Methods and compositions for size-selective elution of xpandomer macromolecules for nanopore sequencing

Abstract

Methods and compositions for size-selective elution of a sample of Xpandomer molecules synthesized on a solid substrate. The methods include releasing the Xpandomers from the solid substrate by cleaving a selectively cleavable bond linking the Xpandomers to the substrate and eluting a sample of Xpandomers with a buffer including an organic co-solvent and a stabilizing agent. The solid substrate includes a coating capable of non-covalent interactions with Xpandomer molecules and functions as a molecular sieve to preferentially retain shorter Xpandomers relative to longer Xpandomers. The sample of eluted Xpandomer molecules therefore has an average length longer than the average length of the non-eluted Xpandomer molecules.

Description

P39616-WO-1METHODS AND COMPOSITIONS FOR SIZE- SELECTIVE ELUTION OF XPANDOMER MACROMOLECULES FOR NANOPORE SEQUENCINGINCORPORATION BY REFERENCE

[0001] All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.BACKGROUND OF THE INVENTION

[0002] Various DNA sequencing technologies have been developed over the past several decades. One of the next generation sequencing technologies involves the use of nanopores, which can be used to determine the sequence of DNA molecules by passing a single strand of the DNA molecule directly through the pore, as is being done by Oxford Nanopore. However, this approach can suffer difficulties from reading long sections of repeat nucleotides since the signal from the nanopore can remain relatively constant for long periods of time, making it difficult to determine the exact number of repeat molecules.

[0003] Another approach which solves this problem involves the use of synthesizing a specialized macromolecule, called an Xpandomer, from a nucleic acid template using a modified polymerase and modified nucleotides, as further described in U.S. Patent No. 7,939,259 Kokoris et al. and International Patent Publication No. WO2020 / 236526A1 O’Connell et al., each of which is incorporated by reference in their entireties for all purposes. The Xpandomer molecule includes a translocation control element that allows the user to advance the Xpandomer in a controlled fashion through the nanopore, which therefore allows the number of repeat nucleotides to be accurately determined.

[0004] The synthesis of the Xpandomer can be relatively complex, time consuming, and difficult to perform manually. Systems capable of performing the solid-state synthesis of the Xpandomer molecule in an automated fashion with minimal input from the end user have been developed. Such systems require methods and compositions to elute and retain a sample of Xpandomer molecules for nanopore sequencing, particularly a sample enriched for substantially full length Xpandomer copies of the DNA template.SUMMARY OF THE INVENTION

[0005] In one aspect, the invention provides a method for size-selective elution of a sample of Xpandomer molecules from a substrate, including the steps of: a) providing a substrate including a plurality of Xpandomer molecules, in which each Xpandomer molecule is covalently joined to the substrate by an extension oligonucleotide polymer including a photocleavable moiety and a oligonucleotide primer, in which the substrate further includes a coating, and in which the coating is capable of non-covalent interactions with the Xpandomer molecules; b) contacting the substrate with a first aqueous buffer, in which the first aqueous buffer includes an organic co-solvent and a stabilizing agent; c) contacting the substrate with UV light, in which the UV light cleaves the photocleavable moiety; and d) eluting a sample of Xpandomer molecules from the substrate, in which the eluted sample includes Xpandomer molecules with an average length greater than the average length of non-eluted Xpandomers In some embodiments, the non-eluted Xpandomer molecules are preferentially retained on the coating by the non-covalent interactions. In some embodiments, the organic co-solvent includes acetonitrile present in an amount of greater than 10% (v / v) to less than 20% (v / v). In further embodiments, the acetonitrile is present in an amount of around 15% (v / v) to around 16% (v / v). In some embodiments, the stabilizing agent includes trehalose. In further embodiments, the trehalose is present in an amount of around 0.5% (w / v) to around 2.0% (w / v). In yet further embodiments, the trehalose is present in an amount of around 1% (w / v). In some embodiments, the first aqueous elution buffer includes 15% (v / v) acetonitrile and 1% (w / v) trehalose. In some embodiments, the substrate is contacted with UV light for around 2 minutes. In some embodiments, the substrate is contacted with UV light at a temperature of around 37 degrees Celsius. In some embodiments, the coating includes denatured protein. In further embodiments, the denatured protein includes denatured nucleic acid polymerase protein or denatured single stranded binding protein. In some embodiments, the denatured nucleic acid polymerase protein is provided by an Xpandomer synthesis termination step, in which the Xpandomer synthesis termination step precedes the step of contacting the substrate with the first aqueous elution buffer, in which the Xpandomer synthesis termination step includes incubating an Xpandomer synthesis reaction at a temperature of around 45 to around 60 degrees Celsiusfor around 5 to around 20 minutes; and in which the Xpandomer synthesis reaction includes a nucleic acid polymerase protein. In some embodiments, the step of providing the substrate including a plurality of Xpandomer molecules includes the ordered steps of a) providing a substrate including a surface functionalized with a polymer including a maleimide moiety on a first end and an alkyne moiety on a second end; b) covalently joining the extension oligonucleotide to the polymer, in which the extension oligonucleotide further includes a terminal azide group capable of forming a covalent bond with the alkyne moiety; c) contacting the extension oligonucleotide with a nucleic acid template under nucleic acid hybridization conditions to provide a hybridized nucleic acid template complex; d) contacting the hybridized nucleic acid template complex with an Xpandomer synthesis reaction mixture under Xpandomer synthesis conditions to provide a plurality of Xpandomer molecules covalently joined to the substrate. In further embodiments, the Xpandomer synthesis reaction mixture includes a nucleic acid polymerase protein. In some embodiments, the method further including the step of e) providing Xpandomer synthesis termination conditions, in which the Xpandomer synthesis termination conditions include incubating the substrate at a temperature of around 45 to around 60 degrees Celsius for around 5 to around 20 minutes. In some embodiments, the extension oligonucleotide further includes a leader sequence and a concentrator sequence. In further embodiments, the leader sequence is a polymer including 2 carbon monomers and the concentrator sequence is a polymer including 12 carbon monomers. In yet further embodiments, the leader sequence includes from around ten to around 1502 carbon monomers. In some embodiments, the concentrator sequence includes from around four to around tenl2 carbon monomers. In some embodiments, the Xpandomer molecules includes a plurality of spermine moieties, in which the spermine moieties include positively charged amine groups. In further embodiments, the method further includes a spermine modification step prior to the step of contacting the substrate with UV light, in which the spermine modification step includes contacting the Xpandomer molecules with a buffer including an anhydride compound. In yet further embodiments, the spermine modification step converts up to 50% of the positively charged amine groups to negatively charged hemi acetyl groups. In some embodiments, the step of eluting a size-selected sample of Xpandomers from the substrate includes collecting the sample of size-selected Xpandomers in a tube.

[0006] In another aspect, the invention provides a method for non-selective elution of a sample of Xpandomer molecules including the steps of the method recited above followed by the steps of contacting the substrate with a second elution buffer, in which the second elution buffer includes an organic co-solvent, a stabilizing agent, and a buffer and collecting a non-size selected sample of Xpandomer molecules. In one embodiments, the second aqueous elution buffer further includes a salt. In some embodiments, the second aqueous elution buffer includes around ImM HEPES. In some embodiments, the organic co-solvent includes acetonitrile present in an amount of around 20% (v / v).

[0007] In another aspect, the invention provides an elution buffer for collecting a size selected sample of Xpandomer molecules including around 15% (v / v) acetonitrile and around 1% (w / v) trehalose.

[0008] In another aspect, the invention provides elution buffer for collecting a non-size selected sample of Xpandomer molecules including from around 15% (v / v) to around 20% (v / v) or over acetonitrile, around 1% w / v trehalose, and around ImM HEPES buffer.BRIEF DESCRIPTION OF DRAWINGS

[0009] The novel features of the invention are set forth with particularity in the claims that follow. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0010] FIGS. 1A and IB illustrate an embodiment of a system for automated synthesis of a macromolecule.

[0011] FIG. 1C illustrates a mixing tube that can be used in the system shown in FIGS. 1A and IB.

[0012] FIG. ID illustrates the synthesis steps that can be performed on the system illustrated in FIGS. lA and IB.

[0013] FIG. 2A illustrates an embodiment of a flow cell.

[0014] FIG. 2B illustrates a functionalized flow channel surface and how a capture probe can be used to immobilize the template to the flow channel surface.

[0015] FIGS. 2C and 2D illustrate an embodiment of the flow cell with a thin film bonded to a molded portion of the flow cell.

[0016] FIG. 3 illustrates an embodiment of a mount for receiving the flow cell.

[0017] FIG. 4A illustrates an embodiment of a holder.

[0018] FIGS. 4B and 4C illustrate embodiments of reagent cartridges that can be placed in the holder shown in FIG. 4A.DETAILED DESCRIPTION OF THE INVENTION

[0019] Automated Solid-State Xpandomer Synthesis Method

[0020] The general structure of the Xpandomer molecule, the xNTPs substrates and the Xpandomer synthesis process are further described, e.g., in U.S. Published Patent Application No. 2022 / 0042075 Merrill et al. and International Patent Publication No. WO2020 / 236526A1 O’Connell et al., each of which is incorporated by reference in their entireties for all purposes. As shown in FIGS. 1A and IB, a system 100 can be used to automate the Xpandomer synthesis process.

[0021] FIG. ID is a flowchart that illustrates an overview of one, non-limiting, embodiment of the synthesis process that can be performed by the system shown in FIGS. 1A and IB. It is to be understood that the particular buffers and other reaction conditions set forth in FIG. ID are intended to simply illustrate one example of how the Xpandomer synthesis process may be implemented. One of skill in the art will appreciate that any single step or combination of steps may be further altered or optimized for particular applications.

[0022] In step 150, a flow channel surface can be functionalized (also shown in FIG. 2B and further described below). This functionalization process may be performed by the system on, e.g., a flow cell by drawing the appropriate reagents into the flow cell using the sipper(s) and reagents on the holder 400. In other embodiments, the flow channel surface can be functionalized prior to shipment to the consumer so that the consumer does not need to perform the functionalization step. The functionalization process includes attaching a plurality of probes, e.g., polymers including an extension oligonucleotide, to the flow channel surface that are capable of binding to a sample.

[0023] In step 152, the sample, which, in certain embodiments, can be template nucleic acid molecules from a single stranded DNA or RNA library, can be introduced into the flow cell 200 and flow channel(s) by using the sipper(s) 224 to draw the template from the holder 400. The template can be allowed to hybridize to the capture probe, which can include apolynucleotide. The temperature during the hybridization step can be controlled by the controller by heating and / or cooling the thermal block attached to the mount 300. For example, the temperature can be varied between about 37 degrees Celsius and 90 degrees Celsius during the hybridization step. Other temperatures can also be used in this step, depending on the probe and template combination used.

[0024] Next, in step 154 a wash can be performed at least one time. In some embodiments, the wash procedure can involve multiple washes (i.e., 1000 uL and 100 uL washes for one minute). This can also be performed at a controlled temperature, such as 37 degrees Celsius, for example. A wash solution can include at least one solvent and at least one detergent. In some embodiments, at least one surfactant can optionally also be included. In some embodiments, at least one buffer can optionally also be included.

[0025] Next, in step 156, an extension reaction can be performed by introducing Mix A solution to the flow channel. Mix A can include solvent(s), buffer(s), salt(s), a polymerase, nucleotide analogs (e.g., XNTPs) and other additives. The controller can control the temperature of this reaction by varying the temperature and holding period duration at each temperature as desired. For example, the extension reaction can include a first incubation period at 37 degrees Celsius for a first duration (i.e., 110 minutes), and second incubation period at 42 degrees Celsius for a second duration (i.e., 10 minutes), and a third incubation period at 50 degrees Celsius for a third duration (i.e., 5 minutes).

[0026] Next, in step 158, a wash can be performed at a controlled temperature using a desired amount of wash buffer (i.e., 1000 uL of Mix B) for a desired duration (i.e., 5 minutes). Mix B can include at least one solvent and at least one detergent. In some embodiments, at least one surfactant can optionally also be included. In some embodiments, at least one buffer can optionally also be included.

[0027] Next, in step 160, a cleavage step is performed using 200uL of Mix C at 23 degrees Celsius for 40 minutes. Mix C can include an acid in solvent.

[0028] Next, in step 162, a wash and neutralization step is performed using 2000uL of Mix B at 23 degrees Celsius for one minute.

[0029] Next, in step 164, a modification step is performed using 300uL of Mix B and Mix Mod at 23 degrees Celsius for five minutes. Mix Mod can be succinic anhydride in solution.

[0030] Next, in step 166, a wash step is performed using 3000uL of Mix D at 23 degrees Celsius for one minute. Mix D can include a solvent and optionally a stabilizer molecule.

[0031] Finally, in step 168, a UV and Elution step is performed using around 50 to 60 uL of Mix D (e.g., a selective elution buffer) at around 37 degrees Celsius for around 2 minutes under UV illumination. The synthesized Xpandomer molecules can be eluted into a tube in the holder using the pump in push operation. The elution step is discussed in further detail herein to selectively elute a sample enriched for a desired size range of Xpandomer copies of a nucleic acid template.

[0032] System for Automated Synthesis

[0033] FIGS. 1A and IB illustrate an embodiment of a system 100 used for macromolecule synthesis. The system 100 includes a removable and disposable flow cell (i.e. card or chip) 200, a mount 300 (including a thermal block described below) for receiving the flow cell 200, a holder / vial rack 400, an XYZ gantry 500, a pump (e.g. syringe pump) 600, a flow and pressure gauge 602, a waste container 604, a buffer container 606, a selectable valve (e.g. rotary valve) 608, a UV source 700, and a controller (not shown). The holder 400 can also be used to hold sample and the end product or anything else that can be contained in a tube, container, vial, or reservoir that can be placed in the holder.

[0034] In some embodiments as shown in FIGS. 1A and IB, a pump 600 can be used in a pull mode to draw fluid into the inlet port of the flow cell 200 via the sipper(s) 22, and the fluid can be drawn out of the flow cell 200 through the outlet ports of the flow cell 200 and into a waste reservoir 604. The pump 600 can also be used in a push mode to pump fluid, such as a wash solution or elution buffer, from a buffer reservoir 606 and into the flow cell 200 through the outlet port, and then the fluid can be pushed out of the flow cell 200 through the inlet port and through the sipper(s) into a product collection tube. A rotary valve 602 or other type of valve can be used with the pump 600 to switch between the waste reservoir 604 and the buffer reservoir 606 in order to allow the pump to operate in both a push and pull configuration. The pump 600 can be a syringe pump or other type of pump that is capable of precisely metering out very small amounts of fluid (i.e., in the microliter to milliliter range).

[0035] In some embodiments, the controller can control the XYZ gantry 500 and pump 600 to perform liquid mixing operations within the flow cell 200 or directly on / in the holder 400.

[0036] Consumable device / cartridge

[0037] FIG. 2A illustrates a perspective view of one embodiment of a flow cell 200 that the user can load into the system. The flow cell 200 can be a consumable device or cartridge that is disposed of after use. The flow cell 200 encloses at least one flow channel 202 thatprovides a functionalized solid phase surface to which a plurality of capture probes can be attached. In some embodiments, the flow cell 200 has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 flow channels. In some embodiments, the flow cell 200 has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 flow channels. In some embodiments, the flow channel 202 can be made of a substrate that is directly functionalized, instead of being coated with a material that is then functionalized. The flow channels can be fabricated using microfluidic techniques and can be used to perform reactions involving fluid volumes in the microliter range. In some embodiments, the flow channel 202 can have a cross-section that is about 0.6 mm x 0.3 mm, or about 0.8 mm x 0.4 mm, or less than about 1 mm x less than 0.5 mm, or less than 2 mm x less than 1 mm. In some embodiments, the volume of the flow channel 202 can be about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 uL. In some embodiments, the surface area to volume ratio can be about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, or 50 mm’1. In some embodiments, the flow cannel 202 can be serpentine to increase the flow length and surface area available to carry out the reactions and / or to aid in mixing.

[0038] In some embodiments, the flow cell 200 can be fabricated from a single injection molded portion 201 that is bonded to a thin film 203, as shown in FIGS. 2C and 2D. For example, the injection molded portion can include the base substrate, the inlet ports, the outlet ports, and a portion of the flow channels 202. The thin film 203 can be disposed over the base substrate and flow channels 202 to enclose and complete the formation of the flow channels 202. The thin film that forms the base of the flow cell 200 can be placed against a thermal block, as further described below. The thin film can have a low thermal resistance which allows efficient heat transfer from a thermal block to the flow cell 200, which allows the rapid heating and cooling of the flow cell 200 to facilitate the different temperature reactions of the Xpandomer synthesis process. In some embodiments, to achieve the desired thermal resistance, the thin film can have a thickness between about 100 pm to about 500 pm. In some embodiments, the thickness can be less than about 500, 400, 300, 200, or 100 pm. In some embodiments, the thickness can be about 100, 200, 300, 400, or 500 pm. In some embodiments, the thin film can be made of a cyclic olefin polymer (COP) or a cyclic olefin copolymer (COC). Other types of polymers may also be used to form the thin film, such UV transparent polymers that can be functionalized (i.e., proton abstractable polymers) and bonded to the injection molded portion of the flow cell. Other polymers that can be usedinclude but are not limited to polypropylene and polyethylene. The thin film can be bonded to the molded portion using a variety of techniques, such as thermal bonding, laser welding, or chemical bonding.

[0039] Each flow channel 202 of the flow cell 200 has an inlet port 220 and an outlet port 222 that provide access to the flow channel 202. Affixed to the inlet port 220 is a sipper 224 that can be used to draw reagents, buffers, wash solution, sample, etc. into the flow cell 200 when the pump is operated in a pull mode. As noted above, the sipper 224 can also be used to deposit the finished product from the flow cell 200 into a collect tube when the pump is operated in a push mode. In some embodiments, the sipper 224 is preattached to the inlet port 220 so that the end user does not need to attach the sipper 224 to the flow cell 200 before use. Instead, the end user can simply insert the preassembled flow cell 200 into the mount 300 as shown in FIGS. 1A and 3. In some embodiments, the sipper 224 has a length that is less than the length of the flow cell 200. In some embodiments, the sipper 224 is less than about 5x, 4x, 3x, 2x, lx, 0.9x, 0.8x, 0.7x, 0.6x, 0.5x, 0.4x, 0.3x, 0.2x, or O.lx the length of the flow cell 200. Reducing or minimizing the length and volume of the sipper 224 is advantageous in reducing the loss and / or carryover of precious reagents, sample, and the synthesized molecule (i.e., the Xpandomer) during the synthesis process. The sipper 224 configuration also provides a direct path to the flow cell that does not need to pass through a valve, which can reduce contamination and leak issues, especially when using corrosive reagents.

[0040] In some embodiments, the inlet port 220 and outlet port 222 extend from the flow cell 200 in an orientation that is normal to a plane that encompasses the flow channel 202. In some embodiments, the sipper 224 also extends from the inlet port 220 in an orientation that is normal to a plane that encompasses the flow channel 202. In some embodiments, the sipper 224 has a 90 degree bend, or whatever angle is needed, to orient the end of the sipper 224 towards the holder 400, as shown in FIG. 1A. In other embodiments, the inlet port 220 can extend from the flow cell 200 in an orientation that is parallel to a plane that encompasses the flow channel, and the sipper 224 can extend from the inlet port is the same orientation towards the holder, and in some embodiments, the sipper 224 will be straight and without a bend.

[0041] FIG. 2B illustrates one embodiment of the flow channel surface (i.e., substrate) 204 that has been functionalized to bind capture probes (e.g., extension oligonucleotides) 206,which can further bind, or hybridize, to a portion of a sample. As shown in FIG. 2B, in step i) a flow channel surface may be functionalized with an alkyne-maleimide linker 204a, which in certain embodiments is a bifunctional linker providing a maleimide moiety on a first end and an alkyne moiety on a second end. The chemical chain between these two reactive groups of the linker may be referred to as the “spacer arm”. The length of the spacer arm determines how flexible the conjugate will be and can be optimized for particular applications. Typically, the spacer arms include hydrocarbon chains or polyethylene glycol (PEG) chains. The terminal maleimide moiety can be converted into a reactive group and subsequently crosslinked to the flow channel surface (e.g., a polyolefin substrate), via a catalyst-free photochemical (e.g., photo-initiated) proton abstraction reaction.Advantageously, the alkyne moiety provided by the end of linker 204a distal to the substrate is capable of reacting with a free azide group provided by, e.g., an extension oligonucleotide in solution.

[0042] To functionalize a substrate with an alkyne-maleimide linker, an exemplary catalyst- free photochemical proton abstraction reaction may include the following steps: 1) priming the substrate (e.g., chip) with an organic solvent, such as DMSO or DMF; 2) adding the linker with a maleimide moiety on one end, such as propargyl maleimide, solubilized in, e.g., DMSO and water; 3) incubating the chip under a UV lamp at 650nM for 20 minutes; 3) washing the chip with a series of solvents, which in certain embodiments may include DMSO, DMF, and a solution of Na2HPO4, Tween-20, and SDS; and 4) washing the chip with aqueous solutions such as water and / or PBS prior to the click reaction.

[0043] As further shown in FIG. 2B, step i) an extension oligonucleotide 206 may be covalently linked to the flow channel surface via formation of triazole group 207 between the terminal alkyne moiety of linker 204a and a terminal azide moiety provided by extension oligonucleotide 206 via a conventional copper-catalyzed click chemical reaction. Thus, in certain embodiments, an extension oligonucleotide may be conveniently “clicked” to an alkyne-functionalized flow channel surface. For simplicity, the product of the reaction that covalently joins linker 204a to extension oligonucleotide 206 may be referred herein to as a “polymer”. In some embodiments, the extension oligonucleotide may be attached to the chip flow channel surface prior to shipping for use. Furthermore, the extension oligonucleotide may be stabilized by hybridization to a complementary oligonucleotide (e.g., a fluorescent hyb oligo) that is linked on one end to a detectable dye, e.g., SIMA, that enables qualityassessment of the functionalized chip.

[0044] In certain embodiments, the extension oligonucleotide may include the following features, from the 5’ to the 3’ direction: a terminal azide group, a polymeric spacer, a photocleavable moiety (e.g., derived from a photocl eavable spacer, or modifier, phosphoramidite commercially available from Glen Research), a leader sequence (here depicted as 206a), a concentrator sequence (here depicted as 206b), and an oligonucleotide primer (here depicted as 206c). As shown in step ii), the oligonucleotide primer provides an initiation site for Xpandomer synthesis when hybridized to nucleic acid template 208 to form a hybridized nucleic acid template complex. In certain embodiments, the nucleic acid template will include a a 5’ end sequence that is complementary to the sequence of oligonucleotide primer 206c, for example, a sequence provided by a heterologous Y adapter component of a nucleic acid template construct.

[0045] The Xpandomer synthesis process is illustrated in step iii), and is initiated from the 3’ end of the oligonucleotide primer using sample polynucleotide 208 as a template. An Xpandomer synthesis reaction includes a nucleic acid polymerase, e.g., a modified DNA polymerase, and XNTP modified nucleotides to synthesize an Xpandomer copy of the sample polynucleotide template 208, which is further described in US provisional patent application 63 / 687,453, filed August 27, 2024, entitled, “Compositions for Replicating a Nucleic Acid Template,” which is herein incorporated by reference in its entirety for all purposes. As discussed with reference to FIG. ID, following synthesis, the Xpandomer molecules are subjected to certain processing steps, including treatment with an acid solution to cleave the selectively cleavable phosphoramidate bonds in the XNTPs and transition the Xpandomer from the constrained to the elongated configuration and a modification step to alter the charge of the spermine constituents of the Xpandomer SSRT. In certain embodiments, the Xpandomer is treated with a solution of succinate anhydride to convert the positively charged amine groups of spermine into negatively charged groups. In certain conditions, such modification of the net charge of spermine facilitates nanopore translocation of the Xpandomer.

[0046] Because Xpandomer synthesis is initiated from extension oligonucleotide 206, the growing Xpandomer is covalently bound to the surface of the flow channel and includes all the features of the extension oligonucleotide, notably the leader and concentrator sequences. The structures of the leader and concentrator features and their roles in nanoporetranslocation of the Xpandomer are described in further detail in, e.g., US Patent no.11,920,184 to Kokoris and McRuer, the entire contents of which are hereby incorporated by reference in its entirety. Briefly, the leader feature is a polymer that includes a high density of negative charge and functions to facilitate capture and threading of the Xpandomer through the nanopore. In certain embodiments, the leader sequence may be synthesized from C2 phosphoramidate monomers, such that the final polymeric feature comprises C2 monomers linked by phosphodiester bonds, which each provide a negative charge. In contrast, the concentrator feature is a polymer with overall lipophilic properties and functions to localize the Xpandomers to the lipid surface of the nanopore sensor. In certain embodiments, the concentrator sequence may be synthesized from C12 phosphoramidate monomers, such that the final polymeric feature comprises in-line Cl 2 monomers, which underlies the overall hydrophobic properties of the concentrator.

[0047] After the Xpandomer extension reaction is complete, UV light (from UV source 700) can be used to cleave the photocleavable moiety in extension oligonucleotide 206, which releases Xpandomer molecule 210 from flow channel surface 204, as depicted in step iV). The released Xpandomers will include leader sequence 206a and concentrators sequence 206b at the 5’ end of the cleaved molecules. However, under certain conditions, the final population of Xpandomers released from the flow channel surface may include a heterogenous mixture of Xpandomer molecules, which differ in length at the 3’ end. For example, the released sample of Xpandomers may include both full length and a variety of truncated copies of the nucleic acid template, the latter resulting from, e.g., incomplete replication of the template or from breakdown of full length Xpandomers during, e.g., the Xpandomer processing steps. Therefore, the sample of Xpandomer molecules collected following elution of photo-cleaved products may, or may not, be optimal for a particular downstream application. For example, for sequencing applications, it would be advantageous to elute a sample enriched for longer, e.g., substantially full length, Xpandomer molecules.

[0048] Elution of Xpandomer Molecules from a Solid Support

[0049] Compared to natural DNA, the Xpandomer is a very large macromolecule that has properties associated with both natural polymers, such as DNA, and properties associated with synthetic polymers. As such, the Xpandomer exhibits complex physico-chemical interactions with the chip environment and buffer components during elution of the photo-cleaved sample from the chip surface. The composition of the elution buffer therefore impacts the ability to collect a sample enriched for a desired size range, for example, a sample enriched for substantially full length Xpandomer molecules.

[0050] In various embodiments, an Xpandomer elution buffer may include one or more of the following reagents: a buffer; an organic solvent or co-solvent; a chaotropic agent; a detergent; and an additive or stabilizing agent.

[0051] In some embodiments, the buffer may include from around ImM to around lOmM or over of HEPES, pH 7.4; NaH2PO4, pH 6.5-8.0; imidazoleCl, pH 6.5-7.5; NH4OAC, pH 6.7; or TrisCi, pH 7.5.

[0052] In some embodiments, the organic solvent or co-solvent may include one or more of ACN, DMF, DMSO, MO A, cyrene, NMP, formamide, nitromethane, sulfolane, IP A, NMS, dioxane, hexanediol, MeOH, or other organic solvents or co-solvents. In some embodiments, organic solvent or co-solvent or combination thereof may be present in an amount of around 5% (v / v) to around 80% (v / v), from around over 5% (v / v) to around under 80% (v / v), from around 10% (v / v) to around 75% (v / v), from around 15% (v / v) to around 70% (v / v), from around 15% (v / v) to around 60% (v / v), from around 15% (v / v) to around 50% (v / v), and the like.

[0053] In some embodiments, the chaotropic agent may be a suitable amount of GuHCl or urea.

[0054] In some embodiments, the detergent may include from around ImM to around 50mM of NaHex, FlHex, Tween, or SDS.

[0055] In some embodiments, the additive or stabilizing agent may be from around ImM to around 500mM of urea, ficoll, glycerol, imidazole, NH4OAc, NaPO4, 4-glyme, GuCl, trehalose, PEG 8k, THF, formamide, spermine, GMP, EDTA., or other stabilizing agent.

[0056] The optimal mixture of reagents for a particular elution buffer will depend on many factors, such as the length range and other properties (e.g., the chemical composition of the features of the SSRT, e.g., the reporter codes, translocation control element, and spermine enhancers) of the target Xpandomer sample.

[0057] In certain embodiments, an Xpandomer elution buffer may include an organic cosolvent in water. The length of the Xpandomer molecule has been found to correlate with the propensity of Xpandomers to aggregate, or precipitate, from aqueous solution under certain conditions. Advantageously, addition of an organic co-solvent to the elution bufferhas been found to increase Xpandomer solubility in aqueous solution. As used herein, the terms “buffer” and “solution” may be used interchangeably and do not imply that either liquid includes any particular reagents, unless such reagents are explicitly called out.

[0058] In some embodiments, Xpandomer molecules have been found to be further stabilized by including a cyroprotectant additive, or stabilizing agent, in the elution buffer. In some embodiments, an Xpandomer elution buffer may include from around 10% (v / v) to around 20% (v / v) acetonitrile (ACN) and from around 0.5% (w / v) to around 2% (w / v) trehalose in H2O. In one embodiment, an Xpandomer elution buffer may include 15% (v / v) ACN and 1% (w / v) trehalose in H2O. In another embodiment, an Xpandomer elution buffer may include 16% (v / v) ACN and 1% (w / v) trehalose in H2O. In another embodiment an Xpandomer elution buffer may include 20% (v / v) ACN and 1% (w / v) trehalose in 1-lOmM HEPES, pH 7.4. As discussed herein, the exact composition of the elution buffer will be determined by the size range of Xpandomers that is optimal for a particular application.

[0059] Through trial and error, the inventors have made the unexpected discovery that certain buffer and other chip conditions can favor elution of a sample of Xpandomers that is enriched for longer molecules, while shorter, e.g., truncated, molecules are not eluted. As discussed herein with reference to the size of Xpandomer molecules, the terms “longer” and “shorter” are relative terms. For example, in certain embodiments, a sample of longer, e.g., eluted, Xpandomers will have an average length that is greater than the average length of shorter , e.g., non-eluted, Xpandomers. In some embodiments, a sample of selectively eluted Xpandomers will have an average length that is greater than the average length of all Xpandomers originally synthesized on the chip. In some embodiments, a sample of selectively eluted Xpandomers will be enriched for substantially full length Xpandomer copies of the nucleic acid template. The average size of two samples of Xpandomer molecules can be easily compared by conventional gel electrophoretic techniques, which are well known in the art. For example, shorter Xpandomer molecules will be seen as low molecular weight bands on a gel, while longer Xpandomer can be easily discriminated as higher molecular weight bands on the gel. In some embodiments, the average size to two samples of Xpandomer molecules can be compared by sequencing a representative population of molecules from each sample.

[0060] In certain embodiments, the chip substrate may include a “coating” of substance that accumulates on the surface of the chip during post-manufacturing processes, e.g., during oneor more of the chip functionalization or Xpandomer synthesis steps. In some embodiments, shorter Xpandomer molecules may be preferentially retained on the surface coating of the chip such that they do not elute with longer Xpandomer molecules. Without being bound by theory, it is speculated that the surface coating of the functionalized chip may have fractionation properties analogous to those of a size exclusion chromatography column, in which larger molecules elute more rapidly than smaller molecules, the latter being preferentially retained by the matrix (e.g., the coating or surface of the chip). In other words, the chip coating may function as a molecular sieve, which preferentially retains smaller molecules from a sample. This phenomenon is referred to herein as “size selective elution” or “size-enrichment”. Again, without being bound by theory, it is speculated that size selective elution reflects complex non-covalent interactions, e.g., hydrophobic, hydrophilic, or ionic interactions, between the Xpandomer molecule and the chip surface coating. One of skill in the art will recognize that such interactions are likely to be strongly influenced by the composition of the elution buffer, the chip coating, and the chip surface. Indeed, the following is a non-limiting list of factors that have been observed to influence the size electivity of Xpandomer elution:

[0061] Solvent. The type of organic solvent, or co-solvent, used in the elution buffer influences size selectivity. The organic co-solvent, acetonitrile (ACN), has been found to favor size selective elution of certain Xpandomer structures to a greater extent than other organic solvents, such as DMSO and DMF. The percentage of ACN in the elution buffer also has an impact on size selectivity, with percentages over around 16% (v / v) exhibiting less size selectivity and percentages of around 20% (v / v) exhibiting no size selectivity. Thus, in certain embodiments, e.g., when a particular application does not require a sample of substantially full length Xpandomer molecules, an elution buffer that includes around 20% (v / v) ACN would be suitable. However, when a particular application does require a sample of substantially full length Xpandomer molecules, e.g., a sequencing application, an elution buffer including around 15% to around 16% (v / v) ACN would be suitable. However, it is to be understood that this description is not intended to be limiting, but rather represents one exemplary embodiment. Any of the solvents disclosed herein may be suitable for eluting a particular Xpandomer structure.

[0062] Denatured protein on the chip surface. The inventors have made the surprising observation that terminating the Xpandomer synthesis reaction with an incubation at anelevated temperature, relative to the temperature of the synthesis reaction, significantly enhances selective elution. Without being bound by theory, it is speculated that higher temperatures denature the polymerase and other proteins present in the Xpandomer synthesis reaction and “coats” them onto the surface of the chip, thereby increasing the chromatographic, or sieving, properties of the chip. Thus, in certain embodiments, the selective elution methods of the present invention may include a “hot finish” step following the Xpandomer synthesis reaction. In some embodiments, the methods include an incubation step at around 45 to around 60 degrees Celsius for around 5 to around 20 minutes following the Xpandomer synthesis reaction. In other embodiments, the incubation step is carried out at temperatures lower than around 45 to higher than around 60 degrees Celsius for less than around 5 to greater than around 20 minutes. The range of suitable temperatures and timing of the incubation step will depend on the particular conditions necessary to denature the particular proteins present in the Xpandomer synthesis reaction.

[0063] In some embodiments, size selectivity may be enhanced by increasing the amount of polymerase or other protein in the Xpandomer synthesis reaction. In some embodiments, the Xpandomer synthesis reaction may include from around 0.05pg / pL or less to around 0.2pg / pL or more of polymerase or other protein. In other embodiments, the Xpandomer synthesis step may be preceded by a wash step using a wash buffer that includes a higher concentration of polymerase or other protein. In some embodiments, a wash buffer may include up to around 0.4pg / pL or more of polymerase or other protein. In some embodiments, any wash step that includes detergents, proteinase K, and similar treatments that could disrupt the coating of denatured protein on the chip would not be suitable for a selective elution protocol.

[0064] In other embodiments, the chip may be pre-treated prior to Xpandomer synthesis to alter the surface chemistry to favor selective elution, for example, to make the surface more hydrophobic. In certain embodiments, the surface chemistry may be altered to increase the density of cross linked maleimide-containing linkers.

[0065] Ionic strength. The concentration of salt in the elution buffer has been found to have a strong impact on Xpandomer size selectivity, with any salt present at a concentration of around 1 OpM or higher in the buffer favoring non-selective elution. Thus, elution buffers including a salt would not be suitable for selective elution protocols, but would be suitable for a protocol intended to maximize recovery of all Xpandomer species.

[0066] Xpandomer features - the leader sequence. As discussed herein, the leader sequence of the Xpandomer is a feature that includes a high density of negative charge. It has been observed that this negative charge favors selective elution of Xpandomer molecules. In certain embodiments, the leader sequence may comprise from around ten to around 150 C2 monomer units, each linked in-line by phosphodiester bonds. In some embodiments, the leader sequence may comprise around 25 C2 monomer units. In other embodiments, the leader sequence may comprise more than around 25 C2 monomer units to favor selective elution of Xpandomer molecules.

[0067] Xpandomer features - the concentrator sequence. As discussed herein, the concentrator sequence of the Xpandomer is a feature with lipophilic, or hydrophobic, properties. It has been observed that this hydrophobic feature favors selective elution of Xpandomer molecules. In certain embodiments, the concentrator sequence may comprise from around four to around ten Cl 2 monomer units, each linked in-line by phosphodi ester bonds. In some embodiments, the concentrator sequence may comprise around six Cl 2 monomer units. In other embodiments, the concentrator sequence may comprise more than around six C12 monomer units to favor selective elution of Xpandomer molecules.

[0068] In some embodiments, either the leader or the concentrator may include polymers of a suitable number of Cl, C2, C3, C4, C5, C6, C7, C8, C9, CIO, Cl l, C12, C13, C14, C15 or higher of carbon containing monomers.

[0069] Xpandomer features - spermine modification. As discussed herein, the SSRT features of the Xpandomer each comprise around four monomers of spermine. Following synthesis, the Xpandomer is treated with a solution of succinate anhydride to modify the positively charged amine groups in the spermine units into negatively charged groups. It has been observed that the degree of spermine modification can impact selective elution of Xpandomer molecules. In certain embodiments, Xpandomers are “under-modified” to favor selective elution. For example, the Xpandomers may be subjected to modification conditions that result in 50% or less of the constituent amines of spermine being modified into negatively charged groups. In other embodiments, other chemical modifiers may be used to convert the positively charged amines in spermine into negatively charged groups. For example, a solution of acetic anhydride may be used.

[0070] In other embodiments, any NHS / PFP ester or acid chloride / fluoride or carboxylic acid (plus activation agent) that could modify a secondary amine in a way that removes thepositive charge may be used as a modifier. In certain embodiments, the modifier may be one or more of NHS-PEGl-OMe, PFP-acetate, NHS methyl carbamate, unprotected glycolic NHS, Bis-PNP-carbonate, diglycolic anhydride, benzoic anhydride, mPEG5-NHS, adamantyl-NHS, trimellitic anhydride, glucuronic-NHS, beta butyrolactone, gamma butyrolactone, sulfo-NHS -acetate, EOM-protected glycolic-NHS, TBS-protected glycolic- NHS, protected gluconic-NHS, protected PEG2-NHS, EOM protected gluconic-NHS, TBS- protected glycolic-PFP, and glycolic-PFP.

[0071] Chip environment - temperature. It has been observed that the temperature of the various Xpandomer synthesis steps can impact selective elution. In general, lower temperatures have been found to favor selective elution of Xpandomer molecules. In certain embodiments, the temperatures of the post-extension wash step, the Xpandomer cleavage step, the neutralization step, the modification step, and the post-modification wash step may be from around 23 to around 30 degrees Celsius. In certain embodiments, the temperature of the elution step may be around 37 degrees Celsius for around from two to around five minutes.

[0072] Chip environment - flow rate. In certain embodiments, the volume of the elution step may be minimized to favor selective elution of Xpandomer molecules. In some embodiments, the volume of the elution step is around from around I OpL to around I OOpL, from around 20pL to around 60pL, or around 55pL and the flow rate is maintained at a level that does not impair size selectivity of Xpandomer elution. For example, increasing the flow rate during the elution step is predicted to make the chip environment more energetic and favor non-selective elution. In some embodiments, the flow rate may be optimized by manipulating the fluidics of the system, for example, by generating turbulence in the flow channel. In some embodiments, air bubbles may be introduced into the stream of liquid, e.g., elution buffer to slow down the rate of flow. In some embodiments a suitable rate of liquid flow may be from around 5pL / minute to around lOOpL / minute, from around1 OpL / minute or over to around 75pL / minute or under, or around 30pL / minute.

[0073] Further Features of the System for Automated Synthesis

[0074] FIG. 3 illustrates an embodiment of the mount 300 for receiving the flow cell 200. As shown, the mount 300 can receive two flow cells 200. In other embodiments, the mount can receive 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 flow cells. A thermal block 302 is thermally coupled to the mount 300 and is used to control the temperature of the flow cell. Thethermal block 302 can be quickly cooled or heated as desired so that the reactions in the flow cell can be performed at temperatures between about 20 Celsius and 60 Celsius. In other embodiments, the temperature range can between about 5 Celsius and 90 Celsius.

[0075] As shown in FIG. 3, the thermal block 302 can include fins to radiate heat and a fan to aid in heat transfer from the thermal block. The thermal block 302 can be thermally attached to the mount 300 or can be integrally formed with the mount 300. For example, one face of the thermal block 302 can be formed to receive the flow cells 200. In some embodiments, the controller can provide real-time thermal control of the thermal block.

[0076] In some embodiments, a thermal pad can be placed between the mount 300 and flow cell 200 to improve heat transfer from the thermal block 302 to the flow cell 200. In some embodiments, the thermal pad can be placed on or pre-attached to the mount. In other embodiments, the thermal pad can be place on or pre-attached to the base of the flow cell 200. In some embodiments, a thermal paste or gel can be used instead of a thermal pad.

[0077] In some embodiments, a portion of the sipper 224 can be disposed against the thermal block 302 or mount 300 in order preheat the fluids as they are drawn into the flow cell 200. In some embodiments, the portion of the sipper 224 that is disposed against the thermal block 302 or mount 300 can be serpentine.

[0078] In some embodiments, the flow cell 200 can be mounted on the mount 300 such that the flow cell 200 is oriented in a vertical direction, such that the sipper 224 is pointed towards the reagents in the holder 400, as shown in FIG. 1A. Depending on the orientation and configuration of the inlet port 220 on the flow cell 200, the sipper 224 can include a bend, such as a 90 degree bend or other angle if needed, to orient the inlet of the sipper 224 towards the reagents. It is advantageous to use the vertical orientation of the flow cell 200 so that the holder 400 can be oriented horizontally such that open reagent containers maintain the fluids within the container with gravity.

[0079] However, in other embodiments, both the flow cell 200 and the holder 400 can be oriented horizontally, and the sipper 224 can include a 90 degree bend or a 180 degree bend, for example, depending on the orientation of the inlet port 220 on the flow cell. In some embodiments, if the inlet port 220 is also oriented horizontally in line with the plane encompassing the flow channels 202, then the sipper 224 can include a 90 degree bend to direct the inlet of the sipper 224 towards the reagents. Similarly, if the inlet port 220 is oriented normally instead, the sipper 224 can have a 180 degree bend.

[0080] In some embodiments, when the flow cell 200 has more than one flow channel and more than one sipper, the spacing between the sippers 224 after being secured to the mount 300 is compatible for use with 96 well plates, which allows the reagents and / or sample to be stored in a 96 well plate, which can be loaded onto the holder 400.

[0081] FIG. 4A illustrates an embodiment of a holder 400. The holder 400 can hold reagents, samples, buffers, and other liquids used in the synthesis method. As shown, a 96 well plate 402 (or plate with another well count) and vial or tube holder 404 can be included in the holder 400. Reagent trays, bottles, and other containers for holding liquids can also be secured to the holder 400. The openings of the containers, such as the openings of the tubes in the tube holder 404 and the wells of the 96 well plate 402 can all be located on the same plane or height. This makes it easier for a covering to be applied over the openings and pierced when needed with a piercing tool 406. Height adapters can be used if needed to adjust the heights of openings so that they are level. Alternatively, custom vial or tube racks and well plates with preadjusted heights can be used with the holder 400 to simplify user operation and reduce user error. The liquid holders can be removably attached to the holder 400 to allow for user customization.

[0082] The holder 400 can be moved in all three axes with the XYZ gantry 500, as shown in FIG. 1A. This configuration allows the flow cell 200 and sipper 224 and the piercing tool 406 to remain in a fixed position while the holder 400 is moved by the gantry 500 to the piercing tool 406 when the covering over the openings needs to be pierced and then to the sippers 224 once the coverings have been pierced. To access different reagents and samples, the gantry 500 can move the holder away from the sippers 224 and then align the sippers 224 with the new reagents and / or samples. One benefit from holding the flow cell 200 in a fixed position is that it reduces the movement of wiring and tubing attached to the flow cell, thermal block, UV source, and other associated components. Repeated movement of these components may cause disconnects or damage to these sensitive components, and therefore, it may be beneficial to adopt a configuration where these components remain stationary.

[0083] In other embodiments, the gantry 500 can be used to move the flow cell 200 and piercing tool 406 while the regent holder and reagents are stationary. This can be accomplished by attaching the mount 300 and flow cell 200 to the gantry 500, while the holder can remain fixed in place on the deck of the instrument. The UV source can stilloptionally remain fixed in one location since the flow cell 200 can be moved in position in front of the UV source when needed.

[0084] FIGS. 4B and 4C illustrate a reagent cartridge 410 that can be loaded onto the holder 400. The reagent cartridge can include a plurality of different types of liquid reservoirs 412 of different sizes and / or shape as well as a cover 414 that can be used to seal the reagent cartridge. In some embodiments, the gantry 500 can move the reagent cartridge 410 and press the reagent cartridge against the cover 414, which can be held in a fixed horizontal position in order to seal reagent cartridge. In some embodiments, the reagent cartridge 410 can also include drop in locations to receive tubes 416 or vials (e.g., Eppendorf tubes). Although described as tubes 416, other containers or reservoirs can be used instead to hold the liquids and reagents. In some embodiments, these tubes can be provided by the manufacturer to contain premade Library or master mix solutions. In addition, a tube can be used to collect the synthesized molecule.

[0085] The gantry 500 can also be optionally used with the sippers 224 or a separate mixing tube 700 to perform liquid mixing and / or bubble mixing operations on the reagent cartridge if desired, as shown in FIG. 1C. The mixing tube 700 can be attached to a dedicated pump 600 and can optionally have an internal volume that is sufficient to aspirate all the fluid in the tube 416 that needs to be mixed. In some embodiments, to achieve the internal volume needed, the mixing tube 700 can optionally have a coiled portion 702 to accommodate a relatively long length of tubing in a small space within the chassis of the device. The inlet of the mixing tube 700 can be fixed to a specific location within the chassis of the device so that the gantry can move tubes 416 to the inlet of the mixing tube 700 when needed.

[0086] In some embodiments, certain reagents that are unstable in water (e.g. succinic anhydride, acetic anhydride, other anhydrides) and need to be used relatively quickly after being formed into a solution can be provided in dry form within a tube or vial. Just before use of these sensitive reagents, a liquid such as water or a buffer can be added to the vial and a mixing procedure, such as bubble mixing, can be performed to dissolve the reagent. Bubble mixing can be performed by inserting a sipper or pipette tip into the liquid and introducing a gas, such as air, into the liquid to agitate the liquid.

[0087] In some embodiments, air gaps can optionally be introduced between the different fluids being introduced into flow cell as part of the Xpandomer synthesis process. Alternatively, an immiscible fluid can be introduced between the different fluids beingintroduced into the flow cell. Separation of the different fluids and reagents going into the flow cell prevents or reduces reactions from occurring outside the target area in the flow cell where the Xpandomer synthesis reactions are occurring. In some embodiments, the channels in the flow cell are relatively narrow in diameter so that the bubbles or immiscible fluid completely fill the diameter of the flow channels so that fragments of the bubbles or the immiscible fluid do not become trapped within the flow cell. In some embodiments, the air gaps or immiscible fluid can be introduced through the sippers that are used to draw reagents into the flow cell. For example, the sipper can draw in the reagent, then be lifted out of the reagent into the air, and then draw in a bolus of air.

[0088] In other embodiments, a train or plurality of bubbles can be introduced into the flow cell, sippers, and other components in order to help clean the components. For example, bubbles can be introduced between small plugs of reagents, wash buffer, or other fluids to help with surface clean up and / or to help control elution of the Xpandomer from the flow cell.

[0089] When a feature or element is herein referred to as being “on” another feature or element, it can be directly on the other feature or element or intervening features and / or elements may also be present. In contrast, when a feature or element is referred to as being “directly on” another feature or element, there are no intervening features or elements present. It will also be understood that, when a feature or element is referred to as being “connected”, “attached” or “coupled” to another feature or element, it can be directly connected, attached or coupled to the other feature or element or intervening features or elements may be present. In contrast, when a feature or element is referred to as being “directly connected”, “directly attached” or “directly coupled” to another feature or element, there are no intervening features or elements present. Although described or shown with respect to one embodiment, the features and elements so described or shown can apply to other embodiments. It will also be appreciated by those of skill in the art that references to a structure or feature that is disposed “adjacent” another feature may have portions that overlap or underlie the adjacent feature.

[0090] Terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. For example, as used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms “comprises”and / or “comprising,” when used in this specification, specify the presence of stated features, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components, and / or groups thereof. As used herein, the term “and / or” includes any and all combinations of one or more of the associated listed items and may be abbreviated as “ / ”.

[0091] Spatially relative terms, such as “under”, “below”, “lower”, “over”, “upper” and the like, may be used herein for ease of description to describe one element or feature’s relationship to another element(s) or feature(s) as illustrated in the figures. It will be understood that the spatially relative terms are intended to encompass different orientations of the device in use or operation in addition to the orientation depicted in the figures. For example, if a device in the figures is inverted, elements described as “under” or “beneath” other elements or features would then be oriented “over” the other elements or features. Thus, the exemplary term “under” can encompass both an orientation of over and under. The device may be otherwise oriented (rotated 90 degrees or at other orientations) and the spatially relative descriptors used herein interpreted accordingly. Similarly, the terms “upwardly”, “downwardly”, “vertical”, “horizontal” and the like are used herein for the purpose of explanation only unless specifically indicated otherwise.

[0092] Although the terms “first” and “second” may be used herein to describe various features / elements (including steps), these features / elements should not be limited by these terms, unless the context indicates otherwise. These terms may be used to distinguish one feature / element from another feature / element. Thus, a first feature / element discussed below could be termed a second feature / element, and similarly, a second feature / element discussed below could be termed a first feature / element without departing from the teachings of the present invention.

[0093] Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising” means various components can be co-jointly employed in the methods and articles (e.g., compositions and apparatuses including device and methods). For example, the term “comprising” will be understood to imply the inclusion of any stated elements or steps but not the exclusion of any other elements or steps.

[0094] As used herein in the specification and claims, including as used in the examples and unless otherwise expressly specified, all numbers may be read as if prefaced by the word“about” or “approximately,” even if the term does not expressly appear. The phrase “about” or “approximately” may be used when describing magnitude and / or position to indicate that the value and / or position described is within a reasonable expected range of values and / or positions. For example, a numeric value may have a value that is + / - 0.1% of the stated value (or range of values), + / - 1% of the stated value (or range of values), + / - 2% of the stated value (or range of values), + / - 5% of the stated value (or range of values), + / - 10% of the stated value (or range of values), etc. Any numerical values given herein should also be understood to include about or approximately that value, unless the context indicates otherwise. For example, if the value “10” is disclosed, then “about 10” is also disclosed. Any numerical range recited herein is intended to include all sub-ranges subsumed therein. It is also understood that when a value is disclosed that “less than or equal to” the value, “greater than or equal to the value” and possible ranges between values are also disclosed, as appropriately understood by the skilled artisan. For example, if the value “X” is disclosed the “less than or equal to X” as well as “greater than or equal to X” (e.g., where X is a numerical value) is also disclosed. It is also understood that the throughout the application, data is provided in a number of different formats, and that this data, represents endpoints and starting points, and ranges for any combination of the data points. For example, if a particular data point “10” and a particular data point “15” are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are considered disclosed as well as between 10 and 15. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0095] Although various illustrative embodiments are described above, any of a number of changes may be made to various embodiments without departing from the scope of the invention as described by the claims. For example, the order in which various described method steps are performed may often be changed in alternative embodiments, and in other alternative embodiments one or more method steps may be skipped altogether. Optional features of various device and system embodiments may be included in some embodiments and not in others. Therefore, the foregoing description is provided primarily for exemplary purposes and should not be interpreted to limit the scope of the invention as it is set forth in the claims.

[0096] The examples and illustrations included herein show, by way of illustration and not of limitation, specific embodiments in which the subject matter may be practiced. As mentioned, other embodiments may be utilized and derived there from, such that structural and logical substitutions and changes may be made without departing from the scope of this disclosure. Such embodiments of the inventive subject matter may be referred to herein individually or collectively by the term “invention” merely for convenience and without intending to voluntarily limit the scope of this application to any single invention or inventive concept, if more than one is, in fact, disclosed. Thus, although specific embodiments have been illustrated and described herein, any arrangement calculated to achieve the same purpose may be substituted for the specific embodiments shown. This disclosure is intended to cover any and all adaptations or variations of various embodiments. Combinations of the above embodiments, and other embodiments not specifically described herein, will be apparent to those of skill in the art upon reviewing the above description.

Claims

- 26 -CLAIMSWhat is claimed is:

1. A method for size-selective elution of a sample of Xpandomer molecules from a substrate, comprising the steps of: a. providing a substrate comprising a plurality of Xpandomer molecules, wherein each Xpandomer molecule is covalently joined to the substrate by an extension oligonucleotide polymer, wherein the extension oligonucleotide polymer comprises a photocleavable moiety and an oligonucleotide primer, wherein the substrate further comprises a coating, and wherein the coating is capable of non-covalent interactions with the Xpandomer molecules; b. contacting the substrate with a first elution buffer, wherein the first elution buffer comprises an organic co-solvent and a stabilizing agent; c. contacting the substrate with UV light, wherein the UV light cleaves the photocleavable moiety; and d. eluting a sample of Xpandomer molecules from the substrate, wherein the eluted sample comprises Xpandomer molecules with an average length greater than the average length of non-eluted Xpandomers.

2. The method of claim 1, wherein the non-eluted Xpandomer molecules are retained on the coating by the non-covalent interactions.

3. The method of claim 1 or 2, wherein the organic co-solvent comprises acetonitrile present in an amount of greater than 10% (v / v) to less than 20% (v / v).

4. The method of claim 3, wherein the acetonitrile is present in an amount of around 15% (v / v) to around 16% (v / v).

5. The method of any one of claims 1 to 4, wherein the stabilizing agent comprises trehalose.

6. The method of claim 5, wherein the trehalose is present in an amount of around 0.5% (w / v) to around 2.0% (w / v).

7. The method of claim 5, wherein the trehalose is present in an amount of around 1% (w / v).

8. The method of any one of claims 1 to 7, wherein the first elution buffer comprises 15% (v / v) acetonitrile and 1% (w / v) trehalose.

9. The method of any one of claims 1 to 8, wherein the substrate is contacted with UV light for around two minutes.

10. The method of any one of claims 1 to 9, wherein the substrate is contacted with UV light at a temperature of around 37 degrees Celsius.

11. The method of any one of claims 1 to 10, wherein the coating comprises denatured protein.

12. The method of claim 11, wherein the denatured protein comprises denatured nucleic acid polymerase protein or denatured single stranded binding protein.

13. The method of claim 12, wherein the denatured nucleic acid polymerase protein is provided by an Xpandomer synthesis termination step, wherein the Xpandomer synthesis termination step precedes the step of contacting the substrate with the first elution buffer, wherein the Xpandomer synthesis termination step comprises incubating an Xpandomer synthesis reaction at a temperature of around 45 to around 60 degrees Celsius for around five to around 20 minutes.

14. The method of any one of claims 1 to 13, wherein the step of providing the substrate comprising a plurality of Xpandomer molecules comprises the ordered steps of: a) providing a substrate comprising a surface functionalized with a polymer comprising a maleimide moiety on a first end and an alkyne moiety on a second end; b) covalently joining the extension oligonucleotide to the polymer, wherein the extension oligonucleotide further comprise a terminal azide moiety capable of forming a covalent bond with the alkyne moiety; c) contacting the extension oligonucleotide with a nucleic acid template under nucleic acid hybridization conditions to provide a hybridized nucleic acid template complex; d) contacting the hybridized nucleic acid template complex with an Xpandomer synthesis reaction mixture under Xpandomer synthesis conditions to provide a plurality of Xpandomer molecules covalently joined to the substrate.

15. The method of claim 14, wherein the Xpandomer synthesis reaction mixture comprises a nucleic acid polymerase protein.

16. The method of claim 15, further comprising the step of: e) providing Xpandomer synthesis termination conditions, wherein the Xpandomer synthesis termination conditions comprise incubating the substrate at a temperature of around 45 to around 60 degrees Celsius for around five to around 20 minutes.

17. The method of any one of claims 1 to 16, wherein the extension oligonucleotide further comprises a leader sequence and a concentrator sequence.

18. The method of claim 17, wherein the leader sequence is a polymer comprising 2 carbon monomers and the concentrator sequence is a polymer comprising 12 carbon monomers.

19. The method of claim 18, wherein the leader sequence comprises from around ten to around 1502 carbon monomers.

20. The method of claim 17 or 18, wherein the concentrator sequence comprises from around four to around ten 12 carbon monomers.

21. The method of any one of claims 1 to 20, wherein the Xpandomer molecules comprise a plurality of spermine moieties, wherein the spermine moieties comprise positively charged amine groups.

22. The method of claim 21, further comprising a spermine modification step prior to the step of contacting the substrate with UV light, wherein the spermine modification step comprises contacting the Xpandomer molecules with a buffer comprising an anhydride compound.

23. The method of claim 22, wherein the spermine modification step converts up to 50% of the positively charged amine groups to negatively charged hemi acetyl groups.

24. The method of any one of claims 1 to 23, wherein the step of eluting a size-selected sample of Xpandomers from the substrate comprises collecting the sample of size-selected Xpandomers in a tube.

25. The method of any one of claims 1 to 24, further comprising the steps of contacting the substrate with a second elution buffer, wherein the second elution buffer comprises an organic co-solvent, a stabilizing agent, and a buffer and collecting a non-size selected sample of Xpandomer molecules.

26. A method for non-selective elution of a sample of Xpandomer molecules comprising the method of claim 1 followed by the steps of contacting the substrate with a second elution buffer, wherein the second elution buffer comprises an organic co-solvent, a stabilizing agent, and a buffer and collecting a non-size selected sample of Xpandomer molecules.

27. The method of claim 25 or 26, wherein the second aqueous elution buffer further comprises a salt.

28. The method of claim 25 or 26, wherein the second aqueous elution buffer comprises around ImM HEPES.

29. The method of any one of claims 25 to 28, wherein the organic co-solvent comprises acetonitrile present in an amount of around 20% (v / v).- 29 -30. An elution buffer for collecting a size selected sample of Xpandomer molecules comprising around 15% (v / v) acetonitrile and around 1% (w / v) trehalose.

31. An elution buffer for collecting a non-size selected sample of Xpandomer molecules comprising from around 15% (v / v) to around 20% (v / v) or over acetonitrile, around 1% (w / v) trehalose, and around ImM HEPES buffer pH 7.4.

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