JP7546345B2 - Methods, compositions and devices for information storage - Patents.com - Google Patents

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62/301,538, filed February 29, 2016, and No. 62/415,430, filed October 31, 2016, the contents of each of which are incorporated herein by reference in their entirety.

FIELD The present invention relates to novel methods, compositions and devices useful for storing and retrieving information using nanopore devices that synthesize and sequence polymers, such as nucleic acids.

There is a continuing desire to store ever more data on or in physical media using ever smaller, but larger capacity, storage devices. It has been reported that the amount of data stored doubles in size every two years, and one study predicts that by 2020, the amount of data we create and copy will reach 44 zettabytes, or 44 trillion gigabytes, per year. Furthermore, existing data storage media (such as hard drives, optical media, and magnetic tape) are relatively unstable and will break down after long-term storage.

Alternative methods for storing large amounts of data for long periods of time (e.g., decades or centuries) are urgently needed.

It has been proposed to use DNA to store data. DNA is extremely stable and can theoretically encode vast amounts of data, allowing data to be stored for very long periods of time. See, for example, Bancroft, C., et al., Long-Term Storage of Information in DNA, Science (2001) 293: 1763-1765. Furthermore, DNA as a storage medium is not subject to the security risks of traditional digital storage media. However, there are no practical methods to implement this idea.

For example, WO2014/014991 describes a method for storing data on DNA oligonucleotides, where the information is coded in binary format with one nucleotide per bit, using a 96-bit (96 nucleotide) data block, a 19 nucleotide address sequence, and flanking sequences for amplification and sequencing. The code is then read by amplifying the sequence using PCR and sequencing it using a high-speed sequencer (such as an Illumina HiSeq instrument). The data block sequences are then placed in the correct order using the address tags, the address sequence and flanking sequences are removed, and the sequence data is translated into a binary code. Such an approach has significant limitations. For example, a 96-bit data block can code only 12 characters (using the conventional 1 byte or 8 bits per character or space). The ratio of useful information stored to "housekeeping" information is low, with about 40% of the sequence information being used for the address and flanking DNA. The specification describes coding a book using 54,898 oligonucleotides. The high-fidelity inkjet-printed DNA microchips used to synthesize oligonucleotides limited the size of the oligos (the described length of 159 nucleotides was the upper limit). Furthermore, reading the oligonucleotides requires amplification and isolation, which introduces further possibilities for error. See also WO2004/088585A2; WO03/025123A2; C. BANCROFT: "Long-Term Storage of Information in DNA", Science (2001) 293 (5536): 1763c-1765; COX J P L: "Long-term data storage in DNA", Trends in Biotechnology (2001)19(7): 247-250.

As has been recognized for over 25 years, the potential information density and stability of DNA makes it an attractive medium for data storage, but there are still no practical techniques for writing and reading large amounts of data in this format.

We have developed a novel approach for nucleic acid storage using a nanofluidic system to synthesize nucleic acid sequences and a nanopore reader to read the sequences. Our approach allows the synthesis, storage and reading of DNA strands hundreds, thousands or millions of bases long. Because the sequences are long, only a relatively small percentage of the sequence is used for identification information, so that the information density is much higher than in the above-mentioned approaches. Furthermore, in some embodiments, the synthesized nucleic acid has a specific location on the nanochip, so the sequence can be identified without identification information. Sequencing performed in the nanochamber is very rapid, and reading the sequence through the nanopore can be extremely rapid, up to the order of a million bases per second. Since only two types of bases are required, this sequencing can be more rapid and accurate than sequencing means that must distinguish between four types of bases (adenine, thymine, cytosine, guanine). In certain embodiments, the two bases do not pair with each other, do not form secondary structures, and are of different sizes. For example, for this purpose, adenine and cytosine are better than adenine and thymine, which tend to hybridize, or adenine and guanine, which are similar sizes.

In some embodiments, the system can be used to synthesize long polymers that encode data, which can be amplified and/or released and then sequenced on a different sequencer. In other embodiments, the system can be used to provide custom DNA sequences. In yet other embodiments, the system can be used to read DNA sequences.

The nanochip used in one embodiment contains at least two separate reaction compartments linked by at least one nanopore, which prevents at least some components from mixing, but allows only single molecules of DNA or other charged polymers (e.g. RNA or peptide nucleic acid (PNA)) to pass in a controllable manner from one reaction compartment to another. The transfer of the polymer (or at least the end of the polymer with added monomers) from one compartment to another allows for successive manipulations/reactions (e.g. addition of bases) to be performed on the polymer, e.g. using enzymes that are prevented from passing through the nanopore because they are too large or because they are anchored to a substrate or bulky moiety. The nanopore sensor reports back on the movement or location of the polymer and its status, e.g. its sequence and whether an attempted reaction was successful or not. This allows data to be written, stored and read, e.g. where the base sequence corresponds to a machine-readable code (e.g. a binary code where each base or group of bases corresponds to a 1 or 0).

Thus, the present invention specifically includes the following embodiments:
- A nanochip for the synthesis of charged polymers (e.g., DNA) containing at least two different monomers, where the nanochip includes two or more reaction chambers separated by one or more nanopores, each reaction chamber including an electrolyte, one or more electrodes for drawing the charged polymer into the chamber, and one or more reagents for facilitating the addition of monomers or oligomers to the polymer. The nanochip may optionally be configured with functional elements to guide, convey, and/or control the DNA, and may optionally be coated or fabricated with a material selected to allow smooth flow of the DNA or to bind to the DNA, and may include nanocircuit elements to provide and control electrodes in close proximity to the nanopore. For example, one or more nanopores may each optionally be coupled to an electrode capable of controlling the movement of the polymer through the nanopore and/or detecting changes in potential, current, resistance, or capacitance at the nanopore-polymer interface, thereby detecting the sequence of the polymer as it passes through one or more nanopores. In a particular embodiment, the oligomers are synthesized using a polymerase or a site-specific recombinase. In some embodiments, the polymers are sequenced during synthesis, allowing for the detection and possibly correction of errors. In some embodiments, the polymers thus obtained can be stored on the nanochip and sequenced when it is desired to access the information encoded in the polymer's sequence.

- A method of synthesizing polymers, such as DNA, using the nanotips described above.
- A single-stranded DNA molecule whose sequence consists essentially of non-hybridizing nucleotides, such as adenine and cytosine nucleotides (A and C), arranged in an order corresponding to a binary code, for use, for example, in methods of data storage.
- Double-stranded DNA comprising a series of nucleotide sequences corresponding to a binary code, where the double-stranded DNA further comprises - A method of reading a binary code encoded in DNA comprising using a nanopore sequencer.
A method and device for data storage using the nanochip to create a charged polymer, such as DNA, containing at least two different monomers, where the monomers are arranged in an order that corresponds to a binary code.

Further aspects and scope of applicability of the present invention will become apparent from the detailed description provided below. It should be understood that the detailed description and specific examples, while indicating preferred embodiments, are for purposes of illustration only and are not intended to limit the scope of the invention.

The present invention will become more fully understood from the detailed description and the accompanying drawings, wherein:
FIG. 1 shows a simple two-chamber nanotip design with a separation membrane perforated by a nanopore and electrodes on either side of the membrane. FIG. 2 shows how a charged polymer, such as DNA, is attracted to an anode. FIG. 3 shows how a charged polymer, such as DNA, is attracted to an anode. FIG. 4 shows that by reversing the polarity of the electrodes, the polymer can be retracted. FIG. 5 shows that by reversing the polarity of the electrodes, the polymer can be retracted. FIG. 6 shows a two-chamber nanotip design for DNA synthesis, where the polymerase enzyme is located in one chamber and the deblocking enzyme is located in the other chamber, neither of which can pass through the nanopore. FIG. 7 shows the addition of an adenine nucleotide when 3′ blocked dATP (A) enters the left chamber, and the current is set in the “forward” direction to drive the DNA into the left chamber. 8 shows the deprotection of an oligonucleotide so that additional nucleotides can be added. Deprotection occurs after moving the DNA into the chamber, for example by setting the current in the "reverse" direction. 9 shows the addition of 3' blocked dCTP (C). In one embodiment, fluid flow is used to exchange the contents of this chamber. For example, as shown, this chamber previously contained "A." FIG. 10 shows how multiple separate holding chambers can be provided while the flow chamber becomes a single lane for providing reagents.

Figure 11 shows how the DNA can be retained in the chamber by either binding it to the chamber (top DNA fragment) or by conjugating it to a bulky group that cannot pass through the nanopore (bottom DNA fragment). In this system, one end of the DNA remains in the holding chamber while it is still able to move into the flow chamber and receive additional nucleotides. FIG. 12 shows a configuration in which DNA is bound to the walls of a chamber and controlled by multiple electrodes. FIG. 13 shows how DNA can be retained in the chamber if desired, simply by controlling the polarity of the electrodes. FIG. 14 shows an array in which DNA is bound to the surface of the chamber with reagents free to flow on either side. FIG. 15 shows an alternative design with the electrode on the side adjacent to the separator, which allows for a cheaper production. Figure 16 shows a three compartment arrangement where DNA can be moved from compartment to compartment by electrodes. This system does not require significant flow of reagents during synthesis. FIG. 17 shows an example of how reagents may be arranged in a three compartment arrangement. FIG. 18 shows an oligonucleotide tethered adjacent to a nanopore, which has electrode elements on either side of the membrane.

FIG. 19 shows a series of DNA molecules bound along a membrane containing a nanopore, each under the control of an electrode adjacent to the nanopore, with flow lanes on either side of the membrane. For example, as shown, the left flow lane provides a flow of buffer wash/3′ blocked dATP(A)/buffer wash/3′ blocked dCTP(C)/buffer wash, where the DNA molecules are directed into the flow chamber only if the desired nucleotide is present. The right lane provides a deblocking reagent, which deprotects the 3′ end of the nucleotide, allowing the addition of additional nucleotides. In one embodiment, the deblocking reagent flows while the left lane is being washed with buffer. In another embodiment, the deprotecting reagent is too bulky to pass through the nanopore to the left lane. FIG. 20 shows a schematic of a proof-of-concept experiment, in which the bits used to encode data are short oligomers linked by topoisomerase. FIG. 21 shows a schematic of a proof-of-concept experiment in which the bits used to encode data are short oligomers linked by topoisomerase. FIG. 22 shows a schematic of a proof-of-concept experiment, in which the bits used to encode data are short oligomers linked by topoisomerase.

FIG. 23 shows a format for a nanopore sequencer, where the polymer sequence is read using capacitance variation. In this capacitance readout scheme, the electrodes form the upper and lower plates of a capacitor, which are separated by a membrane containing the nanopore. The capacitor is embedded in a resonant circuit, where a direct current can be oscillated to draw the charged polymer out of the nanopore. As the polymer, e.g., DNA, passes through the nanopore, the change in capacitance is measured using high frequency impedance spectroscopy. A major advantage of this approach, especially with DNA, is that the measurement frequency can be very high (one measurement is valid for each cycle, so a frequency of 100 MHz corresponds to 100 million measurements per second) and can be much faster than the translocation of monomers through the nanopore (e.g., DNA passes through a nanopore at a rate of the order of 1 million nucleotides per second in response to a current unless somehow inhibited).

FIG. 24 shows a dual addition chamber arrangement suitable for adding two different types of monomers or oligomers, e.g., for two-bit or binary encoding. The top of the figure shows a top view. The bottom shows a side cross-sectional view. The complete device in this embodiment can be assembled from up to three independently fabricated layers and joined by wafer bonding, or can be formed by etching a single substrate. The chip includes an electrical control layer (1), a fluidics layer (2) containing two addition chambers above a storage chamber and in which a charged polymer (e.g., DNA) is tethered between nanopore introductions to the first and second addition chambers, and an electrical ground layer (3).

Figure 25 shows the operation of the double addition chamber arrangement of Figure 24. It is observed that at the bottom of each addition chamber there is a nanopore (4). The nanopore is made by drilling, for example with FIB, TEM, wet or dry etching or by dielectric breakdown. The membrane (5) containing the nanopore is for example one atomic layer to a few tens of nm thick. This membrane is made for example from SiN, BN, SiOx, graphene, transition metal dichalcogenides, for example _WS2 or _MoS2 . Below the nanopore membrane (5) there is a storage or deblocking chamber (6) which contains a reagent for deprotecting the polymer after it has been added in one of the addition chambers (it is recalled that the monomer or oligomer is added in end-protected form so that only one monomer or oligomer is added at a time). The polymer (7) can be moved in and out of the addition chamber by changing the polarity of the electrodes in the electrical control layer (1).

Figure 26 shows a top view of an arrangement similar to Figures 24 and 25, but now there are four additional chambers sharing a common storage or deblocking chamber, and the polymer is anchored at a location (9) that provides access to each of the four chambers. A cross-sectional view of this arrangement is as shown in Figures 24 and 25, and charged polymers can be moved to each of the four additional chambers by manipulating the electrodes in the electrical control layer (1 in Figure 24).

FIG. 27 shows a top view of a nanopore chip with multiple sets of dual addition chambers as shown in FIG. 24 and FIG. 25, which allows multiple polymers to be synthesized in parallel. Monomers (dATP and dGTP nucleotides, here represented as A and G) are loaded into each chamber through a sequential flow path. One or more common deblocking agent flow cells allow the polymer to be deprotected after addition of the monomer or oligomer in one of the addition chambers. This also allows the polymer to be separated on demand (e.g., with a restriction enzyme in the case of DNA, or chemically separated from a surface adjacent to the nanopore) and collected externally. In this particular embodiment, the deblocking agent flow cells are perpendicular to the fluid loading channel used to fill the addition chambers.

FIG. 28 shows further details of the wiring for the dual additional chamber arrangement. The electrical control layer (1) includes wiring made of metal or polysilicon. The density of the wiring is increased by 3D stacking with electrical isolation provided by dielectric deposition (e.g. by PECVD, sputtering, ALD, etc.). Contact to the top electrode by the additional chambers in one embodiment (11) is made using through silicon vias (TSVs) by deep reactive ion etching (DRIE) (cryo-processing or BOSCH processing). Individual voltage control (12) allows each additional chamber to be individually processed, which allows fine control of the arrangement of multiple polymers in parallel. The right side of the figure shows a top view illustrating the wiring for multiple additional cells. The electrical ground layer (3) can be common (as shown) or split to reduce cross-coupling between cells.

FIG. 29 shows an alternative configuration in which the control electrode (13) of the additional chamber may be arranged in a wraparound manner around the side of the chamber instead of at the top of the chamber.

FIG. 30 shows an SDS-PAGE gel confirming that the topoisomerase addition protocol described in Example 3 works, with bands corresponding to the expected A5 and B5 products clearly visible.

Figure 31 shows an agarose gel confirming the correct size of the PCR products of Example 5. Lane 0 is a 25 base pair ladder; lane 1 is the experimental product with lines corresponding to expected molecular weights; lane 2 is negative control #1; lane 3 is negative control #2; lane 4 is negative control #4.

Figure 32 shows an agarose gel confirming that the restriction enzymes described in Example 5 produce the expected products. The ladder on the left is a 100 base pair ladder. Lane 1 is uncut NAT1/NAT9c and lane 2 is cut NAT1/NAT9c. Lane 3 is uncut NAT1/NAT9cl and lane 4 is cut NAT1/NAT9cl.

FIG. 33 shows the fixation of DNA near a nanopore. Panel (1) shows DNA with an origami structure at one end in the left chamber (in a real nanochip, many such origami structures would initially be present in the left chamber). Panel (2) illustrates the system with an anode on the right, which guides the DNA into the nanopore. The DNA strand can pass through the nanopore, but the origami structure is too large to pass through, so the DNA is "stuck". Turning off the current allows the DNA to diffuse (panel 3). When the end of the DNA strand touches a surface near the nanopore, it can be bound by the appropriate chemistry. In panel (4), a restriction enzyme is added, which cuts the origami structure from the DNA. The chamber is washed to remove the enzyme and any remaining DNA. The end result is a single DNA molecule bound near the nanopore, which can travel back and forth through the nanopore.

FIG. 34 shows a basic functional nanopore. In each panel, the y-axis is current (nA) and the x-axis is time (sec). The left panel, "Screening of RF noise," shows the utility of the Faraday cage. A chip without a nanopore is placed in the flow cell and 300 mV is applied. When the lid of the Faraday cage is closed (first arrow), a reduction in noise is seen. When the latch is closed (second arrow), a small spike occurs. The current is seen to be about 0 nA. After creation of the pore (middle panel), application of 300 mV (arrow) results in a current of about 3.5 nA. When DNA is applied to the earth chamber and +300 mV is applied, DNA translocation (right panel) can be observed as a transient decrease in current. (Note, TS buffer is used in this case: 50 mM Tris, pH 8, 1 M NaCl.) Lambda DNA is used for this DNA translocation experiment.

FIG. 35 shows a simplified diagram illustrating the main features of the DNA origami structure: a large single-stranded region, a cubic origami structure, and the presence of two restriction sites (SwaI and AlwN1) near the origami structure.

Figure 36 shows an electron microscope image of the fabricated DNA origami structure, verifying the expected topology. The origami was fabricated in 5 mM Tris base, 1 mM EDTA, 5 mM NaCl, 5 mM MgCl2. To maintain the origami structure, it is preferable to have a Mg ⁺⁺ concentration of about 5 mM or a Na ⁺ /K ⁺ concentration of about 1 M. The origami structures are stored at 500 nM at 4°C.

試験レーン（１）は負の対照である；（２）はＳｗａ１での切断である；（３）はＡｌｗＮ１での切断である；（４）はＳｗａ１／ＡｌｗＮ１での二重切断である。切断は室温で６０分間行い、その後３７℃で９０分間行った。エチジウムブロマイド染色で可視化されたアガロースゲル１／２ｘ ＴＢＥ－Ｍｇ（５ｍＭ ＭｇＣｌ２を含む１／２ｘ ＴＢＥ）。いずれかの酵素での個々の切断はゲルにおいて移動度の効果を示さないが、両方の酵素での切断（レーン４）は、予想されるように異なる長さの２つのフラグメントをもたらす。 FIG. 37 shows restriction enzyme digestion of DNA origami to confirm correct assembly and function. The leftmost lane gives the MW standard. Restriction sites are tested by digesting the origami with AlwN1 and Swa1. The four test lanes contain the following reagents (in microliters):

Test lane (1) is a negative control; (2) is cleavage with Swa1; (3) is cleavage with AlwN1; (4) is a double cleavage with Swa1/AlwN1. Digestion was carried out at room temperature for 60 min followed by 90 min at 37°C. Agarose gel 1/2x TBE-Mg (1/2x TBE containing 5 mM MgCl2) visualized by ethidium bromide staining. Individual cleavage with either enzyme shows no mobility effect in the gel, but cleavage with both enzymes (lane 4) results in two fragments of different lengths as expected.

Figure 38 shows the binding of biotinylated oligonucleotides to streptavidin-coated beads compared to binding to control BSA-coated beads. The y-axis is in units of fluorescence. "Before binding" is the fluorescence of the oligos from the test solution before binding to the beads. (-) control is the fluorescence seen after binding to two batches of BSA-coupled beads. SA-1 and SA-2 are the fluorescence seen after binding to two batches of streptavidin-coupled beads. An apparent small amount of binding is observed to the BSA-coupled beads, but much greater binding is seen to the streptavidin-coupled beads.

FIG. 39 shows the binding of biotinylated oligonucleotides to streptavidin-coated beads in different buffer systems (MPBS and HK buffer) compared to binding to control BSA-coated beads. The left bar "negative control" is the fluorescence of the oligos from the test solution before binding to the beads. The middle column shows the fluorescence of "BSA beads" after binding to BSA beads, and the right column shows the fluorescence of "SA beads" after binding to streptavidin beads. In both buffer systems, the fluorescence is reduced by the streptavidin beads compared to the control, indicating that the biotinylated oligonucleotides bind well to the streptavidin-coated beads in the various buffer systems.

Figure 40 shows a functionalized conjugated _SiO2 nanopore, where the surface is coated with streptavidin on one side and BSA on the other. The x-axis is time and the y-axis is current. Dots indicate when the current was reversed. Upon reversing the current, there is a brief overshoot, after which the current settles to approximately the same absolute value. The nanopore shows a current of approximately +3 nA at 200 mV and approximately -3 nA at -200 mV.

FIG. 41 shows an origami DNA structure inserted into a nanopore. FIG. 42 depicts the binding of single-stranded DNA to a streptavidin-coated surface adjacent to a nanopore.

Figure 43 shows experimental results for origami DNA bound to a surface near a nanopore. The current was + or - 2.5 nA in both directions, lower than the original current of +/- 3 nA, reflecting partial occlusion by the origami structure. The x-axis is time (seconds), the y-axis is current (nA), and the circles indicate the time points when the voltage was switched.

Figure 44 shows the insertion of origami DNA, which results in a slight drop in current. When the current is released, the origami immediately exits the nanopore. The x-axis is time (sec), the y-axis is current (nA), and the circles indicate the time points when the voltage was switched.

Figure 45 displays the control of the movement of a DNA strand back and forth through a nanopore by application of electric current. On the left, the DNA is in the pore so the observed current is lower than when the DNA is not in the pore. When the current is reversed (right), there is no DNA in the pore so the current remains unchanged.

Figure 46 shows experimental results that confirm this indication. When a positive voltage is applied, the current is about 3 nA, which is comparable to the current typically observed when the pore is open. When the voltage is reversed, the current is about -2.5 nA, which is lower than the current typically seen when the pore is open and corresponds to the current typically observed when the pore is blocked by a DNA strand. Several successive voltage switches show consistent results, suggesting that the DNA alternates between the configurations shown in Figure 45. FIG. 47 shows various conjugation chemistries for linking DNA to a surface adjacent to the nanopore.

The following description of the preferred embodiments is merely exemplary in nature and is not intended to limit the invention, its applications, or uses in any manner.

Wherever used, ranges are used as shorthand for describing any value within the range. Any value within the range may be selected as an end point of the range. Additionally, all references cited herein are incorporated by reference in their entirety. In the event of a conflict between a definition in this disclosure and a definition in a cited reference, the present disclosure controls.

Unless otherwise specified, all percentages and amounts expressed herein and elsewhere herein should be understood to refer to percentages by weight. The amounts given are based on the effective weight of the material.

"Nanochip" herein refers to a nanofluidic device that includes multiple chambers containing fluids and optionally channels that allow fluid flow, where the critical dimensions of the nanochip features (e.g., width of elements separating chambers from one another) are between 1 atom and 10 microns thick, e.g., less than 1 micron (e.g., 0.01-1 micron). The flow of materials in the nanochip can be regulated by electrodes. For example, DNA and RNA are negatively charged, so they are attracted to positively charged electrodes. See, e.g., Gershow, M, et al., Recapturing and Trapping Single Molecules with a Solid State Nanopore, Nat Nanotechnol. (2007) 2(12): 775-779, which is incorporated herein by reference. The flow of fluids can also be optionally regulated by gating elements and by flowing, injecting, and/or aspirating fluids into or out of the nanochip. This system allows for accurate multiplex analysis of nucleic acids (DNA/RNA). In some embodiments, the nanotip can be made of a silicon material (e.g., silicon dioxide or silicon _nitride ). Silicon nitride (e.g., _Si3N4 ) is particularly desirable for this purpose because it is relatively chemically inert and provides an effective barrier to the diffusion of water and ions even at a thickness of only a few nm. Silicon dioxide is also useful (as used in the examples herein) because it is an excellent surface for chemical modification. Alternatively, in certain embodiments, the nanotip may be fabricated in whole or in part from a material capable of forming sheets as thin as a single molecule (also referred to as a monolayer material) such as graphene (described, for example, in Heerema, SJ, et al, Graphene nanodevices for DNA sequencing, Nature Nanotechnology (2016) 11: 127-136; Garaj S et al., Graphene as a subnanometre trans-electrode membrane, Nature (2010) 467 (7312), 190-193, the contents of each of which are incorporated herein by reference) or molybdenum disulfide ( _MoS2 ) (described, for example, in Feng, et al., Identification of single nucleotides in _MoS2 Nanopores, Nat Nanotechnol. (2015) 10(12):1070-1076, the contents of which are incorporated herein by reference) or boron nitride (Gilbert, et al. Fabrication of Atomically Transition metal dichalcogenides, such as those described in Precise Nanopores in Hexagonal Boron Nitride, eprint arXiv:1702.01220 (2017).

In some embodiments, the nanotip comprises such a monolayer material that is relatively stiff and inert (e.g., a monolayer material that is at least as inert and stiff as graphene, such as _MoS2 ). The monolayer material can be used, for example, as all or part of a membrane that includes a nanopore. The nanotip can be partially lined with a metal (e.g., the walls can be layered (e.g., metal-silicon nitride-metal) and then the metal is positioned to provide a controllable electrode pair in the vicinity of the nanopore such that the nucleic acid can be moved back and forth through the nanopore by electromotive forces and sequenced by measuring the change in potential as it passes through the nanopore).

Nanotip nanofluidic devices for sequencing DNA are generally known, see, e.g., Li, J., et al, Solid-state nanopore for detecting individual biopolymers, Methods Mol Biol. (2009)544:81-93; Smeets RM, et al. Noise in solid-state nanopores, PNAS (2008)105(2):417-21; Venta K, et al., Differentiation of short, single-stranded DNA homopolymers in solid-state nanopores, ACS Nano. (2013)7(5):4629-36; Briggs K, et al. Automated fabrication of 2-nm solid-state nanopores for nucleic acid analysis, Small (2014)10(10):2077-86; and Chen Z, DNA translocation through an array of kinked nanopores, Nat Mater. (2010) 9(8):667-75, the entire contents of each of which are incorporated herein by reference for their teachings on the design and fabrication of nanochips, e.g., containing nanopores.

A "nanopore" herein is a pore having a diameter of less than 1 micron (e.g., 2-20 nm diameter, e.g., on the order of 2-5 nm). Single-stranded DNA can pass through a 2 nm nanopore; single- or double-stranded DNA can pass through a 4 nm nanopore. Having a very small nanopore (e.g., 2-5 nm) allows the passage of DNA but not larger protein enzymes, thereby allowing for controlled synthesis of DNA (or other charged polymers). When using larger nanopores (or smaller protein enzymes), the protein enzyme may be conjugated to a substrate (e.g., a larger molecule such as a larger protein, a bead, or a surface within the chamber) that prevents the protein enzyme from passing through the nanopore. Various nanopores are known. For example, biological nanopores are formed by the association of pore-forming proteins in a membrane (such as a lipid bilayer). For example, α-hemolysin and similar protein pores are naturally found in cell membranes, which act as channels to transport ions or molecules into and out of cells, and such proteins can be repurposed as nanochannels. Solid-state nanopores are formed in synthetic materials (such as silicon nitride or graphene) by, for example, forming holes in synthetic membranes using, for example, feedback-controlled low-energy ion beam sculpting (IBS) or high-energy electron beam irradiation. Hybrid nanopores can be made by embedding pore-forming proteins in synthetic materials. If metal surfaces or electrodes are at either or both ends of the nanopore, a current along the nanopore can be established through the nanopore via the electrolyte medium. The electrodes can be made of any conductive material (e.g., silver, gold, platinum, copper, titanium dioxide, silver coated with, for example, silver chloride).

Methods for forming nanopores in solids (silicon nitride, membranes) are known. In one approach, a silicon substrate is coated with the membrane material (e.g., silicon nitride) and the overall shape of the membrane is created using photolithography and wet chemical etching to give a silicon nitride membrane of the desired size (e.g., about 25x25 microns) for incorporation into a nanochip. An initial 0.1 micron diameter hole or cavity is drilled in the silicon nitride membrane using a focused ion beam (FIB). Ion beam sculpting can form the nanopores either by shrinking the larger pores (e.g., by lateral mass transport on the membrane surface induced by the ion beam) or by removing membrane material layer by layer from the flat surface of the membrane containing the cavities from the opposite side, such that when the cavities are finally connected, they become sharp-edged nanopores. When the ion beam exposure is turned off, the ion current transmitted through the pores is appropriate for the size of the desired pore. See, e.g., Li, J., et al., Solid-state nanopore for detecting individual biopolymers, Methods Mol Biol. (2009)544:81-93. Alternatively, nanopores can be formed using high energy (200-300 keV) electron beam irradiation in a TEM. Using semiconductor processing techniques, electron beam lithography, reactive ion etching of SiO2 mask layers, and anisotropic KOH etching of Si, pyramidal shaped 20x20 nm and larger pores are created in 40 nm thick membranes. Using an electron beam in a TEM, the larger 20 nm pores are reduced to smaller pores. The TEM allows the reduction process to be observed in real time. With thinner membranes (e.g., <10 nm thick), nanopores can be drilled with a high energy focused electron beam in a TEM. See generally Storm AJ, et al. Fabrication of solid-state nanopores with single-nanometre precision. Nature Materials (2003) 2:537-540; Storm AJ, et al. Translocation of double-stranded DNA through a silicon oxide nanopore. Phys. Rev. E (2005)71:051903; Heng JB, et al. Sizing DNA Using a Nanometer-Diameter Pore. Biophys. J (2004) 87(4):2905-11, the contents of each of which are incorporated herein by reference.

In another embodiment, nanopores are created by dielectric breakdown using a relatively high potential difference across the membrane, where the potential is increased until a current is detected, as described in Kwok, et al., "Nanopore Fabrication by Controlled Dielectric Breakdown," PLOS ONE (2014) 9(3): e92880, the contents of which are incorporated herein by reference.

With these techniques, and depending of course on the actual technique used and the thickness and actual composition of the membrane, the overall shape of the nanopore in a solid material such as silicon nitride may roughly resemble two funnels that meet at their narrowest point (i.e., the actual nanopore). Such a pair of cones is conductive, allowing the polymer to pass in and out through the nanopore. Imaging techniques (e.g., atomic force microscopy (AFM) or transmission electron microscopy (TEM), especially TEM) can be used to verify and measure the size, location and shape of the nanomembrane, the holes or cavities of the FIB, and the final nanopore.

In some embodiments, one end of the polymer (e.g., DNA) is anchored near the nanopore or to the inner wall of a funnel leading to the nanopore. When one end of the polymer is anchored near the nanopore, the polymer approaches the nanopore first by diffusion and then is guided by the electrical gradient, maximizing gradient-guided motion and minimizing diffusional motion, thereby enhancing speed and efficiency. For example, Wanunu M., Electrostatic focusing of unlabeled DNA into nanoscale pores using a salt gradient, Nat Nanotechnol. (2010) 5(2):160-5; Gershow M., Recapturing and trapping single molecules with a solid-state nanopore. Nat Nanotechnol. (2007) 2(12):775-9; Gershow, M., Recapturing and Trapping Single Molecules with a Solid State Nanopore. See Nanotechnol. (2007) 2(12): 775-779.

In one embodiment, one end of a polymer (e.g., DNA) is attached to a bead and the polymer is threaded through the pore. The bead binding prevents the polymer from moving all the way through the nanopore on the other side of the separation membrane in the adjacent chamber. The current is then stopped and the polymer (e.g., DNA) is attached to a surface adjacent to the nanopore in the chamber on the other side of the separation membrane. For example, in one embodiment, one end of the ssDNA is covalently attached to a 50 nm bead and the other end is biotinylated. Streptavidin is attached to a region of the chamber on the other side of the separation membrane at the desired attachment point. The DNA is pulled out of the nanopore by the potential difference and the biotin binds to the streptavidin. The binding to the bead and/or surface adjacent to the nanopore can be either covalent or strong non-covalent (e.g., biotin-streptavidin binding). The beads are then enzymatically cleaved and washed away. In some embodiments, the single-stranded DNA is cleaved with an enzyme that cleaves single-stranded DNA, as described in K. Nishigaki, Type II restriction endonucleases cleave single-stranded DNAs in general. Nucleic Acids Res. (1985) 13(16): 5747-5760, which is incorporated herein by reference. In other embodiments, complementary oligonucleotides are provided to create double-stranded restriction sites, which can then be cleaved with the corresponding restriction enzyme.

As a polymer passes through the nanopore, changes in the potential difference or current across the nanopore caused by partial blockage of the nanopore as the polymer passes through can be detected and used to identify the sequence of monomers within the polymer, as different monomers can be distinguished by their different sizes and electrostatic potentials.

Although the use of nanochips containing nanopores in methods for producing DNA as described herein has not been disclosed in the art, such chips are well known and commercially available for rapid sequencing of DNA. For example, the MinION (Oxford Nanopore Technologies, Oxford, UK) is small and can be attached to a laptop. As single-stranded DNA passes through a protein nanopore 30 bases per second, the MinION measures the current. The DNA strand in the pore obstructs the ion flow, which results in a change in current corresponding to the nucleotides in the sequence. Mikheyev, AS, et al. A first look at the Oxford Nanopore MinION sequencer, Mol. Ecol. Resour. (2014)14, 1097-1102. Although the accuracy of the MinION is poor and requires repeated sequencing, the speed and accuracy of sequencing using the nanochip of the present invention can be significantly improved if the DNA being read contains only two easily distinguishable bases (e.g., A and C).

In some embodiments, the membrane containing the nanopore may have a three-layer morphology, including metal surfaces on either side of an insulating core material (e.g., a silicon nitride membrane). In this embodiment, the metal surfaces are formed, e.g., by lithographic means, to provide a microcircuit including a pair of electrodes at each end of each nanopore, such that a current through the nanopore can be established between the electrodes via an electrolyte medium through the nanopore, which current can drive the polymer through the nanopore and pull the polymer back by reversing the polarity. As the polymer passes through the nanopore, the electrodes can measure the change in potential across the nanopore, identifying the sequence of monomers within the polymer.

In some embodiments, the sequence of the polymer is designed to store data. In some embodiments, the data is stored in a binary code (1s and 0s). In some embodiments, each base corresponds to a 1 or a 0. In other embodiments, an easily recognized sequence of two or more bases corresponds to a 1, and another easily recognized sequence of two or more bases corresponds to a 0. In other embodiments, the data can be stored in a ternary code, a quaternary code, or other code. In a particular embodiment, the polymer is DNA (e.g., single-stranded DNA), where the DNA contains only two bases and does not contain any bases that can self-hybridize (e.g., DNA contains adenine and guanine, adenine and cytosine, thymidine and guanine, or thymidine and cytosine). In some embodiments, the two bases may be interspersed with one or more additional bases, e.g., A and C may contain T at a frequency that does not result in significant self-hybridization, e.g., to "break up" the sequence by presenting a break in the coding sequence. In other embodiments, some or all of the available bases may be used, e.g., when the nucleic acid is double-stranded.

The nucleotide bases may be natural or, in some embodiments, may consist of or include non-natural bases, e.g., as described in Malyshev, D. et al. "A semi-synthetic organism with an expanded genetic alphabet", Nature (2014) 509: 385-388, which is incorporated herein by reference.

In one embodiment, data is stored by the addition of a single monomer (e.g., in the case of DNA, a single nucleotide) to the polymer. In one embodiment, the polymer is DNA and the monomers are adenine (A) and cytosine (C) residues. A and C residues are favored because (i) A and C have a large size difference and therefore should be easily distinguished by the nanopore, (ii) A and C do not pair with each other and therefore do not form significant secondary structure that may complicate interpretation of the nanopore signal, and (iii) G is known to form G-quadruplexes and is therefore less preferred for similar reasons. Nucleotides are added by terminal transferase (or polynucleotide phosphorylase), but are 3' blocked so that only a single nucleotide is added at a time. The blocking is removed prior to the addition of the next nucleotide.

In some embodiments, DNA is placed within the nanochip. In other embodiments, the DNA is removed and optionally converted to double-stranded DNA and/or optionally converted to a crystalline form, e.g., to enhance long-term stability. In yet other embodiments, the DNA can be amplified and the amplified DNA removed for long-term storage, but the original template DNA (e.g., DNA bound to the walls of the chamber within the nanochip) can be left within the nanochip, which can be read and/or used as a template to make additional DNA.

Ａを流す＝３’保護されたｄＡＴＰ
Ｃを流す＝３’保護されたｄＣＴＰ In some embodiments, DNA or other polymers are tethered to a surface proximate to a nanopore during synthesis. For example, in some embodiments, single-stranded DNA molecules are attached at each 5' end to a surface proximate to a nanopore such that the 3' end of the DNA molecule can be pulled from a holding chamber through the nanopore into a flow chamber containing a stream of 3' protected dNTPs along with a polymerase or terminal transferase enzyme that adds the 3' protected dNTPs, or can be held in a holding chamber where the nanopore excludes the enzyme so that it does not add dNTPs, and the current at each nanopore can be independently regulated by an electrode for that nanopore. See, for example, the depictions of Figures 12-16 and Figures 18 and 19. In other embodiments, single-stranded DNA is constructed by addition to the 5' end (with the 3' end attached) using a topoisomerase, as described in more detail below. By controlling whether each DNA molecule participates in each cycle, the sequence of each DNA molecule can be precisely controlled, for example, as follows:

Flow A = 3' protected dATP
Flow C = 3' protected dCTP

Nanopore 1 and nanopore 2 in this diagram are bound to different DNA strands, whose positions (inside or outside the flow chamber) can be controlled separately. The DNA can be deprotected by specific enzymes in the holding chamber or by changing the flow in the flow chamber to effect deprotection by enzymatic, chemical, photocatalytic or other means. In one embodiment, the deblocking reagent flows between the A and C flow cycles (e.g., when the flow chamber is being washed with buffer) so that the deblocking reagent does not deprotect the building block nucleotides. In another embodiment, the deprotecting reagent is too bulky to pass through the nanopore to the flow chamber.

The end result of the above example would be that A and C are added to the DNA of nanopore 1, and C and C are added to the DNA of nanopore 2.

In another embodiment, the chamber arrangement is similar, but with double-stranded DNA tethered to a surface adjacent to the nanopore, oligonucleotide fragments (e.g., two or more types), each corresponding to a binary code, are added sequentially, for example, using a site-specific recombinase (i.e., an enzyme that spontaneously recognizes and cleaves at least one strand of a nucleic acid duplex in a sequence segment known as a site-specific recombination sequence), for example, using a topoisomerase-charged oligonucleotide, as described below.

In certain embodiments, it may be desirable to keep a charged polymer (e.g., DNA) in an aggregated state after synthesis. There are several reasons for this:
- The polymer should be more stable in this shape,
Flocculating the polymer keeps crowding low, allowing the use of longer polymers in smaller volumes.
Ordered aggregation may reduce the likelihood of the polymer forming knots or entanglements;
If any chambers are interconnected, this helps to prevent the polymer from lengthening through pores other than the ones it is supposed to when a current is applied.
• Agglomeration helps to detach the polymer from the electrode where electrochemistry can damage the polymer.

A human cell is about 10 microns but contains 8 billion base pairs of DNA. If this DNA were stretched out it would be over a meter. The DNA is contained within the cell because it is wrapped around histone proteins. In some embodiments, histones or similar proteins serve a similar function in the nanochips of the invention. In some embodiments, the interior surfaces of the nanochips are slightly positively charged so that charged polymers (e.g., DNA) tend to stick weakly to them.

In one embodiment, a charged polymer, such as single- or double-stranded DNA, is attached to a surface in close proximity to the nanopore. This can be accomplished in a variety of ways. In general, the polymer is attached to a relatively bulky structure (e.g., a bead, protein, or DNA origami structure (described below)) that has a diameter too large to pass through the nanopore (e.g., >10 nm, e.g., 20-50 nm), and the polymer is localized to the nanopore by pulling the charged polymer through the nanopore using an electric current, tethering the end of the polymer remote from the bulky structure to a surface adjacent to the nanopore, and decoupling the bulky structure.

Anchoring the end of the polymer away from the bulky structure to the surface adjacent to the nanopore can be accomplished in a variety of ways. In one embodiment, the polymer is single-stranded DNA, and there is a pre-attached DNA strand (approximately 50 bp) that is complementary to a portion of the single-stranded DNA, and the single-stranded DNA and the pre-attached DNA strand can be attached via base pairing. If this base pairing is strong enough, it is enough to keep the DNA anchored even while it is being manipulated. The advantage of this attachment method is that the DNA can be removed from the nanopore chip if desired for long-term storage of the DNA. Alternatively, the chains are covalently attached to the surface using either conjugation chemistry (e.g., the streptavidin-biotin conjugate described in Example 1 below) or "click" chemistry (see Kolb, et al. Angew. Chem. Int. Ed. (2001)40: 2004-2021, which is incorporated herein by reference) and/or enzymatic linkage (e.g., by pre-covalently attaching oligos to a distant surface and then joining them using DNA ligase).

When the distal end of the strand binds to a surface adjacent to the nanopore, the bulky structure is cleaved, for example, using an endonuclease that cleaves a restriction site near the bulky structure.

The bulky structure can be a bead, a bulky molecule (e.g., a protein that reversibly binds to a DNA strand), or a DNA origami structure. DNA origami involves the use of base pairing to create three-dimensional DNA structures. DNA origami technology is generally described in Bell, et al, Nano Lett. (2012)12: 512-517, which is incorporated herein by reference. For example, in the present invention, DNA origami can be used to attach a single DNA molecule to a surface adjacent to a nanopore. In one embodiment, the structure is a "honeycomb cube" (e.g., about 20 nm on each side). This prevents this portion of DNA from passing through the nanopore (as in the accompanying document). There is a long strand of DNA (single-stranded or double-stranded) that is attached to the origami structure. The DNA strand travels through the nanopore until the origami structure encounters the nanopore and blocks further progress. The current is then stopped, and the strand is allowed to attach to the surface adjacent to the nanopore.

In another embodiment, the charged polymer (e.g., DNA) with an origami structure is in the center chamber of a three chamber arrangement. The origami prevents the DNA from completely entering the other two chambers (or the other chamber in the two chamber example). Thus, in this example, the polymer does not need to be tethered to a surface. This reduces the risk of the polymer tying a knot and avoids the need to attach one end of the polymer to a surface and sever the bulky portion at the other end. The volume of the chamber containing the origami should be as small as possible so that the polymer stays relatively close to the pore, which helps ensure that the polymer moves quickly when a current is applied. Note that the center chamber containing the origami portion of the polymer cannot interconnect with the other center chambers (otherwise the various polymers would intermix), but the other chambers (or sets of chambers in the three chamber example) can. These other chambers may have larger volumes, if desired, since the polymer is necessarily near the pore (actually part of it is inside the pore) as the DNA moves into the other chambers.

In some embodiments, the device includes three chambers in series, where the additional chambers are in series to allow flow and have a common electrode, but the "deprotection" chamber is fluidically isolated except for flow through the nanopore and has its own electrode.

In other embodiments, the DNA or other charged polymer is not tethered but can be moved between the synthesis and deprotection chambers under the control of electrodes within the chambers, while the polymerase and deprotection agent are too bulky to pass through the nanopores connecting the chambers and/or are tethered to surfaces within the chambers, thereby restricting movement between the chambers. See, e.g., Figures 1-9 and 16-17.

The current required to translocate a charged polymer through a nanopore depends, for example, on the nature of the polymer, the size of the nanopore, the material of the membrane containing the nanopore, and the salt concentration, and is optimized for a particular system as required. For DNA used in the examples herein, example voltages and currents are 50-500 mV (usually 100-200 mV) and 1-10 nA (e.g., about 4 nA), for example, at salt concentrations on the order of 100 mM to 1 M.

The movement of a charged polymer, e.g. DNA, through a nanopore is typically very fast, e.g. 1-5 μs per base, i.e. on the order of a million bases per second (1 MHz if we adopt frequency terminology), which presents a challenge to obtain accurate readings that are distinguished from noise in the system. Using the method, (i) a nucleotide needs to be repeated in the sequence, e.g. about 100 times in succession, to produce a measurable characteristic change, or (ii) using a protein pore such as alpha hemolysin (αHL) or Mycobacterium smegmatis porin A (MspA), which provides a relatively long pore with the potential for multiple readings as the base passes through the polymer, and in some cases can be tuned to provide a controlled delivery of DNA through the pore one base at a time, in some cases using an exonuclease that cleaves each base as it passes. Various approaches are possible, e.g.
Slowing down the speed of the polymer from about 1 MHz to about 100-200 Hz, for example using a medium that includes an electrorheological fluid that becomes more viscous when a voltage is applied, thereby slowing down the speed of the polymer passing through the nanopore, or a plasmonic fluidic system where the viscosity of the medium can be controlled by light; or using molecular motors or ratchets;
Providing sequences in the polymer, e.g., in single-stranded DNA, that form bulky secondary structures, e.g., "hairpin,""hammerhead," or "dumbbell" configurations, that must be linearized to fit into the nanopore, thereby thinning the information density and providing a signal with a long duration;
- providing multiple reads of the same sequence, for example by using rapidly changing currents to allow multiple reads of the same sequence frame, combined with short bursts of direct current to pull the molecule to the next sequence frame, reading the entire sequence multiple times, or by reading multiple identical sequences in parallel, pooling the reads in each case to provide a common read that amplifies the signal;
Measuring impedance changes in high frequency signals induced by capacitance changes as monomers (e.g., nucleotides) pass through the nanopore, rather than directly measuring changes in current or resistance;
- enhancing the difference in current, resistance or capacitance between different bases, for example by using non-natural bases that differ more in size or are otherwise modified to give different signals, or by forming larger secondary structures in the DNA such as "hairpin", "hammerhead" or "dumbbell" configurations that result in enhanced signals due to their larger size;
Use of an optical readout system, for example an integrated optical antenna adjacent to the nanopore that acts as an optical transducer (or optical signal enhancer) to complement or replace standard ionic current measurements, such as those described in Nam, et al., "Graphene Nanopore with a Self-Integrated Optical Antenna", Nano Lett. (2014)14: 5584-5589, the contents of which are incorporated herein by reference. In some embodiments, monomers, e.g., DNA nucleotides, are labeled with fluorescent dyes such that each different monomer fluoresces with a signature intensity when passing through the junction of the nanopore and its optical antenna. In some embodiments, as the polymer passes through the nanopore at high speed, the solid-phase nanopore strips off the fluorescent label, leading to a series of detectable bursts of photons, as described, for example, in McNally et al., "Optical recognition of converted DNA nucleotides for single molecule DNA sequencing using nanopore arrays", Nano Lett. (2010)10(6): 2237-2244, and Meller A., "Towards Optical DNA Sequencing Using Nanopore Arrays", J Biomol Tech . (2011) 22(Suppl): S8-S9, the contents of each of which are incorporated herein by reference.

In some embodiments, the charged polymer is a nucleic acid, e.g., single-stranded DNA, whose sequence provides the secondary structure. Bell, et al., Nat Nanotechnol. (2016) 11(7): 645-51, incorporated herein by reference, describes labeling antigens in immunoassays using relatively short sequences in a dumbbell configuration that can be detected in a solid-phase nanopore format. The nanopores used by Bell et al. were relatively large to allow the entire dumbbell structure to pass through the pore, but using nanopores smaller than the diameter of the dumbbell configuration causes the DNA to "unzip" and become linear. More complex configurations can be used, e.g., where each bit corresponds to a sequence similar to tRNA (see, e.g., Henley, et al. Nano Lett. (2016) 16: 138-144, incorporated herein by reference). Thus, the invention provides a charged polymer, e.g., single-stranded DNA, that has at least two types of secondary structure, which encode data (e.g., binary data, where one type of secondary structure is a 1 and the second secondary structure is a 0). In other embodiments, the secondary structure is used to slow down the passage of the DNA through the nanopore or to introduce interruptions in the sequence, making it easier to read the sequence.

In another embodiment, the invention utilizes a DNA molecule that includes a series of at least two different DNA motifs, each of which specifically binds to a particular ligand (e.g., a gene regulatory protein for double-stranded DNA, or a tRNA for single-stranded DNA), and the at least two different DNA motifs encode information (e.g., in a binary code where one motif is 1 and the second motif is 0), e.g., the ligand enhances the difference in signal across the nanopore (e.g., a change in current or capacitance) as the DNA passes through the nanopore.

As discussed above, when different monomers pass through the nanopore, they affect the current through the nanopore primarily by physically blocking the nanopore and changing the conductance of the nanopore. Existing nanopore systems measure this change in current directly. The problem with current readout systems is that the systems are quite noisy and, in the case of DNA, for example, relatively long integration times are required to accurately detect differences between different monomers, e.g., different bases, on the order of hundredths of a second when measuring the current fluctuations as different nucleotide units pass through the nanopore. Recently, it has been shown that impedance and capacitance changes can be useful to study cells and biological systems, despite the possibility of complex interactions of salts and biomolecules. For example, Laborde, et al. Nat Nano. (2015)10(9):791-5 (incorporated herein by reference) demonstrate that high frequency impedance spectroscopy can be used to detect small changes in capacitance under physiological salt conditions and image microparticles and living cells beyond the Debye limit.

Thus, in one embodiment of the invention, rather than directly measuring current fluctuations, the sequence of a charged polymer is identified by measuring capacitance fluctuations, e.g., by measuring the phase change of a radio frequency signal induced by the change in capacitance as a monomer (e.g., a nucleotide) passes through a nanopore.

Briefly, capacitance exists in any circuit where there is a gap between one electrical conductor and the other. The current varies directly with the capacitance, but not simultaneously with the capacitance. For example, if you plot the current and voltage over time in an alternating current capacitive circuit, you will see that both the current and the voltage each form a sine wave, but the phases of the waves are out of phase. When there is a change in the current, there is a change in the capacitance, which is reflected in a change in the phase of the signal. Radio frequency alternating current results in a signal of constant frequency and amplitude, but the phase of the signal varies with the capacitance of the circuit. In the present system, rather than alternating current, a pulsating direct current (i.e., the voltage varies between two values but does not cross the "zero" line, so that polarity is maintained and one electrode remains an anode and the other remains a cathode) is used to draw the charged polymer through the nanopore (towards the anode in the case of DNA). When there is nothing in the nanopore, the capacitance is one value, which changes when different monomers of the polymer pass through the nanopore. Suitable frequencies are in the radio frequency range, e.g. 1 MHz to 1 GHz, e.g. 50-200 MHz, e.g. about 100 MHz, e.g. below high microwave frequencies that can cause significant dielectric heating of the medium. To reduce the possibility of interference, different frequencies can be applied to different nanopores, so that multiple nanopores can be measured simultaneously with one radio frequency input line.

Measuring impedance changes (e.g., due to capacitance changes) at high frequencies reduces the effects of 1/f noise, or "pink" noise, inherent in electrical measurement circuits, thus increasing the signal-to-noise available over a given period of time. As many measurements are made in a given period of time, the use of a high frequency signal increases the signal-to-noise ratio, resulting in a more stable signal that is easily distinguished from impedance changes due to environmental or device variations and fluctuations.

Applying these principles to the present invention, the present invention provides, in one embodiment, a method for measuring impedance changes in a radio frequency signal induced by changes in capacitance as monomers (e.g., nucleotides) pass through a nanopore. For example, a method for reading the monomer sequence of a charged polymer, e.g., a DNA molecule, that includes at least two different types of monomers, is provided, which includes applying a radio frequency pulsating direct current to the nanopore, e.g., at a frequency of 1 MHz to 1 GHz, e.g., 50 to 200 MHz, e.g., about 100 MHz, such that the pulsating direct current draws the charged polymer through the nanopore, and reading the monomer sequence by measuring the capacitance fluctuations of the nanopore as the charged polymer passes through the nanopore.

In some embodiments, the invention provides a nanochip for sequencing charged polymers, e.g., DNA, comprising at least a first and a second reaction chamber, each of which contains an electrolyte medium and is separated by a membrane comprising one or more nanopores, and a pair of electrodes connected in a circuit (e.g., in the form of facing plates) disposed on either side of the membrane comprising one or more nanopores, the electrodes being spaced 1-30 microns, e.g., about 10 microns apart, such that when a pulsating direct current radio frequency, e.g., 1 MHz-1 GHz, is applied to the electrodes to draw the charged polymer through the nanopore, e.g., from one chamber to the next, the gap between the electrodes has a capacitance, and the phase of the pulsating direct radio frequency current changes with the change in capacitance as the charged polymer passes through the nanopore, thereby allowing detection of the monomer sequence of the charged polymer. In certain embodiments, the nanochip comprises multiple sets of reaction chambers, the reaction chambers within a set being separated by a membrane having one or more nanopores, and the sets of reaction chambers being separated by a screening layer that minimizes electrical interference between the sets of reaction chambers and/or separates multiple linear polymers to allow them to be sequenced in parallel.

For example, in one embodiment, the electrodes form the upper and lower plates of a capacitor embedded in a resonant circuit, and the change in capacitance is measured as DNA passes through a pore between the plates.

In certain embodiments, the nanochip further comprises reagents for synthesizing a polymer, e.g., DNA, e.g., according to any of nanochip 1 described below.

Thus, in one embodiment, the present invention provides a method (Method 1) for synthesizing a charged polymer [e.g., a nucleic acid (e.g., DNA or RNA)] comprising at least two distinct monomers in a nanochip, the nanochip comprising:
one or more addition chambers containing reagents for adding one or more monomers [e.g., nucleotides] or oligomers [e.g., oligonucleotides] in a buffer in a terminally protected form to the charged polymer such that only a single monomer or oligomer can be added in one reaction cycle; and
one or more storage chambers containing a buffer, but not all of the reagents necessary for the addition of one or more monomers or oligomers, wherein the chambers are separated by one or more membranes containing one or more nanopores;
The charged polymer can pass through the nanopore, but at least one of the reagents for the addition of the one or more monomers or oligomers cannot pass through the nanopore.
Including,
This method is
a) moving the first end of a charged polymer having a first end and a second end to an attachment chamber by electrical attraction, thereby attaching a monomer or oligomer in a blocked form to the first end;
b) transferring the first end of the charged polymer together with the monomer or oligomer attached in blocked form to a storage chamber;
c) deblocking the added monomer or oligomer; and
d) repeating steps a-c until the desired polymer sequence is obtained, where the monomers or oligomers added in step a) are the same or different;
Includes.

For example, the present invention provides the following:
1.1 Method 1, wherein the polymer is a nucleic acid, e.g., the polymer is DNA or RNA, e.g., DNA, e.g., dsDNA or ssDNA.

1.2 Any of the above methods, wherein the second end of the polymer, e.g., the nucleic acid, is protected or attached to a substrate adjacent to the nanopore.

1.3 Any of the methods described above, in which an electrical attraction is provided by applying a voltage between the electrodes in each chamber, and the polarity and current between the electrodes can be controlled, for example, so that the nucleic acid is attracted to the anode.

1.4 The polymer is a nucleic acid,
(i) the first end of the nucleic acid is the 3' end, the addition of the nucleotide is in the 5' to 3' direction, and is catalyzed by a polymerase, e.g., the polymerase is rendered incapable of passing through the nanopore (e.g., because of its size or because it is anchored to a substrate in the first chamber), the nucleotide is 3'-protected at the time of addition, and the 3'-protected nucleotide is added to the 3'-end of the nucleic acid and then the 3'-protecting group on the nucleic acid is removed, e.g., in a storage chamber; or (ii) the first end of the nucleic acid is the 5' end, and the addition of the nucleotide is in the 3' to to 5', the nucleotide being 5'-protected upon addition, and after addition of the 5'-protected nucleotide to the 5'-terminus of the nucleic acid, the 5'-protecting group is removed, e.g., in a second chamber; (e.g., the phosphate on the 5'-protected nucleotide is a nucleoside phosphoramidite attached via the 5'-protecting group to a bulky group that cannot pass through the nanopore, such that following coupling to the nucleic acid, unreacted nucleotides are washed away and the bulky 5'-protecting group is cleaved from the nucleic acid and washed away, allowing the 5'-terminus of the nucleic acid to be transferred to a storage chamber);
wherein addition of nucleotides to the nucleic acid is controlled by movement of a first end of the nucleic acid in and out of one or more addition chambers, and this cycle is continued until the desired sequence is obtained.
Any of the methods mentioned above.

1.5 Any of the above methods, wherein the sequence of monomers or oligomers in the thus synthesized polymer [e.g., the sequence of nucleotides in a nucleic acid] corresponds to a binary code.
1.6 Any of the above methods, wherein the polymer thus synthesized is a single-stranded DNA.
1.7 Any of the methods described above, wherein the sequence of a polymer [e.g., a nucleic acid] is confirmed during processing or synthesis by sequencing monomers or oligomers [e.g., nucleotide bases] as they pass through the nanopore and identifying errors in the sequencing.

1.8 Any of the above methods, wherein the polymer thus synthesized is single-stranded DNA, and at least 95%, such as at least 99%, e.g. substantially all, of the bases in the sequence are selected from two bases that do not hybridize to other bases in the strand, e.g. bases selected from adenine and cytosine.

1.9 Any of the above methods, in which multiple polymers [e.g., oligonucleotides] are independently synthesized in parallel by individually controlling whether the polymers are present in one or more addition chambers or one or more storage chambers, such that polymers [oligonucleotides] with different sequences are obtained.

1.10 Any of the methods described above, wherein there are at least two addition chambers containing reagents suitable for adding different monomers or oligomers, e.g., different nucleotides, e.g., one or more addition chambers containing reagents suitable for adding a first monomer or oligomer, and one or more addition chambers containing reagents suitable for adding a second, different monomer or oligomer, e.g., one or more addition chambers containing reagents suitable for adding an adenine nucleotide, and one or more addition chambers containing reagents suitable for adding a cytosine nucleotide.

1.11 Any of the above methods, wherein at least one addition chamber is a flow chamber, and a flow cycle is provided that includes (i) providing a flow chamber reagent suitable for the addition of a first monomer or oligomer, (ii) rinsing, (iii) providing a flow chamber reagent suitable for the addition of a second, different monomer or oligomer, and (iv) rinsing, and the cycle is repeated until synthesis is complete, and the sequence of the monomers or oligomers in the polymer is controlled by introducing or excluding a first end of the polymer into or from the flow chamber in step (i) or (iii) of each cycle;

1.12 Any of the above methods, wherein the polymer is DNA, at least one addition chamber is a flow chamber, and a flow cycle is provided that includes (i) providing a flow chamber reagent suitable for addition of a first type of nucleotide, (ii) rinsing, (iii) providing a flow chamber reagent suitable for addition of a second type of nucleotide, and (iv) rinsing, and the cycle is repeated until synthesis is complete, and the sequence is controlled by controlling the presence or absence of the first end (e.g., the 3'-end) of the DNA in the flow chamber.

1.13 Any of the above methods, wherein the polymer is DNA, at least one addition chamber is a flow chamber, and a flow cycle is provided that includes (i) providing a flow chamber reagent suitable for the addition of a first type of nucleotide, (ii) rinsing, (iii) providing a flow chamber reagent suitable for the addition of a second type of nucleotide, and (iv) rinsing, (i) providing a flow chamber reagent suitable for the addition of a third type of nucleotide, (ii) rinsing, (iii) providing a flow chamber reagent suitable for the addition of a fourth type of nucleotide, and (iv) rinsing, and repeating the cycle until synthesis is complete, and the sequence is controlled by controlling the presence or absence of a first end (e.g., the 3'-end) of the DNA in the flow chamber when a reagent suitable for the addition of a different type of nucleotide is present.

1.14 The polymer is DNA, the nanotip comprises two addition chambers that are flow chambers, (a) a first flow chamber provides a flow cycle including (i) providing a flow chamber reagent suitable for the addition of a first type of nucleotide, (ii) rinsing, (iii) providing a flow chamber reagent suitable for the addition of a second, different type of nucleotide, and (iv) rinsing, repeating this cycle until synthesis is complete, and (b) a second flow chamber provides (i) a flow chamber reagent suitable for the addition of a second, different type of nucleotide, and (iv) rinsing. any of the above methods, comprising: (i) providing a flow chamber reagent suitable for adding a fourth, different type of nucleotide; (ii) rinsing; (iii) providing a second flow chamber reagent suitable for adding a fourth, different type of nucleotide; and (iv) rinsing, and repeating the cycle until synthesis is complete, where the nucleotides are selected from dATP, dTTP, dCTP, and dGTP, and the sequence is controlled by introducing a first end (e.g., the 3'-end) of the DNA into the flow chamber when the next desired nucleotide is provided.

1.15 Any of the above methods, wherein the polymer is DNA and the nanopore chip includes one or more addition chambers for adding dATP, one or more addition chambers for adding dTTP, one or more addition chambers for adding dCTP, and one or more addition chambers for adding dGTP.

1.16 Any of the above methods, wherein the polymers [e.g., nucleic acids] being synthesized are each attached via their second ends to a surface proximate to the nanopore.

1.17 Any of the methods described above, wherein the sequence of the polymer [e.g., a nucleic acid] is determined following each cycle by detecting changes in voltage, current, resistance, capacitance and/or impedance as the polymer passes through the nanopore.

1.18 Any of the above methods, wherein the polymer is a nucleic acid and the synthesis of the nucleic acid occurs in a buffer, e.g., a buffer to pH 7-8.5, e.g., about pH 8, e.g., a buffer containing tris(hydroxymethyl)aminomethane (Tris), a suitable acid, and optionally a chelator, e.g., ethylenediaminetetraacetic acid (EDTA), e.g., a TAE buffer containing a mixture of Tris base, acetic acid, and EDTA, or a TBE buffer containing a mixture of Tris base, boric acid, and EDTA; e.g., 10 mM Tris pH 8, 1 mM EDTA, 150 mM KCl, or e.g., a solution containing 50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate (pH 7.9 @ 25°C).

1.19 Any of the above methods, wherein the polymer is single-stranded DNA, and further comprising converting the synthesized single-stranded DNA to double-stranded DNA.

1.20 Any of the above methods, further comprising removing the polymer [e.g., nucleic acid] from the nanotip after completion of polymer synthesis.

1.21 Any of the above methods, wherein the polymer is a nucleic acid, further comprising amplifying and recovering copies of the synthesized nucleic acid using appropriate primers and a polymerase (e.g., Phi29).

1.22 Any of the above methods, wherein the polymer is a nucleic acid, further comprising cleaving the synthesized nucleic acid with a restriction enzyme and removing the nucleic acid from the nanochip.

1.23 Any of the above methods, wherein the polymer is a nucleic acid, and further comprising amplifying the thus synthesized nucleic acid.

1.24 Any of the above methods, further comprising removing the polymer [e.g., nucleic acid] from the nanotip and crystallizing the polymer.

1.25 Any of the above methods, wherein the polymer is a nucleic acid, further comprising stabilizing the nucleic acid by drying a solution containing the nucleic acid with one or more buffers (e.g., borate buffer), antioxidants, humectants such as polyols, and optionally chelating agents, e.g., as described in U.S. Pat. No. 8,283,165 B2, incorporated herein by reference; or by forming a matrix between the nucleic acid and a polymer, e.g., poly(ethylene glycol)-poly(l-lysine) (PEG-PLL) AB type block copolymer; or by adding a protein that binds to a complementary nucleic acid strand or DNA.

1.26
(i) reacting a nucleic acid with a 3'-protected nucleotide in an addition chamber in the presence of a polymerase that catalyzes the addition of the 3'-protected nucleotide to the 3' end of the nucleic acid;
(ii) drawing at least the 3′ end of the resulting 3′-protected nucleic acid from the loading chamber through at least one nanopore into a storage chamber, where the polymerase is prevented from passing through the nanopore (e.g., due to its size or because it is anchored to a substrate in the first chamber);
(iii) deprotecting the 3'-protected nucleic acid, e.g., chemically or enzymatically; and (iv) if it is desired to add an additional 3'-protected dNTP to the oligonucleotide, drawing off the 3' end of the oligonucleotide to the same or a different addition chamber so that steps (i) through (iii) are repeated, or if this is not desired, allowing the 3' end of the nucleic acid to remain in the storage chamber until a further cycle in which the desired 3'-protected dNTP is provided in the addition chamber; and (v) repeating steps (i) through (iv) until the desired nucleic acid sequence is obtained.
Any of the methods described above.

1.27 Any of the above methods, wherein the polymer is a nucleic acid, single-stranded DNA (ssDNA), and the one or more nanopores have a diameter that allows passage of ssDNA but not double-stranded DNA (dsDNA), e.g., a diameter of about 2 nm.

1.28 Any of the above methods, wherein the monomer is a 3'-protected nucleotide, e.g., a deoxynucleotide triphosphate (dNTP), e.g., selected from deoxyadenosine triphosphate (dATP), deoxyguanosine triphosphate (dGTP), deoxycytidine triphosphate (dCTP), deoxythymidine triphosphate (dTTP), e.g., dATP or dCTP.

1.29 Any of the methods described above, wherein the polymer is a nucleic acid and the addition of nucleotides to the nucleic acid is catalyzed by a polymerase, e.g., a template-independent polymerase, e.g., terminal deoxynucleotidyl transferase (TdT) or polynucleotide phosphorylase, e.g., the polymerase catalyzes the incorporation of deoxynucleotides at the 3'-hydroxyl end of DNA.

1.30 Any of the above methods, wherein the membrane comprises a plurality of nanopores and a plurality of polymers each attached to a surface proximate the nanopore, e.g., a plurality of nucleic acids attached via their 5' ends to a surface proximate the nanopore.

1.31 Any of the above methods, in which multiple polymers, e.g., multiple nucleic acids, each attached at their 5' ends to a surface adjacent to a nanopore, are synthesized independently, each nanopore associated with an electrode pair, one electrode of the electrode pair positioned adjacent to one end of the nanopore and the other electrode positioned adjacent to the other end of the nanopore, such that a current provided by the electrode pair can move each polymer independently between the first and second chambers.

1.32 Any of the above methods, wherein the polymer is a 3'-protected nucleic acid attached at its 5' end to a surface adjacent to the nanopore, and the 3' end of the 3'-protected nucleic acid is pulled through the nanopore by using an electrical force, e.g., by using an electrical force applied from an electrode in an adjacent chamber.

1.33 The method of 1.20, wherein the new 3'-protected dNTP is the same as or different from the first 3'-protected dNTP.

1.34 The method of 1.20, wherein the 3'-protected dNTP used in step (i) of the cycle alternates between 3'-protected dATP and 3'-protected dCTP in each cycle.

1.35 Any of the above methods, wherein the polymer is a nucleic acid and deprotection of the nucleic acid is carried out with an enzyme that removes the 3'-protecting group on the ssDNA but not the 3'-protecting group on the 3'-protected dNTPs.

For example, the present invention provides a method for synthesizing nucleic acids in a nanochip, the nanochip comprising at least a first chamber and a second chamber separated by a membrane comprising at least one nanopore, the synthesis being carried out in a buffer by cycles of nucleotide addition to a first end of a nucleic acid having a first end and a second end, where the first end of the nucleic acid is moved by electrical attraction between one or more addition chambers (containing reagents capable of adding nucleotides) and one or more storage chambers (not containing reagents necessary for the addition of nucleotides), the chambers being separated by one or more membranes each comprising one or more nanopores, the nanopores being large enough to allow passage of the nucleic acid but too small to allow passage of at least one reagent essential for the addition of nucleotides, for example, the method corresponds to any of the described methods 1.

In certain embodiments, the sequence of the polymer corresponds to a binary code, e.g., the polymer is a nucleic acid and the sequence corresponds to a binary code, where each bit (0 or 1) is represented by a base, e.g., A or C.

In certain embodiments, the polymer is DNA.

In some other embodiments, each bit is represented by a short sequence of monomers rather than a single monomer. For example, in one such embodiment, chunks of DNA are synthesized, with each chunk producing a unique signal through the nanopore, corresponding to a 0 or a 1. This embodiment has certain advantages in that single nucleotides are more difficult to detect in a nanopore, especially in a solid-phase nanopore, so the use of chunks correspondingly reduces the density of information in the polymer, but makes the read less prone to errors.

For example, (double-stranded) nucleotide chunks can be added using site-specific recombinases, i.e. enzymes that spontaneously recognize and cleave at least one strand of a nucleic acid duplex within a sequence segment known as a site-specific recombination sequence. In one such embodiment, the site-specific recombinase is a topoisomerase that is used to ligate topo-conjugated dsDNA oligonucleotide chunks to the sequence. These oligonucleotides themselves have no structure compatible with further ligation until cleaved with a restriction enzyme. Vaccinia virus topoisomerase I specifically recognizes the DNA sequence 5'-(C/T)CCTT-3'. The topoisomerase binds to double-stranded DNA and cleaves it at the 5'-(C/T)CCTT-3' cleavage site. Note that the cleavage is not complete since the topoisomerase only cleaves the DNA at one strand (although the presence of a nearby nick in the other strand results in a sort of double-stranded break), and upon cleavage, the topoisomerase covalently binds to the 3' phosphate of the 3' nucleotide. The enzyme then remains covalently attached to the 3' end of the DNA and can religate the covalently held strand at the same bond that was originally cleaved (as occurs in DNA relaxation) or to a heterologous acceptor DNA with a compatible overhang to generate a recombinant molecule. In this embodiment, a dsDNA donor oligonucleotide (containing one of at least two different sequences, e.g., one "0" and the other "1") is generated, flanked by restriction sites that generate topoisomerase recombination sites and topoisomerase ligation sites. These cassettes are topo-charged; that is, they are covalently attached to a topoisomerase that binds them to the topoisomerase ligation site on the receiver oligonucleotide. Once the extending receiver DNA strand is cut with the restriction enzyme, it can be ligated to the topo-charged cassette. Thus, the extending DNA simply needs to be cycled continuously from the restriction enzyme to the topo-charged cassette, with each cycle adding another donor oligonucleotide. Related approaches have been described for cloning, see, e.g., Shuman S., Novel approach to molecular cloning and polynucleotide synthesis using vaccinia DNA topoisomerase. J Biol Chem. (1994); 269(51):32678-84, incorporated herein by reference.

A similar strategy can be used to add a single base. In the presence of a suitable single-stranded "deprotected" "acceptor" DNA, the topo-charged DNA is enzymatically and covalently linked ("added") to the acceptor by its topoisomerase, which is removed from the DNA in the process. A type IIS restriction enzyme can cleave off all but one base (the one that is "added"). This deprotection-addition process can be repeated to add additional bases (bits). As demonstrated in the examples herein, a topo/type IIS restriction enzyme combination can be used to add a single nucleotide to the 5' end of a target single-stranded DNA. The use of a type IIS restriction enzyme allows for cutting of the DNA at a position different from the recognition sequence (other type IIS restriction enzymes can be found at https://www.neb.com/tools-and-resources/selectioncharts/type-iis-restriction-enzymes). The use of inosine in this system (which acts as a "universal base" and pairs with any other base) allows the reaction to occur without any sequence requirements on the target DNA. The identity of the nucleotide added to the single-stranded target DNA is the 3' nucleotide to which Vaccinia topoisomerase binds via the 3' phosphate. The recognition sequence for Vaccinia topoisomerase is (C/T)CCTT, so this system was used to add a "T" to the target DNA. There is a related topoisomerase, SVF, which can use the recognition sequence CCCTG (https://www.ncbi.nlm.nih.gov/pubmed/8661446). Thus, SVF can be used to add a "G" instead of a "T". When combined with Vaccinia topo, binary data can be coded with T and G.

In other approaches to single base addition, the 5' phosphate provides a blocking group, resulting in the addition of a single base in the 3' to 5' direction. The loading reaction loads the topoisomerase with a single T (or G, or other nucleotide, if desired) bearing a 5' phosphate group. When the loaded topoisomerase "finds" a 5' unblocked (unphosphorylated) single-stranded DNA strand, it adds a T to that strand, resulting in a 5' T-tagged DNA. This addition is facilitated by the presence of an adapter DNA that has a sequence to which the topoisomerase and single-stranded acceptor DNA can bind (note that the adapter DNA is catalytic - it can be reused as a template in repeated reactions). Because the added nucleotide has a 5' phosphate, it is not a substrate for further addition until it is exposed to a phosphatase, which removes the 5' phosphate. This process can be repeated using Vaccinia topoisomerase, which adds a single "T" to the 5' end of the target single-stranded DNA, and SVF topoisomerase, which adds a single "G", thus building up a sequence that codes for the binary information of T and G. Other topoisomerases can be used to add A or C, but the efficiency of this reaction is low.

One advantage of using a topoisomerase-mediated strategy is that the monomers are covalently bound to the topoisomerase and therefore cannot "escape" and interfere with other reactions. With a polymerase, the monomers can diffuse out and the polymerase and/or deblocking agent must be specific (e.g. selective for A vs. C) or the monomers are provided by a flow so that there is no opportunity for mixing.

In one aspect, the invention provides a single nucleotide-loaded topoisomerase, i.e., a topoisomerase bound to a single nucleotide, where, for example, the topoisomerase is bound via the 3'-phosphate of the nucleotide and the nucleotide is protected, e.g., phosphorylated, at the 5'-position.

In another aspect, the present invention provides a method (Method A) for synthesizing a DNA molecule using topoisomerase-mediated ligation by adding a single nucleotide or oligomer to a DNA strand in the 3' to 5' direction, which comprises (i) reacting a DNA molecule with a topoisomerase loaded with a desired nucleotide or oligomer, where the nucleotide or oligomer is blocked from further addition at the 5' end, and then (ii) deblocking the 5' end of the DNA thus formed and repeating steps (i) and (ii) until the desired nucleotide sequence is obtained. For example, the following is provided:

A1.1 Method A, which is a method for synthesizing a DNA molecule by adding a single nucleotide in the 3' to 5' direction, comprising: (i) reacting a DNA molecule with a topoisomerase charged with a desired nucleotide in 5' protected form, e.g., 5' phosphorylated form, such that the desired nucleotide in 5' protected form is added to the 5' end of the DNA, then (ii) deprotecting the 5' end of the DNA thus formed using a phosphatase enzyme, and repeating steps (i) and (ii) until the desired nucleotide sequence is obtained; or

A1.2 Method A, a method for synthesizing a DNA molecule by adding oligomers in the 3' to 5' direction, comprising: (i) reacting a DNA molecule with a topoisomerase loaded with a desired oligomer, thereby ligating the oligomer to the DNA molecule; then (ii) using a restriction enzyme to provide a 5' site for topoisomerase-mediated ligation of another oligomer; and repeating steps (i) and (ii) until the desired oligomer sequence is obtained.

A1.3 Any of the methods described above, including providing ATP and a ligase that seals nicks in the DNA [NB: topoisomerase ligation only joins one strand].

A1.4 Any of the methods described above, wherein the topoisomerase-loaded donor oligonucleotide contains a 5' overhang on the strand complementary to the topoisomerase-bearing strand and contains a polyinosine sequence [NB: inosine acts as a "universal base" and will pair with any other base].

A1.5 Any of the above methods, in which the restriction enzyme is a type IIS restriction enzyme that can cleave off all added DNA except a single base (the "added" base).

A1.6 Any of the above methods, wherein the topoisomerase is selected from vaccinia topoisomerase and SVF topoisomerase I.

A1.7 Any of the above methods using Vaccinia topoisomerase (which recognizes (C/T)CCTT) to add dTTP nucleotides and SVF topoisomerase I (which recognizes CCCTG) to add dGTP nucleotides, e.g., to provide a binary code.

A1.8 Any of the above methods, wherein the DNA is double-stranded and the storage chamber further contains a ligase and ATP to repair the DNA strands not ligated by the topoisomerase.

A1.9 Any of the above methods, including the use of a topoisomerase inhibitor that inhibits the binding and activity of free topoisomerase to DNA oligomers, e.g., the inhibitor is selected from novobiocin and coumermycin.

A1.10 Any of the above methods, wherein the DNA strand thus provided has a sequence that includes thymidine (T) nucleosides and deoxyguanidine (G) nucleosides.

A1.11 Any of the above methods in which the topoisomerase adds a single base, but the restriction enzyme cuts at a position that is one nucleotide 5' from the base added by the topoisomerase.

A1.12 Any of the above methods, wherein the DNA strand thus provided has a sequence that includes the sequences of "TT" and "TG" dinucleotides.

A1.13 Any of the above methods, wherein the DNA is single stranded;
A1.14 Any of the above methods, wherein the DNA is double-stranded.

A1.15 Any of the above methods in which the DNA is on a substrate or magnetic beads and can be selectively exposed to or removed from the reagents required to give the desired sequence.

A1.16 Any of the above methods in which some or all of the reagents for attaching or deblocking DNA are supplied by flow and removed by washing.

A1.17 Any of the above methods, in which binding of a single nucleotide or oligomer to single-stranded DNA is promoted by the presence of a topoisomerase and an adapter DNA having a sequence to which the single-stranded acceptor DNA can bind.

A1.18 Any of the above methods, for example as described in any of methods 2 below, performed in a system in which a nanopore separates a chamber containing a topoisomerase from a chamber containing a phosphatase or a restriction enzyme, and the nanopore allows for movement of DNA by electrical attraction but not for movement of the enzyme.

One concern that may arise is that poly-G sequences may form G-quadruplex secondary structures. By moving the restriction enzyme back one base (5' of the topo sequence) and following a similar topo/IIS strategy, one can add "TT" or "TG", each of which may represent a different bit. This requires two bases to code for one bit, but has the advantage of avoiding the poly-G sequence. In other embodiments, other bases at the 3' end of the topo recognition sequence allow for conjugation using poxvirus topoisomerase, with (C/T)CCTA, (C/T)CCTC, and (C/T)CCTG being used, although less efficiently than (C/T)CCTT (https://www.ncbi.nlm.nih.gov/pubmed/17462694). Protein engineering/selection techniques can be used to improve the efficiency of these reactions as well, and similar approaches can be used to add non-standard bases.

In certain embodiments, the method of synthesizing DNA by this method involves treating the DNA with ligase and ATP. The topoisomerase only joins one side of the DNA (the other is essentially nicked). The ligase repairs the nick and ensures that the topoisomerase itself does not re-cut the reaction product and cleave it off.

In certain embodiments, the method involves the use of a topoisomerase inhibitor to inhibit the binding and activity of free topoisomerase on DNA oligomers. Suitable inhibitors include novobiocin and coumermycin. Note that complete inhibition is not desirable, since low levels of topoisomerase activity aid in the "relaxation" of coiled DNA, which is particularly useful when synthesizing long DNA strands.

Thus, in another embodiment, the disclosure provides a method (Method 2) of synthesizing DNA in a nanochip, the nanochip comprising one or more addition chambers containing a topoisomerase-loaded oligonucleotide (i.e., an oligonucleotide bound to a topoisomerase at its 3′ end) and one or more storage chambers containing a restriction enzyme or a deblocking agent, such as a phosphatase, which chambers also contain a compatible buffer and are separated by a membrane comprising at least one nanopore, where the topoisomerase and the restriction enzyme are prevented from passing through the nanopore (e.g., because they are too large and/or because they are anchored to the substrate of the first and second chambers, respectively), and the synthesis is carried out by cycles of adding single nucleotides or short oligonucleotide chunks to a first end of a nucleic acid having a first end and a second end, where the first end of the nucleic acid is moved between the addition and storage chambers by electrical attraction, e.g., in one embodiment as follows:
(i) transferring the 5' end of a receiver DNA (e.g., double-stranded DNA) to a first loading chamber by electrical force;
(ii) providing in the first addition chamber a topoisomerase-loaded donor oligonucleotide, where the donor oligonucleotide comprises a topoisomerase binding site, an information sequence (e.g., selected from at least two different nucleotides or sequences, e.g., one sequence corresponding to "0" and the other to "1" in binary code), a restriction site that, when cleaved by a restriction enzyme, provides a topoisomerase ligation site;
(iii) allowing sufficient time for the donor oligonucleotide to ligate to the receiver DNA, thus extending it;
(iv) transferring the 5' end of the thus extended receiver DNA by electrical force to a storage chamber, for example for a restriction enzyme to cleave the receiver DNA to provide a topoisomerase junction site, or for the addition of a single nucleotide, for a deblocking agent, for example a phosphatase, to generate a 5' deblocked nucleotide on the single stranded DNA; and (v) repeating the cycle of steps (i) to (iv) and adding oligonucleotides with the same or different information sequences until the desired DNA sequence is obtained.

For example, the present invention provides the following:
2.1 Method 2, in which the 3' end of the receiver DNA is closely bound to a nanopore and the 5' end of the receiver oligonucleotide comprises a topoisomerase attachment site, comprising, after step (iv), flushing the first addition chamber and adding a further oligonucleotide to the 5' end of the receiver DNA by providing a new topoisomerase-loaded donor oligonucleotide to the first addition chamber, where the new donor oligonucleotide has a different information sequence than the previous donor oligonucleotide; if it is desired that a new donor oligonucleotide be added to the receiver DNA, pulling the 5' end of the receiver nucleic acid back to the first chamber and repeating steps (i)-(iii), or if it is not desired, allowing the receiver DNA to remain in the second chamber until the desired donor oligonucleotide has been provided to the first chamber.

2.2 Any of the above methods, in which multiple receiver DNA molecules are synthesized independently and in parallel, and their presence or absence in the first chamber is separately controlled to obtain DNA molecules with different sequences.

2.3 Any of the above methods, in which multiple receiver DNA molecules are independently synthesized, each attached at its 3' end to a surface proximate to a nanopore, and each nanopore is associated with an electrode pair, one electrode of the electrode pair being positioned proximate to one end of the nanopore and the other electrode being positioned proximate to the other end of the nanopore, such that a current provided by the electrode pair can independently move each receiver DNA molecule between the first and second chambers.

2.4 Any of the above methods, wherein the donor oligonucleotides used in step (i) of the cycle alternate between a donor oligonucleotide containing a first information sequence and a donor oligonucleotide containing a second information sequence in each cycle.

2.5 Method 2, which includes adding an additional oligonucleotide to the 5' end of the receiver DNA by returning the 5' end of the receiver DNA to the first addition chamber and adding an oligonucleotide having the same information sequence, or by moving the 5' end of the receiver DNA to a second addition chamber having a donor oligonucleotide bound to topoisomerase at its 3' end, where the donor oligonucleotide in the second addition chamber has a different information sequence than the donor oligonucleotide in the first addition chamber.

［ここで、Ｎは任意のヌクレオチドを表し、制限酵素はＡｃｃ１であり、これは、ＤＮＡ（例えば上記の配列中のＧＴＣＧＡＣ）を切断し、適切なオーバーハングを提供できる］
を有する、上述のいずれかの方法。 2.6 The donor oligonucleotide has the structure:

[where N represents any nucleotide and the restriction enzyme is Acc1, which can cleave DNA (e.g., GTCGAC in the sequence above) and provide the appropriate overhangs]
Any of the methods described above, comprising:

2.7 Any of the above methods in which the donor oligonucleotide has a hairpin structure, e.g., 2.6 in which the NNNNN groups of the top and bottom strands are linked.

を有する、上述のいずれかの方法。 2.8 At least one of the topoisomerase-charged oligonucleotides has the following structure:

Any of the methods described above, comprising:

を有する、上述のいずれかの方法。 2.9 At least one of the topoisomerase-charged oligonucleotides has the following structure:

Any of the methods described above, comprising:

2.10 Any of the methods described above using a topoisomerase-charged oligonucleotide.
2.11 Any of the methods described above, wherein the sequence of the synthesized DNA is determined following each cycle by detecting changes in voltage, current, resistance, capacitance and/or impedance as the oligonucleotide passes through the nanopore.

2.12 Any of the above methods, wherein DNA synthesis occurs in a buffer, e.g., a buffer to pH 7-8.5, e.g., about pH 8, e.g., a buffer containing tris(hydroxymethyl)aminomethane (Tris), a suitable acid, and optionally a chelator, e.g., ethylenediaminetetraacetic acid (EDTA), e.g., a TAE buffer containing a mixture of Tris base, acetic acid, and EDTA, or a TBE buffer containing a mixture of Tris base, boric acid, and EDTA; e.g., 10 mM Tris pH 8, 1 mM EDTA, 150 mM KCl, or e.g., a solution containing 50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate (pH 7.9 @ 25°C).

2.13 Any of the above methods, further comprising removing the DNA from the nanotip.
Any of the methods mentioned above.

2.14 Any of the above methods, further comprising amplifying the DNA thus synthesized.

2.15 Any of the above methods, further comprising removing the DNA from the nanochip and crystallizing the DNA.

2.16 Any of the above methods further comprising drying the solution containing the DNA with one or more buffers (e.g., borate buffer), antioxidants, humectants such as polyols, and optionally chelating agents, as described, for example, in US 8,283,165 B2, which is incorporated herein by reference; or stabilizing the nucleic acid, such as by forming a matrix between the nucleic acid and a polymer, such as poly(ethylene glycol)-poly(l-lysine) (PEG-PLL) AB type block copolymer.

2.17 Any of the methods described above, including providing ATP and a ligase that seals nicks in the DNA [NB: topoisomerase ligation ligates only one strand].

2.18 Any of the methods described above, wherein the topoisomerase-loaded donor oligonucleotide contains a 5' overhang on the strand complementary to the topoisomerase-bearing strand and contains a polyinosine sequence [NB: inosine acts as a "universal base" and will pair with any other base].

2.19 Any of the above methods, in which the restriction enzyme is a type IIS restriction enzyme that can cleave off all of the added DNA except for a single base (the "added" base).

2.20 Any of the above methods, wherein the topoisomerase is selected from vaccinia topoisomerase and SVF topoisomerase I.

2.21 Any of the methods described above, wherein vaccinia topoisomerase (which recognizes (C/T)CCTT) is used to add dTTP nucleotides and SVF topoisomerase I (which recognizes CCCTG) is used to add dGTP nucleotides, e.g., to provide binary code information.

2.22 Any of the above methods, wherein the storage chamber further contains a ligase and ATP to repair DNA strands not ligated by the topoisomerase.

2.23 Any of the above methods, including the use of a topoisomerase inhibitor that inhibits the binding and activity of free topoisomerase to DNA oligomers, e.g., the inhibitor is selected from novobiocin and coumermycin.

2.24 Any of the above methods, wherein the DNA strand thus provided has a sequence that includes thymidine (T) nucleosides and deoxyguanidine (G) nucleosides.

2.25 Any of the above methods in which the topoisomerase adds a single base, but the restriction enzyme cuts at a position that is one nucleotide 5' from the base added by the topoisomerase.

2.26 Any of the above methods, wherein the DNA strand thus provided has a sequence that includes the sequences of "TT" and "TG" dinucleotides.

2.27 Any of the above methods for synthesizing a DNA molecule by adding a single nucleotide in the 3' to 5' direction, comprising: (i) reacting the DNA molecule with a topoisomerase charged with a desired nucleotide in 5' protected form, e.g., 5' phosphorylated form, such that the desired nucleotide in 5' protected form is added to the 5' end of the DNA; then (ii) deprotecting the 5' end of the DNA thus formed using a phosphatase enzyme; and repeating steps (i) and (ii) until the desired nucleotide sequence is obtained.

2.28 Any of the methods described above for synthesizing a DNA molecule by adding oligomers in the 3' to 5' direction, comprising: (i) reacting the DNA molecule with a topoisomerase charged with a desired oligomer, thereby ligating the oligomer to the DNA molecule; and then (ii) using a restriction enzyme to provide a 5' site for topoisomerase-mediated ligation of an additional oligomer; and repeating steps (i) and (ii) until the desired nucleotide sequence is obtained.
2.29 Any of the methods described above, which is a method similar to method A described above.

The products of the synthesis reaction can be detected, interrogated for quality control purposes, and read to extract the data encoded in the polymer. For example, the DNA can be amplified and sequenced by conventional means to confirm the robustness of the nanopore sequencing.

［ここで、情報配列ＡまたはＢは、３～１２個、例えば、約８個のヌクレオチドの配列である］
を含む、オリゴヌクレオチドを提供する。 In another embodiment, the invention provides a method for the preparation of a nucleic acid sequence comprising a topoisomerase binding site, an information sequence (e.g., selected from at least two different sequences, e.g., one sequence corresponding to "0" and the other to "1" in binary code), and a restriction site that provides a topoisomerase linkage site upon cleavage with a restriction enzyme, e.g., the following sequence:

[wherein the information sequence A or B is a sequence of 3 to 12, e.g., about 8, nucleotides]
The present invention provides an oligonucleotide comprising:

［ここで、情報配列ＡまたはＢは、３～１２個、例えば、約８個のヌクレオチドの配列であり、＊は、オリゴヌクレオチドに共有結合したトポイソメラーゼ；例えば、トポイソメラーゼはワクシニアウイルストポイソメラーゼＩである］
を提供する。 In another embodiment, the invention provides a topoisomerase-loaded oligonucleotide that includes a topoisomerase binding site, an information sequence (e.g., selected from at least two different sequences, e.g., one sequence corresponding to "0" and the other to "1" in binary code), and a restriction site that provides a topoisomerase junction site upon cleavage by a restriction enzyme; e.g., a topoisomerase-loaded oligonucleotide having the following structure:

[wherein information sequence A or B is a sequence of 3-12, e.g., about 8, nucleotides, and * is a topoisomerase covalently attached to an oligonucleotide; e.g., the topoisomerase is vaccinia virus topoisomerase I]
to provide.

In another embodiment, the invention provides a single-stranded or double-stranded DNA molecule as described above, where the single strand or the coding sequence consists essentially of non-hybridizing bases, such as adenine and cytosine (A and C), arranged in a sequence that corresponds to a binary code, for example for use in a data storage method. For example, the invention provides DNA (DNA1), where the DNA is single-stranded or double-stranded and at least 1000 nucleotides long, for example 1000-1,000,000 nucleotides long, for example 5,000-20,000 nucleotides long, and the sequence of nucleotides corresponds to a binary code; for example:

1.1 DNA1, where the DNA is single stranded.
1.2 DNA1, where DNA is double stranded.

1.3 Any of the above DNAs, wherein the nucleotides in the single strand or coding sequence are selected from adenine, thymine and cytosine nucleotides, e.g., adenine and cytosine nucleotides, or thymine and cytosine nucleotides.

1.4 Any of the above DNAs that are composed primarily of non-hybridizing nucleotides such that they do not form significant secondary structures when in single-stranded form.

1.5 Any of the above DNAs, wherein the nucleotides are at least 95%, e.g., 99%, e.g., 100%, adenine and cytosine nucleotides.

1.6 Any of the above DNAs containing added nucleotides or nucleotide sequences to separate or disrupt the nucleotides comprising the binary code, e.g., to separate 1's and 0's or groups of 1's and 0's, so that consecutive 1's or 0's can be more easily read.

1.7 Any of the above DNAs, in which (a) each bit of the binary code corresponds to a single nucleotide, e.g., 1 and 0 correspond to A or C, respectively; or (b) each bit of the binary code corresponds to more than one nucleotide, e.g., a sequence of 2, 3 or 4 nucleotides, e.g., AAA or CCC.

1.8 Any of the above DNAs which have been crystallized.
1.9 Any of the above DNAs, wherein the solution containing the nucleic acid is provided in dry form together with one or more buffer salts (e.g. borate buffer), antioxidants, humectants such as polyols, and optionally chelating agents; and/or in a matrix between the nucleic acid and a polymer, e.g. poly(ethylene glycol)-poly(l-lysine) (PEG-PLL) AB type block copolymers; and/or together with a complementary nucleic acid or a protein that binds to the DNA, as described, for example, in US8283165B2, which is incorporated herein by reference.

1.10 Any of the above DNAs produced by any of the described method 1, described method 2, or described method A.

The nanochip can be fabricated, for example, as shown in Figures 23-29. For example, in one format, each polymer chain is associated with two or four addition chambers, with the two addition chamber format being useful for encoding binary codes in the polymer, and the four addition chamber format being particularly useful for creating custom DNA sequences. Each addition chamber contains an individually controllable electrode. The addition chambers contain reagents that add monomers to the polymer in a buffer. The addition chambers are separated from the storage chamber by a membrane that contains one or more nanopores, which may be common to multiple addition chambers, and contain deprotection reagents and buffers that deprotect the protected monomers or oligomers added in the addition chamber. The nanochip contains a set of multiple addition chambers that allows for parallel synthesis of multiple polymers.

High bandwidth and low noise nanopore sensor and detection electronics are important to achieve single DNA base resolution. In certain embodiments, the nanotip is electrically coupled to a complementary metal oxide semiconductor (CMOS) chip. For example, solid-state nanopores can be integrated into a CMOS platform in close proximity to bias electrodes and custom designed amplification electronics, as described in Uddin, et al., "Integration of solid-state nanopores in a 0.5 μm cmos foundry process", Nanotechnology (2013) 24(15): 155501, the contents of which are incorporated herein by reference.

In another embodiment, the present disclosure provides a nanochip (nanochip 1) for synthesizing and/or sequencing a charged polymer, e.g., DNA, comprising at least two distinct monomers, the nanochip comprising at least a first and a second reaction chamber separated by a membrane comprising one or more nanopores, each reaction chamber comprising one or more electrodes for drawing the charged polymer into the chamber, further comprising an electrolyte medium and optionally reagents for adding the monomers to the polymer, e.g., the following:

1.1 A nanotip 1 having a nanopore diameter of 2-20 nm, for example 2-10 nm, for example 2-5 nm.

1.2 Any of the nanotips described above, wherein some or all of the walls of the reaction chamber of the nanotips comprise a silicon material, e.g., silicon, silicon dioxide, silicon nitride, or a combination thereof, e.g., silicon nitride.

1.3 Any of the nanotips described above, wherein some or all of the walls of the nanotip's reaction chamber comprise a silicon material, e.g., silicon, silicon dioxide, silicon nitride, or a combination thereof, e.g., silicon nitride, and some or all of the nanopores are created by ion bombardment.

1.4 Any of the nanochips described above, in which part or all of the nanopore is composed of a membrane, such as a lipid bilayer, that contains the pore-forming protein α-hemolysin.

1.5 Any of the nanochips described above, wherein some or all of the walls of the reaction chamber are coated to minimize interaction with reagents, for example coated with a polymer such as polyethylene glycol or with a protein such as bovine serum albumin.

1.6 Any of the nanotips described above, including an electrolyte medium.
1.7 Any of the above nanotips comprising an electrolyte medium comprising a buffer to pH 7-8.5, e.g., about pH 8, e.g., a buffer comprising tris(hydroxymethyl)aminomethane (Tris), a suitable acid, and optionally a chelator, e.g., ethylenediaminetetraacetic acid (EDTA), e.g., a TAE buffer comprising a mixture of Tris base, acetic acid, and EDTA, or a TBE buffer comprising a mixture of Tris base, boric acid, and EDTA; e.g., 10 mM Tris pH 8, 1 mM EDTA, 150 mM KCl, or e.g., a solution comprising 50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate (pH 7.9 @ 25°C).

1.8 Any of the nanotips described above, comprising a reagent for adding a monomer to a polymer.

1.9 Any of the nanotips described above capable of both polymer synthesis (e.g., "writing" by sequentially adding monomers or groups of monomers to a polymer) and sequencing (e.g., "reading" by measuring the change in current and/or inductance as a monomer passes through a nanopore).

1.10 Any of the nanochips described above, wherein the membrane containing one or more nanopores includes metal surfaces on either side, separated by an insulator, e.g., a silicon nitride membrane, and the metal surfaces are formed, e.g., by means of lithography, to provide electrodes at each end of each nanopore, e.g., such that a current through the nanopore can be established through the nanopore via an electrolyte medium, e.g., a current can be drawn through the nanopore and a change in electrical potential across the nanopore as the polymer passes through the nanopore can be measured and used to identify the sequence of monomers within the polymer.

1.11 Any of the nanotips described above, comprising a charged polymer that is DNA.
1.12 Any of the nanotips described above, comprising a charged polymer that is single stranded DNA (ssDNA).

1.13 Any of the nanochips described above, comprising a charged polymer that is DNA containing a predetermined restriction site.

1.14 Any of the nanochips described above, comprising a charged polymer that is DNA, the DNA being the DNA described in DNA1 above.

1.15 Any of the nanochips described above, comprising a charged polymer that is DNA, the DNA comprising at least 95%, e.g., 99%, e.g., 100% adenines and cytosines.

1.16 Any of the nanochips described above, comprising a charged polymer that is DNA, the DNA containing only adenine and cytosine.

1.17 Any of the nanochips described above, comprising one or more ports allowing for the introduction and washing away of buffers and reagents.

1.18 Any of the nanotips described above, including a buffer, e.g., a buffer to pH 7-8.5, e.g., about pH 8, e.g., a solution comprising tris(hydroxymethyl)aminomethane (Tris), a suitable acid, and optionally a chelator, e.g., a buffer comprising ethylenediaminetetraacetic acid (EDTA), e.g., a TAE buffer comprising a mixture of Tris base, acetic acid, and EDTA, or a TBE buffer comprising a mixture of Tris base, boric acid, and EDTA; e.g., 10 mM Tris pH 8, 1 mM EDTA, 150 mM KCl, or e.g., a solution comprising 50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate (pH 7.9 @ 25°C).

1.19 Any of the nanochips described above that have been or can be lyophilized for storage and subsequent rehydration, e.g., the nanochip structure includes a hydratable or water-permeable polymer.

1.20 Any of the above nanochips that are synthesized in a dry form, for example where the nanochip structure comprises a hydratable or water-permeable polymer, and once the writing process is complete, are hydrated prior to use, and optionally lyophilized for long-term storage.

1.21 Any of the above mentioned nanotips, wherein the charged polymer, e.g. DNA, is stabilized with histones.
1.22 Any of the above nanotips, wherein the inner surface is positively charged.

1.23 Any of the nanochips described above, wherein the electrodes are operably connected to a capacitive circuit capable of applying a radio frequency pulsating direct current to the nanopore, e.g., at a frequency of 1 MHz to 1 GHz, e.g., 50 to 200 MHz, e.g., about 100 MHz, such that, e.g., the pulsating direct current can draw a charged polymer through the nanopore and the monomer sequence can be determined by measuring the capacitance fluctuations of the nanopore as the charged polymer passes through the nanopore.

1.24 Any of the nanotips described above comprising a storage chamber or deblocking agent chamber containing a reagent for deprotecting the polymer after addition of a monomer or oligomer in one of the addition chambers.
1.25 Any of the nanochips described above, comprising a plurality of pairs of additional chambers.

1.26 Any of the nanochips described above, including an electrical control layer, a fluidics layer and an electrical ground layer bonded together by wafer bonding, e.g., as shown in FIG. 24.

1.27 Any of the above nanotips, wherein the nanopore is created by drilling using FIB, TEM, wet etching or dry etching.

1.28 Any of the nanotips described above, wherein the membrane containing the nanopore is between one atomic layer and 30 nm thick.

1.29 Any of the nanotips described above, wherein the membrane containing the nanopore is made of SiN, BN, SiOx, graphene, a transition metal dichalcogenide, such as _WS2 or _MoS2 .

1.30 Any of the above nanochips containing wiring made of metal or polysilicon.

1.31 Any of the above nanotips in which the density of wiring is increased by three-dimensional stacking with electrical shielding provided by dielectric deposition (e.g., by PECVD, sputtering, ALD, etc.).

1.32 Any of the above nanotips, wherein contact with an electrode in an additional chamber is made using a through-silicon via (TSV) by deep reactive ion etching (DRIE), e.g., by cryo-processing or BOSCH processing, or by wet silicon etching.

1.33 Any of the nanochips described above, wherein individual voltage control of the electrodes of each additional chamber allows the electrodes of each additional chamber to be individually controlled and monitored.

1.34 Any of the nanotips described above, wherein each polymer has a first addition chamber, a second addition chamber and a deblocking chamber.

1.35 Any of the above nanochips, wherein one or more chambers have fluid flow.
1.36 Any of the above nanochips, wherein one or more chambers are fluidly separated.
1.37 Any of the nanotips described above, wherein the deblocking chamber has a fluid flow.
1.38 Any of the nanochips described above, wherein the additional chambers have a common fluid flow.

1.39 Any of the nanochips described above, in which the wiring between chambers is common to chambers of similar types (e.g., first additional chamber, second additional chamber, degassing chamber).

1.40 Any of the nanotips described above, wherein the loading chamber has separate voltage control and the deblocking chamber has a common electrical ground.

1.41 Any of the nanotips described above, wherein the deblocking chambers have separate voltage controls, the first additional chamber has a common electrical ground, and the second additional chamber has a common electrical ground.

1.42 Any of the above nanotips, wherein the nanotips are fabricated by wafer bonding and the chambers are pre-filled with the desired reagents prior to bonding.
1.43 Any of the above nanotips, wherein one or more internal surfaces are silanized.

1.44 Any of the nanochips described above, including one or more ports for the introduction or removal of fluids.

1.45 Any of the nanotips described above in which the electrodes in the chamber have limited direct contact with the charged polymer, e.g., the electrodes are positioned far enough away from the nanopore that they cannot be reached by charged polymers bound to the surface adjacent to the nanopore, or the electrodes are protected by a material that allows the passage of water and single-atom ions (e.g., Na+, K+ and Cl- ions) but not the polymer or monomeric or oligomeric reagents that are to join the polymer.

1.46 Any of the above nanochips electrically coupled to a complementary metal oxide semiconductor (CMOS) chip.

For example, in one embodiment, the present invention provides a nanochip, e.g., any of the nanochips described above in Nanochip 1, for sequencing a charged polymer, e.g., DNA, comprising at least two distinct monomers, the nanochip comprising at least a first and a second reaction chamber separated by a membrane comprising an electrolyte medium and comprising one or more nanopores, each reaction chamber comprising at least a pair of electrodes disposed on opposite sides of the membrane, the electrodes being operably coupled to a capacitive circuit capable of providing a radio frequency pulsating direct current to the nanopore, e.g., at a frequency of 1 MHz to 1 GHz, e.g., 50 to 200 MHz, e.g., about 100 MHz, e.g., the pulsating direct current can draw the charged polymer through the nanopore, and the monomer sequence can be read by measuring the capacitance variation of the nanopore as the charged polymer passes through the nanopore.

In another embodiment, the invention provides a method for reading the monomer sequence of a charged polymer, e.g., a DNA molecule, comprising at least two different types of monomers, the method comprising applying a radio frequency pulsating direct current to a nanopore, e.g., at a frequency of 1 MHz to 1 GHz, e.g., 50 to 200 MHz, e.g., about 100 MHz, whereby the pulsating direct current draws the charged polymer through the nanopore, and reading the monomer sequence by measuring the capacitance fluctuation of the nanopore as the charged polymer passes through the nanopore.

In another embodiment, the present invention provides the use of DNA1 as described above in a method for storing information.

In another embodiment, the invention provides the use of single-stranded DNA in a method for storing information, where, for example, the sequence is substantially non-self-hybridizing.

In another embodiment, the invention provides a data storage method and device, which uses a nanochip, e.g., any of nanochips 1 above, to create a charged polymer, e.g., DNA, comprising at least two different types of monomers, e.g., according to any of methods 1 and/or 2 above, where the monomers or oligomers are arranged in an order corresponding to a binary code.

For example, in one embodiment, a nanochip containing the polymers thus synthesized provides a data storage device since the nanochip can be activated and the sequence of the polymer can be detected at any time by passing the polymer through the nanochip. In other embodiments, the polymer can be removed from the nanochip or amplified, and the amplified polymer can be removed from the nanochip and stored until needed and read by a conventional sequencer, e.g., a conventional nanopore sequencing device.

In another embodiment, the present invention provides a method for storing information, comprising synthesizing DNA1 as described above, for example according to either method 1 or method 2 above.

In another embodiment, the invention provides a method for reading a binary code, e.g., encoded in DNA 1, using a nanopore sequencer, e.g., nanochip 1, as described herein.

Any of the above methods, wherein the nanotip is erased using an enzyme that breaks down the charged polymer, e.g., deoxyribonuclease (DNase) that hydrolyzes DNA.

EXAMPLES Example 1 - Anchoring one end of DNA adjacent to a nanopore and controlling forward and backward movement of DNA by electric current An experimental procedure was developed to demonstrate that DNA can be moved back and forth between two chambers separated by a nanopore by electric current under conditions in which the associated protein does not move between the chambers.

Nanochips containing two chambers were fabricated from silicon nitride. 4 nm (dsDNA or ssDNA) and 2 nm (ssDNA only) nanopores were prepared as described in Briggs K, et al. Automated fabrication of 2-nm solid-state nanopores for nucleic acid analysis, Small (2014)10(10):2077-86. The two chambers are referred to as the "far" and "near" chambers, with the far chamber being the chamber to which the 3' end of the DNA is attached.

It is shown that ssDNA (2 nm pore) and ssDNA + dsDNA (4 nm pore) pass through the nanopore, but proteins do not. Nanopore passage is detected by a disruption in the current.

Attachment of DNA to the pore surface: 5' amino modified DNA is attached to carboxy coated polystyrene beads (Fluoresbrite® BB Carboxylate Microspheres 0.05 μm, Polysciences, Inc.) by carbodiimide ^mediated attachment. The DNA is 3' labeled with biotin. The DNA is of a pre-specified length.

Streptavidin binding: Binding was performed on the "far" side of the silicon nitride nanopore-conjugated streptavidin surface as described in Arafat, A. Covalent Biofunctionalization of Silicon Nitride Surfaces. Langmuir (2007) 23 (11): 6233-6244.

Immobilization of DNA close to the nanopore: DNA-bound polystyrene beads in buffer are added to the "near" chamber and buffer is added to the "far" chamber (standard buffer: 10 mM Tris pH 8, 1 mM EDTA, 150 mM KCl). Voltage (approximately 100 mV) is applied (using an Axon Nanopatch200B patch clamp amplifier) until a disruption in the current is observed. A 50 nm bead cannot pass through the nanopore, so the current is greatly disrupted as the DNA strand passes by and the bead is forced against the end of the nanopore. The current is maintained for 1-2 minutes until the DNA is irreversibly bound to the streptavidin immobilized on the far side by biotin binding. To confirm that the DNA is immobilized, the current is reversed. Different currents are observed if the DNA is inside or outside the pore. If the DNA is found not to be immobilized, the process is repeated.

Release of beads with endonuclease: Restriction enzyme in restriction enzyme buffer is added to the chamber where the DNA is bound. In one embodiment, the DNA is single stranded and contains a restriction site that can be cleaved by an enzyme that cleaves single stranded DNA. See, e.g., Nishigaki, K., Type II restriction endonucleases cleave single-stranded DNAs in general. Nucleic Acids Res. (1985) 13(16): 5747-5760. In an alternative embodiment, complementary oligonucleotides are added to the chamber where the DNA is bound, hybridized for 30 minutes to create dsDNA, and then the restriction enzyme is added. Once the bead is released, it is washed away. The current is switched back and forth to ensure that the DNA moves in and out through the pore.

Demonstrate forward and reverse control: Using standard buffer, apply current in the forward direction until disruption of the signal is observed, then return to "normal" after the DNA has passed. Apply reverse current until disruption of the signal is observed. If the DNA remains in the pore, no return to normal of the signal will be observed. Repeat the application of forward and reverse current for several cycles to confirm that the DNA moves forward and backward through the nanopore.

Example 1a: Immobilizing DNA adjacent to a nanopore in a silicon dioxide chip The inner nanochip wall is made of silicon dioxide. Both sides are silanized, but oligonucleotides are attached to only one side of the chip wall and then a nanopore is created.

Silanization: The chip wall surface is treated with piranha solution (available in various brands, typically contains a mixture of _sulfuric acid ( _H2SO4 ) and _hydrogen peroxide ( _H2O2 ) to remove organic residues from the surface) at 30°C and washed with double distilled water. A stock solution of (3-aminopropyl)triethoxysilane (APTES) is prepared in 50% methanol (MeOH), 47.5% APTES, 2.5% nanopure H2O and aged for >1 hour at 4°C. The APTES stock solution is then diluted 1:500 in MeOH and applied to the chip wall at room temperature and incubated. The chip wall is then rinsed with MeOH and dried at 110°C for 30 minutes.

Binding: The chip walls are then incubated in a 0.5% w/v solution of 1,4-phenylenediisothiocyanate (PDC) in dimethylsulfoxide (DMSO) for 5 hours at room temperature. Briefly wash twice with DMSO and twice with double distilled water. The chip walls are then incubated overnight at 37°C with 100 nM amine-modified single-stranded DNA oligomers (approximately 50 mers) in double distilled water (pH 8). The chip walls are then washed twice with 28% ammonia solution to inactivate unreacted material and twice with double distilled water. One or more nanopores are then created in the wall.

Once the nanotip is fabricated, the inner walls are coated with DNA oligomers about 50 bp long. This allows the ssDNA to be attached to a relatively bulky structure (e.g., a bead, protein, or DNA origami structure with a diameter too large to pass through the nanopore) (where the sequence complementary to the surface-bound DNA is away from the bulky structure), and an electric current is used to pull the charged polymer through the nanopore, binding the ssDNA to the complementary surface-bound DNA oligomer adjacent to the nanopore, and detaching the bulky structure, thereby placing a single-stranded DNA in the nanopore with a terminal sequence complementary to the surface-bound DNA.

Example 2: DNA synthesis - addition of a single nucleotide DNA is moved to a "storage" chamber by applying an appropriate electrical current and detecting the movement of the DNA.

Terminal transferase enzyme (TdT, New England Biolabs) in an appropriate buffer (50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, pH 7.9 @ 25°C) and reversibly blocked dATP* are added to the "loading" chamber, and buffer is added to the "storage" chamber.

Nucleotides are added to DNA using dNTPs that have a reversible block at the 3'-OH. Once added to the DNA strand, the next dNTP cannot be added until the blocked dNTP is deblocked.

Deblocking can be chemical or enzymatic. Various approaches are utilized:
a. 3'O-allyl: Allyls are removed by Pd-catalyzed deallylation in aqueous buffer as described in Ju J, Four-color DNA sequencing by synthesis using cleavable fluorescent nucleotide reversible terminators. Proc Natl Acad Sci US A. (2006);103(52):19635-40; or by using iodine (10 mol%) in polyethylene glycol-400 as described in Shankaraiah G., et al., Rapid and selective deallylation of allyl ethers and esters using iodine in polyethylene-400. Green Chem. (2011)13: 2354-2358.

b. 3'O-NH2: Amines are removed in buffered _NaNO2 as described in US8034923.

c. 3'-phosphate. The phosphate is hydrolyzed with endonuclease IV (New England Biolabs). Other possible 3' modifications that can be removed by endonuclease IV include phosphoglycaldehyde and deoxyribose-5-phosphate.

d. 3'-O-Ac: Acetic acid is removed by enzymatic hydrolysis as described in Ud-Dean, A theoretical model for template-free synthesis of long DNA sequence. Syst Synth Biol (2008) 2:67-73.

The DNA is then moved to the "far" chamber by applying an appropriate current and detecting the movement of the DNA. The buffer is exchanged and the DNA is deprotected by adding a deblocking buffer/solution as described above in a-d.
The process is repeated as desired to generate the desired sequence.

Example 3: DNA synthesis: addition of oligonucleotide chunks The 3' end of a double stranded DNA is attached adjacent to a nanopore with a 4 nm opening. The 5' end of the DNA has a CG overhang (reading 5' to 3').

Make oligo cassettes A and B as follows:

Code A and Code B each represent an information sequence. N represents any nucleotide. The 5' sequence contains a topoisomerase recognition site and the 3' sequence contains an Acc1 restriction site. When the oligo is exposed to topoisomerase, the topoisomerase binds to the 3' thymidine:

By applying an appropriate current, the DNA is moved to the "near" chamber and the DNA movement is detected. A topoisomerase-loaded "Code A" oligo is provided to the "addition" chamber. By applying an appropriate current, the DNA is moved to the addition chamber and the DNA movement is detected, whereupon the Code A oligo binds to the DNA. Acc1 is added to the "storage" chamber, where it cleaves the restriction site, resulting in a topoisomerase ligation site.

This process is repeated, adding another "Code A" or "Code B" until the desired sequence is reached. Note that it is not necessary to continually add new Acc1 to the "storage" chamber; simply wash out the Code A or Code B oligo in the "addition" chamber when switching from Code A or Code B.

To sequence in a pore that only passes ssDNA, some modifications are needed to the above protocol. It is already known that when dsDNA encounters a small pore (2 nm) that only passes ssDNA, the complementary strand "drops out". Therefore, if this synthesis is done in a 2 nm pore, we must ensure that the correct dsDNA can reform on the other side. For this, we can add "CGAAGGG<code A or B>GTCGACNNNNN" to the near chamber (so that a restriction site is created) and "CGAAGGG<code A or B>GT" to the far chamber (so that a topo-compatible site is generated).

To elaborate on the above method, we demonstrate that DNA-encoded information is successively "added" to a growing DNA strand in ≧2 successive additions (representing 2 bits of data), each of which involves an "addition" and "deprotection" step. Initial experiments for concept optimization and validation will be performed in microtubes.

In the approach described in this example, one bit of information is encoded in a string of nucleotides. The bit of DNA to be "added" is a short dsDNA sequence bound to Vaccinia topoisomerase I (topo). In the presence of a suitable "deprotected" "acceptor" DNA, the topo-loaded DNA "bit" is enzymatically and covalently linked ("added") to the acceptor by the topoisomerase, a process in which the topoisomerase is subsequently removed from the DNA. A restriction enzyme then cleaves the added bit and "deprotects" it, creating a suitable "acceptor" sequence for adding the next bit.

Topo loading: The general loading scheme is as follows and illustrated in Figure 22 and below, where N represents any nucleotide, and A, T, G and C represent nucleotides with adenine, thymine, guanine and cytosine bases, respectively. Ns above each other are complementary. This example uses the restriction enzyme HpyCH4III, but the basic strategy also holds true with other restriction enzymes, for example as demonstrated in Example 4.

The following oligonucleotides are ordered from Integrated DNA Technologies (IDT). The "b" at the end of some of the oligonucleotides represents biotin):
BAB: CGATAGTCTAGGCACTGTTTGCTGCGCCCTTGTCCGTGTCGCCCTTATCTACTTAAGAGATCATACAGCATTGCGAGTACG
B1: b-CACGTACTCGCAATGCTGTATGATCTCTTAAGTAGATA
B2: ATCTACTTAAGAGATCATACAGCATTGCGAGTACG
TA1: b-CACACTCATGCCGCTGTAGTCACTATCGGAAT
TA2: AGGGCGACACGGACAGTTTGAATCATACCG
TA3b: AACTTAGTATGACGGTATGATTCAAACTGTCCGTGTCGCCCTTATTCCGATAGTGACTACAGCGGCATGAG
TB1: b-CACACTCATGCCGCTGTAGTCACTATCGGAAT
TB2: AGGGCGCAGCAAACAGTGCCTAGACTATCG
TB3b: AACTTAGTATGACGATAGTCTAGGCACTGTTTGCTGCGCCCTTATTCCGATAGTGACTACAGCGGCATGAG
FP1: CACGTACTCGCAATGCT
FP2: CGGTATGATTCAAACTGTCCG
FP3: GCCCTTGTCCGTGTC
Dissolve the oligonucleotide at 100uM in TE buffer and store at -20C.

Hybridized oligonucleotides are made by mixing the oligonucleotides as described below, heating to 95° C. for 5 minutes, then decreasing the temperature at a rate of 5° C. per 3 minutes until a temperature of 20° C. is reached. Hybridized oligonucleotides are stored at 4° C. or −20° C. The oligonucleotide combinations are as follows:

B1/2
48uL B1
48uL B2
4 uL 5M NaCl

A5
20uL TA1
20uL TA2
5 uL TA3b
4 uL 5M NaCl
51uL TE

B5
20uL TB1
20uL TB2
5 uL TB3b
4 uL 5M NaCl
51uL TE

The following buffers and enzymes are used in this example:
TE: 10M Tris pH8.0, 1mM EDTA, pH8.0
WB: 1M NaCl, 10mM Tris pH8.0, 1mM EDTA pH8.0
1xTopo: 20mM Tris pH7.5, 100mM NaCl, 2mM DTT, 5mM _MgCl2
1xRE: 50mM K-acetate, 20mM Tris-acetate, 10mM Mg-acetate, 100ug/ml BSA pH7.9@25C
Vaccinia DNA topoisomerase I (Topo) is purchased from Monserate Biotech (10,000 U/mL). HypCH4III is purchased from NEB. Streptavidin-coated magnetic beads (s-MagBeads) are purchased from ThermoFisher.

The acceptor is prepared as follows: 5uL s-magbeads are washed once with 200uL WB (1 minute binding time). 5uL B1/2 + 195uL WB are added to the beads, incubated for 15 minutes at room temperature, then washed once with 200uL WB, then washed once with 200uL 1x Topo, and resuspended in 150uL 1x Topo.

Prepare Topo-loaded A5 (see Figure 20) as follows: 4uL 10x Topo Buffer + 23uL water + 8uL A5 + 5uL Topo, incubate at 37°C for 30 minutes, add to 5uL s-magbeads (wash 1x with 200uL WB, wash 1x with 200uL 1x Topo, resuspend in 150uL 1x Topo) and allow to bind for 15 minutes at room temperature.

"Add" Load A5 to the acceptor: Remove s-magbeads from Topo Load A5 and add to the acceptor and incubate at 37°C for 60 minutes. Remove an aliquot, dilute 1/200 in TE and store at -20C.

Deprotection: The material is washed once with 200uL WB, once with 200uL 1xRE, and resuspended in 15uL 10xRE and 120uL water. 15uL HypCH4III is added. The mixture is incubated at 37°C for 60 minutes, then washed once with 200uL WB and once with 200uL 1x Topo to produce the product designated "Acceptor-A5".

Prepare Topo-loaded B5 (see Figure 21) as follows: combine 4uL 10x Topo buffer, 23uL water and 8uL B5 + 5uL Topo and incubate at 37°C for 30 minutes. Add product to 5uL s-magbeads (wash 1x with 200uL WB, wash 1x with 200uL Topo, resuspend in 150uL 1x Topo) and allow to bind for 15 minutes at room temperature.

"Add" loaded B5 to acceptor-A5: remove s-magbeads from topo-loaded B5 and add to acceptor-A5 and incubate at 37°C for 60 minutes. Remove an aliquot, dilute 1/200 in TE and store at -20°C.

Deprotection: Wash material once with 200uL WB, once with 200uL 1xRE, and resuspend in 15uL 10xRE and 120uL water. Add 15uL HypCH4III and incubate the mixture at 37°C for 60 minutes.

Verification that the above reaction worked was done by PCR amplification of aliquots of A5 (acceptor with A5 appended: step iii, "A5 appended" in diagram) and B5 (acceptor-A5 with B5 appended: step vi, "B5 appended" in diagram). "No template" is used as a negative control for A5, A5 is used as a negative control for B5, and oligo BAB is used as a positive control for B5. The expected product size for A5 PCR is 68 bp and the expected product size for B5 PCR is 57 bp. (B1/2 with expected size of about 47 bp also runs on the gel, but this may be an approximation since it has an overhang and is biotinylated). PCR reactions (30 cycles of 95/55/68 (1 min each) are performed as follows:

SDS-PAGE using 4-20% Tris-Glycine gels is used to confirm that oligonucleotides of the expected size are produced. Loading is performed as above, but immediately after loading (37°C incubation step), loading buffer is mixed and samples are heated to 70°C for 2 minutes and allowed to cool before running on the gel. Gels are stained with Coomassie. As a negative control, water is added to the reaction instead of Topo. Figure 30 shows the results, clearly showing bands corresponding to the expected product sizes for A5PCR and B5PCR.

Thus, the addition of DNA bits via a topoisomerase-loaded DNA cassette and the deprotection performed by a restriction enzyme are shown to be feasible. In these proof-of-concept experiments, DNA is immobilized by streptavidin-conjugated magnetic beads and moved sequentially to different reaction mixtures, in the form of a nanopore chip, creating separate reaction chambers and using an electric current to move the DNA to these different reaction chambers.

Finally, PCR demonstrates that performing successive additions of DNA sequences corresponding to "bits" of information creates the expected DNA sequence. These reactions work as designed and require minimal optimization.

DNA produced as described in Examples 2 and 3 is recovered and sequenced using a commercially available nanopore sequencer (Oxford Nanopore's MinION) to confirm that the desired sequence is obtained.

を形成する制限酵素ＭｌｕＩを使用して実行する。 Example 4 - DNA synthesis: Addition of oligonucleotide chunks using different restriction enzymes The following synthesis is similar to that of Example 3, except that it cuts with "ACGCGT".

This is carried out using the restriction enzyme MluI which forms the following:

In this example, TOPO is loaded and forms a complex with sequence complementarity that allows the loaded TOPO to transfer DNA to DNA cut with MluI:

By a process similar to the previous example, the loaded TOPO is used to add an oligomer to the 5' end of the strand being synthesized that has a complementary acceptor sequence, thereby releasing the TOPO, and the strand is "deprotected" with MluI, and the cycle is repeated until the desired sequence of the oligomer is obtained.

Example 5 - Adding a Single Base Using a Topoisomerase Strategy We have found that the topoisomerase system can also be designed to add a single base to a single stranded DNA chain (compare Example 3 which describes adding a "cassette"). The DNA bit to be "added" is contained in a short DNA sequence bound to Vaccinia topoisomerase I (topo). In the presence of a suitable single stranded "deprotected""acceptor" DNA, the topo-charged DNA is enzymatically and covalently linked ("added") by the topoisomerase to the acceptor, which is removed from the DNA in the process. A type IIS restriction enzyme can cleave off all of the added DNA except for the single base (the one being "added"). This deprotection-addition process is repeated to add additional bases (bits).

Topo loading: The general loading protocol is similar to Example 3 and is as follows:

As in Example 3, the N's above each other are complementary. I is inosine. Biotin is used to remove unreacted products and by-products. The addition of a single base is carried out as follows:

Deprotection is exemplified using the BciVI restriction enzyme (site in bold) as follows:

The following oligonucleotides are commercially synthesized (B=biotin, P-phosphate, I=inosine):
NAT1 CACGTCAGGCGTATCCATCCCTTCACGTACTCGCAATGCTGTATGGCGAT
NAT1b P-CACGTCAGGCGTATCCATCCCTTCACGTACTCGCAATGCTGTATGGCGAT-B
NAT9cI P-IIIIIAAGGGATGGATACGCCTGACGTG
NAT9x P-ATCGCCATACAGCATTGCGAG
NAT9 ACGTGAAGGGATGGATACGCCTGACGTG
Nat9Acc CACGTAGCAGCAAACAGTGCCTAGACTATCG
Nat1P CACGTCAGGCGTATCCATCC
FP4 CGATAGTCTAGGCACTGTTTG
Oligonucleotides are dissolved at 100 μM in TE buffer and stored at -20°C.

Hybridization: The following hybridized oligonucleotides are made by mixing the listed oligonucleotides and heating to 95° C. for 5 minutes, then decreasing the temperature by 5° C. over 3 minutes until a temperature of 20° C. is reached. Store the hybridized oligonucleotides at 4° C. or -20° C.
NAT1b/NAT9cI/NAT9x
8 μL NAT1B
10 μL NAT9cI
10 μL NAT9x
48 μL TE
4 μL 5M NaCl

NAT1/NAT9cI
10 μL NAT1
10 μL NAT9cI
80 uL PBS

NAT1/NAT9
10 μL NAT1
10 μL NAT9
80 uL PBS

Buffers and enzymes: The following buffers are used:
TE: 10M Tris pH8.0, 1mM EDTA, pH8.0
PBS: phosphate buffered saline (137 mM NaCl, 2.7 mM KCl, 10 mM _Na2HPO4 , 1.8 _{mM KH2PO4} ) (pH _7.4 )
10x Cutsmart: 500mM KAc, 200mM Tris-Ac, 100mM Mg-Ac, 1mg/mL BSA pH 7.9
BciVI is purchased from NEB and streptavidin-coated magnetic beads (s-MagBeads) are purchased from ThermoFisher.

The addition reaction is carried out as follows.
1. Topo Loading: Combine reagents according to the table below:

The reagents are then incubated at 37°C for 30 minutes. Streptavidin magnetic beads (5 uL) in Topo Buffer are used to bind for 10 minutes at room temperature, after which the by-products are removed.

2. Reaction: Combine the reagents according to the table below:

The reagents are then incubated for 30 minutes at 37° C. The addition reaction is expected to proceed as follows:

The asterisk (*) represents the topoisomerase. Note that NAT9cI is phosphorylated but is not shown for illustration purposes.

When the loaded Topo is present in the acceptor sequence, the following reaction proceeds:

PCR amplification and measurement of the molecular weight of the product on an agarose gel confirms that the expected product was produced. A band of the correct size is shown in lane 1 (experimental) and no band in the negative control, see Figure 30.

B. Deprotection reaction: Combine the reagents according to the table below:

The reagents are incubated for 90 minutes at 37° C. For the deprotection reaction, a representative product of the addition reaction is made using purchased oligonucleotides and tested for cleavage with BciVI restriction enzyme:

Because 3' of the cleavage site is a run of inosines opposite "normal" bases, it was unclear whether the restriction enzyme would cleave the DNA as intended. As a positive control, a "proper" base-paired equivalent of NAT1/NAT9cI was made (NAT1/NAT9c):

PCR amplification of the products and subsequent measurement of molecular weight on an agarose gel (Figure 31) indicates that the enzyme works as intended. In the positive control, a large band is observed without cleavage (lane 1), but a small band is observed with cleavage. The same pattern is observed with NAT1/NAT9cI, indicating that the presence of inosine does not abolish or prevent cleavage. A small amount of uncleaved product appears to remain with NAT1/NAT9cI, suggesting that, at least under these conditions, cleavage is not as effective as with NAT1/NAT9c. Cleavage efficiency could be improved by modifying the buffer conditions and/or adding more inosine to the 5' end of NAT9cI.

The above example shows that a combination of topo/type IIS restriction enzymes can be used to add a single nucleotide to the 5' end of a target single-stranded DNA. Using a similar process, a related topoisomerase, SVF, which recognizes the sequence CCCTG (https://www.ncbi.nlm.nih.gov/pubmed/8661446), can be used to add a "G" instead of a "T", thus allowing the construction of sequences that code binary information with T and G.

As mentioned above, when using the topoisomerase strategy to generate dsDNA, nicks in the DNA of the opposite strand can be repaired using ligase with ATP. However, when adding a single nucleotide as in this example, we are constructing single-stranded DNA so that there is no nick to repair and no need to use ligase.

Example 6 - Addition of a Single Base Using a Topoisomerase Strategy Combined with 5' Phosphate Linkage Another approach to single base addition uses the 5' phosphate as a blocking group to effect the addition of a single base pair in the 3' to 5' direction. The charging reaction charges the topoisomerase with a single T (or G, or other nucleotide, as desired) bearing a 5' phosphate group. When the charged topoisomerase "finds" a single stranded DNA strand that is 5' unblocked (unphosphorylated), it adds a T to that strand, resulting in a 5' T-tagged DNA. This addition is facilitated by the presence of an adapter DNA that has a sequence to which the topoisomerase and the single stranded acceptor DNA can bind. (Note that the adapter DNA is catalytic - it can be reused as a template in repeated reactions.) Because the added nucleotide has a 5' phosphate, it is not a substrate for further additions until it is exposed to a phosphatase, which removes the 5' phosphate. This process is repeated using topo to add a single "T" and SVF topoisomerase to add a single "G" to the 5' end of the target single stranded DNA, thus allowing the construction of a sequence that codes for the binary information of T and G. This process is shown diagrammatically as follows:

Example 7 - Use of DNA origami to bind DNA adjacent to a nanopore A DNA strand with a large origami structure at one end is captured in a nanopore and immobilized to surface-bound streptavidin via a biotin moiety at the end of the DNA. After cleavage of the origami structure with a restriction enzyme, the immobilized DNA can traffic through the pore, as confirmed by a disruption in the current. Immobilization allows for the controlled movement of a single DNA molecule through the pore, which allows for the "reading" and "writing" of information to the DNA.

As shown in Figure 35, a bulky double-stranded DNA unit is formed that is too large to fit into the nanopore, and a single-stranded region is linked to the bulky portion by two short double-stranded regions that serve to anchor the DNA to be added during synthesis. The single-stranded regions can then be separated and anchored to a surface adjacent to the nanopore, and the origami structure released. See Figure 33.

Nanopores are fabricated with 50*50 μm openings in a 3 mm chip with 20 nm SiO2. The chips are provided by Nanopore Solutions. The nanopore cassette holder and flow cell are provided by Nanopore Solutions. The amplifier is a Tecella Pico 2 amplifier. This is a USB powered amplifier that uses a USB-to-computer interface for control. Tecella provides the (Windows) software that controls the amplifier. The circuit meter is a FLUKE 17B+ Digital Multimeter capable of detecting currents as low as 0.1 uA. For radio frequency noise screening, a Concentric Technology Solutions TC-5916A Shield Box (Faraday Cage) with USB interface is used. Oligonucleotides are obtained from IDT.com. "PS" is propargylsilane-O-(propargyl)-N-(triethoxysilylpropyl)carbamate, http://www.gelest.com/product/o-propargyl-n-triethoxysilylpropylcarbamate-90/ .

The origami structure is based on single-stranded m13 with a "honeycomb-like" cubic origami structure with sides of approximately 20 nm. There are double-stranded regions adjacent to the honeycomb, each containing a unique restriction site. One of these sites is used to attach modified DNA, allowing binding near the nanopore, and once the DNA is bound, the other site is used to cleave the origami structure.

Nanopore formation: Nanopores are formed in the chip using dielectric breakdown as follows:
1. Carefully mount the chip into the cassette 2. Wetting: carefully pipette 100% ethanol onto the chip. Air bubbles must be removed. However, pipetting the solution directly onto the chip should be avoided or the chip may crack (SiO2 is only 20 nm).
3. Surface treatment: Remove the ethanol and pipette freshly prepared piranha solution (75% sulfuric acid, 25% hydrogen peroxide (30%)) onto the chip. (Allow the piranha solution to reach room temperature.) Leave for 30 minutes.
4. Rinse 4 times with distilled water.
5. Rinse twice with HK buffer (10 mM HEPES pH 8, 1 M KCl).
6. Assemble the cassette into the flow cell.
7. Add 700 μL of HK buffer to each chamber of the flow cell.
8. Insert the silver electrode attached to the amplifier and close the Faraday cage.
9. Test the resistance at 300mV. No current should be detected. If it is detected, the chip is likely cracked and you should start over.
10. Connect the electrodes to a 6V DC current and test the current with a circuit meter. The current should be low and not change. Increase the voltage by 1.5V and hold the voltage until the resistance increases. If the resistance has not increased after 5-10 minutes, increase the voltage by another 1.5V and try again. Repeat until the resistance increases, at which point the applied voltage should be immediately removed. (At sufficient voltage, dielectric breakdown will occur, creating "pores" in the SiO2 film. The pores are small when first formed, but will grow larger as the voltage is maintained.)
11. Use an amplifier to test the pore. You should see a current of a few nA at 300 mV. The higher the current, the larger the pore.

Figure 34 shows a basic functioning nanopore. In each panel, the y-axis is current (nA) and the x-axis is time (sec). The left panel, "Screening RF noise," shows the utility of the Faraday cage. A chip without a nanopore is placed in the flow cell and 300 mV is applied. When the lid of the Faraday cage is closed (first arrow), a reduction in noise is seen. When the latch is closed (second arrow), a small spike occurs. The current is seen to be about 0 nA. After pore fabrication (middle panel), application of 300 mV (arrow) results in a current of about 3.5 nA. When DNA is applied to the earth chamber and +300 mV is applied, DNA translocation (right panel) can be observed as a transient decrease in current. (Note that in this case TS buffer is used: 50 mM Tris, pH 8, 1 M NaCl.) Lambda DNA is used for this DNA translocation experiment.

Silver chloride electrode:
1. Solder the silver wire to the insulated copper wire.
2. Polish the copper wire and soak the silver in fresh 30% sodium hypochlorite for 30 minutes.
3. The silver should acquire a dark grey coating (silver chloride).
4. Rinse the silver wire thoroughly with distilled water and dry it.
5. It's now ready to use.

Silanization of beads: The silanization method is first developed/tested on _SiO2 coated magnetic beads (GBioscience). The following protocol is adopted:
1. Pretreat the beads in fresh Piranha solution for 30 minutes.
2. Wash three times with distilled water.
3. Wash twice with methanol.
4. Dilute the APTES stock solution 1:500 in methanol.
5. Add the diluted APTES to the beads and incubate at RT for 45 minutes.
6. Rinse with methanol.
7. 100°C for 30 minutes.
8. Store under vacuum.

Silanization of Silicon Chip 1. Mount the nanopore-containing chip in a cassette.
2. Rinse with methanol and carefully remove any air bubbles.
3. Add fresh Piranha solution (equilibrated to room temperature) and incubate for 30 minutes.
4. Wash four times with distilled water.
5. Wash with methanol three times.
6. Dilute APTES stock solution 1:500 in methanol and use this to wash the chip twice. Incubate at RT for 45 min.
7. Rinse twice with methanol.
8. Dry under airflow.
9. Store under vacuum overnight.

Streptavidin binding to beads: Streptavidin binding is first developed/tested on the silanized beads prepared above.
1. Wash the silanized beads with modified phosphate buffered saline (MPBS).
2. Make a fresh 1.25% glutaraldehyde solution in MPBS (use frozen 50% glutaraldehyde stock solution).
3. Add 1.25% glutaraldehyde to the beads and let sit for 60 minutes, gently pipetting up and down every 15 minutes.
4. Wash twice with MPBS.
5. Wash twice with water.
6. Dry under vacuum.
7. Add streptavidin (500 μg/mL in MPBS) to the beads and incubate for 60 minutes (for negative control beads use bovine serum albumin (BSA) (2 mg/mL in MPBS) instead of streptavidin).
8. Remove streptavidin and add BSA (2 mg/mL in MPBS). Incubate for 60 minutes.
9. Wash twice with MPBS.
Store at 10.4°C.

Streptavidin Binding to Silicon Chips 1. Rinse the silanized chips twice with ethanol.
2. Rinse the silanized chip twice with MPBS.
3. Make a fresh 1.25% glutaraldehyde solution in MPBS (use frozen 50% glutaraldehyde stock solution).
4. Rinse the chip twice with 1.25% glutaraldehyde and let sit for 60 minutes, gently pipetting up and down every 15 minutes.
5. Wash twice with MPBS.
6. Wash twice with water.
7. Dry under airflow.
8. Add BSA (2 mg/mL in MPBS) to one half of the chip and streptavidin (500 μg/mL in MPBS) to the other. Incubate for 60 minutes. Mark the cassette to indicate which half of the chip is streptavidin modified.
9. Rinse both halves of the chip with BSA (2 mg/mL in MPBS). Incubate for 60 minutes.
10. Wash with MPBS.

The buffers used here are made as follows:
MPBS: 8 g/L NaCl, 0.2 g/L KCl, 1.44 g/L disodium phosphate, 0.240 g/L potassium phosphate, 0.2 g/L polysorbate-20 (pH 7.2)
PBS: 8 g/L NaCl, 0.2 g/L KCl, 1.44 g/L disodium phosphate, 0.240 g/L potassium phosphate TS: 50 mM Tris pH 8.0, 1 M NaCl
HK: 10mM HEPES pH8.0, 1M KCl
TE: 10mM Tris, 1mM EDTA, pH 8.0
Piranha solution: 75% hydrogen peroxide (30%) + 25% sulfuric acid. APTES stock solution: 50% methanol, 47.5% APTES, 2.5% nanopure water. Age at least 1 hour at 4°C. Store at 4°C.
PDC stock solution: 0.5% w/v 1,4-phenylenediisothiocyanate in DMSO

Order oligonucleotides (5' to 3'):
o1 CTGGAACGGTAAATTCAGAGACTGCGCTTTCCATTCTGGCTTTAATG
o3 GGAAAGCGCAGTCTCTGAATTTAC
N1 CTTACTGGAACGGCTATCGATATCGCAGCAGGACAGA
BN1 Biotin-CTTACTGGAACGGCTATCGATATCGCAGCAGGACAGA
N2 GTCCTGCTGCGATATCGATAGCCGTTCCAGTAAG

Hybridization of the oligonucleotide pair is carried out as follows:
1. Make a stock solution of oligos in TE buffer at a concentration of 100 uM. 2. Dilute oligos to 10 μM in PBS. 3. Heat to 85°C for 5 minutes in a thermal cycler. 4. Cool to 25°C with a 5°C gradient over 3 minutes. 5. Store at 4°C or -20°C.

Streptavidin binding: Streptavidin binding to _SiO2 was developed and tested using SiO2 coated magnetic beads, and the protocol was then adapted to _SiO2 chips. Biotinylated oligos are tested for binding to streptavidin and BSA-coupled beads. As expected, negligible binding is observed with BSA-coupled beads, and strong binding is observed with streptavidin-coupled beads. See Figure 38. Since it would be more convenient to perform binding in high salt (DNA transfer is performed in high salt), the ability of the beads to bind in HK buffer is also tested. Binding in HK buffer is comparable to binding in MPBS buffer (Figure 39).

The origami construct is made and confirmed to be operational as described above in Figures 35-37. Oligonucleotides are used to test biotinylation of the origami structure. The AlwNI site is already known to be active from the "origami" results shown in Figure 37 above. An oligonucleotide pair is used below (o1/o3) that recreates a segment of the exact sequence of the origami DNA. The origami molecule is shown below:

The oligo pair o1/o3 is as follows:

The DNA is cleaved with AlwNI in the presence of T4 DNA ligase and a biotinylated oligo complementary to the 3' overhang of the origami sequence (which is attached to a long ssDNA sequence, which is attached to the other side of the origami) according to the following reaction:

In this strategy, AlwN1 cuts the target DNA. Upon addition of ligase, the DNA can be religated, but the restriction enzyme will cut again. However, when the (right) fragment (of o1/o3) is bound to BN1/N2, the restriction site is not recreated and the product is not cut. Specific binding is confirmed by testing with and without the restriction enzyme:

All reagents except ligase are added and the solution is incubated at 37°C for 60 minutes. Ligase is added and the solution is incubated overnight at 16°C. 10xlig buff represents NEB 10x T4 DNA ligase buffer. Ligase is NEB T4 DNA ligase. o1/o3 and n1/n2 represent annealed oligo pairs as above. Units are in microliters. Agarose gel analysis confirmed that in the presence of AlwNI, a larger product was formed, which corresponds to a biotinylated oligonucleotide attached to a long ssDNA arm attached to the origami structure. A similar strategy is used for 3' biotinylation, if desired.

We demonstrate above the ability to create and use nanopores to detect voltage-induced translocation of DNA through the pore, create origami molecules with long ss regions attached at their biotin-distal ends, and bind streptavidin to silicon dioxide and use it to capture biotinylated DNA. Using these tools, we bind single DNA molecules close to nanopores and control their movement.

The first step is to attach streptavidin to one surface of the SiO2 nanopore (and BSA to the other side). This is accomplished by the protocol described above. The resulting pores tend to have lower currents than they had to begin with. After a few short 6v pulses, the current is brought back to near the original current. The functioning nanopore at this point is shown in Figure 40.

Next, the origami DNA is inserted. The origami DNA is added to the appropriate chamber and a current is applied, causing the origami to enter the chamber. A representative example of this is shown in Figure 41. Experimental results when the origami is introduced at a final concentration of 50 pM confirm that the origami-bearing DNA enters the pore relatively quickly (typically within a few seconds), which is detectable by the resulting decrease in current through the nanopore (e.g., in these examples, the current before origami insertion is about 3 nA and after insertion is about 2.5 nA). If the current is allowed to flow for a longer period, a double insertion can be observed. If higher concentrations are used, the insertion occurs too quickly to be observed.

Binding of inserted DNA to the chip. After the origami is inserted into the nanopore, 15 minutes are allowed to pass before voltage is applied again. The ends of the ssDNA regions of the origami contain biotin, and streptavidin is bound to the surface of the nanopore. Streptavidin binds to avidin with a binding constant close to covalent. This 15 minutes allows the DNA to diffuse and allows the biotin end to find and bind to streptavidin. If the DNA was indeed bound to the surface, the current observed when the voltage was reversed should be slightly less than before. Also, switching the current back and forth should result in lower currents in both directions than seen in the empty pore. In the example shown here, the empty pore shows a current of about 3 nA. Figure 42 shows a representative example of bound DNA, and Figure 43 shows experimental results of voltage switching of bound origami DNA. Note that the current seen in both directions is about +/- 2.5 nA, lower than the approximately +/- 3 nA observed in the empty pore. If the DNA was not bound to the surface, switching the voltage would restore the original current (Figure 44).

To remove the origami structure, the buffer in the flow cell chamber containing the origami structure is removed and replaced with 1xSwa1 buffer containing 1uL Swa1/20L. The buffer in the other flow cell chamber is replaced with 1xSwa1 buffer without Swa1. This is incubated at room temperature for 60 minutes, then washed with HK buffer and voltage is applied. The forward and backward movement of DNA shown in Figure 45 is supported by experimental data shown in Figure 46, which indicates controlled movement of the immobilized DNA through the SiO2 nanopore.

Example 8 - Alternative means of attaching polymers to surfaces adjacent to nanopores The above examples describe attachment of DNA to surfaces adjacent to nanopores by biotinylating the DNA and coating the attachment surface with streptavidin. Several alternative means of polymer attachment are shown in Figure 47.
a) DNA Hybridization: In one method, the DNA to be extended in the method of the invention is hybridized to a short oligonucleotide that is bound in close proximity to the nanopore. Once synthesis is complete, the synthesized DNA can be easily removed without the need for a restriction enzyme, or the duplex formed by the bound oligonucleotide and the synthesized DNA can provide a substrate for a restriction enzyme. In this example, short oligomers are bound to the surface using biotin-streptavidin or ligated using 1,4-phenylenediisothiocyanate as follows:

Binding of biotinylated DNA to SiO2:
A. Silane Treatment:
1. Pretreatment: nha solution for 30 min, wash with double distilled _H2O ( _ddH2O ) 2. Preparation of APTES stock solution: 50% MeOH, 47.5% APTES, 2.5% nanopure _H2O : age >1 h at 4°C
3. Dilute APTES stock solution 1:500 in MeOH 4. Incubate chip at room temperature 5. Rinse with MeOH 6. Allow to dry 7. Heat at 110°C for 30 minutes

Combined:
1. Treat the chip with PDC stock solution for 5 hours (room temperature) (PDC stock solution: 0.5% w/v 1,4-phenylenediisothiocyanate in DMSO).
2. Wash twice with DMSO (briefly)
3. Wash twice with _ddH2O (briefly)
4. 100 nM amino-modified DNA in _ddH2O (pH 8), O/N 37°C
5. Wash twice with 28% ammonia solution (inactivation)
6. Wash twice with _ddH2O

Single-stranded DNA having a terminal sequence complementary to the bound oligonucleotide is introduced as described above and allowed to hybridize to the bound oligonucleotide.

b) Click Chemistry: Click chemistry is a general term for reactions that are simple, thermodynamically efficient, do not create toxic or highly reactive by-products, take place in water or biocompatible solvents, and are often used to attach substances of interest to specific biomolecules. Click attachment in this case uses similar chemistry to that used in a) to attach oligonucleotides, but now it is used exclusively to attach polymers that are extended during synthesis in the methods of the invention. In this example, DNA is the polymer, but this chemistry can also be used to attach other polymers that are functionalized by the addition of compatible azide groups.

Silane Treatment:
1. Pretreatment: Piranha solution for 30 min, wash with ddH2O 2. Preparation of PS (propargylsilane) stock solution: 50% MeOH, 47.5% PS, 2.5% Nanopure H2O: Aging >1 h, 4C
3. Dilute APTES stock solution 1:500 in MeOH 4. Incubate chip at room temperature 5. Rinse with MeOH 6. Allow to dry 7. Heat at 110°C for 30 minutes

DNA terminated with an azide functional group is covalently attached to this surface (as shown in Figure 47). Azide terminated oligos are ordered and attached to longer origami DNA as previously described for adding biotin to DNA.

Claims (8)

1. A method for synthesizing a charged polymer in a nanotip, the polymer comprising at least two distinct monomers or oligomers, comprising:
the nanotip comprises one or more loading chambers containing reagents for loading one or more monomers or oligomers in end-capped form in a buffer solution to the charged polymer such that only a single monomer or oligomer can be loaded in one reaction cycle; and one or more storage chambers containing a buffer solution but not all reagents necessary for loading the one or more monomers or oligomers, wherein the chambers are separated by one or more membranes containing one or more nanopores, the nanopores being large enough to allow passage of the charged polymer but too small to allow passage of at least one reagent for loading the one or more monomers or oligomers, such that the charged polymer can pass through the nanopore but not at least one of the reagents for loading the one or more monomers or oligomers, and the nanopores have a diameter of 2-5 nm ;
The method comprises:
(a) moving a first end of a charged polymer having a first end and a second end to an addition chamber by electrical attraction, thereby adding a monomer or oligomer to the first end in a blocked form;
(b) transferring the first end of the charged polymer having the attached monomer or oligomer in blocked form to a storage chamber;
(c) deblocking the added monomer or oligomer; and (d) repeating steps a-c until the desired polymer sequence is obtained, where the monomer or oligomer added in step a) is the same or different.
and
The charged polymer is DNA, the monomers are nucleotides, and the oligomers are oligonucleotides.
method. A method for synthesizing nucleic acids in a nanochip, the nanochip comprising at least an addition chamber and a storage chamber separated by a membrane comprising at least one nanopore, the synthesis being carried out in a buffer by cycles of adding nucleotides to a first end of a nucleic acid having a first end and a second end, where the first end of the nucleic acid moves by electrical attraction between one or more addition chambers (containing reagents capable of adding nucleotides ) and one or more storage chambers (not containing reagents necessary for adding nucleotides ), the chambers being separated by one or more membranes each containing one or more nanopores, the nanopores being large enough to allow the passage of the nucleic acid but small enough to allow the passage of at least one reagent essential for the addition of the nucleotide , and the nanopores having a diameter of 2-5 nm . The method of claim 2, wherein the reagent that adds nucleotides to the nucleic acid comprises a polymerase. The method of claim 2 or 3, wherein the nucleotide is added in a 3'-protected form and the storage chamber comprises a reagent capable of deprotecting the added nucleotide. The method of claim 1, wherein the polymer sequence corresponds to a machine-readable code. The method of claim 5 , wherein the charged polymer or a copy of the charged polymer is removed from the nanotip after synthesis is complete. The method of claim 5 , wherein the charged polymer remains on the nanotip after synthesis is complete. 1. A nanochip for synthesizing charged polymers comprising at least two distinct monomers or oligomers, comprising at least first and second reaction chambers separated by a membrane comprising one or more nanopores, each reaction chamber comprising one or more electrodes for drawing a charged polymer into the chamber, the first reaction chamber further comprising an electrolyte medium and a reagent for adding a monomer or oligomer to the polymer, the monomer or oligomer being protected such that only one can be added at a time, the second reaction chamber further comprising an electrolyte medium and a reagent for deprotecting the monomer or oligomer thus added, the nanopore being large enough to allow the passage of the polymer but small enough to allow the passage of the reagent for adding the monomer or oligomer to the polymer and the reagent for deprotecting the monomer or oligomer thus added, the charged polymer being DNA, the monomers being nucleotides, and the oligomers being oligonucleotides, and the nanopore having a diameter of 2-5 nm .

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