of the above combinations, the formulation can be completely stripped with water, and thus in some embodiments, after exposure, the formulation can be washed off with water.

[0152] In some embodiments, the activation 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 activation formulation comprises 4% by weight of 1,3-diisopropylcarbodiimide (DIC) and 2% by weight of hydroxybenzotriazole (HOBt) dissolved in NMP. In some embodiments, the activation formulation comprises 4% by weight of (O-(7-azabenzotriazol-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 activation formulation comprises 4% by weight of benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) and 2% by weight of DIEA dissolved in NMP.

[0153] In some embodiments, the solvent is water. In some embodiments, the solvent is about 80-90% by weight of the total formulation concentration. In some embodiments, the solvent is about less than 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 more than 99% by weight of the total formulation concentration.

[0154] In some embodiments, the polymer is polyvinylpyrrolidone and / or polyvinyl alcohol. In some embodiments, the polymer is about 0.5-5% by weight of the total formulation. In some embodiments, the polymer is about less than 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, 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 ... %, 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 wt%, 3.9 wt%, 4.0 wt%, 4.1 wt%, 4.2 wt%, 4.3 wt%, 4.4 wt%, 4.5 wt%, 4.6 wt%, 4.7 wt%, 4.8 wt%, 4.9 wt%, 5.0 wt%, or more than 5.0 wt%.

[0155] 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 about 0.5-5% by weight of the total formulation. In some embodiments, the coupling reagent is about less than 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, 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 ... 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, 5.0% by weight, or more than 5.0% by weight.

[0156] Linker formulation Linker formulations are also disclosed herein. The linker formulation may include components such as a solvent, a polymer, a linker molecule, and a coupling reagent. In some embodiments, the polymer is 1% by weight polyvinyl alcohol and 2.5% by weight polyvinylpyrrolidone, the linker molecule is 1.25% by weight polyethylene oxide, the coupling reagent is 1% by weight 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, and the solvent comprises water. In some embodiments, the polymer is 0.5-5% by weight polyvinyl alcohol and 0.5-5% by weight polyvinylpyrrolidone, the linker molecule is 0.5-5% by weight polyethylene oxide, the coupling reagent is 0.5-5% by weight 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, and the solvent comprises water.

[0157] 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, dimethylsulfoxide, or a combination thereof. In some embodiments, the solvent is about 80-90% by weight of the total formulation concentration. In some embodiments, the solvent is about less than 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 more than 99% by weight of the total formulation concentration.

[0158] In some embodiments, the polymer is polyvinylpyrrolidone and / or polyvinyl alcohol. The general structure of polyvinyl alcohol is as follows, where n is any positive integer greater than 1: TIFF2025013596000006.tif24128

[0159] In some embodiments, the polymer is present at about 0.5-5% by weight of the total formulation. In some embodiments, the polymer is present at about 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%, 2.1%, 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 ...7%, 3.8%, 3.9%, 3.0%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, %, 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 wt%, 3.9 wt%, 4.0 wt%, 4.1 wt%, 4.2 wt%, 4.3 wt%, 4.4 wt%, 4.5 wt%, 4.6 wt%, 4.7 wt%, 4.8 wt%, 4.9 wt%, 5.0 wt%, or more than 5.0 wt%.

[0160] A linker molecule may be a molecule that is inserted between the surface disclosed herein and the PNA chain being synthesized via a coupling molecule. The linker molecule does not necessarily provide functionality, such as molecular recognition functionality, to the resulting PNA chain, but instead may increase the distance between the surface and the PNA chain to increase the exposure of the functional region of the PNA chain at the surface. In some embodiments, the linker may be about 4 to about 40 atoms long to provide exposure. The linker molecule may be, for example, aryl acetylene, ethylene glycol oligomer (PEG) containing 2 to 10 monomer units, diamine, diacid, amino acid, and combinations thereof. Examples of diamines include ethylenediamine and diaminopropane. Alternatively, the linker may be the same molecular type as the molecule being synthesized (e.g., nascent polymer or various coupling molecules), for example, a polymer of polypeptides and amino acid derivatives, e.g., aminohexanoic acid, or a PNA polymer. In some embodiments, the linker molecule is a molecule having a carboxyl group at a first end of the molecule and a protecting group at a second end of the molecule. In some embodiments, the protecting group is a t-Boc protecting group or an Fmoc protecting group. In some embodiments, the linker molecule is or comprises an aryl-acetylene, a polyethylene glycol, a nascent polypeptide, a diamine, a diacid, a peptide, a PNA monomer or polymer, or a combination thereof. In some embodiments, the linker molecule is about 0.5-5% by weight of the total formulation concentration.In some embodiments, the linker molecule 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, 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 ... 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, or more than 5.0% by weight.

[0161] The unattached portion (or free end) of the linker molecule may have a reactive functional group that is blocked, protected, or otherwise rendered unavailable for reaction by a removable protecting group. The protecting group may be attached to the linker molecule to protect the reactive functional group on the linker molecule. Protecting groups that may be used include all acid-labile and base-labile protecting groups. For example, the linker amine group may be protected by t-butoxycarbonyl (t-BOC or BOC) or benzyloxycarbonyl (CBZ), both of which are acid-labile, and by 9-fluorenylmethoxycarbonyl (FMOC), which is base-labile.

[0162] Further protecting groups that can be used include acid labile groups: tert-amyloxycarbonyl, adamantyloxycarbonyl, 1-methylcyclobutyloxycarbonyl, 2-(p-biphenyl)propyl(2)oxycarbonyl, 2-(p-phenylazophenylyl)propyl(2)oxycarbonyl, α,α-dimethyl-3,5-dimethyloxybenzyloxycarbonyl, 2-phenylpropyl(2)oxycarbonyl, 4-methyloxybenzyloxycarbonyl, furfuryloxycarbonyl, triphenylmethyl(trityl), p-toluenesulfenylaminocarbonyl, dimethylphosphinothioyl, diphenylphosphinothioyl, 2-benzoyl-1-methylvinyl, o-nitrophenylsulfenyl, and 1-naphthylidene, for protecting amino moieties; These include base labile groups for protecting amino moieties that are unstable when reduced: 9-fluorenylmethyloxycarbonyl, methylsulfonylethyloxycarbonyl, and 5-benzisoazoylmethyleneoxycarbonyl; groups for protecting amino moieties that are unstable when reduced: dithiasuccinoyl, p-toluenesulfonyl, and piperidino-oxycarbonyl; groups for protecting amino moieties that are unstable when oxidized: (ethylthio)carbonyl; groups for protecting amino moieties that are labile to a wide variety of reagents, suitable agents are given in parentheses after the group: phthaloyl (hydrazine), trifluoroacetyl (piperidine), and chloroacetyl (2-aminothiophenol); acid labile groups for protecting carboxylic acids: tert-butyl esters; and acid labile groups for protecting hydroxyl groups: dimethyltrityl. See also Greene, TW, Protective Groups in Organic Synthesis, Wiley-Interscience, NY, (1981).

[0163] For any of the above combinations, the formulation may be completely water-removable.

[0164] support Supports are also disclosed herein. In some embodiments, the support surface is planar (i.e., two-dimensional). In some embodiments, the support surface is functionalized with free amine groups. In some embodiments, the support surface is functionalized with free carboxylic acid groups. The surface functionalized with free amine groups may be converted to free carboxylic acid groups by activating the carboxylic acid groups of a molecule that contains at least two free carboxylic acid groups (e.g., converting the carboxylic acid groups to carbonyl groups using a carbodiimide) and reacting the molecule with the free amine groups attached to the surface of the support. In some embodiments, the molecule that contains multiple carboxylic acid groups is succinic anhydride, polyethylene glycol diacid, benzene-1,3,5-tricarboxylic acid, benzenehexacarboxylic acid, or carboxymethyl dextran.

[0165] In some aspects, the surface is a rigid or semi-rigid material or group of materials. In some embodiments, the surface may be substantially flat, although in some embodiments, it may be desirable to physically separate synthesis areas for different molecules or features, for example, with wells, raised areas, pins, pillars, etched grooves, etc. In certain aspects, the surface may be porous. Surface materials may be, for example, silicon, biocompatible polymers, such as poly(methyl-methacrylate) (PMMA) and polydimethylsiloxane (PDMS), glass, SiO 2(e.g., thermal oxide silicon wafers, e.g., those used in the semiconductor industry), quartz, silicon nitride, functionalized glass, gold, platinum, and aluminum. Functionalized surfaces include, e.g., amino-functionalized glass, carboxy-functionalized glass, and hydroxy-functionalized glass. Additionally, the surface may be optionally coated with one or more layers to provide a second surface for molecular attachment or functionalization, increasing or decreasing reactivity, binding detection, or other specialized applications. The surface material and / or layer may be porous or non-porous. For example, the surface may be composed of porous silicon. Additionally, the surface may be a silicon wafer or chip, e.g., those used in the semiconductor device manufacturing industry. In the case of a wafer or chip, multiple arrays may be synthesized on the wafer.

[0166] In some embodiments, the support may include a porous layer (i.e., a three-dimensional layer) that includes functional groups for binding the first monomer building block. In some embodiments, the support surface includes pillars for attaching or synthesizing PNA. In some embodiments, the porous layer is added to the top of the pillars.

[0167] porous layer support The porous layer that can be used is a flat permeable polymer material that is made of a porous structure with amine groups (either inherent to the component polymer or introduced into the porous layer) for attaching the first PNA building block.For example, the porous layer can be made of porous silicon, and the surface of the porous silicon is provided with functional groups for attaching the polymer building block.In another example, the porous layer can be made of cross-linked polymer materials.In some embodiments, the porous layer can be made of polystyrene, saccharose, dextran, polyacryloylmorpholine, polyacrylate, polymethylacrylate, polyacrylamide, polyacryloylpyrrolidone, polyvinyl acetate, polyethylene glycol, agarose, sepharose, other conventional chromatographic type materials, and their derivatives and mixtures. 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-acrylamido-2-methyl-1-propanesulfonic acid), polysaccharides, and cellulose derivatives. Preferably, the porosity of the porous layer is 10 to 80%. In one embodiment, the thickness of the porous layer is 0.01 μm to about 1,000 μm. The pore size of the porous layer may be 2 nm to about 100 μm.

[0168] According to another aspect of the present invention, there is provided a support comprising a porous polymeric material having a porosity of 10-80%, reactive groups being chemically bonded to the pore surface and adapted for use in interacting with reactive species, e.g., by chemical bonding with deprotected monomeric building blocks or polymer chains. In some embodiments, the reactive groups are free amine groups. The free amine groups are free to bond, e.g., to the activated carboxyl groups of coupling molecules or substituted amino acid molecules.

[0169] In one embodiment, the porous layer is in contact with a support layer. The support layer may comprise, for example, metal, plastic, silicon, silicon oxide, or silicon nitride. In another embodiment, the porous layer may be in contact with a patterned surface, for example, a patterned surface on top of a pillar support, as described below.

[0170] Pillar support In some embodiments, the support may comprise a planar layer having an upper surface and a lower surface; and a plurality of pillars operatively connected to the layer at defined locations, each pillar having a planar surface extending from the layer, the distance between the surface of each pillar and the upper surface of the layer being about 1,000-5,000 angstroms, and the plurality of pillars being about 10,000 / cm 2 It exists at a density exceeding

[0171] In some embodiments, the planar layer comprises a metal, a plastic, a silicon, a silicon oxide, or a 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 a metal, a plastic, a silicon, a silicon oxide, or a silicon nitride. In some embodiments, the planar layer is 100% of a metal, a silicon, a silicon oxide, or a silicon nitride. In some embodiments, the planar layer is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, or more than 99% of a metal, a silicon, a silicon oxide, or a silicon nitride. In some embodiments, the planar layer is a uniform layer of a metal, a silicon, a silicon oxide, or a silicon nitride.

[0172] In some embodiments, the distance between the surface of each pillar and the top surface of the planar layer may be approximately between less than 1,000 angstroms, 2,000 angstroms, 3,000 angstroms, 3,500 angstroms, 4,500 angstroms, 5,000 angstroms, or more than 5,000 angstroms (or any integer in between).

[0173] In some embodiments, the surface of each pillar is parallel to the top surface of the layer, hi some embodiments, the surface of each pillar is substantially parallel to the top surface of the layer.

[0174] In some embodiments, the plurality of pillars is greater than or equal to 500 / cm 2 , 1,000 / cm 2 , 2,000 / cm 2 , 3,000 / cm 2 , 4,000 / cm 2 , 5,000 / cm 2 , 6,000 / cm 2 , 7,000 / cm 2 , 8,000 / cm 2 , 9,000 / cm 2 , 10,000 / cm 2 , 11,000 / cm 2 , or 12,000 / cm 2 In some embodiments, the plurality of pillars is present at a density of greater than 10,000 / cm (or any integer in between). 2 In some embodiments, the plurality of pillars are present at a density of greater than about 10,000 / cm 2 ~About 2.5 million / cm 2 In some embodiments, the plurality of pillars are present at a density of 2.5 million / cm (or any integer in between). 2 It exists at a density exceeding

[0175] In some embodiments, the surface area of ​​each pillar surface is at least 1 μm 2 In some embodiments, the surface area of ​​each pillar surface is at least 0.1 μm 2 , 0.5 μm 2 , 12μm2 , 3 μm 2 , 4 μm 2 , 5 μm 2 , 6 μm 2 , 7 μm 2 , 8 μm 2 , 9 μm 2 , 10μm 2 , 15μm 2 , 20μm 2 , 25μm 2 , 30μm 2 , 35μm 2 , 40μm 2 , 45μm 2 , or 50 μm 2 (or any integer in between). In some embodiments, the surface area of ​​each pillar surface is 10,000 μm 2 In some embodiments, the surface area of ​​each pillar surface is less than 500 μm 2 , 1,000μm 2 , 2,000μm 2 , 3,000μm 2 , 4,000μm 2 , 5,000μm 2 , 6,000μm 2 , 7,000μm 2 , 8,000μm 2 , 9,000μm 2 , 10,000μm 2 , 11,000μm 2 , or 12,000 μm 2 has a total area smaller than (or any integer in between).

[0176] In some embodiments, the distance between the surface of each pillar and the bottom surface of the layer is between 2,000 and 7,000 angstroms, In some embodiments, the distance between the surface of each pillar and the bottom surface of the layer is less than about 500 angstroms, 1,000 angstroms, 2,000 angstroms, 3,000 angstroms, 4,000 angstroms, 5,000 angstroms, 6,000 angstroms, 7,000 angstroms, 8,000 angstroms, 9,000 angstroms, 10,000 angstroms, 11,000 angstroms, 12,000 angstroms, or more than 12,000 angstroms (or any integer in between). In some embodiments, the distance between the surface of each pillar and the bottom surface of the layer is 7,000 angstroms, 3,000 angstroms, 4,000 angstroms, 5,000 angstroms, 6,000 angstroms, or 7,000 angstroms (or any integer in between).

[0177] In some embodiments, the layer is 1,000-2,000 Angstroms thick, hi some embodiments, the layer is approximately less than 500 Angstroms, 1,000 Angstroms, 2,000 Angstroms, 3,000 Angstroms, 4,000 Angstroms, 5,000 Angstroms, 6,000 Angstroms, 7,000 Angstroms, 8,000 Angstroms, 9,000 Angstroms, 10,000 Angstroms, 11,000 Angstroms, 12,000 Angstroms thick, or greater than 12,000 Angstroms thick (or any integer in between).

[0178] In some embodiments, the center of each pillar is at least 2,000 angstroms away from the center of any other pillar. In some embodiments, the center of each pillar is at least about 500 angstroms, 1,000 angstroms, 2,000 angstroms, 3,000 angstroms, or 4,000 angstroms (or any integer in between) away from the center of any other pillar. In some embodiments, the center of each pillar is at least about 2 μm to 200 μm away from the center of any other pillar.

[0179] In some embodiments, the or each pillar comprises silicon. In some embodiments, the or each pillar comprises silicon dioxide or silicon nitride. In some embodiments, the or each pillar is at least 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), 98.5% (by weight), or 99% (by weight) silicon dioxide.

[0180] In some embodiments, the support may include a linker molecule having a free amino terminus attached to the surface of each pillar. In some embodiments, the support may include a linker molecule having a free amino terminus attached to the surface of at least one pillar. In some embodiments, the support may include a linker molecule having a protecting group attached to the surface of each pillar. In some embodiments, the support may include a linker molecule having a protecting group attached to the surface of at least one pillar. In some embodiments, the support may include a coupling molecule attached to the surface of at least one pillar. In some embodiments, the support may include a coupling molecule attached to the surface of each pillar. In some embodiments, the support may include a polymer in contact with at least one surface of the pillar. In some embodiments, the support may include a polymer in contact with the surface of each pillar. In some embodiments, the support may include a gelatinous polymer in contact with at least one surface of the pillar. In some embodiments, the support may include a solid polymer in contact with at least one surface of the pillar.

[0181] In some embodiments, the surface of at least one of the pillars of the support is derivatized. In some embodiments, the support may include a polymer chain attached to the surface of at least one of the pillars. In some embodiments, the polymer chain includes a PNA. In some embodiments, the attachment to the surface of at least one of the pillars is via a covalent bond.

[0182] In some embodiments, the surface of each pillar is square or rectangular in shape. In some embodiments, the support may be coupled to a silicon dioxide layer. The silicon dioxide layer may be about 0.5 μm to 3 μm thick. In some embodiments, the support may be coupled to a wafer, e.g., a silicon wafer. The silicon dioxide layer may be about 700 μm to 750 μm thick.

[0183] array Arrays are also disclosed herein. In some embodiments, the arrays may be two-dimensional arrays. In some embodiments, the arrays comprise a surface comprising a support, the support comprising a planar layer having an upper surface and a lower surface.

[0184] In some embodiments, the two-dimensional array may include features attached to a surface at defined locations, each of which comprises a collection of PNA or PNA-DNA chimeric oligonucleotide strands of determinable sequence and intended length, and the fraction of PNA or PNA-DNA chimeric oligonucleotides in the collection that have the intended length among the individual features is characterized by an average coupling efficiency of about 98% for each coupling step. In some embodiments, the array comprises a plurality of pillars operably coupled to the layer at defined locations, each pillar having a flat surface extending from the layer, the distance between the surface of each pillar and the top surface of the layer being 1,000-5,000 angstroms, and the plurality of pillars being 10,000 / cm 2 It exists at a density exceeding

[0185] In some embodiments, the surface of the array is functionalized with free amine groups. In some embodiments, the surface density of the free amine groups on the array is 10 / cm 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 In some embodiments, the surface density of features on the array is greater than 10 / cm 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 Greater than.

[0186] In some embodiments, the array may be a three-dimensional array, for example a porous array, with features attached to the surface of the porous array. In some embodiments, the surface of the porous array comprises an outer surface and a surface that defines the pore volume within the porous array. In some embodiments, the three-dimensional array may comprise features attached to the surface at a defined location, each of which comprises a collection of PNA strands or PNA-DNA chimeric oligonucleotide strands with determinable sequence and intended length. In one embodiment, the ratio of PNA strands or PNA-DNA chimeric oligonucleotide strands in the collection that have intended length among individual features is characterized by an average coupling efficiency of each coupling step that exceeds 98%.

[0187] In some embodiments, the average coupling efficiency of each coupling step for PNA-PNA binding is at least 98.5%. In some embodiments, the average coupling efficiency of each coupling step for PNA-PNA binding is at least 99%. In some embodiments, the average coupling efficiency of each coupling step for PNA-PNA binding 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%.

[0188] In some embodiments, the average coupling efficiency of each coupling step for PNA-DNA binding is at least 97%. In some embodiments, the average coupling efficiency of each coupling step for PNA-DNA binding 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%.

[0189] In some embodiments, the average coupling efficiency of each coupling step for DNA-DNA binding is at least 98.5%. In some embodiments, the average coupling efficiency of each coupling step for DNA-DNA binding 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%.

[0190] In some embodiments, the average coupling efficiency of each coupling step for full-length PNA or PNA-DNA chimeric oligonucleotide synthesis 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%.

[0191] In some embodiments, the purity of each feature in terms of the ratio of full-length predetermined PNA strands or PNA-DNA chimeric oligonucleotide strands is the ratio F of full-length predetermined PNA strands or PNA-DNA chimeric oligonucleotide strands of each feature having a predetermined sequence and a length N of the predetermined full-length sequence, F=10 (N+1)·log(E / 100%) In some embodiments, F is characterized by an average coupling efficiency E of at least 98.5% when coupling each monomer of the predetermined sequence. In some embodiments, F is characterized by an average coupling efficiency E of at least 98.5% when coupling each monomer of the predetermined sequence. In some embodiments, the average coupling efficiency E of 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%.

[0192] In some embodiments, the distribution of the lengths of the sequences on the array is based on the synthesis of PNA or PNA-DNA chimeric sequences of a defined length (e.g., 64mer). In each coupling step, the length of the sequence where the desired coupling does not occur is defined as the length when the capping solution is used. The length distribution according to the step yield of each sequence length shorter than the complete sequence is calculated according to the following formula: F(N)=10 (N+1)·log(E / 100%) -10 (N)·log(E / 100%) where F(N) is the proportion of sequences on the array at length N that is less than the full length sequence, and E is the average coupling efficiency percent. The exact value of E at each length N can also be used to obtain the exact number of oligomers at each length.

[0193] The ratio of full-length sequences is calculated according to the following formula: F(N)=10 (N)·log(E / 100%) where F(N) is the proportion of sequences on the array that are full-length sequences (no further coupling steps) and E is the average coupling efficiency.

[0194] In some embodiments, the sequence length N is at least 64 monomers in length and the fraction of a predetermined PNA strand or PNA-DNA chimeric oligonucleotide strand that is shorter than the full length is equal to (1-F). In some embodiments, the sequence length N is at least 65 monomers in length.

[0195] In some embodiments, each PNA strand or PNA-DNA chimeric oligonucleotide strand is 5-100 monomers in length. In some embodiments, each PNA strand or PNA-DNA chimeric oligonucleotide strand is at least 64 monomers in length. In some embodiments, each PNA strand or PNA-DNA chimeric oligonucleotide strand is at least 65 monomers in length. In some embodiments, each PNA strand or PNA-DNA chimeric oligonucleotide strand is at least 100 monomers in length or is longer than 100 monomers. In some embodiments, each PNA strand or PNA-DNA chimeric oligonucleotide strand is at least 5 monomers, 10 monomers, 15 monomers, 20 monomers, 25 monomers, 30 monomers, 35 monomers, 40 monomers, 45 monomers, 50 monomers, 55 monomers, 60 monomers, 65 monomers, 70 monomers, 75 monomers, 80 monomers, 85 monomers, 90 monomers, 95 monomers, or 100 monomers in length.In some embodiments, each PNA strand or PNA-DNA chimeric oligonucleotide strand is less than 5 monomers in length or is at least 5 monomers, 6 monomers, 7 monomers, 8 monomers, 9 monomers, 10 monomers, 11 monomers, 12 monomers, 13 monomers, 14 monomers, 15 monomers, 16 monomers, 17 monomers, 18 monomers, 19 monomers, 20 monomers, 21 monomers, 22 monomers, or more. , 23 monomer, 24 monomer, 25 monomer, 26 monomer, 27 monomer, 28 monomer, 29 monomer, 30 monomer, 31 monomer, 32 monomer, 33 monomer, 34 monomer, 35 monomer, 36 monomer, 37 monomer, 38 monomer, 39 monomer, 40 monomer, 41 monomer, 42 monomer, 43 monomer, 44 monomer, 45 monomer, 46 monomer, 47 monomer, 48 monomer, 49 monomer , 50 monomer, 51 monomer, 52 monomer, 53 monomer, 54 monomer, 55 monomer, 56 monomer, 57 monomer, 58 monomer, 59 monomer, 60 monomer, 61 monomer, 62 monomer, 63 monomer, 64 monomer, 65 monomer, 66 monomer, 67 monomer, 68 monomer, 69 monomer, 70 monomer, 71 monomer, 72 monomer, 73 monomer, 74 monomer, 75 monomer, 76 monomer, 77 monomers, 78 monomers, 79 monomers, 80 monomers, 81 monomers, 82 monomers, 83 monomers, 84 monomers, 85 monomers, 86 monomers, 87 monomers, 88 monomers, 89 monomers, 90 monomers, 91 monomers, 92 monomers, 93 monomers, 94 monomers, 95 monomers, 96 monomers, 97 monomers, 98 monomers, 99 monomers, 100 monomers, or longer than 100 monomers. In some embodiments, each PNA strand or PNA-DNA chimeric oligonucleotide strand comprises one or more L-chiral PNA monomers. In some embodiments, each PNA strand or PNA-DNA chimeric oligonucleotide strand comprises one or more D-chiral PNA monomers. In some embodiments, each PNA strand or PNA-DNA chimeric oligonucleotide strand comprises one or more modified nucleotides.

[0196] In some embodiments, each PNA-DNA oligonucleotide chimeric strand is 5-100 monomers in length. In some embodiments, each PNA-DNA oligonucleotide chimeric strand is at least 64 monomers in length. In some embodiments, each PNA-DNA oligonucleotide chimeric strand is at least 65 monomers in length. In some embodiments, each PNA-DNA oligonucleotide chimeric strand is at least 100 monomers in length or is longer than 100 monomers. In some embodiments, each PNA-DNA oligonucleotide chimeric strand is at least 5 monomers, 10 monomers, 15 monomers, 20 monomers, 25 monomers, 30 monomers, 35 monomers, 40 monomers, 45 monomers, 50 monomers, 55 monomers, 60 monomers, 65 monomers, 70 monomers, 75 monomers, 80 monomers, 85 monomers, 90 monomers, 95 monomers, or 100 monomers in length.In some embodiments, each PNA-DNA oligonucleotide chimeric strand is less than 5 monomers in length or is at least 5 monomers, 6 monomers, 7 monomers, 8 monomers, 9 monomers, 10 monomers, 11 monomers, 12 monomers, 13 monomers, 14 monomers, 15 monomers, 16 monomers, 17 monomers, 18 monomers, 19 monomers, 20 monomers, 21 monomers, 22 monomers, 23 monomers, or less. , 24 monomer, 25 monomer, 26 monomer, 27 monomer, 28 monomer, 29 monomer, 30 monomer, 31 monomer, 32 monomer, 33 monomer, 34 monomer, 35 monomer, 36 monomer, 37 monomer, 38 monomer, 39 monomer, 40 monomer, 41 monomer, 42 monomer, 43 monomer, 44 monomer, 45 monomer, 46 monomer, 47 monomer, 48 monomer, 49 monomer, 50 monomer monomer, 51 monomer, 52 monomer, 53 monomer, 54 monomer, 55 monomer, 56 monomer, 57 monomer, 58 monomer, 59 monomer, 60 monomer, 61 monomer, 62 monomer, 63 monomer, 64 monomer, 65 monomer, 66 monomer, 67 monomer, 68 monomer, 69 monomer, 70 monomer, 71 monomer, 72 monomer, 73 monomer, 74 monomer, 75 monomer, 76 monomer, 77 monomer, 78 monomer, 79 monomer, 80 monomer, 81 monomer, 82 monomer, 83 monomer, 84 monomer, 85 monomer, 86 monomer, 87 monomer, 88 monomer, 89 monomer, 90 monomer, 91 monomer, 92 monomer, 93 monomer, 94 monomer, 95 monomer, 96 monomer, 97 monomer, 98 monomer, 99 monomer, 100 monomer, or longer than 100 monomer. In some embodiments, each PNA-DNA oligonucleotide chimeric chain comprises one or more L-chiral PNA monomers. In some embodiments, each PNA-DNA oligonucleotide chimeric chain comprises one or more D-chiral PNA monomers. In some embodiments, each PNA-DNA oligonucleotide chimeric chain comprises one or more modified nucleotides. In some embodiments, each PNA-DNA oligonucleotide chimeric strand contains one or more ribonucleotides at a DNA position.

[0197] In some embodiments, the array may comprise at least 1,000 different features attached to the surface. In some embodiments, the array may comprise at least 10,000 different features attached to the surface. In some embodiments, the array may comprise at least 100, 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, or more than 10,000 (or any integer in between) different features attached to the surface.

[0198] In some embodiments, the array may comprise at least 1,000 different PNA strands or PNA-DNA chimeric oligonucleotide strands attached to the surface. In some embodiments, the array may comprise at least 10,000 different PNA strands or PNA-DNA chimeric oligonucleotide strands attached to the surface. In some embodiments, the array may comprise at least 100, 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, or more than 10,000 (or any integer between) different PNA strands or PNA-DNA chimeric oligonucleotide strands attached to the surface.

[0199] In some embodiments, each feature comprises at least 500 identical full-length PNA strands or PNA-DNA chimeric oligonucleotide strands, each identical full-length PNA strand or PNA-DNA chimeric oligonucleotide strand being at least 64 monomers, 65 monomers, 66 monomers, 67 monomers, 68 monomers, 69 monomers, 70 monomers, 71 monomers, 72 monomers, 73 monomers, 74 monomers, 75 monomers, or more in length. In some embodiments, the amino acid sequence may be 100, 101, 102, 103, 104, 105, 106, 107, 108, 1109, 1120, 1130, 1140, 1150, 1160, 1170, 1180, 1190, 1201, 1210, 1220, 1230, 1240, 1250, 1260, 1270, 1280, 1290, 1301, 1310, 1320, 1330, 1340, 1350, 1360, 1370, 1380, 1390, 1401, 1410, 1420, 1430, 1440, 1450, 1460, 1470, 1480, 1490, 1501, 1510, 1520, 1530, 1540, 1550, 1560, 1570, 1580, 1590, 1610, 1620, 1630, 1640, 1650, 1660, 1670, 1680, 1690, 1701, 1710, 1720, 1730, 1740, 1750, 1760, 1770, 1780, 1790, 1801, 1810, 1820, 1830, 1840, 1850, 1860, 1870, In some embodiments, each feature comprises 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 identical full-length PNA strands or PNA-DNA chimeric oligonucleotide strands, each identical full-length PNA strand or PNA-DNA chimeric oligonucleotide strand being at least 64 monomers, 65 monomers, 66 monomers, 67 monomers, 68 monomers, 69 monomers, 70 monomers, 71 monomers, 72 monomers, 73 monomers, 74 monomers, 75 monomers, 76 monomers, 77 monomers, 78 monomers, 79 monomers, 80 monomers, 81 monomers, 82 monomers, 83 monomers, 84 monomers, 85 monomers, 86 monomers, 87 monomers, 88 monomers, 89 monomers, 90 monomers, 91 monomers, 92 monomers, 93 monomers, 94 monomers, 95 monomers, 96 monomers, 97 monomers, 98 monomers, 99 monomers, 100 monomers, 101 monomers, 102 monomers, 103 monomers, 104 monomers, 105 monomers, 106 monomers, 107 monomers, 108 monomers, 109 monomers, 1100 monomers, 1200 monomers, 1200 monomers, 1300 monomers, 1400 strands, 1500 strands, 1600 strands, 1700 strands, 1 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, 52, 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, 100 or more monomers.

[0200] In some embodiments, each of the defined locations is at a different known location that is physically separated from each of the other defined locations. In some embodiments, each of the defined locations is a distinct location. In some embodiments, each determinable sequence is a known sequence. In some embodiments, each determinable sequence is a distinct sequence.

[0201] In some embodiments, each feature is attached to a different, defined location on the surface of the array, the defined location of each feature corresponds to the defined location of a pillar, and the top surface of each pillar is at least 1 μm in size. 2 It is.

[0202] In some embodiments, the features are covalently attached to the surface, hi some embodiments, the PNA strands or PNA-DNA chimeric oligonucleotide strands are attached to the surface via linker or coupling molecules.

[0203] In certain embodiments, the feature comprises a plurality of distinct nested overlapping PNA strands or PNA-DNA chimeric oligonucleotide strands, which comprise subsequences derived from a source DNA sequence or RNA sequence with known sequence.In some embodiments, the length of each PNA strand or PNA-DNA chimeric oligonucleotide strand in the plurality of overlapping PNA strands or PNA-DNA chimeric oligonucleotide strands is substantially the same.In some embodiments, the length of each PNA strand or PNA-DNA chimeric oligonucleotide strand in the plurality of overlapping PNA strands or PNA-DNA chimeric oligonucleotide strands is the same.

[0204] In some embodiments, each PNA strand or PNA-DNA chimeric oligonucleotide strand in the multiple overlapping PNA strands or PNA-DNA chimeric oligonucleotide strands is at least 5 monomers in length.In some embodiments, each PNA strand or PNA-DNA chimeric oligonucleotide strand in the multiple overlapping PNA strands or PNA-DNA chimeric oligonucleotide strands is at least 5 monomers, 10 monomers, 15 monomers, 20 monomers, 25 monomers, 30 monomers, 35 monomers, 40 monomers, 45 monomers, 50 monomers, 55 monomers, or 60 monomers in length. In some embodiments, each PNA strand or PNA-DNA chimeric oligonucleotide strand within a plurality of overlapping PNA strands or PNA-DNA chimeric oligonucleotide strands is less than 5 monomers in length or is at least 5 monomers, 6 monomers, 7 monomers, 8 monomers, 9 monomers, 10 monomers, 11 monomers, 12 monomers, 13 monomers, 14 monomers, 15 monomers, 16 monomers, 17 monomers in length. 18 monomer, 19 monomer, 20 monomer, 21 monomer, 22 monomer, 23 monomer, 24 monomer, 25 monomer, 26 monomer, 27 monomer, 28 monomer, 29 monomer, 30 monomer, 31 monomer, 32 monomer, 33 monomer, 34 monomer, 35 monomer, 36 monomer, 37 monomer, 38 monomer, 39 monomer, 40 monomer, 41 monomer, 42 monomer, 43 monomer, 44 monomer, 45 monomer, 46 monomer 47 monomer, 48 monomer, 49 monomer, 50 monomer, 51 monomer, 52 monomer, 53 monomer, 54 monomer, 55 monomer, 56 monomer, 57 monomer, 58 monomer, 59 monomer, 60 monomer, 61 monomer, 62 monomer, 63 monomer, 64 monomer, 65 monomer, 66 monomer, 67 monomer, 68 monomer, 69 monomer, 70 monomer, 71 monomer, 72 monomer, 73 monomer, 74 monomer, 75 monomer, 76 monomer, 77 monomer, 78 monomer, 79 monomer, 80 monomer, 81 monomer, 82 monomer, 83 monomer, 84 monomer, 85 monomer, 86 monomer, 87 monomer, 88 monomer, 89 monomer, 90 monomer, 91 monomer, 92 monomer, 93 monomer, 94 monomer, 95 monomer, 96 monomer, 97 monomer, 98 monomer, 99 monomer, 100 monomer or longer.In some embodiments, each PNA strand or PNA-DNA chimeric oligonucleotide strand in a plurality of overlapping PNA strands or PNA-DNA chimeric oligonucleotide strands is less than 5 monomers in length, or is at least 100 monomers, 105 monomers, 110 monomers, 115 monomers, 120 monomers, 125 monomers, 130 monomers, 135 monomers, 140 monomers, 145 monomers, 150 monomers, 155 monomers, 160 monomers, 165 monomers, 170 monomers, 175 monomers, 180 monomers, 185 monomers, 190 monomers, 195 monomers, 200 monomers, or is greater than 200 monomers in length.

[0205] In some embodiments, at least one PNA strand or PNA-DNA chimeric oligonucleotide strand in the multiple overlapping PNA strands or PNA-DNA chimeric oligonucleotide strands is at least 5 monomers in length.In some embodiments, at least one PNA strand or PNA-DNA chimeric oligonucleotide strand in the multiple overlapping PNA strands or PNA-DNA chimeric oligonucleotide strands is at least 5 monomers, 10 monomers, 15 monomers, 20 monomers, 25 monomers, 30 monomers, 35 monomers, 40 monomers, 45 monomers, 50 monomers, 55 monomers, or 60 monomers in length. In some embodiments, at least one PNA strand or PNA-DNA chimeric oligonucleotide strand among a plurality of overlapping PNA strands or PNA-DNA chimeric oligonucleotide strands is less than 5 monomers in length, or at least 5 monomers, 6 monomers, 7 monomers, 8 monomers, 9 monomers, 10 monomers, 11 monomers, 12 monomers, 13 monomers, 14 monomers, 15 monomers, 16 monomers, 17 monomers, or more. Monomer, 18 Monomer, 19 Monomer, 20 Monomer, 21 Monomer, 22 Monomer, 23 Monomer, 24 Monomer, 25 Monomer, 26 Monomer, 27 Monomer, 28 Monomer, 29 Monomer, 30 Monomer, 31 Monomer, 32 Monomer, 33 Monomer, 34 Monomer, 35 Monomer, 36 Monomer, 37 Monomer, 38 Monomer, 39 Monomer, 40 Monomer, 41 Monomer, 42 Monomer, 43 Monomer, 44 Monomer, 45 Monomer, 46 Monomer, 47 Monomer, 48 Monomer, 49 Monomer, 50 Monomer, 51 Monomer, 52 Monomer, 53 Monomer, 54 Monomer, 55 Monomer, 56 Monomer, 57 Monomer, 58 Monomer, 59 Monomer, 60 Monomer, 61 Monomer, 62 Monomer, 63 Monomer, 64 Monomer, 65 Monomer, 66 Monomer, 67 Monomer, 68 Monomer, 69 Monomer, 70 Monomer, 71 Monomer, 72 Monomer, 73 Monomer, 74 Monomer, 7 5 monomers, 76 monomers, 77 monomers, 78 monomers, 79 monomers, 80 monomers, 81 monomers, 82 monomers, 83 monomers, 84 monomers, 85 monomers, 86 monomers, 87 monomers, 88 monomers, 89 monomers, 90 monomers, 91 monomers, 92 monomers, 93 monomers, 94 monomers, 95 monomers, 96 monomers, 97 monomers, 98 monomers, 99 monomers, 100 monomers or longer.

[0206] In some embodiments, the length of each PNA strand or PNA-DNA chimeric oligonucleotide strand in a feature is substantially the same.In some embodiments, the length of each PNA strand or PNA-DNA chimeric oligonucleotide strand in a feature is the same.In some embodiments, a feature comprises a plurality of PNA strands or PNA-DNA chimeric oligonucleotide strands, each strand having a random and determinable monomer sequence.

[0207] method Method for manufacturing a support Methods for making supports are also disclosed herein. In some embodiments, the method for producing a support may include coupling 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 support includes a plurality of amino groups attached to the support for binding PNA monomers during PNA or PNA-DNA chimeric oligonucleotide synthesis and PNA monomer coupling. In some embodiments, the method for producing a support may include coupling a porous layer to a plurality of pillars, the porous layer including functional groups for attaching compounds to the support, wherein the plurality of pillars are attached to the planar layer at defined locations, each pillar having a flat surface extending from the planar layer, the distance between the surface of each pillar and the top surface of the planar layer is about 1,000-5,000 angstroms, and the plurality of pillars are about 10,000 / cm 2 It exists at a density exceeding

[0208] In some embodiments, the surface of each pillar is parallel to the top surface of the planar layer, hi some embodiments, the surface of each pillar is substantially parallel to the top surface of the planar layer.

[0209] surface derivatization The support can be surface derivatized in the semiconductor module as described in U.S. Patent Application Publication No. 2010 / 0240555 (filed Sep. 29, 2006), which is incorporated by reference in its entirety for all purposes. A representative support of the present invention has oxide pillars ready for surface derivatization. Surface derivatization is a method in which aminosilane groups are added to the support so that free amino groups are available for biomolecule coupling. In some aspects, the first molecule attached to the surface-derivatized support is tboc-protected glycine. This coupling procedure is similar to the standard Merrifield solid phase peptide synthesis procedure commonly known to those skilled in the art.

[0210] In some embodiments, the method for preparing the surface of a support may include the steps of obtaining a surface comprising silicon dioxide, and contacting the surface with a photoresist formulation comprising a photoactive compound and a photoprotective compound, and optionally comprising a polymer and a solvent; and applying ultraviolet light to a defined location that is located on the top of the surface and contacts with the photoresist formulation.In some aspects, the method may include removing the photoresist formulation that is outside the defined location.

[0211] Methods for fabricating arrays Also disclosed herein is a method for producing arrays. In some embodiments, the arrays disclosed herein can be synthesized in situ on a surface, for example, on a support disclosed herein. In some cases, the arrays are made using photolithography. For example, the support is contacted with a photoresist formulation. A mask can be used to control the exposure of radiation or light to specific locations on the surface on which free linker or coupling molecules with protective groups are provided. In the exposed locations, the protective groups are removed, resulting in one or more newly exposed reactive moieties on the coupling or linker molecules. The desired linker or coupling molecules are then linked to the unprotected attached molecules, for example, to the free amine groups. This process may be repeated to synthesize multiple features at specific locations or defined positions on the surface (see, e.g., U.S. Pat. No. 5,143,854 to Pirrung et al., U.S. Patent Application Publication Nos. 2007 / 0154946 (filed December 29, 2005), 2007 / 0122841 (filed November 30, 2005), 2007 / 0122842 (filed March 30, 2006), 2008 / 0108149 (filed October 23, 2006), and 2010 / 0093554 (filed June 2, 2008), each of which is incorporated herein by reference).

[0212] In some embodiments, a method for generating a three-dimensional (e.g., porous) array of features may include the steps of obtaining a porous layer attached to a surface; and attaching features to the porous layer, each of the features comprising a collection of PNA strands or PNA-DNA chimeric oligonucleotide strands of determinable sequence and intended length, and the ratio of PNA strands or PNA-DNA chimeric oligonucleotide strands in the collection with intended length among individual features is characterized by an average coupling efficiency of at least about 98% for each coupling step. In some embodiments, the features are attached to the surface using a photoresist formulation comprising a photoactive compound, a photoprotective compound, optionally a polymer and a solvent. In some embodiments, the features are attached to the surface using a photoresist formulation disclosed herein. In some embodiments, the photoresist formulation is removed using water.

[0213] In some embodiments, a process for fabricating an array is described herein. A surface is provided that includes attached free amine groups. The surface is contacted with a photoresist formulation that includes 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, according to a pattern defined by a light mask in a deep UV scanner tool, and photoacid is generated at the locations exposed to the radiation due to the presence of a photoacid generator in the photoresist formulation. To generate sufficient photobase, the exposure energy is 1 mJ / cm2. 2 ~100mJ / cm 2 In some embodiments, the radiation comprises 248 nm ultraviolet light. In some embodiments, the radiation comprises 365 nm ultraviolet light. The radiation that activates the photoacid generator or photobase generator may be of a range of wavelengths and is not limited to the wavelengths disclosed herein.

[0214] Photoprotective compounds shield any molecules attached to the microarray from electromagnetic radiation and prevent possible radiation-induced destruction of the attached molecules. Photoprotective compounds include, but are not limited to, titanium dioxide, zinc sulfide, magnesium fluoride, and the like.

[0215] The surface is post-baked upon exposure in a post-exposure bake module. The post-exposure bake acts as a chemical amplification step. The bake step amplifies the initially generated photoacid and also increases its diffusion rate into the support. The post-bake temperature is at least 60 seconds, usually not more than 120 seconds, and may be between 75°C and 115°C, depending on the thickness of the porous surface or planar layer of the support. The free amine groups are coupled to the activated carboxylic acid groups of the free compound molecules, coupling the free compound molecules to the amine groups attached to the surface. The surface may be a porous surface or a planar layer of the support. The synthesis of the PNA strands or PNA-DNA chimeric oligonucleotide strands coupled to the amine groups attached to the surface is performed in the C→N synthesis direction. Alternatively, a diamine linker may be attached to the free carboxylic acid groups to orient the synthesis in the C→N direction, and the activated carboxylic acid groups of the free compound molecules attach to the amine groups attached to the support surface.

[0216] The photoresist formulation can then be removed. In some embodiments, a method is provided herein for completely removing the photoresist using deionized (DI) water. This process is accomplished in a development module. The wafer is spun on a vacuum chuck for, for example, 60 to 90 seconds, and deionized water is dispensed through a nozzle for about 30 seconds.

[0217] The photoresist formulation may be applied to the surface in a coupling spin module. The coupling spin module may typically have 20 or more nozzles to deliver the photoactive coupling formulation. These nozzles may be made to dispense the photoactive coupling formulation by pressurizing cylinders holding these solutions or by a pump that dispenses the required amount. In some embodiments, a pump is used to dispense 5-8 cc of the photoactive coupling formulation onto the support. The support is spun in a vacuum chuck for 15-30 seconds and the photoactive coupling formulation is dispensed. The spin speed may be set to be 2000-2500 rpm.

[0218] As described under "Compositions," the subsequent steps of the method are illustrated in FIG. 1, Scheme 1, and FIGS. 2A and 2B.

[0219] Optionally, a cap film solution coat is applied on the surface to prevent unreacted amino groups on the support from reacting with the next coupling molecule. The cap film coat solution can be prepared as follows: solvent, polymer, and coupling molecule. The solvent that can be used can be an organic solvent such as N-methylpyrrolidone, dimethylformamide, or a combination thereof. The capping molecule is typically acetic anhydride, and the polymer can be polyvinylpyrrolidone, polyvinyl alcohol, polymethylmethacrylate, poly-(methyl-isopropenyl)-ketone, or poly-(2-methyl-pentene-1-sulfone). In some embodiments, the capping molecule is ethanolamine.

[0220] This process is carried out in a capping spin module. The capping spin module may be equipped with one nozzle that can be made to dispense the cap film coat solution onto the support. The solution may be dispensed by pressurizing a cylinder that stores the cap film coat solution or through a pump that precisely dispenses the required amount. In some embodiments, a pump is used to dispense about 5-8 cc of cap coat solution onto the support. The support is spun in a vacuum chuck for 15-30 seconds and the coupling formulation is dispensed. The rotation speed is set to be 2000-2500 rpm.

[0221] The support containing the capping solution is baked in a capping bake module. The capping bake module is a hot plate that is specifically arranged to receive the wafer immediately after the capping film coat is applied. In some embodiments, a method is provided herein to bake the spin-coated capping coat solution in a hot plate to significantly accelerate the capping reaction. In general, the hot plate bake shortens the capping time to less than 2 minutes.

[0222] The by-products of the capping reaction are removed in a stripper module. The stripper module may include several nozzles, typically up to 10 nozzles, arranged to dispense organic solvents, such as acetone, isopropyl alcohol, N-methylpyrrolidone, dimethylformamide, DI water, etc. In some embodiments, the nozzles may be designated to dispense acetone followed by isopropyl alcohol onto the rotating wafer. The rotation speed is set to be 2000-2500 rpm for about 20 seconds.

[0223] This entire cycle can be repeated as desired, each time with a different coupling molecule, to obtain a PNA or PNA-DNA chimeric oligonucleotide strand of determinable sequence and intended length.

[0224] How to use PNA or PNA-DNA microarrays Also disclosed herein are methods of using the support, formulation, and / or array.The use of the array disclosed herein may include research applications, therapeutic purposes, medical diagnosis, and / or stratification of one or more patients.

[0225] Any of the arrays described herein can be used as a research tool or in research applications.In one aspect, the array can be used in high-throughput screening assays.For example, PNA substrates can be tested by subjecting the array to DNA or RNA molecules and identifying the presence or absence of complementary DNA, RNA or PNA molecules, for example, by detecting at least one change in the features of the array.PNA-DNA chimeric substrates can be tested by subjecting the array to complementary DNA molecules and performing single-base extension reaction to confirm whether the substrate is biologically active and by identifying SNPs in samples.

[0226] In some embodiments, the array can be used to detect sequence variants, such as single nucleotide polymorphisms (SNPs), in a sample. The sequence variants can be detected by observing sequence-specific hybridization of labeled molecules with probes on the array. The sequence variants can also be detected by binding a sequence suspected of having a sequence variant to a probe on the array, followed by a polymerase extension reaction using labeled nucleotides. In a preferred embodiment, a PNA-DNA chimeric oligonucleotide probe is bound to the array and hybridizes to a nucleotide sequence from a sample suspected of containing a sequence variant. The PNA-DNA chimeric oligonucleotide is enzymatically active, i.e., it can serve as a substrate for incorporation of a complementary nucleotide into a growing chain using a polymerase under conditions favorable for polymerization. Figure 7 shows an exemplary scheme for detecting the identity of a sample oligonucleotide hybridized to a PNA-DNA chimeric oligonucleotide covalently attached to an array using a polymerase-based single base extension reaction and labeled nucleotides. An example of a PNA-DNA chimeric oligonucleotide-based SNP detection method is given in US Pat. No. 6,316,230, which is incorporated by reference herein in its entirety.

[0227] Arrays can also be used in screening assays for ligand binding to ascertain substrate specificity, or to identify complementary DNA, RNA, PNA molecules expressed in certain cells in vivo or in vitro. Labeling methods, protease assays, and binding assays useful for carrying out these methods are generally well known to those of skill in the art.

[0228] In some embodiments, array can be used to present a given PNA strand as a sequence of overlapping PNA sequences.For example, the PNA sequence of a known gene is divided into overlapping sequence segments of any length and any suitable overlapping frame, and the PNA strand corresponding to each sequence segment is synthesized in situ as disclosed herein.The individual PNA segments thus synthesized can be arranged starting from the amino terminus of a given PNA strand.

[0229] In some embodiments, sample is applied to the array with multiple random PNA strands.Can screen and BLAST search random PNA strands to confirm the homologous domain with predetermined nucleotide sequence, for example, 90% or more identity.Then, in some aspects, can synthesize and use the whole PNA sequence to identify the potential marker and / or cause of the disease of interest.

[0230] In some embodiments, the array is used for high-throughput screening of one or more genetic factors. The expression of DNA or RNA associated with genes can be investigated upon hybridization of PNA and can be used to evaluate the relationship between genes and diseases.

[0231] In another example, the array can be used to identify one or more biomarkers. Biomarkers can be used in the diagnosis, prognosis, treatment, and management of disease. Biomarkers can be expressed, absent, or at different levels in an individual depending on the disease state, stage of the disease, and response to disease treatment. Biomarkers can be, for example, DNA, RNA, PNA, proteins (e.g., enzymes such as kinases), sugars, salts, fats, lipids, or ions.

[0232] The array can also be used for therapeutic purposes, for example, to identify one or more bioactive agents. The method for identifying a bioactive agent may include applying a plurality of test compounds to the array and identifying at least one test compound as a bioactive agent. The test compound may be a small molecule, an aptamer, an oligonucleotide, a chemical, a natural extract, a peptide, a protein, an antibody fragment, an antibody-like molecule, or an antibody. The bioactive agent may be a therapeutic agent or a modulator of a therapeutic target. In some embodiments, the test compound is a hybridized DNA, RNA, or PNA sequence. The therapeutic target may include a phosphatase, a protease, a ligase, a signaling molecule, a transcription factor, a protein transporter, a protein sorter, a cell surface receptor, a secreted factor, and a cytoskeletal protein.

[0233] In one aspect, the array is also provided for use in medical diagnosis.The array can be used to confirm the response to the administration of a drug or a vaccine.For example, the response of an individual to a vaccine can be confirmed by detecting the gene expression level of the individual using an array with PNA strands or PNA-DNA chimeric oligonucleotide strands that present specific genes related to the induced immune response.Another diagnostic application is to test an individual for the presence of a biomarker.Here, a sample is taken from a subject and tested for the presence of one or more biomarkers.

[0234] Arrays can also be used to stratify patient populations based on the presence or absence of biomarkers that indicate the likelihood that a subject will respond to a therapeutic treatment. Arrays can be used to identify known biomarkers to ascertain appropriate treatment groups. For example, a sample from a subject with a condition can be applied to the array. Binding to the array may indicate the presence of a biomarker for the condition. Previous studies may show that a biomarker is associated with a positive outcome after treatment, whereas the absence of a biomarker is associated with a negative or neutral outcome after treatment. Because a patient has a biomarker, the patient may be stratified by a medical professional into groups that will receive treatment.

[0235] In some embodiments, the method of detecting the presence or absence of expressed gene of interest in a sample may include obtaining the array disclosed in the present invention in contact with a sample suspected of containing the DNA or RNA sequence of the gene of interest; and determining whether the gene of interest is expressed in the sample by detecting the presence or absence of binding with one or more features of the array.In some embodiments, the DNA or RNA sequence of the gene of interest may be obtained from body fluids, such as amniotic fluid, aqueous humor, vitreous humor, bile, serum, breast milk, cerebrospinal fluid, earwax, chyle, endolymph, perilymph, feces, female vaginal fluid, gastric acid, gastric juice, lymph, mucus, ascites, pleural fluid, pus, saliva, sebum, semen, sweat, synovial fluid, tears, vaginal secretions, vomit, or urine.

[0236] In some embodiments, a method for identifying a vaccine candidate may include obtaining an array as disclosed herein in contact with a sample obtained from a subject to whom the vaccine candidate has been previously administered, the sample comprising a plurality of DNA or RNA sequences; and determining the binding specificity of the plurality of DNA or RNA sequences to one or more features of the array. In some embodiments, the features comprise a plurality of distinct nested overlapping PNA strands comprising subsequences derived from a known nucleotide sequence. EXAMPLES

[0237] The following are examples of specific embodiments for carrying out the present invention. The examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any way. Efforts have been made to ensure accuracy with respect to the numerical values ​​used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for.

[0238] The practice of the present invention will employ, unless otherwise specified, conventional methods of protein chemistry, biochemistry, recombinant DNA technology, and pharmacology within the skill of the art. Such techniques are fully explained in the literature. See, for example, TE Creighton, Proteins: Structures and Molecular Properties (WH Freeman and Company, 1993); AL Lehninger, Biochemistry (Worth Publishers, Inc., current addition); Sambrook, et al, Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pennsylvania: Mack Publishing Company, 1990); Carey and Sundberg Advanced Organic Chemistry 3rd Edition (East, Pennsylvania: Mack Publishing Company, 1992); and, 1997, 1999, 10th Edition (East, Pennsylvania: Mack Publishing Company, 1992). rd Ed. (Plenum Press) Vols A and B (1992).

[0239] Example 1: Synthesis of PNA sequences Site-specific PNA sequence synthesis is performed on the array as follows: As described above, the wafer is spin-coated with a photoresist composition containing a photobase generator at 2000-4000 rpm (preferably 2500-3000 rpm) for 10-180 seconds (preferably 60-120 seconds). The wafer and photoresist are exposed to 248 nm UV light in a deep UV scanner tool according to a pattern defined by a photomask, and since the photobase generator is present in the photoactive coupling solution in the photoresist, bases are generated at the locations exposed to the UV light. The exposure energy is 1 mJ / cm. 2 ~100mJ / cm 2 (Preferably 30 to 60 mJ / cm 2 ) is also acceptable.

[0240] After UV exposure, the surface of the wafer is post-baked in a bake module. The post-baking temperature can be 75°C to 115°C for at least 60 seconds (rarely more than 180 seconds). The base generated in the UV-exposed areas removes the protecting groups on the amino groups. The photoresist is then stripped away.

[0241] The free amino groups are then coupled to the activated R2-acetic acid by spin-coating the R2-acetic acid with an activator and reacting in a bake module at a temperature of 55° C. to 115° C. for 60 to 240 seconds. The excess R2-acetic acid is removed.

[0242] After the activated R2-acetic acid is added, a substitution reaction with monoamino-protected ethylenediamine is carried out by adding a substitution mixture to the surface of the wafer. The substitution mixture is spin-coated on the wafer and reacted in a bake module at a temperature of 55° C. to 115° C. for 30 seconds to 300 seconds (preferably 120 seconds). In some embodiments, the monoamino-protected group can be any of the amino-protected groups described above.

[0243] Next, a coupling reaction with peptide nucleic acid monomer acetate is performed. The substituted amine is coupled to the activated PNA monomer acetate by spin-coating the activated PNA monomer acetate with an activator and reacting for 90-300 seconds.

[0244] Optionally, a capping solution coat is applied to the surface to prevent unreacted amino groups on the support from reacting with the next coupling molecule. The capping solution includes a solvent, a polymer, and a coupling molecule. The capping solution is spin-coated on the wafer at 1500-3500 rpm for at least 30 seconds, and reacted in a bake module at a temperature of 55°C-95°C for 30 seconds-90 seconds (preferably 60 seconds) to complete one cycle.

[0245] This entire cycle can be repeated as desired, each time with a different nucleic acid monomer, to obtain a desired PNA sequence at a specific site on the array.

[0246] Example 2: Yield of long PNA sequences As described in Example 1, a 64-mer PNA sequence TIFF2025013596000007.tif12158 was grown on the wafer and the yield of the sequence polymerization was calculated using the fluorescein process as described below. To determine the sequence yield, 2 wt% fluorescein carboxylic acid 5(6)-FAM (Anaspec) activated with 2 equivalents of diisopropylcarbodiimide (VWR) and 2 equivalents of hydroxybenzotriazole (Hobt) (Anapsec) was spin-coated on the wafer at 3000 rpm for 60 seconds and baked at 65°C for 5 minutes in a bake module. The wafer was then washed with ethylenediamine (VWR) for 10 minutes, then washed with N-methyl-pyrrolidone (NMP) for 2 minutes, then washed with IPA for 40 seconds, and spun down by centrifugation. Fluorescence was then measured with a confocal microscope after each addition to determine the sequence yield.

[0247] This example shows step yield data for the 64mer PNA sequence described above. To measure step yield by fluorescence, a fluorescent dye molecule was coupled to the PNA sequence to determine coupling efficiency. The amount of coupled fluorescein dye is a direct measure of the amount of grown sequence.

[0248] The formula used to calculate the step yield is: Step yield = (F n / F 1 ) 1 / n-1 In the formula, F 1 and F n indicates the fluorescein coupling intensity read from a fluorescence scanner device at the 1st step and the nth step. The coupling yield is calculated by the formula: E=10 1 / C·log(F) where F is equal to the full length ratio and C=number of couplings=length-1. Then, the full length ratio F is: F=10 (N+1)·log(E / 100%) As shown by:

[0249] Table 1. Step yields of PNA sequences TIFF2025013596000008.tif77128TIFF2025013596000009.tif184128

[0250] Table 1 shows the fluorescence signal intensity at each monomer layer. Table 2 shows the step yield efficiency of the coupling step, and the overall yield of the 64mer, 75mer, and 100mer. The overall yields of the 75mer and 100mer were calculated based on the individual step yields. The coupling efficiency of each PNA monomer was calculated to be greater than 99.4% in each case over the entire length of each 64mer PNA strand.

[0251] Table 2. Results of PNA sequencing on arrays TIFF2025013596000010.tif39128

[0252] Next, we determined the distribution of the lengths of the sequences on the array based on the synthesis of PNA sequences of a defined length (e.g., 64mer). In each coupling step, the length of the sequence where the desired coupling does not occur is defined as the length when the capping solution is used. The length distribution according to the step yield of each sequence length shorter than the complete sequence is given by the following formula: F(N)=10 (N+1)·log(E / 100%) -10 (N)·log(E / 100%) where F(N) is the proportion of sequences on the array at length N less than the full length sequence, and E is the average coupling efficiency percent. The exact value of E at each length N can also be used to obtain the exact number of oligomers at each length.

[0253] The ratio of full-length sequences is calculated according to the following formula: F(N)=10 (N)·log(E / 100%) where F(N) is the proportion of sequences on the array that are full-length sequences (no further coupling steps) and E is the average coupling efficiency.

[0254] Example 3: Hybridization of PNA sequences with oligonucleotide DNA sequences To confirm the biological activity of the PNA sequence, a 64-mer PNA sequence was TIFF2025013596000011.tif41157 was synthesized on the array according to the method described above.

[0255] Oligonucleotide DNA sequence complementary to the match sequence TIFF2025013596000012.tif12158 (complementary pair sequence) was synthesized (IDT) and labeled with Atto-633 dye (Atto-tec) by techniques well known to those skilled in the art.

[0256] Hybridization of the chips containing matched sequences, single-base mismatched sequences, and mismatched sequences was performed by labeling the complementary pair sequences. The complementary pair sequences were diluted 1:1000 in 0.1X Ssarc buffer (60 mM sodium chloride (Sigma), 6 mM sodium citrate (Sigma), 0.72 wt% N-lauroyl sarcosine sodium salt solution (Sigma)). Hybridization was performed in a hybridization chamber at 45°C for 2 hours. The chips were then washed twice with 0.1X Ssarc buffer at 40°C for 5 minutes, followed by rinsing with DI water. The chips were then examined under a confocal microscope. Results showing that single nucleobase mutations were identified using the above method are shown in Figure 6. The results demonstrated the sensitivity and specificity of identifying specific DNA sequences (including the identification of single-base sequence variants) using hybridization with microarrays containing specific complementary PNA sequences.

[0257] Example 4: Synthesis of PNA-DNA chimeras Part 1 - Synthesis of PNA sequences To generate PNA-DNA chimeras on the array surface, PNA oligos of desired sequences at specific sites were first synthesized on the chip according to the protocol given in Example 1.

[0258] Part 2 - PNA-DNA chimera synthesis process Sequence-specific DNA nucleotides are added to the ends of PNA sequences at specific locations on the array as follows: A wafer containing PNA sequences is spin-coated with photoresist at 2000-4000 rpm (preferably 2500-3000 rpm) for 10-180 seconds, preferably 60-120 seconds, and exposed to 248 nm UV light in a deep UV scanner tool according to a pattern defined by a photomask; a photoactive coupling solution in the photoresist contains a photobase generator, which generates bases at the locations exposed to the UV light. The exposure energy is 1 mJ / cm. 2 ~100mJ / cm 2 (Preferably 30 to 60 mJ / cm 2 ) is also acceptable.

[0259] After UV exposure, the surface of the wafer is post-baked in a bake module. The post-baking temperature can be 75°C to 115°C for at least 60 seconds (rarely more than 180 seconds). The resulting base deprotects the amino-protected PNA sequences in the exposed areas. The photoresist is then stripped away.

[0260] The reverse DNA amidite is activated with a phosphoramidite activating solution (eg, containing a tetrazole catalyst) and then coupled to a free amine.

[0261] Optionally, a capping solution coat is applied to the surface to prevent unreacted amino groups on the support from reacting with the next coupling molecule. The capping solution includes a solvent, a polymer, and a coupling molecule. The capping solution is spin-coated on the wafer at 1500-3500 rpm for at least 30 seconds and reacted in a bake module at a temperature of 55°C-95°C for 30-90 seconds (preferably 60 seconds) to complete one cycle. The phosphite triester formed in the coupling step is then converted to a stable form, which is accomplished by iodine oxidation in the presence of water and pyridine.

[0262] Part 3 - Reverse DNA oligonucleotide synthesis Site-specific DNA sequence reverse synthesis (5'→3') is performed at each PNA-DNA chimera end on the array as follows: As described above, the wafer is spin-coated with a photoresist composition containing a photobase generator at 2000-4000 rpm (preferably 2500-3000 rpm) for 10-180 seconds (preferably 60-120 seconds). The wafer and photoresist are exposed to 248 nm UV light in a deep UV scanner tool according to a pattern defined by a photomask, and since a photoacid generator is present in the photoactive coupling solution, acid is generated at the locations exposed to the UV light. The exposure energy is 1 mJ / cm. 2~100mJ / cm 2 (Preferably 30 to 60 mJ / cm 2 ) is also acceptable.

[0263] After UV exposure, the surface of the wafer is post-baked in a bake module. The post-baking temperature can be 75°C to 115°C for at least 60 seconds (rarely more than 180 seconds). The resulting acid deprotects the hydroxyl group protection at the 3' end of the DNA sequence in the exposed areas. The photoresist is then stripped away.

[0264] The reverse DNA amidite is activated with a phosphoramidite activating solution (eg, containing a tetrazole catalyst) and then coupled to the free hydroxyl group.

[0265] Optionally, a capping solution coat is applied to the surface to prevent unreacted amino groups on the support from reacting with the next coupling molecule. The capping solution includes a solvent, a polymer, and a coupling molecule. The capping solution is spin-coated on the wafer at 1500-3500 rpm for at least 30 seconds and reacted in a bake module at a temperature of 55°C-95°C for 30-90 seconds (preferably 60 seconds) to complete one cycle. The phosphite triester formed in the coupling step is then converted to a stable form, which is accomplished by iodine oxidation in the presence of water and pyridine.

[0266] Example 5: PNA sequences and yields of PNA-DNA chimeras As described in Example 3, a 31-mer PNA sequence and three DNA oligomers were TIFF2025013596000013.tif4130 was grown on a wafer and the step yield of the sequence was determined after each addition using a fluorescein-based step yield detection process as described below. (CGC) is the DNA oligomer portion of the sequence. 2 wt% fluorescein carboxylic acid 5(6)-FAM (Anaspec) activated with 2 equivalents of diisopropylcarbodiimide (VWR) and 2 equivalents of hydroxybenzotriazole (Hobt) (Anapsec) was spin-coated on the wafer at 3000 rpm for 60 seconds and baked at 65°C for 5 minutes in a bake module. To calculate the yield of DNA oligomers, 6-FAM phosphoramidite (Jena Biosciences) was activated with 2 equivalents of benzylthio-tetrazole (TCI) dissolved in acetonitrile, spin-coated on the wafer at 3000 rpm for 60 seconds and allowed to react at room temperature for 5 minutes. The wafers were then washed with ethylenediamine (VWR) for 10 minutes, then washed with N-methyl-pyrrolidone (NMP) for 2 minutes, then washed with IPA for 40 seconds, and spun down. Fluorescence was then measured with a Nikon A1R confocal microscope to determine the sequence yield. The results are shown in Table 3. The results in Table 3 show the following average step yields for each type of addition: - Step yield of each layer of PNA array = 99.4% - Process yield of PNA-DNA chimera step = 97.4% - DNA oligomer step yield = 98.96% was obtained.

[0267] Table 3. Step yields of PNA-DNA chimeric sequences TIFF2025013596000014.tif49128TIFF2025013596000015.tif24455

[0268] Example 6: Hybridization of PNA-DNA chimeras with oligonucleotide DNA sequences and extension with polymerase In order to confirm the biological activity of the PNA-DNA chimeric sequence synthesized according to the above protocol, a 34-mer PNA-DNA chimeric sequence was prepared. TIFF2025013596000016.tif4138 was synthesized according to Example 3. (TAT) is the DNA oligomer portion of the PNA-DNA chimera. Four oligonucleotide DNA sequences complementary to the sequence of the PNA-DNA chimera containing one additional nucleotide Composite image: TIFF2025013596000017.tif26137 (IDT).

[0269] Primer extension reactions were performed to detect the incorporation of the correct complementary nucleotide in the primer extension reaction. Alexa 405 ddATP, Alexa 488 ddCTP, Alexa 555 ddGTP, and Alexa 647 ddTTP were synthesized by techniques well known to those skilled in the art. Hybridization and polymerase extension on each of the four chips was performed as follows: Oligomers S1, S2, S3, and S4 were diluted 1:1000 (100 nM) in 1X DNA Polymerase Buffer (Clontech), 20 nmol MgCl2, 1 unit Titanium Taq DNA Polymerase (Clontech), and monomers (25 pmole each) labeled with all four labeled ddNTPs. Hybridization was carried out in a hybridization chamber at 55° C. for 30 min, after which the chip was washed twice with 0.1× Ssarc buffer at 40° C. for 5 min, followed by rinsing with DI water. The chip was then examined with a Nikon A1R confocal microscope equipped with four wavelengths of the dye used for ddNTPs. The results are shown in Table 4.

[0270] Table 4. Hybridization and extension of PNA-DNA chimeric sequences TIFF2025013596000018.tif42128

[0271] Example 7: Genotyping using PNA sequence hybridization PNA synthesis was tested for genotyping SNP-based applications. Genotyping of the MTHFR region with the known mutations C677T and A1298C was tested using 20 DNA samples that had known genotyping results that were confirmed using real-time PCR.

[0272] The PNA sequence was as follows: TIFF2025013596000019.tif48154

[0273] PNA sequences were synthesized on the chip using the methods outlined above. The locations of the SNPs are indicated in the () areas (enclosed in brackets) and were synthesized in the middle of the sequence.

[0274] DNA was extracted from the samples (buccal swabs) using methods known to those skilled in the art. A standard PCR reaction was performed on the extracted DNA samples using a forward primer and a biotin-labeled reverse primer. Hybridization of the PCR products on the chip was performed using PCR products (20 ul) diluted in hybridization buffer 0.1X Ssarc buffer, 60 mM sodium chloride (Sigma) (80 ul), 6 mM sodium citrate (Sigma), 0.72 wt% N-lauroyl sarcosine sodium salt solution (Sigma). Hybridization was performed in a hybridization chamber at 55°C for 2 hours, after which the chip was washed twice with 0.1X Ssarc buffer at 40°C for 5 minutes. This was followed by incubation with 1 ng / ml Atto 488 streptavidin (Rockland) diluted in PBS buffer, and the chip was washed twice with PBS buffer and rinsed with DI water. The chips were then examined under a Nikon A1R confocal microscope and the results are illustrated in Tables 5 and 6.

[0275] Table 5: Results of 677C>T mutation (PNA) TIFF2025013596000020.tif37153TIFF2025013596000021.tif238153TIFF2025013596000022.tif80153

[0276] Table 6: Results of 1298A>C mutation (PNA) TIFF2025013596000023.tif146157TIFF2025013596000024.tif207157

[0277] In this method, the location of the SNP is ideally near the center of the synthesized sequence.

[0278] Example 8: Genotyping using PNA-DNA chimeras and primer extension The PNA-DNA chimeras were tested for genotyping SNP-based applications. Twenty different DNA samples were tested for genotyping the MTHFR region with the known mutations C677T and A1298C. These DNA samples had known genotyping results that were confirmed using real-time PCR. Following the methodology presented above, the 34-mer PNA-DNA chimera primer sequence for the 677C>T mutation was For TIFF2025013596000025.tif4145 and the 1298A>C mutation, TIFF2025013596000026.tif4139 was synthesized (the DNA nucleotide portions of the PNA-DNA chimera are enclosed in brackets).

[0279] DNA was extracted from the samples (oral swabs) using methods known to those skilled in the art. A standard PCR reaction using forward and reverse primers was performed on the extracted DNA samples. Hybridization and polymerase extension on each chip were performed as follows: PCR products were diluted with 1X DNA Polymerase Buffer (Clontech), 20 nmol MgCl 2The chips were dissolved and mixed with 1 unit of Titanium Taq DNA Polymerase (Clontech) and monomers labeled with all four labeled ddNTPs (25 pmol each). Hybridization was carried out in a hybridization chamber at 55° C. for 30 min, after which the chips were washed twice with 0.1× Ssarc buffer at 40° C. for 5 min, followed by DI water. The chips were then examined with a Nikon A1R confocal microscope equipped with four wavelengths of the dyes used for the ddNTPs, and the results are shown graphically in Tables 7 and 8.

[0280] Table 7: Results of the 677C>T mutation (PNA-DNA chimera) TIFF2025013596000027.tif62170TIFF2025013596000028.tif229170TIFF2025013596000029.tif46170

[0281] Table 8: Results of the 1298A>C mutation (PNA-DNA chimera) TIFF2025013596000030.tif182170TIFF2025013596000031.tif152170

[0282] In this method, the sequences synthesized on the chip contain a region immediately preceding the SNP location, allowing the polymerase to selectively add a matching oligomer that corresponds to the identity of the SNP.

[0283] PNA-DNA chimeras could hybridize to DNA sequences and extend accurately according to the corresponding match DNA monomers. PNA-DNA chimeras can obtain high match / mismatch ratios that accurately pinpoint SNP-based genotyping results. The ratio of match / mismatch sequences is 3.5-4 for PNA sequences, whereas it is 65-70 for PNA-DNA chimera sequences. Thus, with high sequence yields and the ability to perform the polymerase extension step on-chip due to the presence of DNA oligomers, PNA-DNA chimeras provide a highly accurate, high-throughput system for various genomics applications, including but not limited to SNP-based genotyping and DNA sequencing.

[0284] Sequence information SEQUENCE LISTING <110> VIBRANT HOLDINGS, LLC <120> SUBSTRATES, SYSTEMS, AND METHODS FOR NUCLEIC ACID ARRAY SYNTHESIS <150> US 62 / 272,057 <151> 2015-12-28 <160> 19 <170> PatentIn version 3.5 <210> 1 <211> 64 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic PNA oligonucleotide <400> 1 agcaattttg catcttttaa aaggggtttt ccccaatatt atggttcgtc cccctttaaa 60 ggat64 <210> 2 <211> 64 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic PNA oligonucleotide <400> 2 gtggaaattt gacatagtct cagatgccta ttatattacc gagagagtag tctgaaatgc 60 ctta 64 <210> 3 <211> 64 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic PNA oligonucleotide <400> 3 gtggaaattt gacatagtct cagatgccta ttctattacc gagagagtag tctgaaatgc 60 ctta 64 <210> 4 <211> 64 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic PNA oligonucleotide <400> 4 gtggaaattt tgttttgaag gactgtatat aattgtgtca tagtttgtta cctaaaatgc 60 ctta 64 <210> 5 <211> 64 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 5 taaggcattt cagactactc tctcggtaat ataataggca tctgagacta tgtcaaattt 60 ccac 64 <210> 6 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic PNA-DNA chimera oligonucleotide <400> 6 agcaattttg catcttttaa aaggggtttt ccgc 34 <210> 7 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic PNA-DNA chimera oligonucleotide <400> 7 gtggaaattt gacatagtct cagatgccta ttat 34 <210> 8 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 8 cacctttaaa ctgtatcaga gtctacggat aataa 35 <210> 9 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 9 cacctttaaa ctgtatcaga gtctacggat aatac 35 <210> 10 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 10 cacctttaaa ctgtatcaga gtctacggat aatag 35 <210> 11 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 11 cacctttaaa ctgtatcaga gtctacggat aatat 35 <210> 12 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic PNA oligonucleotide <400> 12 ggagaaggtg tctgcgggag ccgatttcat catcacgcag c 41 <210> 13 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic PNA oligonucleotide <400> 13 ggagaaggtg tctgcgggag tcgatttcat catcacgcag c 41 <210> 14 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic PNA oligonucleotide <400> 14 ggaggagctg accagtgaag aaagtgtctt tgaagtcttc g 41 <210> 15 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic PNA oligonucleotide <400> 15 ggaggagctg accagtgaag caagtgtctt tgaagtcttc g 41 <210> 16 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic PNA-DNA chimera primer <400> 16 ctgaagcact tgaaggagaa ggtgtctgcg ggag 34 <210> 17 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic PNA-DNA chimera primer <400> 17 ctgaagatgt ggggggagga gctgaccagt gaag 34 <210> 18 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 18 atacgattac 10 <210> 19 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 19 taggtaatcg tat 13

Claims

1. An array of features attached to a surface at defined locations, each of the features comprising a plurality of PNA polymers of determinable sequence and intended length, the plurality of PNA polymers comprising a distribution of lengths characterized by a coupling efficiency of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, or 99.5%.

2. The distribution of lengths of a plurality of PNA polymers shorter than the intended length is determined according to the formula: F(N)=10 (N+1)・log(E / 100％) -10 (N)・log(E / 100％) 2. The array of claim 1, characterized by: where N=actual length of the PNA polymer, E=coupling efficiency, and E is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, or 99.5%.

3. The ratio of PNA polymers of intended length is determined according to the formula: F(N)=10 (N)・log(E / 100％) 2. The array of claim 1, characterized by: where N=the intended length of the PNA polymer, E=average coupling efficiency, and E is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, or 99.5%.

4. 10. The array of any of the preceding claims, comprising at least 10,000 features.

5. 5. The array of claim 4, comprising at least 100,000 features.

6. 3. The array of claim 2, wherein the intended length of the PNA polymer is at least 30.

7. 3. The array of claim 1, wherein the intended length of the PNA polymer is at least 40.

8. 3. The array of claim 1, wherein the intended length of the PNA polymer is at least 50.

9. 3. The array of claim 2, wherein the intended length of the PNA polymer is at least 75.

10. 3. The array of claim 1, wherein the PNA polymer is a PNA / nucleic acid chimera, the PNA polymer further comprising one or more nucleic acid residues.

11. 11. The array of claim 10, wherein the nucleic acid is a deoxyribonucleic acid.

12. 13. The array of any of the preceding claims, comprising at least 10,000 features per square centimeter.

13. 13. The array of claim 12, comprising at least 20,000, at least 40,000, at least 100,000, at least 200,000, at least 500,000, at least 1 million, at least 2 million, at least 5 million, at least 10 million, at least 20 million, or at least 50 million features per square centimeter.

14. 10. The array of claim 1, wherein the array comprises pillars and the surface is a top surface of the pillars.

15. The top surface of the pillar is at least 1 μm 2 15. The array of claim 14, having an area of

16. Creating a pattern using a photomask; exposing the photoresist to ultraviolet light through the photomask; and generating an acid or base in said pattern on said array as a result of said exposure to said ultraviolet light. A method for making the array of any one of claims 1 to 15, comprising:

17. 17. The method of claim 16, wherein a base is generated from a photobase generator upon exposure to light.

18. The photobase generator is 1,3-Bis[(2-nitrobenzyl)oxycarbonyl-4-piperidyl]propane, 1,3-Bis[1-(9-fluorenylmethoxycarbonyl)-4-piperidyl]propane, 1,5,7-triazabicyclo[4.4.0]dec-5-enyl-phenyl glyoxylate, 1,5,7-triazabicyclo[4.4.0]dec-5-enyl-4-nitrophenyl glyoxylate, 1,5,7-triazabicyclo[4 18. The method of claim 17, wherein the aryl group is selected from the group consisting of 1,8-diazabicyclo[5.4.0]dec-5-enyl-tetraphenylborate, 1,8-diazabicyclo[5.4.0]undec-7-enyl-tetraphenylborate, 1-phenacyl-(1-azonia-4-azabicyclo[2,2,2]octane)-tetraphenylborate, and 1-naphthoylmethyl-(1-azonia-4-azabicyclo[2,2,2]octane)-tetraphenylborate or the like.

19. 20. The method of claim 18, wherein the photobase generator is 1,3-Bis[(2-nitrobenzyl)oxycarbonyl-4-piperidyl]propane.

20. The method of any one of claims 17 to 19, wherein the protecting group is cleaved from the amine group by a base.

21. 21. The method of claim 20, further comprising coupling a PNA monomer to the deprotected amine group.

22. 17. The method of claim 16, wherein an acid is generated from a photoacid generator upon exposure to light.

23. 23. The method of claim 22, wherein the photoacid generator is selected from the group consisting of iodonium salts, polonium salts, and sulfonium salts.

24. The photoacid generator is Bis(4-tert-butylphenyl)iodonium perfluoro-1-butanesulfonate, Bis(4-tert-butylphenyl)iodonium p-toluenesulfonate, Bis(4-tert-butylphenyl)iodonium triflate, Boc-methoxyphenyldiphenylsulfonium triflate, (tert-butoxycarbonylmethoxynaphthyl)-diphenylsulfonium triflate, (4-tert-butylphenyl)diphenylsulfonium triflate, diphenyliodonium hexafluorophosphate, diphenyliodonium perfluoro-1-butanesulfonate, diphenyliodonium triflate, (4-iodophenyl)diphenylsulfonium triflate, (4-methoxyphenyl)diphenylsulfonium triflate, (4-methylphenyl)diphenylsulfonium triflate, 23. The method of claim 22, wherein the arylphenyl sulfonium triflate is selected from the group consisting of (4-methylthiophenyl)diphenylsulfonium triflate, (4-methylthiophenyl)methylphenylsulfonium triflate, Tris(4-tert-butylphenyl)sulfonium triflate, (4-methoxyphenyl)phenylsulfonium triflate, (4-methoxyphenyl)phenyliodonium triflate, 4-methoxyphenyl)phenyliodonium trifluoromethanesulfonate, (4-methoxyphenyl)dimethylsulfonium triflate, and (2,4-dihydroxyphenyl)dimethylsulfonium triflate or the like.

25. 23. The method of claim 22, wherein the photoacid generator is selected from the group consisting of iodonium and sulfonium salts of triflates, phosphates, and antimonates.

26. 23. The method of claim 22, wherein the photoacid generator is (4-iodophenyl)diphenylsulfonium triflate.

27. The method of any one of claims 22 to 26, wherein the protecting group is cleaved from the carboxylic acid group by acid.

28. 28. The method of claim 27, further comprising coupling a DNA monomer to the deprotected carboxylic acid group.

29. The step of coupling the PNA monomers comprises: activating the substituted acetic acid with an activating agent; and Coupling activated acetic acid to unprotected amine groups in selectively exposed areas. and wherein the substitution of the acetic acid comprises a leaving group.

30. 30. The method of claim 29, wherein the step of coupling the PNA monomers is performed simultaneously at multiple sites on an array.

31. 30. The method of claim 29, wherein the leaving group is halo.

32. The coupling step comprises: Replacing the acetic acid leaving group with a diamino-alkane 30. The method of claim 29, further comprising: wherein one amine of the diamino-alkane is protected.

33. 33. The method of claim 32, wherein the diamino-alkane is ethylenediamine.

34. The coupling step comprises: activating the PNA monomer acetate with an activating agent; and Coupling the activated PNA monomer acetate to the unprotected amine of a diamino-alkane 31. The method of claim 29 or 30, further comprising:

35. 35. The method of claim 34, wherein the PNA monomer acetate is R-thymine-1-acetic acid, R-(cytosin-1-yl)-acetic acid, R-adenine-9-yl-acetic acid, R-guanine-9-acetic acid, or R-uracil-1-acetic acid, where R is H or a protecting group.

36. 1. A method for analyzing a sample containing nucleic acid obtained from a subject, the method comprising the steps of: contacting said sample with the array of any one of claims 1 to 15 under conditions that promote hybridization between said sample and said array; detecting a signal from the array, the signal being indicative of the presence, absence, or amount of a sample hybridized to the array at one or more of the characteristic locations; and Analysing the signal, thereby analyzing the sample.

37. 37. The method of claim 36, wherein the analyzing step comprises determining a nucleic acid sequence present in the sample based on the signal.

38. 37. The method of claim 36, wherein the analyzing step comprises determining the presence or absence of SNPs present in the sample based on the signal.

39. 40. The method of claim 38, wherein the array comprises PNA / DNA chimeras, and further comprising the step of performing a primer extension reaction after hybridization between the sample and the array.

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