RU2765308C2 - Methods, compositions and devices for storing information - Google Patents

Abstract

FIELD: biotechnology.SUBSTANCE: invention relates to biotechnology, and it is a method for synthesizing charged polymer containing at least two different monomers in a nanochip, wherein the nanochip contains one or more attachment chambers containing reagents for attaching one or more monomers or oligomers to charged polymer in a buffer solution in a form with protected ends so that only one monomer or oligomer can be attached in one reaction cycle; and one or more backup chambers containing a buffer solution, but not all reagents necessary for the attachment of one or more monomers or oligomers, where chambers are separated by one or more membranes containing one or more nanopores, and where charged polymer can pass through the nanopore and cannot pass through at least one of reagents for the attachment of one or more monomers or oligomers, while the method includes a) moving the first end of charged polymer having the first end and the second end under the action of electric attraction into the attachment chamber, wherein monomers and oligomers are attached to the specified first end in a blocked form, b) moving the first end of charged polymer with added monomer or oligomer in the blocked form to the backup chamber, c) unblocking attached monomer or oligomer, and d) repeating stages a-c, where monomers or oligomers attached at the stage a) are the same or different, until the required polymer sequence is obtained, where charged polymer is DNA, monomers are nucleotides, where oligomers are oligonucleotides, and where the nanopore has a diameter of 2-20 nm.EFFECT: obtaining compositions and devices for information storage.8 cl, 47 dwg, 3 tbl, 7 ex

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[001] This application claims priority to U.S. Provisional Applications Nos. 62/301,538, filed February 29, 2016, and 62/415,430, filed October 31, 2016, the contents of each of which are incorporated herein by reference in their entirety.

FIELD OF THE INVENTION

[002] The invention relates to new methods, compositions and devices for storing and retrieving information using nanopore devices for the synthesis and sequencing of polymers, for example, nucleic acids.

BACKGROUND OF THE INVENTION

[003] There is a constant need to store more and more data on or in physical media, with storage devices becoming increasingly small in size and increasing in capacity. According to available information, the amount of data stored is doubling every two years and, according to one study, by 2020 the amount of data that is created and copied annually will reach 44 zettabytes or 44 trillion gigabytes. Moreover, existing storage media such as hard disks, optical media, and magnetic tapes are relatively unstable and are damaged by long-term storage.

[004] There is a strong need for alternative approaches for storing large amounts of data over long periods of time, such as decades or centuries.

[005] It has been proposed to use DNA for data storage. DNA is extremely stable and in theory can encode huge amounts of data and store data 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. In addition, DNA as a storage medium is not subject to the security risks inherent in traditional digital storage media. However, there is no practical approach to implement this idea.

[006] WO 2014/014991, for example, describes a method for storing data on DNA oligonucleotides, where the information is encoded in binary format, one bit per nucleotide, with a data block of 96 bits (96 nucleotides), an address sequence of 19 nucleotides, and flanking sequences for amplification and sequencing. The code is then read by amplifying the sequences using PCR and sequencing using a high speed sequencer such as the Illumina HiSeq device. The data block sequences are then arranged in the correct order using address marks, the address and flanking sequences are filtered, and the sequence data is translated into binary code. This approach has significant limitations. For example, a 96-bit block of data can only encode 12 letters (using the conventional one byte or 8 bits per letter or space). The ratio of useful information stored to "housekeeping" information is low - approximately 40% of the sequence information is occupied by address and flanking DNA. The description describes the coding of the book using 54898 oligonucleotides. Inkjet-printed high-precision DNA microarrays used for oligonucleotide synthesis limited the size of oligonucleotides (the 159 measures described were the upper limit). Moreover, the reading of oligonucleotides requires amplification and isolation, which leads to additional error potential. See also WO 2004/088585A2; WO 03/025123A2; 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.

[007] While the potential information density and stability of DNA makes it an attractive data storage medium, as has been recognized for twenty-five years, there is still no practical approach to writing and reading large amounts of data in this form.

BRIEF DESCRIPTION OF THE INVENTION

[008] The present inventors have developed a new approach for storing nucleic acids using nanofluidic systems for synthesizing nucleic acid sequences and nanopore readers for reading sequences. The approach developed by the inventors allows the synthesis, storage and reading of DNA strands hundreds, thousands and even millions of bases long. Because the sequences are long, only a relatively small portion of the sequence is dedicated to information identification, so that the information density is significantly higher than in the approach described above. Moreover, in some embodiments, the synthesized nucleic acid has a specific position on the nanochip so that the sequence can be identified even without identifying information. Sequencing carried out in nanochambers is very fast, and reading a sequence through a nanopore can be extremely fast, on the order of up to one million bases per second. Since only two types of bases are required, sequencing can be faster and more accurate than sequencing techniques that must distinguish between four types of nucleotide bases (adenine, thymine, cytosine, guanine). In specific embodiments, the two bases do not pair with each other and do not form secondary structures, and also have different sizes. For example, adenine and cytosine would be better for this purpose than adenine and thymine, which tend to hybridize, or adenine and guanine, which are similar in size.

[009] In some embodiments, this system can be used to synthesize long polymers encoding data that can be amplified and/or extracted and then sequenced on various sequencers. In other embodiments, the system can be used to provide specialized DNA sequences. In other embodiments, the system can be used to read DNA sequences.

[011] Thus, the invention relates, inter alia, to the following embodiments:

- a nanochip for the synthesis of an electrically charged polymer, for example DNA, containing at least two different monomers, and the nanochip contains two or more reaction chambers separated by one or more nanopores, where each reaction chamber contains an electrolytic liquid, one or more electrodes for drawing in an electrically charged polymer into the chamber; and one or more reagents to facilitate the attachment of monomers or oligomers to the polymer. The nanochip may optionally be equipped with functional elements to guide, move and/or control DNA, optionally it may be coated or made with materials to allow smooth flow of DNA or attachment of DNA, and it may contain nanoloop elements to provide and control electrodes near nanopores. For example, each of one or more nanopores can be coupled to electrodes that can monitor the passage of the polymer through the nanopore and/or detect changes in electrical potential, current, resistance, or capacitance at the nanopore-polymer interface, thereby determining the sequence of the polymer as it passes. through one or more nanopores. In specific embodiments, oligomers are synthesized using polymerases or site-specific recombinases. In some embodiments, the polymer is sequenced during synthesis to allow detection and optionally error correction. In some embodiments, the polymer thus obtained is stored on a nanochip and can be sequenced when access to the information encoded in the polymer sequence is desired.

- A method for synthesizing a polymer, such as DNA, using a nanochip, as described.

- Single-stranded DNA molecule, where the sequence essentially consists of non-hybridizing nucleotides, for example, adenine and cytosine (A and C), which are organized in a sequence corresponding to a binary code, for example, for use in a data storage method.

- Double-stranded DNA containing a series of nucleotide sequences corresponding to the binary code, where double-stranded DNA additionally contains

- A method for reading a binary code encoded in DNA, including the use of a nanopore sequencer.

- Data storage method and device for this using the nanochip described above to obtain an electrically charged polymer, for example DNA, containing at least two different monomers, where the monomers are organized in a sequence corresponding to a binary code.

[012] The following aspects and fields of application 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 of the invention, are intended to be illustrative only and are not intended to limit the scope of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[013] The present invention will become more clear from the detailed description and the accompanying drawings, where:

[014] Figure 1 is a diagram of a simple design of a two-chamber nanochip with a separating membrane, a perforated nanopore, and electrodes on each side of the membrane.

[015] Figures 2 and 3 show how a charged polymer, such as DNA, moves towards the anode.

[016] Figures 4 and 5 show that the polymer can move back by changing the polarity of the electrodes.

[017] Figure 6 shows the design of a two-chamber nanochip for DNA systems, in which the polymerase enzyme is located in one chamber, the deblocking enzyme is in the other chamber, and neither of them passes through the nanopore.

[018] Figure 7 shows the attachment of an adenine nucleotide when the 3'-blocked dATP (A) passes through the left chamber and a "forward" current is established to move DNA into this chamber.

[019] FIG. 8 shows the deprotection of an oligonucleotide so that an additional nucleotide can be attached. For example, deprotection occurs after moving the DNA into the chamber by setting the current to "reverse".

[020] Figure 9 shows the attachment of a 3'-blocked dCTP (C). In certain embodiments, a fluid flow is used to replace the contents of this chamber, for example, as shown, there was previously an "A" in this chamber.

[021] FIG. 10 shows how a plurality of separate holding chambers can be provided while the flow chamber becomes a single line for supplying reagents.

[022] Figure 11 shows an approach for retaining DNA associated with its chamber by binding to the chamber (upper DNA fragment in FIG.) or by attaching to a bulky group that cannot pass through the nanopore (lower DNA fragment in FIG. ). In this system, the end of the DNA can still pass into the flow chamber and receive additional nucleotides, however the other end remains in the holding chamber.

[023] FIG. 12 shows a configuration where the DNA is bound to the chamber wall and controlled by a plurality of electrodes.

[024] FIG. 13 shows how DNA can be retained in the chamber, if desired, simply by controlling the polarity of the electrodes.

[025] Figure 14 shows a matrix with free flow of reagents in both directions, where the DNA is associated with the surface of the chamber.

[026] FIG. 15 shows an alternative design with electrodes on the sides adjacent to the separating membrane, allowing for less expensive manufacturing.

[027] FIG. 16 shows a three-compartment system where DNA can move from compartment to compartment via electrodes. This system does not require a significant flow of reagents during the synthesis.

[028] FIG. 17 is an example of how the reagents can be organized in a three-compartment system.

[029] FIG. 18 shows an oligonucleotide bound adjacent to a nanopore, where the nanopore has electrode elements on each side of the membrane.

[030] Figure 19 shows a series of DNA molecules bound along a membrane containing nanopores, each controlled by electrodes adjacent to the nanopore, with a flow line on each side of the membrane. For example, as shown, the left flow line provides a wash buffer/3'-blocked dATP (A)/wash buffer/3'-blocked dCTP (C)/wash buffer flow where DNA molecules move into the flow chamber only when the desired one is present. The right line provides a deblocking agent(s) to remove the protecting group from the 3' end of the nucleotide and allow the addition of another nucleotide. In one embodiment, the flow of deblocking agent(s) occurs when the left line is washed with buffer. In other embodiments, the deprotecting agent(s) is too bulky to pass into the left lane through the nanopores.

[031] Figures 20-22 are schematic representations of proof-of-concept experiments where the bits used to encode data are short oligomers attached using topoisomerase.

[032] FIG. 23 shows a nanopore sequencer format where the polymer sequence is read using capacitive oscillations. In this capacitance reading scheme, the electrodes form the top and bottom plates of a capacitor separated by a membrane containing a nanopore. The capacitor is part of an oscillating circuit where a pulsating direct current can drive the charged polymer through the nanopore. This change in capacitance is measured as a polymer, such as DNA, passes through the nanopore using high frequency impedance spectroscopy. A significant advantage of this approach, particularly with DNA, is that the measurement rate can be very high (in fact, a measurement for each cycle, so 100 MHz corresponds to 100 million measurements per second), and much higher than the travel rate monomers through the nanopore (DNA, for example, unless otherwise limited in any way, passes through the nanopore in response to an electric current at a rate of about 1 million nucleotides per second).

[033] FIG. 24 shows a two-chamber attachment design suitable for attaching two different types of monomers or oligomers, such as for 2-bit or binary coding. On the top of Fig. top view is shown. The lower part shows a cross-sectional side view. The complete device in this embodiment may be assembled from up to 3 independently fabricated layers and combined by plate splicing, or may be formed by engraving a single substrate. The chip contains an electrical control layer (1), a fluid layer (2), which contains two attachment chambers above a backup chamber with a charged polymer (e.g., DNA) anchored between the nanopore inlets in the first and second attachment chambers, and a ground layer ( 3).

[034] FIG. 25 shows the operation of the two-chamber attachment design shown in FIG. 24. It can be observed that at the base of each attachment chamber there is a nanopore (4). The nanopore is obtained, for example, by drilling with FIB, TEM, wet or dry engraving, or by dielectric breakdown. The membrane (5) containing the nanopores has a thickness of, for example, from 1 atomic layer to 10 nm. It is made, for example, from SiN, BN, SiOx, graphene, transition metal dichalcogenides, for example WS ₂ or MoS ₂ . Beneath the nanopore membrane (5) is a reserve or deblocking chamber (6) which contains reagents for deprotecting the polymer after the addition of a monomer or oligomer in one of the attachment chambers (as a reminder, monomers or oligomers are attached in a form with protected ends, so that only one monomer or oligomer is attached at a time). The polymer (7) can be driven into or out of the attachment chambers by changing the polarity of the electrodes in the electrical control layer (1).

[035] FIG. 26 is a plan view of a structure similar to FIGS. 24 and 25, however, in this case, there are four attachment chambers that share a common reserve or deblocking chamber, and the polymer is bonded in position (9) with access to each of the four chambers. The cross section of this design may be as shown in Figures 24 and 25, and the charged polymer may move into each of the four attachment chambers under the control of the electrodes in the electrical control layer (1 in Figure 24).

[036] FIG. 27 is a plan view of a nanopore chip having a plurality of sets of dual chambers for attachment, as shown in FIGS. 24 and 25, allowing the synthesis of multiple polymers in parallel. The monomers (in this case, the dATP and dGTP nucleotides, referred to as as A and G) are loaded into each chamber via serial flow lines. One or more common deblocking flow cells allow deprotection of the polymer after attachment of the monomer or oligomer in one of the attachment chambers. It also allows polymers to be detached when needed (eg using a restriction enzyme in the case of DNA, or chemical detachment from the surface next to the nanopore) and accumulation outside. In this particular embodiment, the release flow cells are perpendicular to the loading channels used to fill the attachment chambers.

[037] FIG. 28 shows the following wiring details for dual mount camera circuits. The electrical control layer (1) includes electrical wiring made of metal or polysilicon. Wiring density is increased by 3D laying, with electrical insulation provided by dielectric coating (eg via PECVD, sputtering, ALD, etc.). Contact (11) with the top of the electrode in the attachment chamber is in one embodiment using silicon vias (TSV) via reactive ion deep engraving (DRIE) (cryo or BOSCH process). Individual voltage control (12) allows each connection chamber to be addressed individually, providing precise control of the sequencing of multiple polymers in parallel. On the right side of Fig. a plan view is shown illustrating wiring for a plurality of bays to be connected. The ground plane (3) can be shared (as shown) or separated to reduce interference between cells.

[038] FIG. 29 shows an alternative configuration where the reference electrodes (13) of the attachment chambers may be positioned on the chamber wall in a wrap-around arrangement instead of being positioned at the top of the chamber.

[039] FIG. 30 is an SDS-PAGE gel confirming that the topoisomerase coupling protocol as described in Example 3 is working, with bands corresponding to expected products A5 and B5 clearly visible.

[040] FIG. 31 shows an agarose gel confirming that the PCR product of Example 5 is the correct size. Lane 0 is a 25 bp molecular weight marker; lane 1 is the product of the experiment - the line corresponding to the expected molecular weight; lane 2 is negative control #1; lane 3 is negative control #2; lane 4 is negative control #4.

[041] FIG. 32 shows an agarose gel confirming that the restriction enzyme as described in Example 5 produces the expected product. The molecular weight marker on the left is a 100 bp marker. Lane 1 corresponds to non-cleaved NAT1/NAT9c, lane 2 corresponds to cleaved NAT1/NAT9c. Lane 3 corresponds to uncleaved NAT1/NAT9cI, lane 4 corresponds to cleaved NAT1/NAT9cI.

[042] Figure 33 shows the immobilization of DNA near the nanopore. Panel (1) shows DNA with an origami structure at one end in the left chamber (in a real nanochip, many such origami structures are initially present in the left chamber). Panel (2) illustrates a system with an anode on the right that drives DNA towards the nanopore. While the DNA strand is able to pass through the nanopore, the origami structure is too large to pass, so the DNA gets stuck. Turning off the current (panel 3) allows the DNA to diffuse. With the right chemistry, the end of a DNA chain is able to bind when it comes into contact with a surface near the nanopore. Panel (4) has a restriction enzyme added that cleaves the origami structure from DNA. The chamber is washed to remove enzyme and residual DNA. The end result is a single DNA molecule attached near the nanopore, capable of moving back and forth through the nanopore.

[043] FIG. 34 shows the basic operation of a nanopore. On each panel, the y-axis represents current (nA) and the x-axis represents time (s). In the left panel, "RF noise screening" illustrates the usefulness of the Faraday cage. The chip without the nanopore is placed in the flow cell and 300 mV is applied. When the Faraday cage cover is closed (first arrow), noise reduction can be observed. A small pulse occurs when the latch is closed (second arrow). It should be noted that the current is ~0 nM. After pore formation (middle panel), application of 300 mV (arrow) results in a current of ~3.5 nM. When DNA is placed in the ground chamber and +300 mV is applied, DNA movement can be observed (right panel) as a temporary decrease in current (note that buffer TS is used in this case: 50 mM Tris, pH 8, 1 M NaCl). Lambda DNA is used for this DNA movement experiment.

[044] Fig. 35 is a simplified picture illustrating the main features of the DNA origami structure: a large single strand region, a cubic origami structure, and the presence of 2 restriction sites (SwaI and AlwN1) near the origami structure.

[045] FIG. 36 is an electron microscope image of the fabricated origami DNA structure and shows the expected topology. Origami was prepared in 5 mM Tris, 1 mM EDTA, 5 mM NaCl, 5 mM MgCl2. To preserve the origami structure, it is preferred that the Mg ⁺⁺ concentrations be ~5 mM or the Na ⁺ /K ⁺ concentrations be 1 M. The origami structure is stored at 4° C. at a concentration of 500 nM.

[046] FIG. 37 shows origami DNA restriction digest to confirm correct assembly and function. On the far left track are the MM standards. Restriction sites are examined by origami cleavage with AlwN1 and Swa1. The four lanes tested contain the following reagents (units are microliters):

[047] Test lane (1) is a negative control; (2) is a cleavage product with Swa1; (3) is a cleavage product with AlwN1; (4) is a product of a double cleavage with Swa1/AlwN1. Cleavage is carried out at room temperature for 60 minutes and then at 37°C for 90 minutes. Agarose gel 1/2x TBE-Mg (1/2x TBE with 5 mM MgCl2) is visualized by staining with ethidium bromide. Individual digestion with each enzyme does not show a mobility effect in the gel, however digestion with both enzymes together (lane 4) results in two fragments of different lengths, as expected.

[048] FIG. 38 shows binding of biotin-labeled oligonucleotides to streptavidin-coated beads versus binding to control BSA-coated beads. Units of fluorescence are shown on the y-axis, "before binding" is the fluorescence of the oligonucleotide in the test solution before binding to the beads, (-) controls are the fluorescence observed after binding to two different lots of BSA-conjugated beads, SA-1 and SA-2 represent the fluorescence observed after binding to 2 different lots of streptavidin-conjugated beads. In the case of BSA-conjugated beads, a small noticeable level of binding is observed, however, much more binding is observed in the case of streptavidin-conjugated beads.

[049] FIG. 39 shows the binding of biotin-labeled oligonucleotides to streptavidin-coated beads versus binding to control BSA-coated beads in various buffer systems: MPBS buffer and HK. The left column "negative control" corresponds to the fluorescence of the oligonucleotide in the test solution before binding to the beads. The middle column shows the fluorescence of "BSA beads" and the right column of "SA beads" after binding to beads with BSA or streptavidin, respectively. In both buffer systems, fluorescence is reduced for streptavidin beads relative to controls, indicating that biotin-labeled oligonucleotides bind well to streptavidin-coated beads in different buffer systems.

[050] FIG. 40 shows the operation of a conjugated SiO ₂ nanopore where the surface is streptate coated on one side and BSA coated on the other. The x-axis represents time and the y-axis represents current. The point shows the place where the current reverses. When the current changes, a short jump occurs, then the current drops to approximately the same absolute value. The nanopore shows a current of ~+3 nA at 200 mV and ~-3 nA at -200 mV.

[051] FIG. 41 shows an origami DNA structure placed in a nanopore.

[052] FIG. 42 shows the attachment of single-stranded DNA to a streptavidin-coated surface next to a nanopore.

[053] FIG. 43 shows the results of an origami DNA experiment bound to a surface near a nanopore. The current is + or - ~2.5 nA in both directions, which is less than the initial current of +/- ~ 3nM, which reflects the partial plugging of the origami structure. The x-axis represents time (s), the y-axis represents current (nA), the circles correspond to voltage switching points.

[054] Figure 44 shows the entry of DNA origami, resulting in a slight decrease in current. Origami immediately exit the nanopore as soon as the current disappears. Time (s) is given on the x-axis, current (nM) is given on the y-axis, the circles correspond to the voltage switching point.

[055] FIG. 45 shows the controlled movement of a DNA strand back and forth through a nanopore by applying a current. On the left side, the DNA is in the pore, so the observed current is lower than if there is no DNA in the pore. When the current is reversed (right side), there is no DNA in the pore, so the current does not change.

[056] FIG. 46 shows experimental results that support this view. When a positive voltage is applied, the current is ~3 nA, which is comparable to the current normally seen when the pore is open. When the voltage is reversed, the current is ~-2.5nA. This is lower than the current normally seen when the pore is open and is in line with the current normally seen when the pore is blocked by a DNA strand. several successive switching voltages show stable results, indicating that the DNA configuration is changing, as shown in Fig.45.

[057] FIG. 47 shows another conjugation chemistry for binding DNA to a surface near the nanopore.

DETAILED DESCRIPTION

[058] The following description of the preferred embodiment(s) is illustrative only and is not intended to limit the invention, its purpose or applications in any way.

[059] As used herein, ranges are used as shorthand for describing each value within a given range. Any value in the range can be selected as the limit of the range. In addition, all references cited in the present description are incorporated herein by reference in their entirety. In the event of a conflict between the definition in the present specification and the definition in the cited reference, the present specification shall govern.

[060] Unless otherwise indicated, all percentages and amounts given in this section and elsewhere in the description should be understood as referring to percentages by weight. The amounts given are based on the active weight of the material.

[061] "Nanochip", as used herein, refers to a nanofluidic device containing a plurality of chambers containing fluid, and optionally fluid flow channels, where the critical dimensions of the nanochip elements, for example, the width of the elements separating the chambers from each other, range from one atom to 10 micrometers, for example, less than one micrometer, for example, 0.01-1 micrometer. The flow of materials in a nanochip can be controlled by electrodes. For example, since DNA and RNA are negatively charged, they will move towards a positively charged electrode. See, for example, Gershow, M, et al., Recapturing and Trapping Single Molecules with a Solid State Nanopore, Nat Nanotechnol. (2007) 2(12): 775-779, incorporated herein by reference. In some cases, fluid flow can also be controlled by restrictive elements and by pumping, injecting and/or pumping fluids into and out of the nanochip. The system is capable of accurate multiplex analysis of nucleic acids (DNA/RNA). In certain embodiments, the implementation of the nanochip can be made of silicon material, such as silicon dioxide or silicon nitride. Silicon nitride (eg Si ₃ N ₄ ) is particularly desirable for this purpose because it is chemically relatively inert and provides an effective barrier against water and ion diffusion, even at a thickness of a few nm. Silica (as used in the examples herein) is also suitable as it is a suitable surface for chemical modification. Alternatively, in certain embodiments, the nanochip may be made wholly or partially from materials that can form sheets as small as one molecule thick (sometimes referred to as single layer materials), such as graphene, such as as described 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 content of each of which is incorporated herein by reference, or a transition metal dichalcogenide such as molybdenum disulfide ( MoS ₂ ) as described in Feng, et al., Identification of single nucleotides in MoS ₂ nanopores, Nat Nanotechnol. (2015) 10(12):1070-1076, the contents of which are incorporated herein by reference, or boron nitride as described in Gilbert, et al. Fabrication of Atomically Precise Nanopores in Hexagonal Boron Nitride, eprint arXiv:1702.01220 (2017).

[062] In some embodiments, the implementation of the nanochip contains a single layer material that is relatively rigid and inert, for example, at least as inert and rigid as graphene, such as MoS ₂ . Single layer materials can be used, for example, as all or part of a membrane containing a nanopore. The nanochip can be partially covered with metal, for example, it can have layers on its walls (for example, metal-silicon-metal), and then the metal can be arranged so that a controlled pair of electrodes is provided in it near the nanopore, so that the nucleic acid can move back and forth through the nanopore under the action of an electromotive force, and could also be cut off by measuring the change in electric potential as the nucleic acid passes through the nanopore.

[063] Nanofluid devices in the form of nanochips for DNA sequencing are well known, for example, as described in 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 full content of each of which is incorporated herein by reference, for example, in relation to their ideas for the design and manufacture of nanochips containing nanopores.

[064] A "nanopore" as used herein is a pore less than 1 micrometer in diameter, eg 2-20 nm in diameter, eg on the order of 2-5 nm. Single-stranded DNA can pass through a 2nm nanopore; single-stranded or double-stranded DNA can pass through the 4nm nanopore. In the case of a very small nanopore, eg 2-5 nm, DNA can pass through it, however, larger protein fragments cannot, thus allowing controlled synthesis of DNA (or other charged polymer). When larger nanopores (or smaller protein enzymes) are used, the protein enzyme can be conjugated to the support, which will prevent it from passing through the nanopore, for example, to a larger molecule, such as a larger protein, to a bead, or to a chamber surface. Various types of nanopores are known. For example, biological nanopores are formed by the assembly of a pore-forming protein in a membrane, such as a lipid bilayer. For example, α-hemolysin and similar protein pores occur naturally in cell membranes where they act as channels for ions or molecules to be transported in and out of cells, and such proteins can be adapted as nanochannels. Solid phase nanopores are formed by synthetic materials such as silicon nitride or graphene, for example, by making holes in a synthetic membrane, for example, using feedback controlled low energy ion beam (IBS) modeling or high energy electron beam illumination. Hybrid nanopores can be made by encapsulating a pore-forming protein in a synthetic material. When a metal surface or electrode is present at either end or either side of the nanopore, an electrical current can be provided through the nanopore in an electrolyte environment. The electrodes can be made from any conductive material, such as silver, gold, platinum, copper, titanium dioxide, such as silver coated with silver chloride.

[065] Methods for forming a solid phase nanopore membrane, for example from silicon nitride, are known. In one approach, a silicon substrate is coated with a membrane material, e.g., silicon nitride, and the overall configuration of the membrane is shaped using photolithography and wet chemical engraving to obtain silicon nitride membranes of the desired size for inclusion in a nanochip, e.g., approximately 25 x 25 micrometers. Initial holes or cavities with a diameter of 0.1 micrometers are punched into the silicon nitride membrane using a focused ion beam (FIB). Ion beam modeling can form a nanopore by shrinking a larger pore, such as through ion beam-induced lateral mass transfer on membrane surfaces, or by removing membrane material with an ion beam peeling layer by layer from the flat side of a membrane containing a cavity on opposite sides, so that when a cavity is eventually formed, the nanopore has sharp edges. When the ion beam is extinguished, then the ion current transmitted through the pore is suitable for the desired pore size. See, for example. 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 illumination in TEM. By using semiconductor processing methods, e-beam lithography, reactive ion etching of SiO2 pattern layers, and anisotropic KOH etching of Si, pyramidal pores of 20×20 nm or more are obtained in a 40 nm thick membrane. The electron beam in TEM is used to downsize larger 20 nm pores to a smaller size. TEM allows real-time observation of the size reduction process. Using a thinner membrane (eg, <10 nm thick), nanopores can be drilled with a high energy focused electron beam in TEM. See especially Storm AJ, et al. Fabrication of solid-state nanopores with single-nanometer 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 content of each of which is included in the present description by reference.

[066] In other embodiments, nanopores are produced using dielectric breakdown using a relatively high potential across the membrane, where the potential rises until a current is detected, e.g., 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.

[067] Using these methods, and depending on the particular method used, and the thickness and composition of the membrane, the final shape of a nanopore in a solid material such as silicon nitride can be broadly similar to two funnels whose tops touch at their narrowest point. , i.e. true nanopore. This shape in the form of two cones allows the polymer to pass through the nanopore and back. Imaging techniques such as atomic force microscopy (AFM) or transmission electron microscopy (TEM), in particular TEM, can be used to confirm and measure the size, position and configuration of nanomembranes, FIB holes or cavities and terminal nanopores.

[068] In some embodiments, the implementation of one end of the polymer, such as DNA, is connected near the nanopore or on the inner wall of the funnel leading into the nanopore. If one end of the polymer is bonded near the nanopore, as the polymer initially approaches the nanopore by diffusion and then moves under the action of an electrical gradient, the movement provided by the gradient is maximized and the diffusion movement is minimized, and thus the speed and efficiency are increased. See, for example, Wanunu M, Electrostatic focusing of unlabelled 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. Nat Nanotechnol. (2007) 2(12): 775-779.

[069] In one embodiment, one end of the polymer, such as DNA, is linked to the bead and the polymer passes through the pore. Bead binding will stop the polymer from moving through the nanopore at the opposite end of the separating membrane in the adjacent chamber. The current is then turned off and the polymer, such as DNA, binds to the surface next to the nanopore, in a chamber on the other side of the separating membrane. For example, in one embodiment, one end of the ssDNA is covalently linked to a 50 nm bead and the other end is biotinylated. The streptavidin is bound to the region at the desired attachment point in the chamber on the other side of the separating membrane. DNA is pulled through the nanopore by an electrical potential, and biotin binds to streptavidin. Binding to the bead and/or surface adjacent to the nanopore can be either through covalent bonds or through strong non-covalent bonds (such as the biotin-streptavidin bond). The pellet is then cut off by the enzyme and washed out. In some embodiments, single-stranded DNA is cleaved with a restriction enzyme that cleaves single-stranded DNA, for example, as described in K. Nishigaki, Type II restriction endonuclease cleave single-stranded DNAs in general. Nucleic Acid Res. (1985) 13(16): 5747-5760, incorporated by reference. In other embodiments, a complementary oligonucleotide is provided to provide a double-stranded restriction site, which can then be cleaved with the appropriate restriction enzyme.

[070] As the polymer passes through the nanopore, the change in electrical potential or current through the nanopore caused by the partial blockage of the nanopore as the polymer passes through can be determined and can be used to identify the sequence of monomers in the polymer, since different monomers can differ in their sizes. and electrostatic potentials.

[071] The use of nanochips containing nanopores in the DNA fabrication method as described herein is not described in the art, however, such chips are well known and commercially available for rapid DNA sequencing. For example, MinION (Oxford Nanopore Technologies, Oxford, UK) is small and can be connected to a laptop computer. Since a single strand of DNA passes through a protein nanopore at a rate of 30 bases per second, MinION measures the electrical current. The DNA strands in the pore disrupt the ion current, resulting in current changes corresponding to the nucleotides in the sequence. Mikheyev, AS, et al. A first look at the Oxford Nanopore MinION sequencer, Mol. ecol. resource. (2014)14, 1097-1102. While the accuracy of MinION is low, requiring repeated sequencing, the speed and accuracy of sequencing using the nanoarrays of the present invention can be greatly improved if the read DNA contains only two readily distinguishable bases, e.g., A and C.

[072] In some embodiments, the nanopore-containing membrane may have a three-layer configuration with a metal surface on each side insulating the core material, such as a silicon nitride membrane. In this embodiment, the metal surfaces are formed, for example, by lithography techniques, to provide a microloop with paired electrodes, one at each end of the nanopore, for example, so that an electrical current can be provided between the electrodes through the nanopore in the electrolyte environment, and this current can be conducted by the polymer. through a nanopore, and when the polarity changed, it could conduct it back. As the polymer passes through the nanopore, the electrodes can measure the change in electrical potential through the nanopore, identifying the sequence of monomers in the polymer.

[073] In some embodiments, a polymer sequence is organized to store data. In some embodiments, data is stored in binary (1s and 0s). In some embodiments, each base corresponds to 1 or 0. In other embodiments, an easily recognizable sequence of two or more bases corresponds to a 1, and another easily recognizable sequence of two or more bases corresponds to a 0. In other embodiments, the data may be stored in a tertiary, quaternary or other code. In a specific embodiment, the polymer is DNA, e.g., single-stranded DNA, where the DNA contains only two types of bases and does not contain any self-hybridizing bases, e.g., where the DNA contains residues of adenine and guanine, adenine and cytosine, thymidine and guanine, or thymidine and cytosine. In some embodiments, one or more additional bases may be inserted between two bases, e.g., A and C may contain T as a "punctuation mark" in the sequence, e.g., indicating a break in the coding sequence, at a frequency that does not result in significant self-hybridization . In other embodiments, for example when the nucleic acid is double stranded, some or all of the available bases may be used.

[074] Nucleotide bases may be natural or, in some embodiments, may consist of or include non-natural bases, for example, as described in Malyshev, D. et al. "A semi-synthetic organism with an expanded genetic alphabet", Nature (2014) 509: 385-388, incorporated herein by reference.

[075] In one embodiment, data is stored by adding single monomers, such as single nucleotides in the case of DNA, to the polymer. In one embodiment, the polymer is DNA and the monomers are adenine (A) and cytosine (C) residues. Residues A and C are advantageous because (i) A and C have a larger difference in size, so their differentiation in the nanopore should be easier, (ii) A and C do not pair with each other, so they do not form a pronounced secondary structure, which could complicate the interpretation of the signal from the nanopore, and (iii) for the same reason, G is less preferred, since it is known to form guanine tetrads. Nucleotides are added by an end transferase (or polynucleotide phosphorylase), however the nucleotides are 3'-locked so that only one nucleotide is added at a time. The block is removed before the next nucleotide is added.

[076] In some embodiments, the implementation of the DNA remains in the nanochip. In other embodiments, it is extracted and optionally converted to double-stranded DNA and/or optionally converted to crystalline form, for example, to improve long-term stability. In other embodiments, the DNA can be amplified and the amplified DNA can be retrieved for long-term storage while the original DNA template, e.g. DNA bound to the chamber wall in the nanochip, can remain in the nanochip where it can be read and/or used in as a template for making additional DNA.

[077] In some embodiments, the DNA or other polymer is anchored to the surface near the nanopore during synthesis. For example, in one embodiment, each single-stranded DNA molecule is connected at the 5' end to a surface near a nanopore, where the electrical current in each nanopore can be independently controlled by electrodes at that nanopore, so that the 3' end of the DNA molecule can be drawn through the nanopore from the holding chamber. into a flow chamber containing a stream of 3'-protected dNTPs along with a polymerase or terminal transferase to attach the 3'-protected dNTPs, or be held in a retention chamber where the nanopore does not allow the enzyme to pass through so that the dNTPs do not attach. See, for example, the images in Figs. 12-16 and also in Figs. 18 and 19. In other embodiments, the implementation of single-stranded DNA is designed to join at the 5' end (with the attached 3' end) using 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:

Stage flow chamber Nanopore 1 Nanopore 2 0 Retention Retention one Stream "A" 2 Moves forward into the flow chamber Attaches "A" Retention 3 Returns to rest chamber; The oligonucleotide protecting group is removed Retention 4 Washing with buffer Retention Retention 5 Stream "C" Direct Attached "C" Direct Connection "C" 6 Reverse Removes the protecting group from the oligonucleotide Reverse Removes the protecting group from the oligonucleotide 7 Retention Direct Attached "C" eight Retention Reverse Removes the protecting group from the oligonucleotide 9 Washing with buffer Retention Retention

Stream A=3'-protected dATP

Stream C=3'-protected dCTP

Nanopore 1 and nanopore 2 in this scheme are associated with different DNA strands, the positions of which (in or outside the flow chambers) are controlled separately. The DNA protecting group can be removed either by a specific enzyme in the retention chamber or by altering the flow in the flow chamber to allow for the removal of the protecting group by enzymatic, chemical, light catalyzed or other means. In one embodiment, flow deblocking agent(s) between cycles of stream A and stream C, such as when the flow chamber is flushed with buffer such that the deblocking agent does not deprotect the nucleotide building blocks. In other embodiments, the deprotecting agent(s) is too bulky to pass into the flow chamber through the nanopores.

[078] The end result in the above example is the attachment of A and C to DNA at nanopore 1, and the attachment of C and C to DNA at nanopore 2.

[079] In another embodiment, the chamber configuration is similar, but with double-stranded DNA anchored to the surface near the nanopore and each of the oligonucleotide fragments, e.g., two or more types corresponding to the binary code, is added sequentially, e.g., using site-specific recombinases , i.e. enzymes that spontaneously recognize and cleave at least one strand of a double strand of nucleic acids with a sequence segment known as a site-specific recombination sequence, for example using topoisomerase-loaded oligonucleotides as described below.

[080] In certain embodiments, it may be desirable to keep an electrically charged polymer, such as DNA, in a condensed state after synthesis. There are several reasons for this:

- the polymer should be more stable in this form,

- polymer condensation will prevent clumping and allow the use of longer polymers in small volumes,

- ordered condensation can reduce the possibility that the polymer forms knots or coils,

- if any of the chambers are interconnected, this will help prevent the polymer from over-elongating and passing into a pore other than intended when an electric current is applied,

- condensation will help keep the polymer away from the electrodes where electrochemistry can damage the polymer.

A human cell is approximately 10 micrometers in size but contains 8 billion base pairs of DNA. In the extended state, they will stretch to a meter. DNA fits into the cell because it is wrapped around histone proteins. In certain embodiments, histones or similar proteins provide a similar function in the nanoarrays of the invention. In some embodiments, the internal surfaces of the nanochips are weakly positively charged, such that an electrically charged polymer, such as DNA, tends to be weakly attached to them.

[081] In certain embodiments, a charged polymer, such as single-stranded or double-stranded DNA, is bound to a surface near the nanopore. This can be done in various ways. Typically, the polymer is localized near the nanopore by associating the polymer with a relatively bulky structure (e.g., bead, protein, or DNA origami structure (described below) having a diameter too large to fit into the nanopore, e.g., >10 nm, e.g., about 20 -50 nm), drawing the charged polymer through the nanopore using an electric current, anchoring the end of the polymer, distal to the bulk structure, on the surface next to the nanopore, and splitting off the bulk structure.

[082] The step of anchoring the end of the polymer distal to the bulk structure at the surface adjacent to the nanopore can be carried out in various ways. In one embodiment, the polymer is single-stranded DNA and there are pre-linked DNA strands (approximately 50 bp) that are complementary to parts of the single-stranded DNA such that the single-stranded DNA and the pre-linked DNA strands can come together via base pairing. If the mating is strong enough, it will be enough to keep the DNA anchored even during manipulation. The advantage of this attachment method is that it allows removal of DNA from the nanopore chip if desired for long-term DNA storage. Alternatively, the chain is attached to the surface covalently, either using conjugation chemistry, e.g., streptavidin-biotin conjugation as described in Example 1 below, or click chemistry (see Kolb, et al. Angew. Chem. Int. Ed. (2001) 40: 2004-2021, incorporated herein by reference) and/or using enzymatic attachment, for example by first covalently linking oligonucleotides to the distal surface and then using DNA ligase to join them.

[083] After binding the distal end of the chain to the surface near the nanopore, the bulk structure is cleaved, for example, using a restriction endonuclease that cleaves at the restriction site near the bulk structure.

[084] The bulk structure can be a granule, a bulky molecule, such as a protein that reversibly binds to a DNA strand, or a DNA origami structure. DNA origami involves the use of base pairing to form three-dimensional DNA structures. DNA origami techniques are generally described in Bell, et al, Nano Lett. (2012)12: 512-517, incorporated herein by reference. For example, within the scope of the present invention, DNA origami can be used to bind a single DNA molecule to a surface adjacent to a nanopore. In one embodiment, this structure is a "honeycomb block", for example, with each side measuring approximately 20 nm. This partially prevents the passage of this part of the DNA through the nanopore (similar to that in the mentioned article). A long DNA chain (single-stranded or double-stranded) is associated with the origami structure. The DNA strand travels through the nanopore until the origami block hits the nanopore and blocks further progress. Then the electric current is turned off and the circuit is connected to the surface next to the nanopore.

[085] In another embodiment, an electrically charged polymer, such as DNA, with an origami structure is in the middle chamber of a three-chamber configuration. Origami keeps the DNA from entering the other 2 chambers entirely (or the other chamber in the case of the 2 chamber example). Thus, in this example, the polymer should not be anchored to the surface. This reduces the risk of polymer entanglement and eliminates the need for the step of bonding one end of the polymer to the surface and cleaving off the bulk portion at the other end. The volume of the origami chamber should be as small as possible to be practical, so that the polymer remains relatively close to the pore, which will help ensure that it moves quickly when the electric current is applied. It should be noted that while the middle chamber containing the origami part of the polymer cannot be connected to other middle chambers (or otherwise different polymers will mix), other chambers (or groups of chambers in the 3 chamber example) can be connected. These other chambers may be larger if desired, since the polymer will necessarily be near the pore (actually, some of it will be in the pore) when the DNA moves into this chamber.

[086] In some embodiments, the apparatus comprises three chambers in line, where the attachment chambers are in series to allow flow and have common electrodes, while the "deprotection" chambers are liquid-sealed except for flow through nanopore and have unique electrodes.

[087] In other embodiments, the DNA or other charged polymer is not anchored, but can move between the synthesis chamber(s) and the deprotection chamber(s) under the control of the electrodes in the chambers, while the movement of the polymerase and agents deprotecting groups between chambers is limited because they are too bulky to pass through the nanopores connecting the chambers and/or are anchored to the surface of the chamber. See, for example, figures 1-9 and 16-17.

[088] The electric current required to move a charged polymer through a nanopore depends on, for example, the nature of the polymer, the size of the nanopore, the material of the membrane containing the nanopore, and the salt concentration, and thus, if necessary, it is optimized for a particular system. In the case of DNA, as used in the examples herein, examples of voltage and current are, for example, 50-500 mV, typically 100-200 mV, and 1-10 nA, such as about 4 nA, with salt concentrations on the order of 100 mM to 1 M.

[089] The movement of a charged polymer, such as DNA, through a nanopore is typically very fast, such as 1 to 5 µs per base, thus on the order of a million bases per second (1 MHz according to the frequency nomenclature), making it difficult to distinguish a correct read from interference in system. Using the methods of the present invention, either (i) the nucleotide must be repeated sequentially, e.g., 100 consecutive times, to provide a measurable signature change, or (ii) protein pores such as alpha-hemolysin (αHL) based, or porin A Mycobacterium smegmatis (MspA) that provide a relatively long pore with the ability to multiple read as the base moves through the polymer, and in some cases, they can be adapted to provide controlled feeding of DNA through the pore one base at a time, in some cases with using an exonuclease to cleave each base as it passes through. Various approaches are possible, for example,

- slowing down the polymer velocity from about 1 MHz to about 100-200 Hz, for example using a medium containing an electrorheological fluid that becomes more viscous when voltage is applied, thereby slowing down the rate of passage of the polymer through a nanopore, or plasmonic fluid system, where the viscosity of the medium can controlled by light; or molecular motor or latch;

- providing a sequence in a polymer, e.g., in single-stranded DNA, that forms a bulky secondary structure, e.g., a "hairpin", "hammerhead" or "dumbbell" configuration, which must be linearized to pass through the nanopore, thus making the information less dense and providing a signal having a higher duration;

- providing multiple reads of the same sequence, e.g. using a rapidly changing current, allowing multiple readings for one loop of the sequence, and in combination with short forward current pulses to pull the molecule up to the next loop of the sequence, by reading the entire sequence multiple times or by reading a plurality of identical sequences in parallel, in each case correlating the read data to provide a consensus read sequence that amplifies the signal;

- measuring the change in the impedance of the high frequency signal induced by the change in capacitance as the monomers (eg nucleotides) pass through the nanopore, instead of measuring changes directly in electric current or resistance;

- amplifying differences in current, resistance, or capacitance between different bases, for example, by using non-natural bases that have a larger size difference or otherwise modified to generate different signals, or by forming larger secondary structures in DNA, such as the "hairpin" configuration ", "hammer heads" or "dumbbells", which provide an enhanced signal due to their larger size;

- use of an optical readout system, for example using an integrated optical antenna next to the nanopore, which acts as an optical converter (or optical signal amplifier), supplementing or replacing the standard ion current measurement, for example, as 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, the monomers, such as DNA nucleotides, are labeled with fluorescent dyes such that each individual monomer fluoresces at a specific intensity as it passes through the junction of the nanopore and its optical antenna. In some embodiments, fluorescent labels are stripped off the solid phase nanopore, resulting in a series of detectable photon pulses as the polymer passes through the nanopore at high speed, for example, as described 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.

[090] In one embodiment, the charged polymer is a nucleic acid, such as single stranded DNA, where the sequences provide the secondary structure. Bell, et al., Nat Nanotechnol.(2016)11(7):645-51, incorporated herein by reference, describes the use of a relatively short sequence of dumbbell configurations detectable in a solid-phase nanopore format to label antigens in an immunoassay. . The nanopores used by Bell, et al. were relatively large so that the entire dumbbell structure could pass through the pore, but when using nanopores smaller than the diameter of the dumbbell configuration, the DNA "unpacks" and becomes linearized. You can use more complex configurations, for example, where each bit corresponds to a sequence similar to tRNA (see, for example, Henley, et al. Nano Lett. (2016)16: 138-144, incorporated herein by reference). Thus, the invention relates to charged polymers, eg single stranded DNA, having at least two types of secondary structure, where the secondary structure encodes data (eg, binary data, where one type of secondary structure is 1 and the second 0). In other embodiments, secondary structures are used to slow down the passage of DNA through a nanopore or to introduce breaks in the sequence, making it easier to read the sequence.

[091] In another embodiment, the invention uses a DNA molecule containing a series of at least two different DNA motifs, where each motif specifically binds to a specific ligand, for example, a gene regulatory protein for double-stranded DNA or tRNA for single-stranded DNA, where at least two different DNA motifs encode information, e.g., in binary, where one motif is a 1 and the second is a 0, e.g., where the ligand amplifies a signal difference (e.g., change in current or capacitance) through the nanopore as the DNA passes through the nanopore .

[092] As described above, when various monomers pass through the nanopore, they affect the current through the nanopore, mainly by physically blocking the nanopore and changing the conductivity through the nanopore. In existing nanopore systems, this change in current is measured directly. The problem of modern reading systems is that there is significant noise in the system and in the case of DNA, for example, when determining current fluctuations as various nucleotide elements pass through a nanopore, a relatively long integration time, on the order of one hundredth of a second, is required to accurately detect differences between different monomers, for example between different bases. It has recently been shown that changes in impedance and capacitance can be useful for studying cells and biological systems, despite the possibility of complex interactions with salts and biological molecules. 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 conditions of salts and for imaging microparticles and living cells beyond the Debye limit.

[093] In one embodiment of the invention, therefore, a change in capacitance is measured, rather than a change in direct current, for example, where the sequence of a charged polymer is identified by measuring the change in phase of the RF signal induced by a change in capacitance as monomers (e.g., nucleotides) pass through through the nanopore.

[094] Simply put, capacitance exists in any circuit where there is a gap between one electrical conductor and another. While the current directly changes with the change in capacitance, it does not change simultaneously with the capacitance. For example, if you plot current and voltage over time in a capacitive AC circuit, you can see that although both current and voltage form a sine wave, the waves are out of phase. When there is a change in current, there is a change in capacitance, which is reflected by a change in the phase of the signal. RF AC provides a signal with a fixed frequency and amplitude, while the phase of the signal varies depending on the capacitance of the loop. In the system developed by the inventors, they use a pulsating direct current instead of alternating current (i.e. the voltage varies between two values, however the voltage does not cross the "zero" line, so that the polarity is maintained and one electrode remains positive and the other negative), so that the charged polymer can be drawn in through the nanopore (toward the positive electrode in the case of DNA). When there is nothing in the nanopore, the capacitance has a single value that changes as different polymer monomers pass through the nanopore. Suitable frequency ranges are in the radio frequency range, eg 1 MHz to 1 GHz, eg 50-200 MHz, eg approximately 100 MHz, eg below higher microwave frequencies which can cause significant dielectric heating of the medium. To reduce the potential for interference, different frequencies can be used for different nanopores so that multiple nanopores can be measured simultaneously with a single RF input line.

[095] Measuring changes in impedance (due to, for example, changes in capacitance) at high frequencies improves the signal-to-noise ratio available in a certain amount of time, as this reduces the effects of 1/f or "pink" noise inherent in electronic measurement circuits. The use of a high frequency signal improves the signal-to-noise ratio as many measurements are taken over a given amount of time, providing a more stable signal that is well distinguishable from impedance changes due to external or device-induced changes and fluctuations.

[096] Using these principles for the present invention, in one embodiment, the invention relates to a method for measuring the change in the impedance of a high frequency signal induced by a change in capacitance as monomers (e.g., nucleotides) pass through a nanopore, for example, a method for reading the monomer sequence of a charged polymer, containing at least two different types of monomers, e.g. DNA molecules, including the use of radio frequency pulsating direct current, for example, with a frequency of 1 MHz to 1 GHz, for example 50-200 MHz, for example, approximately 100 MHz, through a nanopore, where the pulsating direct current draws a charged polymer through the nanopore and the sequence of monomers is read by measuring the change in capacitance through the nanopore as the charged polymer passes through the nanopore.

[097] In some embodiments, the invention relates to a nanochip for sequencing an electrically charged polymer, such as DNA, containing at least two different monomers, and the nanochip contains at least first and second reaction chambers, each of which contains an electrolyte environment and is separated by a membrane, containing one or more nanopores, where a pair of electrodes (e.g. in the form of opposing plates) connected to the circuit are located on each side of the membrane containing one or more nanopores, and the electrodes are separated by a distance of 1-30 micrometers, for example, approximately 10 micrometers, so that the gap between the electrodes has a capacitance when applied to the electrodes of an RF pulsed direct current, for example from 1 MHz to 1 GHz, so that the electrically charged polymer is drawn through the nanopore, for example, from one chamber to the next, and so that the phase of the pulsed direct RF current changes when capacitance as the passage of the electr of the electrically charged polymer through the nanopore, thereby allowing the detection of the sequence of electrically charged polymer monomers. In certain embodiments, the nanochip comprises a plurality of sets of reaction chambers, where the reaction chambers in the set are separated by a membrane having one or more nanopores, and the groups of reaction chambers are separated by a shielding layer to minimize electrical interference and allow them to be sequenced in parallel.

[098] For example, in one embodiment, the electrodes form the top and bottom plates of a capacitor that is part of an oscillating circuit, and the change in capacitance is measured as DNA passes through the pore between the plates.

[099] In certain embodiments, the implementation of the nanochip further contains reagents for the synthesis of polymer, such as DNA, for example, according to nanochip 1, etc., below.

[0100] In one embodiment, the invention thus relates to a method (Method 1) for synthesizing a charged polymer [e.g., a nucleic acid (e.g., DNA or RNA)] containing at least two different monomers, in a nanochip, wherein the nanochip contains

one or more attachment chambers containing reagents for attaching one or more monomers [e.g., nucleotides] or oligomers [e.g., oligonucleotides] to a charged polymer, in a buffer solution in end-protected form, such that only one monomer or oligomer can be attached per one reaction cycle; and

one or more reserve chambers containing a buffer solution, but not all of the reagents needed to attach one or more monomers or oligomers,

where the chambers are separated by one or more membranes containing one or more nanopores, and

where the charged polymer can pass through the nanopore, but at least one of the reagents for attaching one or more monomers cannot,

wherein the method includes

a) moving the first end of the charged polymer, having the first end and the second end, under the influence of electrical attraction into the attachment chamber, and the monomers and oligomers are attached to the specified first end in a blocked form,

b) moving the first end of the charged polymer with the added monomer or oligomer in blocked form to the reserve chamber,

c) deblocking the attached monomer or oligomer, and

d) repeating steps a-c, where the monomers or oligomers attached in step a) are the same or different, until the desired polymer sequence is obtained.

[0101] For example, the invention relates to the following

1.1. Method 1 wherein the polymer is a nucleic acid, eg where the polymer is DNA or RNA, eg where it is DNA, eg dsDNA or ssDNA.

1.2. Any of the above methods, where the second end of the polymer, such as a nucleic acid, is either protected or bonded to a support adjacent to the nanopore.

1.3. Any of the above methods, where electrical attraction is provided by applying an electrical potential between the electrodes in each chamber, where the polarity and current between the electrodes can be controlled, for example, so that the nucleic acid is attracted to the positive electrode.

1.4. Any of the above methods, where the polymer is a nucleic acid and

(i) said first end of the nucleic acid is the 3' end, the attachment of nucleotides occurs in the 5' to 3' direction and is catalyzed by a polymerase, for example, where the passage of the polymerase is difficult (for example, due to its size or due to binding to a substrate in the first chamber ) through the nanopore, the nucleotides are 3'-protected upon attachment and after the attachment of the 3'-protected nucleotide to the 3'-end of the nucleic acid, the 3'-protecting group on the nucleic acid is removed, for example, in a reserve chamber; or

(ii) said first end of the nucleic acid is the 5' end, the attachment of nucleotides is in the 3' to 5' direction, the nucleotides are 5' protected upon attachment and after attachment of the 5' protected nucleotide to the 5' end of the nucleic acid The 5'-protecting group is removed, for example, in the second chamber; (e.g., where the phosphate on the 5'-protected nucleotide is a nucleoside phosphoramidite linked via a 5'-protecting group to a bulky group that cannot pass through the nanopore so that upon binding to the nucleic acid, unreacted nucleotides are washed out, bulky 5' - the protecting group is cleaved from the nucleic acid and washed out, and the 5' end of the nucleic acid can move into the reserve chamber);

where the attachment of nucleotides to the nucleic acid is controlled by moving the first end of the nucleic acid into and out of one or more attachment chambers and the cycle continues until the desired sequence is obtained.

1.5. Any of the above methods, where the sequence of monomers or oligomers in the polymer [eg, the sequence of nucleotides in the nucleic acid] thus synthesized corresponds to the binary code.

1.6. Any of the above methods, where the polymer thus synthesized is a single stranded DNA.

1.7. Any of the above methods where the sequence of a polymer [eg, nucleic acid] is verified during synthesis by sequencing monomers or oligomers [eg, nucleotide bases] as they pass through a nanopore to identify sequencing errors.

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

1.9. Any of the above methods where a plurality of polymers [e.g. oligonucleotides] are synthesized independently in parallel such that polymers [oligonucleotides] having different sequences are obtained by controlling whether they are present in one or more attachment chambers or one or more reserve chambers.

1.10. Any of the above methods, where at least two attachment chambers are present, which contain reagents suitable for attachment of different monomers or oligomers, for example, different nucleotides, for example, where there is one or more attachment chambers, containing reagents suitable for attachment of the first monomer or oligomer, and one or more attachment chambers containing reagents suitable for attachment of a second different monomer or oligomer, for example, where there is one or more attachment chambers containing reagents suitable for attachment of adenine nucleotides, and one or more chambers for attachments containing reagents suitable for attachment of cytosine nucleotides.

1.11. Any of the above methods, where at least one attachment chamber is a flow chamber providing a flow cycle, including (i) feeding into the flow chamber reagents suitable for attaching the first monomer or oligomer, (ii) washing, (iii) feeding into a flow chamber of reagents suitable for attaching a second different monomer or oligomer, and (iv) washing and repeating the cycle to completion of the synthesis, where the sequence of monomers or oligomers in the polymer is controlled by entering or exiting the first end of the polymer from the flow chamber in step (i) or (iii) each cycle;

1.12. Any of the above methods, where the polymer is DNA and at least one attachment chamber is a flow chamber providing a flow cycle comprising (i) feeding into the flow chamber reagents suitable for attachment of the first type of nucleotide, (ii) washing, ( iii) feeding into the flow chamber reagents suitable for attachment of the second type of nucleotide, and (iv) washing and repeating the cycle until the synthesis is completed, where the sequence of monomers or oligomers in the polymer is controlled by the presence or absence of the first end of the DNA (for example, the 3' end) in the flow chamber.

1.13. Any of the above methods, where the polymer is DNA and at least one attachment chamber is a flow chamber providing a flow cycle comprising (i) feeding reagents suitable for attachment of the first type of nucleotide into the flow chamber, (ii) washing, ( iii) feeding into the flow chamber reagents suitable for attachment of the second type of nucleotide, and (iv) washing, (i) feeding into the flow chamber reagents suitable for attachment of the third type of nucleotide, (ii) washing, (iii) feeding into the flow chamber of reagents suitable for attachment of a fourth type of nucleotide, and (iv) washing, and repeating the cycle until the synthesis is complete, where the sequence of monomers or oligomers in the polymer is controlled by the presence or absence of the first DNA end (for example, the 3' end) in the flow chamber, when the reagents, suitable for attachment of various types of nucleotides are present.

1.14. Any of the above methods, where the polymer is DNA and the nanochip contains two attachment chambers, which are flow chambers: (a) a first flow chamber providing a flow cycle, including (i) feeding reagents suitable for the first type of attachment into the flow chamber nucleotide, (ii) washing, (iii) feeding into the flow chamber reagents suitable for attaching the second type of nucleotide, and (iv) washing and repeating the cycle until the synthesis is completed, and (b) a second flow chamber providing a flow cycle including ( i) feeding the flow chamber with reagents suitable for attaching a third nucleotide type, (ii) washing, (iii) feeding the flow chamber with reagents suitable for attaching a fourth nucleotide type, and (iv) washing, and repeating the cycle until the synthesis is complete, where nucleotides are selected from dATP, dTTP, dCTP and dGTP and where the sequence is controlled by directing the first end of the DNA (e.g. 3' end) into the proto another chamber where the next desired nucleotide is provided.

1.15. Any of the above methods wherein the polymer is DNA and the nanopore chip comprises one or more dATP attachment chambers, one or more dTTP attachment chambers, one or more dCTP attachment chambers, and one or more dGTP attachment chambers.

1.16. Any of the above methods, where each synthesized polymer [for example, nucleic acid] is connected through its second end to the surface near the nanopore.

1.17. Any of the above methods, where the sequence of the polymer [eg, nucleic acid] is determined after each cycle by detecting changes in electrical potential, current, resistance, capacitance and/or impedance as the polymer passes through the nanopore.

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

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

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

1.21. Any of the above methods, where the polymer is a nucleic acid, further comprising amplification and extraction of copies of the synthesized nucleic acid using a suitable primer and polymerase (for example, Phi29).

1.22. Any of the above methods, where 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, further comprising amplifying the thus synthesized nucleic acid.

1.24. Any of the above methods, further comprising removing the polymer [eg, nucleic acid] from the nanochip and crystallizing the polymer.

1.25. Any of the above methods, where the polymer is a nucleic acid, further comprising stabilizing the nucleic acid, for example, by drying a solution containing the nucleic acid with one or more of a buffer (for example, a borate buffer), an antioxidant, a humectant, for example, a polyol, and optionally a chelator, for example as described in US 8283165 B2, incorporated herein by reference; or by forming a matrix between the nucleic acid and a polymer such as a polyethylene glycol-poly(l-lysine) (PEG-PLL) type AB block copolymer; or by attaching a complementary strand of nucleic acid or protein that binds DNA.

1.26. Any of the above, including:

(i) reacting the nucleic acid with the 3' protected nucleotide in the attachment chamber in the presence of a polymerase that catalyzes the attachment of the 3' protected nucleotide to the 3' end of the nucleic acid;

(ii) drawing at least the 3' end of the 3' protected nucleic acid thus obtained from the attachment chamber through the at least one nanopore into a reserve chamber where passage of the polymerase through the nanopore is hindered (e.g., due to its size or due to binding to the substrate in the first chamber);

(iii) removing the protecting group from the 3'-protected nucleic acid, for example, chemically or enzymatically; and

(iv) if it is desired to attach an additional 3'-protected dNTP to the oligonucleotide, pull the 3' end of the oligonucleotide into the same or different attachment chamber to repeat steps (i)-(iii), or if this is not desired, provide a 3 the '-end of the nucleic acid in the reserve chamber until the next cycle, where the desired 3'-protected dNTP enters the attachment chamber; and

(v) repeating the cycle of steps (i)-(iv) until the desired nucleic acid sequence is obtained.

1.27. Any of the above methods, where the polymer is single-stranded DNA (ssDNA) and one or more nanopores have a diameter that allows passage of ssRNA, but double-stranded DNA (dsDNA), for example, a diameter of approximately 2 nm.

1.28. Any of the above methods wherein the monomer is a 3'-protected nucleotide, e.g. 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 above methods, where the polymer is a nucleic acid and the attachment of the nucleotide to the nucleic acid is catalyzed by a polymerase, e.g., a template-independent polymerase, e.g., a terminal deoxynucleotidyl transferase (TdT), or a polynucleotide phosphorylase, e.g. end of DNA.

1.30. Any of the above methods, where the membrane contains a plurality of nanopores and a plurality of polymers, each of which is associated with a surface near the nanopore, for example, a plurality of nucleic acids, each of which is associated through its 5'end with a surface near the nanopore.

1.31. Any of the above methods, where a plurality of polymers, each of which is associated with a surface near the nanopore, for example, a plurality of nucleic acids, each of which is 5'-linked to the surface near the nanopore, are synthesized independently, where each nanopore has an associated pair of electrodes, where one electrode in the pair is located near one end of the nanopore, and the other electrode is located near the other end of the nanopore, so that each polymer can independently move between the first and second chambers under the influence of an electric current provided by a pair of electrodes.

1.32. Any of the above methods, wherein the polymer is a 3'-protected nucleic acid 5'-linked to a surface near the nanopore, and the 3'-terminus of the 3'-protected nucleic acid is drawn through the nanopore using electrical force, e.g. force applied through the electrode into the adjacent chamber.

1.33. Method 1.20, where the new 3'-protected dNTP is the same or different from the first 3'-protected dNTP.

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

1.35. Any of the above methods where the polymer is a nucleic acid and deprotection of the nucleic acid is carried out by an enzyme that removes the 3' protecting group on ssDNA but not on the 3' protected dNTP.

[0101] For example, the invention relates to a method for synthesizing a nucleic acid in a nanochip comprising at least a first chamber and a second chamber separated by a membrane containing at least one nanopore, wherein the synthesis is carried out in a buffer solution by a loop of nucleotide addition to the first end of the nucleic acid having a first end and a second end, wherein the first end of the nucleic acid is moved by electrical attraction between one or more attachment chambers (which contain reagents capable of nucleotide attachment) and one or more spare chambers (which do not contain reagents necessary for nucleotide attachment ), the chambers being separated by one or more membranes, each containing one or more nanopores, where the nanopore is large enough to allow the passage of the nucleic acid, but too small to allow the passage of at least one reagent required for attachment i nucleotide, for example, where mode matches any of mode 1, etc.

[0102] In certain embodiments, the polymer sequence corresponds to a binary code, for example, where the polymer is a nucleic acid and the sequence corresponds to a binary code, where each bit (0 or 1) corresponds to a base, for example, A or C.

[0103] In certain embodiments, the polymer is DNA.

[0104] In some other embodiments, each bit corresponds to a short sequence of monomers rather than a single monomer. For example, in one such embodiment, blocks of DNA are synthesized, where each block generates a unique signal through the nanopore and corresponds to zero or one. This embodiment has certain advantages in that single nucleotides are more difficult to detect in nanopores, especially in solid phase nanopores, so that the use of blocks is less prone to read errors, although the density of information in the polymer is correspondingly reduced.

[0105] For example, blocks of (double-stranded) nucleotides can be added using site-specific recombinases, ie. enzymes that spontaneously recognize and cleave at least one strand of a double strand of nucleic acids in a sequence segment known as a site-specific recombination sequence. In one such embodiment, the site-specific recombinase is a topoisomerase used to ligate a topoisomerase-coupled dsDNA oligonucleotide block to the sequence. These oligonucleotides themselves do not have a structure compatible with further ligation unless they are cleaved with a restriction enzyme. Vaccinia topomerase I specifically recognizes the DNA sequence 5'-(C/T)CCTT-3'. Topoisomerase binds to double-stranded DNA and cleaves it at the 5'-(C/T)CCTT-3' cleavage site. It should be noted that the cleavage is incomplete because the topoisomerase cleaves only one DNA strand (although the presence of a single strand near the break causes some double-strand break), and upon cleavage, the topoisomerase covalently binds to the 3'-phosphate of the 3'-nucleotide. The enzyme then remains covalently bound to the 3' end of the DNA and can either re-relate the covalently retained strand at the same bond that was originally cleaved (as occurs during DNA relaxation) or it can re-ligate to a heterologous acceptor DNA having compatible overhangs, creating a recombinant molecule. In this embodiment, dsDNA-based donor oligonucleotides (e.g., containing one of at least two different sequences, one for "0" and the other for "1"), flanked by a topoisomerase recombination site and a restriction site that creates a ligation of topoisomerase. The cassettes are loaded with topoisomerase; those. they are covalently linked to the topoisomerase, which will bind them to the topoisomerase ligation site on the receiving oligonucleotide. When the growing host DNA strand is cleaved by a restriction enzyme, it becomes capable of ligation with the topoisomerase-loaded cassette. So it is only necessary to sequentially cycle the growing DNA from the restriction enzyme to the loaded topoisomerase cassette, with a different donor oligonucleotide added at each cycle. A similar approach is described for cloning, see, for example, Shuman S., Novel approach to molecular cloning and polynucleotide synthesis using vaccinia DNA topoisomerase. J Biol Chem. (1994); 269(51):32678-84, the contents of which are incorporated by reference.

[0106] Single bases can be attached using a similar strategy. In the presence of an appropriate "deprotected" single-stranded "acceptor" DNA, the topoisomerase-loaded DNA is enzymatically and covalently ligated ("attached") to the acceptor topoisomerase, which is removed from the DNA in this process. The type IIS restriction enzyme can then cleave all of the added DNA except for one base (the base that is "added"). This deprotection-addition process can be repeated to add additional bases (bits). As demonstrated in the examples of the present description, it is possible to use a combination of topoisomerase/restriction enzyme type IIS to attach a single nucleotide to the 5' end of a single stranded DNA target. The use of a type IIS restriction enzyme allows cleavage of DNA at a position different from that of 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 residues (which acts as a "universal base" and pairs with any other base) in this system allows this reaction to proceed without any specific target DNA sequence requirements. The nucleotide attached to the single-stranded target DNA is the 3'-nucleotide to which the vaccinia topoisomerase is conjugated via a 3'-phosphate. Because the vaccinia virus topoisomerase recognition sequence is (C/T)CCTT, the inventors used this system to attach "T" to the target DNA. There is a related topoisomerase, SVF, that can be used to recognize the CCCTG sequence (https://www.ncbi.nlm.nih.gov/pubmed/8661446). So SVF can be used to append "G" instead of "T". When paired with vaccinia topoisomerase, binary data can be encoded with T and G.

[0107] In another approach for attaching one base on the 5'-phosphate, a blocking group is provided to ensure that one base is added in the 3' to 5' direction. The loading response provides loading of the topoisomerase T (or G, or other nucleotide as needed) having a 5'-phosphate group. When the loaded topoisomerase "sees" a free 5'-unblocked (non-phosphorylated) single strand of DNA, it adds a T to that strand, providing DNA with a T added to the 5' end. This attachment is facilitated by the presence of adapter DNA having sequences to which topoisomerase and single strand acceptor DNA can bind (note that adapter DNA is catalytic - it can be reused as a template in repeated reactions). The attached nucleotide has a 5'-phosphate on it, so it will not be a substrate for further attachment until exposed to phosphatase, which removes the 5'-phosphate. This process is repeated using vaccinia topoisomerase to attach one "T" to the 5' end of the single-stranded DNA target and SVF topoisomerase to attach one "G", thus allowing the construction of a sequence encoding binary information by T and G. Others topoisomerases can be used to attach A or C, although this reaction is less efficient.

[0108] One advantage of using a topoisomerase-mediated strategy is that the monomer is covalently bound to the topoisomerase, and thus cannot "escape" to interfere with other reactions. When a polymerase is used, the monomers can diffuse, so the polymerases and/or deblocking agents must be specific (eg, selective for A versus C, for example) or alternatively the monomers are provided via a stream so that they do not have the opportunity to mix.

[0109] In one aspect, the invention relates to a topoisomerase loaded with one nucleotide, i.e. to a topoisomerase conjugated to a single nucleotide, eg, wherein the topoisomerase is conjugated via the 3'-phosphate of the nucleotide and the nucleotide is protected, eg, phosphorylated, at the 5' position.

[0110] In another aspect, the invention relates to a method (Method A) for synthesizing a DNA molecule using topoisomerase-mediated ligation by adding single nucleotides or oligomers to a DNA strand in a 3' to 5' direction, comprising (i) reacting a DNA molecule with a topoisomerase, loaded with the desired nucleotide or oligomer, where the nucleotide or oligomer is blocked from further attachment at the 5' end, then (ii) deblocking the 5' end of the DNA thus obtained and repeating steps (i) and (ii) until the desired nucleotide sequence will not be obtained, for example:

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

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

A1.3. Any of the above methods, including the provision of ligase and ATP to repair breaks in the DNA [note: ligation with topoisomerase ligates only one strand].

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

A1.5. Any of the above methods wherein the restriction enzyme is a type IIS restriction enzyme that can cleave all of the attached DNA except for one base (the base that is "attached").

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 where vaccinia topoisomerase (which recognizes (C/T)CCTT) is used to attach dTTP nucleotides and SVF topoisomerase I (which recognizes CCCTG) is used to attach dGTP nucleotides, e.g. to provide a binary code.

A1.8. Any of the above methods, where the DNA is double-stranded and the spare chamber additionally contains ligase and ATP to repair the DNA strand not bound to topoisomerase.

A1.9. Any of the above methods, including the use of a topoisomerase inhibitor to inhibit the binding and activity of free topoisomerase in relation to the DNA oligomer, for example, where the inhibitors are selected from novobiocin and coumermicin.

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

A1.11. Any of the above methods, where the topoisomerase adds one base, however, the restriction enzyme cleaves at a position that is one nucleotide in the 5' direction from the base attached by the topoisomerase.

A1.12. Any of the above methods, wherein the DNA strand thus provided has a sequence containing the dinucleotide sequence "TT" and "TG".

A1.13. Any of the above methods where the DNA is single stranded.

A1.14. Any of the above methods, where the DNA is double-stranded.

A1.15. Any of the above methods, where the DNA is on a support or magnetic bead, where it can be selectively exposed to or removed from reagents, if necessary, to provide the desired sequence.

A1.16. Any of the above methods, wherein some or all of the DNA addition or deblocking reagents are provided via flow and removed by washing.

A1.17. Any of the above methods wherein the attachment of single nucleotides or oligomers to the single stranded DNA is facilitated by the presence of an adapter DNA having sequences to which topoisomerase and the single stranded acceptor DNA can bind.

A1.18. Any of the above methods carried out in a system where the nanopore separates the chamber containing the topoisomerase from the chamber containing the phosphatase or restriction enzyme, where the nanopore allows the movement of DNA by electrical attraction, but does not allow the movement of enzymes, for example, as described for any of the methods 2 etc.

[0111] One possible problem is that poly-G sequences can form G-quatet secondary structures. By shifting the restriction enzyme back one base (5' from the topoisomerase sequence) and following a similar topoisomerase/IIS strategy, "TT" or "TG" can be attached, each of which can correspond to a different bit. Although it requires 2 bases to encode a bit, it has the advantage of avoiding poly-G sequences. In other embodiments, other bases at the 3' end of the topoisomerase recognition sequence - although less efficient than (C/T)CCTT - may allow poxvirus topoisomerase conjugation to (C/T)CCTA, (C/T)CCTC and (C/T)CCTG (https://www.ncbi.nlm.nih.gov/pubmed/17462694). Protein engineering/selection techniques can also be used to increase the efficiency of these reactions, and similar approaches can be used to attach non-canonical bases.

[0112] In certain embodiments, a method for synthesizing DNA using this method includes treating DNA with a ligase and ATP. Topoisomerase joins only one side of the DNA (the other is essentially broken). The ligase repairs the gap and ensures that the topoisomerase itself does not cut the reaction product again and cleave it.

[0113] In certain embodiments, the method includes using a topoisomerase inhibitor to inhibit free topoisomerase binding and activity to the DNA oligomer. Suitable inhibitors include novobiocin and comermicin. It should be noted that complete inhibition is undesirable because low levels of topoisomerase activity can help "relax" coiled DNA, which is especially useful in the synthesis of long DNA strands.

[0114] Thus, in another embodiment, the invention relates to a method (Method 2) for synthesizing DNA in a nanochip containing one or more attachment chambers containing a topoisomerase-loaded oligonucleotide (i.e., an oligonucleotide 3'-linked to a topoisomerase) , and one or more reserve chambers containing a restriction enzyme and a deblocking agent, for example, phosphatase, and these chambers also contain a compatible buffer solution and are separated by a membrane containing at least one nanopore where the passage of topoisomerase and restriction enzyme is difficult (for example, because they are too large and/or because they are associated with the substrate in the first and second chambers, respectively), the synthesis is carried out in one cycle of attachment of single nucleotides or short oligonucleotide blocks to the first end of the nucleic acid having a first end and a second end, where the first end of the nucleic acid moves under the influence of electrical attraction between the connection points and a back-up camera, for example in one embodiment as follows:

(i) driving the 5' end of the host DNA (eg, double-stranded DNA) into the first chamber for attachment under the action of an electrical force,

(ii) providing in the first attachment chamber a topoisomerase-loaded donor oligonucleotide, wherein the donor oligonucleotide contains a topoisomerase binding site, an information sequence (e.g., selected from at least two different nucleotides or sequences, e.g., where one sequence corresponds to "0" and the other corresponds to "1" in binary) and a restriction site which, when cleaved with a restriction enzyme, provides a topoisomerase ligation site;

(iii) allowing sufficient time to ligate the donor oligonucleotide and thereby extend the recipient DNA;

(iv) advancing the 5' end of the recipient DNA thus extended into the reserve chamber by electrical force, such as so that the restriction enzyme cleaves the recipient DNA, providing a topoisomerase ligation site, or in the case of a single nucleotide addition, a deblocking agent, for example phosphatase, forms a 5'-unblocked nucleotide on single-stranded DNA; and

(v) repeating steps (i)-(iv) of the cycle by adding oligonucleotides having the same or different sequence information until the desired DNA sequence or sequences is obtained.

[0115] For example, the invention relates to the following

2.1. Method 2, wherein the 3' end of the receiving DNA is bound near the nanopore and the 5' end of the receiving oligonucleotide contains a topoisomerase ligation site, and comprising the step after step (iv) of attaching an additional oligonucleotide to the 5' end of the receiving DNA by washing the first chamber for attaching and feeding a new topoisomerase-loaded donor oligonucleotide into the first attachment chamber, where the new donor oligonucleotide has a different sequence information from the previous donor oligonucleotide; and, if it is desired that the new donor oligonucleotide be attached to the recipient DNA, pulling the 5' end of the recipient nucleic acid back into the first chamber and repeating steps (i)-(iii), or if not desired, allowing the recipient DNA to remain in the second chamber until the desired donor oligonucleotide enters the first chamber.

2.2. Any of the above methods, where a plurality of receiving DNA molecules are synthesized independently in parallel, so that DNA molecules having different sequences are obtained by checking separately whether they are present in the first chamber.

2.3. Any of the above methods, where a plurality of receiving DNA molecules, each of which is connected at the 3'-end to the surface near the nanopore, is synthesized independently, where each nanopore has a pair of electrodes associated with it, where one electrode in the pair is located near one end of the nanopore, and the other electrode is located near the other end of the nanopore, so that each receiving DNA molecule can independently move between the first and second chambers under the influence of an electric current provided by a pair of electrodes.

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

2.5. Method 2 comprising the step of adding an additional oligonucleotide to the 5' end of the receiving DNA by returning the 5' end of the receiving DNA to the first attachment chamber to add an oligonucleotide having the same sequence information or moving the 5' end of the receiving DNA to the second chamber for an attachment having a donor oligonucleotide linked at the 3' end to a topoisomerase, wherein the donor oligonucleotide in the second attachment chamber has a different sequence information than the donor oligonucleotide in the first attachment chamber.

2.6. Any of the above methods, where the donor oligonucleotide contains the following structure:

5' CGAAGGG <data sequence A or B> GTCGACNNNNN

3' GCTTCCC <---------complementary sequence----------> CAGCTGNNNNN

where N refers to any nucleotide and the restriction enzyme is Acc1, which can cut DNA (eg GTCGAC in the sequence above) to provide a suitable overhang.

2.7. Any of the above methods, where the donor oligonucleotide has a hairpin structure, for example, 2.6, where the NNNNN groups on the upper and lower strands are connected.

2.8. Any of the above methods, wherein at least one of the topoisomerase-loaded oligonucleotides has the following structure:

5' CGAAGGG <data sequence A or B> GTCGACNNNNN

3' *TTCCC <---------complementary sequence----------> CAGCTGNNNNN

(*=topoisomerase)

2.9. Any of the above methods, wherein at least one of the topoisomerase-loaded oligonucleotides has the following structure:

5'pCACGTCAGGCGTATCCATCCCTT*

3' GTGCAGTCCGCATAGGTAGGGAAGGCGC

2.10. The foregoing method, wherein the topoisomerase-loaded oligonucleotide

2.11. Any of the above methods, where the sequence of the synthesized DNA is determined after each cycle by detecting changes in electrical potential, current, resistance, capacitance and/or impedance as the oligonucleotide passes through the nanopore.

2.12. Any of the above methods wherein DNA synthesis takes place in a buffer solution, e.g. a solution containing a pH 7-8.5 buffer, e.g. approximately pH 8, e.g. a buffer containing Tris(hydroxymethyl)aminomethane (Tris), a suitable acid, and optionally a chelator such as ethylenediaminetetraacetic acid (EDTA), such as 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; eg a solution containing 10 mM Tris, pH 8, 1 mM EDTA, 150 mM KCl, or eg 50 mM potassium acetate, 20 mM Tris acetate, 10 mM magnesium acetate, pH 7.9 at 25°C.

2.13. Any of the above methods, additionally including the removal of DNA from the nanochip.

2.14. Any of the above methods, including amplification of the DNA thus synthesized.

2.15. Any of the above methods, additionally including DNA removal from the nanochip and DNA crystallization.

2.16. Any of the above methods, further comprising stabilizing the DNA, for example by drying a solution containing the DNA together with one or more of a buffer (eg, borate buffer), an antioxidant, a humectant, such as a polyol, and optionally a chelator, for example, as described in US 8283165 B2, incorporated herein by reference, or by forming a matrix between the nucleic acid and a polymer such as a polyethylene glycol-poly(l-lysine) (PEG-PLL) type AB block copolymer.

2.17. Any of the above methods, including the provision of ligase and ATP to repair DNA breaks [note: topoisomerase ligation only ligates one strand].

2.18. Any of the above methods wherein the topoisomerase-loaded donor oligonucleotide contains a 5' overhang on a strand complementary to the strand bearing the topoisomerase containing a polyinosine sequence [note: inosine residues act as "universal bases" and pair with any other base].

2.19. Any of the above methods wherein the restriction enzyme is a type IIS restriction enzyme that can cleave all of the attached DNA except for one base (the base that is "attached").

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

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

2.22. Any of the above methods, where the reserve chamber additionally contains a ligase and ATP for the repair of a DNA strand not associated with topoisomerase.

2.23. Any of the above methods, comprising the use of a topoisomerase inhibitor inhibitor to inhibit the binding and activity of free topoisomerase against a DNA oligomer, for example, where the inhibitors are selected from novobiocin and coumermicin.

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

2.25. Any of the above methods, where the topoisomerase adds one base, however, the restriction enzyme cleaves at a position that is one nucleotide in the 5' direction from the base attached by the topoisomerase.

2.26. Any of the above methods, wherein the DNA strand thus provided has a sequence containing the dinucleotide sequence "TT" and "TG".

2.27. Any of the above methods, which is a method of synthesizing a DNA molecule by adding single nucleotides in the 3' to 5' direction, comprising (i) reacting the DNA molecule with a topoisomerase loaded with the desired nucleotide in a 5'-protected form, e.g., 5'- phosphorylated form so that the desired nucleotide in the 5' protected form is attached to the 5' end of the DNA, then (ii) removing the protecting group from the 5' end of the DNA thus formed using the phosphatase enzyme and repeating steps (i) and (ii) until the desired nucleotide sequence is obtained.

2.28. Any of the above methods, which is a method for synthesizing a DNA molecule by adding oligomers in the 3' to 5' direction, comprising (i) reacting the DNA molecule with a topoisomerase loaded with the 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 nucleotide sequence is obtained.

2.29. Any of the above methods, which is the method according to any of Method A, etc.

[0116] The product of the synthesis reactions can be detected, studied for quality control, and read to extract data encoded in the polymer. For example, DNA can be amplified and sequenced by conventional methods to confirm that nanopore sequencing is reliable.

[0117] In another embodiment, the invention relates to an oligonucleotide containing a topoisomerase binding site, an information sequence (for example, selected from at least two different sequences, for example, where one sequence corresponds to "0" and the other corresponds to "1" in binary code ), and a restriction site which, when cleaved with a restriction enzyme, provides a topoisomerase ligation site, for example, containing the following sequence:

5' CGAAGGG <data sequence A or B> GTCGAC

3' GCTTCCC <---------complementary sequence----------> CAGCTG

where the information sequence A or B is a sequence of 3-12, for example, approximately 8 nucleotides.

[0118] In another embodiment, the invention relates to a topoisomerase-loaded oligonucleotide, wherein the oligonucleotide contains a topoisomerase binding site, an information sequence (e.g., selected from at least two different sequences, e.g., where one sequence corresponds to "0" and the other corresponds to "1 " in binary code), and a restriction site which, when cleaved by a restriction enzyme, provides a ligation site for topoisomerase; for example, to a topoisomerase-loaded oligonucleotide having the following structure:

5' CGAAGGG <data sequence A or B> GTCGACNNNNN

3' *TTCCC <---------complementary sequence----------> CAGCTGNNNNN

where the information sequence A or B is a sequence of 3-12, for example, approximately 8 nucleotides and * is a topoisomerase covalently linked to the oligonucleotide; for example, where the topoisomerase is vaccinia topoisomerase I.

[0119] In another embodiment, the invention relates to a single-stranded or double-stranded DNA molecule as described above, wherein the single strand or coding sequence is essentially composed of non-hybridizing bases, e.g., adenine and cytosine residues (A and C), which are organized as a sequence , corresponding to the binary code, for example, for use in a data storage method. For example, the invention relates to DNA (DNA 1), where the DNA is single-stranded or double-stranded, having a length of at least 1000 nucleotides, for example, 1000-1000000 nucleotides or, for example, from 5000 to 20000 nucleotides, where the nucleotide sequence corresponds to the binary code; For example,

1.1. DNA 1, where the DNA is single stranded.

1.2. DNA 1, where the DNA is double stranded.

1.3. Any of the above DNA wherein the nucleotides in the single strand or in the coding strand are selected from adenine, thymine and cytosine nucleotides, for example, selected from adenine and cytosine nucleotides or thymine and cytosine nucleotides.

1.4. Any of the above DNA consisting primarily of non-hybridizing nucleotides, so that it does not form significant secondary structures in the form of a single strand.

1.5. Any of the above DNA wherein the nucleotides are at least 95%, eg 99%, eg 100% adenine and cytosine nucleotides.

1.6. Any of the above DNA containing a nucleotide or sequence of nucleotides added to separate or interrupt nucleotides corresponding to a binary code, for example, to separate 1s and 0s, or groups of 1s and 0s, so that consecutive 1s or 0 was easier to read.

1.7. Any of the above DNA, where (a) each bit in the binary code corresponds to one nucleotide, for example, each of 1 and 0 corresponds to A or C; or (b) each bit in the binary code corresponds to a series of more than 1 nucleotide, eg 2, 3 or 4 nucleotides, eg AAA or CCC.

1.8. Any of the above DNA that is crystallized.

1.9. Any of the above DNA that is provided in dry form along with one or more of a buffer salt (eg, borate buffer), an antioxidant, a humectant, such as a polyol, and optionally a chelator, such as those described in US 8,283,165 B2, incorporated herein as a reference; and/or in a matrix between the nucleic acid and a polymer such as a polyethylene glycol-poly(l-lysine) (PEG-PLL) type AB block copolymer; and/or together with a complementary nucleic acid strand or protein that binds DNA.

1.10. Any of the above DNA obtained by any of Method 1, etc., or Method 2, etc., or Method A, etc.

[0120] Nanochips can be made, for example, as shown in Fig.23-29. For example, in one format, each polymer strand is associated with two or four attachment chambers, where the two attachment chamber format is suitable for encoding a binary code in the polymer and the four attachment chamber format is particularly suitable for obtaining specialized DNA sequences. Each attachment chamber contains a separately controlled electrode. Attachment chambers contain reagents for attaching monomers to polymer in a buffer. Attachment chambers are separated by a membrane containing one or more nanopores from a reserve chamber which may be common to several attachment chambers and which contains deprotection reagents and a buffer to deprotect protected monomers or oligomers attached in attachment chambers. accessions. The nanochips contain multiple groups of attachment chambers to enable parallel synthesis of multiple polymers.

[0121] For resolution corresponding to one DNA base, a high-speed and low-noise nanopore sensor and detection electronics are important. In certain embodiments, the nanochip is electrically coupled to a complementary semiconductor metal oxide (CMOS) chip. Solid-state nanopores can be integrated into a CMOS platform near bias electrodes and dedicated gain electronics, for example, 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 into the present description by reference.

[0122] In another embodiment, the invention relates to a nanochip (nanochip 1) for the synthesis and/or sequencing of an electrically charged polymer, such as DNA, containing at least two different monomers, the nanochip comprising at least first and second reaction chambers separated by a membrane containing one or more nanopores, where each reaction chamber contains one or more electrodes for drawing an electrically charged polymer into the chamber and, in addition, contains an electrolyte medium and optional reagents for attaching monomers to the polymer, for example,

1.1. Nanochip 1, where the nanopore has a diameter of 2-20 nm, such as 2-10 nm, such as 2-5 nm.

1.2. Any of the above nanochips, where some or all of the walls of the reaction chambers of the nanochip contain silicon material, such as silicon, silicon dioxide, silicon nitride, or combinations thereof, such as silicon nitride.

1.3. Any of the above nanochips, where some or all of the walls of the reaction chambers of the nanochip contain silicon material, such as silicon, silicon dioxide, silicon nitride, or combinations thereof, such as silicon nitride, and some or all of the nanopores are made by ion bombardment.

1.4. Any of the above nanoarrays, where some or all of the nanopores are composed of a pore-forming protein, α-hemolysin, in a membrane, such as a lipid bilayer.

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

1.6. Any of the above nanochips containing an electrolyte medium.

1.7. Any of the above nanochips containing an electrolyte medium containing a buffer, e.g. a buffer with pH 7-8.5, e.g. approximately pH 8, e.g. a buffer containing tris(hydroxymethyl)aminomethane (Tris), an appropriate acid and optionally a chelator, e.g , ethylenediaminetetraacetic acid (EDTA), for example TAE buffer containing a mixture of Tris base, acetic acid and EDTA, or TBE buffer containing a mixture of Tris bases, boric acid and EDTA; eg a solution containing 10 mM Tris pH 8, 1 mM EDTA, 150 mM KCl, or eg 50 mM potassium acetate, 20 mM Tris acetate, 10 mM magnesium acetate, pH 7.9 at 25°C.

1.8. Any of the above nanochips containing reagents for attaching monomers to a polymer.

1.9. Any of the above nanochips capable of both synthesizing ('writing', for example, by sequentially attaching monomers or groups of monomers to a polymer) and sequencing ('reading', for example, by measuring changes in current and/or inductance as monomers pass through a nanopore) of a polymer.

1.10. Any of the above nanochips, where the membrane containing one or more nanopores contains a metal surface on both sides, and the metal surface is separated by an insulating material, for example, a silicon nitride membrane, and the metal surfaces are organized, for example, by lithography techniques, to provide electrodes on each side of each nanopore, for example, so that in the electrolyte environment an electrical current can be generated through the nanopore, for example, so that the current can draw the polymer through the nanopore and as the polymer passes through the nanopore, the change in electrical potential through the nanopore can be measured and used to identify the sequence of monomers in polymer.

1.11. Any of the above nanochips containing an electrically charged polymer, which is DNA.

1.12. Any of the above nanochips containing an electrically charged polymer that is single-stranded DNA (ssDNA).

1.13. Any of the above nanochips containing an electrically charged polymer, which is DNA containing a specific restriction site.

1.14. Any of the above nanochips containing an electrically charged polymer that is DNA, where DNA is DNA, as described for any of DNA 1, etc., above.

1.15. Any of the above nanochips containing an electrically charged polymer, which is DNA, where the DNA is at least 95%, for example 99%, for example 100% consists of adenine and cytosine residues.

1.16. Any of the above nanochips containing an electrically charged polymer, which is DNA, where the DNA contains only adenine and cytosine residues.

1.17. Any of the above nanochips containing one or more holes for the possibility of supplying and draining the buffer and reagents.

1.18. Any of the above nanochips containing a buffer solution, for example, a solution containing a buffer for pH 7-8.5, for example, approximately pH 8, for example, a buffer containing tris(hydroxymethyl)aminomethane (Tris), a suitable acid and optionally a chelator, for example , ethylenediaminetetraacetic acid (EDTA), for example TAE buffer containing a mixture of Tris base, acetic acid and EDTA, or TBE buffer containing a mixture of Tris bases, boric acid and EDTA; eg a solution containing 10 mM Tris pH 8, 1 mM EDTA, 150 mM KCl, or eg 50 mM potassium acetate, 20 mM Tris acetate, 10 mM magnesium acetate, pH 7.9 at 25°C.

1.19. Any of the above nanochips that is or is capable of being lyophilized for storage and then rehydration, for example, the nanochip structure contains a hydratable or water-permeable polymer.

1.20. Any of the above nanochips that are synthesized in dry form, for example, where the nanochip structure contains a hydratable or water-permeable polymer, followed by hydration before use, optionally followed by lyophilization for long-term storage after the writing process is complete.

1.21. Any of the above nanochips where an electrically charged polymer such as DNA is stabilized with a histone.

1.22. Any of the above nanochips where the inner surface is positively charged.

1.23. Any of the above nanochips where the electrodes are operatively connected in a capacitive circuit capable of delivering RF pulsating direct current, e.g., 1 MHz to 1 GHz, e.g. 50-200 MHz, e.g. approximately 100 MHz, through a nanopore, e.g. a current can draw a charged polymer through the nanopore, and the sequence of monomers can be determined by measuring the change in capacitance through the nanopore as the charged polymer passes through the nanopore.

1.24. Any of the above nanochips containing a backup or deblocking chamber that contains reagents for deprotecting the polymer after the attachment of a monomer or oligomer in one of the attachment chambers.

1.25. Any of the above nanochips containing multiple pairs of chambers to attach.

1.26. Any of the above nanochips containing an electrical control layer, a fluid layer and a ground layer, for example, as shown in Fig.24, connected by splicing.

1.27. Any of the above nanochips where the nanopore is drilled through FIB, TEM, wet or dry engraving.

1.28. Any of the above nanochips, where the membrane containing nanopores has a thickness from 1 atomic layer to 30 nm.

1.29. Any of the above nanochips, where the membrane containing nanopores is made of SiN, BN, SiOx, graphene, transition metal dichalcogenides, such as WS ₂ or MoS ₂ .

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

1.31. Any of the above nanochips where the wiring density is increased by 3D stacking, with electrical isolation provided by dielectric coating (eg via PECVD, sputtering, ALD, etc.).

1.32. Any of the above nanochips where contact with the electrode in the attachment chamber is made using silicon vias (TSV) via reactive ion deep engraving (DRIE), for example. using cryo process or BOSC process, or wet silicon engraving.

1.33. Any of the above nanochips where individual voltage control for the electrode in each attachment chamber allows individual control and monitoring of the electrode in each attachment chamber.

1.34. Any of the above nanochips, where each polymer is associated with a first attachment chamber, a second attachment chamber, and a deblocking chamber.

1.35. Any of the above nanochips where one or more chambers have fluid flow.

1.36. Any of the above nanochips, where one or more chambers are sealed against liquids.

1.37. Any of the above nanochips where the deblocking chamber has a fluid flow.

1.38. Any of the above nanochips where the attachment chambers share a common fluid flow.

1.39. Any of the above nanochips where the wiring between the chambers is common to chambers of a similar type (eg, first attachment chambers, second attachment chambers, and deblocking chambers).

1.40. Any of the above nanochips where the attachment chambers have individual voltage control and the deblocking chambers share a common ground.

1.41. Any of the above nanochips where the deblocking chambers have individual voltage control, the first attachment chambers have a common ground, and the second attachment chambers have a common ground.

1.42. Any of the above nanochips where the nanochip is made by layer splicing and the chambers are pre-filled with desired reagents prior to splicing.

1.43. Any of the above nanochips where one or more internal surfaces are silanized.

1.44. Any of the above nanochips that has one or more openings for fluid inlet or outlet.

1.45. Any of the above nanochips where the contact of the electrodes in the chambers with the polymer is limited, such as where the electrode is placed too far from the nanopore to be reached by the charged polymer bound to the surface next to the nanopore, or where the electrode is protected by a material that allows the passage of water and monatomic ions (eg Na+, K+ and Cl- ions), but not the passage of the polymer, or monomeric or oligomeric reagents to be bound to the polymer.

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

[0123] For example, in one embodiment, the invention relates to a nanochip, for example, according to any of nanochip 1, etc., for sequencing an electrically charged polymer, such as DNA, containing at least two different monomers, and the nanochip contains at least first and second reaction chambers containing an electrolyte medium and separated by a membrane containing one or more nanopores, where each reaction chamber contains at least one pair of electrodes placed on opposite sides of the membrane, where the electrodes are functionally connected in a capacitive circuit capable of generating a radio frequency pulsating direct current, e.g. 1 MHz to 1 GHz, e.g. 50-200 MHz, e.g. approximately 100 MHz, through a nanopore, e.g. where a pulsating forward current can draw a charged polymer through the nanopore and the sequence of monomers can be determined by measuring the change in capacitance through the nanopore as the charged p polymer through a nanopore.

[0124] In another embodiment, the invention relates to a method for reading the monomer sequence of a charged polymer containing at least two different types of monomers, for example, DNA molecules, including the use of radio frequency pulsed direct current, for example, with a frequency of from 1 MHz to 1 GHz, eg 50-200 MHz, eg approximately 100 MHz, through a nanopore where a pulsating forward current draws a charged polymer through the nanopore and the sequence of monomers is read by measuring the change in capacitance through the nanopore as the polymer passes through the nanopore.

[0125] In another embodiment, the invention relates to the use of any of DNA 1, etc. in the way information is stored.

[0126] In another embodiment, the invention relates to the use of single-stranded DNA in a method for storing information, for example, where the sequence is essentially not self-hybridizing.

[0127] In another embodiment, the invention relates to a data storage method and apparatus using a nanochip, such as any of nanochip 1, etc., to produce an electrically charged polymer, such as DNA, containing at least two different monomers or an oligomer, wherein the monomers or oligomers are arranged in a binary code sequence, for example, according to any of Methods 1 and/or 2 above, etc.

[0128] For example, in one embodiment, a nanochip containing a polymer thus synthesized provides a storage device because the nanochip can be activated and the sequence of the polymer can be determined by passing it through the nanopore at any time. In other embodiments, the polymer is extracted from the nanoarray, or amplified and the amplified polymer is extracted from the nanoarray, stored until needed, and then read using a conventional sequencer, such as a conventional nanopore sequencing device.

[0129] In another embodiment, the invention relates to a method for storing information, including the synthesis of any of the DNA 1, etc., for example, in accordance with any of the methods 1, etc. or ways 2, etc.

[0130] In another embodiment, the invention relates to a method for reading a binary code, e.g., encoded by any of DNA 1, etc., using a nanopore sequencer, e.g., using a nanochip 1, etc., as described herein. description.

[0131] Any of the above methods, where the nanochip is erased using an enzyme that lyses the charged polymer, such as deoxyribonuclease (DNAse) to hydrolyze DNA.

EXAMPLES

Example 1. Immobilization of one end of DNA next to a nanopore and controlled movement of DNA from one side to the other using an electric current

[0132] Experimental techniques have been developed to demonstrate that DNA moves back and forth between two chambers separated by a nanopore using electric current under conditions in which the corresponding protein does not move between chambers.

[0133] A nanochip containing two chambers is made from silicon nitride. Nanopores <4 nm (for dsDNA or ssDNA) and 2 nm (for ssDNA only) are 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. These two chambers are referred to as "near" and "far" chamber, with the far chamber being the chamber in which the 3' end of the DNA is conjugated.

[0134] ssDNA (2 nm pore) and ssDNA+dsDNA (4 nm pore), but not protein, have been shown to pass through the nanopore. Passage through the nanopores is detected by the termination of the electric current.

[0135] Pore Surface DNA Conjugation: Attachment of 5'-amino-modified DNA to carboxy-coated polystyrene beads (Fluoresbrite® BB carboxylate microspheres, 0.05 µm, from Polysciences, Inc.) via carbodiimide-mediated attachment. The 3' end of the DNA is labeled with biotin. DNA has a given length.

[0136] Streptavidin Conjugation: Conjugation was performed on the "far" side of the silicon nitride nanopore, conjugating streptavidin to the surface as described in Arafat, A. Covalent Biofunctionalization of Silicon Nitride Surfaces. Langmuir (2007) 23(11): 6233-6244.

[0137] Immobilization of DNA near the nanopore: DNA-conjugated 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). A voltage (~100 mV) is applied until an intermittent current is observed (using an Axon Nanopatch200B clamping amplifier). The 50nm beads cannot pass through the nanopore, so when the DNA has passed and the bead has pressed down on the end of the nanopore, the current becomes highly discontinuous. The current is maintained for 1-2 min until the DNA binds irreversibly to the immobilized streptavidin on the far side via biotin binding. To confirm DNA immobilization, the current is reversed. A different current is observed if the DNA is inside and outside the pore. If it appears that the DNA is not immobilized, then the process is repeated.

[0138] Endonuclease bead release: 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 is cleavable by an enzyme that cleaves the single stranded DNA. See, for example, Nishigaki, K., Type II restriction endonuclease cleave single-stranded DNAs in general. Nucleic Acids Res. (1985) 13(16): 5747-5760. In an alternative embodiment, the complementary oligonucleotide is added to the chamber where the DNA is bound and allowed to hybridize for 30 minutes to form dsDNA, and then the restriction enzyme is added. After the release of the granule, it is washed off. The current is switched between forward and reverse to confirm that DNA is passing in/through and out of the pore.

[0139] Controlled Forward and Backward Demonstration: Using a standard buffer, current is applied in the forward direction until a signal interruption is observed, and then returns to "normal" after passing through the DNA. The reverse current is applied until an interruption of the signal is observed. It is observed that the signal does not return to normal as long as the DNA remains in the pore. The application of current in the forward and reverse direction is repeated for several cycles to confirm that the DNA passes through the nanopore back and forth.

Example 1a: Immobilization of a DNA strand next to a nanopore in a silicon dioxide chip

[0140] The inner wall of the nanochip is made of silicon dioxide. Both sides are silanized, however, the oligonucleotide is conjugated to only one side of the chip wall and then a nanopore is formed.

[0141] Silanation: The surface of the chip wall is cleaned with a Piranha solution (various commercially available brands, usually containing a mixture of sulfuric acid (H ₂ SO ₄ ) and hydrogen peroxide (H ₂ O ₂ ), which removes organic residues from the surface) at 30°C and washed twice with distilled water. Prepare a stock solution of (3-aminopropyl)triethoxysilane (APTES) with 50% methanol (MeOH), 47.5% APTES, 2.5% nanopure H2O and incubate >1 hour at 4°C. The APTES stock solution is then diluted 1:500 in MeOH and applied to the chip wall and incubated at room temperature. The chip wall is then rinsed with MeOH and dried at 110° C. for 30 minutes.

[0142] Conjugation: The chip wall is then incubated for 5 hours at room temperature in 0.5% w/v. a solution of 1,4-phenylenediisothiocyanate (PDC) in dimethyl sulfoxide (DMSO). It is washed briefly twice with DMSO and washed twice briefly with double-distilled water. The chip wall is then incubated with 100 nM amine-modified single-stranded DNA oligomers (approximately 50-mers) in double-distilled water (pH 8) overnight at 37°C. The chip wall is then washed twice with 28% ammonia to inactivate any unreacted material and washed twice with double distilled water. Then one or more nanopores are formed in the wall.

[0143] After completion of the nanochip fabrication, the inner wall is coated with approximately 50 bp DNA oligomers. This allows single-stranded DNA having a terminal sequence complementary to the DNA surface bound to be localized to the nanopore by binding the ssDNA to a relatively bulky structure (e.g., a granule, protein, or DNA origami structure that is too large in diameter to fit into the nanopore), where the sequence complementary to surface-bound DNA is distal to the bulk structure, drawing the charged polymer through the nanopore using electric current, allowing the ssDNA to bind to the complementary surface-bound DNA oligomer adjacent to the nanopore, and cleaving off the bulk structure.

Example 2 DNA Synthesis - Attachment of a Single Nucleotide

[0144] The DNA is moved to the "backup" chamber by applying an appropriate current and detecting the movement of the DNA.

[0145] Into the "attachment" chamber, add the 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 at 25° C.), and reversibly blocked dATP*. A buffer is also added to the "backup" camera.

[0146] To attach nucleotides to DNA, dNTPs are used that have reversible blocks on the 3'-OH. When attached to a DNA strand, the next dNTP cannot be added until the blocked dNTP is unblocked.

[0147] Deblocking can be chemical or enzymatic. Various approaches are used:

[0148] a. 3'-O-allyl: allyl is 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 U S A. (2006);103(52):19635-40; or 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 glycol-400. Green Chem. (2011)13: 2354-2358.

[0149] b. 3' O-NH2: The amine is removed in buffered NaNO ₂ as described in US 8034923.

[0150] c. 3'-phosphate. phosphate is hydrolyzed with endonuclease IV (New England Biolabs). Other possible 3' modifications that can also be removed by endonuclease IV include phosphoglycoaldehyde and deoxyribose-5-phosphate.

[0151] d. 3'-O-Ac:acetate 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.

[0152] The DNA is then moved to the "far" chamber using the appropriate current and DNA motion detection. The DNA protecting group is removed by changing buffers and adding a deblocking buffer/solution as described in a.-d. above.

[0153] The method is repeated, if desired, to obtain a sequence of interest.

Example 3 DNA Synthesis: Attachment of a Blocked Oligonucleotide

[0154] The 3' end of the double-stranded DNA is tied adjacent to the nanopore with a 4 nm opening. The 5' end of the DNA has an overhanging CG end (reading from 5' to 3').

[0155] Oligonucleotide cassettes A and B are prepared as follows:

5' CGAAGGG <CODE A OR B> GTCGACNNNNN

3' GCTTCCC <COMPLEMENTARY SEQUENCE> CAGCTGNNNNN

[0156] Each of the A code and the B code corresponds to an information sequence. N stands for any nucleotide. The 5' sequence contains the topoisomerase recognition site and the 3' sequence contains the Acc1 restriction site. The oligonucleotide is exposed to topoisomerase and the topoisomerase binds to 3'-thymidine:

5' CGAAGGG <CODE A OR B> GTCGACNNNNN

3' *TTCCC <COMPLEMENTARY SEQUENCE> CAGCTGNNNNN (*=topoisomerase)

[0157] The DNA is moved to the "near" chamber by applying an appropriate current and detecting the movement of the DNA. The topoisomerase-loaded "code A" oligonucleotide is provided in the "attachment" chamber. The DNA is moved into the attachment chamber by applying an appropriate current and detecting movement of the DNA, whereby the Code A oligonucleotide binds to the DNA, Acc1 is added to the "reserve" chamber where it cleaves at the restriction site to provide a topoisomerase ligation site.

[0158] This process is repeated until the desired sequence is reached by adding another "Code A" or "Code B". It should be noted that the continuous addition of a new Acc1 to the "backup chamber" is not required; only washout of code A or code B oligonucleotides in the "attachment" chamber when switching from code A or code B is required.

[0159] Pore sequencing that only allows passage of ssDNA requires some modifications to the protocol described above. It is already known that when dsDNA encounters a small pore (2 nm), only the ssDNA will pass through and the complementary sequence will "split". Thus, when performing this synthesis using a 2 nm pore, it must be ensured that the proper dsDNA is able to "re-form" on the other side. This can be done by adding 'CGAAGGG <CODE A OR B> GTCGACNNNNN' to the near chamber (to ensure the insertion of a restriction site) and 'CGAAGGG <CODE A OR B> GT' to the far chamber (to ensure the formation of a topoisomerase compatible site).

[0160] Describing the above method, the inventors demonstrate the sequential "attachment" of DNA-encoded information into a growing DNA strand with ≥2 consecutive attachments (corresponding to 2 data bits), each of which includes the stage of "attachment" and "deprotection". Initial experiments for optimization and proof of concept are carried out in microtubes.

[0161] In the approach described in this example, one bit of information is encoded into strands of nucleotides. The "attached" DNA bit is a short dsDNA sequence conjugated to vaccinia virus topoisomerase I (topo). In the presence of a suitable "acceptor" "deprotected" DNA, the topoisomerase-loaded DNA "bit" enzymatically and covalently binds ("attaches") to the acceptor via the topoisomerase, which is removed from the DNA in this process. The restriction enzyme can then cleave off the added bit to "remove the protecting group" and create a suitable "acceptor" sequence to attach the next bit.

[0162] Topoisomerase Loading: The general loading scheme is as follows, as schematically represented in Figures 22 and below, where N is any nucleotide and A, T, G, and C correspond to nucleotides with adenine, thymine, guanine, and cytosine bases, respectively. N on top of each other are complementary. While the restriction enzyme HpyCH4III is used in this example, the general strategy will work with other restriction enzymes, for example as demonstrated in example 4.

+

Topoisomerase *

=

+

(loaded with topoisomerase)

(N above each other are complementary)

Accession

General "addition" reaction:

(acceptor)

+

(loaded with topoisomerase)

=

+

*

(free topoisomerase)

Removal of the protecting group

The general "deprotection" reaction is:

+

HpyCH4III

(restriction enzyme)

=

(with the protective group removed)

+

(by-product)

[0163] The following oligonucleotides are ordered from Integrated DNA Technologies (IDT). ʺBʺ at the end of some of the oligonucleotides indicates biotin:

BAB: CGATAGTCTAGGCACTGTTTGCTGCGCCCTTGTCCGTGTCGCCCTTATCTACTTAAGAGATCATACAGCATTGCGAGTACG

B1: b-CACGTACTCGCAATGCTGTATGATCTCTTAAGTAGATA

B2: ATCTACTTAAGAGATCATACAGCATTGCGAGTACG

TA1: b-CACACTCATGCCCGCTGTAGTCACTATCGGAAT

TA2: AGGGCGACACGGACAGTTTTGAATCATACCG

TA3b: AACTTAGTATGACGGTATGATTCAAACTGTCCGTGTCGCCCTTATTCCGATAGTGACTACAGCGGCATGAG

TB1: b-CACACTCATGCCCGCTGTAGTCACTATCGGAAT

TB2: AGGGCGCAGCAAAACAGTGCCTAGACTATCG

TB3b:

AACTTAGTATGACGATAGTCTAGGCACTGTTTGCTGCGCCCTTATTCCGATAGTGACTACAGCGGCATGAG

FP1: CACGTACTCGCAATGCT

FP2: CGGTATGATTCAAACTGTCCG

FP3: GCCCTTGTCCGTGTC

[0164] Oligonucleotides are dissolved to a concentration of 100 μm in TE buffer and stored at -20°C.

[0165] Hybridized oligonucleotides are prepared by mixing the oligonucleotides as described below, heating to 95°C for 5 minutes, and then lowering the temperature by 5°C every 3 minutes until the temperature reaches 20°C. Hybridized oligonucleotides are stored at 4°C or -20°C. The combinations of oligonucleotides are as follows:

B1/2

48 µl B1

48 µl B2

4 µl 5 M NaCl

A5

20 µl TA1

20 µl TA2

5 µl TA3b

4 µl 5 M NaCl

51 µl TE

B5

20 µl TB1

20 µl TB2

5 µl TB3b

4 µl 5 M NaCl

51 µl TE

[0166] The following buffers and enzymes are used in this enzyme:

TE: 10 M Tris pH 8.0, 1 mM EDTA, pH 8.0

WB: 1 M NaCl, 10 mM Tris pH8.0, 1 mM EDTA, pH 8.0

1x Topo: 20 mM Tris, pH7.5, 100 mM NaCl, 2 mM DTT, 5 mM MgCl ₂

1x RE: 50 mM K acetate, 20 mM Tris acetate, 10 mM Mg acetate, 100 μg/ml BSA, pH 7.9, at 25°C.

Vaccinia virus topoisomerase I (topo) DNA was purchased from Monserate Biotech (10,000 U/mL)

HypCH4III purchased from NEB

Streptavidin coated magnetic beads (s-MagBeads) were purchased from ThermoFisher.

[0167] The acceptor is prepared as follows: 5 μl of s-magbeads are washed once with 200 μl of WB (binding time 1 minute). 5 µl B1/2 + 195 µl WB is added to the beads and incubated for 15 minutes at room temperature, and then washed once with 200 µl WB, then washed once with 200 µl 1x Topo, and resuspended in 150 µl 1x Topo .

[0168] Topoisomerase-loaded A5 (see FIG. 20) is prepared as follows: 4 µl 10x topoisomerase buffer+23 µl water+8 µl A5+5 µl topoisomerase incubated at 37°C for 30 minutes, added to 5 µl s -magbeads (washed 1× with 200 μl WB, 1× with 200 μl 1× Topo, resuspended in 150 μl 1× topo) and allowed to bind for 15 minutes at room temperature.

[0169] "Attachment" loaded A5 to the acceptor: s-magbeads are removed from loaded with topoisomerase A5, added to the acceptor and incubated at 37°C for 60 minutes. An aliquot is removed, diluted 1/200 in TE and stored at -20°C.

[0170] Removal of the protecting group: the material is washed once with 200 μl of WB, then washed once with 200 μl of 1x RE and resuspended in 15 μl of 10x RE and 120 μl of water. Add 15 µl of HypCH4III. The mixture is incubated at 37° C. for 60 minutes, then washed once with 200 μl of WB, washed once with 200 μl of 1x topo, to obtain a product, which was called "acceptor-A5".

[0171] Topoisomerase loaded B5 (see FIG. 21) is prepared as follows: 4 µl 10x topoisomerase buffer+23 µl water+8 µl B5+5 µl topoisomerase are combined and incubated at 37° C. for 30 minutes. The product is added to 5 µl s-magbeads (washed 1× with 200 µl WB, 1× with 200 µl 1× Topo, resuspended in 15 0 µl 1× topo) and allowed to bind for 15 minutes at room temperature.

[0172] "Attaching" loaded B5 to acceptor-A5: s-magbeads are removed from topoisomerase-loaded B5, added to acceptor-A5 and incubated at 37°C for 60 minutes. An aliquot is removed, diluted 1/200 in TE and stored at -20°C.

[0173] Deprotection: The material is washed once with 200 μl WB, then washed once with 200 μl 1x RE and resuspended in 15 μl 10x RE and 120 μl water. Add 15 µl of HypCH4III. The mixture is incubated at 37°C for 60 minutes.

[0174] Confirmation that the above reactions are working is provided by PCR amplification of aliquots of A5 (acceptor with A5 attached: step iii, in Scheme "A5 attached") and B5 (acceptor-A5 with B5 attached: step vi, in Scheme "attached B5"). "No template" is used as negative control for A5, A5 is used as negative control for B5, BAB oligonucleotide is used as positive control for B5. The expected product size for A5 PCR is 68 bp, the expected product size for B5 PCR is 57 bp. (Also separate B1/2 on the gel, expected size is ~47 bp, however this may be approximate as there are overhangs and it is biotinylated). PCR reactions (30 cycles 95/55/68 (1 minute each) are performed as follows:

A5 (-) control A5 B5 (-) control B5 Matrix 1 µl 1 µl 1 µl FP1 1 µl 1 µl 1 µl 1 µl FP2 1 µl 1 µl FP3 1 µl 1 µl Water 8 µl 7 µl 7 µl 7 µl Maxima MM 10 µl 10 µl 10 µl 10 µl

[0175] SDS-PAGE using 4-20% Tris-glycine gels is used to confirm that oligonucleotides of the expected size are being produced. Loading is carried out as described above, however immediately after loading (37° C. incubation step), loading buffer is mixed in and the samples are heated to 70° C. for 2 minutes and allowed to cool before gel separation. The gel is stained with Coomassie. For a negative control, water is added to the reaction mixture instead of topoisomerase. Figure 30 shows the results clearly showing the bands corresponding to the expected product sizes for A5 PCR and for B5 PCR.

[0176] It has thus been shown that attachment of a DNA bit via topoisomerase-loaded DNA cassettes and removal of the protecting group by a restriction enzyme is feasible. In these proof-of-concept experiments, DNA is immobilized via streptavidin-conjugated magnetic beads and placed sequentially in different reaction mixtures, however, in the nanopore chip format, the inventors created separate reaction chambers and used electrical current to move the DNA into these different reaction chambers.

[0177] Finally, PCR demonstrates that the expected DNA sequences are generated by sequentially attaching DNA sequences corresponding to the "bits" of information. These reactions worked as intended even with minimal optimization.

[0178] The DNA prepared as described in Examples 2 and 3 was isolated and sequenced using a commercial nanopore sequencer (MinION from Oxford Nanopore), confirming the desired sequence.

Example 4 DNA Synthesis: Attachment of a Blocked Oligonucleotide Using Another Restriction Enzyme

[0179] The following synthesis is carried out analogously to example 3, but using the restriction enzyme MluI, which cuts "ACGCGT" with the formation:

In this example, the topoisomerase is loaded to form a complex with sequence complementarity that will allow the loaded topoisomerase to transfer DNA to DNA cleaved with MluI:

+

topoisomerase

=

("*" indicates 3'-phosphate bound topoisomerase)

+

(b=biotin. It can be removed with streptavidin)

[0180] Through a process similar to the previous example, the loaded topoisomerase is then used to attach the oligomer to the 5' end of the synthesized chain having a complementary acceptor sequence, thereby releasing the topoisomerase, and then the chain is "deprotected" using MluI, and the cycle is repeated until until the desired sequence of oligomers is obtained.

Example 5 Single Base Attachment Using the Topoisomerase Strategy

[0181] The inventors have found that the topoisomerase system can also be designed to attach single bases to a single-stranded DNA strand (compared to Example 3, which describes the addition of "cassettes"). The DNA bit to be "attached" is in a short DNA sequence conjugated to vaccinia topoisomerase I (topo). In the presence of a suitable "acceptor" "deprotected" DNA, the topoisomerase-loaded DNA is enzymatically and covalently ligated ("attached") to the acceptor via the topoisomerase, which is removed from the DNA in this process. The type IIS restriction enzyme can then cleave off the added DNA except for one base (the base that "attaches"). This deprotection-addition process is repeated to add additional bases (bits).

[0182] Topoisomerase loading: The general loading protocol is as follows, similar to example 3:

+

Topoisomerase (*)

=

N-N-N-N-N-N-N-N-N-N-N-N-N-N-N

N-N-N-N-N-N-N-N-N…biotin

(by-product)

+

(loaded with topoisomerase)

As in example 3, N on top of each other are complementary. I is inosine. Biotin is used to remove unreacted product and by-product. Attaching a single base is carried out as follows.

N-N-N-N-N-N-N-N-N-N…

(acceptor sequence nucleotides are in italics)

+

(loaded with topoisomerase)

=

+

*

(free topoisomerase)

The deprotection using the restriction enzyme BciVI is illustrated below (section in bold):

...N-G-T-A-T-C-C-N-N-C-C-C-T-T-N-N-N-N-N-N-N-N-N-N…

...N-N-N-N-N-N-N-N-N-N-N-N-N-N-I-I-I-I-I

+

BciVI

(restriction enzyme)

=

T-N-N-N-N-N-N-N-N-N-N…

(note that "T" is added to the 5' end of the acceptor DNA)

+

N-N-I-I-I-I-I

(dissociated*)

+

...N-G-T-A-T-C-C-N-N-C-C-C-T

...N-N-N-N-N-N-N-N-N-N-N-N

-------------------------------------------------- -------

=

T-N-N-N-N-N-N-N-N-N-N…+N-N-I-I-I-I-I

(NNIIIIII dissociates from a single chain with an attached base)

[0183] The following oligonucleotides have been synthesized commercially (B=biotin, P-phosphate, I=inosine):

NAT1 CACGTCAGGCGTATCCATCCCTTCACGTACTCGCAATGCTGTATGGCGAT

NAT1b P-CACGTCAGGCGTATCCATCCCTTCACGTACTCGCAATGCTGTATGGCGAT-B

NAT9cI P-IIIIIAAGGGATGGATACGCCTGACGTG

NAT9x P-ATCGCCATACAGCATTGCGAG

NAT9 ACGTGAAGGGATGGATACGCCTGACGTG

Nat9Acc CACGTAGCAGCAAAACAGTGCCTAGACTATCG

Nat1P CACGTCAGGCGTATCCATCC

FP4 CGATAGTCTAGGCACTGTTTG

[0184] Oligonucleotides are dissolved to 100 μM in TE buffer and stored at -20°C.

[0185] Hybridization: The following hybridized oligonucleotides are obtained by mixing the oligonucleotides as described, heating to 95°C for 5 minutes, and then lowering the temperature by 5°C every 3' until the temperature reaches 20°C. Hybridized oligonucleotides are stored 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 µl PBS

NAT1/NAT9

10 µl NAT1

10 µl NAT9

80 µl PBS

[0186] Buffers and enzymes: The following buffers are used:

TE: 10 M Tris, pH 8.0, 1 mM EDTA, pH 8.0

PBS: phosphate buffered saline (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4) (pH 7.4)

10× Cutsmart: 500 mM KAc, 200 mM Tris-Ac, 100 mM Mg-Ac, 1 mg/ml BSA pH 7.9.

BciVI was purchased from NEB and streptavidin-coated magnetic beads (s-MagBeads) were purchased from ThermoFisher.

[0187] The addition reaction is carried out as follows.

[0188] 1. Topoisomerase loading: Reagents are combined according to the table:

Experiment (-) control #1 (-) control #2 10x topoisomerase buffer 3 3 3 Water 17 21 23 NAT1B/NAT9cI/NAT9x 6 6 - Topoisomerase 4 - 4

The reagents are then incubated at 37° C. for 30 minutes. By-products are removed using streptavidin magnetic beads (5 μl) in 1x topoisomerase buffer after 10 minutes at room temperature to ensure binding.

[0189] 2. Reaction: reagents are collected according to the table:

Experiment (-) control #1 (-) control #2 From A.1.c one one one NAT9Acc one one one 10x topoisomerase buffer one one one water 7 7 7

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

[0190] An asterisk (*) denotes a topoisomerase. It should be noted that NAT9cI is phosphorylated, however this is not shown for purposes of illustration.

[0191] When an acceptor sequence is present with the loaded topoisomerase, it undergoes the following reaction:

[0192] PCR amplification and measurement of the molecular weight of the product on agarose gel confirms the formation of the expected product. See FIG. 30 for the correct size band in lane 1 (experiment) and the absence of bands in the negative controls.

[0193] B. Deprotection reaction: Reagents are combined according to the table:

one 2 3 4 NAT1/NAT9 one one - - NAT1/NAT9cI - - one one 10x cutsmart 2 2 2 2 water 17 sixteen 17 sixteen BciVI 0 one 0 one

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

It was not known whether the restriction enzyme would cut the DNA in the intended manner, given that the 3' end of the cut site is a series of inosine residues as opposed to "normal" bases. As a positive control, the equivalent of NAT1/NAT9cI with "correspondingly" base pairs (NAT1/NAT9c) is obtained:

Amplification of the product by PCR followed by measurement of molecular weight on agarose gels (FIG. 31) demonstrates that the enzyme works as expected. For the positive control, a larger band is observed in the uncleaved state (lane 1), however, when digested, a smaller band/s are observed. The same pattern is observed for NAT1/NAT9cI, demonstrating that the presence of inosine residues does not suppress or prevent cleavage. As it turns out, a small amount of uncleaved product remains with NAT1/NAT9cI, indicating that cleavage is not as effective, at least under these conditions, as with NAT1/NAT9c. Cleavage efficiency can be increased by changing buffer conditions and/or adding more inosine residues to the 5' end of NAT9cI.

[0194] The above example demonstrates that a topoisomerase/type IIS restriction enzyme combination can be used to add a single nucleotide to the 5' end of a single stranded DNA target. A similar topoisomerase, SVF, which recognizes the CCCTG sequence (https://www.ncbi.nlm.nih.gov/pubmed/8661446) is used to attach "G" instead of "T", using a similar process, thus allowing the construction sequence encoding binary information with T and G.

[0195] As noted above, when dsDNA is produced using topoisomerase strategies, DNA breaks on the opposite strand can be repaired using ligase along with ATP. However, when adding a single nucleotide, as in this example, the inventors designed single-stranded DNA, so that there were no breaks that needed to be repaired, and there was no need to use a ligase.

Example 6 Single base attachment using a topoisomerase strategy coupled with 5'-phosphate binding

[0196] In another approach for attaching one base, the inventors use 5'-phosphate as a blocking group to allow attachment of one base pair in the 3' to 5' direction. The loading reaction loads the topoisomerase with one T (or G, or other nucleotide if desired) having a 5'-phosphate group. When the loaded topoisomerase "sees" a free 5' non-blocked (non-phosphorylated) strand of single-stranded DNA, it adds T to that strand, providing DNA with a T added to the 5' end. This attachment is facilitated by the presence of an adapter DNA having sequences to which a topoisomerase and a single stranded acceptor DNA can bind. (It should be noted that adapter DNA is catalytic and can be reused as a template in repeated reactions). The added nucleotide has a 5'-phosphate on it, so it will not be a substrate for further addition until it is exposed to phosphatase, which removes the 5'-phosphate. This process is repeated using topoisomerase to attach one 'T' to the 5' end of the single-stranded target DNA and SVF topoisomerase to attach one 'G', thus allowing the construction of a sequence encoding binary information with T and G. This process is schematically presented below :

Generally:

Load:

ALTERNATIVE TRANSFER MECHANISM

Example 7 Using DNA Origami to Facilitate DNA Attachment Near the Nanopore

[0197] A DNA strand with a large origami structure at one end is trapped in the nanopore and immobilized on surface-conjugated streptavidin through the biotin end on the DNA. After the restriction enzyme cleaves the origami structure, the immobilized DNA can move back and forth through the pore, as evidenced by the interruption of the current. Immobilization allows controlled movement of a single DNA molecule through a pore, which in turn provides both "reading" and "writing" information on DNA.

[0198] As shown in Figure 35, a bulky double-stranded DNA element is formed that is too large to fit into a nanopore, with a single-stranded region connected to the bulky portion by two short double-stranded regions that serve to anchor the DNA to be attached during synthesis . The single stranded region can then detach and anchor to the surface adjacent to the nanopore and the origami structure can be released. See Fig.33.

[0199] Nanopores are formed in 3 mm chips with 20 nm SiO2, with windows of 50*50 µm. The chips are provided by Nanopore Solutions. Nanopore cassette holders and flow cells are provided by Nanopore Solutions. The amplifier is a Tecella Pico 2 amplifier. This amplifier is a USB powered amplifier that uses a USB computer interface to control. Tecella provides software (Windows) to control the amplifier. The multimeter is a FLUKE 17B+ digital multimeter capable of detecting electrical current as low as 0.1uA. For RF interference screening, the inventors use a Concentric Technology Solutions TC-5916A Shield Box (Faraday Cage) with a USB interface. Oligonucleotides are obtained from IDT.com. "PS" is propargylsilane - O-(PROPARGYL)-N-(TRIETHOXYSILYLPROPYL)CARBAMATE from http://www.gelest.com/product/o-propargyl-n-triethoxysilylpropylcarbamate-90/.

[0200] The origami structure is based on a single strand m13 with a honeycomb origami structure whose side is ~20 nm. Near the cell, there are double-stranded regions, each containing a unique restriction site. One of these sites is used to attach the modified DNA to ensure attachment near the nanopore, and the other is used to cleave the DNA structure.

[0201] Formation of nanopores: Nanopores are formed in chips using dielectric breakdown as follows:

1. Chips are carefully placed in cassettes

2. Wetting: 100% ethanol is gently pipetted onto the chip. Bubbles must be removed. However, direct pipetting of the solution on the chip should be avoided or the chip may crack (SiO2 is only 20 nm).

3. Surface treatment: Ethanol is removed and a freshly prepared Piranah solution (75% sulfuric acid, 25% hydrogen peroxide (30%)) is pipetted onto the chip. (The Piranha solution is allowed to reach room temperature). Leave for 30 minutes.

4. Rinse 4 times with distilled water.

5. Rinse 2x with HK buffer (10 mM HEPES, pH 8, 1 M KCl)

6. Assemble the cassette into the flow cell.

7. Add 700 µl HK buffer to each chamber of the flow cell.

8. Insert the silver electrodes connected to the cycler and close the Faraday cage.

9. Test resistance at 300 mV. The current must not be detected. If it is detected, the chip is probably cracked and you need to start over.

10. Connect the electrodes to DC 6V current and test the current using a multimeter. The current should be low and should not change. Increase the voltage by 1.5 V and hold the voltage until the resistance rises. If the resistance does not increase after 5-10 minutes, increase the voltage by another 1.5 V and try again. Repeat until the resistance rises, at which point the applied voltage should be immediately stopped (with sufficient voltage, a dielectric breakdown occurs and a "hole" is formed in the SiO2 membrane. When the initially created hole is small, its size will increase while maintaining the voltage) .

11. Test the pore using an amplifier. At 300 mV, current from a small amount to several nA should be determined. The greater the current, the larger the pore.

[0202] FIG. 34 shows the basis of the functioning of a nanopore. On each panel, the y-axis represents current (nA) and the x-axis represents time (s). In the left panel, "RF noise screening" illustrates the usefulness of the Faraday cage. The chip without the nanopore is placed in the flow cell and 300 mV is applied. When the Faraday cage cover is closed (first arrow), noise reduction can be observed. A small pulse occurs when the latch is closed (second arrow). It should be noted that the current is ~0 nM. After pore formation (middle panel), application of 300 mV (arrow) results in a current of ~3.5 nM. When DNA is placed in the ground chamber and +300 mV is applied, DNA movement can be observed (right panel) as a temporary decrease in current (note that buffer TS is used in this case: 50 mM Tris, pH 8, 1 M NaCl). Lambda DNA is used for this DNA movement experiment.

[0203] Silver chloride electrodes:

1. The silver wire is soldered to the insulated copper wire.

2. The copper wire is grounded and the silver wire is immersed in fresh 30% sodium hypochlorite for 30 minutes.

3. Silver should acquire a dark gray coating (silver chloride).

4. The silver wire is rinsed thoroughly with distilled water and dried.

5. Now it is ready for application.

[0204] Silanation of Beads: The silanation method is initially developed/tested on SiO ₂ coated magnetic beads (GBioscience). The following protocol is used:

1. Preheat pellets in fresh Pirannah solution for 30 minutes.

2. Rinse 3× with distilled water.

3. Rinse 2× with methanol.

4. Dilute APTES stock solution 1:500 in methanol.

5. Add diluted APTES to beads, incubate at rt. within 45 minutes.

6. Rinse with methanol.

7. 100°C for 30 minutes.

8. Store under vacuum.

[0205] Silanation of the silicon chip

1. Install the nanopore chip into the cassette.

2. Rinse with methanol, carefully removing any air bubbles.

3. Add fresh Pirannah solution (equilibrated to room temperature) and incubate for 30 minutes.

4. Rinse 4× with distilled water.

5. Wash 3× with methanol.

6. Dilute APTES stock solution 1:500 in methanol and use to wash the chip 2×. Incubate at rt. within 45 minutes.

7. Rinse 2× with methanol.

8. Air dry.

9. Store under vacuum overnight.

[0206] Streptavidin Bead Conjugation: Streptavidin Conjugation The conjugation was originally developed/tested on silanated beads prepared as described above.

1. Wash the silanated beads with modified phosphate buffered saline (MPBS)

2. Prepare a fresh solution of 1.25% glutaraldehyde in MPBS (using a 50% stock solution of glutaraldehyde stored frozen).

3. Add 1.25% glutaraldehyde to the beads and leave to stand at 60' while gently pipetting up and down every 15 minutes.

4. Wash 2× with MPBS.

5. Rinse 2× with water.

6. Leave to 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 2× with MPBS.

10. Store at 4°C.

[0207] Conjugation of silicon chichpa with streptavidin

1. Rinse the silanated chip with ethanol 2×

2. Rinse the silanated MPBS chip 2×

3. Prepare a fresh solution of 1.25% glutaraldehyde in MPBS (using a 50% stock solution of glutaraldehyde stored frozen).

4. Rinse the chip with 1.25% glutaraldehyde 2×, leave to stand at 60' while gently pipetting up and down every 15 minutes.

5. Wash 2× with MPBS

6. Rinse 2× with water.

7. Leave to dry under vacuum.

8. Add BSA (2 mg/ml in MPBS) to one half of the chip and streptavidin (500 µg/ml in MPBS) to the other half. Incubate for 60 minutes. Make a marking on the cassette indicating which half of the chip is modified with streptavidin.

9. Rinse both halves of the BSA chip (2 mg/mL in MPBS). Incubate for 60 minutes.

10. Rinse with MPBS.

[0208] The buffers used in this example are obtained 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: 10 mM HEPES, pH 8.0, 1M KCl

TE: 10 mM Tris, 1 mM EDTA, pH 8.0

Pirannah solution: 75% hydrogen peroxide (30%)+25% sulfuric acid

APTES stock solution: 50% methanol, 47.5% APTES, 2.5% nanopure water. Maintain at 4°C for at least 1 hour. Store at 4°C.

PDC Stock Solution: 0.5% w/v 1,4-phenylene diisothiocyanate in DMSO.

[0209] Order oligonucleotides (from 5' to 3'):

o1 CTGGAACGGTAAATTCAGAGACTGCGCTTTCCATTCTGGCTTTAATG

o3 GGAAAGCGCAGTCTCTGAATTTAC

N1 CTTACTGGAACGGCTATCGATATCGCAGCAGGACAGA

BN1 Biotin-CTTACTGGAACGGCTATCGATATCGCAGCAGGACAGA

N2 GTCCTGCTGCGATATCGATAGCCGTTCCAGTAAG

[0210] Hybridization of pairs of oligonucleotides is carried out as follows:

1. Prepare stock solutions of oligonucleotides at a concentration of 100 μM in TE buffer

2. Dilute oligonucleotides to 10 µM in PBS

3. Heat to 85°C for 5' in a thermal cycler

4. Gradually lower the temperature by 5°C every 3' to 25°C

5. Store at 4°C or -20°C.

[0211] Streptavidin Conjugation: Streptavidin SiO ₂ conjugation was developed and tested using SiO 2 coated magnetic beads, and then the protocols were adapted for SiO ₂ chips. The binding of biotinylated oligonucleotides to both streptavidin and BSA conjugated beads is tested. As expected, the BSA-conjugated beads show negligible binding, while the streptavidin-conjugated beads show high binding. See Fig.38. Since it is more convenient to carry out conjugation in a high salt concentration (DNA movement is carried out in a high salt concentration), the ability of the beads to bind in HK buffer is also tested. Binding in HK buffer is comparable to binding in MPBS buffer (FIG. 39).

[0212] Obtain origami designs and confirm their operation, as described above in Fig.35-37. Biotinylation of the origami structure is tested using oligonucleotides. It is already known from the "origami" results described above for FIG. 37 that the AlwNI region is active. Below, a pair of oligonucleotides is used that reproduces a segment of the exact origami DNA sequence (o1/o3). An origami molecule is depicted as follows:

[0213] The o1/o3 oligonucleotide pair is

[0214] DNA is digested with AlwNI in the presence of T4 DNA ligase and a biotinylated oligonucleotide complementary to the overhang on the 3' side of the origami sequence (which is itself linked to a long ssDNA sequence, which is itself linked to the other side of the origami), to according to the following reaction:

In this strategy, AlwN1 cleaves the target DNA. When a ligase is added, this DNA can be ligated, but the restriction enzyme will cleave again. However, if/when the (correct) fragment (o1/o3) binds to BN1/N2, the restriction site is not recreated, thus this product will not be cleaved. Specific attachment is confirmed by testing with and without restriction enzyme:

one 2 o1/o3 one one n1/bn2 one one 10× ligation buffer 2 2 Water 15 14.5 AlwN1 0.5 ligase one one

Add all reagents except ligase and the solution is incubated at 37°C for 60 minutes. Add ligase and the solution is incubated overnight at 16°C. 10x ligation buffer refers to 10x T4 NEB DNA ligase buffer. The ligase is T4NEB DNA ligase. o1/o3 and n1/n2 refer to a hybridized pair of oligonucleotides as shown above. Units are microliters. Agarose gel analysis confirms that in the presence of AlwNI, a larger product is formed corresponding to the biotinylated oligonucleotide associated with the long arm of ssDNA associated with the origami structure. A similar strategy is used for 3'-biotinylation when desired.

[0215] Demonstrated above is the ability to form and use a nanopore to detect voltage-induced DNA movement through the pore, create an origami molecule whose long oc region is bound to biotin at its far end, and conjugate streptavidin to silica and use it to trap biotinylated DNA. These tools are used to attach and control the movement of a single DNA molecule near the nanopore.

[0216] The first step is the conjugation of streptavidin to one surface of the SiO2 nanopore (and BSA on the other side). This is carried out in accordance with the protocol described above. The resulting pores tend to have lower current than they originally had. After several short pulses of 6 V, the current returns to almost the original current. The functioning of the nanopore at this point is shown in Fig.40.

[0217] Next add DNA origami. When DNA origami is added to a suitable chamber and the current is turned on, the origami enters the chamber. An illustration of this is shown in Fig.41. The results of the experiment, when origami is applied at a final concentration of 50 pM, confirm that DNA with origami enters the pore relatively quickly (typically within seconds), which is detected by the resulting decrease in current through the nanopore (for example, in these experiments, the current before origami entry is ~3 nA and ~2.5 nA after entry). If the current is allowed to pass for a longer period of time, a double entry can be observed. If higher concentrations are used, entry is too fast to be observed.

[0218] Linking the entered DNA to the chip. After the origami enters the nanopore, the voltage is applied again 15 minutes later. The end of the region in the form of ss-DNA in origami contains biotin, and streptavidin is conjugated to the surface of the nanopore. Streptavidin binds to avidin with an affinity constant close to a covalent bond. A period of 15 minutes allows the DNA to diffuse and end up with biotin to find and bind to streptavidin. If the DNA has indeed bound to the surface, then when the voltage is reversed, the observed current should be somewhat less than previously observed. Also, switching the current in one direction and the other should result in a current in both directions, which is lower than in the case of a free pore. In the example presented here, the free pore exhibits a current of ~3 nA. Fig. 42 shows an image of the attached DNA, and Fig. 43 shows the results of the voltage switching experiment for the attached DNA origami. It should be noted that the current observed in both directions is ~+/-2.5 nA, which is lower than the ~+/-3 nA observed for the free pore. If the DNA is not bound to the surface, the original current will be restored when the voltage is switched (FIG. 44).

[0219] To remove the origami structure, the buffer in the flow cell chamber containing the origami structures was removed and replaced with 1× Swa1 buffer with 1 μl Swa1/20°L. The buffer in the other flow chamber is replaced with 1x Buffer Swa1 without Swa1. Then, incubation is carried out at room temperature for 60 minutes, and then washed with HK buffer, and a voltage is applied. The forward and backward movement of DNA as shown in Fig. 45 is confirmed by the experimental data presented in Fig. 46 showing the controlled movement of immobilized DNA through the SiO2 nanopore.

Example 8 Alternative way of attaching a polymer to a surface next to a nanopore

[0220] The above examples describe attachment of DNA to a surface adjacent to a nanopore by DNA biotinylation and surface coating for attachment with streptavidin. Some alternative methods of attaching the polymer are presented in Fig.47.

[0221] a) DNA hybridization: In one method, DNA that is elongated in the methods of the invention is hybridized to a short oligonucleotide attached near the nanopore. After synthesis is complete, the synthesized DNA can be readily removed without the need for restriction enzymes, or alternatively, the double strand can be formed by a linked oligonucleotide and the synthesized DNA can provide a substrate for the restriction enzyme. In this example, short oligomers are surface-conjugated using biotin-streptavidin, or ligated using 1,4-phenylenediisothiocyanate as follows:

Conjugation of biotinylated DNA with SiO ₂ :

A. SILANATION:

1. Pretreatment: nha solution for 30 minutes, washing with double distilled H ₂ O (ddH ₂ O)

2. APTES stock solution preparation: 50% MeOH, 47.5% APTES, 2.5% nanopure H ₂ O: holding >1 h at 4°C

3. Dilution of APTES stock solution 1:500 in MeOH

4. Incubation of chips at room temperature

5. MeOH rinse

6. Drying

7. Heating at 110°C for 30 minutes

CONJUGATION:

1. Chip treatment with PDC stock solution for 5 hours (room temperature) (PDC stock solution: 0.5% w/v 1,4-phenylenediisothiocyanate in DMSO)

2. 2 washes in DMSO (short!)

3. 2 washes in ddH ₂ O (short!)

4. 100 nM amino-modified DNA in ddH ₂ O (pH 8) O/N 37°C

5. 2 washes with 28% ammonia solution (deactivation)

6. 2 washes with ddH ₂ O.

[0222] Single-stranded DNA having a terminal sequence complementary to the attached oligonucleotides is introduced as described above and allowed to hybridize to the attached oligonucleotides.

[0223] b) Click chemistry: Click chemistry is a general term for reactions that are simple and thermodynamically efficient, do not form toxic or highly reactive by-products, and are carried out in water or biocompatible solvents and are often used to couple selected substrates to specific biomolecules. . Click conjugation in this case uses a chemistry similar to that used in a) to attach oligonucleotides, only in this case it is used to attach a polymer that is elongated during synthesis in the methods of the invention. While the polymer is DNA in this example, this chemistry can attach other polymers functionalized through the addition of a compatible azide group.

A. SILANATION:

1. Pretreatment: Piranha solution for 30e, washing with ddH ₂ O

2. Preparation of stock solution PS (propargylsilane): 50% MeOH, 47.5% PS, 2.5% nanopure H2O: hold >1 h at 4°C

3. Dilution of APTES stock solution 1:500 in MeOH

4. Incubation of chips at room temperature

5. MeOH rinse

6. Drying

7. Heating at 110°C for 30 minutes

[0224] DNA terminated with an azide functional group covalently binds to this surface (as shown in FIG. 47). Azide-terminated oligonucleotides are custom made and linked to a longer DNA origami as described above for attaching biotin to DNA.

Claims (18)

1. A method for synthesizing a charged polymer containing at least two different monomers in a nanochip, wherein the nanochip contains one or more attachment chambers containing reagents for attaching one or more monomers or oligomers to the charged polymer in buffer solution in end-protected form so that only one monomer or oligomer can be attached in one reaction cycle; and one or more reserve chambers containing a buffer solution, but not all of the reagents needed to attach one or more monomers or oligomers, where the chambers are separated by one or more membranes containing one or more nanopores, and where the charged polymer can pass through the nanopore and at least one of the reactants for attaching one or more monomers or oligomers cannot, wherein the method includes a) moving the first end of the charged polymer, having the first end and the second end, under the influence of electrical attraction into the attachment chamber, and the monomers and oligomers are attached to the specified first end in a blocked form, b) moving the first end of the charged polymer with the added monomer or oligomer in blocked form to the reserve chamber, c) deblocking the attached monomer or oligomer, and d) repeating steps a-c, where the monomers or oligomers attached in step a are the same or different, until the desired polymer sequence is obtained, where the charged polymer is DNA, the monomers are nucleotides, where the oligomers are oligonucleotides, and where the nanopore has a diameter of 2-20 nm. 2. The method according to claim 1, where the nanopore has a diameter of 2-5 nm. 3. The method of claim 1 or 2, wherein the reagents for attaching one or more monomers or oligomers to the charged polymer comprise a polymerase. 4. A method according to any one of claims 1 to 3, wherein the nucleotide is attached in a 3'-protected form and wherein the reserve chamber contains reagents capable of removing a protecting group from the attached nucleotide. 5. The method of claim 1 or 2, wherein the one or more attachment chambers comprise a topoisomerase-loaded nucleotide or oligonucleotide and one or more reserve chambers containing a restriction enzyme or phosphatase, wherein the nanopore is small enough such that the topoisomerase and the restriction enzyme or phosphatase unable to pass through the nanopore. 6. The method of claim 5, wherein one nucleotide is added on each round. 7. A nanochip for the synthesis of an electrically charged polymer containing at least two different monomers, wherein the nanochip contains at least first and second reaction chambers separated by a membrane containing one or more nanopores, where each reaction chamber contains one or more electrodes for electrically drawing in charged polymer into the chamber, and where said first reaction chamber further comprises an electrolyte medium and reagents for attaching monomers or oligomers to the polymer, where said monomers or oligomers are protected so that only one can be attached at a time, and where said second reaction chamber further comprises an electrolyte medium and reagents for deprotecting a monomer or oligomer attached in such a way that the nanopore is large enough to allow passage of the polymer but too small to allow passage of at least one of the monomer attachment reagents. moat or oligomers to the polymer and at least one of the reagents for deprotecting the monomer or oligomer attached in such a way, where the charged polymer is DNA, the monomers are nucleotides and the oligomers are oligonucleotides, and where the nanopore has a diameter of 2-20 nm. 8. The method according to claim 7, where the nanopore has a diameter of 2-5 nm.

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