(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(19) World Intellectual Property
`Organization
`International Bureau
`
`(43) International Publication Date
`08 February 2018 (08.02.2018)
`
`Z=
`
`WIPOIPCT
`
`MURAI ARA UAAAT
`
`(10) International Publication Number
`WO 2018/026920 Al
`
`(51) International Patent Classification:
`CI2N15/10 (2006.01)
`C40B 50/14 (2006.01)
`CI2N15/66 (2006.01)
`C40B 50/18 (2006.01)
`C40B 50/00 (2006.01)
`
`(US). MARSH,Eugene,P.; 147 Sonora Avenue, El Grana-
`da, CA 94018 (US). BANYAI, William; 738 Wayland
`Street, San Francisco, CA 94134 (US). PECK,Bill, J.; 3086
`Carleton Place, Santa Clara, CA 95051 (US).
`
`(21) International Application Number:
`
`PCT/US2017/045105
`
`(74)
`
`Agent: HARBURGER,David; Wilson Sonsini Goodrich
`& Rosati, 650 Page Mill Road, Palo Alto, CA 94304 (US).
`
`(22) International Filing Date:
`
`(25) Filing Language:
`
`(26) Publication Language:
`
`02 August 2017 (02.08.2017)
`
`English
`
`English
`
`(30) Priority Data:
`62/370,548
`
`03 August 2016 (03.08.2016)
`
`US
`
`(71) Applicant: TWIST BIOSCIENCE CORPORATION
`[US/US]; 455 Mission Bay Boulevard South, Suite 545, San
`Francisco, CA 94158 (US).
`
`(81) Designated States (unless otherwise indicated, for every
`kind of national protection available): AE, AG, AL, AM,
`AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, BZ,
`CA, CH, CL, CN, CO, CR, CU, CZ, DE, DJ, Dk, DM, DO,
`DZ, EC, EE, EG, ES, FL GB, GD, GE, GH, GM, GT, HN,
`HR, HU,ID,IL, IN,IR, IS, JO, JP, KE, KG, KH, KN, KP,
`KR, KW, KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME,
`MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI NO, NZ,
`OM,PA,PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA,
`SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN,
`TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW.
`
`(72) Inventors: FERNANDEZ, Andres; 400 Beale Street,
`#1406, San Francisco, CA 94105 (US). INDERMUHLE,
`Pierre, F.; 1817 A Orcgon Strect, Berkeley, CA 94703
`
`(84) Designated States (unless otherwise indicated, for every
`kind of regional protection available): ARIPO (BW, GH,
`GM,KE, LR, LS, MW, MZ, NA, RW,SD,SL, ST, SZ, TZ,
`
`(54) Title: TEXTURED SURFACES FOR POLYNUCLEOTIDE SYNTHESIS
`
`
`
`
`
`310
`
`
`
`_
` 300
`
`_
`
`
`
`
`
`
`
`
`
`
`
`340.
`
`
`
`330
`
`343
`
`
`c—
`
`ye
`
`PECEEECREEEDEEES
`
`
`— i
`
`FIG, 38
`
`(57) Abstract: Methods, devices and systems are provided herein for surfaces for de novo polynucleotide synthesis that provide for
`increased polynucleotide yield. Surfaces described herein comprise a texture that increases surface area provide for increased polynu-
`cleotide yield compared to non-textured surfaces. In addition, the patterned placement of nucleoside coupling reagent spanning such
`surfaces provides for improved synthesis yield, representation, and a reduction in contamination on the surface between different
`polynucleotide species.
`
`[Continued on nextpage]
`
`
`
`wo2018/0269201IIITIMNININNMNNINNAUTENA
`
`

`

`WO 2018/026920 AL IIMIMAIENIIMMINMTUMIL MUMIA ATAU AMM
`
`UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ,
`TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK,
`EE, ES, FI, FR, GB, GR, HR, HU,IE,IS, IT, LT, LU,LV,
`MC, MK,MT,NL, NO,PL, PT, RO,RS,SE, SI, SK, SM,
`TR), OAPI (BF, BJ, CF, CG, CL, CM, GA, GN, GQ, GW,
`KM,ML, MR, NE,SN, TD,TG).
`
`Declarations under Rule 4.17:
`
`— as to applicant's entitlement to apply for and be granted a
`patent (Rule 4.17(ii))
`— as to the applicant's entitlement to claim the priorityofthe
`earlier application (Rule 4.17(iii))
`Published:
`
`— with international search report (Art. 21(3))
`
`

`

`WO2018/026920
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`PCT/US2017/045105
`
`TEXTURED SURFACES FOR POLYNUCLEOTIDE SYNTHESIS
`
`CROSS-REFERENCE
`
`[0001]
`
`This application claims the benefit of U.S. Provisional Application No. 62/370,548,
`
`filed August 3, 2016, which application is incorporated herein by reference in its entirety.
`
`INCORPORATION BY REFERENCE
`
`[0002]
`
`All publications, patents, and patent applications mentionedin this specification are
`
`herein incorporated by reference to the same extent as if each individual publication, patent, or
`
`patent application wasspecifically and individually indicated to be incorporated by reference.
`
`BACKGROUND
`
`[0003]
`
`Highly efficient chemical gene synthesis with high fidelity and low cost has a central
`
`role in biotechnology and medicine, and in basic biomedical research. De novo gene synthesis is a
`
`powerful tool for basic biological research and biotechnology applications. While various methods
`
`are known for the synthesis of relatively short fragments in a small scale, these techniques suffer
`
`from scalability, automation, speed, accuracy, and cost. There is a need for devices for simple,
`
`reproducible, scalable, less error-prone and cost-effective methods that guarantee successful
`
`synthesis of desired genes and are amenable to automation.
`
`BRIEF SUMMARY
`
`[0004]
`
`Provided herein is a device for polynucleotide synthesis, the device comprising: a solid
`
`support comprising a surface; a plurality of loci on the surface, wherein each of the loci comprises:
`
`an inner region, wherein the inner region comprises a plurality of recesses or protrusions; and an
`
`outer region that comprisesa plurality of first molecules, wherein the outer region spans and
`
`extends beyond the inner region, and wherein each ofthe first molecules binds to the surface and
`
`comprises a reactive group capable of binding to a nucleoside. Further provided herein is a device
`
`wherein the plurality of loci are arranged in clusters. Further provided herein is a device wherein
`
`each cluster comprises 50 to 500 loci. Further provided herein is a wherein each cluster comprises
`
`about 121 loci. Further provided herein is a device wherein the outer region has a diameter of up to
`
`100 um. Further provided herein is a device wherein the outer region has a diameter of about 60
`
`um. Further provided herein is a wherein the inner region has a diameter of about 55 um. Further
`
`provided herein is a device wherein the inner region has a diameter 80% to 95% shorter than the
`
`diameter of the outer region. Further provided herein 1s a device wherein the inner region has a
`
`diameter 2 um to 20 um shorter than the diameter of the outer region. Further provided herein is a
`
`device wherein the inner region has a diameter about 5 um shorter than the diameter of the outer
`
`1
`
`

`

`WO 2018/026920
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`PCT/US2017/045105
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`region. Further provided herein is a device wherein each of the recesses or protrusions have an etch
`
`depth of 100 um to 1000 nm. Further provided herein is a device wherein each of the recesses or
`
`protrusions has an etch depth of 200 um to 500 nm. Further provided herein is a device wherein
`
`each of the recesses or protrusions has a width of 100 to 500 um. Further provided herein is a
`
`device wherein each of the recesses or protrusions has a width of 300 to 330 um. Further provided
`
`herein is a device wherein each of the recesses or protrusionshasa pitch length of about 2 to 3
`
`times a width of the recesses or protrusions. Further provided herein is a device wherein each of
`
`the recesses or protrusions has a depth of about 60%to 125% of a pitch length. Further provided
`
`herein is a wherein each of the recesses or protrusions has a patch of up to 1 um. Further provided
`
`herein is device a wherein the solid support has a tensile strength of 1 MPa to 300 MPa. Further
`
`provided herein is a device wherein the solid support has a tensile strength of 1 MPa to 10 MPa.
`
`Further provided herein is a device wherein the solid support has a stiffness of 1 GPa to 500 GPa.
`
`Further provided herein is a device wherein the solid support has a stiffness of 1 GPa to 10 GPa.
`
`Further provided herein is a device wherein the solid support comprises nylon, nitrocellulose, or
`
`polypropylene. Further provided herein is a device wherein the solid support comprises silicon,
`
`silicon dioxide, silicon nitride, polytetrafluoroethylene, polypropylene, polystyrene, polycarbonate,
`
`gold, or platinum. Further provided herein is a device wherein each ofthe first molecules is a
`
`silane. Further provided herein is a device wherein the silane is an aminosilane. Further provided
`
`herein is a device wherein each ofthe first molecules is N-(3-triethoxysilylpropyl)-4-
`
`hydroxybutyramide (HAPS), 11-acetoxyundecyltriethoxysilane, n-decyltriethoxysilane, (3-
`
`aminopropyl)trimethoxysilane, (3-aminopropy])triethoxysilane, 3-
`
`glycidoxypropyltrimethoxysilane, 3-iodo-propyltrimethoxysilane, or octylchlorosilane. Further
`
`provided herein is a device comprising a plurality of second molecules, wherein plurality of second
`
`molecules is located on the surface in a region surrounding the outer region of each of the loci, and
`
`wherein each second molecule binds to the surface and lacks a reactive group capable of binding to
`
`the nucleoside. Further provided herein is a device wherein the second molecule is a fluorosilane.
`
`Further provided herein is a device wherein the fluorosilaneis (tridecafluoro-1,1,2,2-
`
`tetrahydrooctyl)trichlorosilane, perfluorooctyltrichlorosilane, perfluorooctyltriethoxysilane, or
`
`perfluorooctyltrimethoxychlorosilane.
`
`[0005]
`
`Provided herein is a method for polynucleotide synthesis, comprising:
`
`providing predetermined sequences for polynucleotides; providing the device of any one of claims
`
`1 to 27; and synthesizing the polynucleotides. Further provided herein is a method wherein the
`
`polynucleotides comprise at least 30,000 non-identical polynucleotides.
`
`. Further provided herein
`
`is a method wherein the at least 30,000 non-identical polynucleotides encode for at least 750 genes.
`
`

`

`WO 2018/026920
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`PCT/US2017/045105
`
`. Further provided herein is a method wherein the at least 30,000 non-identical polynucleotides
`
`have an aggregate error rate of less than 1 in 1000 bases comparedto the predetermined sequences
`
`for polynucleotides. Further provided herein is a method wherein the at least 30,000 non-identical
`
`polynucleotides have an aggregate error rate of less than 1 in 1500 bases compared to the
`
`predetermined sequences for the polynucleotides. Further provided herein is a method wherein at
`
`least 80% of at least 30,000 non-identical polynucleotides have no errors compared to the
`
`predetermined sequencesfor the polynucleotides. Further provided herein is a method wherein at
`
`least 89% of at least 30,000 non-identical polynucleotides have no errors compared to the
`
`predetermined sequencesfor the polynucleotides.
`
`[0006]
`
`Provided herein is a method for gene synthesis, comprising: providing predetermined
`
`sequences for polynucleotides; providing the device of any one of claims | to 27; synthesizing the
`
`polynucleotides; and assembling the polynucleotides to form a plurality of genes. Further provided
`
`herein is a method further comprising releasing the polynucleotides prior to step (d).
`
`[0007]
`
`Provided herein is a system for polynucleotide synthesis, the system comprising: a
`
`material deposition device comprising plurality of reagents for polynucleotide synthesis and a
`
`plurality of nozzles for depositing the plurality of reagents for polynucleotide synthesis; a computer
`
`for controlling the release of the plurality of reagents for polynucleotide synthesis from the plurality
`
`of nozzles; and the device of any one of claims 1 to 27 for synthesis of polynucleotides.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`FIGURE 1A depicts a 16 x 16 arrayof clusters of loci.
`
`FIGURE 1B depicts an arrangementofclusters of loci.
`
`FIGURE 1C depicts an exemplary arrangementof loci within a cluster of loci.
`
`FIGURE 2Adepicts a cross-section view of an exemplary textured locus comprising an
`
`[0008]
`
`[0009]
`
`[0010]
`
`[0011]
`
`array of raised texture features.
`
`[0012]
`
`FIGURE 2B depicts a cross-section view of an exemplary textured locus comprising an
`
`array of recessed texture features.
`
`
`
`[0013]
`
`[0014]
`
`[0015]
`
`[0016]
`
`[0017]
`
`printing.
`
`[0018]
`
`FIGURE 3A depicts a top view of an exemplary arrayof textured loci.
`
`FIGURE 3B depicts a top view an exemplarytextured locus.
`
`FIGURE 4Adepicts an exemplary device with oneside polished.
`
`FIGURE 4B depicts a processof lithographic printing on an exemplarydevice.
`
`FIGURE 4C recessed features of an exemplary device formed bythe lithographic
`
`FIGURE 4Dillustrates a process of oxidizing an exemplary device.
`
`

`

`WO 2018/026920
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`PCT/US2017/045105
`
`[0019]
`
`FIGURE 4Eillustrates a process of lithographic printing an exemplary device using a
`
`photosensitive lack.
`
`[0020]
`
`FIGURE 4Fillustrates a process of oxide etching an exemplary device to form a
`
`textured silicon surface with fiducial structures.
`
`
`
`[0021] FIGURE5illustrates an exemplary device having a patterned surface comprising loci
`
`coated with a molecule for coupling to a nucleoside, wherein the loci are surrounding by regions
`
`coated with an agent that does not couple a nucleoside.
`
`[0022]
`
`FIGURES 6A-6Fillustrates an exemplary method for generating a surface having a
`
`reduction in nucleoside-coupling agent density.
`
`FIGURE 6Aillustrates a process of cleaning an exemplary device with an oxygen
`
`FIGURE 6Billustrates a process of coated an exemplary device with a photosensitive
`
`FIGURE 6C illustrates a process of optically lithographing an exemplary device.
`
`FIGURE 6Dillustrates a process of depositing a first molecule on the exposed surfaces
`
`of an exemplary device.
`
`
`
`
`[0023]
`
`plasma.
`
`[0024]
`
`lack.
`
`[0025]
`
`[0026]
`
`[0027]
`
`[0028]
`
`FIGURE 6Eillustrates a process of stripping away the photosensitive lack.
`
`FIGURE 6Fillustrates a process of binding a third molecule to the surface of an
`
`exemplary device.
`
`[0029]
`
`FIGURE 7Aillustrates a region of a surface of an exemplary device for polynucleotide
`
`synthesis that is coated with a silane that binds the surface and couples nucleoside.
`
`[0030]
`
`FIGURE 7Billustrates a region of a surface of an exemplary device for polynucleotide
`
`synthesis coated with a mixture ofsilanes, one silane that binds the surface and couples
`
`polynucleotide, and anothersilane that binds the surface and does not couple to nucleoside.
`
`[003 1]
`
`FIGURES 8A-8G illustrates a method a method for generating a surface having a
`
`reduction in nucleoside-coupling agent density.
`
`[0032]
`
`FIGURE 8Aillustrates a process of cleaning an exemplary device with an oxygen
`
`plasma.
`
`[0033]
`
`FIGURE 8Billustrates a process of coating an exemplary device with a first chemical
`
`layer that binds the surface of the device and binds nucleoside.
`
`[0034]
`
`FIGURE 8Cillustrates a process of coating an exemplary device with a photosensitive
`
`lack.
`
`[0035]
`
`[0036]
`
`FIGURE 8Dillustrates a process of optically lithographing an exemplary device.
`
`FIGURE 8Eillustrates a process patterning ofthe first chemical layer.
`
`

`

`WO 2018/026920
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`PCT/US2017/045105
`
`[0037]
`
`FIGURE 8Fillustrates a process of depositing a second chemical layer, comprising a
`
`molecule that binds the surface and does not bind nucleoside on the surface of an exemplary
`
`device, per an embodimentof the disclosure herein
`
`[0038]
`
`FIGURE 8Gillustrates a process of stripping away the photosensitive lack.
`
`
`
`[0039] FIGURE9is a diagram demonstrating an exemplary process workflow from
`
`oligonucleic synthesis to gene shipment.
`
`[0040]
`
`FIGURE 10 illustrates an outline of an exemplary system for nucleic acid synthesis,
`
`including an polynucleotide synthesizer, a device (wafer), schematics outlining the alignment of the
`
`system elements in multiple directions, and exemplary setups for reagent flow.
`
`[0041]
`
`FIGURE 11 illustrates an exemplary phosphoramidite chemistry for oligonucleotide
`
`synthesis.
`
`[0042]
`
`[0043]
`
`[0044]
`
`[0045]
`
`FIGURE 12 illustrates an exemplary device having fiducial markings.
`
`FIGURE 13 illustrates another exemplary device having fiducial markings.
`
`FIGURE 14 illustrates an exemplary computer system.
`
`FIGURE 15 is a block diagram illustrating a first architecture of an exemplary
`
`computer system.
`
`
`[0046]
`
`FIGURE 16 is a diagram demonstrating an exemplary network configured to
`
`incorporate a plurality of computer systems, a plurality of cell phones and personal data assistants,
`
`and Network Attached Storage (NAS).
`
`[0047]
`
`FIGURE 17 is a block diagram of an exemplary multiprocessor computer system using
`
`a shared virtual address memoryspace.
`
`FIGURE 18 is an image of an exemplary textured microfluidic device.
`
`FIGURE 19 is another image of an exemplary textured microfluidic device.
`
`FIGURE 20is a close-up image of an exemplary textured microfluidic device.
`
`FIGURE 21 is a side-view of a slice of an exemplary textured microfluidic device.
`
`FIGURE 272Ais a scanning electron micrograph of an exemplary textured microfluidic
`
`[0048]
`
`[0049
`
`]
`
`[0050]
`
`[0051
`
`[0052
`
`]
`
`]
`
`device.
`
`[0053]
`
`FIGURE 272Bis a scanning electron micrograph of an exemplary textured microfluidic
`
`device.
`
`[0054]
`
`FIGURE 23Ais a low magnification image of an exemplary cluster of non-textured
`
`loci.
`
`[0055]
`
`[0056]
`
`loci.
`
`FIGURE 23Bis alow magnification image of an exemplary cluster of textured loci.
`
`FIGURE 24<Ais a high magnification image of an exemplary cluster of non-textured
`
`

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`WO 2018/026920
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`PCT/US2017/045105
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`[0057]
`
`[0058]
`
`[0059]
`
`[0060]
`
`FIGURE 24Bis a high magnification image of an exemplary cluster of textured loci.
`
`FIGURE 235Aillustrates an exemplary arrangement of outer-textured loci.
`
`FIGURE 25Billustrates an exemplary outer-textured locus.
`
`FIGURE 26Aillustrates a cross-section view of an exemplary outer-textured locus
`
`comprising arrangementof an array of raised texture features.
`
`[0061]
`
`FIGURE 26Billustrates a cross-section view of an exemplary outer-textured locus
`
`comprising an arrangementof an array of recessed texture features.
`
`FIGURES 27Ais a low magnification image of an exemplarycluster of outer-textured
`
`FIGURES 27B is a high magnification image of an exemplary cluster of outer-textured
`
`
`
`
`
`[0062]
`
`loci.
`
`[0063]
`
`loci.
`
`[0064]
`
`FIGURE 28illustrates an exemplary textured microfluidic device with a pattern of
`
`clusters of textured loci.
`
`[0065]
`
`FIGURES 29A-29D are images of droplets dispensed onto an exemplary textured
`
`microfluidic device patterns of clusters of textured loci consistent with the arrangement of FIG.28.
`
`[0066]
`
`FIGURE 29A depicts an image of 200 nL droplets dispensed onto an exemplary
`
`textured microfluidic device.
`
`[0067]
`
`FIGURE 29B depicts an image of 275 nL droplets dispensed onto an exemplary
`
`textured microfluidic device.
`
`[0068]
`
`FIGURE 29C depicts an image of 350 nL droplets dispensed onto an exemplary
`
`textured microfluidic device.
`
`[0069]
`
`FIGURE 29D depicts an image of 425 nL droplets dispensed onto an exemplary
`
`textured microfluidic device.
`
`[0070]
`
`FIGURES 30Ais a chart depicting the dropoutrates for a first exemplary device
`
`comprising textured loci, outer- textured loci and non-texturedloci.
`
`[0071]
`
`FIGURES 30Bis a chart depicting the dropout rates for a second exemplary device
`
`comprising textured loci, outer- textured loci and non-texturedloci.
`
`[0072]
`
`FIGURES 30C is a chart depicting the dropout rates for a third exemplary device
`
`comprising textured loci, outer- textured loci and non-texturedloci.
`
`[0073]
`
`FIGURES 30D is a chart depicting the dropout rates for a fourth exemplary device
`
`comprising textured loci, outer- textured loci and non-texturedloci.
`
`[0074]
`
`[0075]
`
`FIGURE 31Aillustrates an exemplary functionalized surface.
`
`FIGURE 31B displays BioAnalyzer data at five locations on the exemplary
`
`functionalized surface.
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`[0076]
`
`FIGURE 32displays BioAnalyzer data of surface extracted 100-mer oligonucleotides
`
`synthesized on an exemplary silicon oligonucleotide synthesis device.
`
`[0077]
`
`FIGURE 33 represents an exemplary sequence alignment, where “x” denotes a single
`
`base deletion, “star” denotes single base mutation, and “+” denotes low quality spots in Sanger
`
`sequencing.
`
`[0078]
`
`FIGURE 34 represents an exemplary sequence alignment, where “x” denotes a single
`
`base deletion, “star” denotes single base mutation, and “+” denotes low quality spots in Sanger
`
`sequencing.
`
`[0079]
`
`FIGURE 35is an exemplary histogram for oligonucleotides encoding for 240 genes,
`
`with the length of oligonucleotide as the x-axis and numberof oligonucleotide as the y-axis.
`
`[0080]
`
`FIGURE 36is an exemplary histogram for polynucleotides collectively encoding for a
`
`gene, with the length of oligonucleotide as the x-axis and numberof oligonucleotide as the y-axis.
`
`[0081]
`
`FIGURE 37Aillustrates exemplary plots for DNA thickness per device for
`
`polynucleotides of 30, 50, and 80-mers when synthesized a surface.
`
`[0082]
`
`FIGURE 37B and DNA massperdevice for polynucleotides of 30, 50, and 80-mers
`
`when synthesized a surface.
`
`[0083]
`
`FIGURE 38illustrates the deletion rate at a given index of synthesized oligonucleotides
`
`for various silane solutions.
`
`[0084]
`
`FIGURE 39 illustrates average deletion and insertion rates of various textured
`
`microfluidic devices at a depth of 500 um.
`
`[0085]
`
`FIGURE 40illustrates measured and expected yield enhancements of various
`
`exemplary textured microfluidic devices.
`
`[0086]
`
`FIGURE 41 illustrates deletion rates of four nucleic acid bases on exemplary textured
`
`microfluidic devices with different etch depths.
`
`[0087]
`
`FIGURE 42illustrates relative deletion rates by texture type of various exemplary
`
`textured microfluidic devices with different etch depths.
`
`[0088]
`
`FIGURE 43illustrates relative deletion rates by texture depth of various exemplary
`
`textured microfluidic devices with different etch depths.
`
`[0089]
`
`FIGURE 44illustrates insertion rates by base of four nucleic acid bases on exemplary
`
`textured microfluidic devices with different etch depths.
`
`[0090]
`
`FIGURE 45illustrates relative insertion rates by base texture type of exemplary
`
`textured microfluidic devices when compared to an untextured design.
`
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`WO 2018/026920
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`DETAILED DESCRIPTION
`
`[0091]
`
`The present disclosure provides systems, methods, devices for rapid parallel synthesis of
`
`polynucleotide libraries with low error rates. The oligonucleotide synthesis steps described herein
`
`are “de novo,” meaning that oligonucleotides are built one monomerat a time to form a polymer.
`
`During de novo synthesis of polynucleotides, the crowding of single stranded polynucleotides
`
`extending from a surface results in an increase in error rates. To reduce the frequency of crowding-
`
`related errors, methods are provided herein to reduce the density of nucleoside-coupling agent
`
`boundto specific regions of the surface. At the same time, to compensate for the reduced density of
`
`polynucleotides extending from a surface, methods are disclosed herein to increase surface area so
`
`as to increasethe yield of synthesized polynucleotides.
`
`Definitions
`
`[0092]
`
`Throughoutthis disclosure, numerical features are presented in a range format. It should
`
`be understood that the description in range format is merely for convenience and brevity and should
`
`not be construed as an inflexible limitation on the scope of any embodiments. Accordingly, the
`
`description of a range should be considered to have specifically disclosed all the possible subranges
`
`as well as individual numerical values within that range to the tenth of the unit of the lowerlimit
`
`unless the context clearly dictates otherwise. For example, description of a range such as from 1 to
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`6 should be considered to have specifically disclosed subranges such as from | to 3, from 1 to 4,
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`from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual values within that range,
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`for example, 1.1, 2, 2.3, 5, and 5.9. This applies regardless of the breadth of the range. The upper
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`and lowerlimits of these intervening ranges may independently be included in the smaller ranges,
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`and are also encompassed within the invention, subject to any specifically excluded limit in the
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`stated range. Wherethe stated range includes one or both of the limits, ranges excluding either or
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`both of those included limits are also included in the invention, unless the context clearly dictates
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`otherwise.
`
`[0093]
`
`The terminology used herein is for the purpose of describing particular embodiments
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`only andis not intended to be limiting of any embodiment. Asusedherein, the singular forms “a,”
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`“an” and “the” are intended to include the plural forms as well, unless the context clearly indicates
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`otherwise. It will be further understood that the terms “comprises” and/or “comprising,” when used
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`in this specification, specify the presence of stated features, integers, steps, operations, elements,
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`and/or components, but do not preclude the presence or addition of one or more other features,
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`integers, steps, operations, elements, components, and/or groups thereof. As used herein, the term
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`“and/or” includes any and all combinations of one or moreof the associated listed items.
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`[0094]
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`Unless specifically stated or obvious from context, as used herein, the term “about” in
`
`reference to a numberor range of numbersis understood to mean the stated number and numbers
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`+/- 10% thereof, or 10% below the lowerlisted limit and 10% abovethe higherlisted limit for the
`
`valueslisted for a range.
`
`[0095]
`
`Asused herein, the terms “preselected sequence”,
`
`ce
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`2
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`“predefined sequence” or
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`“predetermined sequence” are used interchangeably. The terms meanthat the sequenceof the
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`polymer is known and chosen before synthesis or assembly of the polymer. In particular, various
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`aspects of the invention are described herein primarily with regard to the preparation of nucleic
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`acids molecules, the sequence of the oligonucleotide or polynucleotide being known and chosen
`
`before the synthesis or assemblyof the nucleic acid molecules.
`
`[0001[
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`Provided herein are methods and compositions for production of synthetic (i.e. de novo
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`synthesized or chemically synthesizes) polynucleotides. The term oligonucleotide, oligo, and
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`polynucleotide are defined to be synonymousthroughout. Libraries of synthesized polynucleotides
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`described herein may comprise a plurality of polynucleotides collectively encoding for one or more
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`genes or gene fragments. In someinstances, the polynucleotide library comprises coding or non-
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`coding sequences. In someinstances, the polynucleotide library encodes for a plurality of cDNA
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`sequences. Reference gene sequences from which the cDNA sequences are based may contain
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`introns, whereas cDNA sequences exclude exons. Polynucleotides described herein may encode
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`for genes or gene fragments from an organism. Exemplary organismsinclude, without limitation,
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`prokaryotes (e.g., bacteria) and eukaryotes (e.g., mice, rabbits, humans, and non-human
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`primates).
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`In someinstances, the polynucleotide library comprises one or more polynucleotides,
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`each of the one or more polynucleotides encoding sequences for multiple exons. Each
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`polynucleotide within a library described herein may encodea different sequence,i.e., non-
`
`identical sequence. In someinstances, each polynucleotide within a library described herein
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`comprisesat least one portion that is complementary to sequence of another polynucleotide within
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`the library. Polynucleotide sequences described herein may be, unless stated otherwise, comprise
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`DNA or RNA.
`
`[0002[
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`Provided herein are methods and compositions for production of synthetic (i.e. de novo
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`synthesized) genes. Libraries comprising synthetic genes may be constructed by a variety of
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`methods described in further detail elsewhere herein, such as PCA, non-PCA gene assembly
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`methods or hierarchical gene assembly, combining (“stitching”) two or more double-stranded
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`polynucleotides to produce larger DNA units(i.e., a chassis). Libraries of large constructs may
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`involve polynucleotides that are at least 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70,
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`80, 90, 100, 125, 150, 175, 200, 250, 300, 400, 500 kb long or longer. The large constructs can be
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`bounded by an independently selected upper limit of about 5000, 10000, 20000 or 50000 base
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`pairs. The synthesis of any numberof polypeptide-segment encoding nucleotide sequences,
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`including sequences encoding non-ribosomal peptides (NRPs), sequences encoding non-ribosomal
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`peptide-synthetase (NRPS) modules and synthetic variants, polypeptide segments of other modular
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`proteins, such as antibodies, polypeptide segments from other protein families, including non-
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`coding DNA or RNA,such as regulatory sequences e.g. promoters, transcription factors, enhancers,
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`siRNA, shRNA, RNAi, miRNA,small nucleolar RNA derived from microRNA,or anyfunctional
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`or structural DNA or RNA unit of interest. The following are non-limiting examples of
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`polynucleotides: coding or non-coding regions of a gene or gene fragment, intergenic DNA,loci
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`(locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA,
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`ribosomal RNA,short interfering RNA (siRNA), short-hairpin RNA (shRNA), micro-RNA
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`(miRNA), small nucleolar RNA, ribozymes, complementary DNA (cDNA), which is a DNA
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`representation of mRNA, usually obtained by reverse transcription of messenger RNA (mRNA)or
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`by amplification; DNA molecules produced synthetically or by amplification, genomic DNA,
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`recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any
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`sequence, isolated RNA of any sequence, nucleic acid probes, and primers. cDNA encoding for a
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`gene or gene fragmentreferred to herein, may comprise at least one region encoding for exon
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`sequence(s) without an intervening intron sequence foundin the corresponding genomic sequence.
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`Alternatively, the corresponding genomic sequence to a cDNA maylack intron sequencein the first
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`place.
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`[0096]
`
`Unless otherwise stated, water contact angles mentioned herein correspond to
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`measurements that would be taken on uncurved, smooth, or planar equivalents of the surfaces in
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`question.
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`Clusters and Loci
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`[0097]
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`Provided herein is a device comprising a surface, wherein the surface is modified to
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`support polynucleotide synthesis at predetermined locations and with a resulting low errorrate, a
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`low dropoutrate, a high yield, and a high oligo representation. In some embodiments, the surface
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`comprisesa plurality of loci, wherein each locus comprisesa plurality of first molecules deposited
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`on the locus, wherein the first molecule binds to the surface and comprises a reactive group capable
`
`of binding to a nucleoside.
`
`[0098]
`
`The terms “locus” and “loci,” as used herein, refer to a single discrete active region, and
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`to a plurality of discrete active regions on the surface of the device, respectively, wherein the
`
`plurality of first molecules are deposited on said locus, and wherein the first molecule bindsto the
`
`surface and comprises a reactive group capable of binding to a nucleoside. In some embodiments,
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`the plurality of first molecule comprises one or a mixture of molecule(s), which bindsto the surface
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`and comprises a reactive group capable of binding to a nucleoside.
`
`[0099]
`
`Referring to FIGS. 1A to 1C, an exemplary device 100 provided herein comprises a
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`surface 101, wherein the surface 101 comprises a plurality of loci 110, wherein each locus 110
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`comprisesa plurality of first molecules 120, wherein the plurality of first molecules 120 comprise a
`
`high-energy molecule, and wherein the first molecule binds to the surface 101 and comprises a
`
`reactive group capable of binding to a nucleoside, to synthesize a single sequence polynucleotide.
`
`In this arrangement, the plurality of the first molecules 120 deposited on each locus 110 exhibit a
`
`higher surface energy than the surface 101 of the device, and wherein the variation in the surface
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`energy facilitates localization of droplets of a fluid onto the loci 110. In some embodiments,
`
`localization of droplets onto the loci 110 is altered by adjusting the pattern and geometry ofthe loci
`
`110. In someinstances, the high-energy molecules 120 on one locus 110 are capable of binding to
`
`the surface and comprise a reactive group capable of binding to a certain nucleoside to support the
`
`synthesis of a certain population of polynucleotides having a certain sequence, wherein thefirst
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`molecules on another locus 110 are capable of binding to the surface and comprise a reactive group
`
`capable of binding to a different nucleoside to support the synthesis of a different population of
`
`polynucleotides having a different sequence.
`
`[0100]
`
`In someinstances, the surface 101 of the device 100 comprisesa plurality of loci 110,
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`wherein the plurality of loci 110 are arranged into a plurality of clusters 140, wherein each cluster
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`140 comprises a plurality of loci 110. Referring to FIGS. 1A to 1C, the surface 101 of the device
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`100 comprises a rectilinear array of 16 columns and 16 rowsof cluste