Multiplexed methods for detecting molecules using nanopores

Abstract

1. A method for detecting a plurality of molecules in a sample, comprising: (a) contacting the sample with a support and a nanopore, the support comprising a single-stranded leader, an identifier region, and a molecular binding region specific to a molecule to be detected, wherein a motor protein is bound to the support such that it can control movement of the identifier region within the nanopore; and (b) performing one or more optical or electrical measurements as the support moves through the nanopore to characterize the identifier region and determine whether a molecule is bound to the molecular binding region.

Description

[Technical field]

[0001] The present invention relates to a multiplex method for detecting molecules in a sample using nanopore technology. The present invention also relates to supports for binding and identifying molecules, and populations of such supports, as well as kits and systems comprising such supports. [Background technology]

[0002] Biological sensors are a vital part of medical diagnostics and a rapidly growing part of the industry. Current state-of-the-art detection techniques used for biomarker (e.g., protein) sensing are usually coupled with quantitative optical readout via ELISA assays or qualitative color readout, which are limited by low concentrations and mask rare events. Antibody-based detection techniques are usually limited in scope to polypeptide targets and suffer from low sensitivity at low concentrations. Mass spectrometry (MS)-based techniques generally require extensive sample preparation and may also suffer from low sensitivity at low concentrations, while targeted MS techniques require large sample sizes when analyzing multiple targets.

[0003] Nanopore technology has been used to detect non-polynucleotide molecules. WO2013 / 121201 describes a method for determining the presence or absence of one or more molecules using aptamer-containing probes and transmembrane pore technology, and exemplified by the detection of thrombin. DNA carriers have been used to allow selective label-free detection of targets, but are limited to single analytes and cannot be easily expanded (see, for example, Sze et al. “Nature comms 8.1 (2017): 1-10, and Cai et al. Nature comms 10.1 (2019): 1-9).

[0004] Furthermore, some populations of biomarkers, such as those of miRNAs, are short-lived and present in low concentrations, meaning that they are currently not clinically accessible.

[0005] Therefore, there is a need for analytical methods that can achieve the simultaneous detection of multiple soluble proteins, miRNAs, and other molecules such as biomarkers in complex samples such as biological fluids. Furthermore, there is a need for such techniques to be able to detect very low concentrations of target molecules. Techniques that can achieve this are expected to have far-reaching impacts in medicine, for example, for diagnosis and monitoring of disease progression. Such techniques may also be applied in different fields, such as studying water samples for the presence of pollutants or other contaminants. Summary of the Invention

[0006] The present disclosure relates to methods and methods that utilize nanopore technology to detect proteins, miRNAs, and other biomarkers, as well as other types of molecules, with the potential for highly multiplexed detection directly in unprocessed samples with high sensitivity and rapid readout.

[0007] Thus, the following is provided herein: - a method for detecting a plurality of molecules in a sample, comprising: (a) contacting a sample with a support and a nanopore, the support comprising a single-stranded leader, an identifier region, and a molecular binding region specific for a molecule to be detected, the motor protein being bound to the support such that it can control movement of the identifier region within the nanopore; (b) performing one or more optical or electrical measurements as the carrier translocates through the nanopore to characterize the identifier region and determine whether a molecule is bound to the molecule binding region; a carrier comprising a single-stranded leader, an identifier region and a molecular binding region specific for the molecule to be detected, the motor protein being bound to the carrier at a position between the single-stranded leader and the identifier region; a population of carriers for a plurality of molecules, each carrier comprising a single-stranded leader, an identifier region and a molecular binding region specific for a molecule to be detected, the motor protein being bound to the carrier at a position between the single-stranded leader and the identifier region, and different carriers in the population comprising different identifier regions and different molecular binding regions; A kit for detecting a plurality of molecules in a sample, comprising: (i) a population of carriers, each carrier comprising an identifier region and a molecular binding region specific for a molecule to be detected, and different carriers in the population comprising different identifier regions and different molecular binding regions; (ii) an adaptor comprising a single-stranded leader; (ii) a motor protein; and - a system for detecting a plurality of molecules in a sample, comprising: (i) a population of carriers, each carrier comprising a single-stranded leader, an identifier region, and a molecular binding region specific for a molecule to be detected, the motor protein being bound to the carrier at a position between the single-stranded leader and the identifier region, and different carriers in the population comprising different identifier regions and different molecular binding regions; (iii) a nanopore. [Brief description of the drawings]

[0008] [Figure 1] Schematic of barcode sequencing and biomarker detection. Overall, a custom designed strand consisting mainly of a barcode and a binding region (e.g., cDNA, aptamer, antibody) to which a targeted analyte is bound is tethered to a membrane. The target-bound strand is detected by translocating through a biological nanopore (CsgG). [Figure 2A]Schematic of an exemplary complete carrier including (i) a leader to facilitate threading into the nanopore, (ii) a tether with a cholesterol linker to enhance capture rate, (iii) a motor protein that provides an irregular, curvilinear signal for sequencing the barcode when coupled to the nanopore under an applied voltage, (iv) a polynucleotide identifier segment (e.g., a barcode or multiple barcodes that may be repeated), (v) a spacer that connects the barcodes, and (vi) a molecular binding region such as an aptamer / c-miRNA / antibody that selectively targets, for example, miRNA, protein, or neurotransmitter. [Figure 2B] Schematic of an exemplary complete carrier including an adapter consisting of (i) a leader to facilitate threading into a nanopore, (ii) a tether with a cholesterol linker to enhance capture rate, and (iii) a motor protein that provides an irregular, curvilinear signal for sequencing a barcode when coupled to a nanopore under an applied voltage. The adapter is ligated to a DNA strand consisting of (i) an adapter ligation portion hybridized to a complementary strand with a single A overhang, (ii) a polynucleotide identifier section (barcode or multiple barcodes that may be repeated), (iii) a spacer connecting the barcodes, and (iv) one or more molecular binding regions, e.g., aptamers / c-miRNAs / antibodies that selectively bind target miRNAs, proteins, or neurotransmitters. [Figure 3A]Exemplary methods for determining the presence or absence of a molecule on a carrier and a molecular binding region. Enzymatic digestion is used to remove / digest any molecular binding region that is not bound to a molecule for which it is specific. (a) A nicking enzyme or endonuclease site is included on the carrier such that the molecular binding region is hidden from the nicking enzyme or endonuclease when it is bound to a molecule for which it is specific, but is exposed when the molecule is not bound to the molecular binding region. After contacting the sample with the carrier under conditions suitable for the molecular binding region for which it is specific, a nicking enzyme or endonuclease can be added to introduce a nick into any carrier that is not bound to a molecule for which the molecular binding region is specific. After such digestion, carriers that are bound to a target molecule can be distinguished from carriers that are bound to a target molecule based on the presence or absence of a current signal following the signal generated by the identifier region (barcode sequence). [Figure 3B] Exemplary methods for determining the presence or absence of a molecule on a carrier and a molecular binding region. Enzymatic digestion is used to remove / digest any molecular binding region that is not bound to a molecule that is specific for it. (b) After contacting the sample with the carrier under conditions suitable for the molecular binding region to the molecule that is specific for it, an exonuclease can be added so that the molecular binding region is digested. After such digestion, carriers that are bound to the target molecule can be distinguished from carriers that are bound to the target molecule based on the presence or absence of a current signal after the signal generated by the identifier region (barcode sequence). [Figure 4A] Barcode sequencing and demultiplexing. (a) All sequences are base called with a base calling algorithm (see slides 19-25) and aligned to a reference sequence, which is the barcode sequence. The maximum alignment score is used to classify the barcode. If the maximum alignment score and the second highest score are too close, the event will not be classified. Additionally, a p-value is used to classify the event and eliminate false positive classifications. [Figure 4B] Barcode sequencing and demultiplexing. (b) With this method, 86% of all recorded events are used and classified, achieving an accuracy of 99.95%. [Figure 5A]Sequencing, Alignment, and Barcode Sorting: (a) Barcode sequencing has an accuracy of over 90% with a 0.0001% chance of a false positive. [Figure 5B] Sequencing, alignment, and barcode classification. (b) Confusion matrix showing very low preference for incorrect barcode classification. Barcode 1: TGCTACTCTCCTCATAAGCAGTCCGGTGTATCGAT, Barcode 2: ATCGCTACGCCTTCGGCTCGTAATCATAGTCGAGT, Barcode 3: AGCTCAGGCAGGTCACTCAAGATACGAGCTGCGT, Barcode 4: GTAAGTCTGCATCAGCGCGCGGCTGTGCGAGGATA, Barcode 5: CTACGACAGTACGCTAGCAAGGATAGACACTACGA, Barcode 6: TACTGAACACAAGTTCGTCGTCGAGCAATCACAAT, Barcode 7: AGTCTACCATTACTTGGATCGGATTAGCCTCACTC, Barcode 8: TGCACGAGTGCGTGTCAACCGTCCAGATGCTCGTG, Barcode 9: CTAGTCGCAGTTGTCTCGGCGGAGTTGAGACTGA, Barcode 10: GATCATGGTAGTCTTCAAGATCGAGTATGTCTGTC. [Figure 6] Ten barcoded carriers were identified in a complex mixture. At the same concentrations, no bias was observed in the detection rate of the barcoded carriers. The barcodes used were barcodes 1 to 10 described above. [Figure 7A] Stall analysis was used to determine whether the target was bound to the barcoded strand: (a) If the target analyte is not bound to the barcoded strand, the current signal shows no stall (in dwell time, current amplitude). [Figure 7B] Stalling analysis was used to determine whether the target was bound to the barcoded strand. (b) Bound analyte (exemplified here with complementary miRNA) stalls the carrier, which results in a unique current profile. [Figure 8-1]The carrier without miRNA (barcode 6) stalled significantly less (6.4%) than when miRNA was added (62.18%). [Figure 8-2] This is a continuation of Figure 8-1. [Figure 9-1] Multiplexed detection of miRNAs with concentration dependence. 10 different barcodes allow for the detection and discrimination of 10 different miRNAs. Some heterogeneity in capture rates was observed, but the operating range remained similar from 0 to 5 nM miRNA. The barcodes used were barcodes 1 to 10 as described above. miRNA1: CAGCAGCACACUGUGGUUUGU, miRNA2: AGAGCUUAGCUGAUUGGUGAAC, miRNA3: UAGCUUAUCAGACUGAUGUUG, miRNA4: ACCUGGCAUACAAUGUAGAUUU, miRNA5: UGUAAACAUCCCCGACUGGAAG, miRNA6: UGUAAACAUCCUACACUCUCAGC, miRNA7: AGCUGGUAAAAUGGAACCAAAU, miRNA8: GAGCUUUUGGCCCGGGUUAUAC, miRNA9: AACAUUCAUUGCUGUCGGUGGGU, miRNA10: UAGCACCAUCUGAAAUCGGUUA. [Figure 9-2] This is a continuation of Figure 9-1. [Figure 10-1] Detection of binding between thrombin and 15mer thrombin-binding aptamer. Upon binding with thrombin, the irregular curved events show a much longer residence time, and in terms of current reversal, a significant increase in stall upon binding with 400 nM thrombin is seen, which corresponds to the unwinding of the G-quadruplex and the aptamer-protein interaction. The first thrombin carrier provided in Example 1 was used in this experiment. [Figure 10-2] This is a continuation of Figure 10-1. [Figure 11]Concentration dependence of the binding between thrombin and 15mer thrombin-binding aptamer. Binding was confirmed by increasing thrombin concentration from 0nM to 400nM. As thrombin concentration increases, the increase in carrier stall was observed as more irregular curve events with longer residence time. The first thrombin carrier provided in Example 1 was used in this experiment. [Figure 12] Detection of serotonin using stem-loop aptamers. A barcode sequence related to the serotonin aptamer provided in Example 1 was used. The structure of serotonin and the aptamer on the carrier is shown. Exemplary current traces are provided showing the signal generated when the barcode interacts with the pore and the signal generated when the aptamer interacts with the pore. As shown in the table and graph, the average delay caused by the unfolding of the aptamer increases with serotonin concentration. [Figure 13A-1] Detection of serotonin using stem-loop aptamers. (A) Current traces in the absence and presence of 40 mM serotonin. As shown in the plot of current versus dwell time, the dwell time increases in the presence of serotonin. [Figure 13A-2] This is a continuation of Figure 13A-1. [Figure 13B] Detection of serotonin using stem-loop aptamers. (B) Plots of current versus dwell time for 0 mM, 2.5 mM, 5 mM, 10 mM, 20 mM, and 40 mM serotonin. A concentration-dependent increase in stall events was observed. [Figure 14] Detection of acetylcholine using stem-loop aptamers. The barcode and aptamer sequences used are shown. Exemplary measurements of carriers without and with acetylcholine are provided. A correlation between the concentration of acetylcholine and the percentage of retardation was observed. [Figure 15A-1]Detection of molecules without motor proteins. (A) Detection of increasing concentrations of thrombin using a thrombin-binding aptamer (TBA). The shaded area indicates the signal of the TBA. Events with signal at lower nA indicate TBA-bound thrombin. [Figure 15A-2] This is a continuation of Figure 15A-1. [Figure 15B] Detection of molecules without motor proteins. (B) Detection of increasing serotonin concentrations using serotonin-binding aptamers (SBAs). The shaded areas relate to the signal observed for SBA alone versus SBA-bound serotonin. [Figure 15C-1] Detection of molecules without motor proteins. (C) Detection of barcodes in multiplexed samples. Barcodes are distinguished based on the amplitude of the signal in the barcode region. [Figure 15C-2] This is a continuation of Figure 15C-1. [Figure 16] Example of a multiplexed screening and detection strategy: A population of carriers can be used to generate a biological passport for screening and diagnosis. [Figure 17-1] Carrier strand sequence-miRNA. [Figure 17-2] This is a continuation of Figure 17-1. [Figure 18] Carrier chain sequences - proteins and neurotransmitters. [Figure 19] Confusion matrix for barcode classification. [Figure 20A] Detection of multiple miRNAs. A. Increased translocation time of barcoded samples with 10 nM miRNA (barcode 13) compared to control (0 nM). [Figure 20B] Detection of multiple miRNAs. B. (Top) Characteristic current traces of barcoded events with associated migration standard deviation plots. Events were classified as delayed if the migration standard deviation was below a certain threshold (0.003). (Bottom) Characteristic current traces of delayed events with associated migration standard deviation plots. [Figure 20C] Detection of multiple miRNAs. C. Single barcode (barcode 38) titration curve (n=5). [Figure 20D] Detection of multiple miRNAs. D. Multiplexed titration curves (n=5) of 40 different barcodes with increasing concentrations of (each) miRNA in the same sample. [Figure 20E] Detection of multiple miRNAs. E. Box plots (n=1, dots) showing the delay detected for 10 nM miRNA added in a multiplex experiment (n=5), overlaid with scatter plots of individual experiments for each barcode where 10 nM miRNA was added. [Figure 21A] Quantification of unknown miRNA concentrations in multiplexed experiments. (A) Titration curves for 40 barcode multiplexed experiments were plotted individually. Each curve was fitted with a Hill fit function. True concentrations of spiked miRNA (dark grey) and predicted concentrations of spiked miRNA determined based on the standard curve (light grey). The results show a very high overlap between predicted and actual concentrations. [Figure 21B] Quantification of unknown miRNA concentrations in a multiplexed experiment. (B) Residual analysis showing predicted minus actual values ​​(n=12). [Figure 22A] Detection of cTnI. A: cTnI aptamer sequence. [Figure 22B] Detection of cTnI. B: Comparison of time to event + / - 30ng / ml cTnI [Figure 22C] Detection of cTnI. C: Time to event to C3 peak. [Figure 22D] Detection of cTnI. D: Event time from C3 peak to end. Concentration-delay relationship of cTnI, events are "delayed" with t>95%ile of control events. [Figure 22E] Detection of cTnI. E: Total event time. [Figure 22F] Detection of cTnI. F: Time to event to C3 peak. [Figure 22G] Detection of cTnI. G: Event time from C3 peak to end. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] Molecular Carriers and Multiplex Methods The present disclosure relates to a method for detecting multiple molecules in a sample. The method includes contacting the sample with a carrier, the carrier comprising an identifier region associated with a molecular binding region specific for the molecule to be detected. When the carrier moves through a detector such as a pore under the control of a motor protein, the identifier region is characterized and it is determined whether the molecule is bound to the molecular binding region. Thus, the method identifies the presence or absence of the molecule to be detected from the characterization of the associated identifier region and the determination of whether the molecule is bound to the molecular binding region. Multiple molecules in the sample can be detected. As described in more detail below, the method allows for the correct detection and identification of multiple molecules in the sample.

[0010] As explained above, methods for identifying molecules in a sample are known in the art. One method known in the art includes a probe that includes an aptamer and a tail (see WO2013 / 121201). Different probes are provided with different aptamers and different tails, and each tail can have a different effect on the current flowing through the pore depending on whether the analyte is bound to the aptamer. In this way, the method can detect multiple analytes in a sample. The presence or absence of an analyte bound to an aptamer is detected by the stalling of the movement of the tail through the pore when the analyte is bound to the aptamer, compared to a probe in which the analyte is not bound to the aptamer.

[0011] However, the prior art methods are limited by the variety of probes and the number of different tails that can be generated. Different analytes are simply distinguished from each other by varying the length of the probe, the presence or absence of a double-stranded region in the tail of the probe, and the different binding affinities of different aptamers on each carrier. These limitations limit the number of different analytes in a sample that can be distinguished from each other.

[0012] The inventors have devised a method to distinguish between a large number of different analytes and / or carriers in a sample. The inventors have devised a carrier that includes a motor protein located on the carrier so that the movement of the molecule-specific identifier region in the pore can be controlled. The controlled movement allows for accurate characterization of the identifier region, for example by sequencing. The difference between the identifier regions in a carrier that includes a motor protein designed to detect different molecules can be much smaller if the movement of the carrier through the pore is controlled in this way. Thus, the carrier devised by the inventors allows for the label-free identification of a large number of different identifier regions. Thus, a highly multiplexed method for identifying multiple analytes in a single sample can be implemented using the carrier. By including alternative identifier regions of the carrier in contact with different samples, the method of the present invention also allows for the simultaneous measurement of multiple analytes from multiple samples.

[0013] The improved methods disclosed herein perform well in detecting very low levels of analytes in a sample (i.e., have high sensitivity). The methods disclosed herein allow for efficient detection and screening of, for example, rare proteins and miRNA molecules using ultra-dilute samples (sub-picomolar levels) and can achieve single molecule sensitivity in a high-throughput manner. The methods disclosed herein can be used to detect molecules, such as proteins or polynucleotides, such as miRNA, that are present in a sample at concentrations as low as about 1 pM to about 1 fM. Standard aptamer-based methods of detecting molecules require higher concentrations of analyte to generate a detectable signal. The methods disclosed herein allow for detection at the single molecule level and can be used to determine the concentration of a molecule in a sample. Specific binding of a molecule to a molecule-binding region of a carrier allows for an increase in the effective concentration of the molecule, for example, by localization to a membrane containing a pore via a membrane anchor on the carrier. The carriers disclosed herein also provide the advantage of allowing multiplex detection of molecules in unprocessed samples, such as natural clinical samples.

[0014] Accordingly, provided herein is a method for detecting a plurality of molecules in a sample, the method comprising: (a) contacting a sample with a support and a nanopore, the support comprising a single-stranded leader, an identifier region, and a molecular binding region specific for a molecule to be detected, the motor protein being bound to the support such that it can control movement of the identifier region within the nanopore; (b) performing one or more optical or electrical measurements as the carrier moves through the nanopore to characterize the identifier region and determine whether a molecule is bound to the molecule binding region.

[0015] Characterization of the Identifier Domain The carrier comprises one or more identifier regions. The method requires that the carrier interacts with a detector, e.g., moves within a pore, to characterize the identifier regions. Suitable measurements that can be performed to characterize the identifier regions are discussed below.

[0016] Preferably, the identifier region comprises or is a polynucleotide. Preferably, the polynucleotide sequence of the identifier region is determined. Any suitable technique may be used. The present disclosure is particularly suitable for single molecule characterization and detection of low concentrations of molecules. Exemplary suitable sequencing techniques are discussed in more detail herein. For example, in some preferred embodiments, the sequencing technique is a nanopore sensing method. Nanopore sensing methods are described in detail herein. However, the methods disclosed herein are not limited to nanopore sensing. Other single molecule sequencing techniques can follow the methods disclosed herein.

[0017] In nanopore strand sequencing, the identifier region moves within the pore. The signal recorded as the identifier region moves within the pore allows the sequence of the identifier region to be determined. Other features of the identifier region, such as (i) the length of the polynucleotide, (ii) the identity of the polynucleotide, (iii) the secondary structure of the polynucleotide, and (iv) whether the polynucleotide is modified, can alternatively or additionally be determined to characterize the identifier region.

[0018] The presence of carrier molecules in the nanopore channel affects the open channel ion flow through the pore. This is the essence of the "molecular sensing" of the pore channel. The variation of the open channel ion flow can be measured, for example, by a change in current, using a suitable measurement technique (e.g., WO2000 / 28312, and D. Stoddart et al., Proc. Natl. Acad. Sci., 2010, 106, 7702-7 or WO2009 / 077734). The degree of reduction in ion flow, measured by a reduction in current, is related to the size of the obstacle in or near the pore. Similar information can be obtained, for example, using optical methods disclosed in Huang et al., Nature Nanotechnology 10, 986-991 (2015). Thus, the binding of a molecule of interest (e.g., a target polynucleotide) in or near the pore provides a detectable and measurable event, thereby forming the basis of a "biological sensor".

[0019] When a nucleic acid molecule or individual bases move through the pore (e.g., passing through the nanopore channel), the size difference between the bases causes a directly correlated decrease in ion flow through the channel. The fluctuations in ion flow can be recorded. Suitable electrical measurement techniques for recording the fluctuations in ion flow are described, for example, in WO2000 / 28312 and D.Stoddart et al., Proc.Natl.Acad.Sci., 2010,106,pp7702-7 (single channel recording instruments), and, for example, in WO2009 / 077734 (multi-channel recording techniques). With suitable calibration, the characteristic decrease in ion flow can be used to identify in real time the specific nucleotide and associated bases that cross the channel. In a typical nanopore nucleic acid sequencing, the ion flow of the open channel decreases when the individual nucleotides of the nucleotide sequence of interest pass through the nanopore channel in succession, due to partial blocking of the channel by the nucleotide. It is this decrease in ion flow that is measured using the suitable recording techniques described above. The reduction in ion flux can be calibrated to the reduction in ion flux measured for a known nucleotide passing through the channel, resulting in a means for determining which nucleotide passes through the channel, and thus, if performed sequentially, a method for determining the nucleotide sequence of a nucleic acid passing through the nanopore. Accurate determination of individual nucleotides requires that the reduction in ion flux through the channel typically correlates directly with the size of the individual nucleotide passing through the constriction (or "read head"). It will be appreciated that sequencing can be performed on intact nucleic acid polymers that have been "threaded" through the pore, for example, via the action of an associated motor protein, such as a polymerase or helicase. Suitable motor proteins are described in more detail herein.

[0020] Determining molecular bonds The method determines whether a molecule is bound to a molecular binding region of a carrier. In some embodiments, the method is used to detect the presence or absence of a molecule specifically bound to a carrier. The absence of a molecule specifically bound to a carrier indicates the absence of the molecule in the sample.

[0021] The method can also be used to determine the concentration of a molecule, such as the relative concentration of the molecule-bound carrier to the free carrier, or the absolute concentration of the molecule in a sample. Relative and absolute concentrations can be calculated in any suitable manner from the measurements obtained. Examples of methods for determining relative and absolute concentrations are provided in the Examples and Figures.

[0022] The interaction of the molecular binding region of the carrier with the detector is used to determine whether a molecule is bound to the molecular binding region of the carrier.

[0023] When using nanopore detection, the molecular binding region affects the current flowing through the pore depending on whether or not the molecular binding region is specifically bound to a molecule. The molecular binding region affects the current through the pore in one direction when the molecule is not bound, and affects the current through the pore in a different direction when the molecule is bound. This is important because it allows the presence or absence of a molecule specifically bound to the molecular binding region, and therefore the presence or absence of a molecule in a sample, to be determined using the method.

[0024] The signal generated by the molecule-specific identifier region on the carrier (i.e., an identifier region that is only present on the carrier, which also contains a molecule binding region specific for a given molecule of interest) is used to identify molecules that are bound or unbound to the carrier, and thus, molecules that are present or absent in the sample.

[0025] Depending on whether a molecule is bound to the molecular binding region, the effect of the molecular binding region on the current flowing through the pore can be measured based on the time it takes for the carrier, or the molecular binding region of the carrier, to move through the pore. For example, if the molecular binding region is an aptamer, the secondary and tertiary structure of the aptamer can detectably slow or temporarily stall the progression of the carrier through the pore. If the aptamer is bound to its cognate molecule, the carrier may need to overcome a higher energy barrier to progress through the pore, and the rate of progression of the aptamer through the pore may be detectably slower than when the aptamer is not bound to the molecule (such as an extension interaction).

[0026] In some embodiments, the molecular binding region may be a polynucleotide complementary to a target molecule, such as miRNA. In such embodiments, when the molecular binding region is not bound to a molecule, the carrier may proceed through the pore at a "normal" speed. When the molecular binding region is bound to a target molecule, such as miRNA, then the double-stranded section of the molecular binding region may affect the progression of the carrier through the pore such that progression is detectably delayed or stalled. In this way, the presence or absence of a molecule specifically bound to the molecular binding region of the carrier may be determined.

[0027] In some embodiments, the molecular binding region can be an antibody, an antibody fragment, a nanobody, or an affibody. The presence of such a molecular binding region can prevent the entire carrier from moving through the pore, but the presence or absence of the molecule can be determined. Without wishing to be bound by theory, such a molecular binding region of the carrier acts as a "leaky plug" to the pore. Nanopore measurements are very sensitive to small changes in the system and can distinguish between individual nucleotide bases, so differences in the "leakage" of the plug can be detected depending on whether a molecule is bound to the molecular binding region or not.

[0028] A control experiment may be carried out to determine the effect that the molecule binding region has on the current flowing through the pore and / or the progression of the carrier when the carrier is specifically bound to a molecule compared to when the molecule is not bound. Results from performing the methods described herein on a test sample may then be compared to results derived from such a control experiment to determine whether a particular molecule is present or absent in the test sample. This is described in more detail in WO2013 / 121201.

[0029] Alternative methods can be used to determine whether a molecule is bound to the molecule binding region.For example, a digestion-based method can be used that relies on the difference in digestion of the molecule binding region when the molecule is bound and when the molecule is not bound.In general, the molecule that is bound to the molecule binding region of the carrier can protect the molecule binding region from digestion.

[0030] For example, when the molecule binding region is a polynucleotide, a site-specific endonuclease such as a restriction enzyme or a single-strand nicking enzyme can be used. If the molecule is not bound, the polynucleotide molecule binding region is digested and its absence is detected as the carrier moves within the pore. If the molecule is bound, the polynucleotide molecule binding region is not digested or is simply "nicked", resulting in a slowing or stalling of the progression of the carrier within the pore, and optionally, characterization of the molecule binding region, as described above.

[0031] The digestion method can also be applied when the molecule binding region is a polynucleotide, such as an antibody, an antibody fragment, a nanobody, or an affibody. The binding of the molecule to the molecule binding region can result in protection of the protease target site, and therefore the molecule binding region is not digested. In the absence of the molecule, the molecule binding region can be digested. The difference in signal can be used to determine the presence or absence of the molecule on the carrier.

[0032] In some embodiments, the spacer described herein may be positioned on the leader sequence side of the molecule binding region of the carrier. The spacer retards or stalls the progression of the carrier in the pore, thus providing an indication of the position of the molecule binding region in the measured signal. Furthermore, the presence of the spacer also allows the retardation or stalling of the progression of the carrier in the pore to be exaggerated when the molecule is bound, thus providing a clearer signal for determining the presence or absence of the molecule on the carrier.

[0033] The method also allows the concentration of a molecule to be determined, as described in more detail in the examples.In some embodiments, the relative concentration of the carrier bound to the molecule is determined compared to the free carrier in the sample.This can be useful for determining the relative change in the molecular level between samples.In some embodiments, the absolute concentration of the molecule in the sample can be determined.This can be performed using a standard curve as a reference, as illustrated in the examples.

[0034] The method is preferably a multiplex method allowing for the simultaneous detection of multiple molecules, for example, the method can be for detecting two or more different molecules, such as 5 or more, 10 or more, 50 or more, 100 or more, for example, 200-500, 500 or more, for example, 600-1000, or at least 1000, for example, 1000-10000.

[0035] Motor proteins As one of skill in the art will appreciate, any suitable motor protein can be used in the methods and products provided herein. The motor protein can be any protein that can bind to a polynucleotide and control its movement relative to a detector, such as a nanopore, for example through a pore. In some embodiments, two or more motor proteins are bound to a carrier.

[0036] In some embodiments, the motor protein is or is derived from a polynucleotide handling enzyme. A polynucleotide handling enzyme is a polypeptide capable of interacting with a polynucleotide and modifying at least one property of the polynucleotide. The enzyme can modify the polynucleotide by orienting or moving the polynucleotide to a specific location.

[0037] In some embodiments, the motor protein is derived from any member of Enzyme Classification (EC) groups 3.1.11, 3.1.13, 3.1.14, 3.1.15, 3.1.16, 3.1.21, 3.1.22, 3.1.25, 3.1.26, 3.1.27, 3.1.30, and 3.1.31.

[0038] Typically, the motor protein is a helicase, polymerase, exonuclease, topoisomerase, or a variant thereof.

[0039] The motor protein is typically stalled on the carrier when the carrier is in solution. In some embodiments, the motor protein on the carrier is modified to prevent the motor protein from detaching from the carrier (other than by translocating off the end of the spacer). The motor protein can be adapted in any suitable manner. For example, the motor protein can be loaded onto the carrier and then modified to prevent it from detaching from the spacer. Alternatively, the motor protein can be modified to prevent it from detaching from the carrier before it is loaded onto the carrier. The modification of the motor protein to prevent it from detaching from the carrier can be achieved using methods known in the art, such as those discussed in WO2014 / 013260, which is incorporated herein by reference in its entirety, with particular reference to the passage describing the modification of motor proteins such as helicases to prevent them from detaching from polynucleotide chains.

[0040] For example, a motor protein may have a polynucleotide-unbound opening, e.g., a cavity, groove, or gap, through which a polynucleotide strand can pass when the motor protein disengages from the strand. In some embodiments, the polynucleotide-unbound opening is an opening through which a spacer can pass when the motor protein disengages from the spacer. In some embodiments, the polynucleotide-unbound opening of a given motor protein can be determined by reference to its structure, e.g., by reference to its X-ray crystal structure. The X-ray crystal structure can be obtained in the presence and / or absence of a polynucleotide substrate. In some embodiments, the location of the polynucleotide-unbound opening in a given motor protein can be predicted or confirmed by molecular modeling using standard packages known in the art. In some embodiments, the polynucleotide-unbound opening can be generated transiently by movement of one or more portions of the motor protein, e.g., one or more domains.

[0041] The motor protein can be modified by closing the polynucleotide-free opening. Thus, closing the polynucleotide-free opening can prevent the motor protein from detaching from the spacer. For example, the motor protein can be modified by covalently closing the polynucleotide-free opening. In some embodiments, the preferred motor protein to address in this manner is a helicase. In one embodiment, the motor protein is an exonuclease. Suitable enzymes include, but are not limited to, exonuclease I from E. coli (SEQ ID NO: 1), exonuclease III enzyme from E. coli (SEQ ID NO: 2), RecJ from T. thermophilus (SEQ ID NO: 3) and bacteriophage lambda exonuclease (SEQ ID NO: 4), TatD exonuclease, and variants thereof. Three subunits comprising the sequence shown in SEQ ID NO: 3 or variants thereof interact to form a trimeric exonuclease.

[0042] In one embodiment, the motor protein is a polymerase. The polymerase can be PyroPhage® 3173 DNA polymerase (commercially available from Lucigen® Corporation), SD polymerase (commercially available from Bioron®), Klenow from NEB, or a variant thereof. In one embodiment, the enzyme is Phi29 DNA polymerase (SEQ ID NO: 5) or a variant thereof. Modified versions of Phi29 polymerase that can be used in the present disclosure are disclosed in U.S. Patent No. 5,576,204.

[0043] In one embodiment, the motor protein is a topoisomerase. In one embodiment, the topoisomerase is a member of any of the subclassification (EC) groups 5.99.1.2 and 5.99.1.3. The topoisomerase can be a reverse transcriptase, an enzyme capable of catalyzing the formation of cDNA from an RNA template. They are commercially available, for example, from New England Biolabs® and Invitrogen®.

[0044] In one embodiment, the motor protein is a helicase. Any suitable helicase may be used according to the methods provided herein. For example, the or each motor protein used according to the present disclosure may be independently selected from Hel308 helicase, RecD helicase, TraI helicase, TrwC helicase, XPD helicase, and Dda helicase, or variants thereof. A monomeric helicase may include several domains linked together. For example, TraI helicase and TraI subgroup helicase may contain two RecD helicase domains, one relaxase domain, and one C-terminal domain. The domains typically form a monomeric helicase that is capable of functioning without forming oligomers. Specific examples of suitable helicases include Hel308, NS3, Dda, UvrD, Rep, PcrA, Pif1, and TraI. These helicases typically act on single-stranded DNA. Examples of helicases that can move along both strands of double-stranded DNA include FtfK and hexameric enzyme complexes, or multi-subunit complexes such as RecBCD. Hel308 helicase is described in publications such as WO2013 / 057495, the entire contents of which are incorporated by reference. RecD helicase is described in publications such as WO2013 / 098562, the entire contents of which are incorporated by reference. XPD helicase is described in publications such as WO2013 / 098561, the entire contents of which are incorporated by reference. Dda helicase is described in publications such as WO2015 / 055981 and WO2016 / 055777, the entire contents of which are incorporated by reference.

[0045] In one embodiment, the helicase comprises a sequence set forth in SEQ ID NO:6 (Trwc Cba) or a variant thereof, a sequence set forth in SEQ ID NO:7 (Hel308 Mbu) or a variant thereof, or a sequence set forth in SEQ ID NO:8 (Dda) or a variant thereof. The variant may differ from the native sequence in any of the ways discussed herein. An exemplary variant of SEQ ID NO:8 includes E94C / A360C. A further exemplary variant of SEQ ID NO:8 includes E94C / A360C followed by (ΔM1)G1G2 (i.e., deletion of M1 followed by addition of G1 and G2).

[0046] In some embodiments, a motor protein (e.g., a helicase) can control the movement of a polynucleotide in at least two active modes of operation (when the motor protein is provided with all the components necessary to facilitate movement, e.g., fuel and cofactors such as ATP and Mg2+ as discussed herein) and one inactive mode of operation (when the motor protein does not have the components necessary to facilitate movement).

[0047] When equipped with all the components necessary to facilitate translocation (i.e., in active mode), the motor protein (egelicase) translocates along the polynucleotide in a 5' to 3' or 3' to 5' direction (depending on the motor protein). In embodiments where a motor protein is used to control the translocation of a polynucleotide strand relative to a nanopore, the motor protein can be used to either translocate the polynucleotide away from (e.g., out of) the pore (e.g., against an applied field) or to translocate the polynucleotide toward (e.g., into) the pore (e.g., according to an applied field). For example, when the end of the polynucleotide that the motor protein is translocating toward is captured by the pore, the motor protein acts against the direction of the field resulting from the applied potential to pull the threaded polynucleotide out of the pore (e.g., into the cis chamber). However, when the end that the motor protein is translocating away from is captured within the pore, the motor protein acts according to the direction of the field resulting from the applied potential to push the threaded polynucleotide into the pore (e.g., into the trans chamber).

[0048] When a motor protein (e.g., a helicase) does not have the necessary components to facilitate translocation (i.e., in an inactive mode), the motor protein can bind to a polynucleotide and function as a brake to slow the translocation of the polynucleotide as it translocates relative to the nanopore, for example by being drawn into the pore by a field resulting from an applied potential. In the inactive mode, it does not matter which end of the polynucleotide is captured; it is the applied field that determines the translocation of the polynucleotide relative to the pore, and the motor protein acts as a brake. When in the inactive mode, the control of polynucleotide translocation by the motor protein can be described in several ways, including ratcheting, sliding, and braking.

[0049] In the active mode, motor proteins typically consume fuel molecules. The fuel is typically a free nucleotide or a free nucleotide analog. Free nucleotides include, but are not limited to, adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), guanosine monophosphate (GMP), guanosine diphosphate (GDP), guanosine triphosphate (GTP), thymidine monophosphate (TMP), thymidine diphosphate (TDP), thymidine triphosphate (TTP), uridine monophosphate (UMP), uridine diphosphate (UDP), uridine triphosphate (UTP), cytidine monophosphate (CMP), cytidine diphosphate (CDP), cytidine triphosphate (CTP), cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), deoxyadenosine monophosphate (d ... ), deoxyadenosine diphosphate (dADP), deoxyadenosine triphosphate (dATP), deoxyguanosine monophosphate (dGMP), deoxyguanosine diphosphate (dGDP), deoxyguanosine triphosphate (dGTP), deoxythymidine monophosphate (dTMP), deoxythymidine diphosphate (dTDP), deoxythymidine triphosphate (dTTP), deoxyuridine monophosphate (dUMP), deoxyuridine diphosphate (dUDP), deoxyuridine triphosphate (dUTP), deoxycytidine monophosphate (dCMP), deoxycytidine diphosphate (dCDP), and deoxycytidine triphosphate (dCTP). The free nucleotide is typically selected from AMP, TMP, GMP, CMP, UMP, dAMP, dTMP, dGMP, or dCMP. The free nucleotide is typically adenosine triphosphate (ATP).

[0050] A cofactor for a motor protein is a factor that enables the motor protein to function. The cofactor is preferably a divalent metal cation. The divalent metal cation is preferably Mg 2+ , Mn 2+ , Ca 2+ , or Co 2+ The cofactor is most preferably Mg 2+ It is.

[0051] In the methods described herein, the motor protein is bound to a support such that it can control the movement of an identifier region within a detector, such as a transmembrane pore.

[0052] The movement of the carrier within the detector can be controlled by any suitable means. In some embodiments, the movement of the construct is driven by a physical or chemical force (electric potential). In some embodiments, the physical force is provided by an electrical (e.g., voltage) potential or a temperature gradient, etc.

[0053] In some embodiments, the detector is a nanopore and the construct moves relative to the nanopore when a potential is applied across the nanopore. Since the polynucleotide is negatively charged, when a potential is applied across the nanopore, the polynucleotide moves relative to the nanopore under the influence of the applied potential. For example, when a positive potential is applied to the trans side of the nanopore relative to the cis side of the nanopore, negatively charged analytes are induced to move from the cis side of the nanopore to the trans side of the nanopore. Similarly, when a positive potential is applied to the trans side of the nanopore relative to the cis side of the nanopore, this prevents the movement of negatively charged analytes from the trans side of the nanopore to the cis side of the nanopore. The reverse occurs when a negative potential is applied to the trans side of the nanopore relative to the cis side of the nanopore. Apparatus and methods for applying suitable voltages are described in more detail herein. In some embodiments, the chemical force is provided by a concentration (e.g., pH) gradient.

[0054] sample The sample may be any suitable sample. The sample may be a biological sample. Any of the methods described herein may be performed in vitro on a sample obtained or extracted from any organism or microorganism. The organism or microorganism is typically an archaea, prokaryote, or eukaryote, and typically belongs to one of the five kingdoms Plantae, Animalia, Fungi, Monera, and Protista. In some embodiments, the methods of the various aspects described herein may be performed in vitro on a sample obtained or extracted from any virus.

[0055] The sample is preferably a fluid sample. The sample may be a complex biological fluid. The sample typically comprises a body fluid. The body fluid may be obtained from a human or an animal. The human or animal may have, be suspected of having, or be at risk for a disease. The sample may be urine, lymph, saliva, mucus, semen, cerebrospinal fluid or amniotic fluid, whole blood, plasma, or serum. Typically the sample is from a human, but may alternatively be from another mammal, for example from a commercially farmed animal such as a horse, cow, sheep or pig, or a pet such as a cat or dog.

[0056] Alternatively, plant-derived samples are typically obtained from commercially available crops such as cereals, legumes, fruits, or vegetables, e.g., wheat, barley, oats, rapeseed, corn, soybean, rice, banana, apple, tomato, potato, grapes, tobacco, beans, lentils, sugar cane, cocoa, cotton, tea, or coffee.

[0057] The sample may be a non-biological sample. The non-biological sample is preferably a fluid sample. Examples of non-biological samples include surgical fluids, water, such as drinking water, sea water, or river water, and reagents for clinical testing.

[0058] The sample may typically be processed before being assayed, for example by centrifugation or by passage through a membrane that filters out unwanted molecules or cells, such as red blood cells. The sample may be measured immediately after collection. The sample may also typically be stored, preferably below -70°C, prior to assay.

[0059] In some embodiments, the sample may include genomic DNA. The genomic DNA may be fragmented, or any method described herein may further include fragmenting the genomic DNA. The DNA may be fragmented by any suitable method. For example, methods for fragmenting DNA are known in the art. Such methods may use a transposase, such as MuA transposase, or commercially available G-tube.

[0060] The methods disclosed herein can be used to detect one or more molecules from one or more samples and determine in which samples the molecules are detected using a single assay. This can be achieved when a carrier is used that includes both an identifier region associated with a specific molecule binding region and an identifier region associated with a specific sample. The one or more samples can be two or more, such as at least 3, 4, 5, 10, 20, 50, or 100 samples. The samples can be taken, for example, from different patients, different types of tissues within a patient, or at different times. The different times can be separated by seconds, minutes, days, months, or years. The samples can include one or more control samples.

[0061] Samples can be examined with no or minimal sample preparation, or samples can be treated, for example, to remove impurities or to enrich for the type of molecule being detected, prior to use in the method. The ability to use untreated or minimally treated samples provides for rapid turnover from sample collection to analysis.

[0062] molecule The carriers described herein contain molecular binding regions specific to the molecules to be detected. The methods disclosed herein are for detecting a plurality of molecules. The term "molecule" as provided herein may be used interchangeably with the term "analyte."

[0063] The molecule may be any molecule that can be specifically bound by the molecular binding region. For example, the molecule may be a metal ion, an inorganic salt, a polymer, an amino acid, a peptide, a polypeptide, a protein, a nucleotide, an oligonucleotide, a polynucleotide, a dye, a bleach, a pharmaceutical, a diagnostic agent, a recreational drug, an explosive, and / or an environmental pollutant. The molecule may be a biomarker. The method may include detecting two or more molecules of the same type, such as two or more proteins, two or more nucleotides, or two or more pharmaceuticals. The method may include detecting two or more different types of molecules, such as one or more proteins, one or more nucleotides, and one or more pharmaceuticals.

[0064] The molecule may be secreted from the cell, or alternatively, the molecule may be present intracellularly such that it needs to be extracted from the cell before the method can be carried out.

[0065] In one embodiment, the molecule is selected from an amino acid, a peptide, a polypeptide, a protein, a nucleotide, an oligonucleotide, and / or a polynucleotide.

[0066] In one embodiment, the molecule is selected from an amino acid, a peptide, a polypeptide, and / or a protein. The amino acid, peptide, polypeptide, or protein may be naturally occurring or non-naturally occurring. The polypeptide or protein may include synthetic or modified amino acids therein. Several different types of modifications to amino acids are known in the art. Suitable amino acids and their modifications are discussed below with respect to the transmembrane pore. For the purposes of this disclosure, it is understood that the molecule may be modified by any method available in the art.

[0067] The protein may be a growth regulatory protein such as an enzyme, an antibody, a hormone, a growth factor, or a cytokine. The cytokine may be selected from interleukins such as IL-1, IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, and IL-13, interferons such as IFN-γ, and other cytokines such as TNF-α. The protein may be a bacterial protein, a fungal protein, a viral protein, or a parasite-derived protein.

[0068] In one embodiment, the molecule is selected from a nucleotide, an oligonucleotide, and / or a polynucleotide. A nucleotide typically contains a nucleobase, a sugar, and at least one phosphate group. A nucleobase is typically heterocyclic. Nucleobases include, but are not limited to, purines and pyrimidines, more specifically adenine, guanine, thymine, uracil, and cytosine. A sugar is typically a pentose sugar. Nucleotide sugars include, but are not limited to, ribose and deoxyribose. A nucleotide is typically a ribonucleotide or a deoxyribonucleotide. A nucleotide typically contains a monophosphate, a diphosphate, or a triphosphate. The phosphate can be attached to the 5' or 3' side of the nucleotide.

[0069] Nucleotides include adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), guanosine monophosphate (GMP), guanosine diphosphate (GDP), guanosine triphosphate (GTP), thymidine monophosphate (TMP), thymidine diphosphate (TDP), thymidine triphosphate (TTP), uridine monophosphate (UMP), uridine diphosphate (UDP), uridine triphosphate (UTP), cytidine monophosphate (CMP), cytidine diphosphate (TCP), cytidine triphosphate (TCP), cytidine monophosphate (TCP), cytidine di ... adenosine monophosphate (CDP), cytidine triphosphate (CTP), 5-methylcytidine monophosphate, 5-methylcytidine diphosphate, 5-methylcytidine triphosphate, 5-hydroxymethylcytidine monophosphate, 5-hydroxymethylcytidine diphosphate, 5-hydroxymethylcytidine triphosphate, cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), deoxyadenosine monophosphate (dAMP), deoxyadenosine diphosphate (dADP), deoxyadenosine deoxyguanosine triphosphate (dATP), deoxyguanosine monophosphate (dGMP), deoxyguanosine diphosphate (dGDP), deoxyguanosine triphosphate (dGTP), deoxythymidine monophosphate (dTMP), deoxythymidine diphosphate (dTDP), deoxythymidine triphosphate (dTTP), deoxyuridine monophosphate (dUMP), deoxyuridine diphosphate (dUDP), deoxyuridine triphosphate (dUTP), deoxycytidine monophosphate (dCMP), Nucleotides include, but are not limited to, deoxycytidine diphosphate (dCDP), deoxycytidine triphosphate (dCTP), 5-methyl-2'-deoxycytidine monophosphate, 5-methyl-2'-deoxycytidine diphosphate, 5-methyl-2'-deoxycytidine triphosphate, 5-hydroxymethyl-2'-deoxycytidine monophosphate, 5-hydroxymethyl-2'-deoxycytidine diphosphate, and 5-hydroxymethyl-2'-deoxycytidine triphosphate. The nucleotide is preferably selected from AMP, TMP, GMP, UMP, dAMP, dTMP, dGMP, or dCMP. The nucleotide may be abasic (i.e., lacking a nucleobase). The nucleotide may contain additional modifications.In particular, suitable modified nucleotides include, but are not limited to, 2'-aminopyrimidines (such as 2'-aminocytidine and 2'-aminouridine), 2'-hydroxyl purines (such as 2'-fluoropyrimidines (such as 2'-fluorocytidine and 2'fluorouridine), hydroxylpyrimidines (such as 5'-α-P-boranouridine), 2'-O-methyl nucleotides (such as 2'-O-methyladenosine, 2'-O-methylguanosine, 2'-O-methylcytidine, and 2'-O-methyluridine), 4'-thiopyrimidines (such as 4'-thiouridine and 4'-thiocytidine), and nucleotides having nucleobase modifications (such as 5-pentynyl-2'-deoxyuridine, 5-(3-aminopropyl)-uridine, and 1,6-diaminohexyl-N-5-carbamoylmethyluridine).

[0070] Oligonucleotides are typically short nucleotide polymers having 50 or fewer nucleotides, such as 40 or fewer, 30 or fewer, 20 or fewer, 10 or fewer, or 5 or fewer nucleotides. Oligonucleotides can include any of the nucleotides discussed above, including abasic and modified nucleotides.

[0071] A polynucleotide may be single-stranded or double-stranded. At least a portion of the polynucleotide may be double-stranded. A polynucleotide may be a nucleic acid, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). A polynucleotide may comprise one strand of RNA hybridized to one strand of DNA. A polynucleotide may be any synthetic nucleic acid known in the art, such as peptide nucleic acid (PNA), glycerol nucleic acid (GNA), threose nucleic acid (TNA), locked nucleic acid (LNA), or other synthetic polymers with nucleotide side chains. A polynucleotide may comprise any of the nucleotides discussed above, including modified nucleotides.

[0072] A polynucleotide can be of any length. For example, a polynucleotide can be at least 10, at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, or at least 500 nucleotides or nucleotide pairs in length. A polynucleotide can be 1000 or more nucleotides or nucleotide pairs in length, 5000 or more nucleotides or nucleotide pairs in length, or 100000 or more nucleotides or nucleotide pairs in length.

[0073] In one embodiment, the molecule is a microRNA (miRNA). miRNAs are single-stranded RNA polynucleotide molecules that play a role in the post-transcriptional regulation of gene expression.

[0074] The molecules may be associated with a particular phenotype or a particular type of cell. For example, the molecules may be indicative of bacterial cells. The molecules may be indicative of viruses, fungi, or parasites. These molecules may be specific panels of recreational drugs (such as the SAMHSA 5 panel test), explosives, or environmental contaminants.

[0075] In one embodiment, the molecule is a biomarker that can be used to diagnose or prognose a disease or condition. The biomarker can be any of the above molecules, such as a protein or a polynucleotide. Suitable panels of biomarkers are known in the art, for example, as described in Edwards, AV Get al. (2008) Mol. Cell. Proteomics 7, p1824-1837; Jacquet, S. et al. (2009), Mol. Cell. Proteomics 8, p2687-2699; Anderson N Let al. (2010) Clin. Chem. 56, 177-185. The disease or condition is preferably cancer, coronary heart disease, cardiovascular disease, or sepsis.

[0076] In one embodiment, the molecule is a neurotransmitter. A neurotransmitter is a molecule that transmits signals across a synapse between cells. Examples of neurotransmitters include acetylcholine, dopamine, epinephrine, norepinephrine, nucleotides such as ATP, amino acids such as glutamate, aspartate, and δ-aminobutyric acid, and enkephalins.

[0077] Leader sequence The carrier of the present disclosure comprises a single-stranded leader sequence. The leader sequence typically comprises a polynucleotide, e.g., a polymer such as DNA or RNA, a modified polynucleotide (such as abasic DNA), PNA, LNA, polyethylene glycol (PEG), or a polypeptide. In some embodiments, the leader sequence comprises a single strand of DNA, such as a poly-dT section. The leader sequence can be of any length, but is typically between 10 and 150 nucleotides in length, such as between 20 and 120, 30 and 100, 40 and 80, or 50 and 70 nucleotides in length.

[0078] Identifier Area The carrier of the present disclosure comprises an identifier region. The carrier may comprise two or more identifier regions, such as two or more, three, four, five or more, for example, about ten or more identifier regions. In some embodiments, different identifier regions on the carrier may be associated with different molecular binding regions such that the identity of the associated molecular binding region can be determined when the identifier region passes through a detector. Thus, the carrier may comprise a series of identifier regions and molecular binding regions. The carrier is arranged such that the movement of the identifier region in the detector is controlled by a motor protein bound to the carrier.

[0079] In some embodiments, the presence of two or more identifier regions on a carrier can be used to distinguish the carrier from different samples. In some embodiments, a carrier comprising two or more identifier regions can be used in a method for detecting multiple molecules in multiple samples, and thus the carrier can comprise, for example, one identifier region unique to the sample and one identifier region unique to the molecule to which the carrier binds. The identifier region of the carrier is positioned such that when the carrier contacts the transmembrane pore, the motor protein bound to the carrier controls the movement of the identifier region within the transmembrane pore.

[0080] The purpose of the identifier region is to serve as a unique signal of the identity of the molecule bound to the carrier. A further identifier region may serve as a unique signal of the source of the carrier, for example to identify the sample with which the carrier was contacted. In some embodiments, the identifier region is a polynucleotide or comprises a polynucleotide sequence. The nucleotide may be any of those discussed below. The identifier polynucleotides can be from 2 to 300 nucleotides in length, such as from 2 to 200, 2 to 100, 2 to 75, 2 to 50, 2 to 40, 2 to 30, 2 to 25, 4 to 100, 4 to 75, 4 to 50, 4 to 40, 4 to 30, 4 to 25, 4 to 20, 4 to 15, 4 to 10, 6 to 100, 6 to 75, 6 to 50, 6 to 40, 6 to 30, 6 to 25, 6 to 20, 6 to 15, 6 to 10, 8 to 100, 8 to 75, 8 to 50, 8 to 40, 8 to 30, 8 to 25, 8 to 20, or 8 to 15 nucleotides in length.

[0081] In some embodiments, the molecule binding region or a portion thereof is an identifier region. For example, the identifier region may overlap with the molecule binding region. In such embodiments, the molecule binding region and the identifier region are preferably polynucleotides. When the molecule binding region is a polynucleotide, such as an aptamer or a polynucleotide that hybridizes to a target polynucleotide, the polynucleotide sequence of the molecule binding region is unique to the bound molecule and thus serves to identify the molecule. In some embodiments, the identifier region and the molecule binding region do not overlap.

[0082] In some embodiments, the identifier region comprises a barcode sequence. Polynucleotide barcodes are well known in the art (Kozarewa, I. et al, (2011), Methods Mol. Biol. 733, p279-298). A barcode is a specific sequence of a polynucleotide that affects the current flowing through the pore in a specific and known manner. Barcode sequences are typically 2 nucleotides or more in length, such as 4 nucleotides or more, 8 nucleotides or more, or 12 nucleotides or more in length. In some embodiments, the barcode sequence is 2-50 nucleotides in length, such as 2-45, 2-40, 2-35, 2-30, 2-25, 4-50, 4-45, 4-40, 4-35, 4-30, 4-25, 4-20, 4-15, 4-10, 6-50, 6-45, 6-40, 6-35, 6-30, 6-25, 6-20, 6-15, 6-10, 8-50, 8-45, 8-40, 8-35, 8-30, 8-25, 8-20, or 8-15 nucleotides in length. In some embodiments, the barcode sequence can be 10-50, 10-45, 10-40, 10-35, 10-30, 10-25, or 10-20 nucleotides in length. In some embodiments, the barcode sequence can be 15-50, 15-45, 15-40, 15-35, 15-30, or 15-25 nucleotides in length. In some embodiments, the barcode sequence can be 20-50, 20-45, 20-40, 20-35, or 20-30 nucleotides in length. In some embodiments, the barcode sequence can be 25-50, 25-45, 25-40, or 25-35 nucleotides in length. In some embodiments, the barcode sequence can be about 10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides in length.

[0083] The longer the length of the barcode, the greater the number of unique combinations that can be used. To increase the accuracy of barcode sequencing, the barcode may be repeated in the carrier, allowing the barcode to be proofread. Thus, the identifier region may contain two or more copies of the barcode, such as 3 to 10, e.g., 3 or more, 4 or more, 5 or more, or 6 or more copies.

[0084] Barcoding allows for highlight multiplexed detection. For example, a four-base barcode allows for up to 256 protein or miRNA targets (4 4 = 256) unique configurations. By increasing the number of bases, e.g., 4 8 , 4 12 For example, using an 8-base sequence, 65,536 unique barcodes can be generated. This is a major advance in sensing and diagnostics in general, where typically only one or a handful of molecules, such as a maximum of about five or about ten molecules, can be selectively probed at any one time.

[0085] In some embodiments, the identifier region may comprise a spacer or a series of spacers as described herein. A series of spacers may comprise two or more, e.g., three or more, four or more, five or more, or six or more, seven or more, eight or more, nine or more, or ten or more spacers. A series of spacers may comprise 20 or more, 50 or more, or 100 or more spacers. A series of spacers may comprise 2-1000 spacers, such as 2-100, 2-50, 2-20, or 2-10. The spacers in a series of spacers may be the same or different. When the identifier region moves within the pore, a characteristic signal of the spacer may be measured. The type and number of spacers in different carrier molecules may be distinguished based on the measured signal. The spacer may be any of the spacers described herein, e.g., iSp9 and iSp18 spacers.

[0086] molecular binding region The carrier of the present disclosure comprises a molecular binding region specific to the molecule to be detected. In some embodiments, the carrier may comprise two or more molecular binding regions. The carrier may comprise one or more molecular binding regions of the same type and / or may comprise two or more different molecular binding regions. The two or more molecular binding regions may be three or more molecular binding regions, such as four, five, six or more, for example, about ten. Different molecular binding regions in a carrier typically bind specifically to different molecules. This allows the detection of multiple molecules (analytes) using a single carrier.

[0087] On the carrier, an identifier region may be associated with each molecular binding region. Typically, an identifier region is positioned such that its associated molecular binding region passes through a detector, such as a pore, before interacting with the detector.

[0088] In the carrier, one identifier region may be associated with one or more molecular binding regions, such as two or more, three or more, for example, 4 to 10 molecular binding regions. In this situation, the two or more molecular binding regions typically bind to the same molecule. The two or more molecular binding regions may be specific for different molecules. The two or more molecular binding regions may be separated by a spacer or a series of spacers, such as those defined herein. The spacer may separate the molecular binding region from the associated identifier region.

[0089] Any molecular binding region may be used, provided that it specifically binds to the molecule of interest, such that when the carrier is contacted with the pore, the presence or absence of the molecule bound to the molecular binding region may be determined. For example, the molecular binding region may be an aptamer; a complementary DNA sequence; a peptide or protein, such as an antibody, an antibody fragment, a nanobody, or an affibody; a click chemistry group; biotin or streptavidin, etc.

[0090] In one embodiment, the molecule binding region is an aptamer. An aptamer is a small molecule that binds to one or more molecules. Suitable aptamers and methods for making aptamers are known in the art and are provided, for example, in WO2013 / 121201, which is incorporated herein by reference. Aptamers can be generated using SELEX (Stoltenburg, R. et al., (2007), Biomolecular Engineering 24, p381-403; Tuerk, C. et al., Science 249, p505-510; Bock, LC et al., (1992), Nature 355, p564-566) or NON-SELEX (Berezovski, M. et al. (2006), Journal of the American Chemical Society 128, p1410-1411). The aptamer can be a peptide aptamer or an oligonucleotide aptamer. In one embodiment, the adapter is a peptide aptamer. The peptide aptamer can include any amino acid. The amino acid can be any of those discussed below. In one embodiment, the aptamer is an oligonucleotide aptamer. The oligonucleotide aptamer can include any nucleotide. The nucleotide can be any of those discussed above. The aptamer can be of any length. Aptamers are typically at least 15 amino acids or nucleotides in length, such as about 15 to about 50, about 20 to about 40, or about 25 to about 30 amino acids or nucleotides in length.

[0091] In one embodiment, the molecule binding region is a polynucleotide. In one embodiment, the polynucleotide is an aptamer. In one embodiment, the polynucleotide comprises a sequence complementary to the polynucleotide molecule (target polynucleotide) to be detected. The target polynucleotide can be of any length. For example, the target polynucleotide can be at least 10, at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, or at least 500 nucleotides or nucleotide pairs long. The target polynucleotide can be an oligonucleotide. Oligonucleotides are short nucleotide polymers that typically have 50 or fewer nucleotides, such as 40 or fewer, 30 or fewer, 20 or fewer, 10 or fewer, or 5 or fewer nucleotides. The molecule binding region is preferably complementary to an miRNA. miRNAs are short non-coding RNAs that have a role in post-transcriptional gene regulation and are usually 21-23 nucleotides long, but can be 18-30 nucleotides long, such as 20-25 nucleotides long.

[0092] When the molecule to be detected is a polynucleotide, the molecule binding region may comprise a sequence that is 90% or more identical, such as at least 97%, 98%, or 99%, to the complement of the target polynucleotide. In such a molecule binding region, one or more, for example 2, 3, 4, or 5 nucleotides in the complement may be replaced with non-canonical nucleotides that can base pair with the corresponding nucleotides in the target polypeptide. Preferably, the molecule binding region comprises the complement of the target polynucleotide.

[0093] In one embodiment, the molecular binding region is an antibody, an antibody fragment, a nanobody, or an affibody. The term "antibody" as used herein can refer to a whole antibody (comprising two heavy chains and two light chains), as well as antigen-binding fragments thereof. Antibodies can include, but are not limited to, polyclonal, monoclonal, chimeric, dAb (domain antibody), single chain, Fab, Fab' and F(ab')2 fragments, and scFv. Nanobodies are V H H fragment or V NARAffibodies are single domain antibodies such as fragments. Affibodies are antibody mimics that contain a three helix scaffold domain with amino acid substitutions on two of the three helices, allowing for a large diversity of amino acid sequences and potential antigen binding. Affibodies are discussed in Frejd, Fredrik Y., and Kyu-Tae Kim. (Experimental & molecular medicine 49.3(2017):e306-e306) and Lolom, John, et al. (FEBS letters 584.12(2010): 2670-2680). Suitable antibodies, antibody fragments, nanobodies, and affibodies are known in the art or can be prepared by standard methods.

[0094] Methods for conjugating polypeptides are well known in the art. For example, site-specific C-terminal, N-terminal, or internal loop labeling of proteins using sortase-mediated reactions can be used, as described in Guimaraes et al. Nature protocols 8.9 (2013): 1787, Theile et al. Nature protocols 8.9 (2013): 1800, and Koussa et al. Methods 67.2 (2014): 134-141, all of which are incorporated herein by reference. Those skilled in the art can utilize suitable techniques to conjugate proteins such as antibodies to carriers.

[0095] The molecular binding region specific for the molecule to be detected can bind its intended target molecule (the molecule it is intended to detect) with higher affinity than it binds to an unrelated molecule. If the molecule to be detected is a protein, the unrelated molecule can be an unrelated control protein, such as bovine serum albumin. If the molecule to be detected is a polynucleotide, the unrelated molecule can be a scrambled control polynucleotide (e.g., a random polynucleotide sequence having the same number and type of nucleotides as the intended target molecule). The molecule to be detected preferably binds to the molecular binding region with an affinity at least 10, at least 50, at least 100, at least 500, or at least 1000 times greater than the control. The affinity can be determined by methods known in the art. For example, the affinity can be determined by ELISA assays, biolayer interferometry, surface plasmon resonance, kinetic methods, or equilibrium / solution methods. Those skilled in the art will recognize which molecules specifically bind to the molecular binding region.

[0096] Some cross-reactivity may occur, for example, with miRNA polynucleotides having a similar sequence to the intended target miRNA molecule, or with proteins that share closely related domains. Preferably, a molecule-binding region binds to its target molecule with a higher affinity, e.g., at least 10, at least 50, at least 100, at least 500, or at least 1000 times greater affinity, than a related molecule, such as a related polynucleotide, e.g., a miRNA polynucleotide having a similar sequence, or a related protein, such as a homologue.

[0097] Spacer In some embodiments of the methods provided herein, the carrier comprises a spacer. The spacer is preferably positioned between the bound motor protein and the molecule binding region. When the motor protein interacts with the spacer, the movement of the carrier in the pore is stalled (or, in other words, slowed or delayed). The spacer is more preferably positioned directly adjacent to the molecule binding region. When the carrier moves in the detector, the movement of the motor protein is stalled by the spacer before the molecule binding region interacts with the pore. When the motor protein is stalled at the spacer, an exaggerated optical or electrical signal may be generated when a molecule is bound to the molecule binding region, compared to a similar carrier without a spacer. When the identifier region is separated from the molecule binding region, the spacer is preferably positioned on the carrier between the identifier region and the molecule binding region.

[0098] When the carrier moves through the detector, e.g., when the carrier moves relative to the nanopore, a unique electrical or optical signal is generated when the motor protein encounters the spacer. For example, a spacer positioned between the bound motor protein and the molecular binding region can function as a unique signal that allows the signal generated when the molecular binding region interacts with the detector to be clearly identified, e.g., the signal / trace / irregular curve generated when the carrier moves within the nanopore. Thus, the spacer can be used as a marker to position the signal generated when the molecular binding region moves within the detector, facilitating the determination of the presence or absence of a molecule specifically bound to the molecular binding region.

[0099] The spacer may provide an energy barrier that impedes the movement of the motor protein. For example, the spacer may stall the motor protein by reducing the traction of the motor protein on the polynucleotide. This may be accomplished, for example, by using an abasic spacer, i.e., a spacer in which a base has been removed from one or more nucleotides in the carrier.

[0100] The spacer can physically block the translocation of the motor protein, for example by introducing bulky chemical groups that physically impede the translocation of the motor protein. The spacer can be a double-stranded region of a polynucleotide.

[0101] The spacer may typically comprise a linear molecule such as a polymer. Typically, the linear spacer has a structure different from that of the target polynucleotide. For example, when the target polynucleotide is DNA, the or each spacer typically does not comprise DNA. In particular, when the target polynucleotide is deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), the or each spacer preferably comprises peptide nucleic acid (PNA), glycerol nucleic acid (GNA), threose nucleic acid (TNA), locked nucleic acid (LNA), or a synthetic polymer with nucleotide side chains. In some embodiments, the spacer is one or more nitroindoles, one or more inosines, one or more acridines, one or more 2-aminopurines, one or more 2-6-diaminopurines, one or more 5-bromo-deoxyuridines, one or more inverted thymidines (inverted dT), one or more inverted dideoxy-thymidines (ddT), one or more dideoxy-cytidines (ddC), one or more 5-methylcytidines, one or more 5-hydroxymethylcytidines, one or more 2'-O-methyl RNA bases, one or more isopropyl ethers, one or more tert-butyl ... -deoxycytidine (Iso-dC), one or more iso-deoxyguanosine (Iso-dG), one or more C3(OC3H6OPO3) groups, one or more photocleavable (PC)[OC3H6-C(O)NHCH2-C6H3NO2-CH(CH3)OPO3] groups, one or more hexanediol groups, one or more spacer 9 (iSp9)[(OCH2CH2)3OPO3] groups, or one or more spacer 18 (iSp18)[(OCH2CH2)6OPO3] groups, or one or more thiol bonds. The spacer may include any combination of these groups. Many of these groups are commercially available from IDT® (Integrated DNA Technologies®). For example, C3, iSp9, and iSp18 spacers are all available from IDT®. The spacer may include any number of the above groups as spacer units.

[0102] In some embodiments, the spacer may include one or more chemical groups that stall the motor protein. In some embodiments, suitable chemical groups are one or more pendant chemical groups. One or more chemical groups may be attached to one or more nucleobases in the carrier. One or more chemical groups may be attached to the backbone of the carrier. There may be any number of suitable chemical groups, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more. Suitable groups include, but are not limited to, fluorophores, streptavidin and / or biotin, cholesterol, methylene blue, dinitrophenol (DNPs), digoxigenin and / or antidigoxigenin, and dibenzylcyclooctyne groups. In some embodiments, the spacer may include a polymer. In some embodiments, the spacer may include a polymer that is a polypeptide or polyethylene glycol (PEG).

[0103] In some embodiments, the spacer may contain one or more abasic nucleotides (i.e., nucleotides lacking a nucleobase), such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more abasic nucleotides. The nucleobase may be replaced by -H (idSp) or -OH in the abasic nucleotide. The abasic spacer may be inserted into the target polynucleotide by removing the nucleobase from one or more adjacent nucleotides. For example, the polynucleotide may be modified to include 3-methyladenine, 7-methylguanine, 1,N6-ethenoadenine inosine, or hypoxanthine, and the nucleobase may be removed from these nucleotides using human alkyladenine DNA glycosylase (hAAG). Alternatively, the polynucleotide may be modified to include uracil, and the nucleobase removed with uracil-DNA glycosylase (UDG). In one embodiment, the one or more spacers do not include any abasic nucleotides.

[0104] One or more spacers may be present elsewhere in the carrier. The spacer may include any suitable number of spacers. For example, the carrier may include 2 or more, 3 or more, or 5 or more spacers, such as 1 to about 20 spacers, for example, 1 to about 10 spacers.

[0105] stall area In some embodiments, the carrier comprises a stall region. The stall region of the carrier provides a location where the motor protein is localized on the carrier when the carrier is in solution (i.e., before contacting and translocating within the pore). The stall region typically resides between the leader region and the identifier region. This allows the leader to interact with the detector, such as threading into the pore. It also positions the motor protein on the carrier such that it can control the movement of the identifier region through the detector, e.g., the pore, upon interaction of the carrier with the detector.

[0106] In some embodiments, the stall region is a spacer as described herein and in WO2020 / 234612. The carrier may further comprise a blocking moiety that prevents the motor protein from leaving the spacer.

[0107] A blocking moiety is typically a moiety that prevents the motor protein from moving in a direction opposite to the direction in which the motor protein naturally processes polynucleotides. For example, if the motor protein naturally processes polynucleotide strands in a 5' to 3' direction, a suitable blocking moiety may be a moiety that prevents the motor protein from moving in a 3' to 5' direction. Similarly, if the motor protein naturally processes polynucleotide strands in a 3' to 5' direction, a suitable blocking moiety may be a moiety that prevents the motor protein from moving in a 5' to 3' direction.

[0108] The blocking moiety is typically attached to the carrier to prevent the movement of the motor protein away from the spacer. The prevention of the motor protein from moving away from the spacer can be achieved by providing a steric block to physically prevent the movement of the motor protein. The prevention of the motor protein from moving away from the spacer can be achieved by using a chemical blocking moiety on or across which the motor protein cannot move. In some embodiments, the blocking moiety comprises one or more of the spacer groups discussed herein. In other embodiments, the blocking moiety may comprise a polynucleotide chain.

[0109] The carrier may also include a loading site connected to the stall region or spacer. The loading site is a site for loading the motor protein onto the polynucleotide adaptor. Suitable loading sites are described in more detail in WO2020 / 234612.

[0110] Methods for loading motor proteins onto polynucleotides and stalling motor proteins on polynucleotides in solution, suitable spacers, and suitable blocking moieties are described in more detail in WO2020 / 234612, which is incorporated herein by reference, and WO2014 / 135838, which is incorporated herein by reference.

[0111] anchor In some embodiments, the carrier comprises a membrane anchor or a transmembrane pore anchor bound to the carrier. The anchor can be bound to the carrier covalently or non-covalently. For example, the anchor can be bound to an oligonucleotide hybridized to the polynucleotide region of the carrier. The polynucleotide region of the carrier to which the anchor oligonucleotide is hybridized is different from the molecule binding region in the sense that it does not prevent the specific binding of the molecule to the molecule binding region.

[0112] In some embodiments, the anchor aids in characterizing the target polynucleotide according to the methods disclosed herein. For example, in methods that include contacting a carrier with a transmembrane pore, the membrane anchor or transmembrane pore anchor may facilitate localization of the selected carrier around the transmembrane pore. The terms anchor and tether are used interchangeably herein.

[0113] The anchor can be a polypeptide anchor and / or a hydrophobic anchor that can insert into the membrane. In one embodiment, the hydrophobic anchor is a lipid, a fatty acid, a sterol, a carbon nanotube, a polypeptide, a protein, or an amino acid, such as cholesterol, palmitate, or tocopherol. The anchor can include a thiol, biotin, or a surfactant. In one aspect, the anchor can be biotin (for binding to streptavidin), amylose (for binding to maltose binding protein or fusion protein), Ni-NTA (for binding to poly-histidine or poly-histidine tagged protein), or a peptide (such as an antigen).

[0114] In one embodiment, the anchor comprises one linker, or two, three, four or more linkers. Preferred linkers include, but are not limited to, polymers such as polynucleotides, polyethylene glycol (PEG), polysaccharides, and polypeptides. These linkers can be linear, branched, or cyclic. For example, the linker can be a cyclic polynucleotide. The adaptor can hybridize to a complementary sequence on the cyclic polynucleotide linker. One or more anchors or one or more linkers can include a moiety that can be cleaved or degraded, such as a restriction site or a photolabile group. The linker can be functionalized with a maleimide group for binding to a cysteine ​​residue in a protein. Suitable linkers are described in WO2010 / 086602.

[0115] In one embodiment, the anchor is cholesterol or a fatty acyl chain. Any fatty acyl chain having a length of 6 to 30 carbon atoms, such as hexadecanoic acid, can be used. Examples of suitable anchors and methods for attaching the anchor to the adaptor are disclosed in WO2012 / 164270 and WO2015 / 150786. The same methods can be used to attach the anchor to the carrier.

[0116] Detector Any suitable detector may be used in the methods described herein. The detector may be any detector useful in sequencing methods. For example, nanopore sequencing or single molecule real-time sequencing, e.g., sequencing by synthesis, techniques. Preferably, the detector in the methods used herein is a nanopore. Any suitable nanopore may be used in the methods described herein. In one embodiment, the nanopore is a transmembrane pore.

[0117] A transmembrane pore is a structure that traverses a membrane to some extent. It allows hydrated ions driven by an applied electric potential to flow across or within the membrane. A transmembrane pore typically traverses the entire membrane so that hydrated ions can flow from one side of the membrane to the other side of the membrane. However, a transmembrane pore does not have to traverse the membrane. It may be closed at one end. For example, a pore can be a well, gap, channel, trench, or slit in a membrane along or into which hydrated ions can flow.

[0118] Any transmembrane pore may be used in the methods provided herein. The pore may be biological or artificial. Suitable pores include, but are not limited to, protein pores, polynucleotide pores, and solid pores. The pore may be a DNA origami pore (Langecker et al., Science, 2012; 338: 932-936). Suitable DNA origami pores are disclosed in WO2013 / 083983, WO2018 / 011603, and WO2020 / 025974.

[0119] In one embodiment, the nanopore is a transmembrane protein pore. A transmembrane protein pore is a polypeptide or an assembly of polypeptides that allows hydrated ions, such as polynucleotides, to flow from one side of a membrane to the other side of the membrane. In the methods provided herein, the transmembrane protein pore can form a pore that allows hydrated ions driven by an applied potential to flow from one side of the membrane to the other side. The transmembrane protein pore preferably allows polynucleotides to flow from one side of a membrane, such as a triblock copolymer membrane, to the other side. The transmembrane protein pore allows polynucleotides to translocate through the pore.

[0120] In one embodiment, the nanopore is a transmembrane protein pore that is monomeric or oligomeric. The pore is preferably composed of several repeating subunits, such as at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or at least 16 subunits. The pore is preferably a hexameric, heptameric, octameric, or nanomeric pore. The pore may be a homo-oligomer or a hetero-oligomer.

[0121] In one embodiment, a transmembrane protein pore comprises a barrel or channel through which ions can flow. The subunits of the pore typically surround a central axis and contribute strands to a transmembrane β-barrel or channel or a transmembrane α-helical bundle or channel.

[0122] Typically, the barrel or channel of the transmembrane protein pore comprises amino acids that facilitate interaction with an analyte, such as a target polynucleotide (as described herein). These amino acids are preferably located near the constriction of the barrel or channel. The transmembrane protein pore typically comprises one or more positively charged amino acids, such as arginine, lysine, or histidine, or aromatic amino acids, such as tyrosine or tryptophan. These amino acids typically facilitate interaction between the pore and a nucleotide, polynucleotide, or nucleic acid.

[0123] In one embodiment, the nanopore is a transmembrane protein pore derived from a β-barrel pore or an α-helix bundle pore. A β-barrel pore comprises a barrel or channel formed from β-strands. Suitable β-barrel pores include, but are not limited to, β-toxins such as α-hemolysin, anthrax toxin, and leukocidin, as well as bacterial outer membrane proteins / porins such as Mycobacterium smegmatis porins (Msp), e.g., MspA, MspB, MspC, or MspD, CsgG, outer membrane porin F (OmpF), outer membrane porin G (OmpG), outer membrane phospholipase A, and Neisseria autotransporter lipoprotein (NalP), and other pores such as lysenin. An α-helix bundle pore comprises a barrel or channel formed from α-helices. Suitable α-helix bundle pores include, but are not limited to, inner membrane proteins and α-outer membrane proteins, e.g., WZA and ClyA toxins.

[0124] In one embodiment, the nanopore is a transmembrane pore derived from or based on Msp, α-hemolysin (α-HL), lysenin, CsgG, ClyA, Sp1, or the hemolytic protein Fragaceatoxin C (FraC).

[0125] In one embodiment, the nanopore is derived from CsgG, for example CsgG from E. coli strain K-12 substrain MC4100. Such pores are oligomeric and typically comprise 7, 8, 9 or 10 monomers derived from CsgG. The pore may be a homo-oligomeric pore derived from CsgG comprising identical monomers. Alternatively, the pore may be a hetero-oligomeric pore derived from CsgG comprising at least one monomer that is different from the others. Examples of suitable pores derived from CsgG are disclosed, for example, in WO2016 / 034591, WO2017 / 149316, WO2017 / 149317, WO2017 / 149318, WO2018 / 211241 and WO2019 / 002893.

[0126] In one embodiment, the nanopore is a transmembrane pore derived from lysenin. Examples of suitable pores derived from lysenin are disclosed in WO2013 / 153359.

[0127] In one embodiment, the nanopore is a transmembrane pore derived from or based on α-hemolysin (α-HL). Wild-type α-hemolysin pores are formed of seven identical monomers or subunits (i.e., are heptameric). The α-hemolysin pore may be α-hemolysin-NN or a variant thereof. The variant preferably contains N residues at positions E111 and K147.

[0128] In one embodiment, the nanopore is a transmembrane protein pore derived from an Msp, such as from MspA. An example of a suitable pore derived from MspA is disclosed in WO2012 / 107778.

[0129] In one embodiment, the nanopore is a transmembrane pore derived from or based on ClyA. Examples of suitable pores derived from ClyA are disclosed in WO2014 / 153625.

[0130] In one embodiment, the detector is a nanopipette. Nanopipettes typically have a diameter of about 10 nm, such as about 10 nm to about 12, about 15, about 18, or about 20 nm. Nanopipettes can be made from quartz capillaries, glass, and / or carbon, such glass coated with a carbon layer. Suitable nanopipettes are known in the art.

[0131] film In embodiments involving the use of a nanopore, the nanopore is typically present in a membrane, e.g., the nanopore provides a channel across and / or through the membrane. Any suitable membrane may be used, and suitable membranes are known in the art.

[0132] The membrane is preferably an amphiphilic layer. An amphiphilic layer is a layer formed from amphiphilic molecules, such as phospholipids, that have both hydrophilic and lipophilic properties. The amphiphilic molecules may be synthetic or natural. Non-natural amphiphiles and amphiphiles that form monolayers are known in the art, including, for example, block copolymers (Gonzalez-Perez et al., Langmuir, 2009, 25, 10447-10450).

[0133] The block copolymers can be diblock (consisting of two monomer subunits), but can also be constructed from more than two monomer subunits to form more complex arrangements that behave as amphiphiles. The copolymers can be triblock, tetrablock, or pentablock copolymers. The membrane is preferably a triblock copolymer membrane.

[0134] The membrane may for example be one of the membranes disclosed in WO 2014 / 064443 or WO 2014 / 064444.

[0135] The amphiphilic molecules may be chemically modified or functionalized to facilitate coupling of the anchor. The amphiphilic layer may be a monolayer or a bilayer. The amphiphilic layer is typically planar. The amphiphilic layer may be curved. The amphiphilic layer may be supported.

[0136] Amphiphilic membranes are typically mobile in nature and inherently have a molecular weight of approximately 10 -8 cms -1 The membrane behaves as a two-dimensional fluid with a lipid diffusion rate of 100 s, which means that the pores and the anchored carriers can typically move within the amphiphilic membrane.

[0137] The membrane can be a lipid bilayer. The lipid bilayer can be any lipid bilayer. Suitable lipid bilayers include, but are not limited to, planar lipid bilayers, supported bilayers or liposomes. The lipid bilayer is preferably a planar lipid bilayer. Suitable lipid bilayers are disclosed in WO2008 / 102121, WO2009 / 077734 and WO2006 / 100484.

[0138] The lipid bilayer may be formed from dry lipids as described in WO2009 / 077734. The lipid bilayer may be formed across an aperture as described in WO2009 / 077734.

[0139] Any lipid composition that forms a lipid bilayer may be used. The lipid composition is selected so that a lipid bilayer is formed with the required properties, such as surface charge, ability to support membrane proteins, packing density, or mechanical properties. The lipid composition may contain one or more different lipids. For example, the lipid composition may contain up to 100 lipids. The lipid composition preferably contains 1 to 10 lipids. The lipid composition may contain natural lipids and / or artificial lipids.

[0140] The membrane may include a solid-state layer. The solid-state layer may be formed from both organic and inorganic materials, including but not limited to microelectronic materials, insulating materials such as Si3N4, A12O3, and SiO, organic and inorganic polymers such as polyamides, plastics such as Teflon®, or elastomers such as two-component addition-cured silicone rubber, and glass. The solid-state layer may be formed from graphene. A suitable graphene layer is disclosed in WO2009 / 035647. When the membrane includes a solid-state layer, the pores are typically present in an amphiphilic membrane or layer contained within the solid-state layer, for example, in holes, wells, gaps, channels, trenches, or slits within the solid-state layer. Those skilled in the art can prepare suitable solid-state / amphiphilic hybrid systems. Suitable systems are disclosed in WO2009 / 020682 and WO2012 / 005857. Any of the above-mentioned amphiphilic membranes or layers may be used.

[0141] The methods disclosed herein can be carried out using (i) an artificial amphiphilic layer comprising a pore, (ii) an isolated naturally occurring lipid bilayer comprising a pore. The artificial amphiphilic layer is typically an artificial triblock copolymer layer. In addition to the pore, the layer may contain other transmembrane and / or intramembrane proteins, as well as other molecules. Suitable equipment and conditions are discussed below. The methods disclosed herein are typically carried out in vitro.

[0142] Characterization The methods of the present disclosure include characterizing the identifier region of the carrier, as described in more detail herein, and determining whether a molecule is bound to the molecule binding region.

[0143] The characterization and determination of whether a molecule is bound to the molecular binding region can be performed using any suitable detector system. The characterization and determination of whether a molecule is bound to the molecular binding region can be performed using any device suitable for investigating a membrane / pore system, for example, where a pore is inserted into a membrane. The method can be performed using any device suitable for transmembrane pore sensing. For example, the device can include a chamber containing an aqueous solution and a barrier separating the chamber into two compartments. The barrier can have an opening in which a membrane containing a transmembrane pore is formed. The transmembrane pore is described herein.

[0144] The characterisation method may be carried out using the apparatus described in WO2008 / 102120, WO2010 / 122293 or WO2000 / 028312.

[0145] The characterization method may include performing one or more optical or electrical measurements as the carrier moves through a detector, e.g., a nanopore. The electrical measurement may typically be measuring the flow of ionic current through the pore, by measuring the current. Possible electrical measurements include current measurements, impedance measurements, tunneling or electron tunneling measurements (Ivanov AP et al., Nano Lett. 2011 Jan 12;1 l(l):279-85), and FET measurements (WO 2005 / 124888), e.g., voltage FET measurements. In some embodiments, the signal may be electron tunneling across a solid-state nanopore or voltage FET measurements across a solid-state nanopore.

[0146] Alternatively, the flow of ions through the pore can be measured optically, as disclosed by Heron et al: J. Am. Chem. Soc. 9 Vol. 131, No. 5, 2009. A method for optical polymer sequencing using nanopores is described in WO2016 / 009180.

[0147] Thus, the device may also comprise an electrical circuit capable of applying a potential and measuring the electrical signal across the membrane and pore. The characterization method may be carried out using a patch clamp or a voltage clamp. The characterization method preferably involves the use of a voltage clamp.

[0148] The characterization method may be performed on silicon-based arrays of wells, with each array comprising 128, 256, 512, 1024, 2000, 3000, 4000, 6000, 10000, 12000, 15000 or more wells.

[0149] The characterization method may include measuring the current flowing through the pore. The method is typically carried out with a voltage applied across the membrane and the pore. The voltage used is typically between +2V and -2V, typically between -400mV and +400mV. The voltage used is preferably in a range having a lower limit selected from -400mV, -300mV, -200mV, -150mV, -100mV, -50mV, -20mV, and 0mV, and an upper limit independently selected from +10mV, +20mV, +50mV, +100mV, +150mV, +200mV, +300mV, and +400mV. The voltage used is more preferably in the range of 100mV to 240mV, most preferably in the range of 120mV to 220mV. By using an increased applied potential, it is possible to increase the discrimination between different nucleotides by the pore.

[0150] The characterization method is typically carried out in the presence of any charge carrier, such as metal salts, such as alkali metal salts, halide salts, chloride salts, such as alkali metal chloride salts. The charge carrier may include ionic liquids or organic salts, such as tetramethylammonium chloride, trimethylphenylammonium chloride, phenyltrimethylammonium chloride, or 1-ethyl-3-methylimidazolium chloride. In the exemplary device described above, the salt is present in an aqueous solution in the chamber. Typically, potassium chloride (KCl), sodium chloride (NaCl), or cesium chloride (CsCl) is used. KCl is preferred. The salt may be an alkaline earth metal salt, such as calcium chloride (CaCl2). The salt concentration may be saturated. The salt concentration may be 3M or less, typically 0.1-2.5M, 0.3-1.9M, 0.5-1.8M, 0.7-1.7M, 0.9-1.6M, or 1M-1.4M. The salt concentration is preferably between 150 mM and 1 M. The characterization method is preferably carried out using a salt concentration of at least 0.3 M, such as at least 0.4 M, at least 0.5 M, at least 0.6 M, at least 0.8 M, at least 1.0 M, at least 1.5 M, at least 2.0 M, at least 2.5 M, or at least 3.0 M. High salt concentrations provide a high signal to noise ratio, allowing currents indicative of binding / unbinding to be identified against the background of normal current fluctuations.

[0151] The characterization method is typically carried out in the presence of a buffer. In the exemplary device described above, the buffer is present in the aqueous solution in the chamber. Any suitable buffer may be used. Typically, the buffer is HEPES. Another suitable buffer is Tris-HCl buffer. The method is typically carried out at a pH of 4.0-12.0, 4.5-10.0, 5.0-9.0, 5.5-8.8, 6.0-8.7, or 7.0-8.8, or 7.5-8.5. The pH used is preferably about 7.5.

[0152] The characterization method may be carried out at 0° C. to 100° C., 15° C. to 95° C., 16° C. to 90° C., 17° C. to 85° C., 18° C. to 80° C., 19° C. to 70° C., or 20° C. to 60° C. The characterization method may typically be carried out at room temperature. The characterization method is optionally carried out at a temperature that supports enzyme function, for example, about 37° C.

[0153] Carriers, populations of carriers, kits, and systems Carriers, populations of carriers, kits, and systems are also provided herein.

[0154] The carrier of the present disclosure comprises a single-stranded leader, an identifier region, and a molecular binding region specific to the molecule to be detected, and a motor protein is bound to the carrier at a position between the single-stranded leader and the polynucleotide identifier. The leaders, identifier regions, molecular binding regions, and motor proteins described herein may be applied in any of the carrier, carrier population, kit, and system embodiments discussed. The carrier may further comprise any of the additional features described above.

[0155] A population of carriers described herein for a plurality of molecules is also provided. Different carriers in the population may comprise different identifier regions and different molecular binding regions. For example, each carrier in the population may comprise a unique identifier region and a unique molecular binding region. There may be multiple copies of each carrier in the population. In other words, the identifier region associated with the molecular binding region of a carrier may be different from the identifier region associated with all other molecular binding regions that bind different molecules in the population. In some embodiments, the identifier region of a carrier is different from the identifier region of other carriers in the population that bind different molecules.

[0156] The term "associated with" means that the identifier region and one or more molecular binding regions are present on the same carrier such that the identifier region can be used to uniquely identify one or more molecular binding regions on the same carrier. Typically, the identifier region is positioned on the carrier such that it interacts with the detector under the control of the motor protein before the molecular binding region interacts with the detector. The identifier region may be directly adjacent to the molecular binding region or may be separated by a linker. The linker is typically 2 to about 50, such as 3 to about 20, preferably about 5 to about 10 bases in length. The linker may comprise any nucleotide described herein. One or more spacers may also be positioned between the identifier region and its associated molecular binding region. The spacer may be present with or without a linker on one or both sides of the spacer.

[0157] Also provided is a kit for detecting a plurality of molecules in a sample, comprising: (i) a population of supports as described herein; and (ii) a motor protein.

[0158] There is further provided a system for detecting a plurality of molecules in a sample comprising: (i) a population of supports as described herein; (ii) a motor protein; and (iii) a transmembrane pore.

[0159] The kit or system may comprise two or more populations of carriers, as defined herein. In addition to the identifier region associated with the molecule binding region, each carrier in the population may comprise a further identifier region that is common to all carriers in the population and not present in any other population of carriers in the kit or system, i.e., the further identifier region is unique to the carrier of the population. Thus, the kit or system may comprise two or more carrier populations, such as 3, 4, 5 or more, for example 10 or more, 20 or more, or 50 or more carrier populations, and the carriers in each population may comprise a further identifier region that is unique to the carrier of that population. Such a kit or system may be used to simultaneously analyze multiple samples. The molecules present in each sample detected using the same detector and further identifier region may be used to determine which sample a given molecule is present or absent in.

[0160] definition When an indefinite or definite article is used when referring to a singular noun, e.g., "a" or "an" or "the," this includes the plural of that noun, unless otherwise specified. When the term "comprising" is used in the present specification and claims, it does not exclude other elements or steps. Furthermore, the terms first, second, third, etc. in the present specification and claims are used to distinguish between similar elements and are not necessarily used to describe a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the present invention described herein may be operated in other sequences than those described or illustrated herein. The following terms or definitions are provided solely to aid in the understanding of the present invention. Unless otherwise specifically defined herein, all terms used herein have the same meaning as they would to one skilled in the art of the present invention. Practitioners are particularly referred to Sambrook et al., Molecular Cloning: A Laboratory Manual, 4 thed., Cold Spring Harbor Press, Plainsview, New York (2012), and Ausubel et al., Current Protocols in Molecular Biology (Supplement 114), John Wiley & Sons, New York (2016). The definitions provided herein should not be construed to have a scope less than understood by a person of ordinary skill in the art.

[0161] As used herein, "about" when referring to a measurable value, such as an amount, duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and even more preferably ±0.1% from the particular value, where such variations are appropriate for practicing the disclosed methods.

[0162] As used herein, "nucleotide sequence", "DNA sequence", or "nucleic acid molecule" refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. The term refers only to the primary structure of the molecule. Thus, the term includes double- and single-stranded DNA, as well as RNA. As used herein, the term "nucleic acid" is a single- or double-stranded covalently linked nucleotide sequence in which the 3' and 5' ends on each nucleotide are linked by a phosphodiester bond. Polynucleotides can be composed of deoxyribonucleotide or ribonucleotide bases. Nucleic acids can be produced synthetically in vitro or isolated from natural sources. Nucleic acids can further include modified DNA or RNA, such as methylated DNA or RNA, or RNA that has undergone post-translational modifications, such as 5'-capping with 7-methylguanosine, 3'-processing such as truncation and polyadenylation, and splicing. Nucleic acids can also include synthetic nucleic acids (XNA), such as hexitol nucleic acid (HNA), cyclohexene nucleic acid (CeNA), threose nucleic acid (TNA), glycerol nucleic acid (GNA), locked nucleic acid (LNA) and peptide nucleic acid (PNA). The size of a nucleic acid, also referred to herein as a "polynucleotide", is typically expressed as the number of base pairs (bp) for double-stranded polynucleotides or the number of nucleotides (nt) for single-stranded polynucleotides. 1000 bp or nt is equivalent to a kilobase (kb). Polynucleotides less than about 40 nucleotides in length are typically referred to as "oligonucleotides" and can include primers for use in manipulating DNA, such as via polymerase chain reaction (PCR).

[0163] The term "amino acid" in the context of this disclosure is used in its broadest sense and is meant to include organic compounds containing an amine (NH2) functional group and a carboxyl (COOH) functional group, along with a side chain (e.g., R group) specific to each amino acid. In some embodiments, amino acid refers to a natural Lα-amino acid or residue. Commonly used one-letter and three-letter abbreviations for naturally occurring amino acids are used herein: A=Ala, C=Cys, D=Asp, E=Glu, F=Phe, G=Gly, H=His, I=Ile, K=Lys, L=Leu, M=Met, N=Asn, P=Pro, Q=Gln, R=Arg, S=Ser, T=Thr, V=Val, W=Trp, and Y=Tyr (Lehninger, AL, (1975) Biochemistry, 2d ed., pp. 71-92, Worth Publishers, New York). The general term "amino acid" further includes D-amino acids, retro-inverso amino acids, and chemically modified amino acids such as amino acid analogs, natural amino acids that are not normally incorporated into proteins, such as norleucine, and chemically synthesized compounds that have properties known in the art to be characteristic of amino acids, such as β-amino acids. For example, analogs or mimetics of phenylalanine or proline that allow the same conformational constraints of peptide compounds as natural Phe or Pro are included within the definition of amino acids. Such analogs and mimetics are referred to herein as "functional equivalents" of the respective amino acids. Other examples of amino acids are listed by Roberts and Vellaccio, The Peptides: Analysis, Synthesis, Biology, Gross and Meiehofer, eds., Vol. 5 p. 341, Academic Press, Inc., NY 1983, which are incorporated herein by reference.

[0164] The terms "polypeptide" and "peptide" are used interchangeably herein to refer to a polymer of amino acid residues, as well as variants and synthetic analogs thereof. Thus, these terms apply to amino acid polymers in which one or more amino acid residues are synthetic non-natural amino acids, such as chemical analogs of the corresponding naturally occurring amino acids, as well as to natural amino acid polymers. Polypeptides may also undergo maturation or post-translational modification processes, including, but not limited to, glycosylation, proteolytic cleavage, lipidation, signal peptide cleavage, propeptide cleavage, phosphorylation, and the like. Peptides may be produced using recombinant techniques, for example, through expression of recombinant or synthetic polynucleotides. Recombinantly produced peptides are typically substantially free of culture medium, e.g., culture medium represents less than about 20% of the volume of the protein preparation, more preferably less than about 10%, and most preferably less than about 5%.

[0165] The term "protein" is used to describe a folded polypeptide having a secondary or tertiary structure. A protein may be composed of a single polypeptide or may include multiple polypeptides that assemble to form a multimer. A multimer may be a homo- or hetero-oligomer. A protein may be a naturally occurring or wild-type protein, or a modified or non-naturally occurring protein. A protein may differ from a wild-type protein, for example, by the addition, substitution, or deletion of one or more amino acids.

[0166] A "variant" of a protein includes peptides, oligopeptides, polypeptides, proteins, and enzymes that have amino acid substitutions, deletions, and / or insertions compared to the unmodified or wild-type protein in question and have biological and functional activities similar to the unmodified protein from which they are derived. The term "amino acid identity" as used herein refers to the degree to which sequences are identical amino acid-by-amino acid across a comparison window. Thus, "percentage of sequence identity" is calculated by comparing two optimally aligned sequences across a comparison window, determining the number of positions at which identical amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) appear in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window (i.e., window size), and multiplying the result by 100 to obtain the percentage of sequence identity.

[0167] In all aspects and embodiments of the invention, a "variant" has at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% complete sequence identity with the amino acid sequence of the corresponding wild-type protein. Sequence identity may also be to a fragment or portion of a full-length polynucleotide or polypeptide. Thus, a sequence may have only 50% overall sequence identity with a full-length reference sequence, but the sequence of a particular region, domain, or subunit may share 80%, 90%, or even 99% sequence identity with the reference sequence.

[0168] The term "wild type" refers to a gene or gene product isolated from a natural source. A wild type gene is the gene most frequently observed in a population, and is therefore arbitrarily designated the "normal" or "wild type" form of the gene. In contrast, the terms "modified," "mutant," or "variant" refer to a gene or gene product that exhibits modifications in sequence (e.g., substitutions, truncations, or insertions), post-translational modifications, and / or functional properties (e.g., altered characteristics) when compared to the wild type gene or gene product. It should be noted that naturally occurring mutants can be isolated, which are identified by the fact that they have altered characteristics compared to the wild type gene or gene product. Methods for introducing or substituting natural amino acids are well known in the art. For example, methionine (M) can be substituted with arginine (R) by replacing the codon for methionine (ATG) with the codon for arginine (CGT) at the relevant position in the polynucleotide encoding the mutant monomer. Methods for introducing or substituting non-natural amino acids are also well known in the art. For example, unnatural amino acids can be introduced by including synthetic aminoacyl-tRNA in the IVTT system used to express the mutant monomers. Alternatively, they can be introduced by expressing mutant monomers in E. coli that are auxotrophic for specific amino acids in the presence of synthetic (i.e., non-naturally occurring) analogs of those specific amino acids. They can also be generated by naked ligation when the mutant monomers are generated using partial peptide synthesis. Conservative substitutions replace amino acids with other amino acids of similar chemical structure, similar chemical properties, or similar side chain volume. The amino acids introduced can have similar polarity, hydrophilicity, hydrophobicity, basic, acidic, neutral, or charge as the amino acids they replace. Alternatively, conservative substitutions may introduce another amino acid that is aromatic or aliphatic in place of an existing aromatic or aliphatic amino acid. Conservative amino acid changes are well known in the art and can be selected according to the properties of the 20 major amino acids defined in Table 1 below.If the amino acids have similar polarity, this can also be determined by reference to the hydrophobicity scale for the amino acid side chains in Table 2. [Table 1] [Table 2]

[0169] As described in more detail herein, the mutant or modified protein, monomer or peptide may be chemically modified in any manner and at any site. The mutant or modified monomer or peptide is preferably chemically modified by binding of a molecule to one or more cysteines (cysteine ​​binding), binding of a molecule to one or more lysines, binding of a molecule to one or more unnatural amino acids, enzymatic modification of an epitope, or modification of a terminal. Suitable methods for carrying out such modifications are well known in the art. The modified protein, monomer, or peptide variant may be chemically modified by binding of any molecule. For example, the modified protein, monomer, or peptide variant may be chemically modified by binding of a dye or fluorophore.

[0170] The present invention will be described with respect to certain embodiments and with reference to certain drawings, but the present invention is not limited thereto, but only by the claims. Any reference signs in the claims should not be interpreted as limiting the scope. Of course, it should be understood that not necessarily all aspects or advantages can be achieved in accordance with any particular embodiment of the present invention. Thus, for example, a person skilled in the art will recognize that the present invention can be embodied or performed in a manner that achieves or optimizes one or a group of advantages taught herein, but not necessarily achieves other aspects or advantages that may be taught or suggested herein.

[0171] The present invention, both as to its construction and method of operation, together with its features and advantages, may be best understood by reference to the following detailed description when read in conjunction with the accompanying drawings. Aspects and advantages of the present invention will become apparent from and be elucidated with reference to the embodiments described hereinafter. Throughout this specification, reference to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the appearance of the phrase "in one embodiment" or "in an embodiment" in various places throughout this specification do not necessarily all refer to the same embodiment, but may. Similarly, in describing exemplary embodiments of the present invention, it should be understood that various features of the invention may be grouped together in a single embodiment, figure, or description thereof for the purpose of streamlining the disclosure and aiding in the understanding of one or more of the various aspects of the present invention. However, this method of disclosure is not to be interpreted as reflecting an intention that the claimed invention requires more features than are expressly recited in each claim. Rather, as the following claims reflect, inventive aspects lie in less than all features of a single foregoing disclosed embodiment.

[0172] It is to be understood that "embodiments" of the present disclosure may be specifically combined together, unless the context indicates otherwise. Any specific combination of the disclosed embodiments is a further disclosed embodiment of the claimed invention (unless the context implies otherwise).

[0173] Additionally, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a "polynucleotide" includes two or more polynucleotides, reference to a "motor protein" includes two or more such proteins, reference to a "helicase" includes two or more helicases, reference to a "monomer" refers to two or more monomers, reference to a "pore" includes two or more pores, etc.

[0174] All publications, patents, and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

[0175] Although specific embodiments of the method according to the present invention, specific configurations, and materials and / or molecules have been discussed herein, it should be understood that various changes or modifications in form and details can be made without departing from the scope and spirit of the present invention. The following examples are provided to better illustrate specific embodiments and should not be considered as limiting the present application. The present application is limited only by the claims. EXAMPLES

[0176] In the examples below, the carrier was synthesized in two parts and the parts were ligated together. It is also envisioned that the entire carrier may be synthesized as a single unit without the need for ligation.

[0177] Example 1 In this example, some of the carriers designed so far are described. The sequences provided do not include a leader. A leader can be added to the sequences provided through the use of a ligation C strand (e.g., CCCAGCGGAACTAGGA), which also includes a region complementary to the 3' end of a polynucleotide containing the leader and stall region of the motor protein, as shown in Figure 2A.

[0178] The carrier sequences provided may bind to molecules including proteins (such as thrombin and the SARS-CoV-2 spike protein), neurotransmitters (such as serotonin and dopamine), and miRNA. The identifier region of each carrier is different from that of every other carrier. It is envisioned that different molecule binding regions on different carriers that bind to the same molecule may use the same barcode.

[0179] The sequence includes a 5' ligation strand, an identifier sequence or barcode (underlined), optionally a spacer region including an iSpC3 or iSp18 spacer, and a molecule binding region (italics). The molecule binding region in this example is the complementary sequence of an aptamer or miRNA. Thrombin [ka] Serotonin [ka] Dopamine [ka] SARS-CoV-2, S protein [ka] miRNA (barcodes 11 to 20) Adapter_barcode11_c-has-miR-497-5p [ka] Adapter_barcode12_c-has-miR-27b-5p [ka] Adapter_barcode13_c-has-miR-21-5p [ka] Adapter_barcode14_c-has-miR-221-5p [ka] Adapter_barcode15_c-has-miR-30d-5p [ka] Adapter_barcode16_c-has-miR-30c-5p [ka] Adapter_barcode17_c-has-miR-133a-5p [ka] Adapter_barcode18_c-has-miR-208a-5p [ka] Adapter_barcode19_c-has-miR-181b-5p [ka] Adapter_barcode20_c-has-miR-29a-3p [ka] miRNAS without C3 spacer (barcodes 21-22) Adapter_barcode21_c-has-miR-30c-5p [ka] Adapter_barcode22_c-has-miR-29a-3p [ka]

[0180] Example 2 This example describes the identification of individual carriers by sequencing and demultiplexing their unique barcodes. This example also describes the identification of carriers bound to an analyte by stall analysis.

[0181] method The barcodes described in Example 1 (total concentration 30 nM, equal concentrations of each) were incubated with the ligated c strands in a molar ratio of 1:3 in nuclease-free water for 1 hour. Hybridization was initiated by centrifugation at 4°C for 3 minutes followed by incubation at room temperature for 1 hour. The resulting mixture was mixed with 10 nM of adapters and ligation was performed by adding an equal amount of TA ligase master mix (New England Biolabs) and centrifuging at 4°C for 3 minutes to thoroughly mix the different components while storing the ligase at low temperature. After incubation at room temperature for 20 minutes, 1.4 times the total volume of Ampure XP beads (Beckmann Coutler) were added to absorb the nucleic acids for further purification. The beads were washed with short fragment buffer (Oxford Nanopore Technologies) to selectively remove excess amounts of unhybridized ligated c strands and barcodes that were not ligated. After purification, the beads were washed with nuclease-free water to wash away the purified nucleic acid containing the ligated motor protein-barcode complex. The final solution for the sequencing experiment was made by elution solution, sequencing buffer, tether (100 nM), nuclease-free water, and incubated with a certain concentration of the targeted analyte for at least 30 min.

[0182] All experiments were analyzed with an existing custom-written MATLAB code, the Nanopore app (developed by Joshua Edel between 2006 and 2021). FAST5 sequencing files were uploaded into the app for further processing. The app allowed all 512 channels of the MinION to be uploaded and analyzed individually or in bulk.

[0183] Translocation events were detected using a thresholding algorithm. First, a linear baseline was set between -0.16 and -2 nA, depending on the experiment. A step offset of 1.8 was used to define the onset of an event (green line). If events exceed a threshold of std50 (black line), they are detected by the algorithm. These events are further analyzed by filtering events between 0.025 s and 10 s. All events within this time window are defined as events. In addition, the algorithm is used to position C3 or other spacer elements within the strand design that are used as alignment markers. In this way, the presence of a spacer is not essential for the detection of the presence or absence of a molecule on the support.

[0184] Events detected in the previous step were sequenced and base called using GUPPY or / and MinKNOW software (Oxford Nanopore Technologies).

[0185] After making base calls of the raw read signals, the sequenced events were aligned to a reference sequence, e.g., barcode sequences 1 to 10. The algorithm assigns a barcode to the event with the highest alignment score if the following points are true: 1) The alignment score must be 50 or above, 2) The difference between the maximum alignment score and the second highest score must be at least 5; 3) The difference between the maximum alignment score and the average alignment score of the other barcodes must be at least 10; 4) The p-value must be less than or equal to 0.0001.

[0186] The maximal alignment was classified as a barcode only if all criteria were met; otherwise, the event was deleted and not considered for further analysis. False positives were removed by calculating the p-value of the highest alignment score compared to the rest of the population.

[0187] In the stall analysis, a C3 portion was defined, as well as the start and end of the event. This was important to calculate the average translocation time of all sequenced reads. If the translocation time of an event was significantly longer compared to the average (typically, the std100 bins moved were used), the event was classified as a stall. Furthermore, each stall had to be more than 20 bins to be considered a stall.

[0188] Identification of individual carriers by sequencing and demultiplexing of unique barcodes. All sequences were base called and aligned to a reference sequence, described above, which is the barcode sequence. The maximum alignment score was used to classify the barcode. If the maximum alignment score and the second highest score were too close, the event was not classified. Additionally, a p-value was used to classify the events and remove false positive classifications. This method achieved 99.95% accuracy in identifying individual barcodes, and 86% of all events recorded were used and classified, meaning that relatively little data was wasted compared to previous methods in the art (Figure 4).

[0189] The barcode sequences had an accuracy of over 90%. A false positive rate of only 0.0001% was observed (Figure 5(a)). The method had a very low preference for incorrect barcode classification, as shown in the confusion matrix in Figure 5(b). Improved algorithms and detection techniques will continue to improve barcode classification. Another way to improve barcode classification is to include barcode repetitions in the carrier as a proofreading mechanism. The examples show that 10 barcoded carriers can be successfully distinguished within a complex mixture, enabling highly multiplexed assays.

[0190] Identification of analyte-bound support by stall analysis. In this example, stall analysis was used to determine whether the target is bound to the barcoded strand. If the target analyte is not bound to the barcoded strand, the current signal shows no stall (in dwell time, current amplitude). Bound analyte (exemplified here with complementary miRNA) stalls the carrier, which results in a unique current profile (see FIG. 7). Stalling can be due to, for example, (1) unzipping of a double-stranded nucleotide structure (e.g., miRNA, DNA detection), (2) unraveling of either a G-quadruplex or stem-loop aptamer structure (e.g., for detection of proteins, neurotransmitters, and small binding molecule analytes), or (3) striping of bound antibody-antigen. The relative concentration of the target molecule can be determined by correlating the percentage of stalled carriers versus non-stalled carriers (see FIG. 8). The absolute concentration of carriers in a sample can be determined by measuring the time between individual single molecule detection events (inter-event time), as widely described in the nanopore literature.

[0191] Another method for determining the concentration of a molecule using the method described herein is to determine the concentration dependency of the molecule on the detection of the carrier bound to the molecule.For example, a standard curve can be prepared in a similar manner as shown in Figure 9.

[0192] In another experiment, the detection of binding between thrombin and the 15mer thrombin-binding aptamer was studied. Upon binding with thrombin, the "ragged" events showed much longer residence times, and in terms of current reversal, a significant increase in stall upon binding with 400 nM thrombin was observed, which corresponds to the unwinding of the G-quadruplex and the aptamer-protein interaction. The concentration dependence of binding between thrombin and the 15mer thrombin-binding aptamer was further verified by increasing the thrombin concentration from 0 nM to 400 nM, and an increase in stall was observed as more ragged events with longer residence times (see Figures 10 and 11).

[0193] In another experiment, the detection of bound serotonin using stem-loop aptamers was studied. The increase in stall upon binding with serotonin was attributed to the structural rearrangement of the aptamer from loop to G-quadruplex upon binding with serotonin, resulting in the unwinding of the loop, G-quadruplex, and aptamer-serotonin interactions (see FIG. 12). The concentration dependence of the binding between serotonin and the aptamer was further verified by increasing the serotonin concentration between 0 nM and 40 nM. The increase in stall was observed as more irregular curvilinear events and longer residence times (see FIG. 13).

[0194] A concentration dependence of binding between serotonin and the aptamer was also observed, based on the mean lag time versus concentration, as shown in Table 3 below. [Table 3]

[0195] In another experiment, the detection of bound acetylcholine using step-loop aptamers was studied. The barcode (underlined)-spacer-aptamer (italics) used was as follows: [ka]

[0196] A clear stall event can be seen upon binding with acetylcholine. The concentration dependence of the binding between acetylcholine and the aptamer on the retardation (stall) percentage was observed by increasing the acetylcholine concentration between 0 nM and 40 nM (Figure 14).

[0197] Example 3 Alternative methods for identifying carriers that are bound to an analyte are available.

[0198] In some embodiments, it is not necessary to perform a specific stall analysis: the base-calling software programs GUPPY and MinKNOW (Oxford Nanopore Technologies) can be used to directly analyze the data generated as the carrier moves through the pore.

[0199] Alternatively, and as mentioned above, another option is to use enzymatic digestion to remove / digest any portion of the carrier with unbound molecular binding regions. This can be achieved by using a) an endonuclease or b) an exonuclease that targets the molecular binding regions in the carrier that are not bound to the analyte target (see FIG. 3). Thus, after digestion, carriers that are bound to the target and carriers that are not bound to the target are distinguished based on the presence or absence of a current signal after the barcode sequence. Alternatively, if the molecular binding region is a polypeptide, a protease can be used that digests the portion of the carrier with unbound molecular binding regions, provided that a signal difference can be observed when the carrier with unbound, enzymatically digested molecular binding regions passes through the pore, compared to the carrier with molecules bound to the molecular binding region.

[0200] Example 4 Support assembly protocol Barcodes (total concentration 30 nM, equal concentrations of each) were incubated with ligated c strands in a molar ratio of 1:3. Hybridization was initiated by centrifugation at 4°C for 1 min followed by incubation at room temperature for 1 h. The resulting mixture was mixed with 10 nM adapters and ligation was performed by adding an equal volume of TA ligase master mix (New England Biolabs). The samples were centrifuged at 4°C for 1 min to thoroughly mix the different components while storing the ligase at low temperature. After incubation at room temperature for 20 min, 1.4 times the total volume of Ampure XP beads (Beckmann Coulter) were added to absorb the nucleic acids for further purification. The beads were washed twice with short fragment buffer (Oxford Nanopore Technologies) to selectively remove excess amounts of unhybridized ligated c strands and barcodes that were not ligated. After purification, the beads were washed with nuclease-free water to wash away the purified nucleic acid containing the ligated motor protein-barcode complex. The final solution for sequencing experiments was made by elution solution, sequencing buffer, tether (100 nM), nuclease-free water, and incubated with a specific concentration of the targeted analyte for at least 30 minutes. For miRNA experiments, concentrations of 0.05 nM, 0.1 nM, 0.25 nM, 0.5 nM, 1 nM, 2.5 nM, 5 nM, 10 nM, 25 nM, and 50 nM were used. For protein experiments, concentrations of 10 pg / mL, 50 pg / mL, 100 pg / mL, 500 pg / mL, 1 ng / mL, and 30 ng / mL were used.

[0201] Protocol for running the experiment All experiments were performed at 37° C. for 30 min using research and / or customer scripts.

[0202] Data Analysis Workflow All experiments were analyzed with an existing custom-written MATLAB code, the Nanopore app. FAST5 sequencing files output from the nanopore sequencing device (MinION device, Oxford Nanopore Technologies) were uploaded to the app for further processing. The app allows all 512 channels of the MinION to be uploaded and analyzed individually or in bulk.

[0203] Event Detection Translocation events are detected using a thresholding algorithm. First, a linear baseline is set between -0.16 and -2 nA, depending on the experiment. A step offset of 1.8 is used to define the start of an event. If events exceed a threshold of std30, they are detected by the algorithm. These events are further analyzed by filtering for events longer than 0.1 seconds. All events in this time window are defined as an event.

[0204] Sequencing and base calling Events detected in the previous step were sequenced and base called using GUPPY and / or MinKNOW software (Oxford Nanopore Technologies).

[0205] Alignment After making base calls of the raw read signals, the sequenced events were aligned to a reference sequence, in this case barcode sequences 1 to 40. The algorithm assigned a barcode to the event with the highest alignment score if the following points were true: 1) The sequence must start with "GGG" 2) At least 15 bases must be aligned, 3) only one mismatch in the first 10 bases; 4) Only one mismatch in all aligned bases.

[0206] The maximal alignment was classified as a barcode only if all criteria were met, otherwise the event was removed and not considered for further analysis.

[0207] stall In the stall analysis, a C3 portion was defined, as well as the start and end of the event. This was used to calculate the average translocation time of all sequenced reads. If the translocation time of an event was significantly longer than the average (typically, the moving std75 bin was used), the event was classified as a stall. Furthermore, each stall had to be more than 10 bins to be considered a stall.

[0208] Results: Heatmap of 40 barcodes (Figure 19) Figure 19 presents a confusion matrix showing that the preference for incorrect barcode classification is very low. All 40 barcodes tested were called with greater than 95% accuracy.

[0209] Results: Detection of multiple miRNAs The multiplexed barcode sequencing method described herein enabled the detection of 40 different miRNAs. The results are presented in Figure 20.

[0210] Results: Quantification of unknown miRNA concentrations The methods described herein allowed accurate prediction of miRNA concentrations using blinded studies of multiple different samples of known miRNA concentrations. The results are presented in FIG.

[0211] Results: Detection of cTnI Data regarding the detection of the protein cardiac troponin I (cTnI) is shown in Figure 22. The troponin aptamer sequence shown is: AGTCTCCGCTGTCTCCCGATGCACTTGACGTATGTCTCACTTTCTTTTCATTGACATGGGATGACGCCGTGACTG

[0212] Appendix to Example 4 Carrier strand sequence - miRNA (Figure 17) [ka] [ka] [ka] [ka] [ka]

[0213] Carrier chain sequences - proteins and neurotransmitters (Figure 18) Cardiac Troponin I (cTnI): [ka] Cardiac Troponin T (cTnT): [ka] BNP: [ka] Thrombin: [ka] S-protein: [ka] N-protein: [ka] Serotonin: [ka] acetylcholine: [ka]

[0214] target analyte [Table 4] protein: Cardiac troponin I (cTnI) [Genscript] Cardiac Troponin T (cTnT): TNNT2 Protein Human Recombinant | CTnT Antigen | ProSpec (prospecbio.com) BNP-32 peptide: [Bachem] Human Alpha-Thrombin Native Protein, Biotin (RP-43103) [Thermofisher]

[0215] Reference Example 1 Limitations of multiplex assays without using motor proteins. As described above, the presence of the motor protein on the carrier allows for precise identification of the identifier region and determination of the presence or absence of the molecule on the molecule binding region. If the method is performed in the absence of the motor protein, there are limitations to the measurements that can be obtained.

[0216] In one experiment, increasing concentrations of thrombin were detected by nanopore measurements using a carrier containing an aptamer (underlined) and a 30*T threading strand. [ka]

[0217] With this approach, it is possible to quantify the concentration of thrombin, but there is no way to check for false positives, nor is there an easy way to multiplex the assay: all that is measured is the difference in signal level between the aptamer and the protein-bound aptamer (Figure 15A).

[0218] In a second experiment, increased concentrations of serotonin were detected by nanopore measurements using a carrier containing a stem-loop aptamer (underlined) and a 30*T threading strand. [ka]

[0219] As with the thrombin experiment, it is possible to quantify the concentration of serotonin, but there is no way to check for false positives and no easy way to multiplex the assay: all that is measured is the difference in signal level between the aptamer and the neurotransmitter-bound aptamer (Figure 15B).

[0220] In a third experiment, miRNA levels were detected in a multiplexed format. Barcodes 1-6 (ACGTA, GGACT, TTAAC, GCTAG, CTGAG, and TAGCG) were identified by the unique mean currents observed for each barcode (122.4 pA, 121.8 pA, 111.5 pA, 152.7 pA, 129.3 pA, and 135.4 pA, respectively) (Figure 14C). However, this approach is limited in its multiplexing capabilities and is restricted by amplitude variations in the signals. Realistically, in a multiplexed assay, five barcodes may be used. Barcode assignment and classification is not always precise due to the distribution of amplitudes observed for each barcode overlapping with characteristic signals of other barcodes. Sequence Listing SEQ ID NO:1 - Exonuclease I from E. coli MMNDGKQQSTFLFHDYETFGTHPALDRPAQFAAIRTDSEFNVIGEPEVFYCKPADDYLPQPGAVLITGITPQEARAKGENEAAFAARIHSLFTVPKTCILGYNNVRFDDEVTRNIFYRNFY DPYAWSWQHDNSRWDLLDVMRACYALRPEGINWPENDDGLPSFRLEHLTKANGIEHSNAHDAMADVYATIAMAKLVKTRQPRLFDYLFTHRNKHKLMALIDVPQMKPLVHVSGMFGAWRGN TSWVAPLAWHPENRNAVIMVDLAGDISPLLELDSDTLRERLYTAKTDLGDNAAVPVKLVHINKCPVLAQANTLRPEDADRLGINRQHCLDNLKILRENPQVREKVVAIFAEAEPFTPSDNV DAQLYNGFFSDADRAAMKIVLETEPRNLPALDITFVDKRIEKLLFNYRARNFPGTLDYAEQQRWLEHRRQVFTPEFLQGYADELQMLVQQYADDKEKVALLKALWQYAEEIVSGSGHHHHHH SEQ ID NO:2 - Exonuclease III enzyme from E. coli MKFVSFNINGLRARPHQLEAIVEKHQPDVIGLQETKVHDDMFPLEEVAKLGYNVFYHGQKGHYGVALLTKETPIAVRRGFPGDDEEAQRRIIMAEIPSLLGNVTVINGYFPQGESRDHPIKFPAKAQFYQNLQN YLETELKRDNPVLIMGDMNISPTDLDIGIGEENRKRWLRTGKCSFLPEEREWMDRLMSWGLVDTFRHANPQTADRFSWFDYRSKGFDDNRGLRIDLLLASQPLAECCVETGIDYEIRSMEKPSDHAPVWATFRR SEQ ID NO:3 - RecJ enzyme from T. thermophilus MFRRKEDLDPPLALLPLKGLREAAALLEEALRQGKRIRVHGDYDADGLTGTAILVRGLAALGADVHPFIPHRLEEGYGVLMERVPEHLEASDLFLTVDCGITNHAELRELLENGVEVIVTDHHTPGKTPPPGLVVHPALTPDLKKEKPTGAGVAFLLLWALHERLGLPPPLEYADLAAVGTIADVAPLWGWNRALVKEGLARIPASSWVGLRL LAEAVGYTGKAVEVAFRIAPRINAASRLGEAEKALRLLLTDDAAEAQALVGELHRLNARRQTLEEAMLRKLLPQADPEAKAIVLLDPEGHPGVMGIVASRILEATLRPVFLVAQGKGTVRSLAPISAVEALRSAEDLLLRYGGHKEAAGFAMDEALFPAFKARVEAYAARFPDPVREVALLLDLLPEPGLLPQVFRELALLEPYGEGNPEPLFL SEQ ID NO:4 - Bacteriophage lambda exonuclease MTPDIILQRTGIDVRAVEQGDDAWHKLRLGVITASEVHNVIAKPRSGKKWPDMKMSYFHTLLAEVCTGVAPEVNAKALAWGKQYENDARTLFEFTSGVNVTESPIIYRDESMR TACSPDGLCSDGNGLELKCPFTSRDFMKFRLGGFEAIKSAYMAQVQYSMWVTRKNAWYFANYDPRMKREGLHYVVIERDEKYMASFDEIVPEFIEKMDEALAEIGFVFGEQWR SEQ ID NO:5 - Phi29 DNA polymerase MKHMPRKMYSCAFETTTKVEDCRVWAYGYMNIEDHSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFIINWLERNGFKWSADGLPNTYNTIISRMGQWYMIDICLGYKGKRKIHTVIYDSLKKLPFPVKKIAKDFKLTVLKGDIDYHKER PVGYKITPEEYAYIKNDIQIIAEALLIQFKQGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLLDKEVRYAYRGGFTWLNDRFKEIGEGMVFDVNSLYPAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQI KRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSEGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWARYTTITAAQACYDRIIYCD TDSIHLTGTEIPDVIKDIVDPKKLGYWAHESTFKRAKYLRQKTYIQDIYMKEVDGKLVEGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDDTFTIKSGGSAWSHPQFEKGGGSGGGSGGSAWSHPQFEK SEQ ID NO:6 - Trwc Cba helicase MLSVANVRSPSAAASYFASDNYYASADADRSGQWIGDGAKRLGLEGKVEARAFDALLRGELPDGSSVGNPGQAHRPGTDLTFSVPKSWSLLALVGKDERIIAAYREAVVEALHWAEKNAAETRVVEKGMVVTQATGNLAIGLFQHDTNRNQEPNLHFHAVIANVTQGKDGWRTLKNDRLWQLNTTLNSIAMARFRVAVEKLGYEPGPVLKHGNFEARGISREQVMAFSTRRKEVLEARRGPGGLDAGRIAALDTRASKEGIEDRATLSKQWSEAAQSIGLDLKPLVDRARTKALGQGMEATRIGSLVERGRAWLSRFAAHVRGDPADPLVPPSVLKQDRQTIAAAQAVASAVRHLSQREAAFERTALYKAALDFGLPTTIADVEKRTRALVRSGDLIAGKGEHKGWLASRDAVVTEQRILSEVAAGKGDSSPAITPQKAAASVQAAALTGQGFRLNEGQLAAARLILISKDRTIAVQGIAGKS SVLKPVAEVLRDEGHPVIGLAIQNTLVQMLERDTGIGSQTLARFLGGWNKLLDDPGNVALRAEAQASLKDHVLVLDEASMVSNEDKEKLVRLANLAGVHRLVLIGDRKQLGAVDAGKPFALLQRAGIARAEMATNLRARDPVVREAQAAAQAGDVRKARLHLKSHTVEARGDGAQVAAETWLALDKETRARTSIYASGRAIRSAVNAAVQQGLLASREIGPAKMKLEVLDRVNTTREELRHLPAYRAGRVLEVSRKQQALGLFIGEYRVIGQDRKGKLVEVEDKRGKRFRFDPARIRAGKGDDNLTLLEPRKLEIHEGDRIRWTRNDHRRGLFNADQARVVEIANGKVTFETSKGDLVELKKDDPMLKRIDLAYALNVHMAQGLTSDRGIAVMDSRERNLSNQKTFLVTVTRLRDHLTLVVDSADKLGAAVARNKGEKASAIEVTGSVKPTATKGSGVDQPKSVEANKAEKELTRSKSKTLDFGI SEQ ID NO:7 - Hel308 Mbu helicase MMIRELDIPRDIIGFYEDSGIKELYPPQAEAIEMGLLEKKNLLAAIPTASGKTLLAELAMIKAIREGGKALYIVPLRALASEKFERFKELAPFGIKVGISTGDLDSRADWLGVNDIIVATSEKTDSLLRNGTSWMDEITTVVVDEIHLLDSKNRGPTLEVTITKLMRLNPDVQVVALSATVGNAREMADW LGAALVLSEWRPTDLHEGVLFGDAINFPGSQKKIDRLEKDDAVNLVLDTIKAEGQCLVFESSRRNCAGFAKTASSKVAKILDNDIMIKLAGIAEEVESTGETDTAIVLANCIRKGVAFHHAGLNSNHRKLVENGFRQNLIKVISSTPTLAAGLNLPARRVIIRSYRRFDSNFGMQPIPVLEYKQMAGRAG RPHLDPYGESVLLAKTYDEFAQLMENYVEADAEDIWSKLGTENALRTHVLSTIVNGFASTRQELFDFFGATFAYQQDKWMLEEVINDCLEFLIDKAMVSETEDIEDASKLFLRGTRLGSLVSMLYIDPLSGSKIVDGFKDIGKSTGGNMGSLEDDKGDDITVTDMTLLHLVCSTPDMRQLYLRNTDYTI VNEYIVAHSDEFHEIPDKLKETDYEWFMGEVKTAMLLEEWVTEVSAEDITRHFNVGEGDIHALADTSEWLMHAAAKLAELLGVEYSSHAYSLEKRIRYGSGLDLMELVGIRGVGRVRARKLYNAGFVSVAKLKGADISVLSKLVGPKVAYNILSGIGVRVNDKHFNSAPISSNTLDTLLDKNQKTFNDFQ SEQ ID NO:8 - Dda helicase MTFDDLTEGQKNAFNIVMKAIKEKKHHVTINGPAGTGKTTLTKFIIEALISTGETGIILAAPTHAAKKILSKLSGKEASTIHSILKINPVTYEENVLFEQKEVPDLAKCRVLICDEVSMYDRKLFKILLSTIPPWCTIIGIGDNKQIRPVDPGENTAYISPFFTHKDFYQCELTEVKRSNAPIIDVATDVRNGKWIYDKVVDGHGVRGFTGDTALRDFMVNYFSIVKSLDDLFENRVMAFTNKSVDKLNSIIRKKIFETDKDFIVGEIIVMQEPLFKTYKIDGKPVSEIIFNNGQLVRIIEAEYTSTFVKARGVPGEYLIRHWDLTVETYGDDEYYREKIKIISSDEELYKFNLFLGKTAETYKNWNKGGKAPWSDFWDAKSQFSKVKALPASTFHKAQGMSVDRAFIYTPCIHYADVELAQQLLYVGVTRGRYDVFYV

Claims

1. 1. A method for detecting a plurality of molecules in a sample, comprising: (a) contacting the sample with a support and a nanopore, the support comprising a single-stranded leader, an identifier region, and a molecular binding region specific for a molecule to be detected, and a motor protein bound to the support such that the motor protein can control movement of the identifier region within the nanopore; (b) performing one or more optical or electrical measurements as the carrier moves through the nanopore to characterize the identifier region and determine whether the molecule is bound to the molecule binding region.

2. 2. The method of claim 1, wherein the carrier further comprises a spacer between the bound motor protein and the molecular binding region.

3. The method of claim 2 , wherein the carrier comprises, in order, a single-stranded leader, an identifier region, a spacer, and a molecular binding region.

4. The method of claim 1 , wherein the molecular binding region and / or the identifier region is a polynucleotide.

5. The method of claim 1 , wherein the molecular binding region or a portion thereof is the identifier region.

6. 5. The method of claim 4, wherein the identifier region is a polynucleotide and comprises a barcode sequence.

7. The method of claim 1 , wherein the carrier comprises two or more identifier regions and / or two or more molecular binding regions.

8. 8. The method of claim 7, wherein the carrier comprises two or more identifier regions, and the method is for detecting multiple molecules in multiple samples, one identifier region on the carrier being unique to the sample and one identifier region on the carrier being unique to the molecule to which the carrier binds.

9. The method of claim 1 , wherein the molecule comprises a neurotransmitter, a protein, and / or an miRNA.

10. The method of claim 1 , wherein the molecular binding region is an aptamer.

11. The method of claim 1 , wherein the molecular binding region is an antibody, an antibody fragment, a nanobody, or an affibody.

12. The method of claim 1 , wherein the molecular binding region is complementary to a miRNA.

13. 2. The method of claim 1 , wherein the identifier region is a polynucleotide, and the method comprises determining the polynucleotide sequence of the identifier region.

14. The method of claim 1 , wherein the method is used to detect the presence or absence of the molecule.

15. The method of claim 1 , wherein the method is used to determine the concentration of the molecule.

16. The method of claim 1 , wherein the plurality of molecules is 10 or more different molecules.

17. The method of claim 1 , wherein the motor protein is a helicase, polymerase, nuclease, translocase, or topoisomerase.

18. 18. The method of any one of claims 1 to 17, wherein the nanopore is a protein pore, a solid-state pore, or a DNA origami pore.

19. A carrier comprising a single-stranded leader, an identifier region, and a molecular binding region specific to a molecule to be detected, wherein a motor protein is bound to the carrier at a position between the single-stranded leader and the identifier region.

20. The carrier of claim 19 , further comprising a spacer between the identifier region and the molecular binding region, and / or a molecule specifically bound to the molecular binding region.

21. A population of carriers for a plurality of molecules, said carriers being as defined in claim 19 or 20, and different carriers in said population comprising different identifier regions and different molecule binding regions.

22. 1. A kit for detecting a plurality of molecules in a sample, comprising: (i) a population of carriers, each carrier comprising an identifier region and a molecular binding region specific for a molecule to be detected, and different carriers in the population comprising different identifier regions and different molecular binding regions; (ii) an adaptor comprising a single-stranded leader; (iii) a motor protein.

23. 1. A system for detecting a plurality of molecules in a sample, comprising: (i) a population of carriers according to claim 21; (iii) a nanopore.

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