Patent Application
20220064206
Biomolecules (e.g., nucleic acids) have applications in research, medicine, and information storage. However, there is a need for high-density, scalable, automated, highly accurate and highly efficient systems for generating biomolecules.
Provided herein are devices, methods, and compositions for synthesis of biomolecules.
Provided herein are methods for synthesizing a biomolecule comprising: (a) contacting at least one biomolecule monomer attached to a solid support with a protected biomolecule, wherein the protected biomolecule is configured to form a covalent bond with the at least one biomolecule to generate a protected biomolecule; (b) applying a voltage to a solvent in fluid communication with the protected biomolecule, wherein the voltage results in deprotection of a terminal biomolecule of the protected biomolecule, and wherein the voltage is delivered as at least 2 pulses; (c) repeating steps (a) and (b) to synthesize the biomolecule. Provided herein are methods for synthesizing a polynucleotide comprising: (a) contacting at least one nucleoside attached to a solid support with a protected nucleoside, wherein the protected nucleoside is configured to form a covalent bond with the at least one nucleoside to generate a protected polynucleotide; (b) applying a voltage to a solvent in fluid communication with the protected polynucleotide, wherein the voltage results in deprotection of a terminal nucleoside of the protected polynucleotide, and wherein the voltage is delivered as at least 2 pulses; (c) repeating steps (a) and (b) to synthesize the polynucleotide. Further provided herein are methods wherein the voltage is 0.5-2.5 volts. Further provided herein are methods wherein the voltage is about 2 volts. Further provided herein are methods wherein the total period of time for all pulses is less than 5 seconds. Further provided herein are methods wherein the total period of time for all pulses is less than 1 second. Further provided herein are methods wherein the pulse is 50-500 milliseconds. Further provided herein are methods wherein the pulse is no more than 50 milliseconds. Further provided herein are methods wherein the pulse is 1-50 milliseconds. Further provided herein are methods wherein the pulse is 1-100 microseconds. Further provided herein are methods wherein the voltage is delivered as at least 100 pulses. Further provided herein are methods wherein the voltage is delivered as 50-1000 pulses. Further provided herein are methods wherein the time between any two pulses is 10-2000 milliseconds. Further provided herein are methods wherein the time between any two pulses is 10-500 milliseconds. Further provided herein are methods wherein the polynucleotide is washed between pulses. Further provided herein are methods wherein the polynucleotide is not washed between pulses. Further provided herein are methods wherein at least one of the pulses is a positive voltage. Further provided herein are methods wherein at least one of the pulses is a positive voltage, and at least one of the pulses is a negative voltage. Further provided herein are methods wherein the negative voltage is −0.1 to −1.0 volts. Further provided herein are methods wherein the time between the at least one positive voltage and the at least one negative voltage is less than 10 milliseconds. Further provided herein are methods wherein the time between the at least one positive voltage and the at least one negative voltage is less than 1 millisecond. Further provided herein are methods wherein the solvent comprises a composition for electrochemical acid generation. Further provided herein are methods wherein the solvent comprises hydroquinone, benzoquinone, or a mixture thereof. Further provided herein are methods wherein the mixture of hydroquinone and benzoquinone is present in a 1:1 ratio to 10:1 ratio. Further provided herein are methods wherein the concentration of the mixture of hydroquinone and benzoquinone is 0.5-10 mM. Further provided herein are methods wherein the protected polynucleotide comprises an acid-cleavable protecting group. Further provided herein are methods wherein the protected nucleoside comprises a phosphoramidite or H-phosphonate. Further provided herein are methods wherein the protected nucleoside comprises a phosphate, and contacting comprises use of an enzyme having polymerase activity.
Provided herein are methods for synthesizing a biomolecule comprising: (a) providing a surface having (i) one or more electrodes proximal to the surface and (ii) one or more in-plane cathodes proximal to the surface, wherein the surface comprises a first plurality of protected biomolecules attached thereto; (b) energizing at least one electrode proximal to a first region of the surface to electrochemically generate a deprotection reagent, wherein the deprotection reagent deprotects at least some of the first plurality of biomolecules in the first region; (c) coupling at least one protected biomolecule to at least one deprotected biomolecule in the first region; and (d) repeating steps (a)-(c) to synthesize the biomolecule. Provided herein are methods for synthesizing a polynucleotide comprising: (a) providing a surface having (i) one or more electrodes proximal to the surface and (ii) one or more in-plane cathodes proximal to the surface, wherein the surface comprises a first plurality of protected polynucleotides attached thereto; (b) energizing at least one electrode proximal to a first region of the surface to electrochemically generate a deprotection reagent, wherein the deprotection reagent deprotects at least some of the first plurality of polynucleotides in the first region; (c) coupling at least one protected nucleoside to at least one deprotected polynucleotide in the first region; and (d) repeating steps (a)-(c) to synthesize the polynucleotide. Further provided herein are methods wherein the one or more electrodes comprise at least one anode. Further provided herein are methods wherein the surface comprises a second region comprising a second plurality of protected polynucleotides. Further provided herein are methods wherein the pitch distance between the first region and the second region is no more than 1 micron. Further provided herein are methods wherein the pitch distance between the first region and the second region is no more than 100 nm. Further provided herein are methods wherein no more than 1% of the second plurality of protected polynucleotides is deprotected. Further provided herein are methods wherein at least 99% of the first plurality of protected polynucleotides is deprotected. Further provided herein are methods wherein the voltage is delivered as at least 2 pulses. Further provided herein are methods wherein the first plurality of protected polynucleotides and the second plurality of protected polynucleotides comprise protecting groups capable of removal with acid. Further provided herein are methods wherein the voltage generates acid. Further provided herein are methods wherein the first region and the second region each comprise an addressable anode. Further provided herein are methods wherein a positive voltage is applied to the one or more electrodes, and no voltage is applied to the cathode. Further provided herein are methods wherein a positive voltage is applied to the one or more electrodes, and a negative voltage is applied to the cathode. Further provided herein are methods wherein the absolute voltage difference between the anode and cathode is no more than 2 volts.
Provided herein are devices comprising at least one addressable solid support, wherein the at least one solid support comprises: a base layer comprising silicon; an intermediate layer comprising a conductive material, wherein the intermediate layer is configured to produce an electrochemically generated reagent when energized with a voltage; and a top layer comprising an oxide, wherein the intermediate layer and the top layer are in fluid communication with a solvent, and wherein the intermediate layer is located between the base layer and the top layer, and the top layer is configured for the attachment of molecules, and wherein the solid support comprises a plurality of features. Further provided herein are devices wherein the top layer comprises a plurality of voids configured to allow fluid communication of the solvent with the intermediate layer. Further provided herein are devices wherein at least some of the voids are centered above one or more features. Further provided herein are devices wherein the voids comprise wells or channels. Further provided herein are devices wherein the wells or channels are 1-500 nm deep. Further provided herein are devices wherein the plurality of features are located on the intermediate layer. Further provided herein are devices wherein a smallest dimension of the plurality of features is no more than the diffusion distance of the electrochemically generated reagent. Further provided herein are devices wherein the molecules comprise polymers. Further provided herein are devices wherein the polymers comprise polynucleotides, peptides, or sugars. Further provided herein are devices wherein the plurality of features are located on the top layer. Further provided herein are devices wherein the plurality of features comprises at least 3 features. Further provided herein are devices wherein a smallest dimension of a feature is no more than 225 nm. Further provided herein are devices wherein a smallest dimension of a feature is no more than 100 nm. Further provided herein are devices wherein a device comprises at least two addressable solid supports. Further provided herein are devices wherein the pitch distance between the at least two addressable solid supports is no more than 1 micron. Further provided herein are devices wherein the pitch distance between the at least two addressable solid supports is no more than 150 nm. Further provided herein are devices wherein the device comprises at least 1000 addressable solid supports. Further provided herein are devices wherein the thickness of the intermediate layer is no more than 20 nm. Further provided herein are devices wherein the thickness of the intermediate layer is 5-50 nm. Further provided herein are devices wherein the thickness of the intermediate layer is no more than 100 nm. Further provided herein are devices wherein the thickness of the top layer is no more than 20 nm. Further provided herein are devices wherein the thickness of the top layer is 5-100 nm. Further provided herein are devices wherein the device further comprises a cathode in fluid communication with the solvent. Further provided herein are devices wherein the cathode is a substantially planer surface. Further provided herein are devices wherein the cathode is substantially in the same plane as the intermediate layer. Further provided herein are devices wherein the intermediate layer is a substantially circular or rectangular. Further provided herein are devices wherein the intermediate layer is substantially planer surface. Further provided herein are devices wherein the intermediate layer comprises an anode. Further provided herein are devices wherein the shortest distance between the cathode and the top layer is 10%-30% of the pitch distance. Further provided herein are devices wherein the shortest distance between the cathode and the intermediate layer is 10%-30% of the pitch distance. Further provided herein are devices wherein the smallest width of the cathode is 10%-30% of the pitch distance. Further provided herein are devices wherein the largest dimension of the intermediate layer is 20%-50% of the pitch distance.
Provided herein are devices comprising at least one addressable solid support, wherein the at least one solid support comprises: a base layer comprising silicon; an intermediate layer comprising an oxide; a top layer comprising a conductive material, wherein the top layer is configured to produce an electrochemically generated reagent when energized with a voltage; and wherein the intermediate layer and the top layer are in fluid communication with a solvent, and wherein the intermediate layer is located between the base layer and the top layer, the intermediate layer is configured for the attachment of molecules, and wherein the solid support comprises a plurality of features. Further provided herein are devices wherein the top layer comprises a plurality of voids configured to allow fluid communication of the solvent with the intermediate layer. Further provided herein are devices wherein at least some of the voids are centered above one or more features. Further provided herein are devices wherein the voids comprise wells or channels. Further provided herein are devices wherein the wells or channels are 1-500 nm deep. Further provided herein are devices wherein the plurality of features are located on the intermediate layer. Further provided herein are devices wherein a smallest dimension of the plurality of features is no more than the diffusion distance of the electrochemically generated reagent. Further provided herein are devices wherein the molecules comprise polymers. Further provided herein are devices wherein the polymers comprise polynucleotides, peptides, or sugars. Further provided herein are devices wherein the plurality of features are located on the top layer. Further provided herein are devices wherein the plurality of features comprises at least 3 features. Further provided herein are devices wherein a smallest dimension of a feature is no more than 225 nm. Further provided herein are devices wherein a smallest dimension of a feature is no more than 100 nm. Further provided herein are devices wherein a device comprises at least two addressable solid supports. Further provided herein are devices wherein the pitch distance between the at least two addressable solid supports is no more than 1 micron. Further provided herein are devices wherein the pitch distance between the at least two addressable solid supports is no more than 150 nm. Further provided herein are devices wherein the device comprises at least 1000 addressable solid supports. Further provided herein are devices wherein the thickness of the intermediate layer is no more than 20 nm. Further provided herein are devices wherein the thickness of the intermediate layer is 5-50 nm. Further provided herein are devices wherein the thickness of the intermediate layer is no more than 100 nm. Further provided herein are devices wherein the thickness of the top layer is no more than 20 nm. Further provided herein are devices wherein the thickness of the top layer is 5-100 nm. Further provided herein are devices wherein the device further comprises a cathode in fluid communication with the solvent. Further provided herein are devices wherein the cathode is a substantially planer surface. Further provided herein are devices wherein the cathode is substantially in the same plane as the intermediate layer. Further provided herein are devices wherein the intermediate layer is a substantially circular or rectangular. Further provided herein are devices wherein the intermediate layer is substantially planer surface. Further provided herein are devices wherein the intermediate layer comprises an anode. Further provided herein are devices wherein the shortest distance between the cathode and the top layer is 10%-30% of the pitch distance. Further provided herein are devices wherein the shortest distance between the cathode and the intermediate layer is 10%-30% of the pitch distance. Further provided herein are devices wherein the smallest width of the cathode is 10%-30% of the pitch distance. Further provided herein are devices wherein the largest dimension of the intermediate layer is 20%-50% of the pitch distance.
Provided herein are devices for biomolecule synthesis described herein comprising at least two addressable solid supports, wherein the at least two addressable solid supports form an array. Provided herein are devices for polynucleotide synthesis described herein comprising at least two addressable solid supports, wherein the at least two addressable solid supports form an array. Further provided herein are devices wherein the array comprises at least 1000 addressable solid supports. Further provided herein are devices wherein the array further comprises: a plurality of vias, wherein the plurality of vias are configured to connect at least two vertical layers of the device; and a plurality of routing connections, wherein the plurality of routing connections are configured for addressable control of each device in the array. Further provided herein are devices wherein the vias and routing are no more than 200 nm in length. Further provided herein are devices wherein a via closest in proximity to the intermediate layer has a diameter of no more than 5-30% of the pitch distance. Further provided herein are devices wherein at least one of the vias and routing comprise copper.
All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.
The novel features of the invention are set forth with particularity in the appended claims. 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:
There is a need for higher density synthesis systems, such as those capable of synthesizing large, diverse libraries of biomolecules (e.g., nucleic acids). In some instances, nucleic acids libraries are useful for gene assembly, antibody design, next generation sequencing capture/enrichment, and data storage. In the case of data storage, there is a need for larger capacity storage systems as the amount of information generated and stored is increasing exponentially. Provided herein are methods to increase biomolecule synthesis throughput through increased sequence density, increased step efficiency, and decreased turn-around time using electrochemical control of biomolecule synthesis steps.
Definitions
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which these inventions belong.
Throughout this 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 lower limit unless the context clearly dictates otherwise. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, 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, for example, 1.1, 2, 2.3, 5, and 5.9. This applies regardless of the breadth of the range. The upper and lower limits of these intervening ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention, unless the context clearly dictates otherwise.
The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of any embodiment. 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, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, 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.
Unless specifically stated or obvious from context, as used herein, the term “about” in reference to a number or range of numbers is understood to mean the stated number and numbers+/−10% thereof, or 10% below the lower listed limit and 10% above the higher listed limit for the values listed for a range.
As used herein, the terms “preselected sequence”, “predefined sequence” or “predetermined sequence” are used interchangeably. The terms mean that the sequence of the polymer is known and chosen before synthesis or assembly of the polymer. In particular, various aspects of the invention are described herein primarily with regard to the preparation of nucleic acids molecules, the sequence of the polynucleotide being known and chosen before the synthesis or assembly of the nucleic acid molecules.
Provided herein are methods and compositions for production of synthetic (i.e. de novo synthesized, enzymatically synthesized, chemically synthesized) biomolecules. In some instances, biomolecules are synthesized in a template-independent manner. In some instances, biomolecules comprise polynucleotides. Polynucleotides may also be referred to as oligonucleotides or oligos. Polynucleotide sequences described herein may be, unless stated otherwise, comprise DNA or RNA. In some instances, biomolecules comprise polymers which comprise two or more monomers. Biomolecules in some instances refer to polymers such as nucleic acids (e.g., DNA, RNA), carbohydrates (e.g., sugars), peptides/proteins, lipids, fatty acids, terpenes, peptoids, or mixture thereof. In some instances, biomolecules may be synthesized in an iterative fashion using methods well-known in the art (with or without protecting groups). In some instances, biomolecules may be synthesized in an iterative fashion from monomers, dimers, trimers, or other appropriate building block.
Biomolecule Synthesis
Provided herein are devices, methods, compositions, and systems for biomolecule synthesis on solid supports. In some instances electrochemistry is used to control synthesis of biomolecules, such as through deprotection, coupling, or cleavage steps. Further provided herein are devices, methods, compositions, and systems for nucleic acid storage and synthesis on solid supports. In some instances, solid supports comprise surfaces. In some instances, surfaces comprise one or more features. In some instances, features comprise one or more loci for biomolecule synthesis. Further provided herein are pluralities of devices which are combined to form larger arrays or chips. Further provided herein are devices comprising one or more addressable solid supports. Further provided herein are devices and methods which are configured for electrochemical deprotection or deblocking during biomolecule (e.g., polynucleotide synthesis). Further provided herein are devices comprising one or more addressable solid supports for polynucleotide synthesis. In some instances, devices are charged with an electrical voltage in order to perform one or more steps of polynucleotide synthesis, such as deblocking. Such devices in some instances comprise “active” or “inactive” states, wherein each state comprises one or more voltage states, and/or one or more resistance states (e.g., “on”, “off”, or “disconnect”). Arrays of devices or addressable supports in some instances provide for addressable control of high-density nucleic acid synthesis and/or storage. Further provided herein are devices comprising in-plane cathodes which reduce migration of deprotection reagents to proximal or neighboring devices (i.e., diffusion control). Further provided herein are methods comprising applying a voltage as a series of pulses.
Provided herein are devices for polynucleotide synthesis (e.g.,
Provided herein are devices for polynucleotide synthesis comprising layers of materials. Such devices may comprise any number of layers of materials comprising conductors, semiconductors, or insulative materials. Traditional devices 200 comprise a base layer 201, conducting materials 202a/202b, 205 (one or more conducting layers configured for use as an electrode; conducting materials may be buried in the base layer such as 202a, or above the base layer, such as 202b), and a porous growth layer surface 203 (
A first device 300A provided herein comprises a base layer 301, and a patterned top layer 305 (
A second device 300B provided herein comprises a base layer 301, a buried shield electrode 308, and a patterned top layer 305 (
A third device 400 provided herein comprises a base layer 401, and an intermediate layer 405, and a top layer 406 (
A fourth device 500A provided herein comprises a base layer 501, a first intermediate layer 511, a top layer 505 (
A fifth device 500B provided herein comprises a base layer 501, a first intermediate layer 511, a second intermediate layer 505, a top layer 506 (
A sixth device 700 provided herein comprises a plurality of addressable solid supports 701, which are in fluid communication with the flow cell area 702 (
A seventh device described herein comprises a plurality of device arrays (or addressable solid supports), as shown in
A eight device described herein is shown in
A ninth device described herein is shown in
A tenth device described herein is shown in
Devices may comprise any number of device arrays. In some instances, devices comprise at least 10, 50, 100, 1000, 10,000, 100,000, or more than 100,000 device arrays in a single device. In some instances, devices comprise about 10, 50, 100, 1000, 10,000, 100,000, or about 100,000 device arrays in a single device. In some instances, devices comprise 10-50, 10-5000, 10-10,000, 100-1000, 100-10,000, 100-100,000, 1000-10,000, or 1000-100,000 device arrays in a single device. In some instances, a device comprises an array such as shown in
A eleventh device described herein is shown in
Device arrays may be scaled to any size or dimensions. In some instances, device arrays are about 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 0.8, 1, 2, 5, 8, or about 10 microns in width. In some instances, device arrays are no more than 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 0.8, 1, 2, 5, 8, or no more than 10 microns in width. In some instances, device arrays are 0.01-10, 0.1-10, 0.1-1, 0.5-1, 1-10, or 5-30 microns in width. In some instances, device arrays are separated by about 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 0.8, 1, 2, 5, 8, or about 10 microns. In some instances, device arrays are separated by no more than 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 0.8, 1, 2, 5, 8, or no more than 10 microns. In some instances, device arrays are separated by 0.01-10, 0.1-10, 0.1-1, 0.5-1, 1-10, or 5-30 microns.
Devices with addressable device arrays may be addressed in different patterns or configurations. In some instances, only specific groups (or clusters) of devices in arrays are activated simultaneously. In some instances, device arrays are addressed according to
Devices described herein may be fabricated using numerous methods, such as masking methods. In some instances, a lift-off fabrication method is used (
Devices may be configured a smallest dimension of the plurality of features is no more than the diffusion distance of an electrochemically generated reagent. In some instances, the reagent is an acid. In some instances, the diffusion distance is no more than 1000 nm, 750 nm, 500 nm, 400 nm, 300 nm, 250 nm, 225 nm, 200 nm, 150 nm, 125 nm, 100 nm, 75 nm, 50 nm, 25 nm, 10 nm, or no more than 5 nm.
Devices may be configured with one or more vias or routing components. In some instances, vias and routing are no more than 10, 20, 50, 100, 150, 200, 250, 300, 350, or no more than 500 nm in length. In some instances, vias and routing are about 10, 20, 50, 100, 150, 200, 250, 300, 350, or about 500 nm in length. In some instances, vias and routing are 10-500, 10-350, 10-200, 10-100, 10-50, 50-500, 50-300, 50-250, 50-200, 50-100, 100-300, 100-500, 100-200, 200-500, or 300-500. In some instances, vias comprise a conducting material. In some instances, vias comprise a metal described herein. In some instances, vias comprise copper. In some instances, vias comprise substantially copper.
Electrochemistry
Provided herein are methods of applying voltage to devices described herein. Such voltages may result in any number of different effects, such as electrochemical reaction with solvents or solutes. In some instances, apply a voltage results in deblocking or removal of protecting groups on molecules attached to a synthesis surface. In some instances molecules are polysaccharides, polynucleotides, polypeptides, or other polymer. In some instances, apply a voltage results in deblocking nucleic acids using the devices described herein. Further provided herein are methods wherein the devices described herein are energized with an electrical voltage. In some instances, the electrical voltage is used to deblock oligonucleotides bound to a solid support or surface. Such deblocking in some instances occurs through direct electrochemical reaction of a blocking group on a polynucleotide, or through the generation of deblocking reagents, such as an acid.
Methods described herein may comprising energizing a device with a voltage (applying a voltage) for a period of time. Applied voltages in some instances form a circuit between a cathode and an anode, leading to current flow through the device, solvent, and/or other components. In some instances, a layer of a device is configured as an anode or cathode. In some instances, a device comprises an anode located above the plane of the cathode (“sandwiched”). In some instances, a device comprises a cathode located above the plane of the anode (“opposing cathode”). In some instances, conductive layer is in electrical contact with layer cathode. In some instances, a device comprises an anode located in substantially the same plane of the cathode. Application of voltage in some instances is configured to perform a step of polynucleotide synthesis. In some instances, methods comprise application of a voltage to deblock polynucleotides. In some instances application of a voltage generates a deblocking reagent. In some instances, devices comprise conducting layers in fluid communication with a solvent, wherein the solvent comprises reagents which generate a deblocking reagent. In some instances, the deblocking reagent is an acid. In some instances, the acid is H+.
Methods described herein may comprise applying a voltage to one or more devices described herein. In some instances, such voltages result in deprotection of molecules (polynucleotides, polypeptides, polysaccharides, or other polymer) at one or more devices or regions. In some instances, application of a voltage at one or more devices results in deprotection of polynucleotides at one or more devices or regions within one or more devices. In some instances, a device is described as “inactive” if a deprotection reagent (e.g., acid or other reagent) is not generated at or in the vicinity of a device or region of a device. In some instances, a device is described as “active” if a deprotection reagent (e.g., acid or other reagent) is generated at or in the vicinity of a device or region of a device. In some instances, deprotection of polynucleotides occurs at or near one or more active devices, or regions of one or more active devices. In some instances, both active and inactive devices are energized with voltages. In some instances, voltage is applied to inactive devices in levels which are insufficient to generate a deprotection reagent. In some instances, deprotection comprises application of one or more voltages (or voltage levels) for periods of time. In some instances, a single voltage level is used for deprotection of polynucleotides. In some instances, more than one voltage level is used during deprotection (
Different voltage configurations for activation of a device may be used (
Devices may be described as circuits between an anode and a cathode. In some instances, such circuits are described as being in device states, such as “on”, “off”, or “alternate resistance”. In some instances, alternate resistance is a high resistance state, or “disconnect” state. In some instances, a high resistance state is a resistance state that is higher than an off state (e.g., low/no voltage in off state, but still connected to a ground). In some instances, a high resistance state provides an effective amount of resistance to reduce current flow through one or more inactive devices. Without being bound by theory, the disconnect state in some instances reduces undesired deprotection at areas adjacent to an on device. In some instances, a high resistance state provides an effective amount of resistance to reduce current flow to near zero in one or more inactive devices. In some instances an off state is generated by zero (or near zero) voltage between an inactive device and a common cathode. In some instances an off state exists even with a minimum voltage applied between an inactive device and a common cathode, wherein the minimum voltage is below that amount needed for deprotection. In some instances a high resistance state is generated by zero voltage between an inactive device and the cathode and a higher resistance between the inactive devices and a common cathode. In some instances, an off state indicates zero voltage or negative voltage between the anode and active device (cathode). In some instances, an on state indicates positive voltage between the anode and active device (cathode) which is sufficient for deprotection. In some instances, an inactive device is in the off or alternate resistance state. In some instances, an active device (where deprotection is desired) is cycled (pulsed) between one or more on and off states for a period of time. In some instances, an active device (where deprotection is desired) is cycled between one or more on and off states for a period of time and neighboring inactive devices are maintained in an alternative resistance state. In some instances, methods described herein comprise one or more of (a) providing a surface having (i) one or more electrodes proximal to the surface and (ii) one or more in-plane cathodes proximal to the surface, wherein the surface comprises a first plurality of protected biomolecules attached thereto; (b) energizing at least one electrode proximal to a first region of the surface to electrochemically generate a deprotection reagent, wherein the deprotection reagent deprotects at least some of the first plurality of biomolecules in the first region; (c) coupling at least one protected biomolecule monomer to at least one deprotected biomolecule in the first region; and (d) repeating steps (a)-(c) to synthesize the biomolecule. In some instances the biomolecule comprises a polynucleotide. In some instances, the biomolecule monomer comprises a nucleotide. In some instances, a negative voltage is applied to a cathode, and a positive voltage is applied to an anode. In some instances, the absolute difference in voltage is no more than 2, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, or no more than 0.2 volts. In some instances, the absolute difference in voltage is no more than 0.2-2, 0.5-2, 0.8-2, 1-2, 1.5-2, 0.2-1, 0.2-0.5, 0.5-1, or 0.5-1.5 volts.
A voltage may be applied to the cathode in addition to the anode. In some instances, the cathode is biased with a negative voltage relative to ground. In some instances, biasing the voltage (bias voltage) of the cathode reduces the maximum anode voltage needed for electrochemical deprotection (e.g., the voltage difference between the anode and cathode will equal the anode voltage plus the magnitude of the negative bias voltage at the cathode). In some instances, a device comprises a contact bias on the cathode. In some instances, a bias voltage at the cathode is switched whenever the anode voltage is switched (e.g., synched). In some instances, a cathode controls electrochemistry for a single device. In some instances, a cathode controls electrochemistry for a plurality of devices (“common” cathode). In some instances, use of a common cathode results fewer transistors needed per device. In some instances, the bias voltage is no more than −0.1, −0.2, −0.3, −0.5, −0.7, −0.9, −1.0 −1.1, −1.2, −1.5, −1.8, −2.0, −2.1, −2.2, or no more than −2.5 volts. In some instances, the biased voltage is at least −0.1, −0.2, −0.3, −0.5, −0.7, −0.9, −1.0 −1.1, −1.2, −1.5, −1.8, −2.0, −2.1, −2.2, or at least −2.5 volts. In some instances, the biased voltage is about −0.1, −0.2, −0.3, −0.5, −0.7, −0.9, −1.0 −1.1, −1.2, −1.5, −1.8, −2.0, −2.1, −2.2, or about −2.5 volts. In some instances, the biased voltage is −0.1 to −2.5 volts, −0.2 to −2.5 volts, −0.5 to −2.5 volts, −1.0 to −2.5 volts, −1.5 to −2.5 volts, −1.0 to −2.0 volts, −0.5 to −1.0 volts, −0.2 to −1.5 volts, or −2.0 to −2.5 volts.
The voltage between two layers of a device or surface may be varied. In some instances, a voltage is between the anode and cathode. In some instances the voltage is 0.5-3, 1-3, 1.5-2.5, 1-2.5, or 1.5-2 volts. In some instances, the voltage is at least 0.5, 0.75, 1, 1.2, 1.5, 1.7, 1.9, 2, 2.2, 2.4, or more than 2.4 volts. In some instances, the voltage is about 0.5, 0.75, 1, 1.2, 1.5, 1.7, 1.9, 2, 2.2, 2.4, or about 2.4 volts. In some instances, the voltage is −0.1 to −2.5 volts, −0.2 to −2.5 volts, −0.5 to −2.5 volts, −1.0 to −2.5 volts, −1.5 to −2.5 volts, −1.0 to −2.0 volts, −0.5 to −1.0 volts, −0.2 to −1.5 volts, or −2.0 to −2.5 volts. In some instances, a conducting layer of a device is charged with a positive voltage. In some instances, a conducting layer of a device is charged with a negative voltage. In some instances, a first layer of a device is charged with a positive voltage, and a second layer is charged with a negative voltage at the same time.
The (total) amount of time a voltage is applied may be varied for each synthesis cycle (e.g., deblocking, coupling, etc.). Voltage is applied in some instances for no more than 0.1, 0.2, 0.5, 0.8, 1, 2, 5, or no more than 10 seconds. Voltage is applied in some instances for 0.1-10, 0.5-10, 0.5-5, 0.1-5, 2-5, 2-10, 3-10, or 0.1-2 seconds. Voltage is applied in some instances about 0.1, 0.2, 0.5, 0.8, 1, 2, 5, or about 10 seconds. Voltage is applied in some instances for no more than 0.1, 0.2, 0.5, 0.8. 1, 2, 5, 10, 20, 50, 100, 200, 500, 800, or no more than 1000 milliseconds (ms). Voltage is applied in some instances for about 0.1, 0.2, 0.5, 0.8. 1, 2, 5, 10, 20, 50, 100, 200, 500, 800, or about 1000 milliseconds. Voltage is applied in some instances for 0.1-1000, 0.5-500, 0.5-50, 0.1-5, 2-50, 2-100, 3-200, 0.1-10, 1-100, 1-50, or 0.1-2 milliseconds.
Voltage may be applied as a single “on”/“off” cycle, or applied as a series of alternating “on” and “off” cycles to an active device. In some instances an “on” state is a positive voltage or a negative voltage. The application of voltage in the “on” state followed by an “off” state is in some instances defined as a “pulse.” In some instances, voltage is applied in a series of pulses, such as no more than 1, 2, 3, 4, 5, 6, 7, 8, 10, 20, 50, 80, 100, 110, 120, 150, 180, 200, 220, 250, 300, 500, or more than 500 pulses. In some instances, voltage is applied in a series of pulses, such as at least 1, 2, 3, 4, 5, 6, 7, 8, 10, 20, 50, 80, 100, 110, 120, 150, 180, 200, 220, 250, 300, 500, or at least 500 pulses. In some instances, voltage is applied in a series of pulses, such as 1-1000, 1-500, 1-300, 10-500, 10-100, 50-500, 50-200, 100-1000, 2-10, 2-8, 20-200, or 300-750 pulses. In some instances, voltage is applied in a series of pulses, such as about 1, 2, 3, 4, 5, 6, 7, 8, 10, 20, 50, 80, 100, 110, 120, 150, 180, 200, 220, 250, 300, 500, or about 500 pulses. In some instances, voltage is applied in a series of pulses, such as no more than 100, 200, 500, 800, 1000, 2000, 5000, 8000, 10000, 11000, 12000, 15000, 18000, 20000, 50000, 80000, 100,000, 200,000, 500,000, 800,000, or more than 1,000,000 pulses. In some instances, voltage is applied in a series of pulses, such as at least 100, 200, 500, 800, 1000, 2000, 5000, 8000, 10000, 11000, 12000, 15000, 18000, 20000, 50000, 80000, 100,000, 200,000, 500,000, 800,000, or at least 1,000,000 pulses. In some instances, voltage is applied in a series of pulses, such as about 100, 200, 500, 800, 1000, 2000, 5000, 8000, 10000, 11000, 12000, 15000, 18000, 20000, 50000, 80000, 100,000, 200,000, 500,000, 800,000, or about 1,000,000 pulses. In some instances, voltage is applied in a series of pulses, such as at least 10-1000, 10-5000, 100-10,000, 1000-50,000, 10000-100,000, 50000-500,000, 50000-1,000,000, 10,000-100,000 or 500,000-1,000,000 pulses. In some instances, a method described herein comprises one or more of (a) contacting at least one biomolecule monomer attached to a solid support with a protected biomolecule, wherein the protected biomolecule is configured to form a covalent bond with the at least one biomolecule to generate a protected biomolecule; (b) applying a voltage to a solvent in fluid communication with the protected biomolecule, wherein the voltage results in deprotection of a terminal nucleoside of the protected biomolecule, and wherein the voltage is delivered as at least 2 pulses; (c) repeating steps (a) and (b) to synthesize the biomolecule. In some instances, the biomolecule comprises a polynucleotide. In some instances, the biomolecule comprises a polynucleotide monomer (e.g., nucleotide).
The voltage application time may be divided by the number of pulses to define a pulse time (or pulse width, or time per pulse). In some instances a pulse time is no more than 0.1, 0.2, 0.5, 0.8. 1, 2, 5, 10, 20, 50, 100, 200, 500, 800, or no more than 1000 milliseconds. In some instances a pulse time is about 0.1, 0.2, 0.5, 0.8. 1, 2, 5, 10, 20, 50, 100, 200, 500, 800, or about 1000 milliseconds. The pulse time in some instances is 0.1-1000, 0.5-500, 0.5-50, 0.1-5, 2-50, 2-100, 3-200, 0.1-10, 1-100, 1-50, or 0.1-2 milliseconds. In some instances a pulse time is no more than 0.1, 0.2, 0.5, 0.8. 1, 2, 5, 10, 20, 50, 100, 200, 500, 800, or no more than 1000 microseconds. In some instances a pulse time is about 0.1, 0.2, 0.5, 0.8. 1, 2, 5, 10, 20, 50, 100, 200, 500, 800, or about 1000 microseconds. The pulse time in some instances is 0.1-1000, 0.5-500, 0.5-50, 0.1-5, 2-50, 2-100, 3-200, 0.1-10, 1-100, 1-50, or 0.1-2 microseconds. In some instances, a polynucleotide synthesis surface is washed with a solvent in between pulses. In some instances, a polynucleotide synthesis surface is not washed with a solvent in between pulses. In some instances a series of pulses are used to deliver voltage to a surface, followed by a wash step, followed by another series of pulses. Pulses need not be the same voltage. In some instances, a first pulse is positive, and a second pulse is negative. In some instances, the time between a positive and negative voltage is substantially instantaneous. In some instances, a first pulse is about 2 volts and a second pulse is about −0.6 volts. In some instances, a first pulse is 0.5 to 3 volts and a second pulse is −0.1 to −1.0 volts.
The time period between pulses may be varied to allow, without being bound by theory, electrochemically generated reagents to dissipate. The time between pulses in some instances is no more than 0.1, 0.2, 0.5, 0.8, 1, 2, 5, or no more than 10 seconds. The time between pulses in some instances is 0.1-10, 0.5-10, 0.5-5, 0.1-5, 2-5, 2-10, 3-10, or 0.1-2 seconds. The time between pulses in some instances is about 0.1, 0.2, 0.5, 0.8, 1, 2, 5, or about 10 seconds. The time between pulses in some instances is no more than 0.1, 0.2, 0.5, 0.8. 1, 2, 5, 10, 20, 50, 100, 200, 500, 800, or no more than 1000 milliseconds (ms). The time between pulses in some instances is about 0.1, 0.2, 0.5, 0.8. 1, 2, 5, 10, 20, 50, 100, 200, 500, 800, or about 1000 milliseconds. The time between pulses in some instances is 0.1-1000, 0.5-500, 0.5-50, 0.1-5, 2-50, 2-100, 3-200, 0.1-10, 1-100, 1-50, or 0.1-2 milliseconds. The time between pulses in some instances is no more than 0.1, 0.2, 0.5, 0.8. 1, 2, 5, 10, 20, 50, 100, 200, 500, 800, or no more than 1000 microseconds (ms). The time between pulses in some instances is about 0.1, 0.2, 0.5, 0.8. 1, 2, 5, 10, 20, 50, 100, 200, 500, 800, or about 1000 microseconds. The time between pulses in some instances is 0.1-1000, 0.5-500, 0.5-50, 0.1-5, 2-50, 2-100, 3-200, 0.1-10, 1-100, 1-50, or 0.1-2 microseconds. In some instances, the ratio between on and off times for series of pulses is described as a duty cycle. In some instances, a duty cycle is about 1:100, 1:50, 1:20, 1:10, 1:5, 1:2, 1:1.5, 1:1.05, 1.05:1, 1.5:1, 2:1, or about 3:1. In some instances, a duty cycle is no more than 1:100, 1:50, 1:20, 1:10, 1:5, 1:2, 1:1.5, 1:1.05, 1.05:1, 1.5:1, 2:1, or no more than 3:1. In some instances, a duty cycle is at least 1:100, 1:50, 1:20, 1:10, 1:5, 1:2, 1:1.5, 1:1.05, 1.05:1, 1.5:1, 2:1, or at least 3:1.
Electrochemical deprotection may result in less migration of deprotection reagents to neighboring (or adjacent) addressable devices (or solid supports) where other polynucleotides are synthesized. In some instances, deprotecting comprises energizing an addressable device with two or more pulses. In some instances, an active device comprises a first plurality of protected biomolecules. In some instances, an active device comprises a second plurality of protected biomolecules (proximal) to a neighboring device. In some instances, reduced deprotection reagent migration to neighboring devices results in higher synthesis fidelity on the device. For example, a high percentage of a first plurality of protected biomolecules at an active device are deprotected, while a minimum of percentage of a second plurality of protected biomolecules at a neighboring device are deprotected. In some instances, electrochemical deprotection deprotects a protected nucleoside. In some instances, the deprotection reagent is an acid. In some instances, at least 90%, 95%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.97%, 99.99%, or at least 99.995% of the protected polynucleotides on an addressable device are deprotected. In some instances, no more than 10%, 5%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.05%, 0.02%, 0.01%, 0.005%, 0.004%, 0.001%, or no more than 0.0005% of the protected polynucleotides on an adjacent addressable device are deprotected. In some instances, at least 90%, 95%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.97%, 99.99%, or at least 99.995% of the protected polynucleotides on an addressable device are deprotected, and the pitch distance between addressable devices is no more than 1 micron. In some instances, no more than 10%, 5%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.05%, 0.02%, 0.01%, 0.005%, 0.004%, 0.001%, or no more than 0.0005% of the protected polynucleotides on an adjacent addressable device are deprotected, and the pitch distance between addressable devices is no more than 1 micron. In some instances, at least 90%, 95%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.97%, 99.99%, or at least 99.995% of the protected polynucleotides on an addressable device are deprotected, and the pitch distance between addressable devices is no more than 500 nm. In some instances, no more than 10%, 5%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.05%, 0.02%, 0.01%, 0.005%, 0.004%, 0.001%, or no more than 0.0005% of the protected polynucleotides on an adjacent addressable device are deprotected, and the pitch distance between addressable devices is no more than 500 nm. In some instances, at least 90%, 95%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.97%, 99.99%, or at least 99.995% of the protected polynucleotides on an addressable device are deprotected, and the pitch distance between addressable devices is no more than 200 nm. In some instances, no more than 10%, 5%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.05%, 0.02%, 0.01%, 0.005%, 0.004%, 0.001%, or no more than 0.0005% of the protected polynucleotides on an adjacent addressable device are deprotected, and the pitch distance between addressable devices is no more than 200 nm. In some instances, at least 90%, 95%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.97%, 99.99%, or at least 99.995% of the protected polynucleotides on an addressable device are deprotected, and the pitch distance between addressable devices is no more than 150 nm. In some instances, no more than 10%, 5%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.05%, 0.02%, 0.01%, 0.005%, 0.004%, 0.001%, or no more than 0.0005% of the protected polynucleotides on an adjacent addressable device are deprotected, and the pitch distance between addressable devices is no more than 150 nm. In some instances, at least 90%, 95%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.97%, 99.99%, or at least 99.995% of the protected polynucleotides on an addressable device are deprotected, and the pitch distance between addressable devices is no more than 100 nm. In some instances, no more than 10%, 5%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.05%, 0.02%, 0.01%, 0.005%, 0.004%, 0.001%, or no more than 0.0005% of the protected polynucleotides on an adjacent addressable device are deprotected, and the pitch distance between addressable devices is no more than 100 nm. In some instances, at least 90%, 95%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.97%, 99.99%, or at least 99.995% of the protected polynucleotides on an addressable device are deprotected, and the pitch distance between addressable devices is no more than 50 nm. In some instances, no more than 10%, 5%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.05%, 0.02%, 0.01%, 0.005%, 0.004%, 0.001%, or no more than 0.0005% of the protected polynucleotides on an adjacent addressable device are deprotected, and the pitch distance between addressable devices is no more than 50 nm.
Methods described herein may lead to reduced synthesis times for biomolecules. In some instances, the biomolecules comprise polynucleotides. In some instances, synthesized are synthesized at a rate of at least 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 45, 50, 55, 60, 75, or at least 100 nt/hr. In some instances, synthesized are synthesized at a rate of about 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 45, 50, 55, 60, 75, or at least 100 nt/hr. In some instances, synthesized are synthesized at a rate of 10-100, 10-75, 10-50, 10-25,7 15-25, 15-50, 15-75, 20-80, 20-50, 30-80, 30-50, 50-100, or 75-125 nt/hr.
Various chemical reactions may be used to deblock polynucleotides, directly or indirectly. In some instances electrical voltage oxidizes or reduces a blocking group (protecting group) directly on a polynucleotide, causing the polynucleotide to be deblocked. In some instances, the voltage generates an in-situ reagent which deblocks the blocked nucleotide. In some instances, the polynucleotide comprises an acid-cleavable blocking group. In some instances, the reagent is dissolved in a solvent. In some instances, the reagent is an acid, such as H+. Various reagents may be used to electrochemically generate deblocking reagents such as acids. In some instances, a reagent comprises a quinone. In some instances, the quinone is benzoquinone, hydroquinone, anthraquinone, substituted benzoquinone, a hydrazine, a diazirine, or other reagent configured to generate an acid when a voltage is applied. In some instances, the reagent comprises a mixture of hydroquinone (HQ) and benzoquinone (BQ). In some instances, the reagent comprises a mixture of hydroquinone and benzoquinone, wherein the ratio of HQ:BQ is about 100:1, 50:1, 20:1, 10:1, 8:1, 5:1, 3:1, 2:1, 1:1, 1:2, 1:5, or about 1:10. In some instances, the ratio of HQ:BQ is at least 100:1, 50:1, 20:1, 10:1, 8:1, 5:1, 3:1, 2:1, or at least 1:1. In some instances, the ratio of HQ:BQ is no more than 100:1, 50:1, 20:1, 10:1, 8:1, 5:1, 3:1, 2:1, or no more than 1:1. In some instances, the ratio of HQ:BQ is 100:1-10:1, 50:1-1:1, 20:1-5:1, 15:1-5:1, 10:1-1:1, 10:1-2:1. 20:1-2:1, 1:1-1:5, 1:1-10:1 10:1-1:10, or 5:1-1:5.
Provided herein are methods of fabricating the devices and surfaces for polynucleotide synthesis. Described herein are layers integrated into a solid support. In some instances, layers comprise electrodes or are configured for use as electrodes. In some instances, electrodes are configured as cathodes or anodes. In some instances, an anode comprises a metal oxide. In some instances, nucleic acids are synthesized on an anode. In some instances, nucleic acids are synthesized on a metal oxide layer. In some instances, nucleic acids are synthesized on a porous metal oxide layer comprising a continuous metal layer beneath it. Electrodes in some instances comprise at least one conductor, and are fabricated of materials well known in the art. In some instances, electrodes comprise at least one conductor and one or more insulators or semi-conductors. Materials may comprise metals, non-metals, mixed-metal oxides, nitrides, carbides, silicon-based materials, or other material. In some instances, metal oxides include TiO2, Ta2O5, IrO2, RuO2, RhO2, Nb2O5, Al2O3, BaO, Y2O3, HfO2, SrO or other metal oxide known in the art. In some instances, metal carbides include TiC, WC, ThC2, ThC, VC, W2C, ZrC, HfC, NbC, TaC, Ta2C, or other metal carbide known in the art. In some instances, metal nitrides include GaN, InN, BN, Be3N2, Cr2N, MoN, Si3N4, TaN, Th2N2, VN, ZrN, TiN, HfN, NbC, WN, TaN, or other metal nitride known in the art. In some instances, a device disclosed herein is manufactured with a combination of materials listed herein or any other suitable material known in the art.
Solid supports comprising layers may be coated with additional materials such as semiconductors or insulators. In some instances, a layer is configured for use as an electrode. In some instances, electrodes are coated with materials for biomolecule attachment and synthesis. In some instances, electrodes are coated with materials for polynucleotide attachment and synthesis. Each electrode can control one, or a plurality of different loci for synthesis, wherein each locus for synthesis has a density of polynucleotides. In some instances, the density is at least 1 biomolecule per 10 nm2, 20, 50, 100, 200, 500, 1,000, 2,000, 5,000 or at least 1 oligo per 10,000 nm2. In some instances, the density is about 1 biomolecule per 10 nm2 to about 1 biomolecule per 5,000 nm2, about 1 biomolecule per 50 nm2 to about 1 biomolecule per 500 nm2, or about 1 biomolecule per 25 nm2 to about 1 biomolecule per 75 nm2. In some instances, the density of polynucleotides is about 1 biomolecule per 25 nm2 to about 1 biomolecule per 75 nm2. In some instances, the density is at least 1 biomolecule per 10 nm2, 20, 50, 100, 200, 500, 1,000, 2,000, 5,000 or at least 1 oligo per 10,000 nm2. In some instances, the density is about 1 oligo per 10 nm2 to about 1 oligo per 5,000 nm2, about 1 oligo per 50 nm2 to about 1 oligo per 500 nm2, or about 1 oligo per 25 nm2 to about 1 oligo per 75 nm2. In some instances, the density of polynucleotides is about 1 oligo per 25 nm2 to about 1 oligo per 75 nm2.
Described herein are devices wherein two or more solid supports are assembled. In some instances, solid supports are interfaced together on a larger unit. Interfacing may comprise exchange of fluids, electrical signals, or other medium of exchange between solid supports. This unit is capable of interface with any number of servers, computers, or networked devices. For example, a plurality of solid support is integrated onto a rack unit, which is conveniently inserted or removed from a server rack. The rack unit may comprise any number of solid supports. In some instances the rack unit comprises at least 1, 2, 5, 10, 20, 50, 100, 200, 500, 1000, 2000, 5000, 10,000, 20,000, 50,000, 100,000 or more than 100,000 solid supports. In some instances, two or more solid supports are not interfaced with each other. Nucleic acids (and the information stored in them) present on solid supports can be accessed from the rack unit. See, e.g.,
Solid supports as described herein comprise an active area. In some instances, the active area comprises addressable solid supports, regions, or loci for nucleic acid synthesis. In some instances, the active area comprises addressable regions or loci for nucleic acid storage. In some instances, an active area is in fluid communication with solvents or other reagents. The active area comprises varying dimensions. For example, the dimension of the active area is between about 1 mm to about 50 mm by about 1 mm to about 50 mm. In some instances, the active area comprises a width of at least or about 0.5, 1, 1.5, 2, 2.5, 3, 5, 5, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, or more than 80 mm. In some instances, the active area comprises a height of at least or about 0.5, 1, 1.5, 2, 2.5, 3, 5, 5, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, or more than 80 mm. An exemplary active area within a solid support is seen in
Described herein are devices, compositions, systems and methods for solid support based nucleic acid synthesis and storage, wherein the solid support has a number of sites (e.g., spots) or positions for synthesis or storage. In some instances, the solid support comprises up to or about 10,000 by 10,000 positions in an area. In some instances, the solid support comprises between about 1000 and 20,000 by between about 1000 and 20,000 positions in an area. In some instances, the solid support comprises at least or about 10, 30, 50, 75, 100, 200, 300, 400, 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, 12,000, 14,000, 16,000, 18,000, 20,000 positions by least or about 10, 30, 50, 75, 100, 200, 300, 400, 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, 12,000, 14,000, 16,000, 18,000, 20,000 positions in an area. In some instances the area is up to 0.25, 0.5, 0.75, 1.0, 1.25, 1.5, or 2.0 inches squared. In some instances, the solid support comprises addressable loci having a pitch of at least or about 0.1, 0.2, 0.25, 0.3, 0.4, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5, 6, 7, 8, 9, 10, or more than 10 um. In some instances, the solid support comprises addressable loci having a pitch of about 5 um. In some instances, the solid support comprises addressable loci having a pitch of about 2 um. In some instances, the solid support comprises addressable loci having a pitch of about 1 um. In some instances, the solid support comprises addressable loci having a pitch of about 0.2 um. In some instances, the solid support comprises addressable loci having a pitch of about 0.2 μm to about 10 um, about 0.2 to about 8 um, about 0.5 to about 10 um, about 1 μm to about 10 um, about 2 μm to about 8 um, about 3 μm to about 5 um, about 1 μm to about 3 μm or about 0.5 μm to about 3 um. In some instances, the solid support comprises addressable loci having a pitch of about 0.1 μm to about 3 um. In some instances, the solid support comprises addressable loci having a pitch of at least or about 0.01, 0.02, 0.025, 0.03, 0.04, 0.05, 0.1, 0.15, .02, 0.25, 0.30, 0.35, 0.4, 0.45, 0.5, 0.6, 0.7, 0.8, 0.9, 1, or more than 1 um. In some instances, the solid support comprises addressable loci having a pitch of about 0.5 um. In some instances, the solid support comprises addressable loci having a pitch of about 0.2 um. In some instances, the solid support comprises addressable loci having a pitch of about 0.1 um. In some instances, the solid support comprises addressable loci having a pitch of about 0.02 um. In some instances, the solid support comprises addressable loci having a pitch of about 0.02 μm to about 1 um, about 0.02 to about 0.8 um, about 0.05 to about 0.1 um, about 0.1 μm to about 1 um, about 0.2 μm to about 0.8 um, about 0.3 μm to about 0.5 um, about 0.1 μm to about 0.3 μm or about 0.05 μm to about 0.3 um. In some instances, the solid support comprises addressable loci having a pitch of about 0.01 μm to about 0.3 um. See e.g.
Devices described herein may comprise high-density addressable arrays for synthesis. In some instances, a device array comprises at least 1, 10, 100, 500, 1000, 2000, 5000, 10,000, 20,000, 50,000, 1000,000, or at least 200,000 addressable loci per mm2. In some instances, a device array comprises 1-50,000, 1-10,000, 10-100,000, 50-100,000, 100-100,000, 100-50,000, 100-5000, 100-1000, 500-50,000, 500-10,000, 500-25,000, 1000-200,000, 1000-100,000, 1000-50,000, 1000-25,000, 1000-10,000, 5000-100,000, 5000-200,000, or 5000-50,000 loci per mm2. In some instances, a device array comprises at least 1, 10, 100, 500, 1000, 2000, 5000, 10,000, 20,000, 50,000, 1000,000, or at least 200,000 addressable loci per μm2. In some instances, a device array comprises 1-50,000, 1-10,000, 10-100,000, 50-100,000, 100-100,000, 100-50,000, 100-5000, 100-1000, 500-50,000, 500-10,000, 500-25,000, 1000-200,000, 1000-100,000, 1000-50,000, 1000-25,000, 1000-10,000, 5000-100,000, 5000-200,000, or 5000-50,000 loci per μm2.
The solid support for nucleic acid synthesis or storage as described herein comprises a high capacity for storage of data. For example, the capacity of the solid support is at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or more than 1000 petabytes. In some instances, the capacity of the solid support is between about 1 to about 10 petabytes or between about 1 to about 100 petabytes. In some instances, the capacity of the solid support is about 100 petabytes. In some instances, the data is stored as addressable arrays of packets as droplets. In some instances, the data is stored as addressable arrays of packets as droplets on a spot. In some instances, the data is stored as addressable arrays of packets as dry wells. In some instances, the addressable arrays comprise at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 200, or more than 200 terabytes of data. In some instances, the addressable arrays comprise at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 200, or more than 200 gigabytes of data. In some instances, the addressable arrays comprise at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 200, or more than 200 terabytes of data. In some instances, an item of information is stored in a background of data. For example, an item of information encodes for about 10 to about 100 megabytes of data and is stored in 1 petabyte of background data. In some instances, an item of information encodes for at least or about 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, or more than 500 megabytes of data and is stored in 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, or more than 500 petabytes of background data. In some instances, storage capacity is based on a device pitch (
Nucleic Acid Based Information Storage
Provided herein are devices, compositions, systems and methods for nucleic acid-based information (data) storage. A biomolecule such as a DNA molecule provides a suitable host for information storage in-part due to its stability over time and capacity for enhanced information coding, as opposed to traditional binary information coding. In a first step, a digital sequence encoding an item of information (i.e., digital information in a binary code for processing by a computer) is received. An encryption scheme is applied to convert the digital sequence from a binary code to a nucleic acid sequence. A surface material for nucleic acid extension, a design for loci for nucleic acid extension (aka, arrangement spots), and reagents for nucleic acid synthesis are selected. The surface of a structure is prepared for nucleic acid synthesis. De novo polynucleotide synthesis is then performed. The synthesized polynucleotides are stored and available for subsequent release, in whole or in part. Once released, the polynucleotides, in whole or in part, are sequenced, subject to decryption to convert nucleic sequence back to digital sequence. The digital sequence is then assembled to obtain an alignment encoding for the original item of information.
Items of Information
Optionally, an early step of data storage process disclosed herein includes obtaining or receiving one or more items of information in the form of an initial code. Items of information include, without limitation, text, audio and visual information. Exemplary sources for items of information include, without limitation, books, periodicals, electronic databases, medical records, letters, forms, voice recordings, animal recordings, biological profiles, broadcasts, films, short videos, emails, bookkeeping phone logs, internet activity logs, drawings, paintings, prints, photographs, pixelated graphics, and software code. Exemplary biological profile sources for items of information include, without limitation, gene libraries, genomes, gene expression data, and protein activity data. Exemplary formats for items of information include, without limitation, .txt, .PDF, .doc, .docx, .ppt, .pptx, .xls, .xlsx, .jpg, .gif, .psd, .bmp, .tiff, .png, and .mpeg. The amount of individual file sizes encoding for an item of information, or a plurality of files encoding for items of information, in digital format include, without limitation, up to 1024 bytes (equal to 1 KB), 1024 KB (equal to 1 MB), 1024 MB (equal to 1 GB), 1024 GB (equal to 1 TB), 1024 TB (equal to 1 PB), 1 exabyte, 1 zettabyte, 1 yottabyte, 1 xenottabyte or more. In some instances, an amount of digital information is at least 1 gigabyte (GB). In some instances, the amount of digital information is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more than 1000 gigabytes. In some instances, the amount of digital information is at least 1 terabyte (TB). In some instances, the amount of digital information is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more than 1000 terabytes. In some instances, the amount of digital information is at least 1 petabyte (PB). In some instances, the amount of digital information is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more than 1000 petabytes. In some instances, the digital information does not contain genomic data acquired from an organism. Items of information in some instance are encoded. Non-limiting encoding method examples include 1 bit/base, 2 bit/base, 4 bit/base or other encoding method.
Structures for Biomolecule Synthesis
Provided herein are rigid or flexibles structures for biomolecule synthesis (e.g., polynucleotide synthesis). In the case of rigid structures, provided herein are devices having a structure for the generation of a library of polynucleotides. In some instances, the structure comprises a plate.
In the case of flexible structures, provided herein are devices wherein the flexible structure comprises a continuous loop 3501 wrapped around one or more fixed structures, e.g., a pair of rollers 3503 or a non-continuous flexible structure 3507 wrapped around separate fixed structures, e.g., a pair reels 3505. See
Wells described herein may comprise any sizes or dimensions. In some instances, a well described herein has a width to depth (or height) ratio of 1 to 0.01, wherein the width is a measurement of the width at the narrowest segment of the well. In some instances, a well described herein has a width to depth (or height) ratio of 0.5 to 0.01, wherein the width is a measurement of the width at the narrowest segment of the well. In some instances, a well described herein has a width to depth (or height) ratio of about 0.01, 0.05, 0.1, 0.15, 0.16, 0.2, 0.5, or 1. Provided herein are structures for polynucleotide synthesis comprising a plurality of discrete loci for polynucleotide synthesis. Exemplary structures for the loci include, without limitation, substantially planar regions, channels, wells or protrusions. Structures described herein are may comprise a plurality of clusters, each cluster comprising a plurality of wells, loci or channels. Alternatively, described herein are may comprise a homogenous arrangement of wells, loci or channels. Structures provided herein may comprise wells having a height or depth from about 5 μm to about 500 um, from about 5 μm to about 400 um, from about 5 μm to about 300 um, from about 5 μm to about 200 um, from about 5 μm to about 100 um, from about 5 μm to about 50 um, or from about 10 μm to about 50 um. In some instances, the height of a well is less than 100 um, less than 80 um, less than 60 um, less than 40 μm or less than 20 um. In some instances, well height is about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500 μm or more. In some instances, the height or depth of the well is at least 10, 25, 50, 75, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or more than 1000 nm. In some instances, the height or depth of the well is in a range of about 10 nm to about 1000 nm, about 25 nm to about 900 nm, about 50 nm to about 800 nm, about 75 nm to about 700 nm, about 100 nm to about 600 nm, or about 200 nm to about 500. In some instances, the height or depth of the well is in a range of about 50 nm to about 1 um. In some instances, well height is about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 700, 800, 900 or about 1000 nm.
Structures for polynucleotide synthesis provided herein may comprise channels. The channels may have a width to depth (or height) ratio of 1 to 0.01, wherein the width is a measurement of the width at the narrowest segment of the microchannel. In some instances, a channel described herein has a width to depth (or height) ratio of 0.5 to 0.01, wherein the width is a measurement of the width at the narrowest segment of the microchannel. In some instances, a channel described herein has a width to depth (or height) ratio of about 0.01, 0.05, 0.1, 0.15, 0.16, 0.2, 0.5, or 1.
Described herein are structures for polynucleotide synthesis comprising a plurality of discrete loci. Structures comprise, without limitation, substantially planar regions, channels, protrusions, or wells for polynucleotide synthesis. In some instances, structures described herein are provided comprising a plurality of channels, wherein the height or depth of the channel is from about 5 μm to about 500 um, from about 5 μm to about 400 um, from about 5 μm to about 300 um, from about 5 μm to about 200 um, from about 5 μm to about 100 um, from about 5 μm to about 50 um, or from about 10 μm to about 50 um. In some cases, the height of a channel is less than 100 um, less than 80 um, less than 60 um, less than 40 μm or less than 20 um. In some cases, channel height is about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500 μm or more. In some instances, the height or depth of the channel is at least 10, 25, 50, 75, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or more than 1000 nm. In some instances, the height or depth of the channel is in a range of about 10 nm to about 1000 nm, about 25 nm to about 900 nm, about 50 nm to about 800 nm, about 75 nm to about 700 nm, about 100 nm to about 600 nm, or about 200 nm to about 500. Channels described herein may be arranged on a surface in clusters or as a homogenous field.
The width of a locus on the surface of a structure for polynucleotide synthesis described herein may be from about 0.1 μm to about 500 um, from about 0.5 μm to about 500 um, from about 1 μm to about 200 um, from about 1 μm to about 100 um, from about 5 μm to about 100 um, or from about 0.1 μm to about 100 um, for example, about 90 um, 80 um, 70 um, 60 um, 50 um, 40 um, 30 um, 20 um, 10 um, 5 um, 1 μm or 0.5 um. In some instances, the width of a locus is less than about 100 um, 90 um, 80 um, 70 um, 60 um, 50 um, 40 um, 30 um, 20 μm or 10 um. In some instances, the width of a locus is at least 10, 25, 50, 75, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or more than 1000 nm. In some instances, the width of a locus is in a range of about 10 nm to about 1000 nm, about 25 nm to about 900 nm, about 50 nm to about 800 nm, about 75 nm to about 700 nm, about 100 nm to about 600 nm, or about 200 nm to about 500. In some instances, the width of a locus is in a range of about 50 nm to about 1000 nm. In some instances, the distance between the center of two adjacent loci is from about 0.1 μm to about 500 um, 0.5 μm to about 500 um, from about 1 μm to about 200 um, from about 1 μm to about 100 um, from about 5 μm to about 200 um, from about 5 μm to about 100 um, from about 5 μm to about 50 um, or from about 5 μm to about 30 um, for example, about 20 um. In some instances, the total width of a locus is about 5 um, 10 um, 20 um, 30 um, 40 um, 50 um, 60 um, 70 um, 80 um, 90 um, or 100 um. In some instances, the total width of a locus is about 1 μm to 100 um, 30 μm to 100 um, or 50 μm to 70 um. In some instances, the distance between the center of two adjacent loci is from about 0.5 μm to about 2 um, 0.5 μm to about 2 um, from about 0.75 μm to about 2 um, from about 1 μm to about 2 um, from about 0.2 μm to about 1 um, from about 0.5 μm to about 1.5 um, from about 0.5 μm to about 0.8 um, or from about 0.5 μm to about 1 um, for example, about 1 um. In some instances, the total width of a locus is about 50 nm, 0.1 um, 0.2 um, 0.3 um, 0.4 um, 0.5 um, 0.6 um, 0.7 um, 0.8 um, 0.9 um, 1 um, 1.1 um, 1.2 um, 1.3 um, 1.4 um, or 1.5 um. In some instances, the total width of a locus is about 0.5 μm to 2 um, 0.75 μm to 1 um, or 0.9 μm to 2 um. In some instances, a locus is substantially planer.
In some instances, each locus supports the synthesis of a population of polynucleotides having a different sequence than a population of polynucleotides grown on another locus. Provided herein are surfaces which comprise at least 10, 100, 256, 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000, 20000, 30000, 40000, 50000 or more clusters. Provided herein are surfaces which comprise more than 2,000; 5,000; 10,000; 20,000; 30,000; 50,000; 100,000; 200,000; 300,000; 400,000; 500,000; 600,000; 700,000; 800,000; 900,000; 1,000,000; 5,000,000; or 10,000,000 or more distinct loci. In some cases, each cluster includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 130, 150, 200, 500 or more loci. In some cases, each cluster includes 50 to 500, 50 to 200, 50 to 150, or 100 to 150 loci. In some cases, each cluster includes 100 to 150 loci. In some instances, each cluster includes 109, 121, 130 or 137 loci.
Provided herein are loci having a width at the longest segment of 5 to 100 um. In some cases, the loci have a width at the longest segment of about 30, 35, 40, 45, 50, 55 or 60 um. In some cases, the loci are channels having multiple segments, wherein each segment has a center to center distance apart of 5 to 50 um. In some cases, the center to center distance apart for each segment is about 5, 10, 15, 20 or 25 um.
Provided herein are loci having a width at the longest segment of 5 to 500 nm. In some cases, the loci have a width at the longest segment of about 30, 35, 40, 45, 50, 55, 60, 80, or 100 nm. In some cases, the loci are channels having multiple segments, wherein each segment has a center to center distance apart of 5 to 50 nm. In some cases, the center to center distance apart for each segment is about 5, 10, 15, 20, 25, 50, 100, or 200 nm.
In some instances, the number of distinct polynucleotides synthesized on the surface of a structure described herein is dependent on the number of distinct loci available in the substrate. In some instances, the density of loci within a cluster of a substrate is at least or about 1 locus per mm2, 10 loci per mm2, 25 loci per mm2, 50 loci per mm2, 65 loci per mm2, 75 loci per mm2, 100 loci per mm2, 130 loci per mm2, 150 loci per mm2, 175 loci per mm2, 200 loci per mm2, 300 loci per mm2, 400 loci per mm2, 500 loci per mm2, 1,000 loci per mm2,104 loci per mm2, 105 loci per mm2, 106 loci per mm2, or more. In some cases, a substrate comprises from about 10 loci per mm2 to about 500 mm2, from about 25 loci per mm2 to about 400 mm2, from about 50 loci per mm2 to about 500 mm2, from about 100 loci per mm2 to about 500 mm2, from about 150 loci per mm2 to about 500 mm2, from about 10 loci per mm2 to about 250 mm2, from about 50 loci per mm2 to about 250 mm2, from about 10 loci per mm2 to about 200 mm2, or from about 50 loci per mm2 to about 200 mm2. In some cases, a substrate comprises from about 104 loci per mm2 to about 105 mm2. In some cases, a substrate comprises from about 105 loci per mm2 to about 107 mm2. In some cases, a substrate comprises at least 105 loci per mm2. In some cases, a substrate comprises at least 106 loci per mm2. In some cases, a substrate comprises at least 107 loci per mm2. In some cases, a substrate comprises from about 104 loci per mm2 to about 105 mm2. In some instances, the density of loci within a cluster of a substrate is at least or about 1 locus per um2, 10 loci per um2, 25 loci per um2, 50 loci per um2, 65 loci per um2, 75 loci per um2, 100 loci per um2, 130 loci per um2, 150 loci per um2, 175 loci per um2, 200 loci per um2, 300 loci per um2, 400 loci per um2, 500 loci per um2, 1,000 loci per um2 or more. In some cases, a substrate comprises from about 10 loci per um2 to about 500 um2, from about 25 loci per um2 to about 400 um2, from about 50 loci per um2 to about 500 um2, from about 100 loci per um2 to about 500 um2, from about 150 loci per um2 to about 500 um2, from about 10 loci per um2 to about 250 um2, from about 50 loci per um2 to about 250 um2, from about 10 loci per um2 to about 200 um2, or from about 50 loci per um2 to about 200 μm2.
In some instances, the distance between the centers of two adjacent loci within a cluster is from about 10 μm to about 500 um, from about 10 μm to about 200 um, or from about 10 μm to about 100 um. In some cases, the distance between two centers of adjacent loci is greater than about 10 um, 20 um, 30 um, 40 um, 50 um, 60 um, 70 um, 80 um, 90 μm or 100 um. In some cases, the distance between the centers of two adjacent loci is less than about 200 um, 150 um, 100 um, 80 um, 70 um, 60 um, 50 um, 40 um, 30 um, 20 μm or 10 um. In some cases, the distance between the centers of two adjacent loci is less than about 10000 nm, 8000 nm, 6000 nm, 4000 nm, 2000 nm 1000 nm, 800 nm, 600 nm, 400 nm, 200 nm, 150 nm, 100 nm, 80 um, 70 nm, 60 nm, 50 nm, 40 nm, 30 nm, 20 nm or 10 nm. In some instances, each square meter of a structure described herein allows for at least 107, 108, 109, 1010, 1011 loci, where each locus supports one polynucleotide. In some instances, 109 polynucleotides are supported on less than about 6, 5, 4, 3, 2 or 1 m2 of a structure described herein.
In some instances, a structure described herein provides support for the synthesis of more than 2,000; 5,000; 10,000; 20,000; 30,000; 50,000; 100,000; 200,000; 300,000; 400,000; 500,000; 600,000; 700,000; 800,000; 900,000; 1,000,000; 1,200,000; 1,400,000; 1,600,000; 1,800,000; 2,000,000; 2,500,000; 3,000,000; 3,500,000; 4,000,000; 4,500,000; 5,000,000; 10,000,000 or more non-identical polynucleotides. In some cases, the structure provides support for the synthesis of more than 2,000; 5,000; 10,000; 20,000; 50,000; 100,000; 200,000; 300,000; 400,000; 500,000; 600,000; 700,000; 800,000; 900,000; 1,000,000; 1,200,000; 1,400,000; 1,600,000; 1,800,000; 2,000,000; 2,500,000; 3,000,000; 3,500,000; 4,000,000; 4,500,000; 5,000,000; 10,000,000 or more polynucleotides encoding for distinct sequences. In some instances, at least a portion of the polynucleotides have an identical sequence or are configured to be synthesized with an identical sequence. In some instances, the structure provides a surface environment for the growth of polynucleotides having at least 50, 60, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 bases or more. In some arrangements, structures for polynucleotide synthesis described herein comprise sites for polynucleotide synthesis in a uniform arrangement.
In some instances, polynucleotides are synthesized on distinct loci of a structure, wherein each locus supports the synthesis of a population of polynucleotides. In some cases, each locus supports the synthesis of a population of polynucleotides having a different sequence than a population of polynucleotides grown on another locus. In some instances, the loci of a structure are located within a plurality of clusters. In some instances, a structure comprises at least 10, 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000, 20000, 30000, 40000, 50000 or more clusters. In some instances, a structure comprises more than 2,000; 5,000; 10,000; 100,000; 200,000; 300,000; 400,000; 500,000; 600,000; 700,000; 800,000; 900,000; 1,000,000; 1,100,000; 1,200,000; 1,300,000; 1,400,000; 1,500,000; 1,600,000; 1,700,000; 1,800,000; 1,900,000; 2,000,000; 300,000; 400,000; 500,000; 600,000; 700,000; 800,000; 900,000; 1,000,000; 1,200,000; 1,400,000; 1,600,000; 1,800,000; 2,000,000; 2,500,000; 3,000,000; 3,500,000; 4,000,000; 4,500,000; 5,000,000; or 10,000,000 or more distinct loci. In some cases, each cluster includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 130, 150 or more loci. In some instances, each cluster includes 50 to 500, 100 to 150, or 100 to 200 loci. In some instances, each cluster includes 109, 121, 130 or 137 loci. In some instances, each cluster includes 5, 6, 7, 8, 9, 10, 11 or 12 loci. In some instances, polynucleotides from distinct loci within one cluster have sequences that, when assembled, encode for a contiguous longer polynucleotide of a predetermined sequence.
Structure Size
In some instances, a structure described herein is about the size of a plate (e.g., chip or wafer), for example between about 40 and 120 mm by between about 25 and 100 mm. In some instances, a structure described herein has a diameter less than or equal to about 1000 mm, 500 mm, 450 mm, 400 mm, 300 mm, 250 nm, 200 mm, 150 mm, 100 mm or 50 mm. In some instances, the diameter of a substrate is between about 25 mm and 1000 mm, between about 25 mm and about 800 mm, between about 25 mm and about 600 mm, between about 25 mm and about 500 mm, between about 25 mm and about 400 mm, between about 25 mm and about 300 mm, or between about 25 mm and about 200. Non-limiting examples of substrate size include about 300 mm, 200 mm, 150 mm, 130 mm, 100 mm, 84 mm, 76 mm, 54 mm, 51 mm and 25 mm. In some instances, a substrate has a planar surface area of at least 100 mm2; 200 mm2; 500 mm2; 1,000 mm2; 2,000 mm2; 4,500 mm2; 5,000 mm2; 10,000 mm2; 12,000 mm2; 15,000 mm2; 20,000 mm2; 30,000 mm2; 40,000 mm2; 50,000 mm2 or more. In some instances, the thickness is between about 50 mm and about 2000 mm, between about 50 mm and about 1000 mm, between about 100 mm and about 1000 mm, between about 200 mm and about 1000 mm, or between about 250 mm and about 1000 mm. Non-limiting examples thickness include 275 mm, 375 mm, 525 mm, 625 mm, 675 mm, 725 mm, 775 mm and 925 mm. In some instances, the thickness is at least or about 0.5 mm, 1.0 mm, 1.5 mm, 2.0 mm, 2.5 mm, 3.0 mm, 3.5 mm, 4.0 mm, or more than 4.0 mm. In some cases, the thickness of varies with diameter and depends on the composition of the substrate. For example, a structure comprising materials other than silicon may have a different thickness than a silicon structure of the same diameter. Structure thickness may be determined by the mechanical strength of the material used and the structure must be thick enough to support its own weight without cracking during handling. In some instances, a structure is more than about 1, 2, 3, 4, 5, 10, 15, 30, 40, 50 feet in any one dimension. In some instances, a structure comprises an array of polynucleotide synthesis devices. In some instances, a structure is integrated into a CMOS.
Materials
Provided herein are devices comprising a surface, wherein the surface is modified to support polynucleotide synthesis at predetermined locations and with a resulting low error rate, a low dropout rate, a high yield, and a high oligo representation. In some instances, surfaces of devices for polynucleotide synthesis provided herein are fabricated from a variety of materials capable of modification to support a de novo polynucleotide synthesis reaction. In some cases, the devices are sufficiently conductive, e.g., are able to form uniform electric fields across all or a portion of the devices. In some instances, devices comprises one or more conducting layers. Devices described herein may comprise a flexible material. Exemplary flexible materials include, without limitation, modified nylon, unmodified nylon, nitrocellulose, and polypropylene. Devices described herein may comprise a rigid material. Exemplary rigid materials include, without limitation, glass, fuse silica, silicon, silicon dioxide, silicon nitride, plastics (for example, polytetrafluoroethylene, polypropylene, polystyrene, polycarbonate, and blends thereof, and metals (for example, gold, platinum). Devices disclosed herein may be fabricated from a material comprising silicon, polystyrene, agarose, dextran, cellulosic polymers, polyacrylamides, polydimethylsiloxane (PDMS), glass, or any combination thereof. In some cases, devices disclosed herein are manufactured with a combination of materials listed herein or any other suitable material known in the art.
Devices described herein may comprise material having a range of tensile strength. Exemplary materials having a range of tensile strengths include, but are not limited to, nylon (70 MPa), nitrocellulose (1.5 MPa), polypropylene (40 MPa), silicon (268 MPa), polystyrene (40 MPa), agarose (1-10 MPa), polyacrylamide (1-10 MPa), polydimethylsiloxane (PDMS) (3.9-10.8 MPa). Solid supports described herein can have a tensile strength from 1 to 300, 1 to 40, 1 to 10, 1 to 5, or 3 to 11 MPa. Solid supports described herein can have a tensile strength of about 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 20, 25, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 270, or more MPa. In some instances, a device described herein comprises a solid support for polynucleotide synthesis that is in the form of a flexible material capable of being stored in a continuous loop or reel, such as a tape or flexible sheet.
Young's modulus measures the resistance of a material to elastic (recoverable) deformation under load. Exemplary materials having a range of Young's modulus stiffness include, but are not limited to, nylon (3 GPa), nitrocellulose (1.5 GPa), polypropylene (2 GPa), silicon (150 GPa), polystyrene (3 GPa), agarose (1-10 GPa), polyacrylamide (1-10 GPa), polydimethylsiloxane (PDMS) (1-10 GPa). Solid supports described herein can have a Young's moduli from 1 to 500, 1 to 40, 1 to 10, 1 to 5, or 3 to 11 GPa. Solid supports described herein can have a Young's moduli of about 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 20, 25, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 400, 500 GPa, or more. As the relationship between flexibility and stiffness are inverse to each other, a flexible material has a low Young's modulus and changes its shape considerably under load. In some instances, a solid support described herein has a surface with a flexibility of at least nylon.
In some cases, devices disclosed herein comprise a silicon dioxide base and a surface layer of silicon oxide. Alternatively, the devices may have a base of silicon oxide. Surface of the devices provided here may be textured, resulting in an increase overall surface area for polynucleotide synthesis. Devices disclosed herein in some instances comprise at least 5%, 10%, 25%, 50%, 80%, 90%, 95%, or 99% silicon. Devices disclosed herein in some instances are fabricated from silicon on insulator (SOI) wafer.
The structure may be fabricated from a variety of materials, suitable for the methods and compositions of the invention described herein. In instances, the materials from which the substrates/solid supports of the comprising the invention are fabricated exhibit a low level of polynucleotide binding. In some situations, material that are transparent to visible and/or UV light can be employed. Materials that are sufficiently conductive, e.g. those that can form uniform electric fields across all or a portion of the substrates/solids support described herein, can be utilized. In some instances, such materials may be connected to an electric ground. In some cases, the substrate or solid support can be heat conductive or insulated. The materials can be chemical resistant and heat resistant to support chemical or biochemical reactions such as a series of polynucleotide synthesis reactions. For flexible materials, materials of interest can include: nylon, both modified and unmodified, nitrocellulose, polypropylene, and the like.
For rigid materials, specific materials of interest include: glass; fuse silica; silicon, plastics (for example polytetrafluoroethylene, polypropylene, polystyrene, polycarbonate, and blends thereof, and the like); metals (for example, gold, platinum, and the like). The structure can be fabricated from a material selected from the group consisting of silicon, polystyrene, agarose, dextran, cellulosic polymers, polyacrylamides, polydimethylsiloxane (PDMS), and glass. The substrates/solid supports or the microstructures, reactors therein may be manufactured with a combination of materials listed herein or any other suitable material known in the art.
In some instances, a substrate disclosed herein comprises a computer readable material. Computer readable materials include, without limitation, magnetic media, reel-to-reel tape, cartridge tape, cassette tape, flexible disk, paper media, film, microfiche, continuous tape (e.g., a belt) and any media suitable for storing electronic instructions. In some cases, the substrate comprises magnetic reel-to-reel tape or a magnetic belt. In some instances, the substrate comprises a flexible printed circuit board.
Structures described herein may be transparent to visible and/or UV light. In some instances, structures described herein are sufficiently conductive to form uniform electric fields across all or a portion of a structure. In some instances, structures described herein are heat conductive or insulated. In some instances, the structures are chemical resistant and heat resistant to support a chemical reaction such as a polynucleotide synthesis reaction. In some instances, the substrate is magnetic. In some instances, the structures comprise a metal or a metal alloy.
Structures for polynucleotide synthesis may be over 1, 2, 5, 10, 30, 50 or more feet long in any dimension. In the case of a flexible structure, the flexible structure is optionally stored in a wound state, e.g., in a reel. In the case of a large rigid structure, e.g., greater than 1 foot in length, the rigid structure can be stored vertically or horizontally.
Surface Preparation
Provided herein are methods to support the immobilization of a biomolecule on a substrate, where a surface of a structure described herein comprises a material and/or is coated with a material that facilitates a coupling reaction with the biomolecule for attachment. To prepare a structure for biomolecule immobilization, surface modifications may be employed that chemically and/or physically alter the substrate surface by an additive or subtractive process to change one or more chemical and/or physical properties of a substrate surface or a selected site or region of the surface. For example, surface modification involves (1) changing the wetting properties of a surface, (2) functionalizing a surface, i.e. providing, modifying or substituting surface functional groups, (3) defunctionalizing a surface, i.e. removing surface functional groups, (4) otherwise altering the chemical composition of a surface, e.g., through etching, (5) increasing or decreasing surface roughness, (6) providing a coating on a surface, e.g., a coating that exhibits wetting properties that are different from the wetting properties of the surface, and/or (7) depositing particulates on a surface. In some instances, the surface of a structure is selectively functionalized to produce two or more distinct areas on a structure, wherein at least one area has a different surface or chemical property that another area of the same structure. Such properties include, without limitation, surface energy, chemical termination, surface concentration of a chemical moiety, and the like.
In some instances, a surface of a structure disclosed herein is modified to comprise one or more actively functionalized surfaces configured to bind to both the surface of the substrate and a biomolecule, thereby supporting a coupling reaction to the surface. In some instances, the surface is also functionalized with a passive material that does not efficiently bind the biomolecule, thereby preventing biomolecule attachment at sites where the passive functionalization agent is bound. In some cases, the surface comprises an active layer only defining distinct loci for biomolecule support.
In some instances, the surface is contacted with a mixture of functionalization groups which are in any different ratio. In some instances, a mixture comprises at least 2, 3, 4, 5 or more different types of functionalization agents. In some cases, the ratio of the at least two types of surface functionalization agents in a mixture is about 1:1, 1:2, 1:5, 1:10, 2:10, 3:10, 4:10, 5:10, 6:10, 7:10, 8:10, 9:10, or any other ratio to achieve a desired surface representation of two groups. In some instances, desired surface tensions, wettabilities, water contact angles, and/or contact angles for other suitable solvents are achieved by providing a substrate surface with a suitable ratio of functionalization agents. In some cases, the agents in a mixture are chosen from suitable reactive and inert moieties, thus diluting the surface density of reactive groups to a desired level for downstream reactions. In some instances, the mixture of functionalization reagents comprises one or more reagents that bind to a biomolecule and one or more reagents that do not bind to a biomolecule. Therefore, modulation of the reagents allows for the control of the amount of biomolecule binding that occurs at a distinct area of functionalization.
In some instances, a method for substrate functionalization comprises deposition of a silane molecule onto a surface of a substrate. The silane molecule may be deposited on a high energy surface of the substrate. In some instances the high surface energy region includes a passive functionalization reagent. Methods described herein provide for a silane group to bind the surface, while the rest of the molecule provides a distance from the surface and a free hydroxyl group at the end to which a biomolecule attaches. In some instances, the silane is an organofunctional alkoxysilane molecule. Non-limiting examples of organofunctional alkoxysilane molecules include dimethylchloro-octodecyl-silane, methyldichloro-octodecyl-silane, trichloro-octodecyl-silane, and trimethyl-octodecyl-silane, triethyl-octodecyl-silane. In some instances, the silane is an amino silane. Examples of amino silanes include, without limitation, 11-acetoxyundecyltriethoxysilane, n-decyltriethoxysilane, (3-aminopropyl)trimethoxysilane, (3-aminopropyl)triethoxysilane, glycidyloxypropyl/trimethoxysilane and N-(3-triethoxysilylpropyl)-4-hydroxybutyramide. In some instances, the silane comprises 11-acetoxyundecyltriethoxysilane, n-decyltriethoxysilane, (3-aminopropyl)trimethoxysilane, (3-aminopropyl)triethoxysilane, glycidyloxypropyl/trimethoxysilane, N-(3-triethoxysilylpropyl)-4-hydroxybutyramide, or any combination thereof. In some instances, an active functionalization agent comprises 11-acetoxyundecyltriethoxysilane. In some instances, an active functionalization agent comprises n-decyltriethoxysilane. In some cases, an active functionalization agent comprises glycidyloxypropyltriethoxysilane (GOPS). In some instances, the silane is a fluorosilane. In some instances, the silane is a hydrocarbon silane. In some cases, the silane is 3-iodo-propyltrimethoxysilane. In some cases, the silane is octylchlorosilane.
In some instances, silanization is performed on a surface through self-assembly with organofunctional alkoxysilane molecules. The organofunctional alkoxysilanes are classified according to their organic functions. Non-limiting examples of siloxane functionalizing reagents include hydroxyalkyl siloxanes (silylate surface, functionalizing with diborane and oxidizing the alcohol by hydrogen peroxide), diol (dihydroxyalkyl) siloxanes (silylate surface, and hydrolyzing to diol), aminoalkyl siloxanes (amines require no intermediate functionalizing step), glycidoxysilanes (3-glycidoxypropyl-dimethyl-ethoxysilane, glycidoxy-trimethoxysilane), mercaptosilanes (3-mercaptopropyl-trimethoxysilane, 3-4 epoxycyclohexyl-ethyltrimethoxysilane or 3-mercaptopropyl-methyl-dimethoxysilane), bicyclohepthenyl-trichlorosilane, butyl-aldehydr-trimethoxysilane, or dimeric secondary aminoalkyl siloxanes. Exemplary hydroxyalkyl siloxanes include allyl trichlorochlorosilane turning into 3-hydroxypropyl, or 7-oct-1-enyl trichlorochlorosilane turning into 8-hydroxyoctyl. The diol (dihydroxyalkyl) siloxanes include glycidyl trimethoxysilane-derived (2,3-dihydroxypropyloxy)propyl (GOPS). The aminoalkyl siloxanes include 3-aminopropyl trimethoxysilane turning into 3-aminopropyl (3-aminopropyl-triethoxysilane, 3-aminopropyl-diethoxy-methylsilane, 3-aminopropyl-dimethyl-ethoxysilane, or 3-aminopropyl-trimethoxysilane). In some cases, the dimeric secondary aminoalkyl siloxanes is bis (3-trimethoxysilylpropyl) amine turning into bis(silyloxylpropyl)amine.
Active functionalization areas may comprise one or more different species of silanes, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more silanes. In some cases, one of the one or more silanes is present in the functionalization composition in an amount greater than another silane. For example, a mixed silane solution having two silanes comprises a 99:1, 98:2, 97:3, 96:4, 95:5, 94:6, 93:7, 92:8, 91:9, 90:10, 89:11, 88:12, 87:13, 86:14, 85:15, 84:16, 83:17, 82:18, 81:19, 80:20, 75:25, 70:30, 65:35, 60:40, 55:45 ratio of one silane to another silane. In some instances, an active functionalization agent comprises 11-acetoxyundecyltriethoxysilane and n-decyltriethoxysilane. In some instances, an active functionalization agent comprises 11-acetoxyundecyltriethoxysilane and n-decyltriethoxysilane in a ratio from about 20:80 to about 1:99, or about 10:90 to about 2:98, or about 5:95.
In some instances, functionalization comprises deposition of a functionalization agent to a structure by any deposition technique, including, but not limiting to, chemical vapor deposition (CVD), atomic layer deposition (ALD), plasma enhanced CVD (PECVD), plasma enhanced ALD (PEALD), metal organic CVD (MOCVD), hot wire CVD (HWCVD), initiated CVD (iCVD), modified CVD (MCVD), vapor axial deposition (VAD), outside vapor deposition (OVD), physical vapor deposition (e.g., sputter deposition, evaporative deposition), and molecular layer deposition (MLD).
Any step or component in the following functionalization process be omitted or changed in accordance with properties desired of the final functionalized substrate. In some cases, additional components and/or process steps are added to the process workflows embodied herein. In some instances, a substrate is first cleaned, for example, using a piranha solution. An example of a cleaning process includes soaking a substrate in a piranha solution (e.g., 90% H2SO4, 10% H2O2) at an elevated temperature (e.g., 120° C.) and washing (e.g., water) and drying the substrate (e.g., nitrogen gas). The process optionally includes a post piranha treatment comprising soaking the piranha treated substrate in a basic solution (e.g., NH4OH) followed by an aqueous wash (e.g., water). In some instances, a surface of a structure is plasma cleaned, optionally following the piranha soak and optional post piranha treatment. An example of a plasma cleaning process comprises an oxygen plasma etch. In some instances, the surface is deposited with an active functionalization agent following by vaporization. In some instances, the substrate is actively functionalized prior to cleaning, for example, by piranha treatment and/or plasma cleaning.
The process for surface functionalization optionally comprises a resist coat and a resist strip. In some instances, following active surface functionalization, the substrate is spin coated with a resist, for example, SPR™ 3612 positive photoresist. The process for surface functionalization, in various instances, comprises lithography with patterned functionalization. In some instances, photolithography is performed following resist coating. In some instances, after lithography, the surface is visually inspected for lithography defects. The process for surface functionalization, in some instances, comprises a cleaning step, whereby residues of the substrate are removed, for example, by plasma cleaning or etching. In some instances, the plasma cleaning step is performed at some step after the lithography step.
In some instances, a surface coated with a resist is treated to remove the resist, for example, after functionalization and/or after lithography. In some cases, the resist is removed with a solvent, for example, with a stripping solution comprising N-methyl-2-pyrrolidone. In some cases, resist stripping comprises sonication or ultrasonication. In some instances, a resist is coated and stripped, followed by active functionalization of the exposed areas to create a desired differential functionalization pattern.
In some instances, the methods and compositions described herein relate to the application of photoresist for the generation of modified surface properties in selective areas, wherein the application of the photoresist relies on the fluidic properties of the surface defining the spatial distribution of the photoresist. Without being bound by theory, surface tension effects related to the applied fluid may define the flow of the photoresist. For example, surface tension and/or capillary action effects may facilitate drawing of the photoresist into small structures in a controlled fashion before the resist solvents evaporate. In some instances, resist contact points are pinned by sharp edges, thereby controlling the advance of the fluid. The underlying structures may be designed based on the desired flow patterns that are used to apply photoresist during the manufacturing and functionalization processes. A solid organic layer left behind after solvents evaporate may be used to pursue the subsequent steps of the manufacturing process. Structures may be designed to control the flow of fluids by facilitating or inhibiting wicking effects into neighboring fluidic paths. For example, a structure is designed to avoid overlap between top and bottom edges, which facilitates the keeping of the fluid in top structures allowing for a particular disposition of the resist. In an alternative example, the top and bottom edges overlap, leading to the wicking of the applied fluid into bottom structures. Appropriate designs may be selected accordingly, depending on the desired application of the resist.
In some instances, a structure described herein has a surface that comprises a material having thickness of at least or at least 0.1 nm, 0.5 nm, 1 nm, 2 nm, 5 nm, 10 nm or 25 nm that comprises a reactive group capable of binding nucleosides. Exemplary include, without limitation, glass and silicon, such as silicon dioxide and silicon nitride. In some cases, exemplary surfaces include nylon and PMMA.
In some instances, electromagnetic radiation in the form of UV light is used for surface patterning. In some instances, a lamp is used for surface patterning, and a mask mediates exposure locations of the UV light to the surface. In some instances, a laser is used for surface patterning, and a shutter opened/closed state controls exposure of the UV light to the surface. The laser arrangement may be used in combination with a flexible structure that is capable of moving. In such an arrangement, the coordination of laser exposure and flexible structure movement is used to create patterns of one or more agents having differing nucleoside coupling capabilities.
Described herein are surfaces for polynucleotide synthesis that are reusable. After synthesis and/or cleavage of polynucleotides, a surface may be bathed, washed, cleaned, baked, etched, or otherwise functionally restored to a condition suitable for subsequent polynucleotide synthesis. The number of times a surface is reused and the methods for recycling/preparing the surface for reuse vary depending on subsequent applications. Surfaces prepared for reuse are in some instances reused at least 1, 2, 3, 5, 10, 20, 50, 100, 1,000 or more times. In some instances, the remaining “life” or number of times a surface is suitable for reuse is measured or predicted.
Material Deposition Systems
In some cases, the synthesized polynucleotides are stored on the substrate, for example a solid support. Nucleic acid reagents may be deposited on the substrate surface in a non-continuous, or drop-on-demand method. Examples of such methods include the electromechanical transfer method, electric thermal transfer method, and electrostatic attraction method. In the electromechanical transfer method, piezoelectric elements deformed by electrical pulses cause the droplets to be ejected. In the electric thermal transfer method, bubbles are generated in a chamber of the device, and the expansive force of the bubbles causes the droplets to be ejected. In the electrostatic attraction method, electrostatic force of attraction is used to eject the droplets onto the substrate. In some cases, the drop frequency is from about 5 KHz to about 500 KHz; from about 5 KHz to about 100 KHz; from about 10 KHz to about 500 KHz; from about 10 KHz to about 100 KHz; or from about 50 KHz to about 500 KHz. In some cases, the frequency is less than about 500 KHz, 200 KHz, 100 KHz, or 50 KHz.
The size of the droplets dispensed correlates to the resolution of the device. In some instances, the devices deposit droplets of reagents at sizes from about 0.01 pl to about 20 pl, from about 0.01 pl to about 10 pl, from about 0.01 pl to about 1 pl, from about 0.01 pl to about 0.5 pl, from about 0.01 pl to about 0.01 pl, or from about 0.05 pl to about 1 pl. In some instances, the droplet size is less than about 1 pl, 0.5 pl, 0.2 pl, 0.1 pl, or 0.05 pl.
In some arrangements, the configuration of a polynucleotide synthesis system allows for a continuous polynucleotide synthesis process that exploits the flexibility of a substrate for traveling in a reel-to-reel type process. This synthesis process operates in a continuous production line manner with the substrate travelling through various stages of polynucleotide synthesis using one or more reels to rotate the position of the substrate. In an exemplary instance, a polynucleotide synthesis reaction comprises rolling a substrate: through a solvent bath, beneath a deposition device for phosphoramidite deposition, through a bath of oxidizing agent, through an acetonitrile wash bath, and through a deblock bath. Optionally, the tape is also traversed through a capping bath. A reel-to-reel type process allows for the finished product of a substrate comprising synthesized polynucleotides to be easily gathered on a take-up reel, where it can be transported for further processing or storage.
In some arrangements, polynucleotide synthesis proceeds in a continuous process as a continuous flexible tape is conveyed along a conveyor belt system. Similar to the reel-to-reel type process, polynucleotide synthesis on a continuous tape operates in a production line manner, with the substrate travelling through various stages of polynucleotide synthesis during conveyance. However, in a conveyor belt process, the continuous tape revisits a polynucleotide synthesis step without rolling and unrolling of the tape, as in a reel-to-reel process. In some arrangements, polynucleotide synthesis steps are partitioned into zones and a continuous tape is conveyed through each zone one or more times in a cycle. For example, a polynucleotide synthesis reaction may comprise (1) conveying a substrate through a solvent bath, beneath a deposition device for phosphoramidite deposition, through a bath of oxidizing agent, through an acetonitrile wash bath, and through a block bath in a cycle; and then (2) repeating the cycles to achieve synthesized polynucleotides of a predetermined length. After polynucleotide synthesis, the flexible substrate is removed from the conveyor belt system and, optionally, rolled for storage. Rolling may be around a reel, for storage. In some instances, a flexible substrate comprising thermoplastic material is coated with nucleoside coupling reagent. The coating is patterned into loci such that each locus has diameter of about 10 um, with a center-to-center distance between two adjacent loci of about 21 um. In this instance, the locus size is sufficient to accommodate a sessile drop volume of 0.2 pl during a polynucleotide synthesis deposition step. In some cases, the locus density is about 2.2 billion loci per m2 (1 locus/441×10−12 m2). In some cases, a 4.5 m2 substrate comprise about 10 billion loci, each with a 10 μm diameter.
In some arrangements, a device for application of one or more reagents to a substrate during a synthesis reaction is configured to deposit reagents and/or nucleoside monomers for nucleoside phosphoramidite based synthesis. Reagents for polynucleotide synthesis include reagents for polynucleotide extension and wash buffers. As non-limiting examples, the device deposits cleaning reagents, coupling reagents, capping reagents, oxidizers, de-blocking agents (including agents which form deblocking agents using electrochemistry), acetonitrile, gases such as nitrogen gas, and any combination thereof. In addition, the device optionally deposits reagents for the preparation and/or maintenance of substrate integrity. In some instances, the polynucleotide synthesizer deposits a drop having a diameter less than about 200 um, 100 um, or 50 μm in a volume less than about 1000, 500, 100, 50, or 20 pl. In some cases, the polynucleotide synthesizer deposits between about 1 and 10000, 1 and 5000, 100 and 5000, or 1000 and 5000 droplets per second.
Described herein are devices, methods, systems and compositions where reagents for polynucleotide synthesis are recycled or reused. Recycling of reagents may comprise collection, storage, and usage of unused reagents, or purification/transformation of used reagents. For example, a reagent bath is recycled and used for a polynucleotide synthesis step on the same or a different surface. Reagents described herein may be recycled 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times. Alternatively or in combination, a reagent solution comprising a reaction byproduct is filtered to remove the byproduct, and the reagent solution is used for additional polynucleotide synthesis reactions.
Many integrated or non-integrated elements are often used with polynucleotide synthesis systems. In some instances, a polynucleotide synthesis system comprises one or more elements useful for downstream processing of synthesized polynucleotides. As an example, the system comprises a temperature control element such as a thermal cycling device. In some instances, the temperature control element is used with a plurality of resolved reactors to perform nucleic acid assembly such as PCA and/or nucleic acid amplification such as PCR.
De Novo Polynucleotide Synthesis
Provided herein are systems and methods for synthesis of a high density of polynucleotides on a substrate in a short amount of time. In some instances, methods comprise use of electrochemical deprotection. In some instances, the substrate is a flexible substrate. In some instances, at least 1010, 1011, 1012, 1013, 1014, or 1015 bases are synthesized in one day. In some instances, at least 10×108, 10×109, 10×1010, 10×1011, or 10×1012 polynucleotides are synthesized in one day. In some cases, each polynucleotide synthesized comprises at least 20, 50, 100, 200, 300, 400 or 500 nucleobases. In some cases, these bases are synthesized with a total average error rate of less than about 1 in 100; 200; 300; 400; 500; 1000; 2000; 5000; 10000; 15000; 20000 bases. In some instances, these error rates are for at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.5%, or more of the polynucleotides synthesized. In some instances, these at least 90%, 95%, 98%, 99%, 99.5%, or more of the polynucleotides synthesized do not differ from a predetermined sequence for which they encode. In some instances, the error rate for synthesized polynucleotides on a substrate using the methods and systems described herein is less than about 1 in 200. In some instances, the error rate for synthesized polynucleotides on a substrate using the methods and systems described herein is less than about 1 in 1,000. In some instances, the error rate for synthesized polynucleotides on a substrate using the methods and systems described herein is less than about 1 in 2,000. In some instances, the error rate for synthesized polynucleotides on a substrate using the methods and systems described herein is less than about 1 in 3,000. In some instances, the error rate for synthesized polynucleotides on a substrate using the methods and systems described herein is less than about 1 in 5,000. Individual types of error rates include mismatches, deletions, insertions, and/or substitutions for the polynucleotides synthesized on the substrate. The term “error rate” refers to a comparison of the collective amount of synthesized polynucleotide to an aggregate of predetermined polynucleotide sequences. In some instances, synthesized polynucleotides disclosed herein comprise a tether of 12 to 25 bases. In some instances, the tether comprises 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more bases.
Described herein are methods, systems, devices, and compositions wherein chemical reactions used in polynucleotide synthesis are controlled using electrochemistry. Electrochemical reactions in some instances are controlled by any source of energy, such as light, heat, radiation, or electricity. For example, electrodes are used to control chemical reactions as all or a portion of discrete loci on a surface. Electrodes in some instances are charged by applying an electrical potential to the electrode to control one or more chemical steps in polynucleotide synthesis. In some instances, these electrodes are addressable. Any number of the chemical steps described herein is in some instances controlled with one or more electrodes. Electrochemical reactions may comprise oxidations, reductions, acid/base chemistry, or other reaction that is controlled by an electrode. In some instances, electrodes generate electrons or protons that are used as reagents for chemical transformations. Electrodes in some instances directly generate a reagent such as an acid. In some instances, an acid is a proton. Electrodes in some instances directly generate a reagent such as a base. Acids or bases are often used to cleave protecting groups, or influence the kinetics of various polynucleotide synthesis reactions, for example by adjusting the pH of a reaction solution. Electrochemically controlled polynucleotide synthesis reactions in some instances comprise redox-active metals or other redox-active organic materials. In some instances, metal or organic catalysts are employed with these electrochemical reactions. In some instances, acids are generated from oxidation of quinones.
Control of chemical reactions with is not limited to the electrochemical generation of reagents; chemical reactivity may be influenced indirectly through biophysical changes to substrates or reagents through electric fields (or gradients) which are generated by electrodes. In some instances, substrates include but are not limited to nucleic acids. In some instances, electrical fields which repel or attract specific reagents or substrates towards or away from an electrode or surface are generated. Such fields in some instances are generated by application of an electrical potential to one or more electrodes. For example, negatively charged nucleic acids are repelled from negatively charged electrode surfaces. Such repulsions or attractions of polynucleotides or other reagents caused by local electric fields in some instances provides for movement of polynucleotides or other reagents in or out of region of the synthesis device or structure. In some instances, electrodes generate electric fields which repel polynucleotides away from a synthesis surface, structure, or device. In some instances, electrodes generate electric fields which attract polynucleotides towards a synthesis surface, structure, or device. In some instances, protons are repelled from a positively charged surface to limit contact of protons with substrates or portions thereof. In some instances, repulsion or attractive forces are used to allow or block entry of reagents or substrates to specific areas of the synthesis surface. In some instances, nucleoside monomers are prevented from contacting a polynucleotide chain by application of an electric field in the vicinity of one or both components. Such arrangements allow gating of specific reagents, which may obviate the need for protecting groups when the concentration or rate of contact between reagents and/or substrates is controlled. In some instances, unprotected nucleoside monomers are used for polynucleotide synthesis. Alternatively, application of the field in the vicinity of one or both components promotes contact of nucleoside monomers with a polynucleotide chain. Additionally, application of electric fields to a substrate can alter the substrates reactivity or conformation. In an exemplary application, electric fields generated by electrodes are used to prevent polynucleotides at adjacent loci from interacting. In some instances, the substrate is a polynucleotide, optionally attached to a surface. Application of an electric field in some instances alters the three-dimensional structure of a polynucleotide. Such alterations comprise folding or unfolding of various structures, such as helices, hairpins, loops, or other 3-dimensional nucleic acid structure. Such alterations are useful for manipulating nucleic acids inside of wells, channels, or other structures. In some instances, electric fields are applied to a nucleic acid substrate to prevent secondary structures. In some instances, electric fields obviate the need for linkers or attachment to a solid support during polynucleotide synthesis.
A suitable method for polynucleotide synthesis on a substrate of this disclosure is a phosphoramidite method comprising the controlled addition of a phosphoramidite building block, i.e. nucleoside phosphoramidite, to a growing polynucleotide chain in a coupling step that forms a phosphite triester linkage between the phosphoramidite building block and a nucleoside bound to the substrate. In some instances, the nucleoside phosphoramidite is provided to the substrate activated. In some instances, the nucleoside phosphoramidite is provided to the substrate with an activator. In some instances, nucleoside phosphoramidites are provided to the substrate in a 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100-fold excess or more over the substrate-bound nucleosides. In some instances, the addition of nucleoside phosphoramidite is performed in an anhydrous environment, for example, in anhydrous acetonitrile. Following addition and linkage of a nucleoside phosphoramidite in the coupling step, the substrate is optionally washed. In some instances, the coupling step is repeated one or more additional times, optionally with a wash step between nucleoside phosphoramidite additions to the substrate. In some instances, a polynucleotide synthesis method used herein comprises 1, 2, 3 or more sequential coupling steps. Prior to coupling, in many cases, the nucleoside bound to the substrate is de-protected by removal of a protecting group, where the protecting group functions to prevent polymerization. Protecting groups may comprise any chemical group that prevents extension of the polynucleotide chain. In some instances, the protecting group is cleaved (or removed) in the presence of an acid. In some instances, the protecting group is cleaved in the presence of a base. In some instances, the protecting group is removed with electromagnetic radiation such as light, heat, or other energy source. In some instances, the protecting group is removed through an oxidation or reduction reaction. In some instances, a protecting group comprises a triarylmethyl group. In some instances, a protecting group comprises an aryl ether. In some instances, a protecting comprises a disulfide. In some instances a protecting group comprises an acid-labile silane. In some instances, a protecting group comprises an acetal. In some instances, a protecting group comprises a ketal. In some instances, a protecting group comprises an enol ether. In some instances, a protecting group comprises a methoxybenzyl group. In some instances, a protecting group comprises an azide. In some instances, a protecting group is 4,4′-dimethoxytrityl (DMT). In some instances, a protecting group is a tert-butyl carbonate. In some instances, a protecting group is a tert-butyl ester. In some instances, a protecting group comprises a base-labile group.
Following coupling, phosphoramidite polynucleotide synthesis methods optionally comprise a capping step. In a capping step, the growing polynucleotide is treated with a capping agent. A capping step generally serves to block unreacted substrate-bound 5′-OH groups after coupling from further chain elongation, preventing the formation of polynucleotides with internal base deletions. Further, phosphoramidites activated with 1H-tetrazole often react, to a small extent, with the O6 position of guanosine. Without being bound by theory, upon oxidation with I2/water, this side product, possibly via O6-N7 migration, undergoes depurination. The apurinic sites can end up being cleaved in the course of the final deprotection of the polynucleotide thus reducing the yield of the full-length product. The O6 modifications may be removed by treatment with the capping reagent prior to oxidation with I2/water. In some instances, inclusion of a capping step during polynucleotide synthesis decreases the error rate as compared to synthesis without capping. As an example, the capping step comprises treating the substrate-bound polynucleotide with a mixture of acetic anhydride and 1-methylimidazole. Following a capping step, the substrate is optionally washed.
Following addition of a nucleoside phosphoramidite, and optionally after capping and one or more wash steps, a substrate described herein comprises a bound growing nucleic acid that may be oxidized. The oxidation step comprises oxidizing the phosphite triester into a tetracoordinated phosphate triester, a protected precursor of the naturally occurring phosphate diester internucleoside linkage. In some instances, phosphite triesters are oxidized electrochemically. In some instances, oxidation of the growing polynucleotide is achieved by treatment with iodine and water, optionally in the presence of a weak base such as a pyridine, lutidine, or collidine. Oxidation is sometimes carried out under anhydrous conditions using tert-Butyl hydroperoxide or (1S)-(+)-(10-camphorsulfonyl)-oxaziridine (CSO). In some methods, a capping step is performed following oxidation. A second capping step allows for substrate drying, as residual water from oxidation that may persist can inhibit subsequent coupling. Following oxidation, the substrate and growing polynucleotide is optionally washed. In some instances, the step of oxidation is substituted with a sulfurization step to obtain polynucleotide phosphorothioates, wherein any capping steps can be performed after the sulfurization. Many reagents are capable of the efficient sulfur transfer, including, but not limited to, 3-(Dimethylaminomethylidene)amino)-3H-1,2,4-dithiazole-3-thione, DDTT, 3H-1,2-benzodithiol-3-one 1,1-dioxide, also known as Beaucage reagent, and N,N,N′N′-Tetraethylthiuram disulfide (TETD).
For a subsequent cycle of nucleoside incorporation to occur through coupling, a protected 5′ end (or 3′ end, if synthesis is conducted in a 5′ to 3′ direction) of the substrate bound growing polynucleotide is be removed so that the primary hydroxyl group can react with a next nucleoside phosphoramidite. In some instances, the protecting group is DMT and deblocking occurs with trichloroacetic acid in dichloromethane. In some instances, the protecting group is DMT and deblocking occurs with electrochemically generated protons. Conducting detritylation for an extended time or with stronger than recommended solutions of acids may lead to increased depurination of solid support-bound polynucleotide and thus reduces the yield of the desired full-length product. Methods and compositions described herein provide for controlled deblocking conditions limiting undesired depurination reactions. In some instances, the substrate bound polynucleotide is washed after deblocking. In some cases, efficient washing after deblocking contributes to synthesized polynucleotides having a low error rate.
Methods for the synthesis of polynucleotides on a substrate described herein may involve an iterating sequence of the following steps: application of a protected monomer to a surface of a substrate feature to link with either the surface, a linker or with a previously deprotected monomer; deprotection of the applied monomer so that it can react with a subsequently applied protected monomer; and application of another protected monomer for linking. One or more intermediate steps include oxidation and/or sulfurization. In some instances, one or more wash steps precede or follow one or all of the steps.
Methods for the synthesis of polynucleotides on a substrate described herein may comprise an oxidation step. For example, methods involve an iterating sequence of the following steps: application of a protected monomer to a surface of a substrate feature to link with either the surface, a linker or with a previously deprotected monomer; deprotection of the applied monomer so that it can react with a subsequently applied protected monomer; application of another protected monomer for linking, and oxidation and/or sulfurization. In some instances, one or more wash steps precede or follow one or all of the steps.
Methods for the synthesis of polynucleotides on a substrate described herein may further comprise an iterating sequence of the following steps: application of a protected monomer to a surface of a substrate feature to link with either the surface, a linker or with a previously deprotected monomer; deprotection of the applied monomer so that it can react with a subsequently applied protected monomer; and oxidation and/or sulfurization. In some instances, one or more wash steps precede or follow one or all of the steps.
Methods for the synthesis of polynucleotides on a substrate described herein may further comprise an iterating sequence of the following steps: application of a protected monomer to a surface of a substrate feature to link with either the surface, a linker or with a previously deprotected monomer; and oxidation and/or sulfurization. In some instances, one or more wash steps precede or follow one or all of the steps.
Methods for the synthesis of polynucleotides on a substrate described herein may further comprise an iterating sequence of the following steps: application of a protected monomer to a surface of a substrate feature to link with either the surface, a linker or with a previously deprotected monomer; deprotection of the applied monomer so that it can react with a subsequently applied protected monomer; and oxidation and/or sulfurization. In some instances, one or more wash steps precede or follow one or all of the steps.
In some instances, polynucleotides are synthesized with photolabile protecting groups, where the hydroxyl groups generated on the surface are blocked by photolabile-protecting groups. When the surface is exposed to UV light, such as through a photolithographic mask, a pattern of free hydroxyl groups on the surface may be generated. These hydroxyl groups can react with photoprotected nucleoside phosphoramidites, according to phosphoramidite chemistry. A second photolithographic mask can be applied and the surface can be exposed to UV light to generate second pattern of hydroxyl groups, followed by coupling with 5′-photoprotected nucleoside phosphoramidite. Likewise, patterns can be generated and oligomer chains can be extended. Without being bound by theory, the lability of a photocleavable group depends on the wavelength and polarity of a solvent employed and the rate of photocleavage may be affected by the duration of exposure and the intensity of light. This method can leverage a number of factors such as accuracy in alignment of the masks, efficiency of removal of photo-protecting groups, and the yields of the phosphoramidite coupling step. Further, unintended leakage of light into neighboring sites can be minimized. The density of synthesized oligomer per spot can be monitored by adjusting loading of the leader nucleoside on the surface of synthesis.
The surface of a substrate described herein that provides support for polynucleotide synthesis may be chemically modified to allow for the synthesized polynucleotide chain to be cleaved from the surface. In some instances, the polynucleotide chain is cleaved at the same time as the polynucleotide is deprotected. In some cases, the polynucleotide chain is cleaved after the polynucleotide is deprotected. In an exemplary scheme, a trialkoxysilyl amine such as (CH3CH2O)3Si—(CH2)2—NH2 is reacted with surface SiOH groups of a substrate, followed by reaction with succinic anhydride with the amine to create an amide linkage and a free OH on which the nucleic acid chain growth is supported. Cleavage includes gas cleavage with ammonia or methylamine. In some instances cleavage includes linker cleavage with electrically generated reagents such as acids or bases. In some instances, once released from the surface, polynucleotides are assembled into larger nucleic acids that are sequenced and decoded to extract stored information.
The surfaces described herein can be reused after polynucleotide cleavage to support additional cycles of polynucleotide synthesis. For example, the linker can be reused without additional treatment/chemical modifications. In some instances, a linker is non-covalently bound to a substrate surface or a polynucleotide. In some embodiments, the linker remains attached to the polynucleotide after cleavage from the surface. Linkers in some embodiments comprise reversible covalent bonds such as esters, amides, ketals, beta substituted ketones, heterocycles, or other group that is capable of being reversibly cleaved. Such reversible cleavage reactions are in some instances controlled through the addition or removal of reagents, or by electrochemical processes controlled by electrodes. Optionally, chemical linkers or surface-bound chemical groups are regenerated after a number of cycles, to restore reactivity and remove unwanted side product formation on such linkers or surface-bound chemical groups.
Assembly
Polynucleotides may be designed to collectively span a large region of a predetermined sequence that encodes for information. In some instances, larger polynucleotides are generated through ligation reactions to join the synthesized polynucleotides. One example of a ligation reaction is polymerase chain assembly (PCA). In some instances, at least of a portion of the polynucleotides are designed to include an appended region that is a substrate for universal primer binding. For PCA reactions, the presynthesized polynucleotides include overlaps with each other (e.g., 4, 20, 40 or more bases with overlapping sequence). During the polymerase cycles, the polynucleotides anneal to complementary fragments and then are filled in by polymerase. Each cycle thus increases the length of various fragments randomly depending on which polynucleotides find each other. Complementarity amongst the fragments allows for forming a complete large span of double-stranded DNA. In some cases, after the PCA reaction is complete, an error correction step is conducted using mismatch repair detecting enzymes to remove mismatches in the sequence. Once larger fragments of a target sequence are generated, they can be amplified. For example, in some cases, a target sequence comprising 5′ and 3′ terminal adapter sequences is amplified in a polymerase chain reaction (PCR) which includes modified primers that hybridize to the adapter sequences. In some cases, the modified primers comprise one or more uracil bases. The use of modified primers allows for removal of the primers through enzymatic reactions centered on targeting the modified base and/or gaps left by enzymes which cleave the modified base pair from the fragment. What remains is a double-stranded amplification product that lacks remnants of adapter sequence. In this way, multiple amplification products can be generated in parallel with the same set of primers to generate different fragments of double-stranded DNA.
Error correction may be performed on synthesized polynucleotides and/or assembled products. An example strategy for error correction involves site-directed mutagenesis by overlap extension PCR to correct errors, which is optionally coupled with two or more rounds of cloning and sequencing. In certain instances, double-stranded nucleic acids with mismatches, bulges and small loops, chemically altered bases and/or other heteroduplexes are selectively removed from populations of correctly synthesized nucleic acids. In some instances, error correction is performed using proteins/enzymes that recognize and bind to or next to mismatched or unpaired bases within double-stranded nucleic acids to create a single or double-strand break or to initiate a strand transfer transposition event. Non-limiting examples of proteins/enzymes for error correction include endonucleases (T7 Endonuclease I, E. coli Endonuclease V, T4 Endonuclease VII, mung bean nuclease, Cell, E. coli Endonuclease IV, UVDE), restriction enzymes, glycosylases, ribonucleases, mismatch repair enzymes, resolvases, helicases, ligases, antibodies specific for mismatches, and their variants. Examples of specific error correction enzymes include T4 endonuclease 7, T7 endonuclease 1, S1, mung bean endonuclease, MutY, MutS, MutH, MutL, cleavase, CELI, and HINF1. In some cases, DNA mismatch-binding protein MutS (Thermus aquaticus) is used to remove failure products from a population of synthesized products. In some instances, error correction is performed using the enzyme Correctase. In some cases, error correction is performed using SURVEYOR endonuclease (Transgenomic), a mismatch-specific DNA endonuclease that scans for known and unknown mutations and polymorphisms for heteroduplex DNA.
Sequencing
After extraction and/or amplification of polynucleotides from the surface of the structure, suitable sequencing technology may be employed to sequence the polynucleotides. In some cases, the DNA sequence is read on the substrate or within a feature of a structure. In some cases, the polynucleotides stored on the substrate are extracted is optionally assembled into longer nucleic acids and then sequenced.
Polynucleotides synthesized and stored on the structures described herein encode data that can be interpreted by reading the sequence of the synthesized polynucleotides and converting the sequence into binary code readable by a computer. In some cases the sequences require assembly, and the assembly step may need to be at the nucleic acid sequence stage or at the digital sequence stage.
Provided herein are detection systems comprising a device capable of sequencing stored polynucleotides, either directly on the structure and/or after removal from the main structure. In cases where the structure is a reel-to-reel tape of flexible material, the detection system comprises a device for holding and advancing the structure through a detection location and a detector disposed proximate the detection location for detecting a signal originated from a section of the tape when the section is at the detection location. In some instances, the signal is indicative of a presence of a polynucleotide. In some instances, the signal is indicative of a sequence of a polynucleotide (e.g., a fluorescent signal). In some instances, information encoded within polynucleotides on a continuous tape is read by a computer as the tape is conveyed continuously through a detector operably connected to the computer. In some instances, a detection system comprises a computer system comprising a polynucleotide sequencing device, a database for storage and retrieval of data relating to polynucleotide sequence, software for converting DNA code of a polynucleotide sequence to binary code, a computer for reading the binary code, or any combination thereof.
Provided herein are sequencing systems that can be integrated into the devices described herein. Various methods of sequencing are well known in the art, and comprise “base calling” wherein the identity of a base in the target polynucleotide is identified. In some instances, polynucleotides synthesized using the methods, devices, compositions, and systems described herein are sequenced after cleavage from the synthesis surface. In some instances, sequencing occurs during or simultaneously with polynucleotide synthesis, wherein base calling occurs immediately after or before extension of a nucleoside monomer into the growing polynucleotide chain. Methods for base calling include measurement of electrical currents/voltages generated by polymerase-catalyzed addition of bases to a template strand. In some instances, synthesis surfaces comprise enzymes, such as polymerases. In some instances, such enzymes are tethered to electrodes or to the synthesis surface. In some instances, enzymes comprise terminal deoxynucleotidyl transferases, or variants thereof.
Computer Systems
In various aspects, any of the systems described herein are operably linked to a computer and are optionally automated through a computer either locally or remotely. In various instances, the methods and systems of the invention further comprise software programs on computer systems and use thereof. Accordingly, computerized control for the synchronization of the dispense/vacuum/refill functions such as orchestrating and synchronizing the material deposition device movement, dispense action and vacuum actuation are within the bounds of the invention. In some instances, the computer systems are programmed to interface between the user specified base sequence and the position of a material deposition device to deliver the correct reagents to specified regions of the substrate.
The computer system 3700 illustrated in
Software and data are stored in external storage 3824 and can be loaded into RAM 3810 and/or cache 3804 for use by the processor. The system 3800 includes an operating system for managing system resources; non-limiting examples of operating systems include: Linux, Windows™, MACOS™, BlackBerry OS™, iOS™, and other functionally-equivalent operating systems, as well as application software running on top of the operating system for managing data storage and optimization in accordance with example embodiments of the present invention. In this example, system 3800 also includes network interface cards (NICs) 3820 and 3821 connected to the peripheral bus for providing network interfaces to external storage, such as Network Attached Storage (NAS) and other computer systems that can be used for distributed parallel processing.
In some example embodiments, processors can maintain separate memory spaces and transmit data through network interfaces, back plane or other connectors for parallel processing by other processors. In other embodiments, some or all of the processors can use a shared virtual address memory space.
The above computer architectures and systems are examples only, and a wide variety of other computer, cell phone, and personal data assistant architectures and systems can be used in connection with example embodiments, including systems using any combination of general processors, co-processors, FPGAs and other programmable logic devices, system on chips (SOCs), application specific integrated circuits (ASICs), and other processing and logic elements. In some embodiments, all or part of the computer system can be implemented in software or hardware. Any variety of data storage media can be used in connection with example embodiments, including random access memory, hard drives, flash memory, tape drives, disk arrays, Network Attached Storage (NAS) and other local or distributed data storage devices and systems.
In example embodiments, the computer system can be implemented using software modules executing on any of the above or other computer architectures and systems. In other embodiments, the functions of the system can be implemented partially or completely in firmware, programmable logic devices such as field programmable gate arrays (FPGAs), system on chips (SOCs), application specific integrated circuits (ASICs), or other processing and logic elements. For example, the Set Processor and Optimizer can be implemented with hardware acceleration through the use of a hardware accelerator card.
Provided herein are numbered embodiments 1-87: 1. A device comprising at least one addressable solid support, wherein the at least one solid support comprises: a base layer comprising silicon; an intermediate layer comprising an oxide, wherein the intermediate layer and the top layer are in fluid communication with a solvent, and wherein the intermediate layer is located between the base layer and the top layer, and is configured for the attachment of molecules, and a top layer comprising a conductive material; wherein the top layer is configured to generate an electrochemically generated reagent when energized with a voltage; wherein the solid support comprises a plurality of features, and wherein a smallest dimension of the plurality of features is no more than the diffusion distance of the electrochemically generated reagent. 2. The device of embodiment 1, wherein the molecules comprise polymers. 3. The device of embodiment 2, wherein the polymers comprise polynucleotides, peptides, or sugars. 4. The device of any one of embodiments 1-3, wherein a smallest dimension of a feature is no more than 5 microns. 5. The device of embodiment 4, wherein a smallest dimension of a feature is no more than 1 micron. 6. The device of any one of embodiments 1-5, wherein the conductive material comprises platinum. 7. The device of any one of embodiments 1-6, wherein the oxide comprises a nitride or carbide. 8. The device of any one of embodiments 1-6, wherein the oxide comprises silicon oxide. 9. The device of any one of embodiments 1-8, wherein the base layer comprises a complementary metal-oxide-semiconductor. 10. The device of any one of embodiments 1-9, wherein the device comprises at least 100 addressable solid supports. 11. The device of embodiment 10, wherein the device comprises at least 1000 addressable solid supports. 12. The device of embodiment 10, wherein the at least 100 addressable solid supports are separated by no more than a 1 micron gap. 13. The device of any one of embodiments 1-12, wherein the thickness of the top layer is no more than 200 nm. 14. The device of embodiment 13, wherein the thickness of the top layer is no more than 100 nm. 15. The device of any one of embodiments 1-14, wherein the top layer is patterned with a plurality of wells, wherein the wells provide fluid communication of the intermediate layer with the solvent. 16. The device of embodiment 15, wherein the diameter of the wells is no more than 5 microns. 17. The device of embodiment 15, wherein the diameter of the wells is 3-50 microns. 18. The device of embodiment 15, wherein the pitch distance between wells is no more than 20 microns. 19. The device of embodiment 15, wherein the pitch distance between wells is no more than 10 microns. 20. The device of any one of embodiments 1-19, wherein the device further comprises a cathode, wherein the cathode is in fluid communication with the solvent. 21. The device of embodiment 20, wherein the cathode is a substantially planer surface. 22. The device of embodiment 21, wherein the cathode is located in the same plane as the top layer. 23. The device of embodiment 21, wherein the cathode is located in a different plane relative to the top layer. 24. The device of embodiment 22, wherein the distance between the cathode and the top layer is 0.1-5 microns. 25. The device of embodiment 23, wherein the distance between the cathode and the top layer is 10-500 nm. 26. The device of any one of embodiments 20-26, wherein the cathode comprises glass. 27. The device of embodiment 26, wherein at least one of the surfaces is configured as a cathode. 28. The device of embodiment 1, wherein the cathode comprises a conductive material. 29. The device of embodiment 28, wherein the conductive material comprises platinum. 30. The device of any one of embodiments 1-29, wherein the device further comprises a buried electrode, wherein the buried electrode is not in fluid communication with the solvent or the top layer. 31. A device for polynucleotide synthesis comprising an array of the devices of embodiments 1-30, wherein the array comprises at least 10 devices. 32. The device of embodiment 31, wherein the array comprises at least 100 devices. 33. The device of embodiment 31, wherein the array comprises at least 1000 devices. 34. The device of embodiment 31, wherein the array further comprises a plurality of vias, wherein the plurality of vias are configured to connect at least two vertical layers of the device. 35. The device of embodiment 31, wherein the array further comprises a plurality of routing connections, wherein the plurality of routing connections are configured for addressable control of each device in the array. 36. The device of embodiment 31, wherein the vias and routing are less than 1 micron in length. 37. A device for polynucleotide synthesis comprising at least one surface, wherein the at least one surface comprises: a base layer comprising silicon; a first intermediate layer comprising an oxide, wherein the first intermediate layer, the second intermediate layer, and the top layer are in fluid communication with a solvent; and a second intermediate layer comprising a conductive material; a top layer comprising an oxide, wherein the top layer is configured for the attachment of polynucleotides, wherein the first intermediate layer is between the base layer and the second intermediate layer, the second intermediate layer is between the first intermediate layer and the top layer, and wherein the solid support comprises a plurality of features. 38. The device of embodiment 37, wherein a smallest dimension of a feature is no more than 5 microns. 39. The device of embodiment 37, wherein a smallest dimension of a feature is no more than 3 microns. 40. The device of embodiment 37, wherein a smallest dimension of a feature is 1-5 microns. 41. The device of embodiment 37, wherein the conductive material comprises platinum. 42. The device of embodiment 37, wherein the oxide comprises a nitride or carbide. 43. The device of embodiment 37, wherein the oxide comprises silicon. 44. The device of embodiment 37, wherein the base layer comprises a complementary metal-oxide-semiconductor. 45. The device of embodiment 37, wherein the device comprises at least 10 addressable solid supports. 46. The device of embodiment 37, wherein the device comprises at least 100 addressable solid supports. 47. The device of embodiment 37, wherein the top layer and the second intermediate layer are patterned as an array of cylinders. 48. The device of embodiment 37, wherein the top layer and the second intermediate layer are patterned randomly on the first intermediate layer. 49. The device of embodiment 37, wherein the device further comprises a buried electrode, wherein the buried electrode is not in fluid communication with the solvent, the second intermediate layer, or the top layer. 50. A device for polynucleotide synthesis comprising an array of the devices of embodiments 1-30, wherein the array comprises at least 10 devices. 51. The device of embodiment 31, wherein the array comprises at least 1000 devices. 52. The device of embodiment 31, wherein the array further comprises a plurality of vias, wherein the plurality of vias are configured to connect at least two vertical layers of the device. 53. The device of embodiment 31, wherein the array further comprises a plurality of routing connections, wherein the plurality of routing connections are configured for addressable control of each device in the array. 54. The device of embodiment 31, wherein the vias and routing are less than 1 micron in length. 55. A method for polynucleotide synthesis comprising: (a) contacting a nucleoside attached to a solid support with a protected nucleoside, wherein the protected nucleoside is configured to form a covalent bond with the nucleoside to generate a protected polynucleotide; and (b) applying a voltage to a solvent in fluid communication with the protected polynucleotide, wherein the voltage results in deprotection of the terminal nucleoside of the protected polynucleotide, and wherein the voltage is delivered as at least 2 pulses. 56. The method of embodiment 55, wherein the voltage is 1-2.5 volts. 57. The method of embodiment 55, wherein the voltage is about 2 volts. 58. The method of embodiment 55, wherein the total period of time for all pulses is less than 5 seconds. 59. The method of embodiment 55, wherein the total period of time for all pulses is less than 1 second. 60. The method of embodiment 55, wherein the pulse is no more than 50-500 milliseconds. 61. The method of embodiment 55, wherein the pulse is no more than 50 milliseconds. 62. The method of embodiment 55, wherein the pulse is 1-50 milliseconds. 63. The method of embodiment 55, wherein the voltage is delivered as at least 100 pulses. 64. The method of embodiment 55, wherein the voltage is delivered as 50-1000 pulses. 65. The method of embodiment 55, wherein the time between any two pulses is 10-2000 milliseconds. 66. The method of embodiment 55, wherein the time between any two pulses is 10-500 milliseconds. 67. The method of embodiment 55, wherein the polynucleotide is washed between pulses. 68. The method of embodiment 55, wherein the polynucleotide is not washed between pulses. 69. The method of embodiment 55, wherein at least one of the pulses is a positive voltage. 70. The method of embodiment 55, wherein at least one of the pulses is a positive voltage, and at least one of the pulses is a negative voltage. 71. The method of embodiment 70, wherein the negative voltage is −0.1 to −1.0 volts. 72. The method of embodiment 70, wherein the time between the at least one positive voltage and the at least one negative voltage is less than 10 milliseconds. 73. The method of embodiment 70, wherein the time between the at least one positive voltage and the at least one negative voltage is less than 1 millisecond. 74. The method of any one of embodiments 55-73, wherein the solvent comprises a composition for electrochemical acid generation. 75. The method of embodiment 74, wherein the solvent comprises hydroquinone, benzoquinone, or a mixture thereof 76. The method of embodiment 75, wherein the mixture of hydroquinone and benzoquinone is present in about a 1:1 ratio. 77. The method of embodiment 75, wherein the mixture of hydroquinone and benzoquinone is present in about a 10:1 ratio. 78. The method of embodiment 75, wherein the concentration of the mixture of hydroquinone and benzoquinone is 0.5-10 mM. 79. The method of embodiment 75, wherein the concentration of the mixture of hydroquinone and benzoquinone is 1.5-5 mM. 80. The method of any one of embodiments 70-79, wherein the protected polynucleotide comprises an acid-cleavable protecting group. 81. A method for polynucleotide synthesis comprising: (a) inactivating a first region of a solid support by increasing the resistance between a first electrode in proximity to the first region and an electrical ground; (b) activating a second region of the support by applying a voltage to a second electrode in fluid communication with the second region of the solid support, wherein the second region comprises a first plurality of protected polynucleotides attached to the solid support, and wherein the voltage results in deprotection of at least one protected polynucleotide. 82. The method of embodiment 81, wherein increasing the resistance comprising disconnecting the first electrode from the electrical ground. 83. The method of embodiment 81, wherein the resistance is increased by connection to an electrical resistor or transistor. 84. The method of embodiment 81, wherein the first region comprises a second plurality of protected polynucleotides. 85. The method of embodiment 81, wherein the voltage is delivered as at least 2 pulses. 86. The method of embodiment 81, wherein the first plurality of protected polynucleotides and the second plurality of protected polynucleotides comprise protecting groups capable of removal with acid. 87. The method of embodiment 81, wherein the voltage generates acid.
The following examples are set forth to illustrate more clearly the principle and practice of embodiments disclosed herein to those skilled in the art and are not to be construed as limiting the scope of any claimed embodiments. Unless otherwise stated, all parts and percentages are on a weight basis.
A device was functionalized to support the attachment and synthesis of a library of polynucleotides. The device surface was first wet cleaned using a piranha solution comprising 90% H2SO4 and 10% H2O2 for 20 minutes. The device was rinsed in several beakers with DI water, held under a DI water gooseneck faucet for 5 min, and dried with N2. The device was subsequently soaked in NH4OH (1:100; 3 mL:300 mL) for 5 min, rinsed with DI water using a handgun, soaked in three successive beakers with DI water for 1 min each, and then rinsed again with DI water using the handgun. The device was then plasma cleaned by exposing the device surface to O2. A SAMCO PC-300 instrument was used to plasma etch O2 at 250 watts for 1 min in downstream mode.
The cleaned device surface was actively functionalized with a solution comprising N-(3-triethoxysilylpropyl)-4-hydroxybutyramide using a YES-1224P vapor deposition oven system with the following parameters: 0.5 to 1 torr, 60 min, 70° C., 135° C. vaporizer. The device surface was resist coated using a Brewer Science 200× spin coater. SPR™ 3612 photoresist was spin coated on the device at 2500 rpm for 40 sec. The device was pre-baked for 30 min at 90° C. on a Brewer hot plate. The device was subjected to photolithography using a Karl Suss MA6 mask aligner instrument. The device was exposed for 2.2 sec and developed for 1 min in MSF 26A. Remaining developer was rinsed with the handgun and the device soaked in water for 5 min. The device was baked for 30 min at 100° C. in the oven, followed by visual inspection for lithography defects using a Nikon L200. A cleaning process was used to remove residual resist using the SAMCO PC-300 instrument to O2 plasma etch at 250 watts for 1 min.
The device surface was passively functionalized with a 100 μL solution of perfluorooctyltrichlorosilane mixed with 10 μL light mineral oil. The device was placed in a chamber, pumped for 10 min, and then the valve was closed to the pump and left to stand for 10 min. The chamber was vented to air. The device was resist stripped by performing two soaks for 5 min in 500 mL NMP at 70° C. with ultrasonication at maximum power (9 on Crest system). The device was then soaked for 5 min in 500 mL isopropanol at room temperature with ultrasonication at maximum power. The device was dipped in 300 mL of 200 proof ethanol and blown dry with N2. The functionalized surface was activated to serve as a support for polynucleotide synthesis.
A two dimensional polynucleotide synthesis device was assembled into a flowcell, which was connected to a flowcell (Applied Biosystems (ABI394 DNA Synthesizer”). The polynucleotide synthesis device was uniformly functionalized with N-(3-TRIETHOXYSILYLPROPYL)-4-HYDROXYBUTYRAMIDE (Gelest) was used to synthesize an exemplary polynucleotide of 50 bp (“50-mer polynucleotide”) using polynucleotide synthesis methods described herein.
The sequence of the 50-mer was as described in SEQ ID NO.: 1. 5′AGACAATCAACCATTTGGGGTGGACAGCCTTGACCTCTAGACTTCGGCAT##TTTTTTT TTT3′ (SEQ ID NO.: 1), where # denotes Thymidine-succinyl hexamide CED phosphoramidite (CLP-2244 from ChemGenes), which is a cleavable linker enabling the release of polynucleotides from the surface during deprotection.
The synthesis was done using standard DNA synthesis chemistry (coupling, capping, oxidation, and deblocking) according to the protocol in Table 1 and an ABI synthesizer.
The phosphoramidite/activator combination was delivered similar to the delivery of bulk reagents through the flowcell. No drying steps were performed as the environment stays “wet” with reagent the entire time.
The flow restrictor was removed from the ABI 394 synthesizer to enable faster flow. Without flow restrictor, flow rates for amidites (0.1M in ACN), Activator, (0.25M Benzoylthiotetrazole (“BTT”; 30-3070-xx from GlenResearch) in ACN), and Ox (0.02M I2 in 20% pyridine, 10% water, and 70% THF) were roughly ˜100 uL/sec, for acetonitrile (“ACN”) and capping reagents (1:1 mix of CapA and CapB, wherein CapA is acetic anhydride in THF/Pyridine and CapB is 16% 1-methylimidizole in THF), roughly ˜200 uL/sec, and for Deblock (3% dichloroacetic acid in toluene), roughly ˜300 uL/sec (compared to ˜50 uL/sec for all reagents with flow restrictor). The time to completely push out Oxidizer was observed, the timing for chemical flow times was adjusted accordingly and an extra ACN wash was introduced between different chemicals. After polynucleotide synthesis, the chip was deprotected in gaseous ammonia overnight at 75 psi. Five drops of water were applied to the surface to recover polynucleotides. The recovered polynucleotides were then analyzed on a BioAnalyzer small RNA chip (data not shown).
A device comprising the layout of
Polynucleotide synthesis steps were conducted to generate products having the sequence TTTTTT, following the general methods of Examples 1 and 2 with modification; the chemical deblocking step was replaced with an electrochemical deblocking step comprising 5 mM (hydroquinone/benzoquinone “HQ/BQ” in acetonitrile,
A synthesis device was prepared following the general procedure of Example 3, wherein the synthesis surfaces comprised uniform holes with diameters of 5 μm and a pitch of 9 μm (
A synthesis device was prepared following the general procedure of Example 3 with modification; the number of deblocking cycles and concentration of HQ/BQ was modified to that shown in
A synthesis device was prepared following the general procedure of Example 3 with modification; either 1 mM HQ/BQ or 5 mM HQ/BQ was used with a single pulse time of 1.5 seconds to deblock. The synthesis surfaces comprised uniform holes with diameters of 5 μm and a pitch of 9 um. 1 mM HQ/BQ conditions resulted in higher peaks (
A synthesis device was prepared following the general procedure of Example 3 with modification; the electrochemical deblocking time (or pulse time) was modified. 5 mM HQ/BQ was used for the deblocking step. The synthesis surfaces comprised uniform holes with diameters of 5 μm and a pitch of 9 um, and the sequence TT6 was synthesized. A fluorescence image of the device and enlargements of the top of five of the synthesis surfaces are shown in
A synthesis device was prepared following the general procedure of Example 7 with modification; the electrochemical pulse times were modified, and the concentration of HQ/BQ was varied at 5 mM (
A synthesis device was prepared following the general procedure of Example 7 with modification; both single pulse and multiple pulse deblocking conditions were used (
A synthesis device was prepared and used to synthesize a polynucleotide having the sequence TTT6, following the general procedure of Example 7 with modification; pulse times of 60-540 milliseconds were employed, and a single 2 sec pulse was used as a control (
A synthesis device was prepared and used to synthesize a polynucleotide having the sequence TTT6, following the general procedure of Example 7 with modification; off times of 2-95 milliseconds were employed, multiple 5 millisecond pulse times were used, the total no of pulses was fixed to be 4 seconds in total, and a single 4 sec pulse was used as a control (
A synthesis device was prepared and used to synthesize a polynucleotide having the sequence TTT6, following the general procedure of Example 11 with modification; 100% hydroquinone, 1:1 benzoquinone/hydroquinone, or 1:10 hydroquinone/benzoquinone was used for the deblocking step. Halo widths (um) for various pulse-off times and oxidation ratios are shown in
A synthesis device was prepared and used to synthesize a polynucleotide having the sequence TTT6, following the general procedure of Example 7 with modification; off times of 2000 milliseconds were employed, multiple 10-2000 millisecond pulse times were used, the total no of pulses was fixed to be 4 seconds in total, and a single 4 sec pulse was used as a control (
A synthesis device was prepared and used to synthesize a polynucleotide having the sequence TTT6, following the general procedure of Example 7 with modification; deblocking was performed in three steps, using a secondary backside voltage (
After staining, a fluorescence image of the device was acquired and is shown in
A synthesis device was prepared and used to synthesize a polynucleotide having the sequence TTT6, following the general procedure of Example 7 with modification; deblocking was only performed on surfaces 6 and 10, and the top metal cathode was replaced with a glass cathode (
A surface comprising a base layer of silicon and a top layer comprising an oxide was patterned with a removable masking material, such as a photoresist (
A surface comprising a base layer of silicon, a first layer comprising an oxide, a second layer of titanium nitride, a third layer comprising platinum, a fourth layer comprising titanium nitride, (from bottom to top) is patterned with a removable masking material, such as a photoresist (
Following the general procedures of Example 17, a surface comprising the configuration of
Following the general procedures of Example 18, devices comprising an array of patterned synthesis surfaces are fabricated into chips (
Devices of
The chips fabricated using the general methods of Example 19 are networked into a larger, rack mounted array. Each synthesis surface of each array on each chip is individually addressable and in communication with a central control unit.
Following the general procedures of Example 17, a device is fabricated wherein the synthesis surface comprises an array of uniform lines with a thickness of 3 μm (
Following the general procedures of Example 17, a device is fabricated comprising a plurality of synthesis surfaces, wherein the synthesis surfaces are separated by 3 um. Surfaces separated by less than 3 μm may also be employed.
Following the general procedures of Example 17, a device is fabricated wherein the synthesis surface comprises an in-plane cathode. The gap between the cathode and the synthesis surfaces is between 10-500 um.
A device of Example 19 is used following the general procedures of Example 3 to synthesize an array comprising a plurality of polynucleotides, where the plurality of polynucleotides collectively encode for digital information. The polynucleotides are optionally cleaved from the device surface and sequenced, or sequenced directly from the device. The sequences obtained are then converted into the digital information.
A chip is fabricated according to
A chip comprising an array of devices and in-plane cathodes is fabricated according to
Following the general procedure of Example 7, polynucleotides were synthesized on the device array using three different deprotection conditions. In the first configuration (“B”), +2V was applied to device 4 in the array, an above-plane (or opposing) cathode was connected to ground (0V), and neighboring devices were disconnected (
Chips (
A device array having the general design shown in
Different layouts of device patterns were then examined (
A device array having the general design shown in
The device arrays having the dimensions according to Table 5 are also fabricated.
Such devices are integrated into flow cells, and then evaluated for polynucleotide synthesis using the methods described herein, such as synthesis of 100mers, 150mers, 200mers, 250mers, or 300mers. Devices in some instances can encode 21 bytes per oligo.
While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.
This application claims the benefit of U.S. Provisional Application No. 63/071,646, filed Aug. 28, 2020, which is incorporated by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 63071646 | Aug 2020 | US |
