US-12624392-B2 - Molecular array generation using photoresist

Abstract

Provided in some aspects are methods for light-controlled in situ surface patterning of a substrate. Compositions such as nucleic acid arrays produced by the methods are also disclosed. In some embodiments, a method disclosed herein comprises using photoresist for photocontrollable hybridization and/or ligation of nucleic acid molecules, wherein photoresist removal allows hybridization and/or ligation of nucleic acid molecules at the exposed area. A large diversity of barcodes can be created in molecules on the substrate via sequential rounds of light exposure, hybridization, and ligation.

Inventors

• David Michael Patterson
• Denis Pristinski
• Preyas Shah
• Steven William Short
• Dieter Wilk
• Siyuan Xing

Assignees

• 10X GENOMICS, INC.

Dates

Publication Date

20260512

Application Date

20211229

Claims (20)

1. 1 . A method for providing an array, comprising: (a) irradiating a substrate comprising a layer of positive photoresist to expose a first region and not expose a second region, wherein oligonucleotide molecules attached to the substrate at their 5′ ends in the first region and in the second region are embedded in the layer of positive photoresist and comprise 3′ hydroxyl functional groups that are not protected by a protecting group; (b) treating the layer of positive photoresist with a developer solution to dissolve and remove the layer of positive photoresist in the first region, thereby rendering 3′ hydroxyl functionalized ends of the oligonucleotide molecules attached to the substrate in the first region available for hybridization and/or ligation, whereas 3′ hydroxyl functionalized ends of the oligonucleotide molecules attached to the substrate in the second region are protected by the layer of positive photoresist in the second region from hybridization and/or ligation; and (c) attaching an oligonucleotide comprising a barcode sequence to a 3′ hydroxyl functionalized end of an oligonucleotide molecule of the oligonucleotide molecules attached to the substrate in the first region via hybridization and/or ligation, wherein the 3′ hydroxyl functionalized ends of the oligonucleotide molecules attached to the substrate in the second region do not receive the barcode sequence, thereby providing, on the substrate, an array comprising different oligonucleotide molecules in the first region and the second region.
2. 2 . The method of claim 1 , wherein the oligonucleotide molecules attached to the substrate in the first region and the oligonucleotide molecules attached to the substrate in the second region comprise one or more common sequences.
3. 3 . The method of claim 1 , further comprising, prior to (a), forming a pattern of oligonucleotide molecules on an initial substrate to generate the substrate.
4. 4 . The method of claim 3 , wherein forming the pattern of oligonucleotide molecules comprises: irradiating the initial substrate, wherein the initial substrate comprises a plurality of functional groups and an initial layer of positive photoresist, through a patterned mask and treating the initial layer of positive photoresist with a developer solution, thereby producing a treated substrate, whereby the initial layer of positive photoresist in a first region of the initial substrate is dissolved and removed, thereby rendering functional groups in the first region of the initial substrate available for reacting with 5′ functional groups in functionalized oligonucleotide molecules, whereas functional groups in a second region of the initial substrate are protected by the initial layer of positive photoresist in the second region from reacting with the 5′ functional groups in the functionalized oligonucleotide molecules; and contacting the treated substrate with the functionalized oligonucleotide molecules, thereby coupling the 5′ functional groups in the functionalized oligonucleotide molecules to the functional groups in the first region but not to the functional groups in the second region, thereby forming the pattern of oligonucleotide molecules on the treated substrate and generating the substrate.
5. 5 . The method of claim 4 , wherein the 5′ functional groups in the functionalized oligonucleotide molecules are amino groups.
6. 6 . The method of claim 4 , further comprising blocking unreacted functional groups of the treated substrate that have not been coupled to the functionalized oligonucleotide molecules.
7. 7 . The method of claim 4 , wherein the irradiating and contacting are repeated in one or more cycles.
8. 8 . The method of claim 7 , wherein the initial layer of positive photoresist is not removed prior to the one or more cycles.
9. 9 . The method of claim 1 , wherein (a) comprises irradiating the substrate through a patterned mask.
10. 10 . The method of claim 9 , further comprising, after the irradiating, removing the patterned mask from the substrate, and re-using the patterned mask in a subsequent cycle of the irradiating and the attaching.
11. 11 . The method of claim 1 , wherein the barcode sequence is between about 4 and about 25 nucleotides in length.
12. 12 . The method of claim 1 , wherein the oligonucleotide comprising the barcode sequence is between about 10 and about 50 nucleotides in length.
13. 13 . The method of claim 1 , wherein the oligonucleotide comprising the barcode sequence is hybridized to a 3′ hydroxyl functionalized end of an oligonucleotide molecule attached to the substrate in the first region.
14. 14 . The method of claim 1 , wherein (c) comprises ligating the oligonucleotide comprising the barcode sequence to the 3′ hydroxyl functionalized end of the oligonucleotide molecule attached to the substrate in the first region.
15. 15 . The method of claim 1 , wherein the oligonucleotide comprising the barcode sequence is hybridized to a splint which is in turn hybridized to the 3 ′ hydroxyl functionalized end of the oligonucleotide molecule attached to the substrate in the first region.
16. 16 . The method of claim 15 , further comprising ligating the oligonucleotide comprising the barcode sequence to the 3′ hydroxyl functionalized end of oligonucleotide molecule to generate a barcoded oligonucleotide molecule attached to the substrate in the first region.
17. 17 . The method of claim 16 , further comprising blocking 3′ termini of barcoded oligonucleotide molecules and/or unligated oligonucleotide molecules attached to the substrate in the first region from ligation.
18. 18 . The method of claim 1 , wherein the method comprises performing (a)-(c) for N cycles, wherein N is an integer of 2 or greater.
19. 19 . The method of claim 18 , wherein the barcode sequences received by oligonucleotide molecules in a feature on the substrate in cycle I and in the feature on the substrate in cycle J are different, wherein I and J are integers and 1 ≤I <J ≤N.
20. 20 . The method of claim 19 , wherein the layer of positive photoresist is not removed between cycles of the N cycles.

Description

CROSS-REFERENCE TO RELATED APPLICATION This application claims priority to U.S. Provisional Patent Application No. 63/132,385, filed Dec. 30, 2020, entitled “MOLECULAR ARRAY GENERATION USING PHOTORESIST,” which is herein incorporated by reference in its entirety for all purposes. FIELD The present disclosure relates in some aspects to methods for manufacturing a molecular array using photoresist and the molecular array generated in situ on a substrate. BACKGROUND Arrays of nucleic acids are an important tool in the biotechnology industry and related fields. These nucleic acid arrays, in which a plurality of distinct or different nucleic acids are positioned on a solid support surface in the form of an array or pattern, find use in a variety of applications, including gene expression analysis, drug screening, nucleic acid sequencing, mutation analysis, and the like. A feature of many arrays that have been developed is that each of the distinct nucleic acids of the array is stably attached to a discrete location on the array surface, such that its position remains constant and known throughout the use of the array. Stable attachment is achieved in a number of different ways, including covalent bonding of a nucleic acid polymer to the support surface and non-covalent interaction of the nucleic acid polymer with the support surface. There are two main ways of producing nucleic acid arrays in which the immobilized nucleic acids are covalently attached to the substrate surface, i.e., via in situ synthesis in which the nucleic acid polymer is grown on the surface of the substrate in a step-wise, nucleotide-by-nucleotide fashion, or via deposition of a full, presynthesized nucleic acid/polypeptide, cDNA fragment, etc., onto the surface of the array. While nucleic acid arrays have been manufactured using in situ synthesis techniques, applications in the field of genomics and high throughput screening have fueled the demand for precise chemistry and high fidelity of the synthesized oligonucleotides. Accordingly, there is continued interest in the development of new methods for producing nucleic acid arrays in situ. Provided herein are methods, uses and articles of manufacture that meet such needs. SUMMARY In some aspects, disclosed herein is a method for providing an array, comprising: (a) irradiating a substrate comprising an unmasked first region and a masked second region, whereby a photoresist in the first region is degraded to render oligonucleotide molecules in the first region available for hybridization and/or ligation, whereas oligonucleotide molecules in the second region are protected by a photoresist in the second region from hybridization and/or ligation; and (b) attaching an oligonucleotide of at least four nucleotide residues in length to oligonucleotide molecules in the first region via hybridization and/or ligation, wherein oligonucleotide molecules in the second region do not receive a sequence of the oligonucleotide, thereby providing on the substrate an array comprising different oligonucleotide molecules in the first and second regions. In some embodiments, the oligonucleotide comprises a barcode sequence, and all or a portion of the barcode sequence is attached to oligonucleotide molecules in the first region, wherein oligonucleotide molecules in the second region do not receive the barcode sequence or portion thereof. In any of the embodiments herein, the oligonucleotide molecules on the substrate can comprise one or more common sequences. In any of the embodiments herein, the one or more common sequences can comprise a homopolymeric sequence, such as a poly(dT) sequence, of three, four, five, six, seven, eight, nine, ten or more nucleotide residues in length. In any of the embodiments herein, the one or more common sequences can comprise a common primer sequence. In some embodiments, the common primer sequence is between about 10 and about 35 nucleotides in length. In any of the embodiments herein, the one or more common sequences can comprise a partial primer sequence. For example, a terminal sequence of an oligonucleotide molecule on the substrate together with a sequence of an oligonucleotide attached to the oligonucleotide molecule on the substrate can form the hybridization sequence for a primer. In this example, the terminal sequence of the oligonucleotide molecule on the substrate can be viewed as a partial primer sequence. In any of the embodiments herein, oligonucleotide molecules in the first region and oligonucleotide molecules in the second region can be identical in sequence. In any of the embodiments herein, oligonucleotide molecules on the substrate prior to the irradiating step can be identical in sequence. In any of the embodiments herein, oligonucleotide molecules in the first region and oligonucleotide molecules in the second region can be different in sequences, optionally wherein oligonucleotide molecules in the first region and oligonucleotide molecules in the second regi