A method for characterizing polynucleotides that travel through nanopores.

Abstract

This provides a method for characterizing target polynucleotides. [Solution] This specification provides a method for characterizing a target polypeptide as it moves toward a nanopore using a motor protein. Polynucleotide adapters and kits comprising such adapters are also provided. The method, kit, and adapter are found to have applications in polynucleotide characterization, e.g., sequencing.

Description

[Technical Field]

[0001] This disclosure relates to target polynucleotides when they move toward a detector such as a transmembrane nanopore. This disclosure provides a method for characterizing [something]. This disclosure also provides a novel polyn for use in such a method. Cleotide adapters and kits are also provided. This disclosure also provides information on rereading polynucleotides. We will also provide methods for obtaining it. [Background technology]

[0002] Nanopore sensing involves the individual bonds or interactions between analyte molecules and ion conduction channels. This is an approach to the detection and characterization of analytes that relies on the observation of action events. The sensor places a single pore of nanometer size within an insulating film and, in the presence of analyte molecules, the pore... It can be fabricated by measuring the voltage-driven ion current passing through it. (Inside the nanopore) Alternatively, if the analyte is nearby, the ion flow through the pore changes, and measurements can be taken across the channel. A fixed ionic current or a change in current will result. The identity of the analyte is determined by its solid The current signature, in particular the duration and extent of the current block, and the interaction with the pore. This becomes apparent through fluctuations in the current level during the process.

[0003] Polynucleotides are important analytes for sensing in this manner. Nanopore sensing of analytes reveals their identity and the sensed analytes. It is possible to count single molecules, but their composition such as their nucleotide sequences, and Furthermore, it provides information on the presence of features such as base modification, oxidation, reduction, decarboxylation, and deamination. It is also possible to do so. Nanopore sensing enables rapid and inexpensive polynucleotide sequencing. It has the potential to enable single molecular sequence reads of polynucleotides with lengths of tens of thousands to tens of thousands of base pairs. provide.

[0004] Two of the key components of polymer characterization using nanopore sensing are, 1) Control of polymer movement through pore, and (2) when polymer moves through pore This is the identification of the building blocks of the component. Nanopores of analytes such as polynucleotides. During synthesis, it is important to control the movement of polynucleotides to the pore. If this is not controlled, accurate characterization of polynucleotides may be hindered or obstructed. It has a sex. For example, if the movement of polynucleotides to pores is not controlled, homo Precisely distinguishing each nucleotide within a polymer polynucleotide becomes problematic.

[0005] By using motor proteins that control the movement of polynucleotides, nanoparticles It is known to control the movement of polynucleotides relative to detectors such as A. Motor proteins include helicases, exonucleases, topoisomerases, and other types of motor proteins. It contains polynucleotide handling enzymes. Motor proteins are polynucleotides. To process in a controlled manner. Therefore, using motor proteins, nanopores The movement of polymers such as polynucleotides relative to any detector can be controlled.

[0006] When the detector is a nanopore, the disclosed method typically involves detecting motor proteins. This includes using to supply polynucleotides to nanopores. This operation is as described herein. It is described in more detail. Methods for supplying polynucleotides to nanopores have been widely developed. has been proven to be extremely useful for characterizing polynucleotides.

[0007] However, there remains a need for additional methods for characterizing polypeptides . One problem is that when supplying a polynucleotide to a detector such as a nanopore , there may be cases where it is desirable to obtain data different from that obtained from methods comprising such supply . For example, in a method comprising supplying a polynucleotide to a detector, the error profile of data resulting from characterizing the polynucleotide may, in some circumstances, not be optimal for accurate characterization of the polynucleotide. Another problem is that when a motor protein is used to supply a polynucleotide to a detector such as a nanopore, the motor protein may move forward in an uncontrolled manner along the polynucleotide strand, a phenomenon that is also known as slippage. When slippage occurs, for example, one or more nucleotides within the polynucleotide may not be accurately characterized , which can cause problems when characterizing the polynucleotide. This is particularly problematic when characterization of the polynucleotide involves determining its sequence . As a strategy for reducing slippage, conventional approaches have focused on modifying motor proteins to minimize the tendency of the protein to slip along the polynucleotide strand . However, alternative methods for moving a polynucleotide relative to a detector such as a nanopore that can reduce slippage would also be useful.

[0008] There also exists a need for approaches to improve the data obtained when characterizing polynucleotides It exists. One problem is that, in some cases, when characterizing polynucleotides, It is desirable to improve the accuracy of the feature-recognition data. Several known people In the method, multiple polynucleotides are characterized from a polynucleotide sample, and obtained The collected data is aggregated, which improves overall accuracy. However, this can result in Possible problems exist. For example, heterogeneity in a sample can be difficult to characterize when multiple polynucleotide chains are being identified. When aggregating data obtained from these sources, there is a possibility that useful information regarding differences between the chains may be lost. This could mean that there is a further step after the first strand has been processed for characterization. Because it is necessary to capture new chains, inefficiencies can occur. Therefore, Alternative and / or improved methods for characterizing creotides are needed.

[0009] For these and other reasons, transferring polynucleotides to detectors such as nanopores is difficult. Novel and / or improved methods for moving it are needed. [Overview of the project]

[0010] This disclosure describes how to move a target point relative to a detector using a motor protein. This disclosure relates to a method for characterizing renucleotides. More specifically, this disclosure relates to a motor protein This concerns how the substance moves polynucleotides out of the detector. The direction of rheotide movement is opposite to the known way in which polynucleotides move into nanopores. Yes, it exists. This is described in more detail in this specification.

[0011] In the disclosed method, the motor protein stalls on a polynucleotide. The ing part initially stalls, and the method provided herein This involves a motor protein detecting the movement of polynucleotides from a detector (e.g., a nanopore). This involves causing the motor protein to lose momentum so that it can control movement. Methods for stalling and destalling ter proteins are described in detail herein.

[0012] This disclosure provides nanopores as exemplary detectors, but the methods provided herein (i) zero-mode waveguide, (ii) field-effect transistor, optionally a Noyar field (iii) effect transistor, (iv) AFM chip, (iv) nanotube, optionally car The disclosed method is suitable for detectors such as von nanotubes and (v) nanopores. , polynucleotides pass through the detector or include a detector such as a well in the detector chip. It is particularly suitable for methods of movement through structures.

[0013] Therefore, this specification provides a method for characterizing a target polypeptide, and this method , (i)(i) a detector having a first aperture and a second aperture, or (ii) a detector including A structure having a first opening and a second opening is brought into contact with a target polynucleotide. This causes the target polynucleotide to move the stalled motor protein onto it. The motor protein is stalled at the stalled region, and contact is necessary. (ii) The stalled portion is brought into contact with the nanopore, thereby causing the motor protein to destall. to make contact, (iii) The motor protein passes through the detector or structure from the second opening to the first opening When controlling movement in the direction of the target polynucleotide, one or more characteristic measurements of the target polynucleotide This involves measuring a constant value, thereby characterizing the target polynucleotide. This includes the act of doing something.

[0014] Furthermore, this specification provides a method for characterizing a target polynucleotide, and this method is (i) Contact the detector with the target polynucleotide to which the motor protein is bound. Therefore, the target polynucleotide is at the polynucleotide binding site of the motor protein. It is bound to the motor protein, and contact is made. (ii) The motor protein moves the target polynucleotide in the first direction relative to the detector. When controlling, obtain one or more measurements characteristic of the target polynucleotide. , (iii) The motor moves the target polynucleotide in a second direction relative to the detector. - Debinding target polynucleotides from the polynucleotide binding site of proteins , (iv) Reattach the target polynucleotide to the polynucleotide binding site of the motor protein. Combined, the motor protein moves the target polynucleotide in a first direction relative to the detector. When controlling, obtain one or more measurements characteristic of the target polynucleotide. This includes characterizing the target polynucleotide.

[0015] Furthermore, this specification provides a method for characterizing a target polynucleotide, and this method is (i) The first opening of a transmembrane nanopore having a first opening and a second opening is used as a target opening. The process involves contacting the polynucleotide, causing the target polynucleotide to stall on it. It has a motor protein, and the motor protein is stalled at the stalled region, contact to, (ii) The stalled portion is brought into contact with the nanopore, thereby causing the motor protein to destall. to make contact, (iii) The motor protein moves from the second opening of the nanopore to the first opening of the nanopore. When controlling the movement of target polynucleotides through nanopores in the direction of the target polynucleotide, This involves obtaining one or more measurements characteristic of the creotide, thereby targeting the poly Characterizing nucleotides, including obtaining them.

[0016] In some embodiments, the nanopore extends across a film having a cis side and a trans side. The first opening of the nopore is on the cis side of the membrane, and the second opening of the nanopore is on the trans side. Yes, the motor protein moves from the trans side to the cis side of the membrane through the target polymer pore via the nanopore. Controlling nucleotide movement. In some embodiments, the nanopore is cis-side and trans-side. In a film having a trans side, the first opening of the nanopore is on the trans side of the film, and the nanopore The second opening of A is on the cis side, and the motor protein moves from the cis side of the membrane to the trans side. This controls the movement of target polynucleotides through nanopores.

[0017] In some embodiments, the method involves applying force across nanopores. The target polynucleotide passes through the nanopore in the opposite direction to the applied force. The movement is controlled, and the force preferably includes an electric potential applied across the nanopore.

[0018] In some embodiments, the motor protein is a helicase. Morphologically, motor proteins are DNA-dependent ATPase (Dda) helicases. ru.

[0019] In some embodiments, the adapter is located at one or both ends of the target polynucleotide. It is attached to it. In some embodiments, the motor protein stalls on the adapter. They are doing it.

[0020] In some embodiments, the nanopore is a leader at the first end of the target polynucleotide. The motor protein captures the sequence and, at the second end of the target polynucleotide, or at the target It is stalled on the adapter attached to the second end of the polynucleotide.

[0021] In some embodiments, - The target polynucleotide is single-stranded, - The target polynucleotide contains a leader sequence, and the leader sequence is the target polynucleotide. The ada is located at the first end of the tide or attached to the first end of the target polynucleotide. It is included in the putter, and, - The motor protein is stalled at the second end of the target polynucleotide, or The target polynucleotide is stalling on the adapter at its second end.

[0022] In some embodiments, the target polynucleotide is double-stranded.

[0023] In some embodiments, - The target polynucleotide is double-stranded and comprises a first strand and a second strand. - The target polynucleotide contains a leader sequence, and the leader sequence is a polynucleotide Located at the first end, and included in the first chain, or included in an adapter attached to the first chain. Rare, and, - The motor protein stalls at the second end of the target polynucleotide.

[0024] In some embodiments, the motor protein is the first chain of the target polynucleotide It stalls at the second end, or the adapter at the second end of the first strand of the target polynucleotide. - Stalls above. In some embodiments, the first chain and the second chain are the second of the first chain The motor protein attaches together with the hairpin adapter at the end, and the hairpin adapter It stalls at the chute. In some embodiments, the first chain and the second chain are (i) the first chain (ii) by a hairpin adapter attached to the second end of the second chain and (ii) to the first end of the second chain They attach together, and the motor protein is at the second end of the second strand of the double-stranded polynucleotide. It stalls either at the end or at the second end of the second chain on the adapter.

[0025] In some embodiments, the target polynucleotide includes a portion complementary to the tag sequence. In some embodiments, the target polynucleotide is hybridized to an oligonucleotide. The oligonucleotide contains a creotide-containing moiety, and (a) the target polynucleotide (b)(i) The hybridization part for hybridization and the part complementary to the tag sequence. (ii) an affinity molecule that can bind to the tag. Several embodiments So, the target polynucleotide is double-stranded, and the part complementary to the tag sequence is the polynucleotide. The first chain portion of Otid, and / or an oligonucleotide hybridized thereto The portion containing tide is the first chain of the polynucleotide.

[0026] In some embodiments, the motor protein is one independently selected from the following: The vehicle stalls at the stall region, which includes the stall unit shown above: -Polynucleotide secondary structure, preferably hairpin or G-quadrivalent (TBA), - Preferably, peptide nucleic acid (PNA), glycerol nucleic acid (GNA), threose nucleus Acids (TNA), locked nucleic acids (LNA), cross-linked nucleic acids (BNA), and debasalized nucleotides nucleic acid analogs selected from, - Nitroindole, inosine, acridine, 2-aminopurine, 2-6-diaminopurine Phosphorus, 5-bromodeoxyuridine, inverted thymidine (inverted dTs), inverted dideoxy- Thymidine (ddTs), dideoxycytidine (ddCs), 5-methylcytidine, 5- Hydroxymethylcytidine, 2'-O-methylRNA base, isodeoxycytidine (Is o-dCs), isodeoxyguanosine (Iso-dGs), C3(OC3H6OPO3) ) group, photocleavable (PC) [OC3H6-C(O)NHCH2-C6H3NO2-CH( CH3)OPO3 group, hexanediol group, spacer 9 (iSp9)[(OCH2C H2)3OPO3] base, spacer 18 (iSp18) [(OCH2CH2)6OPO3 A spacer unit selected from ] groups and thiol linkages, and -Fluorophores, traptabidine, streptavidin, and neutraavidin, etc. avidin and / or biotin, cholesterol, methylene blue, dinitrophenol (DNP), digoxigenin and / or anti-digoxigenin and dibenzylcyclooctane n base.

[0027] In some embodiments, the motor protein is destamped, and polynucleotides This includes applying a destall force to the drive, wherein the destall force is less than the reading force, and / or In the opposite direction, the reading power is controlled by the motor protein's ability to control the movement of the target polynucleotide. This method is applied while measurements are being taken to determine one or more characteristics of a polynucleotide. It is the force that causes the motor protein to lose speed. In some embodiments, the force that causes the motor protein to lose speed is The applied force includes stepping one or more times between the destabilization force and the reading force.

[0028] In some embodiments, the motor protein comprises one or more stall units and one or more The vehicle stalls in the stalled region, which includes the pausing portion, and one or more of the pausing portions become nanopores. Contact delays the movement of polynucleotides through the nanopore, thereby slowing down the movement of the nanonucleotides. The lore protein destalls from one or more stall units. In some embodiments, it stops. The section includes one or more stopping units selected independently from the following: -Polynucleotide secondary structure, preferably hairpin or G-quadrivalent (TBA), - Preferably, peptide nucleic acid (PNA), glycerol nucleic acid (GNA), threose nucleus Acids (TNA), locked nucleic acids (LNA), cross-linked nucleic acids (BNA), and debasalized nucleotides nucleic acid analogs selected from, -Fluorophores, traptabidine, streptavidin, and neutraavidin, etc. avidin and / or biotin, cholesterol, methylene blue, dinitrophenol (DNP), digoxigenin and / or anti-digoxigenin and dibenzylcyclooctane n group, and -Polynucleotide-binding protein.

[0029] In some embodiments, the target polynucleotide is a motor protein polynucleotide. It includes a blocking portion that prevents detachment from the ocid. In some embodiments, The target polynucleotide contains a leader sequence at the first end of the target polynucleotide, The target protein is at the second end of the target polynucleotide, or the target polynucleotide The motor stalls on the adapter attached to the second end, and the blocking part is the motor protein It is located between the protein and the second end of the polynucleotide, thereby allowing the motor protein to Prevents the target polynucleotide from unassociating at its second terminus. .

[0030] The first end and second end contain attachment sites for attachment to the double-stranded polynucleotide analyte. A polynucleotide adapter having ends is also provided, and the polynucleotide adapter is, (i) A stalled motor on top of the adapter in an orientation for processing the adapter in the direction of the attachment point. (ii) Located between the motor protein and the second end of the adapter This includes the blocking portion.

[0031] Furthermore, the first adapter described herein includes a single-stranded leader sequence at the first end. , a second adapter containing an attachment site at the second end for attaching to the double-stranded polynucleotide analyte A kit including the pod is also offered.

[0032] In some embodiments of the polynucleotide adapter or kit provided herein The polynucleotide adapter, the motor protein, and / or the blocking The parts are as defined herein. [Brief explanation of the drawing]

[0033] [Figure 1]A schematic diagram illustrating the distinction between (A) the direction of polynucleotide (PN) transfer from a nanopore under the control of a motor protein by the method provided herein, and (B) the direction of polynucleotide transfer to a pore by a contrasting method. The open arrows indicate the direction of transfer of the motor protein (MP) and PN. In both cases, the MP is, for example, a 5'-3' helicase. [Figure 2] This is a schematic diagram of one embodiment of the method provided herein, in which the target polynucleotide is single-stranded, the target polynucleotide comprising a leader sequence located at the first end of the target polynucleotide, and a motor protein stalled at the second end of the target polynucleotide by a stall region (x). The leader sequence is captured by a nanopore, and the single-stranded polynucleotide moves through the nanopore until it reaches the stalled motor protein. Once destalled, the motor protein controls the movement of the polynucleotide from the pore. [Figure 3] This is a schematic diagram of one embodiment of the method provided herein, in which the target polynucleotide is double-stranded, the target polynucleotide comprising a leader sequence (wavy line) located at the first end of the first strand of the target polynucleotide, and a motor protein stalled at a stalled region (x) at the second end of the first strand of the target polynucleotide. The leader sequence is captured by a nanopore, and the first strand of the target polynucleotide moves through the nanopore until it reaches the stalled motor protein. After destallation, the motor protein (MP) controls the movement of the first strand of the target polynucleotide (PN) from the pore. [Figure 4]This is a schematic diagram of one embodiment of the method provided herein, wherein the target polynucleotide is double-stranded, and the target polynucleotide includes a leader sequence (wavy line) located at the first end of the first strand of the target polynucleotide, and a motor protein (MP) is stalled at a stall portion (x) of a hairpin adapter connecting the second end of the first strand of the target polynucleotide to the first end of the second strand of the target polynucleotide. The leader sequence is captured by a nanopore, and the first strand of the target polynucleotide moves through the nanopore until it reaches the stalled motor protein. After destallation, the motor protein controls the movement of the first strand of the target polynucleotide (PN) from the pore. [Figure 5] This is a schematic diagram of one embodiment of the method provided herein, wherein the target polynucleotide is double-stranded, the target polynucleotide comprising a leader sequence located at the first end of the first chain of the target polynucleotide, and a hairpin adapter ligating the second end of the first chain of the target polynucleotide to the first end of the second chain of the target polynucleotide. A motor protein (MP) stalls at a stall region (x) at the second end of the second chain of the target polynucleotide. The leader sequence (wavy line) is captured by a nanopore, and the first chain of the target polynucleotide, the hairpin adapter, and the second chain of the target polynucleotide pass through the nanopore until they reach the stalled motor protein. After destallation, the motor protein controls the movement of the second chain of the target polynucleotide (PN), the hairpin adapter, and the first chain as they exit the pore. [Figure 6]This is a nanopore sequencing adapter with a DNA helicase that moves from 5' to 3', where the 3' strand is preferentially captured in the nanopore. The adapter contains two oligonucleotides known as the top strand (A) and the bottom strand (B). The top strand contains a 5' biotin moiety (C) complexed with monovalent traptabidine (D), where the DNA motor (direction 5'-3') is loaded into a closed poly(dT) binding site (E), stalled by an internal spacer 18 moiety (F), and the 3' dT base is offered for ligation to a double strand (G) with a dA tail. The bottom strand contains a 5' phosphate moiety (H), a double-stranded region (I) containing a BNA base as a stalling chemical group, 20 consecutive 3' terminal thymidine bases as a leader (wavy line, J), and a site (K) for hybridizing a hydrophobic tether. See Example 1. [Figure 7] Figure 6 is a conceptual diagram showing how a sequencing adapter (A) is ligated to both ends of a double-stranded DNA polynucleotide (B) with a dA tail to generate a continuous double helix. [Figure 8] This is a schematic diagram of the experiment in Example 1, showing the capture, destallation, and sequencing of polynucleotide analytes. Vs: sequencing potential, Vu: deblocking potential. The polarity of the applied potential is indicated by the arrow. The direction of the applied force is the same as the direction of the arrow. (A) Applying the sequencing potential (120mV). Capture of polynucleotide analytes via the open pore and 3' leader (from Figure 7). Separation of the double helix by the nanopore, and the complementary strand is removed. (B) The polynucleotide reaches the enzyme, which is stalled in the spacer region. The enzyme cannot move over the spacer region. (C) Applying the deblocking potential (0mV) so that the enzyme moves away from the nanopore and moves freely over the spacer region. (D) Applying the sequencing potential (120mV). The polynucleotide moves through the nanopore until the enzyme reaches the nanopore, and then the enzyme controls the movement of the polynucleotide from the nanopore. (E) The DNA motor reaches the leader and becomes idle. (F) The deblocking potential is applied, and the DNA motor and analyte are expelled from the nanopore. Repeat the cycle from (A). [Figure 9]Top: Representative current-time traces from Example 1. States A-F correspond to the states described in Figure 8. Bottom: Magnified view (1 second) of the area enclosed by the rectangle in the trace above, showing the controlled movement of polynucleotides from the nanopore. The applied potentials are as follows: A and B: 120 mV, C: 0 mV, D, E and F: 120 mV, and the cycle is repeated. [Figure 10] The components of the experiment described in Example 2, in which both chains of the polynucleotide analyte are first rearranged through the nanopore without enzyme, then the enzyme “de-stalls”, and then the enzyme controls the movement of both chains of the polynucleotide analyte from the nanopore. A. Adapter including a hairpin portion and a 3'-TCCT overhang that specifically binds to one end of the polynucleotide analyte. B. Sequencing adapter identical to that described in Example 1 and Figure 6. C. Polynucleotide analyte with asymmetric ends, one having a 3'dA tail and the other having a 3'-AGGA overhang. The template chain and complementary chain are shown by dashed and solid lines, respectively. Ligation of DA, B, and C yields library molecule D. [Figure 11]This is a schematic diagram of the experiment in Example 2, showing the capture of both strands of a polynucleotide analyte, "de-stalling," and sequencing. Vs: sequencing potential, Vu: deblocking potential. The polarity of the applied potential (if not zero) is indicated by the arrow. The direction of the applied force is the same as the direction of the arrow. (A) Applying the sequencing potential (120mV). Capture of the polynucleotide analyte via the open pore and 3' leader (from Figure 7). Separation of the double strand by the nanopore, the template and complementary strands move into the transcompartment. (B) The polynucleotide reaches the enzyme, which is stalled in the spacer region. The enzyme cannot move over the spacer region. (C) Applying the deblocking potential (variable, 0mV to -120mV) so that the enzyme leaves the nanopore and moves freely over the spacer region. (D) Applying the sequencing potential (120mV). The polynucleotide moves through the nanopore until the enzyme reaches the nanopore, and then the enzyme controls the movement of the polynucleotide from the nanopore. (E) The DNA motor moves through the template portion and reaches the hairpin. (F) The DNA motor moves through the complementary portion, and the template and complementary strands refold in the cis-compartment. The motor reaches the leader section and idles in the nanopore. A deblocking potential is applied, and the DNA motor and analyte are ejected from the nanopore. [Figure 12] (a) A typical current-time trace of the data from Example 2, where the destamping voltage varies between 0 and -120mV. When the discharge potential increases above -60mV, no event is observed, suggesting that the hairpin formed in the transformer provides resistance to chain discharge up to this voltage. The portion where movement is controlled is shown as a box enclosed by a dashed line. (b) A typical current-time trace of the event described in Example 2. States A to G correspond to those described in Figure 11. [Figure 13]This is a representative current-time trace from Example 3, showing the capture of polynucleotide analytes into the nanopore and controlled movement from the nanopore. The DNA motor was “de-stalled” using the “active de-stalling” process described in Example 3. Asterisks indicate where the active stall potential was applied, with a 3-second pause between de-stalling attempts, initially up to 5 times at 5 seconds, then up to 5 times at 25 seconds. After the first attempt at 5 seconds, the enzyme de-stalled, as in Examples 1 and 2, controlling the movement of polynucleotides out of the nanopore with the template (Temp.) and complementary (Comp.) sections, followed by the leader state. A: Current-time trace showing the behavior of a “1D DNA library” similar to that described in Example 1, de-stalled after the first attempt. B: Current-time trace showing the behavior of a bound template-complementary polynucleotide (“2D DNA library”) de-stalled after 4 attempts, similar to that described in Example 2, bound by a hairpin portion. [Figure 14] The hairpin portion of the experiment described in Example 4 is used, in which both strands of the polynucleotide analyte are first rearranged through the nanopore without enzyme, then the enzyme is “de-stalled”, and then the enzyme controls the movement of both strands of the polynucleotide analyte from the nanopore. Additional portions of the hairpin introduce an additional signal during the initial enzyme-free capture step. These portions are illustrated as follows: (A) No portion in the hairpin as a control. (B) Hairpin with oligonucleotide i hybridized into the hairpin loop. (C) Three consecutive fluorescein dT bases ii in the hairpin loop, indicated by the asterisk. (D) As in (C), but using oligonucleotide hybridized into the hairpin loop. [Figure 15]This schematic diagram illustrates the capture and enzyme-free rearrangement of a double-stranded polynucleotide analyte with a hairpin portion, where the hairpin portion has an optionally large fluorophore and optionally an oligonucleotide that hybridizes into a hairpin loop. The schematic diagram shows two additional detectable intermediates, A1 and A2, which correspond to the oligonucleotide hybridized into the hairpin loop at the top of the nanopore using the fluorophore in the lumen of the nanopore, and the fluorophore in the lumen of the nanopore only. An additional state D1 corresponds to the fluorophore in the lumen of the nanopore and an enzyme moving along the fluorophore. [Figure 16(a)](a) Data showing the identification of the aenzymatic transfer of polynucleotides in which the template and complementary strands are linked via a hairpin portion. The polynucleotides are guided through the nanopore via an applied potential before the enzyme-controlled transfer step. The schematic diagram of the experiment is similar to that described in Example 2 and Figure 11. The hairpin is shown in Figure 14A. (i) A sequencing adapter containing only DNA and a polynucleotide library linked to the hairpin adapter. (ii) Representative current-time traces of the molecules shown in (i). The assignment of components A-G is based on states A-G described in Figure 11. (iii) A magnified view of the area enclosed by a rectangle shown in (ii), showing the identification of open pore level A and stall level B. The area enclosed by an asterisk has a different shape and noise than B, and also differs in relation to other representative molecules described in this example, and is presumed to arise from the aenzymatic rearrangement portion. (b) Data showing the identification of aenzymatic transfer of polynucleotides, where the template and complementary strands are linked via a hairpin portion, and the oligonucleotide hybridizes to the hairpin. The polynucleotide is guided through the nanopore via an applied potential before an enzyme-controlled transfer step. The schematic diagram of the experiment is similar to that described in Example 2 and Figure 11. The hairpin is shown in Figure 14B. (i) A polynucleotide library linked to a sequencing adapter and a hairpin adapter containing DNA hybridized with oligonucleotides (ON). (ii) Representative current-time traces of the molecules shown in (i). The assignment of components A-G is based on states A-G described in Figure 11. (iii) A magnified view of the area enclosed by a rectangle shown in (ii), showing the identification of open pore level A and stall level B. Compared to the example shown in Figure 16a, an additional level A2 (described in Figure 15) arises from the hybridized oligonucleotide. Thus, the asterisked region corresponds to aenzymatic rearrangement. (c) This data shows the identification of the non-enzymatic transfer of polynucleotides in which the template and complementary strands are linked via a hairpin region, and the hairpin has three bulky groups (three consecutive fluorescein dT bases, FAM).The polynucleotides are guided through the nanopore via an applied potential before the enzyme-controlled migration step. The schematic diagram of the experiment is similar to that described in Example 2 and Figure 11. The hairpin is shown in Figure 14C. (i) Polynucleotide library linked to a sequencing adapter containing fluorescein bases and a hairpin adapter. (ii) Representative current-time trace of the molecule shown in (i). The assignment of components A-G is based on states A-G described in Figure 11. The additional level D1 is presumed to result from the slow migration of the enzyme through the bulky FAM region. (State F is not seen in this example because the complementary region E is reduced for the efflux step G). (iii) Enlarged view of the region enclosed in the rectangle shown in (ii), showing the identification of open pore level A and stall level B. Compared to the example shown in Figure 16a, an additional downtick current level A1 of approximately 20 pA (see Figure 15) is generated from the FAM group. Thus, the asterisked region corresponds to an aenzymatic rearrangement. (d) Data showing the identification of non-enzymatic transfer of polynucleotides in which the template and complementary strands are linked via a hairpin region, with three bulky groups (three consecutive fluorescein-dT bases, FAM) present on the hairpin, where oligonucleotides (ON) hybridize. The polynucleotides are guided through the nanopore via an applied potential before an enzyme-controlled transfer step. The schematic diagram of the experiment is similar to that described in Example 2 and Figure 11. The hairpin is shown in Figure 14D. (i) Oligonucleotides (ON) hybridize to a polynucleotide library ligated to a sequencing adapter containing fluorescein bases (FAM) and a hairpin adapter. (ii) Representative current-time trace of the molecule shown in (i). The assignment of components A-G is based on states A-G described in Figure 11. An additional level D1 with a downtick in the current level is presumed to result from the slow movement of the enzyme in the bulky FAM region. (iii)(ii) is an enlarged view of the area enclosed by the rectangle, showing the distinction between open pore level A and stall level B.Compared to the examples shown in Figures 16a and 16c, an additional downtick current level A1 (see Figure 15) of approximately 20 pA is generated from the FAM group. Compared with Figure 16b, an additional level A2 due to hybridized ON is also observed. Therefore, the asterisked region corresponds to an enzymatic rearrangement. (e) Measurement of the duration of enzymatic rearrangement in the E. coli test library. (i) Four representative examples from the random E. coli test library described in Example 4, where double-stranded polynucleotides are ligated to a sequencing adapter at one end and to a hairpin portion at the other end. Oligonucleotides are hybridized to the hairpin portion. Therefore, the resulting polynucleotides are similar to those in Figure 16b, except that the polynucleotides are of random length. The four examples shown are event-fitted current-time traces to simplify the raw data. Level A2 and the enzymatic portion (indicated by an asterisk) are shown in each example. A threshold of 60 pA (dotted line) was used to distinguish the enzyme-free portion A2. Therefore, the periods marked with an asterisk were measured as the time during which the threshold of 60 pA between the pore level A (where the current opened) and the oligonucleotide level A2 exceeded. (ii) The relationship between the enzyme-controlled chain duration (measured as the sum of periods D and E shown in Figure 16b, ii) and the unenzymatic capture duration (measured as described in Part i of this figure) was measured for 30 examples and is shown as a scatter plot. A linear regression line with an R2 value of 0.414 is shown, indicating a positive correlation. [Figure 16(b)] (As stated above.) [Figure 16(c)] (As stated above.) [Figure 16(d)] (As stated above.) [Figure 16(e)] (As stated above.) [Figure 17(a)](a) A nanopore sequencing adapter having a DNA helicase that moves from 5' to 3', where the 3' strand is preferentially captured by the nanopore. The enzyme stalls via another blocker strand containing a BNA region, and the helicase stalls via a spacer portion on the strand loaded with it. The adapter contains oligonucleotides known as the top strand (A), bottom strand (B), blocker strand (C), and back blocker (D). Both the blocker strand and the back blocker hybridize to the double-stranded top strand forming region. The DNA motor (direction 5'-3') is loaded into a closed poly(dT) binding site (E) in the single-stranded region between C and D and stalls via an internal spacer 18 portion (F). The top strand has a 3'dT base for ligation into a dA-tailed double strand. The bottom chain includes a 5' phosphate moiety (circled P), 20 consecutive thymidine bases as a leader (wavy line G), and a site for hybridizing the hydrophobic tether (H). (b) A schematic diagram showing the sequencing adapter (A) described in Figure 17a, ligated at both ends to a double-stranded polynucleotide analyte (B). (c) A schematic diagram of the experiment in Example 5 showing the capture, destallation, and sequencing of the polynucleotide analyte. Vs: sequencing potential, Vu: deblocking potential. The polarity of the applied potential is indicated by the arrow. The direction of the applied force is the same as the direction of the arrow. (A) Applying the sequencing potential (120mV). Capture of the polynucleotide analyte via the open pore and 3' leader (from Figure 7). Separation of the double strand by the nanopore, and the complementary strand is removed. (B) The nanopore is temporarily stalled at the blocker strand portion. (C) The polynucleotide reaches the enzyme stalled at the spacer portion. The enzyme cannot move over the spacer portion. (D) A deblocking potential is applied (0mV) to allow the enzyme to move away from the nanopore and move freely over the spacer portion. (E) A sequencing potential is applied (120mV). The nanopore moves polynucleotides until the enzyme reaches the nanopore, after which the enzyme controls the movement of polynucleotides from the nanopore. (F) The DNA motor reaches the leader and becomes idle. (G) A deblocking potential is applied, and the DNA motor and analyte are ejected from the nanopore. Repeat the cycle from (A).(d)i, a representative current-time trace of Example 5, showing the capture of polynucleotide analytes into and controlled movement from the nanopore using an adapter in which the biotin-traptabidine backblocker is replaced with another backblocker oligonucleotide, as described in Figures 17a and 17b. The DNA motor was “destalled” using the “activation destallation” process described in Example 5 and the earlier Example 3. Levels A to G (described in Figure 17c) are assigned in relation to the previous example. The squared regions ii (enzyme-free rearrangement) and iii (enzyme-controlled rearrangement) are shown in magnified view. [Figure 17(b)] (As stated above.) [Figure 17(c)] (As stated above.) [Figure 17(d)] (As stated above.) [Figure 18(a)](a) Schematic diagram of Example 6, showing the capture, destallation, and sequencing of both strands of the polynucleotide analyte, with occasional rereading of the strands. Vs: sequencing potential, Vu: deblocking potential. The polarity of the applied potential is indicated by the arrow. The direction of the applied force is the same as the direction of the arrow. (A) Applying the sequencing potential (120mV). Capture of the polynucleotide analyte via the open pore and 3' reader (from Figure 7). Separation of the double strand by the nanopore, the template and complementary strands move into the transcompartment. (B) The polynucleotide reaches the enzyme stalled in the spacer region. The enzyme cannot move over the spacer region. (C) Applying the deblocking potential (variable, 0mV to -120mV) so that the enzyme detaches from the nanopore and moves freely over the spacer region. (D) Applying the sequencing potential (120mV). The nanopore moves the polynucleotide until the enzyme reaches the nanopore, and then the enzyme controls the movement of the polynucleotide from the nanopore. (E) The DNA motor moves the template portion and reaches the hairpin. (F) The DNA motor moves the complementary portion, and the template and complementary strands refold in the cis-compartment. The motor reaches the leader section and idles in the nanopore. State (F) is pushed back to state (E) by the enzyme from 3'-5', enabling a strand reread (RR). (G) A deblocking potential is applied, and the DNA motor and analyte are ejected from the nanopore. (b) A representative current-time trace from Example 6, showing an example where the polynucleotide enzyme is read twice via the enzyme being pushed backward from the C3 reader under the applied potential. Enlarged view of enzyme-controlled portions (i) and (ii) and the C3 level is also identified. (c) Six representative reread examples from the experiment described in Example 6. The enzyme-regulated regions were mapped using an HMM model trained with data from the pore and enzyme combinations used. The example readings indicate that the same strand of the bacteriophage lambda DNA mixture of seven restriction enzyme fragments is mapped at least twice. [Figure 18(b)] (As stated above.) [Figure 18(c)] (As stated above.) [Figure 19(a)] (a) An example of a typical HMM mapping of the data described in Example 7, using data collected at a sequencing potential of 120 mV. (b) An example of a typical HMM mapping of the data described in Example 7, using data collected at a sequencing potential of 140 mV. (c) An example of a typical HMM mapping of the data described in Example 7, using data collected at a sequencing potential of 160 mV. (d) Histograms of single-molecule enzyme rates extracted from the data in Figures 19a, 19b, and 19c. The number of molecules in each population is shown. The medians for each population are as follows: 120 mV, 319 bp / sec, 140 mV, 259 bp / sec, 160 mV, 196 bp / sec. [Figure 19(b)] (As stated above.) [Figure 19(c)] (As stated above.) [Figure 19(d)] (As stated above.) [Figure 20(a)] (a) This is an experimental diagram, identical to Figure 18a / Example 6. In addition, the “entry” stage used to measure the enzyme-free rearrangement (between steps A and C) is marked with an asterisk. (b) These are representative current-time traces of the three library examples shown in Example 8, with a 10kb PCR fragment (top), bacteriophage lambda DNA (center), and T4 DNA (bottom). Full-length readings of T4 DNA are not recorded; examples of partial fragments are shown. In each example, the “entry” stage is marked with an asterisk, and the enzyme-controlled stage is marked with an E. The duration of each portion is measured manually and marked on the trace. A magnified view of the entry stage of the T4 example is shown. The portion marked with B in Figure 20a (blocker oligonucleotide at the top of the pore) cannot be reliably detected. (c) This is a log-log scatter plot of measured capture times measured from the traces of the 31 examples described in Example 8. Markers are colored grayscale according to the library of origin. [Figure 20(b)] (As stated above.) [Figure 20(c)] (As stated above.) [Modes for carrying out the invention]

[0034] The present invention will be described in relation to specific embodiments and with reference to certain drawings, however The invention is not limited to those, but is limited only by the claims. None of these reference symbols should be interpreted as limiting their scope. Needless to say, Not necessarily all aspects or advantages of the present invention are achieved according to any particular embodiment of the present invention. It is important to understand that this is not always possible. Therefore, for example, a person skilled in the art would understand this specification. Without necessarily achieving other aspects or advantages that may be taught or suggested in this document, The present invention is embodied or executed in a manner that achieves or optimizes one or a group of the indicated advantages. They will recognize that this is possible.

[0035] This invention relates to both organization and operation methods, along with its features and advantages, and is accompanied by the attached drawings. When read together, the following modes for carrying out the invention will be most clearly understood. This can be understood. The aspects and advantages of the present invention will become clear with reference to the embodiments described below. This will be clarified. Throughout this specification, references to "one embodiment" or "a certain embodiment" are not limited to , the specific features, structures, or properties described in relation to the embodiments are at least the present invention This means that it is included in one embodiment. Therefore, in various places throughout this specification The occurrences of the phrases "in one embodiment" or "in a certain embodiment" are not necessarily all the same. This does not necessarily refer to an embodiment, but it may. Similarly, exemplary embodiments of the present invention The description of the embodiments simplifies this disclosure and helps in understanding one or more of the various embodiments of the invention. To that end, various features of the present invention are not summarized in a single embodiment, figure, or description thereof. Please understand that this may sometimes be the case. However, the method disclosed herein is the claim of The intention is that Akira requires more features than are explicitly enumerated in each patent claim. It should not be interpreted as reflecting the following claims. Rather, it should reflect the following claims. Therefore, the embodiments of the invention do not include all the features of a single, previously disclosed embodiment.

[0036] Unless otherwise indicated by the context, the “embodiments” of this disclosure may be specifically combined together. Please understand that all disclosed embodiments, in any particular combination, may differ depending on the context. (Unless otherwise implied) these are further disclosed embodiments of the claimed invention.

[0037] In addition, as used herein and in the appended claims, the singular "a", "an The pronouns ", and "the" refer to multiple objects unless the context explicitly indicates otherwise. Therefore, for example, a reference to "polynucleotide" includes two or more polynucleotides. The reference to "motor protein" includes two or more such proteins, "helicase". A reference to "helicase" includes two or more helicases, and a reference to "monomer" includes two or more monomers. A reference to "poa" can include two or more instances of "poa."

[0038] All publications, patents, and patent applications cited above or below in this specification are subject to the terms of their respective rights. The entire text is incorporated herein by reference.

[0039] definition When referring to a singular noun, use either the indefinite or definite article, for example, "a" or "an," or "the." When used, unless otherwise specified, it includes the plural form of the noun. Where the term "including" is used in this description and claims, it means that other elements or The steps are not excluded. Furthermore, the first, second, third, etc. in this description and claims. The term is used to distinguish similar elements and does not necessarily refer to the order in which they occurred or the order in which they occurred. It is not always used to explain the appropriateness of those terms when used in that way. It is interchangeable under certain circumstances, and embodiments of the present invention described herein are described herein. Or, please understand that it is possible to operate in an order other than the order illustrated. The following terms or definitions The definitions provided herein are for the sole purpose of aiding in the understanding of the present invention. Unless otherwise specifically defined herein, Furthermore, all terms used herein have the same meaning as those understood by those skilled in the art. Experts should refer to definitions and technical terms, particularly Sambrook et al., M. Olecular Cloning:A Laboratory Manual,4 th ed.,Cold Spring Harbor Press,Plainsview , New York (2012), and Ausubel et al., Current Protocols in Molecular Biology(Suppleme (nt 114), John Wiley & Sons, New York (2016) Refer to the definitions provided herein. The definitions provided herein may be understood to be narrower in scope than those understood by those skilled in the art. It should not be treated badly.

[0040] As used herein, "about" refers to measurable values ​​such as quantity and duration. From the specified value, ±20% or ±10%, more preferably ±5%, and even more preferably ± This means that it includes a variation of 1%, more preferably ±0.1%, and such variation is open It is appropriate to implement the methods shown.

[0041] The terms "nucleotide sequence," "DNA sequence," or "nucleic acid molecule" as used herein are: A nucleotide of any length, either ribonucleotide or deoxyribonucleotide This refers to the polymer form. This term refers only to the primary structure of the molecule. Therefore, this The terminology includes double-stranded and single-stranded DNA and RNA. The term "nucleic acid" as used herein refers to nucleic acids. The term refers to the 3' and 5' ends of each nucleotide being linked by a phosphodiester bond. Polynucleotides are single-stranded or double-stranded covalently bonded nucleotide sequences that are linked together. Rheotides can be composed of deoxyribonucleotide bases or ribonucleotide bases. Nucleic acids can be produced synthetically in vitro or isolated from natural sources. , modified DNA or RNA, for example, methylated DNA or RNA , or post-translational modifications, e.g., 5'-capping, cleavage and porting with 7-methylguanosine. RNA used for 3'-processing such as rear-denylation, and for splicing. It may also contain: Nucleic acids include hexitol nucleic acid (HNA), cyclohexene nucleic acid (CeN). A) Threose nucleic acid (TNA), glycerol nucleic acid (GNA), locked nucleic acid (LNA) This may also include synthetic nucleic acids (XNAs) such as peptide nucleic acids (PNAs). The size of nucleic acids, also called "polynucleotides," is typically that of a double-stranded polynucleotide. The number of base pairs (bp), or in the case of a single-stranded polynucleotide, the number of nucleotides (nt). It is expressed in terms of the number of units. 1000 bp or nt corresponds to kilobases (kb). The length is approximately 4 Polynucleotides with fewer than 0 nucleotides are typically called "oligonucleotides". Ply for use in DNA manipulation, such as via polymerase chain reaction (PCR). It may include Ma.

[0042] In connection with this disclosure, the term “amino acid” is used in its broadest sense, and each ami Along with a side chain specific to ano acids (e.g., an R group), amines (NH2) and carboxyls (CO2) are also present. It is intended to include organic compounds containing an OH) functional group. In some embodiments, Mino acids refer to naturally occurring L-α-amino acids or residues. Commonly used one- and three-letter abbreviations: A=Ala, C=Cys, D=Asp, E=Glu, F=Phe, G=Gly, H=His, I=Ile, K=Lys, L=Le u, M=Met, N=Asn, P=Pro, Q=Gln, R=Arg, S=Ser, T= Thr, V=Val, W=Trp, and Y=Tyr are used herein (Lehni Nger, AL, (1975) Biochemistry, 2d ed., pp.7 1-92, Worth Publishers, New York). "Amino acids" Common terms include D-amino acids, retroinversoamino acids, and amino acid analogs. Chemically modified amino acids, such as norleucine, which are not normally incorporated into proteins, are natural In the art, the characteristics of naturally occurring amino acids and amino acids such as β-amino acids have been demonstrated. It further includes chemically synthesized compounds possessing the properties of knowledge. For example, natural Phe or Pro Phenylalanine or proline allows for the same structural restriction of peptide compounds. Analogues or imitations are included within the definition of amino acids. Such analogues and imitations are included in this specification. These are referred to as the "functional equivalents" of each amino acid in the book. For other examples of amino acids, see the references. Roberts and Vellaccio, The P eptides:Analysis,Synthesis,Biology,Gross and Meiehofer, eds.,Vol.5 p.341,Academic Listed by Press, Inc., NY, 1983.

[0043] The terms "polypeptide" and "peptide" refer to polymers of amino acid residues, and also to polymers of amino acid residues. This term is used interchangeably herein to refer to variants and synthetic analogues of [the specified term]. These terms refer to chemical analogs of naturally occurring amino acids, where one or more amino acid residues correspond to the same amino acid. Amino acid polymers, which are synthetic amino acids that do not exist in nature, as well as naturally occurring amino acids Applicable to amino acid polymers. Polypeptides undergo glycosylation, proteolytic cleavage, This may include lipidization, signal peptide cleavage, propeptide cleavage, phosphorylation, etc. Peptides may also undergo maturation or post-translational modification processes, but are not limited to these. Recombination techniques are used to process the peptides. For example, it can be produced by the expression of recombinant or synthetic polynucleotides. The peptides produced typically contain substantially no culture medium, for example, culture medium This is less than about 20% of the volume of the protein preparation, more preferably less than about 10%, most preferably This corresponds to less than approximately 5%.

[0044] The term "protein" refers to a folded polypeptide having a secondary or tertiary structure. Used to explain. A protein may consist of a single polypeptide, or It may contain multiple polypeptides that aggregate to form a multimer. The multimer is homo-oligopolysaccharide. It can be either a gomer or a heterooligomer. Proteins are naturally occurring proteins. It may be protein or wild-type protein, or modified protein or naturally occurring It may be a protein that does not exist. A protein is, for example, a protein with one or more amino acids attached. Additions, substitutions, or deletions may result in a protein that differs from the wild-type protein.

[0045] The "mutant form" of the protein is compared to the unmodified or wild-type protein in question. Unmodified proteins having amino acid substitutions, deletions, and / or insertions, and derived therefrom. Peptides, oligopeptides, polypeptides, etc., having similar biological and functional activity. This includes proteins and enzymes. The term "amino acid identity" as used herein refers to amino acid identity. This refers to the degree to which the sequence is identical amino acid by amino acid across the comparison window. Therefore, The "percentage of sequence identity" is two optimally aligned values ​​across the comparison window. The sequences are compared, and identical amino acid residues (e.g., Ala, Pro, Ser, Thr, G) are identified. ly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, A The number of positions in both sequences where sp, Glu, Asn, Gln, Cys, and Met occur The number of determined and matching positions is calculated, and this number is compared to the total number of positions in the comparison window. Divide by (i.e., window size), multiply the result by 100 to get the sequence identity parsing. It is calculated by calculating the vintage.

[0046] In all aspects and embodiments of the present invention, "mutant" refers to the corresponding wild-type protein. For the amino acid sequence, at least 50%, 60%, 70%, 80%, 90%, 95%, Or it has 99% complete sequence identity. Sequence identity is full-length polynucleotide or poly It may also be a fragment or part of a peptide. Therefore, the sequence may be the complete sequence. While it may have only 50% sequence identity overall with the reference sequence, it may have sequence identity in specific regions, domains, or sub-regions. The sequence of a unit may share 80%, 90%, or even 99% sequence identity with the reference sequence. ru.

[0047] The term "wild type" refers to a gene or gene product isolated from a naturally occurring source. The wild-type gene is the most frequently observed gene in a given population, and therefore, any of that gene This is the designed "normal" or "wild-type" form. In contrast, it is a "modified" or "mutant." The term "mutant" refers to a modification of the sequence compared to the wild-type gene or gene product. For example, substitution, cleavage, or insertion), post-translational modification, and / or functional properties (for example, modification). This refers to a gene or gene product that exhibits altered characteristics. It involves isolating naturally occurring mutants. It should be noted that these can be modified compared to the wild-type gene or gene product. Identified by the fact that it has altered characteristics. By introducing naturally occurring amino acids or Methods for substitution are well known in the art. For example, methionine (M) is a mutation. The methionine codon (AT) at the relevant position in the polynucleotide encoding the monomer. By replacing G) with the arginine codon (CGT), it is replaced with arginine (R). It is possible. Methods for introducing or substituting amino acids that do not exist in nature are also circulating in the art. It is knowledge. For example, amino acids that do not exist in nature are used to express mutant monomers. This can be introduced by including synthetic aminoacyl-tRNA in the IVTT system being used. Alternatively, they are synthetic (i.e., non-naturally occurring) analogues of specific amino acids. In the presence of these specific amino acids, mutant E. coli molecules have nutritional requirements. They can be introduced by expressing mutant monomers. These mutant monomers partially pept When produced using cytoplasmic synthesis, it is called naked ligation. Conservative substitutions can also be produced by (tion). Conservative substitutions involve replacing amino acids with similar chemical structures. Replace with other amino acids having similar chemical properties or similar side chain volume. No acids are similar in polarity, hydrophilicity, hydrophobicity, basicity, acidity, and neutrality to the amino acids they replace. It may have properties or be charged. Alternatively, conservative substitutions may be existing aromatic or aliphatic amino acids. Another amino acid, which is aromatic or aliphatic, can be introduced instead. Conservative amino acid changes are These are well known in the relevant field and follow the characteristics of the 20 main amino acids defined in Table 1 below. If amino acids have similar polarity, this is also the amino acid in Table 2. This can be determined by referring to the hydrophobicity scale of the side chains. [Table 1] [Table 2]

[0048] Mutant or modified proteins, monomers, or peptides may be used in any manner and at any site. They can also be chemically modified. Mutants or modified monomers or peptides are preferably, Molecular attachment to one or more cysteine ​​molecules (cysteine ​​bond), molecular attachment to one or more lysine molecules Attachment, attachment of molecules to one or more non-natural amino acids, enzymatic modification of epitopes, or terminal repair It is chemically modified by ornamentation. A preferred method for carrying out such modification is in the art. This is well known. Modified protein, monomer, or peptide variants can attach to any molecule. They can be chemically modified by, for example, modified proteins, monomers, or peptides. The variant can be chemically modified by the attachment of dyes or fluorophores.

[0049] As used herein, the alkylene group may be aliphatic or alicyclic in its hydrocarbon properties. In a compound, two hydrogen atoms from the same carbon atom, or one hydrogen atom from each of two different carbon atoms. Unsubstituted or substituted by removing any two hydrogen atoms , it is a saturated bidentate portion. Hydrocarbon compounds may have 1 to 20 carbon atoms, In this case, the alkylene group is C 1-20 It is an alkylene. The alkylene group is C 1-10 Alki If it is a lente, it may have, for example, 1 to 10 carbon atoms. Typically, C 1-6 Alkylene, or C 1-4 Alkylenes, e.g., methylene, ethylene, i-propylene, n -Propylene, t-butylene, s-butylene, or n-butylene.

[0050] The alkenylene group may be an aliphatic or alicyclic hydrocarbon compound, or it may be the same carbon atom as the other carbon atom. Remove two hydrogen atoms, or remove one hydrogen atom from each of two different carbon atoms. An unsubstituted or substituted bidentate moiety obtained by doing so, comprising one or more carbon atoms -Contains a carbon double bond. Hydrocarbon compounds may have 2 to 20 carbon atoms, in which case, The alkenylene group is C 2-20is alkenylene. When the alkenylene group is a C 2-10 alkenyl ene, it may have, for example, 2 to 10 carbon atoms. Usually, it is C 2-6 alkenylene, or C 2-4 alkenylene.

[0051] An alkynylene group is an unsubstituted or substituted bidentate moiety obtained by removing two hydrogen atoms from the same carbon atom of a hydrocarbon compound which may be aliphatic or alicyclic, or removing one hydrogen atom from each of two different carbon atoms, and comprises one or more carbon-carbon carbon triple bonds. The hydrocarbon compound may have 2 to 20 carbon atoms, in which case the alkynylene group is C alkynylene. When the alkynylene group is a C alkynylen e, it may have, for example, 2 to 10 carbon atoms. Usually, it is C 2-20 alkynylene. When the alkynylene group is C 2-10 alkynylene , it may have, for example, 2 to 10 carbon atoms. Usually, it is C 2-6 al kynylene, or C 2-4 alkynylene.

[0052] An arylene group is an unsubstituted or substituted monocyclic or fused polycyclic bidentate moiety obtained by removing two hydrogen atoms, one from each of two different aromatic ring atoms of an aromatic compound, said moiety having (unless otherwise specified) 5 to 14 ring atoms . Typically, each ring has 5 to 7 or 5 to 6 ring atoms. The arylene group may be unsubstituted or substituted.

[0053] A heteroarylene group is a bidentate moiety obtained by removing two hydrogen atoms, one from each of two different ring atoms of a heteroaryl group. The heteroaryl group is obtained by removing two hydrogen atoms, one from each of two different ring atoms of a heteroaryl group. The heteroaryl group is ​A substituted or unsubstituted monocyclic or fused polycyclic (e.g., bicyclic or tricyclic) aromatic group. Typically, the ring portion contains at least one heteroatom, for example, O, S, N, P, Se and It contains one, two, or three heteroatoms, more typically selected from Si, O, S, and N. It contains 5 to 14 ring atoms. Examples include pyridyl, pyrazinyl, pyrimidinyl, and py Ridadinil, Furanil, Thienyl, Pyrazolidinil, Pyrrolyl, Oxadiazolyl, Iso Oxazolyl, thiadiazolyl, thiazolyl, imidazolyl, triazolyl, pyrazolyl Oxazolyl, isothiazolyl, benzofuranil, isobenzofuranil, benzothioff Enyl, indolyl, indazolyl, carbazolyl, acridinyl, urinyl, sinnoly Nyl, quinoxalinyl, naphthilidinyl, benzimidazolyl, benzoxazolyl, ki Examples include norinyl, quinazolinyl, and isoquinolinyl.

[0054] The carbocyclylene group, also known as the cycloalkylene group, is unsubstituted or substituted. The process involves removing two hydrogen atoms, one from each of the two carbon atoms in the cyclic alkyl group. This is the two-seat portion obtained by [the process]. Typically, this portion is (unless otherwise specified) It contains 3 to 10 ring atoms and has 3 to 10 carbon atoms. An example is cyclop Ropane (C3), cyclobutane (C4), cyclopentane (C5), cyclohexane (C 6) Cycloheptane (C7), Methylcyclopropane (C4), Dimethylcyclopropane C5, methylcyclobutane (C5), dimethylcyclobutane (C6), methylcyclobutane Lopentane (C6), Dimethylcyclopentane (C7), Methylcyclohexane (C7) Examples include dimethylcyclohexane (C8) and menthane (C10).

[0055] The heterocyclylene moiety consists of two hydrogen atoms from two different ring atoms of the heterocyclyl group. This is the bidentate portion obtained by removing the heterocyclyl group. The heterocyclyl group is unsubstituted or A substituted cyclic group, typically having at least one heteroatom in the ring portion, for example, One element selected from O, S, N, P, Se, and Si, more typically from O, S, and N. It contains 5 to 14 atoms, including 2 or 3 heteroatoms. For example, piperaz. N, piperidine, morpholin, 1,3-oxazinane, pyrrolidine, imidazolidine, Oxazolidine, tetrahydropyrazine, tetrahydropyridine, dihydro-1,4-O Xazine, tetrahydropyrimidine, dihydro-1,3-oxazine, dihydropyrrole Examples include dihydroimidazole and dihydrooxazole groups.

[0056] The arylene-alkylene group is a combination of an arylene group and an alkylene group as defined herein. A group formed by the formation of a bond between two elements. Heteroarylene-alkylene group This involves forming a bond between a heteroarylene group and an alkylene group as defined herein. It is a group formed by and . The carbocyclylene-alkylene group is defined herein as It is formed by forming a bond between the carboxyl group and the alkylene group. It is a group. The heterocyclylene-alkylene group is a heterocyclylene as defined herein. It is a group formed by forming a bond between a group and an alkylene group.

[0057] When it is stated that a group is substituted, it is typically one, two, or three such groups. Substituted by one or more substituents, typically one or two, usually one. The substituent can be independently selected from halogens, -OR', and -NR'2 (wherein R' is Typically, H or unsubstituted C 1-2 (Alkyl and unsubstituted C1-C2 alkyl groups).

[0058] Methods for characterizing analytes This disclosure describes how to use motor proteins to target detectors such as nanopores. The present invention relates to a method for characterizing target polynucleotides that move along any suitable motor. The protein may be used in the manner provided herein. An exemplary motor protein is described herein. More details are provided in the specifications.

[0059] This disclosure also involves contacting a detector with polynucleotides, and the polynucleotides against the detector. This includes rereading the target polynucleotide, such as moving back and forth. Methods for characterizing rheotides are described in more detail herein.

[0060] More specifically, in some embodiments, the present disclosure describes how the motor protein is polynucleotide This concerns a method for moving rheotide out of the detector (for example, out of the nanopore). In this embodiment, the direction of movement of the polynucleotide is such that the polynucleotide moves to a nanoparticle. This is the opposite of known methods of moving into A. This is described in more detail herein.

[0061] This disclosure provides nanopores as exemplary detectors, but the methods provided herein (i) zero-mode waveguide, (ii) field-effect transistor, optionally a Noyar field (iii) effect transistor, (iv) AFM chip, (iv) nanotube, optionally car The disclosed method is suitable for detectors such as von nanotubes and (v) nanopores. , polynucleotides pass through the detector or include a detector such as a well in the detector chip. It is particularly suitable for methods of movement through structures.

[0062] In the disclosed method, the motor protein typically has a polynucleotide at the stalled portion. The engine first stalls on the octagon. Preferred stall points are described in detail herein. Stalling of motor proteins on a renucleotide has several advantages. For example, stalling While this is happening, motor proteins typically do not stall, for example, polynucleotides. It consumes less fuel when moving freely than when moving freely with respect to the Chido. This is unproductive fuel consumption. Reducing it could be advantageous.

[0063] The methods provided herein typically involve a motor protein detecting a detector (e.g., a Motor proteins can control the movement of polynucleotides from nopores. This includes destalling the motor protein. A method for destalling a motor protein is described herein. It is described in more detail. Controlled loss-stalling of motor proteins is a motor protein Quality can precisely determine the point at which polynucleotide processing begins. It has various advantages, including, for example, the ability to perform data recording on polynucleotides before the start of data recording. To prevent data loss as a result of undesirable motor protein movement, This may be useful in characterizing creotides.

[0064] The disclosed method is obtained when polynucleotides are moved from a detector such as a nanopore. The data obtained is obtained when the same polynucleotide is moved into a detector (e.g., a nanopore). This is at least partially based on the recognition that the data obtained may differ from what is expected. How many data characteristics are there, including error profiles, noise profiles, and error profiles? In one embodiment, the same polynucleotide is transferred to a contrasting detector such as a nanopore. The methods may all be different. In some embodiments, the data obtained by the disclosed methods , has advantages compared to data obtained by other known methods. Therefore, the disclosed The law increases the available options when polynucleotide characterization is required. Therefore, users who wish to characterize polynucleotides will need to address the specific use of the polynucleotide in question. You can choose the most suitable method for your journey.

[0065] As described above, the disclosed method, in some embodiments, involves the detection of nanopores and other materials. This relates to the transfer of target polynucleotides from an extractor. This specification describes exemplary detection. While nanopores are considered as a container, this method is not limited to them.

[0066] Nanopores typically have two openings, namely a first opening and a second opening. These openings are often referred to as cis and trans openings of nanopores. The first opening is a cis opening, and the second opening is a transformer opening, but several actual In this configuration, the first opening is a transformer opening, and the second opening is a cis opening. The notation of "cis" and "trans" openings in nanopores is routine in this technical field. For example, the cis opening of a nanopore typically has cis and trans chambers. The transformer opening faces the cis chamber of the nanopore device, such as the apparatus described in the document, and is typically It faces the transformer chamber.

[0067] In the particular method provided herein, the first opening of the nanopore is a stalled motor This method involves contact with a polynucleotide that has a protein on top. Using this, the nanopore is moved in the direction from the second opening of the nanopore to the first opening of the nanopore. This includes controlling the movement of target polynucleotides.

[0068] Therefore, from the perspective of motor proteins, the target polynucleotide is a nanopore To move outwards. The notation "outwards" refers to the movement of polynucleotides toward the motor protein. Regarding the overall movement. This direction of movement is when the target polynucleotide moves to the motor protein. Therefore, this may be in contrast to another mode of movement that involves moving "into" the nanopore.

[0069] The differences in these movement schemes are significant. Polynucleotides "exit" from the pore. In the method provided herein, the direction of movement is the nano furthest from the motor protein. From the pore entrance (i.e., the distal entrance), the entrance of the nanopore closest to the motor protein This is the direction toward the mouth (proximal entrance). In contrast, polynucleotides move "inward" into the pore. In this method, the direction of movement is the entrance of the nanopore closest to the motor protein (proximal entrance). From the opening, it moves towards the nanopore entrance (distal entrance) furthest from the motor protein. It is the direction.

[0070] Therefore, in some embodiments of the provided method, the nanopore is cis-side and tra-side. In a membrane having a cis side, the first opening of the nanopore is on the cis side of the membrane, and the second opening of the nanopore The opening of 2 is on the trans side. In this embodiment, the motor protein is in the cis position of the membrane. Located on the side, it facilitates the movement of target polynucleotides through nanopores from the trans side to the cis side of the membrane. Control.

[0071] In another embodiment of the provided method, the nanopore is a film having a cis side and a trans side. For example, the first opening of the nanopore is on the trans side of the film, and the second opening of the nanopore is on the cis side. It is located on the side. In such embodiments, the motor protein is located on the trans side of the membrane. This controls the movement of target polynucleotides through nanopores from the cis side to the trans side.

[0072] In contrast to the transfer of polynucleotides to the pore in contrasting methods, the method provided herein Figure 1 schematically illustrates the differences in the direction of polynucleotide movement from the pore in the methods described. show.

[0073] Reread In some embodiments, the methods provided herein characterize polynucleotides This involves rereading polynucleotides. When polynucleotides move back and forth relative to the detector, the polynucleotides exhibit characteristic features. This includes obtaining one or more specific measurements.

[0074] In one embodiment, this specification provides a method for characterizing a target polypeptide. The law is, (i) Contact the detector with the target polynucleotide to which the motor protein is bound. Therefore, the target polynucleotide is at the polynucleotide binding site of the motor protein. It is bound to the motor protein, and contact is made. (ii) The motor protein moves the target polynucleotide in the first direction relative to the detector. When controlling, obtain one or more measurements characteristic of the target polynucleotide. , (iii) The motor moves the target polynucleotide in a second direction relative to the detector. - Debinding target polynucleotides from the polynucleotide binding site of proteins , (iv) Reattach the target polynucleotide to the polynucleotide binding site of the motor protein. Combined, the motor protein moves the target polynucleotide in a first direction relative to the detector. When controlling, obtain one or more measurements characteristic of the target polynucleotide. This includes characterizing the target polynucleotide.

[0075] In related embodiments, this specification provides a method for characterizing a target polypeptide. This method, (i) Contact the detector with the target polynucleotide to which the motor protein is bound. Therefore, the target polynucleotide is at the polynucleotide binding site of the motor protein. It is bound to the motor protein, and contact is made. (ii) The motor protein moves the target polynucleotide in the first direction relative to the detector. When controlling, obtain one or more measurements characteristic of the target polynucleotide. , (iii) The target polynucleotide moves in a second direction relative to the detector. This allows the renucleotide to dissociate from the polynucleotide binding site of the motor protein. Making it into a Noh play, (iv) Reattach the target polynucleotide to the polynucleotide binding site of the motor protein. Combined, the motor protein moves the target polynucleotide in a first direction relative to the detector. When controlling, obtain one or more measurements characteristic of the target polynucleotide. This includes characterizing the target polynucleotide.

[0076] The disclosed method has many advantages compared to conventionally known methods. For example, target poly Each nucleotide reading uses the same strand and the same detection region, thus achieving equivalent accuracy. It should be. This would allow the same base calling model to be used for each reading. This becomes possible. Furthermore, combining data from multiple readings becomes easier. In addition, natural distribution Because the column is reread multiple times, (for example) it retains epigenetic information. This makes it possible. This method is also adaptive, and will reread until the data with the required accuracy is obtained. The process can be repeated multiple times.

[0077] More specifically, this method involves the motor protein targeting the detector in a first direction. When controlling nucleotide movement, one or more measurements characteristic of the target polynucleotide are used. This may include obtaining a value. The first direction is that the motor protein is a polynucleotide This could be the direction driving the motion. The first direction could be the direction of the force applied to the detector. The first direction may be opposite to the direction of the force applied across the detector.

[0078] In many cases, the detector is included in a structure having a first aperture and a second aperture, or The nanopore comprises a transmembrane having a first opening and a second opening, and step (i) is the first This involves contracting the opening of 1 with a target polynucleotide. Typically, motortan The protein facilitates the movement of target polynucleotides in the direction from the second opening to the first opening. It controls the target polynucleotide of the motor protein. Typically, the target polynucleotide controls the polynucleotide of the motor protein. When debinding occurs from the bond site, the target polynucleotide moves from the first opening to the second opening. Move in the direction of the section.

[0079] Therefore, if the detector is a nanopore or contains a nanopore, the first direction is this It may be “into” the nanopore as described in the specification. Therefore, in some embodiments, The movement of polynucleotides while one or more measurements are being performed is toward the outside of the nanopore. In some embodiments, the nanopore is made into a film having a cis side and a trans side. The first opening of the nanopore is on the cis side of the film, and the second opening of the nanopore is on the cis side. Located on the lance side, motor proteins are located on the cis side of the membrane and are trans from the cis side of the membrane. Controls the movement of target polynucleotides through nanopores toward the side. In other embodiments, nano The pore extends across the film, having both a cis and a trans side, and the first opening of the nanopore is the cis side of the film. The second opening of the nanopore is on the trans side, and the motor protein is on the membrane. Located on the trans side, the target polynucleo passes through the nanopore from the trans side to the cis side of the membrane. Control the movement of the cydoid.

[0080] More often, if the detector is a nanopore or contains a nanopore, the first direction is , which is the direction toward the "outward" of the nanopore as described herein. Therefore, several implementations Morphologically, the movement of polynucleotides between one or more measurements is outside the nanopore. is carried out towards. In some embodiments, the nanopore spans a membrane having a cis side and a trans side , the first opening of the nanopore is located on the cis side of the membrane, the second opening of the nanopore is located on the trans side, and the motor protein is located on the cis side of the membrane, and controls the movement of the target polynucleotide through the nanopore from the trans side of the membrane to the cis side. In other embodiments the nanopore spans a membrane having a cis side and a trans side, the first opening of the nanopore is located on the cis side of the membrane, the second opening of the nanopore is located on the trans side, and the motor protein is located on the trans side of the membrane, and controls the movement of the target poly nucleotide through the nanopore from the cis side of the membrane to the trans side.

[0081] The provided method may comprise unbinding the target polynucleoti de from the polynucleotide binding site of the motor protein. This is described in detail hereinafter herein . When the target polynucleotide unbinds from the polynucleotide binding site of the motor protein , the target polynucleotide moves in a second direction relative to the detector. The second direction is usual ly opposite to the first direction.

[0082] Accordingly, in some embodiments of the method wherein the detector is or comprises a nanopore , the first direction in which the target polynucleotide moves relative to the detector is into the nanopore , and the second direction in which the target polynucleotide moves relative to the detector is out of the nanopore . In other embodiments, the first direction in which the target polynucleotide moves relative to the detector is out of the nanopore, and the second direction in which the target polynucleotide moves relative to the detector is into the nanopore.

[0083] ​ Next, the provided method involves targeting polynucleotides to the polynucleotides of motor proteins. This may include rebinding to the ocidal binding site. The motor protein then... When one or more measurements characteristic of creotides are performed, the first of the target polynucleotides Controls movement in a specific direction. The first direction is the same as the first direction described above.

[0084] Therefore, in one embodiment, this specification describes a method for characterizing a target polynucleotide. Provided, this method, (i) The first opening of a transmembrane nanopore having a first opening and a second opening, This involves contacting a target polynucleotide containing a bound motor protein, The target polynucleotide is attached to the polynucleotide binding site of the motor protein. Binding to a protein, making contact, (ii) The motor protein moves from the first opening of the nanopore to the second opening of the nanopore. When controlling the movement of target polynucleotides in a particular direction, the target polynucleotide has characteristics that are characteristic of the target polynucleotide. Obtaining one or more measurements, (iii) The target polynucleotide passes from the second opening of the nanopore to the first opening of the nanopore. Move in the direction of the target port from the polynucleotide binding site of the motor protein. Debinding of the nucleotide, (iv) Reattach the target polynucleotide to the polynucleotide binding site of the motor protein. Combined, the motor protein moves from the first opening of the nanopore to the second opening of the nanopore. When controlling the movement of a target polynucleotide in a certain direction, the target polynucleotide Obtain one or more characteristic measurements, This includes characterizing the target polynucleotide. Characterizing a target polynucleotide may include, for example, determining its sequence. .

[0085] For example, in some embodiments, the nanopore is a film having a cis side and a trans side. For example, the first opening of the nanopore is on the cis side of the film, and the second opening of the nanopore is on the tra. Located on the trans side, the motor protein passes through nanopores from the cis side to the trans side of the membrane. Controls the movement of target polynucleotides. In other embodiments, the first opening of the nanopore is Located on the trans side of the membrane, the second opening of the nanopore is on the cis side, and is a motor protein This controls the movement of target polynucleotides from the trans side to the cis side of the membrane through nanopores. In some embodiments, this method applies a force (e.g., electric potential) to the entire nanopore. This includes the motor protein passing through the target polypore in the same direction as the applied force. It controls the movement of nucleotides.

[0086] In another embodiment, this specification provides a method for characterizing a target polypeptide. The method is, (i) The first opening of a transmembrane nanopore having a first opening and a second opening, This involves contacting a target polynucleotide containing a bound motor protein, The target polynucleotide is attached to the polynucleotide binding site of the motor protein. Binding to a protein, making contact, (ii) The motor protein moves from the second opening of the nanopore to the first opening of the nanopore. When controlling the movement of target polynucleotides in a particular direction, the target polynucleotide has characteristics that are characteristic of the target polynucleotide. obtaining one or more measurement values, (iii) unbinding the target polynucleotide from the polynucleotide binding site of the motor protein such that the target polynucleotide moves in a direction from the second opening of the nanopore to the first opening of the nanopore ; (iv) rebinding the target polynucleotide to the polynucleotide binding site of the motor protein, obtaining one or more characteristic measurement values for the target polynucleotide when the motor protein controls movement of the target polynucleotide in the direction from the second opening of the nanopore to the first opening of the nanopore, obtaining one or more characteristic measurement values for the target polynucleotide, thereby characterizing the target polynucleotide. Characterizing the target polynucleotide may comprise, for example, determining the sequence of the target polynucleotide .

[0087] For example, in some embodiments, the nanopore spans a membrane having a cis side and a trans side, the first opening of the nanopore is on the cis side of the membrane, the second opening of the nanopore is on the trans side, and the motor protein controls movement of the target polynucleotide through the nanopore from the trans side to the cis side of the membrane. In other embodiments, the first opening of the nanopore is on the trans side of the membrane, the second opening of the nanopore is on the cis side, and the motor protein on the trans side of the membrane, the second opening of the nanopore is on the cis side, and the motor protein controls movement of the target polynucleotide through the nanopore from the cis side to the trans side of the membrane In some embodiments, the method comprises applying a force (for example, a potential) across the nanopore and the motor protein controls movement of the target polynucleotide through the nanopore in a direction opposite the applied force In some embodiments, the method comprises applying a force (e.g., an electric potential) across the nanopore,

[0088] The movement of polynucleotides in a second direction relative to the detector may spontaneously occur. It is important to distinguish this from typical slipping. For example, a slip of one or two bases is This is not an example of a reread as described herein. Typically, in step (iii) The distance the target polynucleotide travels relative to the detector is at least 10 nucleotides. It is long. In some embodiments, the distance at which the target polynucleotide moves relative to the detector The distance is at least 20 nucleotides long, for example at least 30 nucleotides long, for example a small At least 40 nucleotides long, for example, at least 50 nucleotides long, for example, at The length is 100 nucleotides. Longer distances may be used. In some embodiments, The distance the target polynucleotide travels relative to the detector in step (iii) is, At least 1000 nucleotides (1 kb) in length, for example, at least 2 kb in length, for example, It is at least 5kb or at least 10kb in length, for example, at least 100kb or less It is at least 1000kb long.

[0089] Steps (iii) and (iv) of this method involve rereading the target polynucleotide multiple times. Steps (iii) and (iv) may be repeated multiple times. 1 time, for example at least 2 times, for example at least 3 times, for example at least 4 times, for example few At least 5 times, for example at least 10 times, for example at least 20 times, for example at least 5 0 times, for example at least 100 times, for example at least 1000 times, for example at least 10 This can be repeated 0,000 times, for example, at least 100,000 times or more. Therefore, this The method may include "flooring" polynucleotides back and forth with respect to the detector. .

[0090] Therefore, steps (iii) and (iv) are repeated once (and only once), As a result, if the method includes steps (iii) and (iv) only twice and only twice, then this method Steps (i), (ii), (iii), (iv), (iii1), and (iv1) This includes three parts of the polynucleotide, namely in step (ii) The first part, the second part in steps (iii) and (iv), and step (i A characteristic measurement is performed in the third part of ii1) and (iv1). Step (i Repeat steps (ii) and (iv) twice (and only twice), and the method is as follows: If (iv) is to be included three times and only three times, this method is as follows: (iii), (iv), (iii1), (iv1), (iii2), and (iv2) As a result, the four parts of the polynucleotide, namely the first in step (ii) The part of step (iii) and the second part in (iv), step (iii1) and The third part in (iv1), and in steps (iii2) and (iv2) The fourth part is characterized by the measurement performed. In other words, steps (iii) and (iv) If this is repeated n times, each repetition will result in a characteristic at the (n+2) portion of the polynucleotide. A precise measurement can be obtained. Repeating steps (iii) and (iv) multiple times will result in nano Because the polynucleotide portion analyzed by the pore is sampled multiple times, the characteristics This could lead to improvements in the analysis, and the probabilistic errors that may be recorded in the analysis are statistical. The significance is lost. Therefore, the accuracy of the characteristic data obtained in this way is improved. These methods can achieve, for example, at least 99% accuracy, at least 99%. Achieving a very high level of accuracy, such as 0.9% accuracy or at least 99.99% accuracy. This makes it possible to do so. Therefore, in some embodiments, step (iii) and ( iv) At least 99.9% accuracy, or at least 99.99% accuracy, etc. The process is repeated until a 99% accuracy level is reached.

[0091] The portion of the polynucleotide read in step (ii) of this method and step (iv) The polynucleotide portions read by this method typically overlap. In other words, this method This includes rereading at least a portion of the polynucleotide multiple times. Therefore, In some embodiments, in step (ii), the motor protein targets the poly Control the movement of the first portion of the nucleotide in a first direction relative to the detector, step ( iv) In this step, the motor protein detects the second portion of the target polynucleotide. Controlling movement in a first direction relative to the first part, the first part at least partially and the second part They overlap. In some hair measurements, the second portion is at least 20% of the first portion, and less At least 30%, at least 40%, at least 50%, at least 60%, at least 7 Overlaps with 0%, at least 80%, at least 90%, or at least 95%. In this embodiment, the first part is the same as the second part. Therefore, several embodiments In this state, a portion of the polynucleotide is repeatedly characterized in the manner provided. Each reaction In the repeat, the second part of the polynucleotide is the first part of the polynucleotide of the previous repeat. If the polynucleotides do not completely overlap with the fractions, they will appear to the detector as a dip. It ratchets in a zigzag pattern. In each iteration, the second part of the polynucleotide is the same as the previous part. If the first part of the repeating polynucleotide completely overlaps with the same part of the polynucleotide The minutes are flossed back and forth relative to the detector.

[0092] Forces applied during movement In some embodiments of the disclosed method, force is applied to the entire detector, for example, the entire nanopore. It can be used. To control this method, the force can be controlled. For example, by increasing the force By doing so, the movement of polynucleotides passing through the detector (e.g., nanopore) is increased. This can be reduced, for example by controlling the rate at which polynucleotides pass through the pore. It is possible.

[0093] In the methods provided herein, any suitable force can be applied. The force is detected. This can be a potential applied to the entire container, for example, the entire nanopore. In some embodiments, No external force is applied to the entire nopore. For example, in some embodiments, no potential is applied. i. In some embodiments, the polynucleotide is applied to the nanopore. It is particularly suitable for methods in which optical measurements are performed while the device is in motion.

[0094] In other embodiments, the force may be a voltage force applied to the entire nanopore. It can be applied using any suitable apparatus, such as the apparatus described in the details. The suitable potential is as described in the details. This will be described in more detail in the detailed document.

[0095] In some embodiments, a force is applied across a membrane in which nanopores are embedded. Force typically flows from the cis side of the film to the trans side, i.e., from the cis side of the nanopore to the trans side. , is applied. The force is a positive voltage applied to the nanopore, or a negative voltage applied to the nanopore. It could be pressure.

[0096] Typically, the force is positive on the trans side of the pore relative to the cis side of the pore in the nanopore. It is a positive voltage applied over a certain range. In such embodiments, the force is thus negatively charged. It attracts the polynucleotide and moves from the cis side to the trans side of the pore. In the application form, the method provided herein typically involves a motor tamper on the cis side of the pore. Using a material, against the applied force, from the transform side of the pore to the cis side of the pore, In other words, it involves controlling the movement of polynucleotides in the opposite direction to the applied force. However, in some embodiments, the methods provided herein (e.g., polynuclear The method of rereading the rheotide involves using a motor protein on the cis side of the pore, From the cis side of A to the trans side of pore, the polynucleotide moves in the same direction as the applied force. This may include controlling movement.

[0097] In other embodiments, the force is such that the transform side of the pore is negative relative to the cis side of the pore. This is a negative voltage applied across the nanopore. In such embodiments, the force is applied to the negatively charged nanopore. It attracts the renucleotide and moves from the trans side to the cis side of the pore. The method provided herein typically involves the motor protein on the transformer side of the pore. Using quality, against the applied force, in the direction from the cis side of the pore to the transform side of the pore, Furthermore, this involves controlling the movement of polynucleotides in the opposite direction to the applied force. However, in some embodiments, the methods provided herein (e.g., polynucleotides) The method for rereading the ocide involves using a motor protein on the trans side of the pore. From the trans side of the pore to the cis side of the pore, the polynucleotide is moved in the same direction as the applied force. This may include controlling movement.

[0098] However, as described below, the methods provided herein involve the applied force and It does not rely on moving polynucleotides in the opposite direction. In some embodiments, The direction of movement may be the same as the applied force, but still in the direction of exiting the pore. Yes. In such embodiments, the motor protein is typically based solely on the applied force. The movement of polynucleotides from the pore is controlled at a rate faster than the rate at which they are generated.

[0099] Therefore, in some embodiments, the force is such that the transform side of the pore is relative to the cis side of the pore. This method applies a positive voltage across the nanopore so that it is positive. The movement of polynucleotides from the cis side of the pore to the trans side of the pore is controlled by force. This may involve using a motor protein on the trans side of the pore. In the application configuration, the force is negative on the transform side of the pore relative to the cis side of the pore. This is a negative voltage applied over a certain distance, and this method is applied by the force on the transformer side of the pore. To control the movement of polynucleotides from the pore towards the cis side, on the cis side of the pore This may include using motor proteins.

[0100] setting In some embodiments of the provided method, the leader sequence is connected to the target polynucleotide. It is included or attached to a polynucleotide. The leader sequence is provided herein. In this method, it can be captured by a detector (e.g., a nanopore).

[0101] The leader sequence is described in more detail herein. Typically, the leader sequence is It is a single-stranded polynucleotide region that does not have a prominent secondary structure. For example, the leader sequence Typically, these do not form hairpins or G-quadrilaterals and are therefore trapped by nanopores. It is easy to do.

[0102] The leader sequence is typically provided at the first end of a polynucleotide, or at the end of a polynucleotide. The adapter is contained in the adapter attached to the first end of the renucleotide. It is described in more detail there.

[0103] Typically, the leader sequence is located at the first end of a polynucleotide (e.g., the target polynucleotide). By being contained in the first end of the rheotide, or at the first end of the target polynucleotide Provided by being contained in the attached polynucleotide adapter, motor protein The nucleotide is at the second end of the target polynucleotide, or at the second end of the target polynucleotide. It stalls on the adapter to which it is coupled. For example, the leader sequence is a single-stranded polynucleotide. Motor proteins can be located at the 3' end of a single-stranded polynucleotide, and at the 5' end of a single-stranded polynucleotide. It can be located at the 5' end of a single-stranded polynucleotide. Alternatively, the leader sequence may be located at the 5' end of a single-stranded polynucleotide. Motor proteins can be located at the 3' end of a single-stranded polynucleotide. Therefore, the first end of the polynucleotide is captured by the nanopore and passes through the nanopore. For example, it becomes possible to pass from the first end to the second end. (Polynucleotide) The motor protein at the second terminal is typically composed of polynucleotides that completely fill the nanopore. To prevent movement to the second end of the polynucleotide. In the method provided herein, End motor proteins typically carry polynucleotides from the second end to the first end. By processing in this direction, the nanopores move toward the motor protein. The movement of cleotides can be controlled.

[0104] In some embodiments, the target polynucleotide is single-stranded, and the target polynucleotide The dot contains the leader sequence, which is located at the first end of the target polynucleotide. Either it is contained in an adapter attached to the first end of the target polynucleotide, and it is a motor The protein stalls at the second end of the target polynucleotide, or the target polynucleotide The second end of the chid stalls on the adapter. In such embodiments, the leader array is, In terms of structure, it is captured by nanopores, and the single-stranded polynucleotide is a stall motor protein. It moves through the nanopore until it reaches its target. Once it loses momentum, the motor protein This controls the movement of polynucleotides from the pore. This is illustrated in Figure 2.

[0105] In some embodiments, the target polynucleotide is double-stranded.

[0106] In some embodiments, the target polynucleotide is double-stranded, with a first strand and a second strand The chain contains the target polynucleotide, the leader sequence contains the polynucleotide An adapter located at the first end of Otide, either included in the first chain or attached to the first chain. In this case, the motor protein stalls at the second end of the target polynucleotide. Due to this configuration, the first end of the first strand of the double-stranded polynucleotide is trapped by the nanopore. This allows the material to pass through the nanopore from the first end to the second end. The motor protein at the second terminal of the rheotide is typically composed of polynucleotides in nanoparticles. This prevents A from moving completely. The first strand of a double-stranded polynucleotide is the template strand. The first strand of a double-stranded polynucleotide can be a complementary strand.

[0107] In some embodiments, the motor protein is the first chain of the target polynucleotide It stalls at the second end, or the adapter at the second end of the first strand of the target polynucleotide. -It stalls above. In some embodiments, the target polynucleotide is double-stranded, and the first The target polynucleotide comprises a chain and a second chain, and includes a leader sequence, and the leader sequence It is located at the first end of the polynucleotide and is included in the first chain or attached to the first chain. The motor protein contained within the attached adapter is located in the first chain of the target polynucleotide. It stalls at the second end, or the adapter at the second end of the first strand of the target polynucleotide. —It stalls above. For example, the leader sequence is the 3' end of the first strand of a double-stranded polynucleotide. Motor proteins can be located at the ends, and are located at the 5' end of the first strand of a double-stranded polynucleotide. It can be located at the 5' end of the first strand of a double-stranded polynucleotide. Alternatively, the leader sequence may be at the 5' end of the first strand of the double-stranded polynucleotide. Motor proteins can be present in the 3' end of the first strand of a double-stranded polynucleotide. It can be placed. In such embodiments, the leader array is typically captured by a nanopore. The single-stranded polynucleotides pass through the nanopore until they reach the stall motor protein. Movement. After destallation, the motor protein moves from the pore of the first strand of the polynucleotide. This controls the movement of the object. This is illustrated in Figure 3.

[0108] In some embodiments, the first and second chains are connected by a hairpin at the second end of the first chain. The motor proteins, which attach together by the adapter, stall at the hairpin adapter. In some embodiments, the hairpin adapter has three of the first chains at its 5' end. It binds to the end and, at its 3' end, attaches to the 5' end of the second strand of the target double-stranded polynucleotide. In some embodiments, the hairpin adapter is connected to the 5' end of the first chain at its 3' end. It binds to the end and, at its 5' end, attaches to the 3' end of the second strand of the target double-stranded polynucleotide. Therefore, the hairpin adapter connects the first chain to the second chain. A typical example is a double-stranded polynucleotide where the second end of the first strand is connected to the double-stranded polynucleotide. It is linked to the first end of the second chain of creotide.

[0109] In some embodiments, the target polynucleotide is double-stranded, with a first strand and a second strand The chain contains the target polynucleotide, the leader sequence contains the polynucleotide An adapter located at the first end of Otide, either included in the first chain or attached to the first chain. -Included, the first chain and the second chain are connected by a hairpin adapter at the second end of the first chain And when they attach together, the motor protein stalls at the hairpin adapter. In the application morphology, the leader sequence is typically captured by nanopores and double-stranded polynucleotides. The first strand of Otid moves through the nanopore until it reaches the stalled motor protein. After destallation, the motor protein pores the first strand of the double-stranded polynucleotide. We control their movement. This is shown in Figure 4.

[0110] In some embodiments, the first and second chains are (i) the second end of the first chain, and (ii) The first end of the second chain is attached together by a hairpin adapter that is attached to it. Furthermore, the motor protein stalls at the second end of the second strand of the double-stranded polynucleotide. Or, stall at the second end of the second chain on the adapter. In some embodiments, The apin adapter connects its 5' end to the 3' end of the first strand of the target double-stranded polynucleotide. It attaches to the target double-stranded polynucleotide, and at its 3' end, it attaches to the 5' end of the second strand of the target double-stranded polynucleotide. The ter protein stalls at the 3' end of the second chain. In some embodiments, hairp The adapter attaches to the 5' end of the first strand of the target double-stranded polynucleotide at its 3' end. It attaches, and at its 5' end, it attaches to the 3' end of the second strand of the target double-stranded polynucleotide, and The tar protein stalls at the 5' end of the second chain. Therefore, the hairpin adapter The first chain is then connected to the second chain.

[0111] In some embodiments, the target polynucleotide is double-stranded, with a first strand and a second strand The chain contains the target polynucleotide, the leader sequence contains the polynucleotide An adapter located at the first end of Otide, either included in the first chain or attached to the first chain. -Included, the first chain and the second chain are (i) the second end of the first chain and (ii) the second The first end of the chain is attached together with a hairpin adapter attached to the motor. The protein stalls at the second end of the second strand of the double-stranded polynucleotide, or adapts to the Stalls at the second end of the second chain on the terminal. In such embodiments, the leader arrangement is typical. It is captured by nanopores, the first strand of the double-stranded polynucleotide, hairpin adapter - and the second strand of the double-stranded polynucleotide reach the stalled motor protein. It then moves through the nanopore. After destallation, the motor protein controls the movement of the second chain. In some cases, a hairpin adapter may also be used, and in even more cases, a double-strand polynucleotide may be used. The first chain of the ion also controls its exit from the pore. This is illustrated in Figure 5.

[0112] Instead of the motor protein stalling at the end of the polynucleotide, the polynucleotide It is clear that the motor may stall along the way. - In such embodiments, the protein is characterized in the manner provided herein. It stalls at the end of the nucleotide portion. Those skilled in the art will know that in the method provided herein, The characteristic polynucleotide portion is the motor protein on the polynucleotide. This parameter can be determined by the configuration and controlled by the user of this method. They will understand.

[0113] Embodiments of the disclosed method, which include rereading a target polynucleotide (e.g.) The motor protein controls the movement of the target polynucleotide in the first direction relative to the detector. When controlling the target polynucleotide, it is necessary to obtain one or more measurements characteristic of the target polynucleotide, and The motor protein moves the target polynucleotide in a second direction relative to the detector. Debinding of the target polynucleotide from the polynucleotide binding site, and the target polynucleotide Rebinding rheotide to the polynucleotide binding site of motor protein, and motor - When the protein controls the movement of the target polynucleotide in the first direction relative to the detector A method comprising obtaining one or more measurements characteristic of the target polynucleotide. ) When the motor protein is near the leader sequence (the motor protein is near the leader sequence) When it comes into contact with the motor sequence, the polynucleotide binding site of the motor protein is targeted. To promote the debonding of target polynucleotides, a leader sequence can be constructed or designed. can.

[0114] In such embodiments, the motor protein is typically paired with a target polynucleotide. Rather than doing so, the part of the target polynucleotide to be characterized relative to the leader sequence. It has a lower affinity than the affinity for the target. In some embodiments, the reader targets the target It has a different structure from polynucleotides. In some embodiments, the leader is a target It contains nucleotides of a different type than polynucleotides.

[0115] For example, in some embodiments, the target polynucleotide is a deoxyribonucleonucleotide. It contains DNA. In such embodiments, the reader includes nucleic acid bases and sugar moieties (for example, It may contain one or more nucleotides lacking both of the spacer portion. Suitable spacer portion The minutes are described in detail herein, including C2 spacers, C3 spacers, C6 spacers Includes Pacers, iSP9 spacers, iSP18 spacers, etc. Alternatively or additionally The leader is ribonucleotide (RNA), peptide nucleotide (PNA), and glycans. Cerol nucleotide (GNA), threose nucleotide (TNA), locked nucleotide It may contain osides (LNA), cross-linked nucleotides (BNA), or baseless nucleotides. In some embodiments, the leader is a modified phosphate bond (e.g., methylphosphonate). It may contain one or more nucleotides having a nucleotide or phosphothiolate bond.

[0116] In some other embodiments, the target polynucleotide is a ribonucleotide (RNA). Includes. In such embodiments, the leader includes one or more spacers, deoxygenated elements as defined above. Siribonucleotides (DNA), peptide nucleotides (PNA), glycerol nucleotides Otide (GNA), threose nucleotide (TNA), locked nucleotide (LNA) ), cross-linked nucleotides (BNA), debasic nucleotides, or nucleotides containing modified phosphate bonds May include Ochido

[0117] Typically, the target polynucleotide contains deoxyribonucleotides (DNA), The leader has one or more spacer parts (e.g., C3 spacers) and / or one or more Contains ribonucleotides.

[0118] The leader uses only one type of polynucleotide that is different from the target polynucleotide. It may include. For example, if the target polynucleotide is DNA, the leader may include spacers. It may contain a portion or RNA. The leader is of multiple types different from the target polynucleotide. It may contain polynucleotides. For example, if the target polynucleotide is DNA, The leader may contain a spacer portion and RNA. The leader contains the target polynucleotide and It may contain a portion that is the same type of polynucleotide. For example, the target polynucleotide is If it is DNA, the leader is DN in addition to spacer polynucleotides or RNA. It may include part A. Such part may be called a "trap," i.e., a spacer (e.g. For example, C3 spacer) and / or RNA (e.g., 2'-methoxyuridine) polynucleotide A leader based on rheotide may contain one or more DNA traps. A trap is typical. In general, 1 to 6 nucleotides, for example, 1, 2, 3, 4 or 5 nucleotides, for example It contains 1 to 10 nucleotides, such as 1 to 3 nucleotides. Therefore, the target If the polynucleotide is DNA, the leader is one or more RNAs (e.g., 2'- Methoxyuridine) and / or spacer (e.g., C3 spacer) portion, and 1-10 It may include one or more DNA (e.g., thymidine) traps of nucleotide length.

[0119] Those skilled in the art will know that if the leader contains a polynucleotide chain, the leader sequence is typical. It is not definitive, and motor proteins and any polynucleotides to be characterized It will also be understood that other experimental conditions, such as D, may be controlled or selected. The sequences are provided for illustrative purposes only in the examples, particularly in Example 10. For example, the leader This is an array such as one or more sequence numbers 70, 71, or 72, or sequence numbers 70, 71, or 7 One or more of 2 and at least 20%, for example at least 30%, for example at least 40%, For example, at least 50%, for example, at least 60%, for example, at least 70%, for example, a small At least 80%, for example at least 90%, for example at least 95% sequence similarity or the same It may contain a monochromatic polynucleotide sequence. The leader sequence is typically as described in this specification. The methods provided in this document may be modified without adversely affecting their effectiveness.

[0120] Stalling of motor proteins As explained above, the method provided herein involves a motor tank stalled in the stalled section. This includes characterizing target polynucleotides that have protein on top.

[0121] Any suitable stall portion may be used in the manner provided herein. Several implementations In this state, the stall portion includes the stall portion described herein. In some embodiments, stall The part includes one or more stall units.

[0122] Any suitable stall unit can be used. The stall unit is typically a motor protein. It provides an energy barrier that hinders the movement of quality. For example, a stall unit is on a polynucleotide. This reduces the traction force of the motor protein, causing it to stall. This is obtained, for example, from a debase spacer, i.e., a polynucleotide adapter. - Achieved by using spacers removed from one or more nucleotides in the mixture. Spacers can be used, for example, to introduce larger chemical groups for polynucleotide handling. By physically hindering the movement of proteins, polynucleotide handling proteins It is possible to physically block the movement of the substance.

[0123] In some embodiments, the stall unit may include linear molecules such as polymers. In this case, the stall unit has a structure different from that of the target polynucleotide. For example, the target If the polynucleotide is DNA, then the or each stall unit typically contains DNA. No. Specifically, the target polynucleotide is deoxyribonucleic acid (DNA) or ribonucleic acid. If it is RNA, then the stall unit or each stall unit is preferably peptide nucleic acid (PNA), Glycerol nucleic acid (GNA), threose nucleic acid (TNA), locked nucleic acid (LNA), The invention includes a synthetic polymer having bridged nucleic acid (BNA) or nucleotide side chains. Several implementations Morphologically, the stall unit consists of one or more nitroindoles, one or more inosine, and one or more Acridine, one or more 2-aminopurines, one or more 2-6-diaminopurines, one or more The above 5-bromodoxyuridine, one or more inverted thymidines (inverted dT), one or more Inverted dideoxythymidine (ddT), one or more dideoxycytidines (ddC), 1 One or more 5-methylcytidines, one or more 5-hydroxymethylcytidines, one or more 2 -O-methylRNA base, one or more iso-deoxycytidines (Iso-dC), one The above iso-deoxyguanosine (Iso-dG), one or more C3(OC3H6OPO 3) One or more light-cutting (PC) units [OC3H6-C(O)NHCH2-C6H3 NO2-CH(CH3)OPO3] group, one or more hexanediol groups, one or more hexanediol groups A 9(iSp9)[(OCH2CH2)3OPO3] group, or one or more spaces A 18 (iSp18)[(OCH2CH2)6OPO3] group, or one or more thiols It may include bonds. The stall site may include any combination of these groups. Many of ours are IDT (registered trademark) (Integrated DNA Technology). It is commercially available from gies(registered trademark). For example, C3, iSp9, and iSp18 All spacers are available from IDT (registered trademark). The stall area is defined as a stall unit. It may contain any number of the above bases. For example, the stall site may have 1 to about 12 or more (for example, about 1 This can include approximately 8 stall units, for example, 1 to 4 units (or 1 to 6 units).

[0124] In some embodiments, the stall unit is one or more compounds that stall the motor protein. It may include chemical groups. In some embodiments, preferred chemical groups are one or more pendant chemical groups. It is a group. One or more chemical groups can bind to one or more nucleic acid bases in a polynucleotide. One or more chemical groups may be attached to the polynucleotide backbone. 2, 3, 4, 5, 6, 7 Any number of suitable chemical groups may be present, such as 8, 9, 10, 11, 12 or more. The base contains fluorophores, streptavidin and / or biotin, cholesterol, and Chilen blue, dinitrophenol (DNP), digoxigenin and / or anti-digoxigenin This includes, but is not limited to, nin and dibenzylcyclooctin groups.

[0125] In some embodiments, the stall unit may include a polymer. In some embodiments, The stall unit contains a polymer that is a polypeptide or polyethylene glycol (PEG). obtain.

[0126] In some embodiments, the stall unit is one or more debaselated nucleotides (i.e., nuclei Nucleotides lacking an acid-base pair, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, It may contain 12 or more debasalized nucleotides. Nucleic acid bases are present in the debasalized nucleotides. It can be replaced by -H(idSp) or -OH. A debase residue is one or more adjacent By removing nucleic acid bases from the target nucleotide, insertion into the target polynucleotide occurs. It is possible. For example, polynucleotides include 3-methyladenine, 7-methylguanine, 1 , may be modified to include N6-ethenoadenine inosine or hypoxanthine, nucleic acid salt The base uses human alkyladenine DNA glycosylase (hAAG) to convert these nuclei It can be removed from the rheotide. Alternatively, the polynucleotide can be modified to contain uracil. The nucleic acid bases, which may be embellished, can be removed by uracil DNA glycosylase (UDG). In one embodiment, one or more stall units do not contain any debasalized nucleotides.

[0127] Suitable stall units are those with polynucleotide / polynucleotide adapter properties, and motors. The protein and the conditions under which this method should be performed can be designed or selected accordingly. For example, many polynucleotide processing proteins process DNA in vivo. And such proteins typically lose their DNA using something other than DNA. It can be done quickly.

[0128] Therefore, in some embodiments of the provided method, the motor protein is as follows: The vehicle stalls in a stall region that includes one or more stall units selected independently of the vehicle: -Polynucleotide secondary structure, preferably hairpin or G-quadrivalent (TBA), - Preferably, peptide nucleic acid (PNA), glycerol nucleic acid (GNA), threose nucleus Acids (TNA), locked nucleic acids (LNA), cross-linked nucleic acids (BNA), and debasalized nucleotides nucleic acid analogs selected from, - Nitroindole, inosine, acridine, 2-aminopurine, 2-6-diaminopurine Phosphorus, 5-bromodeoxyuridine, inverted thymidine (inverted dTs), inverted dideoxy- Thymidine (ddTs), dideoxycytidine (ddCs), 5-methylcytidine, 5- Hydroxymethylcytidine, 2'-O-methylRNA base, isodeoxycytidine (Is o-dCs), isodeoxyguanosine (Iso-dGs), C3(OC3H6OPO3) ) group, photocleavable (PC) [OC3H6-C(O)NHCH2-C6H3NO2-CH( CH3)OPO3 group, hexanediol group, spacer 9 (iSp9)[(OCH2C H2)3OPO3] base, spacer 18 (iSp18) [(OCH2CH2)6OPO3 A spacer unit selected from ] groups and thiol linkages, and -Fluorophores, traptabidine, streptavidin, and neutraavidin, etc. avidin and / or biotin, cholesterol, methylene blue, dinitrophenol (DNP), digoxigenin and / or anti-digoxigenin and dibenzylcyclooctane n base.

[0129] The stall portion described herein is suitable for use in the disclosed rereading method. It is also possible to configure a commander. As explained above, there are several implementations of this method. In this state, when the motor protein is near the leader (for example, the motor protein When it comes into contact with the leader sequence, the polynucleotide binding site of the motor protein To promote the dissociation of target polynucleotides, the leader sequences described herein are constructed. Or design. In some embodiments, the leader array is any of the above spacer portions It may include any of the following.

[0130] Motor protein destallation In some embodiments, the method provided herein involves detecting the stall portion with a detector (e.g., This involves contacting the motor protein with a nanopore, thereby causing it to lose momentum. After stalling, the motor protein exits the detector, as will be described in more detail herein. It is possible to control the movement of polynucleotides (for example, those emerging from nanopores).

[0131] In its simplest form, the stalled portion is brought into contact with a detector, such as a nanopore, to detect the loss. The motor protein can be de-stamped from the fast portion. However, in some cases... In this application, the method actively destabilizes the motor protein as described herein. This includes causing someone to do something.

[0132] In some embodiments, the motor protein is destamped, and polynucleotides This includes applying a destall force to the drive, wherein the destall force is less than the reading force, and / or In the opposite direction, the reading power is controlled by the motor protein's ability to control the movement of the target polynucleotide. This method is applied while measurements are being taken to determine one or more characteristics of a polynucleotide. It is a power that is exerted.

[0133] For example, the reading range is typically +2V to -2V, and the potential range is typically -400mV to +400mV. It can be provided as follows. The voltage used is preferably -400mV, -300mV, - Select from 200mV, -150mV, -100mV, -50mV, -20mV, and 0mV. The lower limit to be selected is +10mV, +20mV, +50mV, +100mV, +150mV, It has upper limits that can be independently selected from +200mV, +300mV, and +400mV. It is within the range. The voltage used is more preferably in the range of 100mV to 240mV. The voltage is preferably in the range of 120mV to 220mV. Typically, the destabilization force is greater than the reading force. It is also small. For example, the destall force is approximately -100mV to +100mV, for example, approximately -50mV to It could be approximately +50mV, for example, between approximately -25mV and approximately +25mV.

[0134] For example, in some embodiments, the reading power is +50mV to +300mV, more preferably. More specifically, in the range of +100mV to +200mV, such as +120mV to +150mV. Yes, the de-stall force is -40mV to +40mV, or -50mV to +50mV, for example, 0mV. The potential range is between -20mV and +20mV.

[0135] In some embodiments, the destabilization force is in the opposite direction to the reading force. For example, several In that embodiment, the reading force is applied as a positive potential, and the destabilization force is applied as a negative potential. In other embodiments, the reading force is applied as a negative potential and the destabilization force is a positive potential. It is applied as a position. If the destabilization force is in the opposite direction to the reading force, then it is the reading force and They may be the same size, or they may be smaller than the reading power.

[0136] In some embodiments, the destall force is applied at zero potential. For example, in some embodiments In this configuration, the reading force is applied as a positive potential, and the destabilization force is applied at a zero potential. In other embodiments, the reading force is applied as a negative potential, and the destabilization force is the zero applied potential. It applies to [the system / platform].

[0137] In some embodiments, the destalling force is the destalling force of the motor protein from the stalled region. It is applied for a sufficient amount of time. In some embodiments, the destuck force is applied for about 10 ms to about 1 s For example, 1ms to about 10s, for example, about 300ms to about 500ms, or about 100ms to about 700ms, applied.

[0138] In some embodiments, the destalling of a motor protein is read as the destalling force. This includes changing the force applied between the take force and the take force one or more times. In some embodiments, Changing the applied force in this way affects the applied potential between the stall force and the reading force. This includes stepping or sloping between the intervals. If sloping, any suitable waveform can be used. For example, the slope can be linear, exponential, or sigmoid. .

[0139] In some embodiments, the applied force varies between a single destabilization force and a reading force. In some embodiments, the applied force is a series of different destabilization forces and reading forces. It varies between. In some embodiments, the applied force is a series of increasing destuck forces and The destall force changes in stages between the reading force and the destall force. The destall force at each step is any suitable destall force. The stall force may be any of the de-stall forces described herein, and each step is performed as It may be applied for a suitable period of time, for example, any period as described herein.

[0140] In some embodiments, the destalling force is the same as the reading force. This is also referred to as "escaping stall" in the "ning" setting.

[0141] In some embodiments, the motor protein comprises one or more stall units and one or more The vehicle stalls at a stalled area that includes a stopping section, and when one or more stopping sections come into contact with a nanopore, the nano The movement of polynucleotides through the pore is delayed, and as a result, one motor protein The vehicle de-stalls from the above-mentioned stall units. This embodiment is suitable for use in self-propelled settings.

[0142] In some embodiments, the stopping portion prevents the movement of polynucleotides through the nanopore. It provides an energy barrier. For example, the stopping portion allows polynucleotides to pass through the nanopore. By providing physical blocks that need to be removed before it becomes possible to do so This could hinder the movement of polynucleotides through nanopores.

[0143] While not bound by theory, the inventors believe that the stopping portion is where the motor protein is lost. The movement of polynucleotides through the nanopore is slowed down enough time to overcome the speed unit and stall. I think it should be delayed.

[0144] In some embodiments, the termination portion is a polynucleotide secondary structure, preferably a hairpin. It contains one or more stopping units including a polynuclear or G quadruple chain (TBA). Such secondary structures are polynuclear. This prevents the creotide from freely passing through the nanopore. When the stopping part comes into contact with the nanopore... The secondary structure dissociates (for example, unravels). Depending on the time it takes for the secondary structure to dissociate, This allows the motor protein to destall from the stall unit.

[0145] In some embodiments, the stop portion includes a hybridized oligonucleotide. Contains one or more stop units. The oligonucleotide is hybridized to the target polynucleotide. This can prevent the target polynucleotide from moving through the nanopore. The stopping portion is the nanopore. When brought into contact with the target polynucleotide, the hybridized oligonucleotide is separated from the target polynucleotide. To separate. The hybridized oligonucleotide dissociates from the target polynucleotide. The time required determines whether the motor protein can destall from the stall unit.

[0146] In some embodiments, the stop portion is preferably peptide nucleic acid (PNA), glycero Selected from granular nucleic acid (GNA), threose nucleic acid (TNA), and locked nucleic acid (LNA) One or more nucleotide analogs, cross-linked nucleic acids (BNA), and debasalized nucleotides are included. The unit includes the nucleic acid analog, which may be provided together with the target polynucleotide, or labeled They may hybridize to or otherwise bind to target polynucleotides. Nucleic acids When the analogue is provided along with the target polynucleotide, the stop portion is brought into contact with the nanopore. The nucleic acid analog passes through the nanopore. Depending on the time it takes for the nucleic acid analog to pass through the pore, This allows the motor protein to escape stall from the stall unit. Nucleic acid analogs target the port When hybridizing to a renucleotide, if the stop portion is brought into contact with a nanopore, typically In this, nucleic acid analogs are targeted so that the target polynucleotide can pass through the nanopore. It dissociates from the polynucleotide. It takes time for nucleic acid analogs to dissociate from polynucleotides. Depending on the time elapsed, the motor protein can destall from the stall unit.

[0147] In some embodiments, the stop portion is fluorophore, traptavidin, strep Avidins such as toavidin and neutraavidin, and / or biotin, cholesterol Methylene blue, dinitrophenol (DNP), digoxigenin and / or anti-digoxigenin It contains one or more termination units that include chemical groups such as xygenin and dibenzylcyclooctin groups. The chemical group attaches to the target polynucleotide, and the target polynucleotide passes through the nanopore. This can prevent that from happening. In some embodiments, when the stopping portion is brought into contact with the nanopore, the target A chemical group is removed from the target polynucleotide. In some embodiments, the termination portion is nano When brought into contact with the pore, the chemical group passes through the nanopore. The chemical group is removed from the target polynucleotide. Depending on the time it takes for the motor protein to be removed and / or to pass through the nanopore, This makes it possible to escape stall from the stall unit.

[0148] In some embodiments, the termination portion contains one or more polynucleotide-binding proteins. Includes the above-mentioned resting unit. Suitable polynucleotide-binding proteins are described in more detail herein. It is described. Polynucleotide-binding proteins bind to polynucleotides, nano This can prevent the movement of polynucleotides through the pore. When the stopping portion is brought into contact with the nanopore, For example, when a polynucleotide-binding protein moves and comes into contact with a motor protein. This delays the movement of polynucleotides through the nanopore. Depending on the time taken for execution, This allows the ter protein to escape stall from the stall unit.

[0149] Although not bound by theory, the inventors also believe that the stopping portion is a point in the stopping unit. We believe that this often determines the three-dimensional structure of a renucleotide. The upper spacer 18 (iSp18) [(OCH2CH2)6OPO3] contains linear groups. This is especially true in the case of [unspecified]. Although not bound by theory, such stall units are nanopores and If in contact, the force applied across the pore (e.g., the applied voltage field) will stall. It is thought that the part can be extended almost in a straight line. In this three-dimensional structure, the motor tank Proteins typically cannot pass through the stalled region and lose stall. However, target polymers When a nucleotide is stopped at a stop region, the environment of the stalled unit is the same as the environment in solution. It is thought that the stall unit can employ a more compact pseudo-random coil configuration. In this configuration, it may become easier for motor proteins to overcome stall units and destall. .

[0150] Therefore, in some embodiments, the motor protein is selected independently from the following: The vehicle stalls in a stall section that includes one or more stall units and one or more stopping units: -Polynucleotide secondary structure, preferably hairpin or G-quadrivalent (TBA), - Preferably, peptide nucleic acid (PNA), glycerol nucleic acid (GNA), threose nucleus Acids (TNA), locked nucleic acids (LNA), cross-linked nucleic acids (BNA), and debasalized nucleotides nucleic acid analogs selected from, -Fluorophores, traptabidine, streptavidin, and neutraavidin, etc. avidin and / or biotin, cholesterol, methylene blue, dinitrophenol (DNP), digoxigenin and / or anti-digoxigenin and dibenzylcyclooctane n group, and -Polynucleotide-binding proteins, Furthermore, when one or more stopping points come into contact with the nanopore, the polynucleotide passing through the nanopore... The movement is delayed, causing the motor protein to lose stall from one or more stall units. .

[0151] Motor protein As those skilled in the art will understand, any suitable motor protein is provided herein. It can be used in the methods and products described.

[0152] Motor proteins can bind to polynucleotides and to detectors, for example. Any protein that can control the movement of a nanopore, for example, through the pore. It could be a quality.

[0153] More specifically, motor proteins such as helicases typically have at least two The active operating mode (all components necessary to promote transport, e.g., ATP and Mg 2+ (if provided) and one inactive operating mode (components necessary to facilitate movement) If not provided, or if the motor protein is modified to prevent active mode (In the case where this is the case) DNA movement can be controlled.

[0154] Once all the necessary components to facilitate movement are provided, the motor protein DN Moves along polynucleotides such as A in either the 5'-3' or 3'-5' direction. Many motor proteins can do this. Many motor proteins use polynucleotides such as DNA to form 5' Processing is performed in the -3' direction. This is how the motor controls the movement of polynucleotides. Proteins are typically suitable for use in the methods provided herein.

[0155] However, motor proteins lack the components necessary to facilitate movement. In cases where this is not possible, or where it hinders the active control of the movement of polynucleotides relative to the nanopore. If modified for this purpose, it still affects the movement of polynucleotides relative to nanopores. It can be passively controlled. For example, motor proteins can control polynucleotides. The polynucleotide binds and is applied at the site (for example, by the method provided herein). When pulled into the pore by the first force in the pore, the movement of the polynucleotide is slowed down. It can act as a brake. In "inactive" mode, the applied force is nano Normally, DNA is 3' long to provide the propulsion force that moves polynucleotides through the pore. Or is it captured in either the 5' or 5' direction (i.e., the nanopore in the 5'-3' direction or the 3'-5' direction) Whether it moves in the direction is not an issue. However, in such embodiments, the motor tamper The substance acts, for example, as a brake, to form polynucleotides against nanopores. Its movement can still be controlled. In inactive mode, the motor protein controls the polygon The control of the cleotide's movement includes ratcheting, sliding, and braking. It can be explained in several ways. Typically, the methods provided herein are passive modes. It does not involve the use of motor proteins that operate on a motor. However, it does not involve polynucleotides. Embodiments of the methods provided herein that use binding proteins include polynucleotides. The binding protein may be a motor protein that operates in a passive mode.

[0156] As described above, some embodiments of the methods provided herein involve nanopores Polynucleotide-binding proteins act as stopping points that prevent the movement of polynucleotide chains through them. This also includes the use of protein. In some embodiments, the polynucleotide-binding protein is the present invention It may be a motor protein as described in the details. In other embodiments, polynucleotide binding Proteins bind to polynucleotides, but they lack the ability to process polynucleotides. It can be a protein, that is, in some embodiments it is a motor protein do not have.

[0157] Polynucleotide handling enzymes can interact with polynucleotides. It is a polypeptide. Enzymes are composed of individual nucleotides or shorter chains of nucleotides, for example. By cleaving polynucleotides in order to form dinucleotides or trinucleotides... The polynucleotide may be modified. The enzyme may orient it or position it at a specific position. Polynucleotides may be modified by moving them to a specific position. Even if the motor protein is a polynucleotide handling enzyme, the origin of it It may also be the case that polynucleotide-binding proteins are polynucleotide-handling enzymes. However, it could also be derived from that.

[0158] In one embodiment, the motor protein is more preferably an enzyme classification (EC) group 3.1.11, 3.1.13, 3.1.14, 3.1.15, 3.1.16, 3.1.2 1, 3.1.22, 3.1.25, 3.1.26, 3.1.27, 3.1.30, and 3 It originates from one of the members in .1.31.

[0159] Typically, motor proteins are helicases, polymerases, and exonucleases. , topoisomerase, or a variant thereof.

[0160] In some embodiments, motor proteins and / or polynucleotide-binding proteins The modification allows the motor protein to detach from the polynucleotide. This can be prevented. This includes, as specified herein, rereading the target polynucleotide. This is particularly useful in the disclosed method. Therefore, some embodiments of such method Therefore, the target polynucleotide does not detach from the motor protein.

[0161] As used herein, the term "disassociation" refers to the disassociation of motor proteins from a target polynucleotide. This refers to the dissociation of a molecule. Therefore, motor proteins are examples of molecules that dissociate from their target polynucleotides. For example, it can be modified to prevent dissociation into a reaction medium. Potential of motor proteins Distinguishing "detachment" from the "unbinding" of motor proteins from target polynucleotides This is important. As used herein, "debonding" refers to the motor of the target polynucleotide. This refers to the temporary release of the active site of a protein (described in more detail herein), but does not mean detachment. It does not have a taste. Therefore, for example, motor proteins are motor proteins It prevents the motor protein from unassociating with the polynucleotide, but the motor protein is polynucleotide It may be modified so as not to prevent detachment from the cyd. If it is not coupled, the motor - The protein remains bound to the target polynucleotide. For example, motor proteins The quality is such that it can maintain binding to the target polynucleotide (i.e., the target polynucleotide or (This can prevent their disassociation), but it is topologically closed around the target polynucleotide. This is because the polynucleotide binding site is where the motor protein targets the polynucleotide. While bound to the rheotide, the motor protein binds to the target polynucleotide or It remains freely bound to or detached from the target polynucleotide, allowing for detachment. Obtained. When a motor protein detaches from its target polynucleotide, the applied force It can move along (for example, along) the target polynucleotide below, and the target poly It can rebind to nucleotides. It associates with the target polynucleotide, but the target polynucleotide When detached from the ocide, the motor protein dissociates from the target polynucleotide. It is not possible.

[0162] Motor proteins and / or polynucleotide-binding proteins are used in any preferred manner. This can be adapted to prevent detachment. For example, motor proteins and / or The polynucleotide-binding protein is loaded onto the polynucleotide, and then it is... It can be modified to prevent deassociation from renucleotides. Alternatively, motor Proteins and / or polynucleotide-binding proteins are loaded onto polynucleotides. Previously, it may be modified to prevent it from deassociating with polynucleotides. - Proteins and / or polynucleotide-binding proteins deassociate from polynucleotides Modification of motor proteins to prevent this can be done using methods known in the art, for example. If so, the entirety of WO2014 / 013260 is incorporated herein by reference. Using the method described, the motor protein unassociates from the polynucleotide chain. A section describing the modification of motor proteins such as helicases to prevent this. This can be achieved by referencing, for example, motor proteins and / or polynucleotides. Otidyl binding proteins are treated with tetramethylazodicarboxamide (TMAD). It can be modified by and. Various other closing parts are described in detail herein. It can be done.

[0163] For example, motor proteins and / or polynucleotide-binding proteins are motor proteins. When a protein and / or polynucleotide-binding protein detaches from a chain, the chain A polynucleotide debonding opening through which it can pass, for example, a cavity, groove, or void It may have. In some embodiments, the polynucleotide debinding opening is a motor protein When a nucleotide and / or polynucleotide-binding protein unassociates from a nucleotide, This is an opening through which creotide can pass. In some embodiments, a given mo Debonding of polynucleotides in ter proteins and / or polynucleotide-binding proteins The mouth part can be described by referring to its structure, for example by referring to its X-ray crystal structure. The X-ray crystal structure can be determined in the presence and / or absence of a polynucleotide substrate. It can be obtained under certain conditions. In some embodiments, a given motor protein and / or The position of the polynucleotide debinding opening in polynucleotide-binding proteins is Estimated or confirmed by molecular modeling using standard packages known in the art. Obtained. In some embodiments, the polynucleotide debinding opening is located in the motor protein. One or more parts of it may be transiently generated, for example, by the migration of one or more domains.

[0164] Motor proteins and / or polynucleotide-binding proteins are polynucleotides It can be modified by closing the debinding opening. The polynucleotide debinding opening is It can be closed at the closed portion. Therefore, to close the polynucleotide debonding opening Therefore, motor proteins and / or polynucleotide-binding proteins are polynucleotides Disassociation from the nucleotide can be prevented. For example, motor proteins and / or polynucleotides. Otid binding proteins covalently close polynucleotide debonding openings. Therefore, it can be modified. However, as explained above, polynucleotide debonding opening Closing the part means that the target polynucleotide is bound to the motor protein's polynucleotide bond. This does not necessarily prevent detachment from the site. In some embodiments, this is addressed. The preferred protein for this purpose is helicase.

[0165] In some embodiments, the disclosure includes, in particular, rereading a target polynucleotide. In embodiments of the method, the motor protein is released from the target polynucleotide. The motor protein can be modified to prevent the deassociation of renucleotides. It can be modified in any way.

[0166] Although not bound by theory, the inventors believe that they can promote debonding and delay rebonding. I believe this could facilitate reinterpretation. It is not bound by theory. However, the inventors believe that this is because motor proteins perform actions on target polynucleotides. Each step is related to the probability that the motor protein will detach from the polynucleotide. This is because we believe that such decoupling is possible at what is called an off-rate. It can be separated. An increase in the off-speed is due to the drag of the motor protein against the polynucleotide chain. It is thought to promote a back-and-forth. Similarly, although not bound by theory again, this The inventors found that when the target polynucleotide is detached, the motor protein reattaches. The distance that can be traveled along the target polynucleotide before it is related to the on (on) velocity. It is thought that they are related. Therefore, rereading is motor with respect to the target polynucleotide. This can be facilitated by increasing the off-speed of the protein and decreasing the on-speed. Adjust the off-speed and on-speed of motor proteins for this type of polynucleotide. Doing so is within the capabilities of a person skilled in the art, taking into consideration the disclosures herein. Motor proteins are targeted by target polynucleotides from the polynucleotide binding site of motor proteins. To promote the debinding of rheotides and / or the polynucleotide binding of motor proteins Modifications may be made to delay the rebinding of target polynucleotides to the binding site. In this embodiment, the motor protein is the polynucleotide binding site of the motor protein. To promote the debinding of target polynucleotides from the position, and the polynucleotides of motor proteins It promotes both delaying the rebinding of target polynucleotides to nucleotide binding sites. It is modified to mean "to advance".

[0167] In some embodiments, the motor protein is located around (i) the target polynucleotide. (ii) The polynucleotide binding site of the motor protein is topologically closed, Promotes the debinding of target polynucleotides from the polynucleotide binding site of a target protein. The target polynucleotides of the motor protein to the polynucleotide binding site. The motor protein may be modified with a closure region to delay ocide rebinding. Such closed portions may be modified in any preferred manner to facilitate adhesion.

[0168] In some embodiments, the closed portion may include a bifunctional crosslinked portion. It may contain a functional crosslinking agent. The bifunctional crosslinking agent attaches to the motor protein at two points. , by closing the polynucleotide debinding opening of the motor protein, thereby the motor While preventing the detachment of polynucleotides from proteins, the polynucleotides of motor proteins This enables the detachment of polynucleotides from nucleotide binding sites.

[0169] The closure portion can attach to any suitable position on the motor protein. For example, the closure portion These can crosslink two amino acid residues in a motor protein. Typically, in the closure portion, Therefore, at least one of the crosslinked amino acids is cysteine ​​or a non-natural amino acid. Cysteine ​​or non-natural amino acids are naturally occurring amino acid residues in motor proteins. It can be introduced into motor proteins by substitution or modification. The law is well known in the art, for example, synthetic polymers containing such non-natural amino acids It contains natural chemical ligation with the motor chain. It introduces cysteine ​​into the motor protein. Methods for doing so include, for example, Sambrook et al., Molecular Cl Oning: A Laboratory Manual, 4 th ed., Cold S pring Harbor Press, Plainsview, New York(2 012), and Ausubel et al.,Current Protocols in Molecular Biology (Supplement 114), John References such as Wiley & Sons, New York (2016) are cited. The technology is used within the scope of the skills of those skilled in the art.

[0170] In some embodiments, the closed portion has a length of about 1 Å to about 100 Å. The length is determined according to the static bond length, or more preferably using molecular dynamics simulations. It can be calculated as follows. The length is, for example, approximately 2 Å to approximately 80 Å, for example, approximately 5 Å to approximately 50 Å, for example, about 8 to about 30 Å, for example, about 10 to about 25 Å or about 20 Å, for example, about 1, 2, 3 , 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, Alternatively, it could be 19 Å.

[0171] While not bound by theory, the inventors generally believe that a longer closing portion is possible. Increase the off-rate of motor proteins from renucleotides, and therefore re-read We believe this could facilitate the process.

[0172] In some embodiments, the closing portion includes a joint. In some embodiments, the closing portion It contains disulfide bonds. Disulfide bonds connect motor proteins such as TMAD. It can be formed by treatment with any suitable reagent.

[0173] In some embodiments, the closure portion consists of two click chemises on the motor protein. The reagents include those that form bonds between tree groups. Examples of click chemistry reagents are given herein. It will be provided to.

[0174] In some embodiments, the closed portion contains a protein. For example, a biotin group is a motor. - It may be present on a protein, and the closed portion may contain streptavidin. Tags such as p-tags or spy-tags may be present on motor proteins, and the closed portion is Each may contain proteins such as snoop catcher or spy catcher.

[0175] In some embodiments, the closing portion includes the structure of formula [ABC], where A and Each C is an independent reactive functional group that reacts with amino acid residues in the motor protein. And B is the connecting portion. In some embodiments, the closing portion is a thio group, for example This includes bonds between thiol groups on cysteine ​​residues. Therefore, in some embodiments, A and C are cysteine-reactive functional groups. In some embodiments, the linking portion B is Linear or branched unsubstituted or substituted alkylenes, alkenylenes, and alkynylenes , containing allylene, heteroarylene, carbocyrene or heterocyclene moiety, The part is one or more of O, N(R), S, C(O), C(O)NR, C(O)O, non-position Substitution or substitution allirene, allirene-alkylene, heteroarirene, heteroar Len-alkylene, carbocyclylene, carbocyclylene-alkylene, heterocyclylene Atoms or groups selected from ¹¹ and heterocyclylene-alkylenes, which are optionally interrupted. or terminates, in the formula R is H, unsubstituted or substituted alkyl, and unsubstituted or Selected from substituted aryls. Typically, R is H or methyl, and more typically H That is the case.

[0176] Typically, the alkylene group is C 1-20 It is an alkylene group. Typically, an alkylene group. The n group is C 2-20 It is an alkenylene group. Typically, an alkenylene group is C 2-20 Al It is a quinylene group. Typically, an allerene group is C 6-12 It is an arylene group. Typical. In this context, the heteroarylene group is a heteroarylene group with 5 to 12 members. Typically, The rubocyclylene group is C 5-12 It is a carbocyclylene group. Typically, heterocyclylene The 'n' group is a heterocyclylene group with 5 to 12 members.

[0177] Typically, alkylene, alkenylene, or alkynylene moieties consist of O, N(R), and S. Selected from C(O), C(O)NR, and C(O)O and unsubstituted or substituted arylenes. It may be interrupted or terminated at the atom or group being treated. Typically alkylenes, alkenylenes, or The alkynylene moiety is selected from O, N(R), and unsubstituted or substituted arylenes. Not interrupted by, or being interrupted by, one or more atoms or groups It may terminate. More often, the alkylene, alkenylene, or alkynylene portion is 1 Not interrupted by more than one oxygen atom, or interrupted by one or more oxygen atoms It can end there.

[0178] For example, the bonding portion is often unsubstituted or substituted C. 1-10 Alkylene, C 2-10 Alkenylene, or C 2-10 This is the alkynylene moiety, interrupted by one or more oxygen atoms. It either does not occur, is interrupted, or terminates at an oxygen atom.

[0179] In some embodiments, the connecting portion B is alkylene, oxyalkylene, or polyoxy It contains an alkylene group and / or A and C are each maleimide groups. Xyalkylene or polyoxyalkylene groups are, for example, about 5 Å to about 50 Å, for example, about They can have lengths of approximately 8 to 30 Å, such as 10 to approximately 25 Å.

[0180] For example, the connecting part is (CH2CH2O) xThe formula may include PEG parts such as, x is 1 to 10, for example 1 to 5, for example 1, 2, or 3. The exemplary connecting portion is in Example 9. It is described in, for example, BMOE (1,2-bismaleimideethane), BMOP (1 ,3-bismaleimidepropane), BMB (1,4-bismaleimidebutane), BM (P EG)2(1,8-bismaleimide-diethylene glycol) and BM(PEG)3(1 Examples include 11-bismaleimide-triethylene glycol.

[0181] Motor proteins suitable for closure using the above-described closure portion are as described in this specification. This will be discussed in more detail in the book. In some preferred embodiments, the motor protein is Licase, for example, Dda helicase as described herein.

[0182] In one embodiment, the motor protein and / or polynucleotide-binding protein are It is either an exonuclease or derived from an exonuclease. Preferred enzymes include E Exonuclease I (SEQ ID NO: 1) derived from E. coli, exonuclease I derived from E. coli Rease III enzyme (SEQ ID NO: 2), RecJ (derived from T. thermophilus) Column number 3) and bacteriophage lambda exonuclease (SEQ ID NO: 4), TatD This includes, but is not limited to, exonucleases and their variants. Three subunits, including the sequence shown in number 3 or its variant, interact to form a trimer. - Forms exonuclease.

[0183] In one embodiment, the motor protein and / or polynucleotide-binding protein are It is derived from polymerase. Polymerase is PyroPhage® 3173 DNA polymerase (commercially available from Lucigen (registered trademark) Corporation) (is available), SD polymerase (commercially available from Bioron®), NEB It may be Klenow, or a variant thereof. In one embodiment, the enzyme is Phi2 9 DNA polymerase (SEQ ID NO: 5) or a variant thereof. P that can be used in the present invention A modified version of hi29 polymerase is disclosed in U.S. Patent No. 5,576,204. It is being done.

[0184] In one embodiment, the motor protein and / or polynucleotide-binding protein are It is derived from topoisomerase. In one embodiment, the topoisomerase is preferably partially A member of either of the classification (EC) groups 5.99.1.2 and 5.99.1.3 Topoisomerase is an enzyme that can catalyze the formation of cDNA from an RNA template. These could be reverse transcriptases, for example, New England Biola It is commercially available from bs (registered trademark) and Invitrogen (registered trademark).

[0185] In one embodiment, the motor protein and / or polynucleotide-binding protein are It is derived from a helicase. Any suitable helicase is used according to the method provided herein. It can be used. For example, the enzymes used in accordance with this disclosure, or each enzyme independently, may be H el308 helicase, RecD helicase, TraI helicase, TrwC helicase XPD helicase and Dda helicase, or their variants, may be selected. Nomeric helicases may contain several domains attached together. For example, TraI Helicases and TraI subgroup helicases have two RecD helicase domains. These may include a relaxase domain and a C-terminal domain. These domains are typical It forms monomer helicases that can function without forming oligomers. Specific examples of suitable helicases include Hel308, NS3, Dda, UvrD, and R Examples include ep, PcrA, Pif1, and TraI. These helicases are typical It acts on single-stranded DNA. It can move along both strands of double-stranded DNA. Examples of helicases include the FtfK and hexamer enzyme complex, or RecBCD and other multifilaments. A subunit complex is one example. In one embodiment, the motor protein is Dda( It is a DNA-dependent ATPase (helicase).

[0186] Hel308 helicase is incorporated in its entirety by reference in WO2013 / 057. It is described in publications such as 495. The full details of RecD helicase are compiled by reference. It is described in publications such as WO2013 / 098562. XPD Helicar Ze is described in publications such as WO2013 / 098561, which incorporates its full details by reference. It is listed. Dda helicase is incorporated by reference in WO20, with the full details of each of them included. This is documented in publications such as 15 / 055981 and WO2016 / 055777.

[0187] In one embodiment, the helicase is the sequence shown in Sequence ID No. 6 (TrwcCba) This is its variant, the sequence shown in Sequence ID No. 7 (Hel308Mbu), or its variant. Alternatively, it may include the sequence (Dda) shown in Sequence ID No. 8 or a variant thereof. The variant is this The sequence may differ from the natural sequence in any of the methods discussed in the specification. Example of Sequence ID No. 8 Typical variants include E94C / A360C. Further exemplary variants of Sequence ID No. 8 are E 94C / A360C, then (ΔM1)G1G2 (i.e., deletion of M1, then G1 and (Includes the addition of G2).

[0188] Typically, motor proteins or polynucleotide-binding proteins have a fuel binding site. It may have a position. Active unwinding of DNA is, for example, due to hydrolysis in motor proteins. It can be coupled with the acceleration of the solution.

[0189] The fuel is typically a free nucleotide or a free nucleotide analog. Reotide is adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine ATP (triphosphate), GMP (guanosine monophosphate), GDP (guanosine diphosphate) , guanosine triphosphate (GTP), thymidine monophosphate (TMP), thymidine diphosphate (T DP), thymidine triphosphate (TTP), uridine monophosphate (UMP), uridine diphosphate (UDP), Uridine triphosphate (UTP), Cytidine monophosphate (CMP), Cytidine diphosphate Adenosine triphosphate (CDP), cytidine triphosphate (CTP), cyclic adenosine monophosphate (cAMP), Cyclic guanosine monophosphate (cGMP), deoxyadenosine monophosphate (dAMP), deo Xyadenosine diphosphate (dADP), deoxyadenosine triphosphate (dATP), deo Deoxyguanosine monophosphate (dGMP), deoxyguanosine diphosphate (dGDP), deo Xyguanosine triphosphate (dGTP), deoxythymidine monophosphate (dTMP), deoxy Cythymidine diphosphate (dTDP), deoxythymidine triphosphate (dTTP), deoxy Lysine monophosphate (dUMP), deoxyuridine diphosphate (dUDP), deoxyuridine Deoxycytidine triphosphate (dUTP), deoxycytidine monophosphate (dCMP), deoxycytidine diphosphate One or more of the following: phosphate (dCDP) and deoxycytidine triphosphate (dCTP) These are possible, but not limited to, free nucleotides, typically AMP, TMP, GMP Selected from CMP, UMP, dAMP, dTMP, dGMP, or dCMP. Free A typical nucleotide is adenosine triphosphate (ATP).

[0190] Motor protein cofactors are factors that enable motor proteins to function. The cofactor is preferably a divalent metal cation. Mg 2+ Mn 2+ Ca 2+ , or Cole 2+ The cofactor is most preferably Mg 2+ That is the case.

[0191] In some embodiments, the polynucleotide-binding protein used herein Other than motor proteins. When used herein, polynucleotide bonds The terms synthetic protein and polynucleotide binding site can be used interchangeably. Cut.

[0192] For example, polynucleotide-binding proteins or polynucleotide-binding moieties are helic S-hairpin-helix (HhH) domain, eukaryotic single-strand binding protein (SSB), Bacterial SSBs, archaeal SSBs, viral SSBs, double-strand binding proteins, sliding Ramp, progression factor, DNA binding loop, replication initiation protein, telomere binding protein, Selected independently from repressors, zinc fingers, and proliferating cell nuclear antigens (PCNAs) It may include one or more domains.

[0193] The helix-hairpin-helix (HhH) domain is a sequence-nonspecific DN. It is a polypeptide motif that binds to A. A preferred domain is Metanopyrus ca Domain H (residues 696-751) and Domain H derived from topoisomerase V from Ndreli H1 (residues 696-802) (SEQ ID NO: 54) is an example. Polynucleotide binding site The minutes represent the domain HL of SEQ ID NO: 54 as shown in SEQ ID NO: 55, or its polynucleotide. It may be a binding mutant. The HhH domain is the compound shown in SEQ ID NO: 40, 48, or 49. It may include a row, or a polynucleotide linkage variant thereof.

[0194] SSBs bind to single-stranded DNA with high affinity in a sequence-nonspecific manner. Classified into the following series: Class: All beta proteins, Fold: OB fold Superfamily: nucleic acid-binding proteins, Family: single-stranded DNA-binding domains, SSB. SSB refers to eukaryotes such as humans, mice, rats, fungi, protozoa, or plants. They can originate from prokaryotes such as fungi and archaea, or from viruses. Eukaryotic SSBs are replication-dependent. Also known as protein A (RPA). In most cases, they are of different sizes. It is a heterotrimmer formed from a unit. It is part of a larger unit (e.g., Sacc RPA70 of Haromyces cerevisiae is stable and monomeric. It binds to ssDNA in a stable state. Bacterial SSBs have stable homotetramers (e.g., E. co.). li, Mycobacterium smegmatis and Helicobacter pylori) or homodimer (e.g., Deinococcus radiodu It binds to DNA as rans and Thermotoga maritima. Encoded by the archaeon Sulfolobus solfataricus Some SSBs, such as others, are homotetramers. Some SSBs from other species are homotetramers. It has been shown to be nomar (Methanococcus jannaschii) and Methanothermobacter thermoautotrophicus m). Archaeoglobus fulgidus and Methanococcoi Other archaeal species, including des burtonii, have sequences similar to RPA. It contains two open reading frames. The viral SSB is linked to DNA as monomers. To combine.

[0195] SSB typically has no net negative charge compared to wild-type protein, or a net negative charge. It is selected or modified to have a carboxyl-terminal (C-terminal) region with reduced charge. SSBs typically do not block transmembrane pores. The C-terminal region of an SSB is typically C This is the last approximately one-third, one-quarter, one-fifth, or one-eighth of the terminal SSB. The end region is typically the last approximately 10 amino acids to the last approximately 60 amino acids of the C-terminus of the SSB. Up to the acid, for example, the last approximately 30 amino acids of the C-terminus of an SSB, and the last approximately 20 amino acids of an SSB. From the first amino acid to the last 40 amino acids.

[0196] An example of a SSB containing a C-terminal region with no net negative charge is human mitochondrial S1. SB(HsmtSSB; Sequence ID No. 50, Human replication protein A 70kDa subunit, Human replication protein A14kDa subunit, telomere terminus, Oxytrichano VA-derived binding protein α subunit, Oxytrichanova-derived telomere The core domain of the terminal-binding protein β subunit, Schizosaccharomy Protection of telomere protein 1 (Pot1) derived from Ces pombe, human Pot1, ma OB-fold domain of BRCA2 derived from rat or cow, p5-tan derived from phi29 Examples include the protein (SEQ ID NO: 51) and its polynucleotide-linked variants. An example of an SSB whose terminal region can be modified to reduce the net negative charge is Ec oli's SSB (EcoSSB; SEQ ID NO: 52, Mycobacterium tube) SSB of rculosis, SSB of Deinococcus radiodurans SSB derived from Thermus thermophiles, Sulfolobus s SSB derived from Olfataricus, human replication protein A32kDa subunit ( RPA32) fragment, CDC1 from Saccharomyces cerevisiae 3SSB, Primosomal replication protein N (PriB) from E. coli, A PriB derived from Rabidopsis thaliana, virtual protein At4g28 440, SSB derived from T4 (gp32; SEQ ID NO: 53), SSB derived from RB69 (gp3 2; SEQ ID NO: 41), SSB derived from T7 (gp2.5; SEQ ID NO: 42), and polynucleotides Otid bonds and their variants are examples. A suitable modification for reducing the net negative charge is W This information is disclosed in O2014 / 013259.

[0197] Double-strand binding proteins bind to double-stranded DNA with high affinity. The protein contains the mutator S (MutS; NCBI reference sequence: NP_417213). 1; Sequence ID 56), Sso7d(Sufolobus solfataricus P 2;NCBI reference sequence: NP_343889.1;SEQ ID NO: 57;Nucleic Ac ids Research,2004,Vol 32,No.3,1197-1207) ,Sso10b1 (NCBI reference sequence: NP_342446.1; sequence number 58), Ss o10b2 (NCBI reference sequence: NP_342448.1; sequence number 59), tryptoph Fan repressor (Trp repressor; NCBI reference sequence: NP_291006.1; SEQ ID NO: 60), lambda repressor (NCBI reference sequence: NP_040628.1; distribution Column number 61), Cren7 (NCBI reference sequence: NP_342459.1; Sequence ID 62) ), major histone classes H1 / H5, H2A, H2B, H3, and H4 (see NCBI). Array: NP_066403.2, Sequence ID 63), dsbA (NCBI reference array: NP_ 049858.1; Sequence ID 64), Rad51 (NCBI reference sequence: NP_00286) 6.2; Sequence ID 65), sliding clamp and topoisomerase VMka (sequence ID 65) Number 54) or polynucleotide-linked mutants of any of these proteins are examples. However, it is not limited to these.

[0198] Another example of a polynucleotide-binding protein is the sliding clamp. Sliding clamps are typically multimer proteins (homozygous) that surround dsDNA. It is a dimer or homotrimer. Sliding clamp is usually ATP-dependent. To assemble them around the DNA helix, accessory proteins ( It requires a clamp loader. Also, it does not come into direct contact with the DNA and is used as a topology tether. It functions as such. The processivity factors involved in DNA sliding clamp are This is a viral protein that fixes the same type of polymerase to DNA, and is produced The length of the fragments increases dramatically. They are monomers (derived from herpes simplex virus 1). (In the case of L42) or (the UL44 of cytomegalovirus is a multimer dimer) It is possible. UL42 is typically shown in Sequence ID No. 43 or Sequence ID No. 47. Includes sequences or their polynucleotide-linked variants.

[0199] Another polynucleotide-binding protein is bacteriophage T7 DNA polymerase. This is the thioredoxin-binding domain (TBD) (residues 258-333). Binding to redoxin (e.g., from E. coli) allows polypeptides to bind to DNA. This causes a change in its three-dimensional structure. Other polynucleotide-binding proteins include , accessory protein cisA derived from phage Φx174 and phage M13 Gene II proteins are examples of proteins with unique DNA-binding capabilities. Yes, and some of them recognize specific DNA sequences. Other polynucleotide-binding proteins In terms of quality, telomere-binding proteins are a prime example.

[0200] Small DNA binding motifs (such as helix-turn-helix) are specific to certain DNA Recognizes the sequence. In the case of bacteriophage 434 repressor, a 62-residue fragment is manipulated. It was shown that zinc fingers retain DNA binding ability and specificity. It is composed of about 30 amino acids that bind to DNA in a specific manner. Typically, each zinc finger - It recognizes only three DNA bases, but it can link multiple fingers to create longer sequences. It can be recognized.

[0201] Proliferating cell nuclear antigen (PCNA) is a very fast dsDNA or ssDNA that slides up and down. It forms a tight clamp. PCNA derived from Cleon archaea, as shown in SEQ ID NOs. 44, 45 and It is a heterotrimer of 46. Therefore, polynucleotide-binding proteins are sequence Trimers containing the sequences shown in numbers 44, 45, and 46, or their polynucleotide linkage variants It may be an anomaly. Another PCNA sliding clamp (NCBI reference sequence: ZP_06) 863050.1 (SEQ ID NO: 66) forms a dimer. Therefore, polynucleotide The tide-binding protein is a dimer containing SEQ ID NO: 66 or its polynucleotide-binding mutation. It can take shape.

[0202] The polynucleotide binding motif can be selected from the following: [Table 3-1] [Table 3-2] [Table 3-3]

[0203] Polynucleotides The present invention relates to the process when a target polynucleotide moves toward a detector such as a nanopore. This includes characterizing target polynucleotides.

[0204] Polynucleotides, such as nucleic acids, are macromolecules containing two or more nucleotides. Rheotides can be single-stranded or double-stranded. Double-stranded polynucleotides are formed together. It is made from two hybridized single-stranded polynucleotides. Target polynucleotide This can be a single-stranded polynucleotide or a double-stranded polynucleotide.

[0205] A polynucleotide can contain any combination of any nucleotides. The substance can be naturally occurring or artificially created.

[0206] A nucleotide typically consists of a nucleic acid base, a sugar, and at least one phosphate group. Nucleic acid bases and sugars form nucleosides.

[0207] Nucleic acid bases are typically heterocyclic. Nucleic acid bases include purines and pyrimidines, and more Specifically, adenine (A), guanine (G), thymine (T), uracil (U), and cysteine. This includes, but is not limited to, tosine (C).

[0208] Sugars are typically pentoses. Nucleotide sugars include ribose and deoxyribohydrate. This includes, but is not limited to, sugars. The sugar is preferably deoxyribose. The polynucleotide is preferably the following nucleoside: deoxyadenosine (dA), Deoxyuridine (dU) and / or thymidine (dT), deoxyguanosine (dG), It also contains deoxycytidine (dC).

[0209] Nucleotides are typically ribonucleotides or deoxyribonucleotides. Nucleotides typically contain monophosphate, diphosphate, or triphosphate. The compound may contain more than three phosphoric acid molecules, for example, four or five phosphoric acid molecules. Phosphoric acid is nutrient-rich. It can bind to the 5' or 3' side of the creotide. The nucleotide contains adenosine monophosphate (A MP), guanosine monophosphate (GMP), thymidine monophosphate (TMP), uridine monophosphate Acid (UMP), 5-methylcytidine monophosphate, 5-hydroxymethylcytidine monophosphate, Cytidine monophosphate (CMP), cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate Phosphate (cGMP), deoxyadenosine monophosphate (dAMP), deoxyguanosine monophosphate Phosphate (dGMP), deoxythymidine monophosphate (dTMP), deoxyuridine monophosphate Acid (dUMP), deoxycytidine monophosphate (dCMP), and deoxymethylcytidine The nucleotides include, but are not limited to, monophosphates. The nucleotides are preferably AMP, TMP, GMP, CMP, UMP, dAMP, dTMP, dGMP, dCMP, and dU Selected from MP.

[0210] Nucleotides can be debased (i.e., lacking nucleic acid bases). Nucleotides are Nucleic acid bases and sugars may also be absent (i.e., they are C3 spacers).

[0211] Nucleotides in a polynucleotide can adhere to each other in any manner. Nucleotides typically attach to other molecules by their sugar and phosphate groups, similar to nucleic acids. These can be linked via their nucleic acid bases, similar to pyrimidine dimers.

[0212] Polynucleotides are nucleic acids such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). It is possible. A polynucleotide is one of the RNA molecules hybridized to a single strand of DNA. Polynucleotides can include strands of any synthetic nucleic acid known in the art. For example, peptide nucleic acids (PNA), glycerol nucleic acids (GNA), threose nucleic acids (T NA), cross-linked nucleotides (BNA), locked nucleic acids (LNA), or nucleotide side chains It may be another synthetic polymer having the following: The PNA backbone is a repeating polymer linked by peptide bonds. It is composed of N-(2-aminoethyl)-glycine units. The GNA skeleton is phosphodi It is composed of repeating glycol units linked by ester bonds. The TNA skeleton is made up of LNA is composed of repeating threose sugars linked together by sphodiester bonds. As discussed above, the extra link between the 2' oxygen and 4' carbon in the ribose moiety It is formed from ribonucleotides that have crosslinks.

[0213] Polynucleotides are preferably DNA, RNA, or DNA or RNA hybrids. DNA is most preferably DNA. DNA / RNA hybrids have DNA on the same strand. It may include DNA and RNA. Preferably, the DNA / RNA hybrid is hybridized with the RNA strand. It contains a single strand of DNA derived from soybeans.

[0214] By modifying the polynucleotide backbone, the possibility of chain breaks can be reduced. For example, DNA is known to be more stable than RNA under many conditions. The main chain of the chain avoids damage caused by radical chemicals such as free radicals. It can be modified for that purpose.

[0215] DNA or RNA containing non-natural or modified bases may be modified using an appropriate polymerase. Produced by amplifying native DNA or RNA polynucleotides in the presence of TP. It is possible.

[0216] Nucleotides in polynucleotides can be modified. Nucleotides can be oxidized or methylated. It can be modified. One or more nucleotides in a polynucleotide may be damaged. For example, polynucleotides may contain pyrimidine dimers. Such dimers are Typically associated with UV damage, it is a major cause of cutaneous melanoma. One or more nucleotides in a nucleotide can be modified, for example, by a label or tag. .

[0217] Single-stranded polynucleotides are strong secondary structures such as hairpin, quadruple-stranded, or triple-stranded DNA. These may include regions with structures. These types of structures are polynucleotides relative to nanopores. It can be used to control the movement of, for example, using a secondary structure, as specified herein. As explained in more detail, stopping the movement of polynucleotides through nanopores. This is possible. Each continuous secondary structure along the chain stops the movement of the chain relative to the nanopore. Polynucleotides can reshape their secondary structure after migrating through nanopores. Using the following structure, when the negative voltage is low or not applied (on the transformer side of the nanopore), (Application) This can prevent polynucleotides from returning through the nanopore, and therefore, The methods provided herein are relevant because they help control the movement of renucleotides. In this step, it occurs only in a controlled manner.

[0218] When used herein, a double-stranded polypeptide refers to a single-stranded region and other structures, for example. The secondary structure may include regions having hairpin loops, triple helix, and / or quadruple helix. As mentioned above, this may be useful in the context of single-stranded polynucleotides.

[0219] In a double-stranded molecule, the two strands can, for example, have the 5' end of one strand connect to the 3' end of the other strand at the end of the molecule. By connecting the ends with a hairpin structure, a covalent bond can be formed.

[0220] The target polynucleotide can be of any length. For example, the target polynucleotide is: At least 10, at least 50, at least 100, at least 150, at least 2 00, at least 250, at least 300, at least 400, or at least 500 The length may be a nucleotide or a pair of nucleotides. The target polynucleotide is 1000 More than nucleotides or nucleotide pairs, or 5000 or more nucleotides or Length of a nucleotide pair, or 100,000 or more nucleotides or nucleotide pairs Length, or the length of 500,000 or more nucleotides or nucleotide pairs, or 1, Length of nucleotides or nucleotide pairs of 000,000 or more, or 10,000,00 A length of 0 or more nucleotides or nucleotide pairs, or 100,000,000 or more. The length of a nucleotide or nucleotide pair, or more than 200,000,000 nucleotides This could be the length of a rheotide or nucleotide pair, or the total length of a chromosome.

[0221] The target polynucleotide may be an oligonucleotide. Oligonucleotides are, Typically, the number of nucleotides is 50 or less, for example, 40 or less, 30 or less, 20 or less, 10 or less, or It is a short nucleotide polymer having 5 or fewer nucleotides. Target oligonucleotide The nucleotides are preferably about 15 to about 30 nucleotides long, for example, about 20 to about 25 nucleotides. It is long. For example, oligonucleotides are approximately 15, 16, 17, 18, and 19 , approximately 20, approximately 21, approximately 22, approximately 23, approximately 24, approximately 25, approximately 26, approximately 27, approximately 28, approximately 29 It may be approximately 30 nucleotides long.

[0222] The target polynucleotide may be a long polynucleotide fragment. In this embodiment, A long target polynucleotide is typically a combination of multiple shorter target polynucleotides. It is fragmented into multiple parts.

[0223] Target polynucleotides are the products of PCR reactions, genomic DNA, and endonuclease digestion. This may include the products of and / or DNA libraries.

[0224] Target polynucleotides can occur naturally. Target polynucleotides are secreted from cells. It is possible. Alternatively, the target analyte may be an analyte present inside the cell, and therefore, the method The analyte must be extracted from the cells before the procedure can be performed.

[0225] Target polynucleotides are common organisms such as viruses, bacteria, archaea, plants, or animals. It may originate from. Such organisms, in order to adjust the sequence of target polynucleotides, for example, By adjusting the base composition and removing undesirable sequence elements, Selection or modification of organisms to achieve desired polynucleotide characteristics. This is routine for those skilled in the art.

[0226] Source organisms for target polynucleotides can be selected based on the desired characteristics of the sequence. Desirable characteristics include single-stranded polynucleotides and double-stranded polynucleotides produced by living organisms. The ratio of creotides, the complexity of the polynucleotide sequences produced by organisms, and the organisms The composition of the polynucleotides produced (GC composition, etc.), or the composition of the polynucleotides produced by organisms. One example is the length of a continuous polynucleotide chain. For example, a continuous polynucleotide chain of approximately 50kb. If a nucleotide chain is needed, lambda phage DNA can be used. If a continuous chain is needed, polynucleotides can be produced using other organisms, for example. For example, E. coli produces approximately 4.5 Mb of continuous dsDNA.

[0227] Target polynucleotides are often obtained from humans or animals, for example, from urine, lymph, or saliva. Obtained from fluid, mucus, semen, or amniotic fluid, or from whole blood, plasma, or serum. Target Polynucleotides can be obtained from plants, such as cereals, legumes, fruits, or vegetables. The target polynucleotide may contain genomic DNA. Genomic DNA can be fragmented. DNA can be fragmented by any preferred method. For example, a method for fragmenting DNA This is known in the art, and such a method involves a transposase such as MuA transposase. Zarze can be used. Genomic DNA is often not fragmented.

[0228] In some embodiments, the polynucleotide is synthetic or semi-synthetic. For example, D NA or RNA is synthesized by conventional DNA synthesis methods such as phosphoramidite-based chemical reactions. It may be a pure synthetic product. The synthetic polynucleotide subunit is ligate. They can be linked together by known means such as ions or chemical bonds to form longer chains. In some embodiments, internal self-forming structures (e.g., hairpins, quadruples) are formed, for example. Therefore, by ligating the appropriate sequence, it can be designed within the substrate. Polynucleotides are known in the art for applications including PCR and integration into bacterial factories. It can be replicated and scaled up for production by the means described.

[0229] In some embodiments, the polynucleotide has a simplified nucleotide composition. In some embodiments, polynucleotides are obtained as repeating patterns of the same subunits. It has, for example, the iterative unit can be (AmGn)q, where m, n and q are positive integers. For example, m is often 1-20, for example 1-10, for example 1-5, for example 1 It is 2, 3, 4, or 5. n is often 1 to 20, for example 1 to 10, for example 1 to 5 For example, 1, 2, 3, 4, or 5. m and n may be the same or different. In many cases, q is between 1 and approximately 100,000. A typical repeating unit is, for example, ( It may be AAAAAAGGGGGG)q. Repeat polynucleotides are known in the art. Many means are used, for example, having adhesive ends that enable ligation. It can be fabricated by linking together synthetic subunits. Several implementations Morphologically, polynucleotides can be linked polynucleotides. The method for linking CHIDS is described in PCT / GB2017 / 051493.

[0230] In some embodiments, the polynucleotide may contain a base with a reactive side chain. It can be done. If necessary, any suitable reactive functional group can be incorporated into the side chain. A suitable example of a functional group is a click chemistry reagent. Suitable examples of Lee include, but are not limited to, the following: (a) The azide reacts with a strained alkyne, for example, on a cyclooctane ring. , copper-free variant of the 1,3-bipolar cycloaddition reaction, (b) The reaction between the oxygen nucleophile on one linker and the epoxide or aziridine on the other. Reaction with the part, and (c) The alkyne moiety is replaced with an arylphosphine to achieve a specific reaction with azide. A Staudinger linkage can be formed by adding an amide bond.

[0231] Polypolymer adapter In some embodiments, motor proteins and / or polynucleotide-binding proteins The nucleotide, if present, may be provided on the polynucleotide adapter. WO2015 / 110813 is a loading mechanism for target polynucleotides, such as motor protein adapters. This document contains information about the ding, which is incorporated herein by reference in its entirety.

[0232] Adapters are typically polynucleotides that can be attached to the ends of target polynucleotides. The nucleotide chain is typically one of those disclosed herein. The purpose is to characterize by law.

[0233] Polynucleotide adapters can be attached to both ends of the target polynucleotide. Specifically, different adapters can be attached to the two ends of the target polynucleotide. The adapter can be attached to only one end of the target polynucleotide. Methods for adding to renucleotides are known in the art. The adapter is, for example, By ligation, by click chemistry, by tagmentation Polynucleotides by topoisomerase conversion or by any other suitable method It can adhere to it.

[0234] The adapter may be a synthetic or artificial material. Typically, the adapter is as described herein. The polymers described are included. In some embodiments, the adapter contains polynucleotides. In some embodiments, the adapter may include a single-stranded polynucleotide chain. In some embodiments, the adapter may include a double-stranded polynucleotide. Otido adapters can handle DNA, RNA, modified DNA (basic DNA, etc.), RNA, and PN. May include A, LNA, BNA, and / or PEG. Typically, the adapter is single-stranded and / Or it contains double-stranded DNA or RNA.

[0235] The adapter may include a stall section as described herein. The adapter is motor It may contain a loading site for a protein or polynucleotide-binding protein. Adapters may include tags.

[0236] The adapter could be a Y-adapter. A Y-adapter is typically double-stranded. (a) A region at one end where the two strands are hybridized together, and (b) at the other end The two chains contain regions that are not complementary. The non-complementary parts of those chains have an overhang. The hybridized stem of the adapter is typically a double-stranded polynucleotide. It attaches to the 5' end of the first strand of the nucleotide and to the 3' end of the second strand of the double-stranded polynucleotide. or, the 3' end of the first strand of the double-stranded polynucleotide and the double-stranded polynucleotide It attaches to the 5' end of the second strand. The presence of a non-complementary region in the Y adapter is distinct from the double-stranded portion. Since two different chains typically do not hybridize with each other, the adapter is given a Y shape. Motor proteins or polynucleotides are adapters such as Y-adapters. It can bind to motor hangs. In another embodiment, motor proteins or polynucleotides The binding protein can bind to the double-stranded region. In other embodiments, motor proteins Alternatively, the polynucleotide-binding protein has a single-stranded region and / or double-stranded region of the adapter. It can bind to the first motor protein or polynucleotide. In other embodiments, it can bind to the first motor protein or polynucleotide. The protein can bind to the single-stranded region of the adapter, and to a second motor protein or Polynucleotide-binding proteins can bind to the double-stranded region of their adapter.

[0237] In one embodiment, the adapter includes a membrane anchor or a pore anchor. Several embodiments Morphologically, anchors are to which motor proteins or polynucleotide-binding proteins are bound. Polynucleus that are complementary to the overhang and therefore hybridized to it It can bind to rheotides.

[0238] In some embodiments, the non-complementary polynucleotide adapters such as Y adapters One of the chains is a leader that can pass through nanopores when it comes into contact with transmembrane pores. It may include columns.

[0239] The leader sequence is typically a polynucleotide, such as DNA or RNA, with modifications. Dinucleotides (such as denucleotide-denucleotide DNA), PNA, LNA, polyethylene glycol (PE G), or a polymer such as a polypeptide. In some embodiments, a leader sequence It contains single-stranded DNA such as polydT sections. The leader sequence is of any length. However, they are typically 10-150 nucleotides long, for example, 20-120, 30-10 The length is 0, 40-80, or 50-70 nucleotides.

[0240] In one embodiment, the polynucleotide adapter is a hairpin loop adapter. A hairpin loop adapter is an adapter containing a single polynucleotide chain, Can the ends of a nucleotide chain hybridize with each other, or can they hybridize with each other? Soybeans are used, and the central section of the polynucleotide forms a loop. Suitable hairpin The adapter can be designed using methods known in the art. Typical The 3' end of the hairpin loop adapter is connected to the first strand of the double-stranded polynucleotide. The '5' end of the hairpin loop adapter is attached to the terminal of the double-stranded polynucleotide. Either the 3' end of the double strand is attached, or the 5' end of the hairpin loop adapter is attached to the double strand. The first strand of the renucleotide is bound to the 3' end, and the 3' end of the hairpin loop adapter is It binds to the 5' end of the second strand of a double-stranded polynucleotide. This is explained in more detail below. Thus, the polynucleotide adapter is used to characterize the target polynucleotide. It can bind to target polynucleotides.

[0241] Those skilled in the art will know that if the adapter contains a polynucleotide chain, the sequence of the adapter is typical. Typologically, it is not definitive, and motor proteins and any polynucleotides that should be characterized It will also be understood that other experimental conditions, such as ocidal content, may be controlled or selected according to the circumstances. Typical sequences are provided for illustrative purposes only in the examples. For example, the adapter is sequence number 21. Sequences such as ~26 or 28~33, or sequence numbers 21~26 or 28~33 One or more and at least 20%, for example at least 30%, for example at least 40%, for example so at least 50%, for example at least 60%, for example at least 70%, for example less Both have 80%, for example, at least 90%, for example, at least 95% sequence similarity or identity. The adapter sequence may include a polynucleotide sequence having the following characteristics. The adapter sequence is typically as specified herein. The method provided can be modified without adversely affecting its effectiveness.

[0242] In some embodiments, the polynucleotide adapter is a motor protein and / Alternatively, it may include a loading site for loading polynucleotide-binding proteins. The loading site is, for example, a motor protein or a polynucleotide-binding protein. It may be a single-stranded region that can be targeted by. The loading site is as specified herein. A motor protein or polynucleotide is evaluated using the method provided. To rearrange the nucleotide-binding protein, a motor protein or polynucleotide-binding protein is used. Polynucleotide adapters that can bind exogenous polynucleotide chains containing protein. It could be the domain of Pter.

[0243] Therefore, the motor protein used in the method provided herein is polynucleated It may stall on the rheotide adapter. In other embodiments, the motor protein targets the port It stalls on the renucleotide but not on the polynucleotide adapter.

[0244] Blocking section In some embodiments, a blocking moiety is used to allow the motor protein to target a port This can prevent the renucleotide from unassociating.

[0245] In some embodiments, the blocking portion is included in the target polynucleotide. In one embodiment, the blocking portion is a polynucleotide attached to the target polynucleotide. Included in the ocide adapter. In some embodiments, a polynucleotide adapter, For example, the polynucleotide adapter described herein includes a blocking portion.

[0246] The blocking portion prevents the motor protein from unassociating with the target polynucleotide. It can be used to prevent, for example, a motor protein from targeting a polynucleotide or If present at the 3' end of the polynucleotide chain in the polynucleotide adapter, block The King's moiety is typically located between the motor protein and the 3' end of the chain. - The protein targets the polynucleotide or polynucleotide adapter polynucleotide. When present at the 5' end of the motor chain, the blocking portion is typically associated with the motor protein. It is located between the 5' end of the chain and the other end.

[0247] For example, in some embodiments, the polynucleotide adapter is a target polynucleotide. It may include a first end containing an attachment point for adhering to the nitrate analyte, and a second end, and a motor - The protein is oriented to process the adapter in the direction of the attachment site, polynucleotide Stalling may occur on the rheotide adapter. In such embodiments, the motor protein is polynucleated. To prevent unassociation from the second end of the creotide adapter, the motor protein A blocking portion may be placed between the adapter and the second end of the adapter.

[0248] For example, in some embodiments, the polynucleotide adapter is a target polynucleotide. The tide analyte may include a 3' end containing an attachment site for binding to the 5' end, and a 5' end. Motor proteins process the adapter in the direction of the 3' end, i.e., from 5' to 3'. In an orientation for singling, it may stall on the polynucleotide adapter. Such embodiments So, how does the motor protein detach from the 5' end of the polynucleotide adapter? To prevent this, a blocking portion is placed between the motor protein and the 5' end of the adapter. In other embodiments, the polynucleotide adapter may be positioned as a target polynucleotide. The analyte may include a 5' end containing an attachment point for binding to the 3' end, and a 3' end. The adapter protein processes the adapter in the direction of the 5' end, i.e., from 3' to 5'. In an orientation for stabilization, it may stall on the polynucleotide adapter. In such embodiments, This prevents the motor protein from unassociating from the 3' end of the polynucleotide adapter. To achieve this, a blocking region is positioned between the motor protein and the 3' end of the adapter. It is possible.

[0249] In some embodiments, the target polynucleotide is the first end of the target polynucleotide. The motor protein contains a leader sequence at its end, and the second end of the target polynucleotide or It stalls on the adapter bound to the second end of the target polynucleotide, blocking the blocking region The part is the motor protein and the second end of the polynucleotide (i.e., the polynucleotide It is located between the end of the polynucleotide at the second end of the motor, thereby providing a motor The protein disassociates from the target polynucleotide at the second end of the target polynucleotide. To prevent that from happening.

[0250] For example, in some embodiments, the target polynucleotide is located at the 5' end of the first chain. The motor protein contains the command sequence and is located at the 3' end of the first strand of the target polynucleotide. The first chain stalls at its 3' end on the adapter attached to the motor, and the blocking part is It is located between the protein and the 3' end of the first chain of the polynucleotide, thereby mo The ter protein is at the 3' end of the first chain of the target polynucleotide. This prevents deassociation. In another embodiment, the target polynucleotide is part of the first chain. The motor protein contains a leader sequence at its terminal, and the motor protein is the first strand of the target polynucleotide. The first chain stalls at the 5' end on the adapter attached to the 5' end of the blocking portion. It is located between the motor protein and the 5' end of the first chain of the polynucleotide, and Therefore, the motor protein is located at the 5' end of the first chain of the target polynucleotide. It prevents unassociation from the rheotide. Of course, there are two polynucleotide adapters. Can be bound to chain polynucleotides or single-stranded polynucleotides. Target polynucleotide If it is a double-stranded polynucleotide, the blocking portion is typically a motor protein. It is located on the same chain as the protein. Motor proteins are present on each chain of a double-stranded polynucleotide. In cases where (for example, when a double-stranded polynucleotide is rotationally symmetric), the polynucleotide Each chain typically contains blocking regions.

[0251] Any suitable blocking portion can be used in the provided manner. Suitable blocking portions include many of the same groups that can be used as stopping portions as described herein. This includes: For example, the blocking portion may include one or more of the following: -Polynucleotide secondary structure, preferably hairpin or G-quadrivalent (TBA), - Preferably, peptide nucleic acid (PNA), glycerol nucleic acid (GNA), threose nucleus Acids (TNA), locked nucleic acids (LNA), cross-linked nucleic acids (BNA), and debasalized nucleotides nucleic acid analogs selected from, -Fluorophores, traptabidine, streptavidin, and neutraavidin, etc. avidin and / or biotin, cholesterol, methylene blue, dinitrophenol (DNP), digoxigenin and / or anti-digoxigenin and dibenzylcyclooctane n group, and -Polynucleotide-binding protein. These elements are described in more detail herein in the context of the stopping portion. It is.

[0252] Spacer In some embodiments, the polynucleotide or polynucleotide adapter is, for example, For example, 1 to about 10 spacers, for example, 1 to about 5 spacers, for example, 1, 2, 3 It may include 4 or 5 spacers. The spacers may be any suitable number of spacers. It may include positions. Spacers typically hinder the movement of polynucleotide-binding proteins. It provides an energy barrier. For example, a spacer can be used, for example, a debase spacer. By reducing the traction force of the protein, the motor protein or polynucleotide Spacers can hinder the movement of ocidal binding proteins. For example, spacers can introduce bulky chemical groups. By physically hindering the movement of proteins, polynucleotide-binding proteins Its movement can be physically blocked.

[0253] In some embodiments, one or more spacers are polynucleotides or polynucleotides. It is contained in the cido adapter and provides a specific signal as they pass through the nanopore. Use one or more spacers to define or divide one or more regions of a polynucleotide. For example, the adapter can be separated from the target polynucleotide.

[0254] In some embodiments, the spacer is made of, for example, polypeptide or polyethylene glycoside. It may contain linear molecules such as polymers that are PEG. Typically, such spaces The sir has a different structure from the target polynucleotide. For example, the target polynucleotide If the spacer is DNA, then the spacer or each spacer typically does not contain DNA. The target polynucleotide is deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). In this case, the spacer or each spacer is preferably a peptide nucleic acid (PNA), glycerol nucleus Acids (GNA), threose nucleic acids (TNA), locked nucleic acids (LNA), or nucleotides The material comprises a synthetic polymer having side chains. In some embodiments, the spacer is one or more Nitroindole, one or more inosine, one or more acridine, one or more 2-amino acids Purine, one or more 2-6-diaminopurines, one or more 5-bromodeoxyuridines , one or more inverted thymidines (inverted dT), one or more inverted dideoxythymidines (ddT) ), one or more dideoxycytidines (ddC), one or more 5-methylcytidines, one The above 5-hydroxymethylcytidine, one or more 2'-O-methylRNA bases, one or more The above iso-deoxycytidine (Iso-dC), one or more iso-deoxyguanosines ( Iso-dG), one or more C3(OC3H6OPO3) groups, one or more photocleavable ( PC)[OC3H6-C(O)NHCH2-C6H3NO2-CH(CH3)OPO3] group, one or more hexanediol groups, one or more spacer 9 (iSp9)[(OCH2 CH2)3OPO3] group, or one or more spacers 18 (iSp18) [(OCH It may contain a [2CH2)6OPO3] group or one or more thiol bonds. The spacer is It may include any combination of these groups. Many of these groups are IDT (registered trademark). Commercially available from Integrated DNA Technologies (registered trademark). For example, C3, iSp9, and iSp18 spacers are all made by IDT (Registered Trademark). Available from (standard). A spacer contains any number of the above bases as a spacer unit. It is visible.

[0255] In some embodiments, the spacer is one or more chemical groups, for example, one or more pens. It may contain a dant chemical group. One or more chemical groups may be present in one of the polynucleotide adapters. It can attach to the nucleic acid bases above. One or more chemical groups can be attached to the backbone of the polynucleotide adapter. They can adhere. Any number, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more. Suitable chemical groups may exist. Suitable groups include fluorophores, streptavidins, and / or biotin, cholesterol, methylene blue, dinitrophenol (DNP), di Contains goxigenin and / or anti-digoxigenin, as well as dibenzylcyclooctin groups. However, it is not limited to these.

[0256] In some embodiments, the spacer is one or more debaset nucleotides (i.e., Nucleotides lacking nucleic acid bases, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 It may contain 12 or more debasalized nucleotides. Nucleic acid bases are present in the debasalized nucleotides. It can be replaced by -H(idSp) or -OH. The debase spacer is one or more By removing nucleic acid bases from the adjacent nucleotides above, the target polynucleotide It can be inserted inside. For example, polynucleotides such as 3-methyladenine and 7-methylgua It may be modified to contain nin, 1,N6-ethenoadenine inosine, or hypoxanthine. Nucleic acid bases are obtained using human alkyladenine DNA glycosylase (hAAG). These nucleotides can be removed. Alternatively, polynucleotides containing uracil can be used. The nucleic acid bases may be modified in such a way that they are removed by uracil DNA glycosylase (UDG). It is possible that in one embodiment, one or more spacers include any debasalized nucleotides. No.

[0257] Suitable spacers have properties of polynucleotides or polynucleotide adapters, and The protein can be designed or selected depending on the conditions under which this method is to be performed. ru.

[0258] tag In some embodiments, the polynucleotide or polynucleotide adapter is a tag Or it may include a tether. For example, a polynucleotide may, for example, be tethered via its adapter. It binds to a tag on a nanopore, for example, during the characterization of polynucleotides by nanopores. It can be released at a single point. Strong non-covalent bonds (e.g., biotin / avidin) still remain It is reversible and is useful in some embodiments of the method described herein.

[0259] The pore tag and polynucleotide adapter pair allows the polynucleotide to be attached to the tag on the nanopore. Binding sites on the cyd (for example, by the anchor or leader array of the adapter, or The binding strength of the binding site (provided by the capture array within the double-stranded stem of the adapter) or When an applied force is applied, the bound polynucleotide is released from the nanopore. Until then, it is sufficient to maintain the coupling between the nanopore and the polynucleotide. It can be composed of the following.

[0260] In some embodiments, the tag or tether is uncharged. This allows for a potential difference. This ensures that the tag or tether is not drawn into the nanopore under its influence.

[0261] A polynucleotide or adapter is attracted to or bound to one or more molecules, It can be linked to the adapter and / or any hybridize to the target polynucleotide. Molecules may be used. Molecules attached to the pore include PNA tags, PEG linkers, and short o Ligonucleotides, positively charged amino acids, and aptamers may be selected. Such molecules are It is known in this field that it binds to A. For example, a short oligonucleotide. Pores to which it adheres are described in Howarka et al (2001) Nature Biote Disclosed in ch.19:636-639 and WO2010 / 086620, poa Pores containing PEG adhering to the lumen are described by Howarka et al (2000). This is disclosed in J.Am.Chem.Soc.122(11):2411-2416.

[0262] Short oligonucleotides attached to a detector (e.g., transmembrane pore), with a leader distribution Use oligonucleotides that contain a sequence complementary to the sequence of the column or another single-stranded sequence of the adapter. The capture of target polynucleotides may be enhanced in the method described herein by using the following method.

[0263] In some embodiments, the tag or tether is an oligonucleotide (e.g., DNA, Even if it contains RNA, LNA, BNA, PNA, or morpholino) Good. Oligonucleotides (e.g., DNA, RNA, LNA, BNA, PNA, or mo Rufolino) has a length of approximately 10-30 nucleotides or approximately 10-20 nucleotides. It is possible. In some embodiments, oligonucleotides for use in tags or tethers. Ocides (e.g., DNA, RNA, LNA, BNA, PNA, or morpholino) are other Modified area or, for example, a modified area for bonding to a solid substrate surface including beads, at least It may have one terminal (e.g., a 3'- or 5'-terminus). Terminal modifiers are linked Reactive functional groups that can be used in combination may be added. Functional groups that can be added Examples include amino, carboxyl, thiol, maleimide, aminooxy, and so Any combination of these is possible, but is not limited to them. Functional groups include oligonucleotides. To add up the physical distance from the end of the ocidal sequence to the functional group, spacers of different lengths are used (e.g., For example, it can be combined with C3, C9, C12, and spacers 9 and 18.

[0264] In some embodiments, the tag or tether comprises a morpholino oligonucleotide. However, it may also be a morpholino oligonucleotide. Cleotides have a length of approximately 10-30 nucleotides or approximately 10-20 nucleotides. This is possible. Morpholino oligonucleotides can be modified or unmodified. This is also fine. For example, in some embodiments, the morpholino oligonucleotide is oligo The nucleotide may be modified at its 3' and / or 5' ends. Morpholinooligonucle An example of 3' and / or 5' terminal modification of ocides is 3' affinity for chemical bonding. Tags and functional groups (e.g., 3'-biotin, 3'-primary amine, 3'-disulfide amine) (including 3'-pyridyldithio and any combination thereof); 5'-terminal modification (e.g.) For example, including 5'-primary ammine and / or 5'-dapsil); click chemistry Modifications for (e.g., 3'-azide, 3'-alkyne, 5'-azide, 5'-alkyne) Examples include, but are not limited to, the above, and any combination thereof.

[0265] In some embodiments, the tag or tether is connected to, for example, a detector, such as a nanopore. A polymer linker may be further included to facilitate bonding. Exemplary polymer linker - includes, but is not limited to, polyethylene glycol (PEG). The kara is approximately 500Da to 10kDa (including both ends), or approximately 1kDa to 5kDa (both ends). It may have a molecular weight (including the ends). The polymer linker (e.g., PEG) is, for example, However, these include, maleimide, NHS ester, and dibenzocyclooctin (DB CO), azides, biotins, amines, alkynes, aldehydes, and any combination thereof It can be functionalized with different functional groups including wase. In some embodiments, tags or The tether also contains 1 kDa PEG having a 5'-maleimide group and a 3'-DBCO group. In some embodiments, the tag or tether may be a 5'-maleimide group and a 3'-D The PEG may further contain a 2kDa PEG having a BCO group. In some embodiments, the tag Alternatively, the tether further modifies 3kDa PEG having a 5'-maleimide group and a 3'-DBCO group. It may also be included. In some embodiments, the tag or tether is a 5'-maleimide group and The PEG may further contain a 5 kDa PEG having a 3'-DBCO group.

[0266] Other examples of tags or tethers include His tags, biotin or streptavidin, and specimens. Antibodies that bind, aptamers that bind to the sample, and sample-binding domains such as DNA-binding domains. (For example, peptide zippers like leucine zippers, single-stranded DNA binding proteins ( This includes, but is not limited to, SSBs, and any combination thereof. .

[0267] Using any method known in the art, a tag or tether is attached to the outer surface of a nanopore, for example, Alternatively, they may be attached to the cis side of the membrane. For example, one or more tags or tethers may be attached to one or more surfaces. Cysteine ​​(cysteine ​​bond), one or more primary amines such as lysine, one or more non-crystalline amines Natural amino acids, one or more histidines (His-tagged), one or more biotins or streptin Avidin, a tag based on one or more antibodies, one or more enzymatic modifications of an epitope (e.g., (including acetyltransferases) and any combination thereof, through nanopores It can adhere to. A preferred method for carrying out such modification is available in the art. It is knowledge. Suitable non-natural amino acids include 4-azido-L-phenylalanine (Faz), and Liu CCand Schultz PG, Annu.Rev.Bioc. Any of the numbers 1 to 71 in Figure 1 of hem., 2010, 79, 413-444 It includes, but is not limited to, amino acids.

[0268] One or more tags or tethers are attached to the nanopore via cysteine ​​bonds. In this embodiment, one or more cysteine ​​molecules are added to one or more monomers that form nanopores. It can be introduced by substitution. In some embodiments, the nanopore is attached to the following It can be chemically modified by: (i) 4-phenylazomareinanyl, 1.N-(2- Hydroxyethyl maleimide, N-cyclohexyl maleimide, 1,3-maleimide Ropionic acid, 1,1-4-aminophenyl-1H-pyrrole,2,5,dione,1,1- 4-Hydroxyphenyl-1H-pyrrole,2,5,dione,N-ethylmaleimide,N -Methoxycarbonylmaleimide, N-tert-butylmaleimide, N-(2-amino Ethyl)maleimide, 3-maleimide-proxyl, N-(4-chlorophenyl)maleimide Mido, 1-[4-(dimethylamino)-3,5-dinitrophenyl]-1H-pyrrole- 2,5-dione, N-[4-(2-benzimidazolyl)phenyl]maleimide, N-[ 4-(2-benzoxazolyl)phenyl]maleimide, N-(1-naphthyl)-maleimide Mido, N-(2,4-xylyl)maleimide, N-(2,4-difluorophenyl)male Imide, N-(3-chloro-para-tolyl)-maleimide, 1-(2-amino-ethyl) -Pyrrole-2,5-dione hydrochloride, 1-cyclopentyl-3-methyl-2,5 -Dihydro-1H-pyrrole-2,5-dione, 1-(3-aminopropyl)-2,5- Dihydro-1H-pyrrole-2,5-dione hydrochloride, 3-methyl-1-[2-o Xo-2-(piperazin-1-yl)ethyl]-2,5-dihydro-1H-pyrrole-2 ,5-dione hydrochloride, 1-benzyl-2,5-dihydro-1H-pyrrole-2, 5-dione, 3-methyl-1-(3,3,3-trifluoropropyl)-2,5-dihydr Ro-1H-pyrrole-2,5-dione, 1-[4-(methylamino)cyclohexyl]- 2,5-Dihydro-1H-pyrrole-2,5-dionetrifluoroacetic acid, SMILES O=C1C=CC(=O)N1CC=2C=CN=CC2, SMILES O=C1C= CC(=O)N1CN2CCNCC2,1-benzyl-3-methyl-2,5-dihydro- 1H-pyrrole-2,5-dione, 1-(2-fluorophenyl)-3-methyl-2,5 -Dihydro-1H-pyrrole-2,5-dione, N-(4-phenoxyphenyl)maleimi Maleimides containing diabromomaleimides such as N-(4-nitrophenyl)maleimide (ii)3-(2-iodoacetamide)-proxyl, N-(cyclopropylmethicone) (L)-2-iodoacetamide, 2-iodo-N-(2-phenylethyl)acetamide , 2-iodo-N-(2,2,2-trifluoroethyl)acetamide, N-(4-acetamide) (Tylphenyl)-2-iodoacetamide, N-(4-(aminosulfonyl)phenyl) -2-iodoacetamide, N-(1,3-benzothiazole-2-yl)-2-iod Acetamide, N-(2,6-(diethylphenyl)-2-iodoacetamide, N-( iodoacetamide such as 2-benzoyl-4-chlorophenyl)-2-iodoacetamide Mido, (iii)N-(4-(acetylamino)phenyl)-2-bromoacetamide, N-(2-acetylphenyl)-2-bromoacetamide, 2-bromo-n-(2-shea Nophenyl)acetamide, 2-bromo-N-(3-((trifluoromethyl)phenyl ) Acetamide, N-(2-benzoylphenyl)-2-bromoacetamide, 2-bro Mo-N-(4-fluorophenyl)-3-methylbutanamide, N-benzyl-2-bro Mo-N-phenylpropionamide, N-(2-bromo-butyl)-4-chloro-benz Sulfonamide, 2-bromo-N-methyl-N-phenylacetamide, 2-bromo- N-phenethylacetamide, 2-adamantan-1-yl-2-bromo-N-cyclo Hexyl acetamide, 2-bromo-N-(2-methylphenyl)butanamide, mono Bromoacetamide such as bromoacetanilide, (iv) aldrithiol-2, aldrithiol Dolichiol-4, Isopropyl Disulfide, 1-(Isobutyl Disulfanil)-2 -Methylpropane, dibenzyl disulfide, 4-aminophenyl disulfide, 3-( 2-Pyridyldithio)propionic acid, 3-(2-Pyridyldithio)propionic acid hydra D, 3-(2-pyridyldithio)propionic acid N-succinimidyl ester, am6a Disulfides such as mPDP1-βCD, and (v)4-phenylthiazole-2-thio 5,6,7,8-tetrahydroquinazoline-2-thiol and other thiols All.

[0269] In some embodiments, the tag or tether is directly attached to the nanopore or to one or more linkers. They may be attached via [a specific method]. Tags or tethers may be attached via [a specific method] as described in WO2010 / 086602. Brilinkers may be used to attach to the nanopores. Alternatively, peptide linkers may be used. It may be done. The peptide linker is an amino acid sequence. Peptide linker length, flexibility The properties of the monomer and hydrophilicity are typically designed not to interfere with the function of the monomer and pores. The number of flexible peptide linkers is 2 to 20, for example, 4, 6, 8, 10, or 16. A stretch of serine and / or glycine amino acids. A more preferred flexible linker. (SG)1, (SG)2, (SG)3, (SG)4, (SG)5, and (SG)8 Inclusions include, S is serine and G is glycine. Preferred rigidity linkers are 2-30. For example, stretches of 4, 6, 8, 16, or 24 proline amino acids. A rigid linker is one in which P is proline, (P) 12 Includes.

[0270] anchor In one embodiment, the polynucleotide or polynucleotide adapter is a membrane anchor or This may include a transmembrane pore anchor. In one embodiment, the anchor is disclosed herein. It assists in the characterization of target polynucleotides according to the method. For example, membrane anchor - Or transmembrane pore anchors localize selected polynucleotides around nanopores It can be promoted.

[0271] Anchors are polypeptide anchors and / or hydrophobic anchors that can be inserted into membranes. - This is possible. In one embodiment, the hydrophobic anchor is lipids, fatty acids, sterols, carbohydrates These are nanotubes, polypeptides, proteins, or amino acids, for example, cholesterol. The anchor is thiol, palmitate, or tocopherol. or may include a surfactant. In one embodiment, the anchor is (binding to streptavidin Biotin (for binding to maltose-binding proteins or fusion proteins) Amylose, (for binding to polyhistidine or polyhistidine-tagged proteins) It may be Ni-NTA or a peptide (such as an antigen).

[0272] In one embodiment, the anchor includes one linker, or two, three, four or more linkers. It is possible. Preferred linkers include polymers, such as polynucleotides and polyethylene. This includes, but is not limited to, PEG, polysaccharides, and polypeptides. These linkers may be linear, branched, or annular. For example, the linker may be annular It may also be a dinucleotide. The adapter is complementary on the cyclic polynucleotide linker. It can hybridize into a specific arrangement. One or more anchors or one or more linkers can be cut or It may contain components that can be decomposed, such as restricting sites or photodissociable groups. The linker is Functionalized with maleimide groups, it attaches to cysteine ​​residues of proteins. Suitable linker This is described in WO2010 / 086602.

[0273] In one embodiment, the anchor is cholesterol or a fatty acyl chain. For example, hex Any fatty acyl chain having a carbon atom length of 6 to 30, such as sadecanoic acid, can be used. Examples of suitable anchors and methods for attaching anchors to adapters can be found in WO2012 / 164. Disclosed in 270 and WO2015 / 150786.

[0274] In another embodiment, the anchor is connected to a polynucleotide or polynucleotide adapter. It consists of, or may contain, a hydrophobic modification. The hydrophobic modification is a polynucleotide or This may include modified phosphate groups contained within the polynucleotide anchor. Hydrophobic modifications are, for example, For example, phosphorothioates such as alkyl phosphorothioates (PPT) whose charge has been neutralized. This may include, and these are Jones et al, J.Am.Chem.Soc.2021. It is described in 143,22,8305, and its entire contents are incorporated herein by reference. Suitable alkyl groups include, for example, C1-C6 alkyl groups. 10 a Examples of propyl groups include methyl, ethyl, propyl, butyl, pentyl, and hexyl groups. This can be done. Incorporation of charge-neutralized alkyl-phosphorothioates into polynucleotides. This allows polynucleotides to engage with hydrophobic regions such as lipid bilayers.

[0275] detector In the method provided herein, polynucleotides are subjected to a detector such as a nanopore. Move. The detector consists of (i) a zero-mode waveguide, (ii) a field-effect transistor, and an optional select Selectively Noyre field-effect transistors, (iii) AFM chips, (iv) nanotubes , optionally selected from carbon nanotubes and (v) nanopores. Preferably The detector is a nanopore.

[0276] Polynucleotides are characterized in any preferred manner in the methods provided herein. It can be kicked. In one embodiment, the polynucleotide is a polynucleotide against the nanopore. This is characterized by detecting ion currents or optical signals during movement. This will be described in more detail. This method is suitable for these and other methods for detecting polynucleotides. They are doing it.

[0277] In another non-limiting example, in one embodiment, polynucleotides are sequenced by a synthetic reaction. Characterized by detecting by-products of polynucleotide processing reactions such as sterilization. Therefore, this method involves the (poly)nucleopolymerization of nucleic acid chains by enzymes such as polymerase. This may include detecting the products of the sequential addition of rheotides. The products may include the three-dimensional structure of the enzyme, etc. This could involve a change in one or more properties of the enzyme. Therefore, such a method can affect polymerase or Enzymes such as reverse transcriptase are used as templates for nucleotide bases into elongated oligonucleotide chains. Dependent integration triggers conformational changes in the enzyme in response to successively encountered templates. Under such conditions, the double-stranded polynucleotide is subjected to the incorporation of stranded nucleic acid bases and / or This involves the incorporation of template-specific native or analogous bases (i.e., incorporation events), such Detection of enzyme conformational changes in response to uptake events, and the resulting sequence changes in the template chain. Detection may include, in such a method, the polynucleotide chain is, in the method provided herein. It may be moved according to the following. Such a method is described in US2017 / 0044605. Detect and / or measure the capture event using methods known to those skilled in the art. This may include doing so.

[0278] In another embodiment, when a nucleotide is added to a synthetic nucleic acid chain complementary to the template chain, a phosphate group is formed. The labeled species is released and the phosphate-labeled species is detected using the detector described herein. The by-products can be labeled. Polynucleotides characterized in this way are used in the methods of this specification. It can be moved according to. Suitable labels can be made using nanopores or zero-mode waveguides, or It may be an optical label detectable by Raman spectroscopy or other detectors. A suitable label is a n It may be a non-optical label that can be detected using a nopore or other detector.

[0279] In another approach, the nucleoside phosphate (nucleotide) is not labeled and is phased into the template chain. When nucleotides are added to a complementary synthetic nucleic acid chain, natural by-product species are detected. The detector may be an ion-sensitive field-effect transistor or another type of detector.

[0280] These and other detection methods are suitable for use in the methods described herein. As the octide moves relative to the detector, the detector can be used to perform any suitable measurement. can.

[0281] nanopore In embodiments of the present invention where the detector is a nanopore, any suitable nanopore can be used. This is possible. In one embodiment, the nanopore is a transmembrane pore.

[0282] Transmembrane pores are structures that extend to some extent across the membrane. Therefore, they are driven by the applied potential. The hydrated ions can then flow across or within the membrane. Transmembrane pore Typically, this involves traversing the entire membrane, thereby allowing hydrated ions to move from one side of the membrane to the other. It becomes possible for it to flow. However, transmembrane pores do not need to cross the membrane. One end may be closed. For example, a pore is a well, gap, channel, or groove in a membrane. , or it may be a slit, through which hydrated ions can flow.

[0283] In the method provided herein, the nanopore typically has a first opening and a second opening. It has a section. The first opening is typically a cis opening, and the second opening is typically a to This is a lance opening. However, in some embodiments, the first opening is a transformer An opening, and a second opening is a cis opening. Used in the manner provided herein. The motor protein is typically supplied to the first opening of the nanopore, and therefore , target polynucleotide in the direction from the second opening of the nanopore toward the first opening of the nanopore Control the movement of the cydoid.

[0284] Any transmembrane pore can be used in the manner provided herein. The pore is biological and It may be artificial or otherwise suitable. Suitable pores include protein pores and polynucleotide pores. This includes, but is not limited to, solid pores. Pores can be DNA origami pores. (Langecker et al.,Science,2012;338:932- 936). A preferred DNA origami pore is disclosed in WO2013 / 083983.

[0285] In one embodiment, the nanopore is a transmembrane protein pore. This allows hydrated ions such as polynucleotides to flow from one side of the membrane to the other. It is a polypeptide or an aggregate of polypeptides. In the method provided herein, the membrane is penetrated. Through protein pores, hydrated ions driven by the applied potential flow from one side of the membrane to the other. A pore can be formed that allows for this. Transmembrane protein pores are preferably This refers to the flow of polynucleotides from one side to the other of a membrane, such as a triblock copolymer membrane. This makes it possible. Transmembrane protein pores allow polynucleotides to move through the pore. This makes it possible.

[0286] In one embodiment, the nanopore is a transmembrane protein pore that is a monomer or oligomer. Yes. The pores are preferably at least 6, at least 7, at least 8, and at least 9 , at least 10, at least 11, at least 12, at least 13, at least 14 several repeating subunits, such as at least 15 or at least 16 subunits It is made of knitted material. The pore is preferably hexamer, heptamer, octamer , or nanomer pore. The pore is either a homo-oligomer or a hetero-oligomer. That's fine.

[0287] In one embodiment, the transmembrane protein pore is a barrel through which ions can pass and flow. Or it may include channels. The pore subunits typically surround a central axis and transcend the membrane. Contributes to transmembrane β-barrels or channels or transmembrane α-helix bundles or channels do.

[0288] Typically, the barrel or channel of a transmembrane protein pore is a target polynucleotide ( It contains amino acids that promote interaction with analytes such as those described herein. Amino acids are preferably located near the barrel or channel constriction. Transmembrane protein The porosity typically contains one or more positively charged elements, such as arginine, lysine, or histidine. These contain untreated amino acids, or aromatic amino acids such as tyrosine or tryptophan. The amino acids are typically located between pores and nucleotides, polynucleotides, or nucleic acids. It promotes the interaction between them.

[0289] In one embodiment, the nanopore is derived from a β-barrel pore or an α-helix bundle pore. It is a transmembrane protein pore. A β-barrel pore is a barrel or channel formed from a β-chain. It contains β-barrel pores, such as β-toxins, α-hemolytic toxins, anthrax toxins, and Leucosidines, as well as bacterial outer membrane proteins / porins, such as Mycobacteri um smegmatis porin (Msp), for example, MspA, MspB, MspC, or MspD, CsgG, outer membrane porin F (OmpF), outer membrane porin G (OmpG), outer membrane Phospholipase A, and Neisseria autotransporter lipoprotein (N This includes, but is not limited to, alP, and other pores, such as lysenine. - Helix bundle pores include barrels or channels formed from α-helices. Suitable α-helix bundle pores include inner membrane proteins and α-outer membrane proteins, for example. This includes, but is not limited to, WZA and ClyA toxins.

[0290] In one embodiment, the nanopore contains Msp, α-hemolysin (α-HL), lysenin, and CsgG. , due to ClyA, Sp1, or the hemolytic protein fragaceatoxin C (FraC) These are transmembrane pores that result from or are based on them.

[0291] In one embodiment, the nanopore is CsgG, for example, E. coli strain K-12 sub-strain MC4 It originates from CsgG derived from 100. Such pores are oligomers, and typically Csg It contains 7, 8, 9, or 10 monomers derived from G. The pore contains the same monomer. It could be a homo-oligomeric pore derived from CsgG. Alternatively, the pore is slightly different from others. It may be a heterooligomeric pore derived from CsgG containing at least one monomer. Examples of suitable pores derived from sgG are disclosed in WO2016 / 034591.

[0292] In one embodiment, the nanopore is a transmembrane pore derived from lysenine. Examples of suitable pores are disclosed in WO2013 / 153359.

[0293] In one embodiment, the nanopores are derived from or based on α-hemolysin (α-HL). It is a transmembrane pore. The wild-type α-hemolytic toxin pore consists of seven identical monomers or subunits. It is formed from (i.e., it is a nanomer). The α-hemolysin pore is α-hemolysin- It may be NN or a variant thereof. The variant preferably has N at positions E111 and K147. Contains residues.

[0294] In one embodiment, the nanopore is a transmembrane derived from Msp, for example, MspA. This is a protein pore. An example of a suitable pore derived from MspA is WO2012 / 1077. It is disclosed in 78.

[0295] In one embodiment, the nanopore is derived from or based on ClyA, and is a transmembrane pore. be.

[0296] film In the disclosed method, the detector is typically a nanopore present in the film. A suitable film may be used.

[0297] The membrane is preferably an amphiphilic layer. The amphiphilic layer possesses both hydrophilic and lipophilic properties. It is a layer formed from amphiphilic molecules such as phospholipids. Amphiphilic molecules are synthesized or These may be naturally occurring. They may form amphiphilic substances and monolayers that do not exist naturally. Amphiphilic substances are known in the art, and among them are, for example, block copolymers. -(Gonzalez-Perez et al., Langmuir, 2009, 25 Block copolymers include ,10447-10450). This is a polymer material in which units are polymerized together to form a single polymer chain. Copolymers typically possess properties contributed by each monomer subunit. However, block copolymers are polymers formed from individual subunits. It may have unique properties that it does not possess. Block copolymers are composed of one monomer subunit. One subunit is hydrophobic (i.e., lipophilic), while the other subunit is hydrophilic in aqueous media. It can be manipulated to be so. In this case, the block copolymer may have amphiphilic properties and It can form structures that mimic body membranes. Block copolymers are diblocks (two monomers) It may consist of subunits, but is constructed from three or more monomer subunits. It can form more complex arrangements that behave as amphiphilic materials. Copolymers are triblock The film may be a tetrablock or pentablock copolymer. The film is preferably a tri It is a block copolymer film.

[0298] Archaeal bipolar tetraether lipids are constructed such that the lipids form a monolayer membrane. These are naturally occurring lipids. These lipids are generally the extreme conditions that allow organisms to survive in harsh environments. It can be found in halophilic bacteria, thermophilic bacteria, halophilic bacteria, and acidophilic bacteria. Their stability is determined by the final bilayer. It is thought to be obtained from the fusion properties of these elements. The general motif has hydrophilic-hydrophobic-hydrophilic properties. By creating triblock polymers, we can mimic these biological entities. Constructing goji polymers is straightforward. This material behaves similarly to lipid bilayers. It can form monomer films and encompass a wide range of phase behaviors, from vesicles to layered films. The membranes formed from these triblock copolymers have several advantages over biological lipid membranes. Therefore, since the triblock copolymer is synthesized, its exact structure must be carefully controlled. The correct chain length is necessary to control the formation of membranes and to interact with pores and other proteins. And can provide characteristics.

[0299] Block copolymers are constructed from subunits that are not classified as lipid submaterials. In some cases, hydrophobic polymers may be derived from siloxanes or other non-hydrocarbon monomers. It can be produced. The hydrophilic subsection of the block copolymer also has low protein binding properties. This may allow for the creation of membranes that are highly resistant when exposed to raw biological samples. This head group unit may also originate from an unclassified lipid head group.

[0300] Triblock copolymer membranes exhibit increased mechanical and environmental stability compared to biological lipid membranes. For example, they also have a much higher operating temperature or pH range. Synthetic properties of block copolymers. This provides a foundation for customizing polymer-based films for a wide range of applications.

[0301] In some embodiments, the film is related to International Application No. WO2014 / 064443 or the same WO2014 / 064443. It is one of the membranes disclosed in O2014 / 064444.

[0302] Amphiphilic molecules are chemically modified to facilitate the coupling of polynucleotides. It may be functionalized or otherwise. The amphiphilic layer may be a monolayer or a dilayer. The amphiphilic layer is typically planar. The amphiphilic layer may also be curved. The sex demographic may be supported.

[0303] Amphiphilic membranes are typically about 10 -8 cm s -1 Secondary lipid diffusion rate It is naturally mobile, acting essentially as a fluid. This is due to pores and couplings. This means that polynucleotides can typically move within an amphipathic membrane.

[0304] The membrane can be a lipid bilayer. The lipid bilayer is a model of the cell membrane and is suitable for a wide range of experiments. It serves as an excellent foundation for research. For example, lipid bilayers can be used for single-channel recording. It can be used for in vitro investigations of membrane proteins. Alternatively, lipid bifurcation The sublayer can be used as a biosensor to detect the presence of a wide range of substances. The lipid bilayer can be any lipid bilayer. Suitable lipid bilayers include planar lipids. This includes, but is not limited to, a bilayer, a supporting bilayer, or liposomes. The molecular layer is preferably a planar lipid bilayer. A suitable lipid bilayer is WO2008. Disclosed in / 102121, WO2009 / 077734, and WO2006 / 100484. It is being done.

[0305] Methods for forming lipid bilayers are known in the art. Lipid bilayers are, Generally Montal and Mueller(Proc.Natl.Acad.Sci Formed by the method described in .USA.,1972;69:3561-3566), So, the lipid monolayer crosses both sides of the opening perpendicular to the interface at the aqueous solution / air interface. Lipids are typically first dissolved in an organic solvent, and then a small amount of solvent is added. By evaporating the medium at the interface of the aqueous solution on both sides of the opening, the electrolyte aqueous solution is formed on the surface. It is added. When the organic solvent evaporates, a bilayer is formed at the solution / air interface on both sides of the opening. It moves physically up and down across the opening until it is reached. The planar lipid bilayer crosses the opening and forms a membrane. It may be formed inside or across the opening within a recess.

[0306] The Montal & Mueller method is suitable for protein pore insertion and involves the bifurcation of high-quality lipids. It is popular because it is a cost-effective and relatively simple method for forming a sub-layer. Other common methods for forming molecular layers include tip-dipping and bilayer formation. Painting bilayers and liposome bilayer patch clans It includes "pu".

[0307] Tip immersion bilayer formation involves placing an open surface (for example) on the surface of a test solution supporting a monolayer of lipids. This involves contacting the pipette tip. In this case as well, the lipid monolayer is the most Initially, a small amount of lipid dissolved in the organic solvent is evaporated at the solution surface, thereby creating a solution / air environment. It is generated as a plane. Subsequently, the bilayer is formed by the Langmuir-Shafer method. Mechanical automation is required to move the opening relative to the solution surface.

[0308] In the case of bilayer coating, a small amount of lipid dissolved in an organic solvent is immersed in the open solution of the test aqueous solution. It is applied directly. The lipid solution is thinly spread across the opening using a paint brush or equivalent. It can be spread out. By diluting the solvent, the formation of a lipid bilayer is brought about. However, However, it is difficult to completely remove the solvent from the bilayer, and as a result, The bilayer formed by this method has low stability and is prone to generating noise during electrochemical measurements. .

[0309] Patch clamps are commonly used in the study of biological cell membranes. The cell membrane is pinpointed by suction. The membrane patch is attached to the end of the pet, and adheres across the opening. The liposomes are retained and then ruptured to seal the lipid bilayer across the pipette opening. This method is adapted to produce a lipid bilayer. It is necessary to create small openings in monolayer liposomes and materials having a glass surface. Let's assume that.

[0310] Liposomes are processed by sonication, extrusion, or the Mozafari method (Colas et al.) It can be formed by (2007) Micron 38:841-847).

[0311] In some embodiments, the lipid bilayer is specified in International Application No. WO2009 / 077734 It is formed as described. This method is advantageous in that the lipid bilayer is formed from dry lipids. In the most preferred embodiment, the lipid bilayer is described in WO2009 / 077734. As shown, it is formed across the opening.

[0312] A lipid bilayer is formed from two opposing layers of lipids. These two lipid layers are These hydrophobic tail groups are arranged to face each other, forming a hydrophobic interior. The hydrophilic head groups of the bilayer are oriented outward toward the aqueous environment on both sides of the bilayer. The layers include a liquid disordered phase (fluid layered), a liquid ordered phase, and a solid ordered phase (layered gel phase, comb-type gel phase). This includes, but is not limited to, planar bilayer crystals (layered subgel phase, lamellar crystal phase). No, they can be present in several lipid phases.

[0313] Any lipid composition that forms a lipid bilayer can be used. Required properties, such as surface charge, ability to support membrane proteins, packing density, or mechanical properties. A lipid bilayer having properties is selected. The lipid composition is one or more different It may contain lipids. For example, a lipid composition may contain up to 100 lipids. The lipid composition preferably contains 1 to 10 lipids. It may contain lipids and / or artificial lipids.

[0314] Lipids typically consist of a head group, an interface, and two parts that may be the same or different. Contains hydrophobic tail groups. Suitable head groups include neutral head groups, such as diacylglycerides. DG and ceramide (CM), zwitterionic head group, for example, phosphatidylcholine (PC), phosphatidylethanolamine (PE), and sphingomyelin (SM) , negatively charged head groups, for example, phosphatidylglycerol (PG), phosphatidyl Lucerin (PS), phosphatidylinositol (PI), phosphatidic acid (PA), and cardiolipin (CA), and positively charged head groups, for example, trimethylammonate This includes, but is not limited to, nium-propane (TAP). Suitable interface portions include It includes naturally occurring interface parts, such as glycerol-based or ceramide-based parts. However, it is not limited to these. Suitable hydrophobic tail groups include saturated hydrocarbon chains, for example, lau Phosphate (n-dodecanolic acid), myristic Acid (n-Tetradecononic acid), pal Mitic acid (n-hexadecanoic acid), stearic acid (n-octadecanoic acid), and Arakidi eicosanoic acid (n-eicosanoic acid), unsaturated hydrocarbon chain, for example, oleic acid (cis-9-octa This includes, but is not limited to, decanoic acid, and branched hydrocarbon chains, such as phytanoyl. It is not done. The length of the chain and the position and number of double bonds in an unsaturated hydrocarbon chain can vary. The length of the methyl groups and other chains within the branched hydrocarbon chain, as well as the position and number of branches, can vary. The tail group can be linked to the interface as an ether or ester. Lipids are mycolic acid It is possible.

[0315] Lipids can also be chemically modified. The head group or tail group of a lipid can be chemically modified. This is possible. Suitable lipids in which the head group is chemically modified include PEG-modified lipids, for example. , 1,2-diacyl-sn-glycero-3-phosphoethanolamine-N-[methoxy( Polyethylene glycol)-2000], functionalized PEG lipids, e.g., 1,2-distea Royl-sn-glycero-3phosphoethanolamine-N-[biotinyl(polyethylene)] Glycols (2000), as well as lipids modified for conjugation, for example, 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine-N-(succini (Lu) and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N- (Biothinyl) is included, but not limited to, these. The tail group is chemically modified. Suitable lipids include polymerizable lipids, such as 1,2-bis(10,12-tricosadiino (tricosadiynoyl)-sn-glycero-3-phosphocholine, lipid fluoride Quality, for example, 1-palmitoyl-2-(16-fluoropalmitoyl)-sn-glycerol -3-phosphocholine, deuterated lipids, e.g., 1,2-dipalmitoyl-D62-sn- Glycerol-3-phosphocholine and ether-binding lipids, such as 1,2-di-O-phyta Lipids include, but are not limited to, nyl-sn-glycero-3-phosphocholine. Even if chemically modified to facilitate polynucleotide coupling, functionalized That's fine.

[0316] An amphiphilic layer, such as a lipid composition, will typically affect the properties of the layer. It contains one or more additives. Preferred additives include fatty acids, such as palmitic acid and myristic acid. Acids, and oleic acid, fatty alcohols, such as palmitic alcohol, myristic alcohol Cole, and oleic alcohol, sterols, such as cholesterol, ergosterols. L, lanosterol, sitosterol, and stigmasterol, lysophospholipids, for example Then, 1-acyl-2-hydroxy-sn-glycero-3-phosphocholine and ceramide These are included, but not limited to.

[0317] In another embodiment, the film includes a solid layer. The solid layer is a microelectronic material, an insulating material, for example. For example, Si3N4, A12O3, and SiO, organic and inorganic polymers, such as polyamides. Plastics, for example, Teflon®, or elastomers, for example, two Organic materials and, including but not limited to, component-cured silicone rubber and glass. It can be formed from both inorganic and solid materials. The solid layer can be formed from graphene. A suitable graphene layer is disclosed in WO2009 / 035647. The film includes a solid layer. In this case, a pore is typically a hole, well, gap, or channel within a solid layer. It is present in the amphiphilic film or layer contained within the groove or slit. Those skilled in the art will know that suitable A solid / amphiphilic hybrid system can be prepared. A suitable system is WO2009 / 0 Disclosed in 20682 and WO2012 / 005857. The amphiphilicity discussed above. Either a film or a layer can be used.

[0318] The methods disclosed herein typically include (i) an artificial amphiphilic layer containing a pore, (ii) ) an isolated, naturally occurring lipid bilayer containing a pore, or (iii) a pore inserted inside This method is performed using cells that have been modified. Typically, artificial triblock copolymers are used. This is carried out using an artificial amphiphilic layer, such as a single layer. In addition to the pore, the layer also contains other transmembrane tangents. It may contain proteins and / or intramembrane proteins, as well as other molecules. Suitable apparatus and conditions are: This will be discussed below. The method of the present invention is typically carried out in vitro.

[0319] General method As described above, the method provided herein is operated using any suitable detector. Therefore, any suitable apparatus for detecting polynucleotides can be used.

[0320] In some embodiments, this method is suitable for sensing transmembrane pores in any device. This can be carried out using a device that includes a chamber containing an aqueous solution and two chambers. It may include a barrier that separates the components. The barrier has openings where a membrane is formed, including transmembrane pores. It may be possible. Transmembrane pores are described herein.

[0321] This method is based on WO2008 / 102120, WO2010 / 122293, or WO00 This can be carried out using the apparatus described in / 28312. In short, fractions within the channel of the pore The binding of the child (e.g., target polynucleotide) to the open channel ion flow passing through the pore. This involves influencing something, and this is the essence of "molecular sensing" in pore channels. The fluctuations in channel ion flow can be measured using a suitable measurement technique based on the change in current. This can be done. The degree of decrease in ion flow, measured by the decrease in current, can be measured inside or near the pore. It is related to the size of the obstacle. Therefore, the target portion within or near the pore is The binding of a child (e.g., a target polynucleotide) provides a detectable and measurable event. This forms the basis for "biological sensors," which detect the presence of biomolecules. This enables personalized drug development, medicine, diagnostics, life science research, and environmental monitoring. Applications are found in security and / or defense industries.

[0322] When used to characterize polynucleotides, the presence or absence of the target polynucleotide, or This determines one or more features. This method involves the presence of at least one target polynucleotide. This method may be used to determine presence, absence, or one or more characteristics. This may relate to determining the presence, absence, or one or more characteristics of a target polynucleotide. The method involves any number of target polynucleotides, e.g., 2, 5, 10, 15, 20, 30, The presence, absence, or one or more characteristics of 40, 50, or 100 or more target polynucleotides are determined. This may include defining any number of features of one or more target polynucleotides, for example, 1 Two, three, four, five, or more features can be determined by the methods provided herein. The characteristics obtained include the identity or sequence of the polynucleotide, the length of the polynucleotide, and poly Examples include whether or not the nucleotides are modified. In some embodiments, the present The method provided in the document is a method for sequencing target polynucleotides. In the embodiment, the polynucleotide sequence is a real-time signal or By aligning the base calling, it can be determined in real time. An exemplary method for determining the Otid sequence is incorporated herein by reference in WO2016. It is listed in / 059427.

[0323] When used for characterizing polynucleotides, this method typically involves measuring electric current. This may include measuring the flow of ionic current through the pore. Alternatively, the ions through the pore The flow of this information is from Heron et al: J.Am.Chem.Soc.9 Vol.13. As disclosed in 1, No. 5, 2009, it can be measured optically. Therefore The device can apply an electric potential and measure electrical signals across the film and pore. It may also be equipped with a gas circuit. Characterization is performed using a patch clamp or voltage clamp. This is possible. The characterization method preferably involves the use of voltage clamping.

[0324] This method was described by Chen et al. in Nature Communications (2018). As described in 9:1733, it may include measuring the optical signal, and all of it The contents are incorporated herein by reference. For example, optically designed nanopore structures ( Using nanopores such as plasmonic nanoslits, single-molecule surface-enhanced Raman spectroscopy is performed. By locally enabling spectroscopy (SERS), polynucleotides can be detected by direct Raman spectroscopy. It is possible to characterize the "D" part.

[0325] This method uses arrays with 128, 256, 512, 1024, 2000, 3000, and 40 Silicone substrates with 00, 6000, 10000, 12000, 15000 or more wells This can be done in a well array.

[0326] This method may include measuring the current flowing through a pore. This method typically involves measuring the current. The process is performed with pressure applied across the membrane and pore. The voltage used is typically The voltage used is between +2V and -2V, typically between -400mV and +400mV. The values ​​are -400mV, -300mV, -200mV, -150mV, -100mV, A lower limit selected from -50mV, -20mV, and 0mV, and +10mV, +20mV, +50mV, +100mV, +150mV, +200mV, +300mV, and +400 It is within a range having an upper limit that is independently selected from mV. The voltage used is more preferably Or, within the range of 100mV to 240mV, most preferably within the range of 120mV to 220mV. It is inside. By using an increased applied potential, different nucleotides are separated for each pore. It is possible to increase the number of identifications.

[0327] The disclosed methods, in particular, include methods involving the rereading of target polynucleotides as described herein. In some embodiments of the method, the polynucleotide binding site of the motor protein To provide conditions for promoting the debinding of target polynucleotides, and / or Delays the rebinding of target polynucleotides to the polynucleotide binding site of the ter protein. This includes causing something to happen.

[0328] This method typically involves metal salts, such as alkali metal salts, halogen salts, and chloride salts. This is carried out in the presence of any charge carrier, such as alkali metal chloride salts. This refers to ionic liquids or organic salts, such as tetramethylammonium chloride and trimethyl chloride. Phenylammonium, phenyltrimethylammonium chloride, or 1-ethyl-3-methylammonium It may contain chylimidazolium chloride. In the exemplary apparatus considered above, the salt is chylimidazolium chloride. It is present in aqueous solutions within the chamber. Potassium chloride (KCl), sodium chloride (NaCl), Alternatively, cesium chloride (CsCl) is typically used. KCl is preferred. The salt is C. It may be an alkaline earth metal salt such as calcium (CaCl2). The salt concentration is the concentration at saturation. It is possible. The salt concentration can be 3M or less, typically 0.1-2.5M, 0.3-1 0.9M, 0.5~1.8M, 0.7~1.7M, 0.9~1.6M, or 1M~1.4M The salt concentration is preferably 150 mM to 1 M. The characterization method is preferably , at least 0.3M, for example, at least 0.4M, at least 0.5M, at least 0.6M, at least 0.8M, at least 1.0M, at least 1.5M, at least This is performed using a salt concentration of 2.0 M, at least 2.5 M, or at least 3.0 M. High salt concentration provides a high signal-to-noise ratio and a normal background for current fluctuations. This allows for the identification of currents indicating coupling / non-coupling.

[0329] In some embodiments, providing the motor protein polynucleotide Provide a salt concentration that increases the rate at which the target polynucleotide dissociates from the tide bond site. This includes providing the motor protein It slows down the rate of rebinding of target polynucleotides to the polynucleotide binding site. This includes providing sea urchin salt concentration from the polynucleotide binding site of motor proteins. To promote the debinding of target polynucleotides and / or delay their rebinding. Determining a suitable salt concentration is within the scope of the skill of a person skilled in the art, taking into consideration the disclosures herein. ru.

[0330] In some embodiments, providing the conditions is the polynucleotide of the motor protein. The osmotic pressure is adjusted to increase the rate at which the target polynucleotide dissociates from the oxidative binding site. This includes providing. In some embodiments, providing the conditions is motor tamper It reduces the rate of rebinding of target polynucleotides to the polynucleotide binding site of the substance. This includes providing osmotic pressure to the polynucleotide binding site of the motor protein. To promote the debinding and / or delay the rebinding of their target polynucleotides. Determining a suitable osmotic pressure is within the capabilities of those skilled in the art, taking into consideration the disclosures herein. be.

[0331] This method is typically carried out in the presence of a buffer. In the exemplary apparatus considered above, The buffer solution is present in the aqueous solution within the chamber. Any suitable buffer solution can be used. Typical The buffer is HEPES. Another suitable buffer is Tris-HCl buffer. This method is typically used for 4.0-12.0, 4.5-10.0, 5.0-9.0, and 5. Perform at pH levels of 0.5-8.8, 6.0-8.7, 7.0-8.8, or 7.5-8.5. The pH used is preferably about 7.5.

[0332] The methods are: 0°C to 100°C, 15°C to 95°C, 16°C to 90°C, 17°C to 85°C, and 18°C. It can be carried out at ~80°C, 19°C to 70°C, or 20°C to 60°C. The method is typically: The procedure is carried out at room temperature. This method can optionally be performed at a temperature that supports enzyme function, for example, around 37°C. It will be implemented.

[0333] In some embodiments, providing the conditions is the polynucleotide of the motor protein. Increase the temperature to increase the rate at which the target polynucleotide dissociates from the ocidal bond site. This includes causing the motor tamper It reduces the rate of rebinding of target polynucleotides to the polynucleotide binding site of the substance. This includes raising the temperature. While not strictly bound by theory, the inventors They found that increasing the temperature increases the rate of dissociation of motor proteins from polynucleotides, for example. We believe that by increasing the poly of motor proteins, re-reading can be promoted. To promote the debinding of target polynucleotides from nucleotide binding sites, and / or Determining a suitable temperature for delaying recombination is, in consideration of the disclosures herein, It is within the scope of the contractor's skills.

[0334] Temperature to facilitate rereading Provide Examples of conditions are provided herein; see, for example, Example 11. In some embodiments, the target port from the polynucleotide binding site of the motor protein Polynucleotides for promoting the debonding of dinucleotides and / or motor proteins Provides conditions for delaying the rebinding of target polynucleotides to the ocidal binding site. This means that the temperature range is approximately 20°C to 50°C, for example, approximately 30°C to 45°C, for example, approximately 34°C to 40°C. For example, it provides temperatures of approximately 31, 32, 33, 34, 35, 36, 37, 38, or 39°C. This may include doing so.

[0335] Further aspects of the methods to be disclosed The following are further aspects of the disclosed method: 1. A method for characterizing a target polynucleotide, (i) Contact the detector with the target polynucleotide to which the motor protein is bound. Therefore, the target polynucleotide is at the polynucleotide binding site of the motor protein. It is bound to the motor protein, and contact is made. (ii) The motor protein moves the target polynucleotide in the first direction relative to the detector. When controlling, obtain one or more measurements characteristic of the target polynucleotide. , (iii) The motor moves the target polynucleotide in a second direction relative to the detector. - Debinding target polynucleotides from the polynucleotide binding site of proteins , (iv) Reattach the target polynucleotide to the polynucleotide binding site of the motor protein. Combined, the motor protein moves the target polynucleotide in a first direction relative to the detector. When controlling, obtain one or more measurements characteristic of the target polynucleotide. A method comprising characterizing a target polynucleotide thereby.

[0336] 2. The method described in Embodiment 1, which includes repeating steps (iii) and (iv) multiple times. Law.

[0337] 3. In step (ii), the motor protein moves in the first direction relative to the detector. Controlling the movement of the first portion of the target polynucleotide, in step (iv), The ter protein directs the second portion of the target polynucleotide towards the first direction relative to the detector. Controlling the movement of the first part such that the first part overlaps with the second part in at least partially, in embodiment 1 or 2 Methods used.

[0338] 4. The method according to any one of the prior art, wherein the first part is the same as the second part.

[0339] 5. In step (iii), the distance the target polynucleotide moves relative to the detector. The method according to any one of the prior embodiments, wherein the separation is at least 100 nucleotides long.

[0340] 6. The detector is included in a structure having a first aperture and a second aperture, or the first The nanopore comprises a first opening and a second opening, and step (i) is a first opening The method according to any one of the prior art, which includes contracting a portion with a target polynucleotide. Law.

[0341] 7. (i) The motor protein targets the direction from the second opening to the first opening. (ii) Control the movement of polynucleotides, and target polynucleotides in motor proteins When the target polynucleotide detaches from the polynucleotide binding site, the target polynucleotide opens to the first opening. The method according to embodiment 6, wherein the object moves in the direction of the second opening.

[0342] 8. The process involves applying a force to the detector, and the motor protein reacts to the applied force in the opposite direction. In the direction of controlling the movement of the target polynucleotide relative to the detector, any one of the preceding embodiments The method used.

[0343] 9. The detector includes a transmembrane nanopore spanning a film having a cis side and a trans side. (i) The first opening of the nanopore is on the cis side of the film, and the second opening of the nanopore is on the cis side. Located on the lance side, the motor protein passes through a nanopore from the trans side to the cis side of the membrane. Controls the movement of target polynucleotides, and the target polynucleotides are located on the motor protein When the target polynucleotide detaches from the cis side of the membrane, It moves towards the transformer side through the nanopore, and, (ii) The first opening of the nanopore is on the transformer side of the film, and the second opening of the nanopore However, it is located on the cis side, and the motor protein passes through the nanopore from the cis side to the trans side of the membrane. It controls the movement of target polynucleotides, and the target polynucleotides move through motor proteins. When the target polynucleotide is detached from its polynucleotide binding site, it moves trans-trans to the membrane. A method according to any one of the prior embodiments, wherein movement occurs from the side to the cis side through a nanopore.

[0344] 10. The target polynucleotide is a motor from the target polynucleotide in the vicinity of the leader. - A leader designed to promote the debonding of polynucleotide binding sites in proteins. The method according to any one of the prior embodiments, comprising an adhering or leader.

[0345] 11. When the target polynucleotide comes into contact with the motor protein, The method according to embodiment 10, wherein the protein is detached from the polynucleotide binding site.

[0346] 12. Motor proteins become leaders of the target polynucleotide. The method according to embodiment 10 or embodiment 11, which has a low affinity for the other party.

[0347] 13. The leader contains a different type of nucleotide than the target polynucleotide. The method described in one of the following 10-12.

[0348] 14. (i) The target polynucleotide contains deoxyribonucleotide (DNA), The leader is a nucleo lacking both a nucleic acid base and a sugar moiety (spacer moiety). Tides, ribonucleotides (RNA), peptide nucleotides (PNA), glycerol Cleotide (GNA), threose nucleotide (TNA), locked nucleotide (L NA), cross-linked nucleotides (BNA), debasic nucleotides, or modified phosphate bonds (ii) The target polynucleotide contains one or more nucleotides, or (ii) the target polynucleotide is ribo It contains nucleotides (RNA), and the leader is the nucleic acid base and sugar portion (spacer portion) Nucleotides lacking both, deoxyribonucleotides (DNA), peptide nucleotides (PNA), glycerol nucleotide (GNA), threose nucleotide (T NA), locked nucleotide (LNA), cross-linked nucleotide (BNA), debased nucleotide Embodiments 10-13, comprising a nucleotide having a creotide or a modified phosphate bond. The method described in any one of the following ways.

[0349] 15. The target polynucleotide contains deoxyribonucleotide (DNA) and the leader Embodiment 1, which includes one or more spacer portions and / or one or more ribonucleotides. A method described in one of the following 0-14.

[0350] 16. The target polynucleotide does not disassociate from the motor protein, unlike in previous models. Either one of the methods.

[0351] 17. The motor protein is modified, and the target polynucleotide is modified. A method according to any one of the prior embodiments for preventing disassociation from D.

[0352] 18. The motor protein is modified, and the polynucleotide bond of the motor protein Promotes the debinding of target polynucleotides from the site, and / or the target polynucleotide The prior embodiment delays the rebinding of motor proteins to polynucleotide binding sites. Any one of the following methods.

[0353] 19. Motor proteins (i) motor proteins around target polynucleotides (ii) motors for topologically closing the polynucleotide binding sites of quality It promotes the debinding of target polynucleotides from the polynucleotide binding site of proteins, and / or the target polynucleotide to the polynucleotide binding site of the motor protein The method described in any one of the preceding embodiments, modified by a closed portion to delay recombination Law.

[0354] 20. The motor protein is modified, and the binding of the closure portion to the motor protein becomes less efficient. The method described in embodiment 19, which makes it easy.

[0355] 21. Motor proteins have at least one amino acid in the motor protein. The method according to embodiment 20, which is modified by substitution with a stain or a non-natural amino acid. .

[0356] 22. The method according to any one of embodiments 19 to 21, wherein the closed portion contains a bifunctional crosslinking agent. Law.

[0357] 23 The closed portion cross-links two amino acid residues of the motor protein, and the closed portion Embodiment 1, in which at least one cross-linked amino acid is cysteine ​​or a non-natural amino acid. The method described in any one of the following 9-22.

[0358] 24. The closed portion has a length of approximately 1 Å to approximately 100 Å, any one of embodiments 19 to 23. The method used.

[0359] 25. The closed portion includes a bond, preferably a disulfide bond, according to any of embodiments 19 to 21. The method described in one of the following options.

[0360] 26. The closing part includes the structure of formula [ABC], where A and C are motortan Each of these is an independent reactive functional group for reacting with amino acid residues in the protein, and B is linked. The method according to any one of embodiments 19 to 24, which is a part of the method.

[0361] 27. The method according to embodiment 26, wherein A and C are each independently cysteine-reactive functional groups. Law.

[0362] 28. The connecting portion B is a linear or branched, unsubstituted or substituted alkylene, Kenylene, alkynylene, arylene, heteroarylene, carbocyclylene or hetero It contains a cyclylene moiety, and the moiety consists of one or more of O, N(R), S, C(O), and C(O)N. R, C(O)O, unsubstituted or substituted arylenes, arylene-alkylenes, hetero-arylenes -Lene, heteroarylene-alkylene, carbocyclylene, carbocyclylene-alkyl Atom or group selected from lene, heterocyclylene and heterocyclylene-alkylene , optionally interrupted or terminated, where R is H, unsubstituted or substituted alkyl The method according to embodiment 26 or 27, which is selected from unsubstituted or substituted aryls.

[0363] 29. The connecting portion B contains alkylene, oxyalkylene, or polyoxyalkylene groups. M AND / OR A and C are each maleimide groups, one of any one of embodiments 26 to 28. Methods used.

[0364] 30. The closed portion has a length of approximately 5 Å to approximately 50 Å, as in aspects 19-25 or 26-29. The method described in any one of the following ways.

[0365] 31. From the polynucleotide binding site of motor proteins to the target polynucleotide Conditions for promoting debinding, and / or polynucleotide binding of motor proteins. This includes providing conditions for delaying the rebinding of target polynucleotides to a site. The method according to any one of the prior art embodiments.

[0366] 32. Providing the conditions from the polynucleotide binding site of the motor protein This includes increasing the temperature to increase the rate at which the target polynucleotide dissociates. The method according to embodiment 31.

[0367] 33. Providing the conditions for the polynucleotide binding site of the motor protein This includes increasing the temperature to slow down the rate of recombination of the target polynucleotide. The method described in Specifications 31 or 32.

[0368] 34. The motor protein is a helicase, as described in any one of the preceding embodiments. Law.

[0369] These embodiments relate to features that will be described in more detail herein.

[0370] Polypolymer adapter Polynucleotide adapters containing motor proteins are also provided. Any of the polynucleotide adapters shown can be used to implement the methods described herein and above. It will be understood that this can be applied to form.

[0371] In one embodiment, a first including an attachment site for attaching to a double-stranded polynucleotide analyte A polynucleotide adapter having a terminal and a second terminal is also provided, and the polynucleotide The adapter is (i) oriented in the direction of the attachment point for processing the adapter. (ii) The motor protein that stalled above, and the second of the motor protein and adapter It includes a blocking portion located between the end and the end.

[0372] In one embodiment, the polynucleotide adapter is described in more detail herein. It is a renucleotide adapter. In one embodiment, the motor protein is as described herein. The motor protein described herein. In one embodiment, the blocking portion is described herein. This is the blocking section of the image.

[0373] Motor proteins attach to adapters for binding to double-stranded polynucleotides. The polynucleotide adapter is oriented to process in the direction toward the point. The ter protein controls the movement of target polynucleotides from trans to cis. They can be oriented on a polynucleotide adapter.

[0374] Motor proteins are directed towards detectors such as nanopores. To control the movement of the target polynucleotide on the polynucleotide adapter, Oriented, that is, as described in more detail herein, exiting the detector, for example, nano Exiting the pore.

[0375] In some embodiments, the polynucleotide adapter is the stall portion described herein. Includes. In some embodiments, the polynucleotide adapter is a stop as described herein. Including the stopper portion.

[0376] kit A kit containing polynucleotide adapters and motor proteins is also provided. Any of the polynucleotide adapters disclosed in this document are not considered in this specification and above. It will be understood that this can be applied to the embodiment of the kit.

[0377] In one embodiment, a kit for modifying a target polynucleotide is provided, and the kit This includes a first adapter provided in the present invention and a first terminal comprising a single-stranded leader array, The second end contains an attachment point for attaching to the double-stranded polynucleotide analyte. Includes "tar".

[0378] In one embodiment, the second adapter is an adapter as described in detail herein. be.

[0379] system The system also includes polynucleotide adapters, motor proteins, and nanopores. Provided. Any of the polynucleotide adapters disclosed herein are provided herein and above. It will be understood that this can be applied to the embodiments of the system discussed.

[0380] In one embodiment, a system for characterizing a target double-stranded polynucleotide is provided, and the system teeth, - A stop portion and a polynucleotide adapter that optionally includes a stop portion, -When the target polynucleotide moves relative to the nanopore, the target polynucleotide Nanopores for marking, - A motor for moving double-stranded polynucleotides in a first direction relative to the nanopore. It contains protein.

[0381] In one embodiment, the polynucleotide adapter is described in more detail herein. It is a renucleotide adapter. In one embodiment, the motor protein is as described herein. The motor protein described herein. In one embodiment, the nanopore is as described herein. It is a nanopore. The system may further include a film, a control device, and other components.

[0382] Specific embodiments, specific configurations, and materials and / or molecules may be used in the methods according to the present invention. As discussed herein, various changes or modifications of form and detail may extend beyond the scope of the present invention. Please understand that it may be done without deviating from the purpose. The following examples are specific Provided to better illustrate embodiments and deemed to limit this application This application should not be limited by the claims alone. [Examples]

[0383] Example 1 This example uses a DNA modifier that unwinds dsDNA while transferring the 5'-3' of ssDNA. Using a ter, we demonstrate the controlled movement of DNA polynucleotide chains through nanopores. The NA motor first stalled on the Y adapter linked to the polynucleotide. Nucleotides traveled through the nanopore at the following different stages: (1) polynucleotides The 3' end of DN is trapped by a nanopore, the nanopore displaces, and DN stalls at its 5' end. (2)D The naturally aspirated motor initially failed to overcome the stall under positive bias, but when reverse voltage was applied... The motor, activated by use ("de-stalled"), enters the "de-stalled" stage, (3) the motor DNA has begun to move 5'-3' from the nanopore against the applied potential, DNA mo In the control phase, (4) after reaching the end of the polynucleotide, the potential to expel the chain is reversed. A certain level of blockage was observed that could be removed by doing so.

[0384] Asymmetric 3.6 kilobase double-stranded DNA analytes (bacteriophage lambda DNA) The fragment (SEQ ID NO: 20) was obtained by PCR, and NEBNext end repair and NEBNe repair were performed. xt dA-tailing module (New England Biolabs (N Using EB)) and USER digests, add a 3'dA overhang to one end. Generate it, and create a 3'AGGA overhang at the opposite end.

[0385] The Y adapter anneals DNA oligonucleotides (SEQ ID NO: 21, SEQ ID NO: 22). It was prepared by ringing. A DNA motor (Dda helicase) was used with an adapter. Loaded. Added monomer traptabidine to the adapter and used 5' bio as a blocker. By binding to the chin portion, (1) the DNA motor is prevented from diffusing in the reverse direction from the 5' end. (2) This prevented the 5' end of the library from being unintentionally captured by nanopores.

[0386] Oxford Nanopore Technologies Sequence Determination Kit SKQ- LSK109 (also referred to as LSK-SQK109 in this specification, https: / / co mmunity.nanoporetech.com / protocols / gDNA- sqk-lsk109 / v / gde_9063_v109_revt_14aug201 Using LNB and T4 DNA ligase (NEB) from (see details), double-stranded DN The A analyte was ligated to the dA end of the Y adapter. The sample was Agencou Purified using rt AMPure XP (Beckman Coulter) beads. Oxford Nanopore Technologies Sequencing Kit (L The substrate was washed twice with LFB (SK-SQK109). The ligated substrate was then treated with 10 mM Elute with TrisCl and 50mM NaCl (pH 8.0) to create a "DNA library". — was obtained.

[0387] FLO-MIN106 from Oxford Nanopore Technologies Electrical measurements were obtained using the MinION flow cell and MinION Mk1b. 120 0 μL FB (Oxford Nanopore Technologies sequencing agent) A 50nM DNA tether was added to the (SQK-LSK109) and the tether mixer was then used. We obtained the solution. After running 800 μL of tether mix through the system, we waited for 5 minutes and then SpO With the tON port open, an additional 200 μL of tether mix was flowed into the system. Ox Ford Nanopore Technologies Sequencing Kit (SQK-LS) 37.5 μL SQB from K109, 15 μL DNA library, 0.7 μL Excess monomer traptabidine (approximately 100 nM tetramer), and Oxford Nano Two results from the pore Technologies sequencing kit (SQK-LSK109) 2.5 μL of LB was mixed to obtain a "sequencing mix". 75 μL of sequencing Add the mixing mix to the MinION flow cell via the SpotON flow cell port. did.

[0388] The custom sequencing script was prepared to control the applied potential as follows: 0.5 seconds destall phase (0mV), 85.5 seconds sequence determination (+120mV), ejection phase ( (Varying between 0mV and -120mV over 1 second, then -120mV over 3 seconds). This series of applicable potentials I repeated it multiple times.

[0389] Raw data is obtained using MinKNOW software (Oxford Nanopore Tech). The data was collected in bulk FAST5 files using hnologies.

[0390] Figure 6 shows the adapter used in this embodiment. Figure 7 shows the double-stranded polynucleotide. The adapter attached to the analyte is shown. Figure 8 shows how the polynucleotide analyte is captured and detached. A schematic diagram of the experiment in this embodiment shows the pattern of applied potentials necessary for characterization. Figure 9 shows an example of current-vs-time traces in this embodiment. The data was obtained using nanopores. By capturing the polynucleotide analyte and then lowering the application potential to 0 to -120 mV, After the DNA is "de-stalled," it is controlled and moves stepwise from the nanopore. This indicates that enzyme-mediated events exceeding the -40mV destall potential were rarely recorded. This suggests that, between 0 and -40 mV, the single strand is retained in the nanopore during the destallation phase. Yes, they are.

[0391] Example 2 This example uses a DNA modifier that unwinds dsDNA while transferring the 5'-3' of ssDNA. Using a ter, both strands of the DNA polynucleotide double helix are controlled as they pass through the nanopore. This indicates movement. The DNA motor initially moves on the Y adapter linked to the polynucleotide. It stalled. The template chain and complementary chain were linked via a hairpin portion. Polynucleotides , it moved through the nanopore in the following different stages: (1) the 3' end of the polynucleotide The DNA motor is captured by a nanopore, the nanopore undergoes rearrangement, and then stalls at the 5' end. Until it reaches the target, the two strands are separated under a positive potential, with the complementary strand passing through first, then the template strand. (2) The DNA motor is unable to overcome the stall under the initial positive bias. However, by applying the reverse potential, the "de-stalled" stage was activated ("de-stalled"). (3) The motor moves the DNA 5'-3' from the nanopore against the applied potential. To begin with, the DNA motor moves along the template strand first, then through the hairpin, and then along the complementary strand. (4) After moving, DNA motor control stage, after reaching the end of the polynucleotide, A certain block level can be removed by reversing the potential that discharges it. Being seen.

[0392] Asymmetric 3.6 kilobase double-stranded DNA analytes (bacteriophage lambda DNA) The fragment (SEQ ID NO: 20) was obtained by PCR, and NEBNext end repair and NEBNe repair were performed. xt dA-tailing module (New England Biolabs (N Using EB)) and USER digests, add a 3'dA overhang to one end. Generate it, and create a 3'AGGA overhang at the opposite end.

[0393] The Y adapter anneals DNA oligonucleotides (SEQ ID NO: 21, SEQ ID NO: 22). Prepared by ringing. DNA motor (Dda helicase) adapter Loaded into. Monomer traptabidine was added to the adapter as a blocker for 5' It binds to the otin portion, (1) preventing the DNA motor from diffusing in the reverse direction from the 5' end. (2) This prevented the 5' end of the library from being unintentionally captured by nanopores.

[0394] Hairpins with a 3'-TCCT overhang are DNA oligonucleotides (sequence) Number 23) is used in the double-stranded annealing buffer (Integrated DNA Techno). (logies, Inc.) Heat 1 μM ~ 95°C for 2 minutes, then with wet ice It was prepared by cooling.

[0395] Oxford Nanopore Technologies' sequencing kit (LS Using LNB and T4 DNA ligase (NEB) from K-SQK109, double-stranded The DNA analyte and hairpin were connected to a Y-adapter. The sample was sent to Agentcourt. Refined using AMPure XP (Beckman Coulter) beads, Oxford Nanopore Technologies Sequencing Kit (LSK) The substrate was washed twice with LFB (-SQK109). The ligated substrate was then treated with 10 mM T Elute with ris-Cl and 50 mM NaCl (pH 8.0) to create a "DNA library". I obtained it.

[0396] FLO-MIN106 from Oxford Nanopore Technologies Electrical measurements were obtained using the MinION flow cell and MinION Mk1b. 120 0 μL FB (Oxford Nanopore Technologies sequencing agent) A 50nM DNA tether was added to the (SQK-LSK109) and the tether mixer was then used. We obtained the solution. After running 800 μL of tether mix through the system, we waited for 5 minutes and then SpO With the tON port open, an additional 200 μL of tether mix was flowed into the system. Ox Ford Nanopore Technologies Sequencing Kit (SQK-LS) 37.5 μL SQB from K109, 15 μL DNA library, 0.7 μL Excess monomer traptabidine (approximately 100 nM tetramer), and Oxford Nano Two results from the pore Technologies sequencing kit (SQK-LSK109) 2.5 μL of LB was mixed to obtain a "sequencing mix". 75 μL of sequencing Add the mixing mix to the MinION flow cell via the SpotON flow cell port. did.

[0397] A custom sequence script is prepared to control the applied potential as follows: Furthermore, a 0.5-second destall phase (variable depending on the experiment, in the range of 0mV to -120mV), 85 Sequence determination in 0.5 seconds (+120mV), ejection phase (0mV, 1 second, -120mV, 3 seconds). This series of applied potentials was repeated multiple times. Raw data is obtained from MinKNOW software (Oxford Nanopore Tech The data was collected in bulk FAST5 files using nologies.

[0398] Figure 10 shows the components used in this embodiment: hairpin (A), adapter (B) and polynucleotide analytes (C), (D) all ligated together Components. Figure 11 shows the capture and destabilization of hairpin-derivatized polynucleotide analytes. This is a schematic diagram of the experiment in this embodiment, showing the pattern of applied potentials necessary for characterization. This is shown. Figure 12a shows some examples of current-time traces in this embodiment. The data is from Capture of polynucleotide analytes by nopores, and the subsequent "de-stalled" nanopores This shows controlled stepwise movement of DNA from A. The loss-stall potential is 0mV to -120mV. The voltage was varied between -60mV and -60mV, but no enzyme-mediated events exceeding -60mV were observed, which indicates that Tran The hairpin, folded in the compartment, is ejected during destallation down to a potential of -60mV. This suggests resistance to and additional resistance compared to single-stranded DNA alone (implementation) (From Example 1). Figure 12b shows states A to G assigned to the current trace example in Figure 11. Compared to Example 1, an additional state E is observed in Figure 12b, which is the mold portion D This may be due to the subsequent enzyme-mediated transfer of the complementary portion of the polynucleotide from the nanopore.

[0399] Example 3 This example demonstrates controlled destallation of a DNA motor via an "active destallation" process. . DNA modulo, which unwinds one or both strands of a double helix of DNA polynucleotides. The DNA was passed through a nanopore using a ter. The DNA motor consists of Y nucleotides linked to polynucleotides. The first stall occurred on the adapter. Optionally, at the distal end of the polynucleotide, the template strand and The complementary chain was connected via a hairpin section, but in other cases, the template and complement chains are connected via a hairpin. The apin was omitted and not linked. Polynucleotides undergo nanopore processing at the following different stages. It moved through: (1) The 3' end of the polynucleotide was captured by the nanopore, The nopore rearranged and separated the double helix until it reached the DNA motor that had stalled at the 5' end. (2) The DNA motor can first overcome the stall under a positive bias. However, activation was achieved by repeatedly applying the efflux potential, followed by returning to the sequencing potential. (3) The motor is in an active "de-stall" phase, against the applied potential. Then the DNA motor control stage begins, where the DNA starts moving 5'-3' from the nanopore, and ( 4) By reversing the potential that ejects the chain after reaching the end of the polynucleotide A certain level of blockage that could be removed was observed.

[0400] Asymmetric 3.6 kilobase double-stranded DNA analytes (bacteriophage lambda DNA) The fragment (SEQ ID NO: 20) was obtained by PCR, and NEBNext end repair and NEBNe repair were performed. xt dA-tailing module (New England Biolabs (N Using EB)) and USER digests, add a 3'dA overhang to one end. Generate it, and create a 3'AGGA overhang at the opposite end.

[0401] Symmetrical 3.6 kilobase double-stranded DNA analytes (bacteriophage lambda DNA) The fragment (SEQ ID NO: 20) was obtained by PCR, and NEBNext end repair and NEBNe repair were performed. xt dA-tailing module (New England Biolabs (N Use EB)) to generate 3'dA overhangs at both ends.

[0402] The Y adapter anneals DNA oligonucleotides (SEQ ID NO: 21, SEQ ID NO: 22). It was prepared by ringing. A DNA motor (Dda helicase) was used with an adapter. Loaded. Added monomer traptabidine to the adapter and used 5' bio as a blocker. By binding to the chin portion, (1) the DNA motor is prevented from diffusing in the reverse direction from the 5' end. (2) This prevented the 5' end of the library from being unintentionally captured by nanopores.

[0403] Hairpins with a 3'-TCCT overhang are DNA oligonucleotides (sequence) Number 23) is used in the double-stranded annealing buffer (Integrated DNA Techno). (logies, Inc.) Heat 1 μM ~ 95°C for 2 minutes, then with wet ice It was prepared by cooling.

[0404] Oxford Nanopore Technologies' sequencing kit (LS Using LNB and T4 DNA ligase (NEB) from K-SQK109, double-stranded The DNA analyte was ligated to a Y-adapter. The sample was from Agentcourt AMPur. Refined using e XP (Beckman Coulter) beads, Oxford Nanopore Technologies Sequencing Kit (LSK-SQK10) 9) The substrate was washed twice with LFB. The ligated substrate was then treated with 10 mM Tris-Cl. The DNA was eluted in 50 mM NaCl (pH 8.0) to obtain a "1D DNA library".

[0405] Oxford Nanopore Technologies Sequence Determination Kit (LSK) - Using SQK109) and T4 DNA ligase (NEB) LNB, asymmetric double-stranded DNA DNA analytes were attached to a Y-adapter and a hairpin. The sample was taken from an Agentcourt. Refined using AMPure XP (Beckman Coulter) beads, Oxford Nanopore Technologies Sequencing Kit (LSK) The substrate was washed twice with LFB (-SQK109). The ligated substrate was then treated with 10 mM T Elute with ris-Cl and 50 mM NaCl (pH 8.0) and then "2D DNA library" He obtained "Lee".

[0406] FLO-MIN106 from Oxford Nanopore Technologies Electrical measurements were obtained using the MinION flow cell and MinION Mk1b. 120 0 μL FB (Oxford Nanopore Technologies sequencing agent) A 50nM DNA tether was added to the (SQK-LSK109) and the tether mixer was then used. We obtained the solution. After running 800 μL of tether mix through the system, we waited for 5 minutes and then SpO With the tON port open, an additional 200 μL of tether mix was flowed into the system. Ox Ford Nanopore Technologies Sequencing Kit (SQK-LS) 37.5 μL SQB from K109, 15 μL 1D library or 2D DNA Library, 0.7 μL of excess monomer traptabidine (approximately 100 nM tetramer), and Oxford Nanopore Technologies sequencing kit (SQ Mix 22.5 μL of LB from K-LSK109 to create the "sequencing mix". Obtained. 75 μL of sequencing mix was transferred via the SpotON flow cell port. Added to inION flow cell.

[0407] To control the applied potential using MinION's activated unblocking circuit, I prepared the sequence script. I set the sequence determination voltage to 120mV and activated the block. The release potential ("activation-destabilization" stage, step (2) above) is -12m in the 1D library. V, set to -48mV in the 2D library. Stall level and chain (sequence determination) level The classification was programmed into the configuration file of the MinKNOW device control software, and it stalled. This enables the detection of species, and using the knowledge of static block release potential from Examples 1 and 2, We applied an unblocking potential that does not cause a complete release of the chain. The script is as follows: Function activated: MinKNOW unblocks the chain if it detects that the chain is at a stall level. The potential is applied for 5 seconds initially, then the sequencing potential is returned to 120mV to activate sequencing. Check the chain 5 times. If a stall level still exists, further check the possibility of unblocking. Apply for 25 seconds and repeat 5 times. Include a 3-second break between each unblocking attempt. That's right. When MinKNOW returns a sequencing potential, it detects an active sequencing strand. At this point, the unblocking attempt is stopped, and only the sequencing potential is applied. If a sex-specific sequencing decision strand is not generated, MinKNOW turns off the channel. 15 minutes Apply "mux scan" each time to reset the system and all the flow cells We completely unblocked Nell and checked for active nanopores at 120mV.

[0408] Raw data is obtained using MinKNOW software (Oxford Nanopore Tech). The data was collected in bulk FAST5 files using hnologies.

[0409] Figures 7 and 10, D show the polynucleotide analytes used in this embodiment. The preparation is described in Examples 1 and 2. Figure 13 shows the 1D DNA library (A). An example of current tracing of a 2D DNA library (B) is shown. The region where destamping was attempted. Mark it with an asterisk. The data is then processed using these methods in a 1D library and It is possible to destamp both 2D libraries, and to destamp the enzyme and nanoparticles. Several repeated attempts were made to confirm the enzymatically controlled transfer of polynucleotides from A. To show that it is possible.

[0410] Example 4 In this example, the portion of the DNA transposition via nanopore that does not contain the first enzyme (3'-5') Using the signal period from ), the template strand and complementary strand are connected to the terminal 5'-3' DNA motor. Before the DNA strand is actively moved in the opposite direction from the nanopore, the hairpin portion is connected This document describes a method for estimating the size of double-stranded DNA molecules when they are joined. In addition, this example describes... This demonstrates how to distinguish signals using markers added to hairpins.

[0411] The DNA motor first stalled on the Y-adapter linked to the polynucleotide. The template chain and complementary chain were joined together via a hairpin portion according to Example 2. Selectively, the hairpin portion contains a bulky fluorophore group or debasic group, and / or additional The oligonucleotide was hybridized to a hairpin.

[0412] Asymmetric 3.6 kilobase double-stranded DNA analytes (bacteriophage lambda DNA) A fragment (SEQ ID NO: 20) was obtained by PCR using primers, and one of them contained multiple It contains dUTP bases, and performs NEBNext end repair and NEBNext dA tailings. The New England Biolabs (NEB) module, followed by NEB End repair and dA tailing are performed using USER digest, with a 3'dA tail added to one end. This generates an overhang, and then generates a 3'AGGA overhang at the opposite end.

[0413] E. col sheared to a shear size of approximately 20kb using Covaris gTube. We will ligate a generic adapter to i SCS110 DNA and amplify it by PCR. From this, a random library of Escherichia coli double-stranded DNA was generated. The fragment was then modified to generate 3'dA overhangs at both ends, specifically at the NEBNext terminal. Repair and NEBNext dA tailing module (New England Bio End repair and dA tail formation were performed by labs(NEB).

[0414] The Y adapter anneals DNA oligonucleotides (SEQ ID NO: 21, SEQ ID NO: 22). It was prepared by ringing. A DNA motor (Dda helicase) was used with an adapter. Loaded. Added monomer traptabidine to the adapter and used 5' bio as a blocker. By binding to the chin portion, (1) the DNA motor is prevented from diffusing in the reverse direction from the 5' end. (2) This prevented the 5' end of the library from being unintentionally captured by nanopores.

[0415] Hairpins with a 3'-TCCT or 3'-T overhang are shown in Sequence ID 24, Sequence ID 24. Add DNA number 25 or sequence number 26 to duplex annealing buffer (I) at a concentration of 1 μM. (Integrated DNA Technologies, Inc.) 95°C for 2 minutes It was prepared by heating it and then rapidly cooling it on wet ice.

[0416] Oxford Nanopore Technologies Sequence Determination Kit (LSK) - Using LNB and T4 DNA ligase (NEB) from SQK109, asymmetric 3 0.6 kg base double-stranded DNA analytes and hairpin (SEQ ID NO: 24 or SEQ ID NO: 26) It was connected to the adapter. The sample is Agentcourt AMPure XP (Bec Purified using KMAN Coulter beads, Oxford Nanopor e Technologies sequencing kit (LSK-SQK109) LFB 2 The substrate was washed multiple times. The ligated substrate was then treated with 10 mM Tris-Cl and 50 mM NaC. The DNA was eluted in pH 8.0 to obtain a "3.6kb DNA library".

[0417] Oxford Nanopore Technologies Sequence Determination Kit (LSK) - Using LNB and T4 DNA ligase (NEB) from SQK109, Esch Ericia coli double-stranded DNA and hairpin (SEQ ID NO: 25) into a Y-adapter Linked. The sample is Agentcourt AMPure XP (Beckman C Purified using oulter beads, Oxford Nanopore Tech Washed twice with LFB from the Nologies sequencing kit (LSK-SQK109). The ligated substrate was treated with 10 mM Tris-Cl and 50 mM NaCl (pH 8.0) Elution was performed to obtain a "randomized E. coli study library".

[0418] FLO-MIN106 from Oxford Nanopore Technologies Electrical measurements were obtained using the MinION flow cell and MinION Mk1b. 120 0 μL FB (Oxford Nanopore Technologies sequencing agent) A 50nM DNA tether was added to the (SQK-LSK109) and the tether mixer was then used. We obtained the solution. After running 800 μL of tether mix through the system, we waited for 5 minutes and then SpO With the tON port open, an additional 200 μL of tether mix was flowed into the system. Ox Ford Nanopore Technologies Sequencing Kit (SQK-LS) 37.5 μL SQB from K109, 15 μL 3.6 kb library or random 0.7 μL of excess monomer traptabidine from any of the E. coli test libraries. (Approximately 100 nM tetramer), Oxford Nanopore Technology Mix 22.5 μL of LB from the ess sequencing kit (SQK-LSK109) and, A sequencing mix was obtained. A portion of the reaction solution contained the oligonucleotide of SEQ ID NO: 27. D was also added at 50 nM. 75 μL of sequencing mix was used in SpotON FlowSample. Added to MinION flow cell via report.

[0419] I tested the two libraries with different execution scripts. The 3.6kb library was To control the applied potential, a custom sequencing script is used. The following steps were taken: a 0.5-second destall phase (-40mV) and an 85.5-second sequence determination phase (+120mV). The discharge phase (0mV, 1 second, -120mV, 3 seconds). This series of applied potentials is repeated multiple times. The random E. coli test library was modified according to the custom activity-induced loss of rate of change described in Example 3. The process was performed using a script with a capture / arrangement voltage of 120mV and an output voltage of -48mV. .

[0420] Raw data is obtained using MinKNOW software (Oxford Nanopore Tech). The data was collected in bulk FAST5 files using hnologies.

[0421] Figure 14 shows the hairpin and oligonucleotide combinations used in this embodiment. Using a 3.6kb DNA library, we first characterized the capture-stage signal. Figure 15 shows the intermediates expected to be detected by electrical measurements of enzyme-free and enzyme-mediated rearrangements. A schematic diagram of the body is shown. Compared with Figure 11, the bulky groups within the nanopore and the blocks on the nanopore are shown. Two additional states A1 and A2 correspond to each of the Kerr oligonucleotides (see Figure 15). (Illumination) is expected during the initial enzyme-free capture. Additional state D1 is bulky hairpin portion. Corresponding to enzymes that move via a group, the template (D) step and complementary chain (E) step of enzyme-mediated transfer This is expected to occur in betwee...

Claims

1. A method for characterizing a target polynucleotide, (i) The first opening of a transmembrane nanopore having a first opening and a second opening, The method involves contacting the target polynucleotide, wherein the target polynucleotide is It has a motor protein that has stalled above, and the motor protein stalls at the stalled portion It involves making contact, (ii) Bringing the stalled portion into contact with the nanopore, thereby the motor The process of causing the tar protein to lose momentum and making contact with it, (iii) The motor protein enters the nanopore through the second opening of the nanopore Movement of the target polynucleotide through the nanopore in the direction toward the first opening of A. When controlling the target polynucleotide, one or more characteristic measurements of the target polynucleotide are obtained. And thereby characterizing the target polynucleotide, obtaining, Includes, method.

2. The nanopore extends across a film having a cis side and a trans side, and the first of the nanopores The opening is on the cis side of the film, and the second opening of the nanopore is on the tra Located on the trans side, the motor protein moves from the trans side to the cis side of the membrane. The method according to claim 1, which controls the movement of the target polynucleotide through the nanopore. Law.

3. The nanopore extends across a film having a cis side and a trans side, and the first of the nanopores The opening is on the transformer side of the film, and the second opening of the nanopore is Located on the cis side, the motor protein moves from the cis side to the trans side of the membrane. The method according to claim 1, which controls the movement of the target polynucleotide through the nanopore. Law.

4. The process involves applying force across the nanopore, and the motor protein is subjected to Controlling the movement of the target polynucleotide through the nanopore in the direction opposite to the applied force. Control, The force preferably includes a potential applied across the nanopore, according to the prior claims. The method described in any one of the items.

5. The motor protein is a helicase, as described in any one of the prior claims. Law.

6. The motor protein is a DNA-dependent ATPase (Dda) helicase. The method according to any one of the prior claims.

7. The adapter is attached to one or both ends of the target polynucleotide, according to the prior claim. The method described in any one of the items.

8. The method according to claim 7, wherein the motor protein is stalled on the adapter. 。

9. The nanopore captures the leader sequence at the first end of the target polynucleotide, The motor protein is at the second end of the target polynucleotide, or the target poly The prior claim is stalled on the adapter attached to the second end of the nucleotide. The method described in either of the above terms.

10. - The target polynucleotide is single-stranded, - The target polynucleotide includes a leader sequence, and the leader sequence is the target Located at the first end of the polynucleotide, or the first end of the target polynucleotide It is included in the adapter attached to the end of, - The motor protein stalls at the second end of the target polynucleotide. A dolphin, or stalled on the adapter at the second terminus of the target polynucleotide. , the method according to any one of the prior claims.

11. The method according to any one of claims 1 to 9, wherein the target polynucleotide is double-stranded. Law.

12. - The target polynucleotide is double-stranded and comprises a first strand and a second strand, - The target polynucleotide includes a leader sequence, and the leader sequence is the poly Located at the first end of a nucleotide, and included in the first chain, or attached to the first chain. Included in the attached adapter, - The motor protein is stalled at the second end of the target polynucleotide. The method according to claim 11.

13. The motor protein is at the second end of the first chain of the target polynucleotide. The adapter at the second end of the first strand of the target polynucleotide is either stalled or - The method according to claim 12, which is stalled above.

14. The first chain and the second chain are connected by a hairpin adapter at the second end of the first chain. -They adhere together, and the motor protein stalls at the hairpin adapter. The method according to claim 12 or 13.

15. The first chain and the second chain are (i) the second end of the first chain and (ii) The hairpin adapter attached to the first end of the second chain is attached together with the mo The receptor protein stalls at the second end of the second strand of the double-stranded polynucleotide. Claim 1, either the second chain is stalled on the adapter at the second end of the second chain. The method described in 2.

16. The target polynucleotide includes a portion that is complementary to the tag sequence, according to any of the prior claims. The method described in any one of the items.

17. The target polynucleotide has an oligonucleotide hybridized thereto. The oligonucleotide includes a portion thereof, and the oligonucleotide is a hybrid of (a) the target polynucleotide (b) A hybridized portion for issuance, and (i) a portion complementary to the tag sequence or (ii) The prior art claims include an affinity molecule that can bind to a tag. The method.

18. The target polynucleotide is double-stranded, and the portion complementary to the tag sequence is The portion of the first chain of the polynucleotide and / or hybridizes thereto The portion having the oligonucleotide is the portion of the first chain of the polynucleotide. A method according to claim 16 or 17.

19. The motor protein comprises one or more stall units independently selected from the following: - Polynucleotide secondary structure, preferably hairpin or G-quadrilateral (TBA), - Preferably, peptide nucleic acid (PNA), glycerol nucleic acid (GNA), threose nucleus Acids (TNA), locked nucleic acids (LNA), cross-linked nucleic acids (BNA), and debasalized nucleotides nucleic acid analogs selected from, - Nitroindole, inosine, acridine, 2-aminopurine, 2-6-diaminopurine Phosphorus, 5-bromodeoxyuridine, inverted thymidine (inverted dTs), inverted dideoxy- Thymidine (ddTs), dideoxycytidine (ddCs), 5-methylcytidine, 5- Hydroxymethylcytidine, 2'-O-methylRNA base, isodeoxycytidine (Is o-dCs), isodeoxyguanosine (Iso-dGs), C3 (OC ３ H ６ OPO ３ )Base, optical cutting may (PC) [OC] ３ H ６ -C(O)NHCH ２ -C ６ H ３ NO ２ -CH ( CH ３ ) OPO ３ ] group, hexanediol group, spacer 9 (iSp9) [(OCH ２ C H ２ ) ３ OPO ３ ] Base, Spacer 18 (iSp18) [(OCH ２ CH ２ ) ６ OPO ３ A spacer unit selected from ] groups and thiol linkages, and - Fluorophores, traptabidine, streptavidin, and neutraavidin, etc. avidin and / or biotin, cholesterol, methylene blue, dinitrophenol (DNP), digoxigenin and / or anti-digoxigenin and dibenzylcyclooctane The method according to any one of the prior claims, wherein the vehicle is stalled at a stall region containing an ion group.

20. Destalling the motor protein provides the polynucleotide with appropriate destalling force. This includes using such a method, where the destabilization force is less than and / or in the opposite direction to the reading force. The reading power allows the motor protein to control the movement of the target polynucleotide. While measurements are being taken to determine one or more characteristics of the polynucleotide, The method according to any one of the prior claims, which is the force to be applied.

21. The motor protein is destabilized, and the applied force is the destabilization force. The method according to claim 20, comprising stepping one or more times between the reading force and the reading force.

22. The motor protein comprises a stall section including one or more stall units and one or more stopping sections. It stalls at a certain position, and the one or more stopping parts come into contact with the nanopore, The movement of the polynucleotide through the motor protein is delayed, thereby delaying the movement of the motor protein The method according to any one of the prior claims, wherein the vehicle de-stalls from one or more stall units.

23. The aforementioned stopping portion is one or more stopping portions independently selected from the following: - Polynucleotide secondary structure, preferably hairpin or G-quadrilateral (TBA), - Preferably, peptide nucleic acid (PNA), glycerol nucleic acid (GNA), threose nucleus Acids (TNA), locked nucleic acids (LNA), cross-linked nucleic acids (BNA), and debasalized nucleotides nucleic acid analogs selected from, - Fluorophores, traptabidine, streptavidin, and neutraavidin, etc. avidin and / or biotin, cholesterol, methylene blue, dinitrophenol (DNP), digoxigenin and / or anti-digoxigenin and dibenzylcyclooctane n group, and - The method according to claim 22, comprising a polynucleotide-binding protein.

24. The target polynucleotide is removed from the motor protein by the polynucleotide. A blocking portion for preventing assembly, as described in any one of the prior claims The method.

25. The target polynucleotide has a leader sequence at its first end. The motor protein includes the second end of the target polynucleotide or the label The polynucleotide stalls on the adapter attached to the second end, blocking The g portion is located between the motor protein and the second end of the polynucleotide. By placing the motor protein at the second end of the target polynucleotide, the motor protein is positioned at the second end of the target polynucleotide. The method according to claim 24, which prevents the target polynucleotide from unassociating at its end.

26. The first end contains an attachment site for attaching to the double-stranded polynucleotide analyte, and the second end A polynucleotide adapter having ends, (i) Stall on the adapter in a direction for processing the adapter in the direction of the attachment point. (ii) the motor protein and the adapter A polynucleotide adapter including a blocking portion located between the two ends.

27. The first adapter according to claim 26, and the first end comprising a single-stranded leader array, A second adapter with an attachment site at the end of 2 for attaching to a double-stranded polynucleotide analyte. A kit that includes - and.

28. The polynucleotide adapter, the motor protein, and / or the block Claim 26 or Claim The polynucleotide adapter or kit described in 27.

29. A method for characterizing a target polynucleotide, (i) Contact the detector with the target polynucleotide to which the motor protein is bound. The target polynucleotide is the polynucleotide of the motor protein. The motor protein is bound at the attachment site, and contact is made. (ii) The motor protein moves in the first direction relative to the detector and the target polynucle When controlling the movement of ocide, one or more measurements characteristic of the target polynucleotide. To obtain, (iii) The target polynucleotide moves in a second direction relative to the detector. , from the polynucleotide binding site of the motor protein to the target polynucleotide Debinding the do, (iv) The target polynucleotide is the polynucleotide bond of the motor protein The motor protein is recombined at the binding site and moves forward in the first direction relative to the detector. When controlling the movement of the target polynucleotide, the target polynucleotide has a characteristic 1 Obtain more than one measurement value, A method comprising characterizing the target polynucleotide thereby.

30. The method according to claim 29, comprising repeating steps (iii) and (iv) multiple times. Law.

31. In step (ii), the motor protein is the first to the detector Control the movement of the first portion of the target polynucleotide in the direction of step (iv) In this case, the motor protein is the second portion of the target polynucleotide, Controlling the movement of the dispenser in the first direction, the first part, at least partially The method according to claim 29 or 30, which overlaps with the second part described above.

32. The first part is the same as the second part, in any one of claims 29 to 31. Method of description.

33. In step (iii), the target polynucleotide moves relative to the detector. The distance is at least 100 nucleotides long, according to any one of claims 29 to 32. Method of description.

34. The detector is included in a structure having a first aperture and a second aperture, or the first The nanopore comprises a transmembrane having an opening and a second opening, and step (i) is the first Any of claims 29 to 33, comprising shrinking the opening with the target polynucleotide. The method described in item 1.

35. (i) The motor protein moves in the direction from the second opening to the first opening (ii) the target polynucleotide controls the movement of the target polynucleotide When the motor protein detaches from the polynucleotide binding site, the target poly Claim 34, wherein the nucleotide moves from the first opening toward the second opening. Methods used.

36. The process includes applying a force to the detector, wherein the motor protein reacts to the applied force. In the opposite direction, the movement of the target polynucleotide relative to the detector is controlled, The method described in any one of paragraphs 29 to 35.

37. The detector includes a transmembrane nanopore spanning a film having a cis side and a trans side, (i) The first opening of the nanopore is on the cis side of the film, and the front of the nanopore The second opening is located on the trans side, and the motor protein is located on the trans side of the membrane. Controlling the movement of the target polynucleotide through the nanopore from the cis side to the cis side, The target polynucleotide is the polynucleotide binding site of the motor protein When debonding occurs, the target polynucleotide moves from the cis side of the membrane to the trans side. It moves toward the side through the nanopore, (ii) The first opening of the nanopore is on the transformer side of the film, and the The second opening of the nopore is on the cis side, and the motor protein is of the membrane The movement of the target polynucleotide through the nanopore from the cis side to the trans side. Controlling the target polynucleotide of the motor protein When the target polynucleotide detaches from the binding site, the trans side of the membrane The movement then proceeds to the cis side through the nanopore, as described in any one of claims 29 to 36. The method.

38. The target polynucleotide is in the vicinity of the leader, It is configured to promote the debinding of the polynucleotide binding site of the motor protein. Adhering to the leader, or including the leader, any one of claims 29 to 37 The method described in section [section number].

39. The target polynucleotide is released when the motor protein comes into contact with the leader. Claim 3, wherein the motor protein is detached from the polynucleotide binding site. The method described in 8.

40. The motor protein, in relation to the target polynucleotide, The method according to claim 38 or 39, which also has low affinity to the reader.

41. The leader includes a nucleotide of a different type from the target polynucleotide, The method according to any one of claims 38 to 40.

42. (i) The target polynucleotide comprises a deoxyribonucleotide (DNA) and The leader is a nucleotide lacking both a nucleic acid base and a sugar portion (spacer portion). Otide, ribonucleotide (RNA), peptide nucleotide (PNA), glycerol Nucleotides (GNAs), threose nucleotides (TNAs), locked nucleotides ( LNA), cross-linked nucleotides (BNA), debasic nucleotides or modified phosphate bonds (ii) The target polynucleotide is , comprising ribonucleotides (RNA), the leader comprises nucleic acid bases and sugar portions (spacers) Nucleotides lacking both (the - portion), deoxyribonucleotides (DNA), peptides Cleotide (PNA), Glycerol nucleotide (GNA), Threose nucleotide Contains (TNA), locked nucleotides (LNA), and cross-linked nucleotides (BNA) , including debasalized nucleotides or nucleotides having modified phosphate bonds, The method described in any one of paragraphs 38 to 41.

43. The target polynucleotide comprises deoxyribonucleotide (DNA), and the Lee The claim includes one or more spacer portions and / or one or more ribonucleotides. The method described in any one of paragraphs 38 to 42.

44. Claim 29, wherein the target polynucleotide does not disassociate from the motor protein. The method described in any one of items ~43.

45. The motor protein is modified, and the target polynucleotide is the target polynucleotide The method according to any one of claims 29 to 44, which prevents disassociation from rheotide.

46. The motor protein is modified, and the polynucleotide of the motor protein To promote the debinding of the target polynucleotide from the bond site, and / or the target poly Delays the rebinding of nucleotides to the polynucleotide binding site of the motor protein. The method according to any one of claims 29 to 45, which extends the duration.

47. The motor protein (i) the motor around the target polynucleotide For topologically closing the polynucleotide binding sites of the protein, and (ii ) The target polynucleotide from the polynucleotide binding site of the motor protein Promotes the debinding of tide and / or the polynucleotide binding of the motor protein. Modified with a closed portion to delay the rebinding of the target polynucleotide to the site. The method according to any one of claims 29 to 46.

48. The motor protein is modified, and the closure portion is attached to the motor protein. The method according to claim 47, which facilitates combination.

49. The motor protein has at least one amino acid in the motor protein The modification according to claim 48, which is modified by substitution with cysteine ​​or a non-natural amino acid. method.

50. The method according to any one of claims 47 to 49, wherein the closed portion contains a bifunctional crosslinking agent. Law.

51. The closed portion cross-links two amino acid residues of the motor protein, and the closed portion At least one amino acid that is crosslinked by the fraction is cysteine ​​or a non-natural amino acid. The method according to any one of claims 47 to 50.

52. The closed portion has a length of approximately 1 Å to approximately 100 Å, any one of claims 47 to 51. The method described in section [section number].

53. The closing portion includes a bond, preferably a disulfide bond, according to any of claims 47 to 49. The method described in any one of the items.

54. The closing portion includes the structure of formula [A-B-C], where A and C are the motor Each of these is an independent reactive functional group for reacting with amino acid residues in the protein, and B is a linked The method according to any one of claims 47 to 52, which is the connection part.

55. The method according to claim 54, wherein A and C are each independently cysteine-reactive functional groups.

56. The connecting portion B is a linear or branched, unsubstituted or substituted alkylene, alkenile , alkynylene, allylene, heteroarylene, carbocyclylene or heterocycline The portion includes a ren portion, and the portion is one or more of O, N(R), S, C(O), C(O)NR , C(O)O, unsubstituted or substituted arylene, arylene-alkylene, hetero-arylene Len, heteroarylene-alkylene, carbocyclylene, carbocyclylene-alkylene Atom or group selected from , heterocyclylene and heterocyclylene-alkylene, It may be optionally interrupted or terminated, where R is H, unsubstituted or substituted alkyl, The method according to claim 54 or 55, and selected from unsubstituted or substituted aryls.

57. The connecting portion B contains alkylene, oxyalkylene, or polyoxyalkylene groups. , and / or A and C are each maleimide groups, according to any one of claims 54 to 56 Method of description.

58. Claims 47-53 or 54-57, wherein the closed portion has a length of approximately 5 Å to approximately 50 Å. The method described in any one of the items.

59. The target polynucleotide from the polynucleotide binding site of the motor protein Conditions for promoting the debinding of the cydore, and / or the poly of the motor protein Conditions for delaying the rebinding of the target polynucleotide to the nucleotide binding site The method according to any one of claims 29 to 58, including providing.

60. The above conditions are provided by the polynucleotide binding site of the motor protein. To increase the rate at which the target polynucleotide dissociates, the temperature is increased. The method according to claim 59, including the method described in claim 59.

61. The above conditions are provided by the polynucleotide binding site of the motor protein. The temperature is increased to reduce the rate of rebinding of the target polynucleotide to the The method according to claim 59 or 60, including the act of doing so.

62. The motor protein is a helicase, as described in any one of claims 29 to 61. Method of loading.

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