Protein translocation through nanopores

Abstract

Methods of translocating an analyte through a nanopore and corresponding systems are described. An example method comprises translocating an analyte through a nanopore in a structure defining an internal cavity and a barrier dividing the interior cavity into a first chamber and a second chamber. The barrier comprises a channel structure defining a nanopore therethrough fluidically coupling the first and second chambers. The first chamber comprises the analyte and the method comprises modifying side groups of the analyte. Said modifying changes at least one of an electrical a chemical property of the analyte. The method further comprises applying a voltage gradient between the first and second chambers via the nanopore. Applying the voltage causes a translocation of the analyte through the nanopore from the first chamber and the second chamber. Embodiments described herein can be helpful for enabling translocation or ensuring smooth translocation of an analyte through a nanopore.

Description

5200.2441001Protein Translocation through NanoporesRELATED APPLICATION

[0001] This application claims the benefit of U.S. Provisional Application No.63 / 746,120, filed on January 16, 2025. The entire teachings of the above application(s) are incorporated herein by reference.GOVERNMENT SUPPORT

[0002] This invention was made with government support under Grant No.R01HG012553 from National Institutes of Health. The government has certain rights in the invention.BACKGROUND

[0003] Efficient single-molecule protein translocation through biological nanopores may be critical for advancing technologies such as single-molecule proteomics and protein sequencing for non-limiting examples. Proteins or polypeptides, which may comprise 20 distinct amino acids, can exhibit diverse charge distributions and complex structures due to intra- and intermolecular interactions, which may pose significant challenges for nanopore translocation.SUMMARY

[0004] Methods of translocating an analyte through a nanopore and corresponding systems are described herein. Embodiments of the methods and systems can be useful for enabling translocation for a broad range of analytes, including, as non-limiting examples, proteins or peptides with positive charges or a capacity for forming disulfide bonds. The embodiments may further be useful for ensuring smooth translocation of the analytes, which can facilitate more robust measurements of the analyte as it passes through the nanopore.

[0005] An example embodiment is directed toward a method of translocating an analyte through a nanopore in a structure defining an interior cavity and a barrier dividing the interior cavity into a first chamber and a second chamber. The barrier comprises a channel structure defining the nanopore therethrough fluidically coupling the first and second chambers. The first chamber comprises the analyte and the method comprises modifying at least one side groups of the analyte. Said modifying changes at least one of an electrical property or a- 1 - 5001986. vl5200.2441001chemical property of the analyte. The method further comprises applying a voltage gradient between the first chamber and the second chamber via the nanopore. Said applying causes a translocation of the analyte through the nanopore from the first chamber to the second chamber. Said modifying the at least one side group further causes a corresponding change in the translocation of the analyte in the presence of the voltage gradient relative to translocation absent the modifying of the at least one side group of the analyte in the presence of the voltage gradient

[0006] According to some embodiments, the modifying the at least one side groups of the analyte can comprise introducing a reagent to the analyte. The reagent can react selectively with the at least one side groups. In some embodiments, the reagent can react selectively with at least one of: a charged side group of the at least one side groups and a thiol side group of the one or more side groups. In other embodiments, the reagent can comprise at least one of: an anhydride, wherein the anhydride can comprise a maleic anhydride or a carbonic anhydride, or a precursor or derivative thereof; an activated ester, wherein the activated ester can comprise an N-hydroxysuccinimide ester, a pentafluorophenyl ester, a tetrafluorophenyl ester, a sulfo-tetrafluorophenyl ester, or a precursor of derivative thereof; an isocyanate; an acyl halide; an epoxide; a Michael acceptor, wherein the Michael acceptor can comprise maleimide, acrylamide, methacrylmide, propiolamide, or a precursor or derivative thereof; and an alpha-halo carbonyl, wherein the alpha-halo carbonyl can comprise iodoacetamide, bromoacetamide, or chloroacetamide. In some embodiments, the method can further comprise introducing a reducing agent to the analyte and the reagent. The reducing agent can comprise one or more of tris(2-carboxyethyl)phosphine, diothitreitol, or 2-mercaptoethanol.

[0007] According to some embodiments, modifying the at least one side groups of the analyte can comprise tuning a pH of a solution of the first chamber or the second chamber to change a charge of the at least one side groups.

[0008] According to other embodiments, the analyte can be a polypeptide or a protein.

[0009] According to some embodiments, the method can further comprise observing an electronic signature of the analyte produced by the analyte passing through the nanopore and determining a property of the analyte based on the electronic signature observed.

[0010] According to some embodiments, the method can further comprise controlling a rate of translocation of the analyte through the nanopore based on at least one of a salt concentration of the first or the second solution, a salt species of the first or the second solution, pH of the first or the second solution, or the voltage gradient applied.- 2 - 5001986. vl5200.2441001

[0011] Another example embodiment is directed toward a method of translocating an analyte through a nanopore in a structure defining an interior cavity and a barrier dividing the interior cavity into a first chamber and a second chamber. The barrier comprises a lipid bilayer disposed on a side of the barrier facing the second chamber and a channel structure defining the nanopore therethrough fluidically coupling the first and second chambers. The method comprises introducing the analyte to the first chamber. Said first chamber contains a first solution, the second chamber contains a second solution, and the first solution and the second solution are compositionally different. The method further comprises applying a voltage gradient between the first chamber and the second chamber via the nanopore. The applying the voltage gradient causes a translocation of the analyte through the nanopore from the first chamber to the second chamber.

[0012] According to some embodiments, the first solution can comprise a denaturing agent and wherein the denaturing agent comprises at least one of guanidinium chloride, urea, or sodium dodecyl sulfate (SDS). In some embodiments, the first solution can comprise the denaturing agent at a concentration that does not disrupt a lipid bilayer or the channel structure.

[0013] According to some embodiments a pH of the first solution can be sufficiently different from a pH of the second solution such that a charge of a side group of the analyte in the first solution is different to a charge of the side group in the second solution. The side group of the analyte can undergo a change in the charge of the side group as it passes through the nanopore.

[0014] According to some embodiments, a concentration of an electrolyte in the first chamber can be different from a concentration of the electrolyte in the second chamber and the electrolyte can comprise at least one of potassium (K+) ions or chloride (C1-) ions.

[0015] According to other embodiments, the method can further comprise inserting the channel structure into the barrier. The channel structure can define a first aperture and a second aperture of the nanopore, the first aperture having a smaller circumference than the second aperture. The channel structure can be inserted such that the first aperture is in fluidic contact with one of the first or the second solution and the second aperture is in fluidic contact with the other of the first or the second solution. In some embodiments, the method can further comprise controlling an orientation of the channel structure in the barrier by containing, by the second solution, the channel structure. The channel structure being contained by the second solution can cause the channel structure to be inserted such that the- 3 - 5001986. vl5200.2441001first aperture is in fluidic contact with the first solution and the second aperture is in fluidic contact with the second solution. In some embodiments, inserting the channel structure can further include positioning the channel structure such that a distance between the smaller aperture and the barrier is smaller than a distance between the larger aperture and the barrier.

[0016] According to some embodiments, the method can further comprise observing an electronic signature of the analyte produced by the analyte passing through the nanopore and determining a property of the analyte based on the electronic signature observed.

[0017] Another example embodiment is directed to a system for translocating an analyte through a nanopore. The system comprises a structure defining an interior cavity and a barrier dividing the interior cavity into a first chamber configured to contain a first solution and a second chamber configured to contain a second solution compositionally different from the first solution. The barrier comprises a lipid bilayer disposed on a side of the barrier facing the second chamber and a channel structure defining the nanopore therethrough fluidically coupling the first and second chambers. The system further comprises voltage source electrodes configured to apply a voltage gradient to the first and second chambers via the nanopore. The voltage gradient applied causes a translocation of the analyte of the first solution or the second solution through the nanopore.

[0018] In some embodiments, the channel structure can define a first aperture and a second aperture of the nanopore. The first aperture can have a smaller circumference than the second aperture. The first aperture can be in fluidic contact with the first solution and the second aperture can be in fluidic contact with the second solution.BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The foregoing will be apparent from the following more particular description of example embodiments, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating embodiments.

[0020] FIGS. 1 A and IB are flow diagrams of example embodiments of methods of translocating an analyte through a nanopore.

[0021] FIGS. 2A and 2B illustrate example embodiments of systems 200a, 200b configured for translocating analytes in a trans-Xo-cis configuration and a cis-Xo-trans configuration, respectively.- 4 - 5001986. vl5200.2441001

[0022] FIG. 3 illustrates schematically an example embodiment of a system for translocating an analyte through a nanopore using asymmetric buffer conditions.

[0023] FIG. 4 illustrates schematically an example embodiment of a system for translocating an analyte through a nanopore, the system comprising chemical modifications to the analyte.

[0024] FIG. 5 illustrates schematically an example embodiment of a system for translocating an analyte capable of forming disulfide bonds through a nanopore, the system including asymmetric buffer conditions.

[0025] FIG. 6 illustrates an example embodiment of a system for translocating an analyte capable of forming disulfide bonds through a nanopore, the system including asymmetric buffer conditions and chemical modification of the analyte.

[0026] FIG. 7 is a plot illustrating current-voltage (I-V) curves in a system for translocating an analyte through a nanopore using different buffer conditions, according to an example embodiment.

[0027] FIGS. 8 A and 8B are plots illustrating current trajectories and translocation events for translocating an analyte through a nanopore from a vestibule side to a stem side, according to an example embodiment.

[0028] FIGS. 9A and 9B are plots illustrating current trajectories and translocation events for translocating an analyte through a nanopore from a stem side to a vestibule side, according to an example embodiment.

[0029] FIGS. 10A-10C are plots illustrating metrics of translocating an analyte through a nanopore using trans-to-cis and cis-to-trans configurations, according to example embodiments.

[0030] FIG. 11 A is a plot illustrating dwell time (logarithmic) versus change in current (AI / Io) of translocation of an analyte through a nanopore in an absence of a denaturing agent, according to an example embodiment.

[0031] FIGS. 1 IB and 11C are histograms of the dwell time and the change in current, respectively, of FIG. 11 A.

[0032] FIG. 12A is a plot illustrating dwell time (logarithmic) versus change in current (AI / Io) of translocation of an analyte through a nanopore in a presence of a denaturing agent, according to an example embodiment.

[0033] FIGS. 12B and 12C are histograms of the dwell time and the change in current, respectively, of FIG. 12 A.- 5 - 5001986. vl5200.2441001

[0034] FIGS. 13 A and 13B are plots illustrating voltage / current trajectories of MBP-D10 and BirA-DlO, respectively, translocating through a nanopore under symmetrical pH of 7.5 conditions, according to example embodiments.

[0035] FIGS. 14A and 14B are plots illustrating voltage / current trajectories of MBP-D10 and BirA-DlO, respectively, translocating through a nanopore under pH asymmetric conditions, according to example embodiments.

[0036] FIG. 15 is a plot illustrating dwell time versus change in current (AI / Io) of translocation of analytes through a nanopore, with histograms of the dwell time and the change in current, according to an example embodiment.

[0037] FIG. 16 is a plot illustrating translocation time of MBP-D10 at pH 7.5 and pH 8.6 under different applied voltages, according to an example embodiment.

[0038] FIG. 17 is a plot illustrating translocation time of BirA-DlO at pH 8.6 and pH 9.5 under different applied voltages, according to an example embodiment.

[0039] FIGS. 18 and 19 illustrate schematically example embodiments of reactions for chemical modification of analytes using Boc anhydride.

[0040] FIG. 20 is a plot illustrating translocation time in log scale of MBP-D10 and Boc anhydride treated MBP-D10 using different applied voltages, according to an example embodiment.

[0041] FIG. 21 is a plot illustrating translocation time in log scale of BirA-DlO and Boc-anhydride-treated BirA-DlO using different applied voltages, according to an example embodiment.

[0042] FIGS. 22A and 22B are plots illustrating dwell time versus current blockade and a histogram of dwell time, respectively, of translocation of Boc anhydride-treated BirA-DlO at pH 7.5.

[0043] FIG. 23 illustrates schematically an example embodiment of a reaction for chemical modification of an analyte using acrylamide.

[0044] FIG. 24 is a plot illustrating dwell time versus change in current (AI / Io) of translocation of BSA-D10 through a nanopore, with histograms of the dwell time and the change in current, according to an example embodiment.

[0045] FIG. 25 is a plot illustrating dwell time versus change in current (AI / Io) of translocation of acrylamide-treated BSA-D10 through a nanopore, with histograms of the dwell time and the change in current, according to an example embodiment.- 6 - 5001986. vl5200.2441001

[0046] FIGS. 26A and 26B illustrate example embodiments of reactions for chemical modification of an analyte.

[0047] FIG. 27 is a table illustrating example analytes that can be chemically modified, according to an example embodiment.

[0048] FIGS. 28A and 28B are plots illustrating current traces of translocation of unmodified and modified BSA-D10, respectively, through a nanopore, according to example embodiments.

[0049] FIGS. 28C and 28D are plots illustrating current traces of translocation of unmodified and modified hPLCZl-DlO, respectively, through a nanopore, according to example embodiments.

[0050] FIGS. 28E and 28F are plots illustrating current traces of translocation of unmodified and modified HS105-D10, respectively, through a nanopore, according to example embodiments.

[0051] FIG. 29A is a plot illustrating current traces corresponding to translocation events of analytes, according to an example embodiment.

[0052] FIGS. 29B-D are stacked plots illustrating analyses of the current traces of FIG.29A, the analyses including translocation time (log scale) versus current blockade (FIG. 29B), histograms of translocation time (log scale, FIG. 29C), and histograms of current blockade (FIG. 29D) for each of the analytes.DETAILED DESCRIPTION

[0053] A description of example embodiments follows.

[0054] Efficient single-molecule protein translocation through biological nanopores can be critical for advancing single-molecule proteomics and protein sequencing technologies. Proteins, which can be composed of 20 distinct amino acids, can exhibit diverse charge distributions and complex structures due to factors such as intra- and intermolecular interactions, which may pose significant challenges for nanopore translocation. Embodiments of methods and systems described herein can include techniques to enhance protein translocation efficiency and smoothness.

[0055] According to some embodiments, nanopores, which can be alternatively referred to herein as a pore, a channel, or a void, can comprise biological or synthetic nanopores. Biological nanopores can include, for example, biomacromolecules (including but not limited to, nucleic acids or proteins, or their artificial analogs with unnatural modifications, e.g.,- 7 - 5001986. vl5200.2441001nucleic acid structure comprising 2’-methxoynucleotides, protein structure comprising alkyne-pyrrolysine) that form a nanometer-sized (for example, having a diameter less than lOOnm, typically less than lOnm) channel that can permit flow of ions and passage of analytes (for example, analyte proteins or polypeptides). The biological nanopores may be disposed in an insulative barrier (for example, a barrier comprising amphiphilic lipids or block copolymers) that does not permit the flow of ions and the passage of the analytes. In some embodiments, biological nanopores can comprise wild-type or mutant forms of protein pores such as such as a-hemolysin (aHL), cytotoxin K (CytK), Mycobacterium smegmatis porin A (MspA) and related homologs. Mutant forms may include, but are not limited to, substitutions, insertions, deletions and / or chemical modifications of amino acids or nucleic acids at construction sites or other regions of the pore, including charged, neutral, or functionalized residues and / or domains that alter pore geometry, electrostatics, or analyte interactions. In some embodiments, such nanopores can have an alpha-helix-rich domain extending outwards from the barrier that can be distinct in geometry and secondary structure from the beta-barrel -rich domain that may be located inside the barrier. The mutant forms of nanopores can be engineered.

[0056] In some contexts used herein, a nanopore can reference a channel, pore, or void defined by a channel structure. In some instances, as used herein, a nanopore can refer to a pore (e.g., a channel or void) in combination with a channel-forming structure (e.g., the channel structure).

[0057] As used herein, a vestibule and a stem side of a nanopore may be defined as follows. As described hereinabove, a nanopore may be disposed in a barrier, e.g., an insulative barrier. Additionally, the nanopore can have structures that extend beyond the barrier (for example, as illustrated and described with respect to FIGS. 2 A and 2B, wherein a nanopore extends transversally through a barrier, from a first face of the barrier through a second face of the barrier, and further extends beyond the barrier). A side with respect to the barrier including a larger portion of an extended structure of the nanopore can be referred to as the vestibule side. A side with respect to the barrier that does not include significant extended structures of the nanopore (with respect to the barrier) or that includes a volume enclosed by a narrowest site along the channel of the nanopore can be referred to as the stem side. In some embodiments, a vestibule side can comprise a side with respect to the barrier that includes a volume enclosed by a widest site along the channel of the nanopore. In other embodiments, a side of a nanopore having an the alpha-helix-rich domain extending outwards- 8 - 5001986. vl5200.2441001from the barrier, as described hereinabove, may be defined as the vestibule side. The side that is opposite to the vestibule side can be defined as the stem side (and vice versa).

[0058] As used herein, “smooth translocation” or “translocate smoothly,” or variations thereof, can refer to achieving substantially consistent and well-defined translocation events. In some embodiments, smooth translocation may be described or characterized by, for each analyte species and / or each conformational state thereof, a monomodal distribution in a scatter plot of dwell time or a mean fractional blockade (e.g., current blockade) with at least about 50% (e.g., 50%) of events within about 0.1 tolerance (e.g., 0.1 tolerance) of fractional blockade and about 1 tolerance of log (base 10) dwell time. While examples of values for assessing dwell time a fractional blockade are provided herein, it should be understood that such values are general benchmarks and may vary based upon factors such as experimental parameters or analyte.

[0059] According to some embodiments, an example of dwell time can comprise time during which a residue of an analyte passes through a nanopore. Improved dwell times may include, for example, dwell times with a more monomodal distribution or a smaller variance of a monomodal distribution. According to some embodiments, an example of current blockade or fractional blockade can include a temporary change in measured current, e.g., ionic current, as a residue of the analyte passes through the nanopore.

[0060] In some embodiments, current or fractional blockade can be affected by a clogging of a nanopore by the analyte. Such clogs may result in altered dwell times of blockade events, e.g., extended dwell times. In some embodiments, a clog may need to be unclogged by a voltage reversal, as further described hereinbelow. Improving translocation or smooth translocation can include decreasing a duration (e.g., with respect to dwell time) or a frequency of such clogging events.

[0061] According to some embodiments, examples of translocation efficiency, which can include protein translocation efficiency, can comprise one or more of capture rate (e.g., a rate at which analytes are captured by a nanopore for translocation) or translocation rate (e.g., rate of translocation of analyte through the nanopore, which can be linked to dwell time of each residue of the analyte) as non-limiting examples. In some embodiments, improving translocation efficiency can include, for example, increasing a capture rate. Improving translocation of an analyte through a nanopore can further comprise optimizing a translocation rate (which may comprise increasing a translocation rate or decreasing a translocation rate based on a given analyte). In other embodiments, translocation efficiency- 9 - 5001986. vl5200.2441001can also be affected by clogging of a nanopore by an analyte, as further described hereinbelow. In such embodiments, an example of improving translocation efficiency can include decrease a frequency or duration of clogging events during the translocation of the analyte through the nanopore.

[0062] According to some embodiments, improving translocation or smooth translocation can relate to the factors described hereinabove, or any combination thereof. Such improvements may correspond to specific experimental parameters, for example, analytes, buffer conditions, solutes, or nanopores as non-limiting examples, or any combination thereof.

[0063] Embodiments of methods and systems described herein may be useful for translocating an analyte, which can include a polypeptide or a protein, through a nanopore. The methods and systems may be useful for improving translocation of the analyte, which may include ensuring smooth translocation of the analyte through the nanopore, as described hereinabove. Smooth translocation of the analyte may be advantageous, as non-limiting examples, for ensuring successful translocation of the analyte through the nanopore (e.g., as opposed to clogging the nanopore) or for facilitating processing of measurements acquired from the nanopore as the analyte is translocated through. The embodiments described herein may further be used independently or in combination with one another.

[0064] FIGS. 1 A and IB are flow diagrams of example embodiments of methods for translocating an analyte through a nanopore. As illustrated in FIG. 1 A, a method 101a can be performed in conditions 103a comprising a structure defining an interior cavity with a barrier dividing the interior cavity into a first chamber and a second chamber. The barrier can comprise a channel structure defining a nanopore therethrough fluidically coupling the first and second chambers. The first chamber can comprise the analyte. The method 101a can comprise modifying 105a at least one side group of the analyte. Said modifying can change at least one of an electrical property or a chemical property of the analyte. The method 101a can further comprise applying 107a a voltage gradient between the first chamber and the second chamber via the nanopore. Applying the voltage gradient can cause a translocation of the analyte through the nanopore from the first chamber to the second chamber. Said modifying the at least one side group can cause a corresponding change in the translocation of the analyte in the presence of the voltage gradient relative to translocation absent the modifying of the at least one side group of the analyte in the presence of the voltage gradient.- 10 - 5001986. vl5200.2441001

[0065] Another example embodiment can be directed to translocating an analyte through a nanopore in a structure defining an interior cavity and a barrier dividing the interior cavity into a first chamber and a second chamber. The barrier can comprise or define a channel structure defining a nanopore therethrough, the channel structure fluidically coupling the first and second chambers. The first chamber can comprise the analyte. The method can comprise applying a voltage between the first chamber and the second chamber via the nanopore. Said applying can cause translocation of the analyte through the nanopore from the first chamber to the second chamber. The method can further comprise modifying at least one side group of the analyte. Said modifying can change at least one of an electrical property or a chemical property of the analyte that causes a corresponding change of the translocation in the presence of the voltage gradient.

[0066] FIG. IB illustrates a method 101b that can be performed in conditions 103b comprising a structure defining an interior cavity with a barrier dividing the interior cavity into a first chamber and a second chamber. The barrier comprises a lipid bilayer disposed on a side of the barrier facing the second chamber and a channel structure defines a nanopore therethrough fluidically coupling the first and second chambers. The method 101b comprises introducing 105b the analyte to the first chamber, wherein said first chamber contains a first solution and the second chamber contains a second solution. The first solution and the second solution are compositionally different. The method 101b further comprises applying 107b a voltage gradient between the first chamber and the second chamber via the nanopore.Applying the voltage gradient can cause a translocation of the analyte through the nanopore from the first chamber to the second chamber.

[0067] In some embodiments, the first solution and the second solution being compositionally different can cause a corresponding change in the translocation of the analyte in the presence of the voltage gradient relative to translocation in the presence of the voltage gradient wherein the first solution and the second solution are compositionally the same or substantially similar. In some embodiments, the first solution and the second solution being compositionally similar or dissimilar can be in reference to the composition of the first solution (without the presence of the analyte) with respect to the composition of the second solution.

[0068] FIGS. 2A and 2B illustrate example embodiments of systems 200a, 200b configured for translocating analytes in a trans-Xo-cis configuration and a cis-Xo-trans configuration, respectively. FIG. 2A illustrates a system 200a configured for trans-Xo-cis- 11 - 5001986. vl5200.2441001translocation, which can also be referred to as stem-to-vestibule (or stem side-to-vestibule side) translocation, as defined hereinabove. A portion of the system 200a is illustrated, and the portion can comprise a barrier 202a, which can comprise a lipid bilayer, and a channel structure 208a. The channel structure 208a can define a nanopore 210a therethrough fluidically coupling a first chamber 204a and a second chamber 206a. An analyte 212a, e.g., a protein, can be disposed on the trans or stem side of the barrier 202a, that is, within the first chamber 204a. The second chamber 206a can be a cis or vestibule side of the barrier 202a. The system 200a can further include voltage source electrodes 214a, 216a configured to apply a voltage gradient to the first and second chambers 204a, 206a via the nanopore 210a. The voltage gradient applied can cause a translocation of the analyte 212a from the first chamber 204a to the second chamber 206a. In some embodiments, a side or chamber with the analyte can include a denaturing agent 218a, e.g., GdmCl, which can be useful for linearizing the analyte for translocation. In some embodiments, the denaturing agent 218a can be added to only one of the chambers, e.g., the first chamber 204a, which can create asymmetric buffer conditions from the stem to the vestibule side.

[0069] FIG. 2B illustrates a system 200b configured for cis-lo-trans translocation, which can also be referred to as vestibule-to-stem (or vestibule side-to-stem side) translocation, as defined hereinabove. A portion of the system 200b is illustrated (which can be similar to the portion of the system of FIG. 2 A), and the portion can comprise a barrier 202b, which can comprise a lipid bilayer, and a channel structure 208b. The channel structure 208b can define a nanopore 210b therethrough fluidically coupling a first chamber 204b and a second chamber 206b. An analyte 212b, e.g., a protein, can be disposed on the cis or vestibule side of the barrier, that is, within the first chamber 204b. Similar to the system of FIG. 2A, the system can include voltage source electrodes 214b, 216b and / or a denaturing agent 218b.

[0070] Further exemplification of embodiments of methods and systems for translocation of analytes, including the embodiments described herein with reference to FIGS. 1 A-2B, are disclosed hereinbelow.

[0071] Tuning Translocation Based on Experimental Conditions

[0072] According to an example embodiment, experimental parameters of protein or polypeptide translocation can be tuned or controlled by employing asymmetric buffer conditions or optimizing protein entry orientation. Example experimental parameters can include, for non-limiting examples, buffer pH, salt species or concentration, denaturant species or concentration, reducing agent species or concentration, and chemical- 12 - 5001986. vl5200.2441001modifications, for example, modifications to adjust protein charge or disrupt disulfide bonds. Tuning the experimental parameters can achieve marked improvements in, according to an example embodiment, nanopore-based biomolecular sensing. The improvements may be helpful for facilitating more efficient protein analysis, which may have broad applications in single-molecule protein sequencing, biomarker detection, and proteomics. According to some embodiments, improving or improvements in translocating an analyte through a nanopore or nanopore-based sensing can include, as a non-limiting example, achieving (or achieving relative improvement in) smooth translocation of an analyte through a nanopore, as described hereinabove. In other embodiments, improvements may also be directed to other factors, such as analyte capture rate or translocation rate as non-limiting examples, or any combination thereof.

[0073] According to some embodiments, a nanopore system can include a two-chamber flow cell, with vestibule side and stem side, as defined hereinabove, chambers containing optimized asymmetric buffers. Guanidinium chloride (GdmCl), a potent denaturant, can be critical for unfolding proteins and enhancing translocation efficiency, for example, by generating a robust electro-osmotic flow within biological nanopores [1, 2] (bracketed references are made in reference to citations provided within the Reference section hereinbelow). However, a presence of high concentrations of GdmCl, for example, concentrations of 3M or higher, on both sides of a barrier, for example, a planar lipid bilayer, can destabilize the lipid bilayer membrane [3] and significantly reduce biological nanopore insertion efficiency, which can create a burden on experiments involving the nanopore system.

[0074] In some embodiments, high concentrations of GdmCl may be employed in a nanopore system by using an asymmetric buffer strategy, for example, as described herein with respect to FIGS. 2A and 2B, wherein a high concentration of GdmCl in a first chamber of a two-chamber system can be useful for facilitating analyte translocation through biological nanopores while minimizing negative effects of the high concentration of GdmCl on the nanopore or the lipid bilayer membrane in a second chamber of the two-chamber system. For example, in an embodiment wherein an analyte, e.g., a polypeptide, can translocate from a stem side to a vestibule side of a nanopore (stem-to-vestibule side direction, for example, as further described with respect to FIG. 2A), GdmCl and the analytes can be introduced into the stem side chamber while biological nanopores can be added to the vestibule side chamber. This separation of the analyte and the biological nanopore can allow- 13 - 5001986. vl5200.2441001the GdmCl and the biological nanopores to be introduced simultaneously without interference.

[0075] Additionally, a planar lipid bilayer can be formed on the vestibule side chamber, for example, using the Montal-Mueller technique, which may be useful for ensuring that the lipid bilayer can remain protected from GdmCl exposure. In other embodiments, GdmCl can be introduced into a desired chamber (e.g., a vestibule or stem chamber) after biological nanopores have been successfully inserted into a lipid bilayer. This versatile approach can support both stem-to-vestibule side and vestibule side-to-stem side translocation setups (as described herein with respect to FIGS. 2A and 2B). Both strategies can be useful for enabling use of GdmCl concentrations up to 3 M in a specific chamber, which may be helpful for fully linearizing protein analytes while maintaining lipid bilayer stability and nanopore insertion efficiency (as described herein with respect to Example 1).

[0076] In some embodiments, asymmetric buffer conditions can include variations in salt (such as KC1, NaCl) or denaturant (such as GdmCl, urea) composition, differences in salt and denaturant concentrations, or distinct pH values between the two chambers (further described hereinbelow with respect to Example 1). These conditions can be tailored to optimize specific experimental objectives, such as enhancing protein unfolding, promoting efficient translocation, or maintaining nanopore stability.

[0077] According to some embodiments, a direction of translocation of an analyte through a nanopore, e.g., a biological nanopore, can affect a smoothness or success of the translocation. In some embodiments, the direction of translocation can include the trans-to-cis configuration and a cis-to-trans configuration described herein with respect to FIGS. 2 A and 2B. Biological nanopores, for example, a-Hemolysin, MspA and CytK, can have an hourglass-like shape characterized by a broad vestibule, which may facilitate an initial capture of analytes, and a narrow stem or constriction, which may be useful for translocation and sensing of the analytes. Biomolecules like deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) may typically be introduced to the vestibule side of these nanopores for analysis due to ease of access provided by the wide opening and enhanced molecular capture efficiency, which may be driven by a higher electric field strength near the vestibule.

[0078] However, according to some embodiments of methods and systems for translocating an analyte, vestibule-to-stem translocation may not be optimal for protein analytes. Proteins, which can be composed of 20 distinct amino acids, can exhibit varying charge distributions. In some embodiments, translocation of polypeptides may be dominantly- 14 - 5001986. vl5200.2441001driven by electro-osmotic force (EOF) instead of electrophoretic force (EPF) [4-6], Vestibule-to-stem translocation direction can result in challenges such as incomplete unfolding of large proteins or formation of blob-like structures prior to entry of an analyte protein into the constriction, which can result in inconsistent or inefficient translocation [7], Additionally, aggregation or jamming of analytes near a vestibule can affect translocation of the analytes through a nanopore and further complicate analysis, particularly for complex protein analytes.

[0079] According to some example embodiments, a strategy of translocating analytes, e.g., proteins or polypeptides, from a stem side to a vestibule side (stem-to-vestibule side) of a nanopore system may be useful for improving analyte translocation. In some embodiments, the stem-to-vestibule translocation can be combined with an asymmetric buffer strategy, as described hereinabove, wherein proteins can be unfolded in a high concentration of GdmCl and a narrow constriction at the stem side can promote initial protein unfolding, which may help ensure full linearization of an analyte before entering a sensing region of a nanopore. The linearization may help enhance detection accuracy. Moreover, starting translocation of analytes from the stem side may further be advantageous by reducing a risk of protein aggregation or clogging in the vestibule of a nanopore, which may ensure smoother and single-file translocation (for example, as described hereinbelow with respect to Example 2). Additionally, according to some embodiments, using a high concentration of GdmCl on the stem side may generate a strong EOF within the biological nanopore [1], which can enhance biomolecule capture rates and further improve analysis efficiency (further described herein with respect to Example 2). According to some embodiments, electroosmotic force (EOF) may arise from a surface charge of a nanopore’s inner wall. Guanidinium (e.g., Gdm+) adsorption at the stem side can render the constriction positively charged, thereby inducing EOF. Increasing the GdmCl concentration can enhance this positive surface charge and can correspondingly strengthen the EOF.

[0080] FIG. 3 illustrates schematically an example embodiment of a system 300 for translocating an analyte 312 through a nanopore 310 using asymmetric buffer conditions. The system can include a structure 320 defining an interior cavity 322 and can comprise a barrier 302, e.g., a barrier including a lipid bilayer, dividing the interior cavity 322 into a first chamber 304 and a second chamber 306. The system can further comprise a channel structure 308 defining the nanopore 310 therethrough fluidically coupling the first chamber 304 and the second chamber 306. The system 300 can further comprise voltage source electrodes 314,- 15 - 5001986. vl5200.2441001316 configured to apply a voltage gradient to the first chamber 304 and the second chamber 306 via the nanopore 310 for translocating the analyte 312.

[0081] As illustrated, the analyte 312 can be translocated from the first chamber 304 to the second chamber 306 in a stem-to-vestibule configuration (trans to cis). The first chamber 304, or the stem side chamber, can include a denaturing agent 318 (e.g., GdmCl, which can be helpful for unfolding the analyte for translocation) and a higher concentration of chloride ions 324. The second chamber 306, or the vestibule side chamber, can comprise the chloride ions 324 and a higher pH (higher concentration of hydroxide ions 326). As such, the system can include asymmetric buffer conditions, wherein a buffer solution of the first chamber 304 is compositionally different from a buffer solution of the second chamber 306.

[0082] The analyte 312 can undergo translocation based on one or more forces 328, 330, 332 applied to the analyte. For example, the analyte can experience an electroosmotic force 328, which can be associated with a concentration gradient of solutes, e.g., the chloride ions, within solutions of the first chamber or the second chamber. In the illustrated embodiment, the EOF 328 can act in favor of translocation in a stem-to-vestibule direction. The analyte 312 can also experience electrophoretic forces 330, 332 based on interactions between heterogenous charges from various residues 334, 336 of the analyte and the voltage gradient applied. The charges can act in favor or disfavor of the translocation. For example, in the illustrated irans-to-cis configuration, wherein the positive voltage electrode 316 is in the cis chamber, a negatively charged residue 334 may experience an EPF 330 toward the cis chamber and a positively charged residue 336 may experience an EPF 332 away from the cis chamber. Example EPFs and residues are labeled with reference numbers, but it should be understood that similar relationships can exist for other residues and EPFs.

[0083] According to some embodiments, using a buffer in the vestibule side chamber 306 with higher pH can cause a larger portion of lysine and histidine residues to be deprotonated and to no longer possess positive charges. Such embodiments can reduce counter-directional EPF, e.g., the EPF 334, against translocation and can result in smoother translocation.

[0084] In some embodiments of methods and systems for translocating an analyte, analyte, e.g., protein, translocation dynamics may be regulated by adjusting at least one of electrolyte species and concentration, buffer pH, or salt species and concentration. Efficient and controlled polypeptide translocation through biological nanopores can be vital for advancing applications such as single-molecule protein sequencing and proteomics for nonlimiting examples. Single-file transport of full-length proteins may typically be achieved- 16 - 5001986. vl5200.2441001using denaturants like urea, guanidinium chloride (GdmCl) or sodium dodecyl sulfate (SDS) [2, 8], However, these approaches may fail to eliminate or sufficiently account for electrostatic interactions between non-uniformly charged polypeptides and other factors of an experimental environment, for example, a nanopore wall, which may lead to clogging or nonsmooth translocation. A potential strategy to overcome such interactions can include engineering nanopore lumen charges to tune an electro-osmotic force (EOF) [5, 9] to modulate polypeptide translocation speed. However, such nanopore engineering can be timeconsuming, and may potentially compromise pore stability and limit adaptability of pores for analyzing diverse analytes.

[0085] Successful translocation of various protein analytes has been previously demonstrated. However, many such demonstrations have used model proteins, which may not accurately represent the spectrum of naturally existing proteins. Some analytes, for example, model proteins such as maltose-binding protein (MBP), may contain moderate negative charges and may be easier to pass through biological nanopores at physiological pH conditions. For example, in some embodiments, proteins with a higher isoelectric point (pl), such as biotin protein ligase (BirA, pl=7.684), may often clog biological nanopores at neutral pH, leading to unsuccessful translocation (Fig 11). Approximately one quarter to one third of human proteins may be positively charged and a small portion of proteins can be highly negatively charged at physiological pH. Highly negatively charged polypeptides may undergo translocation that occurs too quickly, which may be due a strong EPF and which can compromise resolution of measurements of the analyte. Fast translocation may cause distinguishing subtle features of analytes, e.g., polypeptides or proteins, to become more difficult to discern. Therefore, it may be critical to find a solution to facilitate the translocation, including an optimized translocation, of those polypeptides.

[0086] According to some example embodiments of methods and systems for translocating an analyte, protein translocation may be regulated by optimizing an electrolyte, a pH, a salt species, and / or a concentration of solutes used within solutions of a nanopore system (as described hereinbelow with respect to Example 3). In some embodiments, adjusting electrolyte buffer pH can alter charge properties of protein analytes and a nanopore, which may be useful for modulating electrostatic interactions to reduce nonspecific binding. The reduction of nonspecific binding may be helpful for ensuring smooth translocation. In other embodiments, adjusting pH may also be helpful for enabling tuning of a translocation speed of protein analytes (as described herein with respect to Example 3). Higher pH can- 17 - 5001986. vl5200.2441001deprotonate amino acids such as lysine and arginine, which may be useful for increasing protein negativity and enhancing electrophoretic force for faster translocation. Lower pH, by contrast, may reduce net negative charge, which may be useful for slowing translocation and improving detection sensitivity. In some embodiments, salt species and concentration of the salts may also be useful for regulating protein translocation. Adjusting the salt species and the concentration of the salts can allow for optimization of ionic strength, reducing nonspecific interactions and aggregation while modulating the translocation speed.

[0087] Facilitating translocation by chemical modification of protein analytes

[0088] Proteins with complex structures and intricate intra- or intermolecular interactions may present significant challenges during nanopore translocation. Previous reports have indicated that up to 92% percent of human proteins may contain one or more cysteine residues. Those cysteine residues can form disulfide bonds either intramolecularly or intermolecularly, which can induce structural complexity and aggregation, and can significantly hinder efficient polypeptide translocation. Chemical modifications, such as altering specific charges or eliminating disulfide bonds, can enhance protein conformational flexibility and minimize structural impediments, which may be useful for facilitating smoother and more controlled translocation. According to some embodiments, a strategy for facilitating translocation of protein molecules through a nanopore by modifying a protein chemically can comprise processes for altering a charge of the protein, for eliminating disulfide bonding capability, or a combination thereof.

[0089] According to some embodiments, to alter a charge of proteins, reagents modifying residues of the protein, for example, positively charged residues (e.g., histidine, lysine and arginine), can be used to convert a side chain of the residue into a nonionic, zwitterionic, or negatively charged resultant moiety. Selection of the reagents for modifying the residues of the proteins may depend on a target residue. While examples are provided hereinbelow for modification of positively charged residues, e.g., histidine, lysine, and arginine, it should be understood that other reagents may be used for modifying other groups of residues based on chemical characteristics of the residues, for example, a charge or a reactivity.

[0090] In some embodiments, modification of a charge of the residues of the proteins may be useful for avoiding or mitigating an opposing force toward a translocation direction (which may be an EPF) associated with positive charges in an applied electrical field. Here the charge of a residue refers to its capability of ionizing into a charged state in a range of pH of the nanopore experiment. According to some embodiments, various reagents, such as N-- 18 - 5001986. vl5200.2441001hydroxysuccinimide acetate (NHS) esters, may exist for facile modification of residues (e.g., lysine). Additional methods may be available for modification of other residues, for example, histidine or arginine. In some embodiments, a series of reactions using various reagents targeting various residues may be used.

[0091] FIG. 4 illustrates schematically an example embodiment of a system 400 for translocating an analyte 412 through a nanopore 410, the system 400 comprising chemical modifications to the analyte 412. The system 400 of FIG. 4 can be similar to the system 300 of FIG. 3 and similar elements are labeled with like reference numbers but incremented by 100. The system 400 can comprise a structure 420 defining an interior cavity 422 and a barrier 402 dividing the interior cavity 422 into a first chamber 404 and a second chamber 406. The barrier 406, which can include a lipid bilayer, can comprise a channel structure 408 defining the nanopore 410 fluidically coupling therethrough the first chamber 404 and the second chamber 406. The system 400 can further comprise analytes, e.g., the analyte 412, denaturing agents 418 (e.g., Gdm ions), and / or chloride ions 424. The system 400 can further comprise voltage source electrodes 414, 416 for applying a voltage gradient in the system via the nanopore.

[0092] As described hereinabove, chemical modifications may be used to reduce a charge of the analyte 412 by reacting a residue, e.g., a charged residue, with a reagent. Examples of charged residues can include the residues 334 and 336 illustrated herein with respect to FIG.3. In some embodiments, the reagent can include di-tert-butyl decarbonate anhydride (Boc anhydride) and the charged residue can be a lysine residue, an arginine residue, or a histidine residue. Reacting a charged residue with Boc anhydride can result in a charge-neutral residue 436. Such embodiments may be useful for smooth translocation of the analyte by enabling EOFs 428 and EPFs 430 (which may act on negatively charged residues 434) to act in favor of translocation.

[0093] In other embodiments, reagents may be useful for preventing disulfide bond formation by reaction with thiol groups of cysteine residues. While reducing agents like dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP) can temporarily disrupt disulfide bonds, the reducing agents can be often insufficient to keep cysteine-rich proteins, such as bovine serum albumin (BSA), which contains up to 35 cysteines, fully linearized. In some embodiments, permanent chemical modifications, such as covalently blocking thiol groups with reagents, could be used to prevent disulfide reformation. This strategy can reduce structural impediments and can ensure smoother and more consistent protein translocation- 19 - 5001986. vl5200.2441001through the nanopore. Example criteria for selection of such reagents are described hereinbelow.

[0094] According to some embodiments, criteria for selecting reagents for modifying residues of an analyte, for example, a protein or a polypeptide, can include one or more of:a) The reagent can include small resultant moi eties (for example, <lkDa additional MW, and may be selected to be as small as possible, which may be helpful for avoiding: i) hindrance of translocation due to clogging; ii) slower modification kinetics due to steric hindrance; and / or iii) compromise of current signal of the adjacent segments of the protein due to volume of the moiety.b) The reagent can feature quantitative reaction efficiency.c) The reagent can have reasonable costs.d) The resultant moiety may not resemble any naturally occurring post- translational modification (PTM) of a residue that is of interest of a particular nanopore experiment. This criterion may be useful for avoiding ambiguity in nanopore detection of PTMs. For example, NHS acetate can acetylate the side chain of lysine. However, lysine acetylation may be a PTM widely found in nature

[0010] , Thus, NHS acetate should not be used in embodiments wherein acetyl-lysine PTMs are to be studied in the experiment.e) The reagent may not need to be amino-acid-specific. That is, the reagent may modify one or more types of residues. Specifically, a reagent can be used to both alter a charge of an analyte and alkylate cysteines of the analyte.

[0095] According to some embodiments, examples of reagents to alter a charge of an analyte can include but are not limited to: anhydrides (e.g., maleic anhydrides, including carbonic anhydrides / bicarbonates such as diethtyl pyrocarbonate (DEPC) and Boc anhydride), activated esters (e.g., NHS esters, sulfo-NHS esters, pentafluorophenyl esters, tetrafluorophenyl esters, sulfo-tetrafluorophenyl esters), isocyanates, isothiocyanates, acyl halides, and epoxides.

[0096] According to some embodiments, examples of reagents to alkylate cysteines can include but are not limited to: Michael acceptors (e.g., alkenes such as maleimides, acrylamides and methacrylamides; alkynes such as propiolamides); alpha-halo carbonyls (e.g., alpha-halo carboxyls, such as iodoacetamide, bromoacetamide and chloroacetamide); and epoxides.- 20 - 5001986. vl5200.2441001

[0097] In some embodiments, Boc anhydride can be used to modify lysine residues on a protein (as further described herein with respect to Example 4). Boc anhydride can react readily with primary amines, for example, a primary amine of a side chain of lysine (as described hereinbelow with respect to FIG. 18). Hydrolysis of this anhydride may cause no problems when a highly excessive amount (>10mM) is used. Evidence may also exist showing that decarbonate modifying histidine [11, 12], and Boc anhydride modifying imidazole

[0013] , which may support that Boc anhydride can modify histidine residues (as described hereinbelow with respect to FIG. 19). Example 4, as described hereinbelow, can demonstrate modification of an analyte using Boc anhydride. In some embodiments, lysines of unfolded proteins can be modified by NHS acetate through an NHS ester reaction, where an NHS ester reacts with a primary amine to form a stable amide bond (as described hereinbelow with respect to Example 6). NHS ester reactions may be a common method for labeling lysine residues of proteins because the reactions can be efficient and can form a strong covalent linkage.

[0098] In some embodiments, proteins can be treated with reducing agents to eliminate disulfide bonds. The reducing agents can cleave disulfide bonds into free cysteines, and the free cysteines can be reacted with alkylating agents to prevent the free cysteines from forming disulfide bonds again (as described hereinbelow with respect to FIG. 23). Examples of such reducing agents can include TCEP, DTT, and 2-mercaptoethanol (2 -ME). Examples of such alkylating agents can include iodoacetamide, acrylamide and N-ethylmaleimide (NEM). Example 5 shows modification of protein by TCEP and then acrylamide. Example 6, as described hereinbelow, can demonstrate modification of complex protein analytes by TCEP and then NEM.

[0099] FIG. 5 illustrates schematically an example embodiment of a system 500 for translocating an analyte 512 through a nanopore 510, the system 500 including asymmetric buffer conditions. The system 500 of FIG. 5 can be similar to the system 300 of FIG. 3 and similar elements are labeled with like reference numbers but incremented by 200. The system 500 can comprise a structure 520 defining an interior cavity 522 and a barrier 502 dividing the interior cavity 522 into a first chamber 504 and a second chamber 506. The barrier 502, which can include a lipid bilayer, can comprise a channel structure 508 defining the nanopore 510 fluidically coupling therethrough the first chamber 504 and the second chamber 506. The system 500 can further comprise analytes 512-1, 512-2, denaturing agents 518 (e.g., Gdm- 21 - 5001986. vl5200.2441001ions), and chloride ions 524. The system can further comprise voltage source electrodes 514, 516 for applying a voltage gradient in the system 500 via the nanopore 510.

[0100] A presence of cysteine residues 534 may result in a formation of disulfide bonds 538-1, 538-2 by thiol groups of the cysteine residues 534. As illustrated in FIG. 5 and as described hereinabove, the denaturing agents 518, e.g., the Gdm ions, can be helpful for breaking disulfide bonds, e.g., the disulfide bonds 538-1, 538-2, in the analyte 512-1 polypeptide or protein but disulfide bonds, e.g., the intramolecular disulfide bond 538-1 or the intermolecular disulfide bond 538-2 between the analytes 512-1, 512-2, can reform. The disulfide bonds 538-1, 538-2 can affect the translocation of the analyte 512-1 and may result in clogging of the nanopore 510. In some embodiments, the analyte 512-1 may not translocate smoothly because the nanopore 510 cannot pass multiple peptide chains at once and thus may be unable to pass the analyte 512-1 with the disulfide bonds.

[0101] FIG. 6 illustrates an example embodiment of a system 600 for translocating an analyte 612-1 through a nanopore 610, the system 600 including asymmetric buffer conditions and chemical modification of the analyte 612-1. The system 612 of FIG. 6 can be similar to the system 500 of FIG. 5 and similar elements are labeled with like reference numbers but incremented by 100. The system 600 can comprise a structure 620 defining an interior cavity 622 and a barrier 602 dividing the interior cavity 622 into a first chamber 604 and a second chamber 606. The barrier 602, which can include a lipid bilayer, can comprise a channel structure 608 defining the nanopore 610 fluidically coupling therethrough the first chamber 604 and the second chamber 606. The system 600 can further comprise analytes 612-1, 612-2, denaturing agents 618 (e.g., Gdm ions), and chloride ions 624. The system 600 can further comprise voltage source electrodes 614, 616 for applying a voltage gradient in the system 600 via the nanopore 610.

[0102] The analyte 612-1, 612-2 can comprise cysteine residues 634 can be reacted with an agent, for example, with acrylamide. Alkylation of the cysteine residues 634 can be helpful for preventing formation of disulfide bonds, e.g., the disulfide bonds 538-1, 538-2 of FIG. 5, by reacting with thiol groups from residues of the analyte 612-1, 612-2. Accordingly, the analyte 612-1 may remain unfolded and may remain independent from other analytes, e.g., the analyte 612-2, as opposed to the analyte 512-1 of FIG. 5, which can be helpful for translocating the analyte 612-1 without clogging.Exemplification- 22 - 5001986. vl5200.2441001

[0103] Exemplifications of experimental parameters for translocating an analyte through a nanopore are provided hereinbelow. According to some embodiments, experimental parameters (e.g., buffer concentration, electrolyte concentration, or pH as non-limiting examples) may include the values described herein. In other embodiments, experimental parameters can include slight variations to one or more of the parameters described hereinbelow. In some embodiments, the slight variations can include a range of, for example, ±5% from the described values, ±10% from the described values, or ±20% from the described values, as non-limiting examples.

[0104] Example 1 - Buffer Parameters

[0105] According to an example embodiment, a nanopore, e.g., M2MspA, may demonstrate robust pore stability and consistent current conductivity under various asymmetric buffer conditions, including but not limited to:

[0106] i) Symmetric conditions with 1 M KC1, 10 mM Tris, pH 7.5 on both vestibule side and stem sides. (Figure 6, 9)

[0107] ii) Asymmetric conditions with vestibule side: 0.6 M KC1, 3 M GdmCl, 10 mM Tris, pH 7.5; trans: 1 M KC1, 10 mM Tris, pH 7.5. (Figure 6, 7)

[0108] iii) Asymmetric conditions with vestibule side: 1 M KC1, 10 mM Tris, pH 7.5; trans: 0.6 M KC1, 3 M GdmCl, 10 mM Tris, pH 7.5. (Figure 6, 7)

[0109] iv) Asymmetric conditions with vestibule side: 1 M KC1, 10 mM CHES, pH 8.6; trans: 0.6 M KC1, 3 M GdmCl, 10 mM Tris, pH 7.5. (Figure 12)

[0110] v) Asymmetric conditions with vestibule side: 1 M KC1, 10 mM CHES, pH 9.5; trans: 0.6 M KC1, 3 M GdmCl, 10 mM Tris, pH 7.5. (Figure 15)

[0111] vi) Asymmetric conditions with vestibule side: 0.8 M KC1, 1.5 M GdmCl, 10 mM Tris, pH 7.5; trans: 0.6 M KC1, 3 M GdmCl, 10 mM Tris, pH 7.5.

[0112] vii) Asymmetric conditions with vestibule side: 0.64 M KC1, 2.7 M GdmCl, 10 mM Tris, pH 7.5; trans: 0.6 M KC1, 3 M GdmCl, 10 mM Tris, pH 7.5.

[0113] FIG. 7 is a plot 740 illustrating current-voltage (I-V) curves in a system for translocating an analyte through a nanopore using different buffer conditions, according to an example embodiment. The nanopore can be a M2MspA biological nanopore. Three buffer conditions are illustrated, including buffer conditions (i) 1 M KC1, 10 mM Tris, pH 7.5 at both vestibule side and stem side, illustrated using squares; buffer condition (ii): vestibule side-0.6 M KC1, 3 M GdmCl, 10 mM Tris, pH 7.5, and stem side:l M KC1, 10 mM Tris, pH 7.5, illustrated using triangles; and buffer condition (iii): vestibule side: 1 M KC1, 10 mM- 23 - 5001986. vl5200.2441001Tris, pH 7.5, and stem side: 0.6 M KC1, 3 M GdmCl, 10 mM Tris, pH 7.5, illustrated using circles. The plot 740 of FIG. 7 may indicate that asymmetric buffer conditions can result in greater current flow through the nanopore when using asymmetric buffer conditions.

[0114] Example 2 - Translocation Direction

[0115] As described hereinabove, for example, at least with respect to FIGS. 2A and 2B, a direction of translocation of an analyte may influence efficiency of translocation. According to some example embodiments, analyte translocation for nanopore-based experiments can include stem side-to-vestibule side translocation and vestibule side-to-stem side translocation. Effects of translocation direction, such as translocation efficiency, can be experimentally determined, as described hereinbelow.

[0116] According to an example embodiment, a protein analyte, e.g., MBP-D10, can be added to a stem side chamber along with 3 M GdmCl for stem side-to-vestibule side translocation while protein nanopores, e.g., MspA, can be added to a vestibule side chamber. The D10 of the protein analyte (MBP-D10) can represent an amino acid sequence attached to an analyte (for example, the D10 can comprise a sequence of 10 aspartate amino acids). The sequence can be helpful for facilitating capture of the analyte by the nanopore. The analysis can be initiated once a single nanopore has been inserted into the planar lipid bilayer.According to another example embodiment, a protein analyte and GdmCl can be added to the vestibule side and a nanopore can be inserted on the stem side chamber for vestibule side-to-stem side translocation.

[0117] FIGS. 8 A and 8B are plots 840a, 840b illustrating current trajectories (840a) and translocation events (840b) for translocating an analyte through a nanopore from a vestibule side to a stem side, according to an example embodiment. The nanopore can be defined by a channel structure, e.g., MspA, which can be inserted into a lipid bilayer of a barrier in an absence of GdmCl, and the analyte can include an MBP-D10 protein. A voltage of 90 mV may be applied to translocate the analyte through the nanopore using buffer condition (ii) described hereinabove with respect to Example 1 (vestibule side: 0.6 M KC1, 3 M GdmCl, 10 mM Tris, pH 7.5; and stem side: 1 M KC1, 10 mM Tris, pH 7.5).

[0118] FIGS. 9A and 9B are plots 940a, 940b illustrating current trajectories (940a) and translocation events (940b) for translocating an analyte through a nanopore from a stem side to a vestibule side, according to an example embodiment. The nanopore can be defined by a channel structure, e.g., MspA, which can be inserted into a lipid bilayer of a barrier in an absence of GdmCl, and the analyte can include an MBP-D10 protein. A voltage of 90 mV- 24 - 5001986. vl5200.2441001may be applied to translocate the analyte through the nanopore using buffer condition (iii) described hereinabove with respect to Example 1 (vestibule side: 1 M KC1, 10 mM Tris, pH 7.5; and stem side: 0.6 M KC1, 3 M GdmCl, 10 mM Tris, pH 7.5).

[0119] The buffer conditions (ii) and (iii) described herein with reference to Example 1 may be reversed (i.e., a buffer solution of the stem side of the buffer condition (ii) is a buffer solution of the vestibule side of buffer condition (iii), and vice versa). For the experiments of FIGS. 8A-9B, 200 nM of protein analyte MBP-D10 can be added to the chamber containing the GdmCl and a 90mV voltage can be applied to cause translocation of the analyte.

[0120] As illustrated by the plots 840a, 940a of the current trajectories of FIGS. 8A and 9A, respectively, the trans-to-cis configuration (940a) may be associated with significantly more translocation events when compared to the cis-to-trans configuration (840a). The translocation events can be indicated by the peaks corresponding in time in the applied voltage (V) and current (I) plots. The voltage trace can show discrete voltage steps applied over time. The current trace can display the ionic current response under these conditions, wherein each transient downward spike can correspond to a molecule translocation event. The plots 840b, 940b of FIGS. 8B and 9B, respectively, may illustrate that a width of given peaks is shorter for the trans-to-cis configuration than for the cis-to-trans configuration, which can be indicative of smoother translocation of the respective analytes.

[0121] These experiments may reveal that at 90 mV, the translocation efficiency of 200 nM DIO-tagged MBP protein through the MspA nanopore can be significantly lower for vestibule side-to-stem side translocation (840a) compared to stem-to-vestibule side (940a). For vestibule side-to-stem side translocation, fewer events can be observed at voltages below 90 mV, whereas stem-to-vestibule side translocation may exhibit frequent events even at 40 mV (not shown).

[0122] FIGS 10 A- 10C are plots 1040a-c illustrating metrics of translocating an analyte through a nanopore using trans-to-cis and cis-to-trans configurations, according to example embodiments. The metrics can include dwell time in log-scale (log td, 1040a), a relative change in current (AI / Io, 1040b), or a capture rate (1040c), e.g., a capture rate of an analyte in a nanopore. The experimental conditions used for the plots 1040a-c of FIGS. 10 A- 10C can be similar to the cis-to-trans configuration and the trans-to-cis configuration described hereinabove with respect to FIGS. 8A-9B. FIGS. 10B and 10C share a legend with FIG. 10A.

[0123] With respect to translocating an analyte through a nanopore using the cis-to-trans configuration, translocating an analyte using the trans-to-cis configuration may be associated- 25 - 5001986. vl5200.2441001with: a) shorter dwell times with respect to an applied voltage (FIG. 10A), b) larger changes in currents at lower applied voltages (FIG. 10B), and c) higher capture rates at all applied voltages.

[0124] The capture rate of stem-to-vestibule side translocation may increase significantly with voltage, rising from 5.1 ± 0.2 s ' pM1at 80 mV to 26.5 ± 4.3 s ' pM1at 150 mV. In contrast, the capture rate for vestibule side-to-stem side translocation was much lower, increasing from 0.4 s ' pM1at 80 mV to 2.3 s ' pM1at 150 mV, but dropping slightly to 2.0 s1pM1at 170 mV.

[0125] For stem-to-vestibule side translocation, the dwell time of MBP-D10 decreased with increasing voltage, consistent with smooth protein translocation. Additionally, the slight decrease in current blockade with increasing voltage further confirmed efficient translocation. Conversely, for vestibule side-to-stem side translocation, the dwell time decreased between 90 mV and 120 mV, then slightly increased at higher voltages. The current blockade for vestibule side-to-stem side translocation increased with higher voltages, suggesting that MBP-D10 was unable to translocate effectively from vestibule side to stem side under high applied voltage, likely due to crowding in the nanopore vestibule.

[0126] FIG. 11 A is a plot 1140a illustrating dwell time (logarithmic) versus change in current (AI / Io) of translocation of an analyte through a nanopore in an absence of a denaturing agent, according to an example embodiment. The change in current can also be referred to as a current blockade. The analyte, which can include MBP-D10, can be translocated through a nanopore in a trans-to-cis direction (stem side to vestibule side) at 90mV. An electrolyte buffer containing 1 M KC1 and 10 mM Tris, pH 7.5 can be added to both sides.

[0127] FIGS. 1 IB and 11C are histograms 1140b, 1140c of the dwell time (1140b) and the change in current (1140c), respectively, of FIG. 11 A.

[0128] FIG. 12A is a plot 1240a illustrating dwell time (logarithmic) versus change in current (AI / Io) of translocation of an analyte through a nanopore in a presence of a denaturing agent, according to an example embodiment. The analyte, which can include MBP-D10, can be translocated through a nanopore in a trans- -cis direction (stem side to vestibule side) at 90mV applied voltage. The vestibule side can contain an electrolyte buffer containing 1 M KC1 and 10 mM Tris at pH 7.5 and the stem side can contain 0.6 M KC1, 3 M GdmCl and 10 mM Tris at pH 7.5.

[0129] FIGS. 12B and 12C are histograms 1240b, 1240c of the dwell time (1240b) and the change in current (1240c), respectively, of FIG. 12 A.- 26 - 5001986. vl5200.2441001

[0130] FIGS. 11 A-12C may highlight a critical role of GdmCl in facilitating stem-to-vestibule side translocation. According to some embodiments, in the absence of GdmCl on the stem side, protein analytes can frequently clog the nanopore, which can result in two distinct dwell-time populations (as illustrated in FIGS. 11 A and 1 IB versus FIGS. 12A and 12B). A shorter dwell-time population may correspond to pore collisions caused by incomplete protein unfolding. Conversely, when 3 M GdmCl was present on the stem side (FIGS. 12A-12C), MBP-D10 translocation can exhibit a single, well-defined distribution of dwell time and current blockade, which may indicate smooth, single-file translocation through the nanopore.

[0131] Example 3 - Effect of Asymmetric pH on Analyte Translocation

[0132] According to some embodiments, a charge of an analyte, which may be based on charges of residues, e.g., amino acids or side chains of the amino acids, of the analyte, can affect translocation of the analyte through a nanopore. As described hereinbelow, modifying the charge of the analyte, for example, by using asymmetric buffer conditions or pH, can be useful for improving analyte translocation.

[0133] FIGS. 13A and 13B are plots 1340a, 1340b illustrating voltage / current trajectories of MBP-D10 (1340a) and BirA-DlO (1340b), respectively, translocating through a nanopore under symmetrical pH of 7.5 conditions, according to example embodiments. The MBP-D10 and BirA-DlO can be analytes for translocation in a nanopore system with a vestibule chamber comprising a buffer with 1 M KC1 and 10 mM Tris at pH 7.5 and a stem chamber comprising a buffer with 0.6 M KC1, 3 M GdmCl, 10 mM Tris, pH 7.5. Translocation of the analytes may be performed by applying a voltage of 60 mV between the stem and vestibule chambers. As illustrated, the MBP-D10 may exhibit smooth translocation at pH 7.5 (1340a) while BirA-DlO, which can be slightly positively charged, can tend to clog a nanopore (1340b). The clogging can be indicated by the troughs 1342 in the plot of the current trajectory. The voltage plot can show a voltage change over time, wherein each spike can indicate a voltage switch. The current trace can display an ionic current response under such conditions, wherein each transient downward spike can correspond to a molecule translocation event.

[0134] FIGS. 14A and 14B are plots 1440a, 1440b illustrating voltage / current trajectories of MBP-D10 and BirA-DlO, respectively, translocating through a nanopore under pH asymmetric conditions, according to example embodiments. Similar to the plots 1340a, 1340b of FIGS. 13A and 13B, respectively, analytes for translocating through a nanopore can- 27 - 5001986. vl5200.2441001comprise MBP-D10 and BirA-DlO. Buffer conditions on a vestibule side of the nanopore can comprise 1 M KC1 and 10 mM CHES at pH 8.6 and buffer conditions on a stem side can comprise 0.6 M KC1, 3 M GdmCl, and 10 mM Tris at pH 7.5. As compared to the plots 1340a, 1340b of FIGS. 13A and 13B, respectively, the plots 1440a, 1440b may indicate smoother translocation based on the increased number of spikes and decreased frequency or duration of troughs in the current trajectory. These results may suggest that altering a pH of the vestibule side for stem-to-vestibule translocation, which can include the use of asymmetric buffer conditions, can improve translocation. As described hereinabove, improving translocation can comprise one or more of improving smoothness of translocation (as described hereinabove), decreasing a frequency or duration of clogging (for example, as indicated by the trough 1342), increasing a number of translocation effects (which may be related to translocation efficiency), and increasing capture rate.

[0135] FIG. 15 is a plot 1540a illustrating dwell time in log-scale versus change in current (AI / Io) of translocation of analytes through a nanopore, with histograms 1540b, 1540c of the dwell time in log-scale (1540a) and the change in current (1540c), according to an example embodiment. Experimental conditions used to acquire data in the plot 1540a can be similar to the experimental conditions used for acquiring the data of the plots 1440a, 1440b of FIGS. 14A and 14B, respectively. In particular, the analytes, which can include MBP-D10 and BirA-DlO, can be translocated from a stem side comprising buffer conditions of 0.6 M KC1, 3 M GdmCl, and 10 mM Tris at pH 7.5 to a vestibule side comprising buffer conditions 1 M KC1 and 10 mM CHES at pH 8.6 by applying a voltage of 60 mV. Using the asymmetric buffer conditions, translocation of BirA-DlO may exhibit a single population on the plot 1540a, which can indicate more consistent translocation. Additionally, as illustrated by FIG.15, a current blockade (AI / Io) for BirA-DlO (84.1 ± 0.1%) can be slightly higher than that of MBP-D10 (83.3 ± 0.1%) at 60 mV. However, the translocation time (dwell time or log td) of BirA-DlO (132.9 ± 45.2 ms) can be approximately two orders of magnitude longer than that of MBP-D10 (1.4 ± 0.3 ms), which may reflect slower movement of BirA-DlO through the nanopore.

[0136] FIG. 16 is a plot 1640 illustrating translocation time of MBP-D10 at pH 7.5 and pH 8.6 under different applied voltages, according to an example embodiment. In particular, the pH 7.5 and pH 8.6 can be with respect to a vestibule side for stem side-to-vestibule side translocation. Buffer conditions can comprise, for the vestibule side, 1 M KC1 and 10 mM Tris at pH 7.5 or 1 M KC1 and 10 mM CHES at pH 8.6. Buffer conditions for the stem side- 28 - 5001986. vl5200.2441001can comprise 0.6 M KC1, 3 M GdmCl, and 10 mM Tris at pH 7.5 for both vestibule side buffer conditions. Voltages between 60 mV and 90 mV were applied to cause translocation of the MBP-D10 through a nanopore fluidically coupling the vestibule side and the stem side.

[0137] As illustrated in FIG. 16, a higher vestibule side pH can be helpful for decreasing translocation times, particularly at lower applied voltages. For translocation of MBP-D10, the translocation times decrease for both vestibule side buffer conditions (pH 7.5 and pH 8.6) with increasing voltage, which can be consistent with smooth translocation. At 60 mV, the translocation time of MBP-D10 at pH 7.5 (vestibule side) was approximately 3.9 ± 1.7 ms, which decreased by 2.8-fold to 1.4 ± 0.3 ms when the pH increased to 8.6.

[0138] FIG. 17 is a plot 1740 illustrating translocation time of BirA-DlO at pH 8.6 and pH 9.5 under different applied voltages, according to an example embodiment. In particular, the pH 8.6 and pH 9.5 can be with respect to a vestibule side for stem side-to-vestibule side translocation. Buffer conditions can comprise, for the vestibule side, 1 M KC1 and 10 mM CHES at pH 8.6 or 1 M KC1 and 10 mM CHES at pH 9.5. Buffer conditions for the stem side can comprise 0.6 M KC1, 3 M GdmCl, and 10 mM Tris at pH 7.5 for both vestibule side buffer conditions. Voltages between 60 mV and 90 mV were applied to cause translocation of the BirA-DlO through a nanopore fluidically coupling the vestibule side and the stem side.

[0139] As illustrated in FIG. 17, the translocation of BirA-DlO exhibited decreased translocation times with increasing voltage across pH conditions, which can be consistent with smooth translocation. Additionally, the dwell time of BirA-DlO can drop significantly from 132.9 ± 45.2 ms to 6.5 ± 0.2 ms as the vestibule side-side pH increased from 8.6 to 9.5.

[0140] The plots 1640, 1740 of FIGS. 16 and 17, respectively, can highlight a critical role of vestibule-side pH in modulating translocation dynamics. In some embodiments, the plots of FIGS. 16 and 17 may indicate improvements in translocating an analyte through a nanopore, as described hereinabove. For example, the results can further demonstrate that higher pH conditions can significantly reduce or decrease dwell times and enhance translocation efficiency (e.g., faster translocation rate, which can be due to shorter overall dwell times for each residue of an analyte) , particularly for positively charged proteins like BirA-DlO. As described hereinabove, the improvement in translocation may be attributed in part to a localized neutralization of the positive charge at the nanopore due to the higher pH on the vestibule side.

[0141] Example 4 - Boc Anhydride Treatment of Analyte Proteins- 29 - 5001986. vl5200.2441001

[0142] As described hereinabove, chemical modification of residues of analytes, e.g., chemical modification of side groups of analytes such as polypeptides or proteins, can be helpful for improving translocation of the analytes through a nanopore. While specific embodiments are presented hereinbelow, including Boc anhydride treatment of positively charged polypeptides and acrylamide treatment of cysteine residues, it should be understood that other chemical modifications targeting other properties of analyte molecules that affect translocation can be used.

[0143] According to some embodiments, a positively charged analyte, for example, BirA-D10, can be treated with Boc anhydride. The Boc anhydride can react with amine groups, for example, the amine groups of lysine. Products of the Boc anhydride reacting with the amine groups may not be positively charged at a given pH, for example, a pH of about 7.5, which can be helpful for translocation of the positively charged analyte.

[0144] FIGS. 18 and 19 illustrate schematically example embodiments reactions 1844, 1944 for chemical modification of analytes using Boc anhydride. FIG. 18 illustrates a reaction 1844 of Boc anhydride 1846 with an analyte 1848-1, 1848-2 comprising a side chain of lysine 1836-1, 1836-2, which can be in a charged 1836-1 or uncharged 1836-2 state, to form an uncharged product 1850. FIG. 19 illustrates a reaction 1944 of Boc anhydride 1946 with an analyte 1948-1, 1948-2 comprising a side chain of histidine 1936-1, 1936-2, which can be in a charged 1936-1 or uncharged 1936-2 state, to form an uncharged product 1950-1, 1950-2.

[0145] According to an example embodiment, analytes such as BirA-DlO and MBP-D10 can be treated with Boc anhydride. A solution comprising ~56 pM of BirA-DlO in a buffer comprising 1 M GdmCl, 2 mM TCEP and 33 mM Tris at pH 7.5 or a solution comprising ~82 pM MBP-D10 in a buffer comprising 150 mM NaCl and 20 mM at Tris pH 7.5 can be diluted 20* into a buffer of 1 x PBS and 6 M GdmCl. 50 mM of Boc anhydride can be added to the dilution and the dilution can be incubated at 37° C for 1 hour. Subsequently, a reaction between Boc anhydride and the analyte can be quenched by addition of 500 mM ethanolamine / 250 mM HC1 an incubated at 37° C for 1 hour. Samples including the analyte can be exchanged into a buffer of 3 M GdmCl, 0.6 M KC1, and 10 mM Tris at pH 7.5 to remove small molecules using a desalting column, e.g., Zeba desalting columns. 2 mM TCEP can be added to the sample and the sample can be stored at 4° C until tested.

[0146] Boc anhydride-treated MBP-D10 and BirA-DlO can be evaluated using a nanopore system with asymmetric buffer conditions comprising, at a vestibule side, a pH of- 30 - 5001986. vl5200.24410018.6. The analytes can be introduced to the stem side in an electrolyte buffer containing 0.6 M KC1 and 3 M GdmCl at pH 7.5, while a nanopore, e.g., a MspA nanopore, can be inserted from the vestibule side in a buffer containing 1 M KC1 at pH 8.6.

[0147] FIG. 20 is a plot 2040 illustrating translocation time in log scale of MBP-D10 and Boc anhydride treated MBP-D10 using different applied voltages, according to an example embodiment. The MBP-D10 and Boc-anhydride treated MBP-D10 can be translocated using voltages from 60 mV to 90 mV through a nanopore in a stem-to-vestibule direction. A vestibule chamber can include a buffer comprising 1 M KC1 and 10 mM CHES at pH 8.6 and a stem side can include a buffer comprising 0.6 M KC1, 3 M GdmCl, and 10 mM Tris at pH 7.5. As illustrated, Boc anhydride-treated MBP-D10 can exhibit slightly faster translocation compared to the untreated protein, particularly at lower applied voltages.

[0148] FIG. 21 is a plot 2140 illustrating translocation time in log scale of BirA-DlO and Boc-anhydride-treated BirA-DlO using different applied voltages, according to an example embodiment. The BirA-DlO and Boc-anhydride treated BirA-DlO can be translocated using voltages from 60 mV to 90 mV through a nanopore in a stem-to-vestibule direction. A vestibule chamber can include a buffer comprising 1 M KC1 and 10 mM CHES at pH 8.6 and a stem side can include a buffer comprising 0.6 M KC1, 3 M GdmCl, and 10 mM Tris at pH 7.5. As illustrated, Boc anhydride-treated BirA-DlO can exhibit a notably faster translocation compared to the untreated analyte, particularly at lower applied voltages. For example, at 60 mV, the translocation speed of Boc anhydride-treated BirA-DlO was 10 times faster than its untreated counterpart.

[0149] According to some embodiments, charge neutralization via Boc anhydride treatment can enable BirA-DlO to successfully translocate through the nanopore at pH 7.5.

[0150] FIGS. 22A and 22B are plots 2240a, 2240b illustrating dwell time versus current blockade (2240a) and a histogram (2240b) of dwell time, respectively, of translocation of Boc anhydride-treated BirA-DlO at pH 7.5. Boc anhydride-treated BirA-DlO, which can be an analyte, can be translocated through a nanopore using an applied voltage of 80 mV from a stem side including a buffer comprising 0.6 M KC1, 3 M GdmCl and 10 mM Tris at pH 7.5 to a vestibule side including a buffer comprising 1 M KC1 and 10 mM Tris at pH 7.5 via a nanopore, e.g., MspA. The plots 2240a, 2240b illustrate a translocation time of 2.2 ± 0.4 ms at 80 mV. As compared to the plot 1340b of FIG. 13B, Boc anhydride treated BirA-DlO may exhibit significant improvements with respect to dwell time and clogging of the nanopore. The improvements can include, for example, less clogging of the nanopore or smoother- 31 - 5001986. vl5200.2441001translocation, which may be indicated by the more monomodal distribution of the samples as illustrated in FIGS. 22 A and 22B.

[0151] These results can highlight a significant impact of Boc anhydride treatment on enhancing translocation efficiency, particularly for positively charged proteins like BirA-D10, which may likely be due to neutralization of surface charges that otherwise impede nanopore passage.

[0152] Example 5 - Acrylamide Treatment of Analyte Proteins

[0153] According to some embodiments, analytes comprising residues with cysteine molecules, which can form disulfide bonds, can be treated to reduce a likelihood of disulfide bond formation, which can be helpful for smooth translocation of the analyte.

[0154] FIG. 23 illustrates schematically an example embodiment of a reaction 2344 for chemical modification of an analyte using acrylamide. An analyte 2348-1 can comprise a disulfide bond 2338, which can be formed, for example, from cysteine residues 2334. A reaction 2342 for chemically modifying the analyte 2348-1 can comprise reacting the analyte 2348-1 with the disulfide bond 2338 with a reducing agent 2352 to cleave the disulfide bond 2338 and to form an analyte 2348-2 with thiol groups of the cysteine residues 2334. The reaction 2342 can further comprise reacting the analyte 2348-2 comprising the cysteine residues 2334 with acrylamide 2346 to form a product 2350 without thiol groups. The product 2350 may be incapable of forming disulfide bonds.

[0155] According to an example embodiment, BSA-D10, which can be included at a concentration of 3.2 mg / mL in a buffer comprising 6 M GdmCl, 5 mM TCEP, and Tris-HCl at pH 7.5 buffer can be treated with TCEP to cleave disulfide bonds. The treatment can include addition of 20 mM TCEP-HC1, neutralization by 20 mM NaOH, and incubation at 37°C for 1 hour. Samples, which can include the BSA-D10 with cleaved disulfide bonds, can be treated for alkylation of cysteine by addition of 100 mM acrylamide and incubation in darkness at room temperature overnight. The acrylamide-treated samples can be buffer exchanged into a buffer of 3 M GdmCl, 0.6 M KC1, and 10 mM Tris at pH 7.5 to remove small molecules using Zeba desalting spin columns and stored at 4°C until tested.

[0156] Acrylamide-treated B S A-D 10 and untreated B SA-D 10 can be evaluated in using an asymmetric nanopore system, as described hereinabove. Analytes, which can include the B SA-D 10 and the acrylamide-treated B SA-D 10, can be introduced to the stem side in an electrolyte buffer containing 0.6 M KC1, 3 M GdmCl, and 6 mM TCEP at pH 7.5, while the- 32 - 5001986. vl5200.2441001MspA nanopore cam be inserted from the vestibule side in a buffer containing 1 M KC1 at pH 8.6 or 7.5.

[0157] FIG. 24 is a plot 2440a illustrating dwell time versus change in current (AI / Io) of translocation of BSA-D10 through a nanopore, with histograms 2440b, 2440c of the dwell time (2440b) and the change in current (2440c), according to an example embodiment. As described hereinabove, translocation of the BSA-D10 can be carried out in a stem-to-vestibule configuration wherein the stem side includes a buffer comprising 0.6 M KC1, 3 M GdmCl, 6 mM TCEP, and 10 mM Tris at pH 7.5 and the vestibule side includes a buffer comprising 1 M KC1 and 10 mM Tri at pH 8.6. Untreated BSA-D10, as illustrated in the plot 2440a and the histogram 2440c, can display a broad and heterogeneous distribution in translocation times, with no distinct population, which can be indicative of inconsistent translocation through the nanopore.

[0158] FIG. 25 is a plot 2540a illustrating dwell time versus change in current (AI / Io) of translocation of acrylamide-treated BSA-D10 through a nanopore, with histograms 2540b, 2540c of the dwell time and the change in current, according to an example embodiment. As described hereinabove, the experimental conditions for the translocation of acrylamide-treated BSA-D10 can be similar to those of the translocation of BSA-D10 of FIG. 24. In contrast, acrylamide-treated BSA-D10 can show a well-defined population with an average current blockade of 84.2% and an average dwell time of 490.9 ps. The dwell time of acrylamide-treated BSA-D10 can be 687 ps at 70 mV, which can be consistent with protein translocation. These results can highlight an importance of chemical modification of cysteine in smooth protein translocation.

[0159] Example 6 - NHS acetate and NEM Treatment of Complex Protein Analytes

[0160] According to some example embodiments, one or more of the strategies described hereinabove can be utilized for translocation of an analyte through a nanopore. For example, an analyte protein can include one or more of positively charged groups, e.g., lysine groups, or disulfide bond-forming groups, e.g., cysteine groups. Without treatment or modification, such proteins may be difficult to translocate smoothly through a nanopore.

[0161] FIGS. 26A and 26B illustrate example embodiments of reactions 2644a, 2644b for chemical modification of an analyte. The chemical modifications described can be similar to the chemical modifications of FIGS. 18 and 23. Continuing with respect to the reaction 2644a of FIG. 26A, the analyte 2648a, which can contain a positively charged or amine side group 2636 such as lysine, can be reacted with N-hydroxy succinimide acetate (NHS acetate) 2646a- 33 - 5001986. vl5200.2441001to form a product 2650a with neutralized lysine residues. As illustrated in FIG. 26B, the analyte 2648b, which can contain cysteine residues 2634 capable of forming disulfide bonds, can be reacted with N-ethylmaleimide (NEM) 2646a to form a product 2650b without thiol groups.

[0162] FIG. 27 is a table 2740 illustrating analytes that can be chemically modified, according to an example embodiment. The table 2740 includes names, length (in amino acid residues), charges (net), number of cysteine residues (#C), number of lysine residues (#K), and charge after modification (-K / C). The analytes can include as non-limiting examples bovine serum albumin (BSA), human phospholipase C zeta 1 (hPLCZl), and HS105. The molecular weights of the foregoing analytes may all exceed 50 kDa. These proteins can possess markedly different net charges: BSA and HS105 can be strongly negatively charged, with net charges of -22.1 and -28.9, respectively, while hPLCZl can comprise abundant lysine and arginine residues, which can result in a highly positive net charge of +13.2. The diverse charge states of these analytes can profoundly affect their capture efficiency and translocation dynamics in the nanopore system. Moreover, complex tertiary structures and the presence of multiple cysteine residues of analytes can make them prone to forming disulfide bonds and aggregating under partially denaturing conditions.

[0163] According to an example embodiment, chemical modification of analytes can be carried out by combining an analyte with a DIO-tag to a C-terminal of the analyte to form a tagged analyte (i.e., BSA to BSA-D10, hPLCZl to hPLCZl-DlO, and HS105 to HS105-D10), which can be helpful for increasing pore capture efficiency. Chemical modification of the tagged analytes can include mixing samples of the tagged analytes (concentration in parenthesis), e.g., BSA-D10 (1.65mg / mL), hPLCZl-DlO (7.74mg / mL), HS105-D10 (2.92mg / mL), in a buffer with 6 M GdmCl and 40 mM 4-(2-hy droxy ethyl)- 1-piperazineethanesulfonic acid (HEPES) at pH 7.5. The samples can be treated with TCEP to cleave disulfide bonds by addition of 20 mM TCEP-HC1 (of which the pH can be preadjusted to pH 7) and incubation at 37° C for 1 hour. The samples can be diluted with DMSO into 80% DMSO, after which 50 mM NHS aetate and 20 mM NEM can be added for incubation in darkness at 37° C overnight. The samples can be buffer exchanged into a buffer of 6 M GdmCl and 10 mM Tris at pH 7.5 using successive (3x) dilution and concentration cycles. The exchange can be performed using an ultrafilter unit to remove all small molecules and the samples can be stored at 4° C until tested.- 34 - 5001986. vl5200.2441001

[0164] According to example embodiments, experiments translocating analytes through a nanopore, as further described hereinbelow, can carried out under the following conditions. Final concentrations of analytes within a buffer solution can comprise 21 nM unmodified BSA-D10, 400 nM unmodified hPLCZl-DlO, 100 nM unmodified HS105-D10, 20 nM modified BSA-D10, 337.5 nM modified hPLCZl-DlO, and 50 nM modified HS105-D10. In some embodiments, each analyte can be in a separate buffer solution to be translocated in an individual experiment. Other embodiments can comprise mixing multiple analytes within a given buffer solution. The analytes can be translocated using a stem-to-vestibule configuration. A buffer solution of the stem side chamber (trans side) can comprise 0.6 M KC1, 3 M GdmCl, and 10 mM Tris at pH 7.5 and a buffer solution of the vestibule side chamber (cis side) can comprise 0.6 M KC1, 0.4M GdmCl, and 10 mM Tris at pH 7.5. The translocation may be carried out under an applied voltage of 90 mV through a nanopore such as MspA.

[0165] FIGS. 28A and 28B are plots 2840a, 2840b illustrating current traces of translocation of unmodified (2840a) and modified (2840b) BSA-D10, respectively, through a nanopore, according to example embodiments. The nanopore may become frequently clogged after introducing 21 nM of BSA-D10 to the stem side chamber (2840a) containing 3 M GdmCl and 10 mM Tris. The cloggings could not be removed by applying higher voltages or reversing the voltage polarity (not illustrated), which can suggest that the clogging likely originated from incompletely unfolded BSA-D10 molecules or residual disulfide linkages. After introducing chemically modified BSA-D10 (using NHS acetate and NEM), pore clogging can be much less frequent (2840b) and the nanopore could be readily reopened by voltage reversal, after which translocation events could be observed (not shown).

[0166] FIGS. 28C and 28D are plots 2840c, 2840d illustrating current traces of translocation of unmodified (2840c) and modified (2840d) hPLCZl-DlO, respectively, through a nanopore, according to example embodiments. Addition of unmodified hPLCZl-D10, even at concentrations up to 400 nM, can produce very few capture events (2840c). Such low capture rates can be due to a strong positive charge of unmodified hPLCZl-DlO, which can be electrophoretically repelled from a nanopore under positive bias. In contrast, addition of chemically modified hPLCZl-DlO (which can have a net charge of about -35.3) can show a markedly increased capture rate under similar or identical experimental conditions (2840d), which can indicate improved solubility or charge neutralization of the hPLCZl-DlO analyte after modification.- 35 - 5001986. vl5200.2441001

[0167] FIGS. 28E and 28F are plots 2840e, 2840f illustrating current traces of translocation of unmodified (2840e) and modified (2840f) HS105-D10, respectively, through a nanopore, according to example embodiments. HS105-D10 may be the largest analyte of those described herein with respect to FIGS. 28A-28F. Moderate pore clogging may be observed at 100 nM of unmodified HS105-D10 in a nanopore system (2840e). Addition of modified HS105-D10 can result in more abundant and well-defined translocation events (2840f).

[0168] FIG. 29A is a stacked plot illustrating current traces 2940a- 1, 2940a-2, 2940a-3 corresponding to translocation events of analytes, according to an example embodiment. As described herein with reference to FIGS. 27-28F, the current traces can correspond with analytes including chemically modified BSA-D10 (2940a- 1), chemically modified hPLCZl-D10 (2940a-2), and chemically modified HS105-D10 (2940a-3). The current traces 2940a- 1, 2940a-2, 2940a-3can be associated with protein translocation at +90 mV applied voltage under denaturing conditions, e.g., vestibule side including a buffer comprising 0.6 M KC1, 0.4 M GdmCl, and 10 mM Tris at pH 7.5 and stem side including a buffer comprising 0.6 M KC1, 3 M GdmCl, and 10 mM Tris at pH 7.5.

[0169] FIGS. 29B-D are stacked plots illustrating corresponding analyses of the current traces 2940a- 1, 2940a-2, 2940a-3 of FIG. 29 A, the corresponding analyses including translocation time (log scale) versus current blockade 2940b- 1, 2940b-2, 2940b-3 (FIG. 29B), histograms of translocation time 2940c-l, 2940c-2, 2940c-3 (log scale, FIG. 29C), and histograms of current blockade 2940d-l, 2940d-2, 2940d-3 (FIG. 29D) for each of the analytes. The plots of FIGS. 29B-D generally show a single population with respect to dwell time and current blockade for each of the modified analytes, which can be indicative of smooth translocation of analyte molecules through a nanopore. The smooth translocation may be associated with the modification of the analytes.

[0170] The teachings of all patents, published applications and references cited herein are incorporated by reference in their entirety.

[0171] While example embodiments have been particularly shown and described, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the embodiments encompassed by the appended claims.References- 36 - 5001986. vl5200.24410011. Mehrafrooz B, Yu L, Pandey L, Siwy ZS, Wanunu M, Aksimentiev A. Electroosmotic Flow Generation via a Sticky Ion Action. ACS Nano. 2024 Jul9; 18(27): 17521-17533. doi: 10.1021 / acsnano.4c00829. Epub 2024 Jun 4. PMID: 38832758; PMCID: PMC11233251.2. Yu L, Kang X, Li F, Mehrafrooz B, Makhamreh A, Fallahi A, Foster JC, Aksimentiev A, Chen M, Wanunu M. Unidirectional single-file transport of full-length proteins through a nanopore. Nat Biotechnol. 2023 Aug;41(8): 1130-1139. doi:10.1038 / s41587-022-01598-3. Epub 2023 Jan 9. Erratum in: Nat Biotechnol. 2023 Oct;41(10):1483. doi: 10.1038 / s41587-023-01995-2. PMID: 36624148; PMCID: PMC 10329728.3. Yu L, Kang X, Alibakhshi MA, Pavlenok M, Niederweis M, Wanunu M. Stable polymer bilayers for protein channel recordings at high guanidinium chloride concentrations. Biophys J. 2021 May 4; 120(9): 1537-1541. doi:10.1016 / j.bpj.2021.02.019. Epub 2021 Feb 20. PMID: 33617833; PMCID:PMC8204206.4. Huang G, Willems K, Soskine M, Wloka C, Maglia G. Electro-osmotic capture and ionic discrimination of peptide and protein biomarkers with FraC nanopores. Nat Commun. 2017 Oct 16;8(1):935. doi: 10.1038 / s41467-017-01006-4. PMID:29038539; PMCID: PMC5715100.5. Sauciuc A, Morozzo Della Rocca B, Tadema MJ, Chinappi M, Maglia G.Translocation of linearized full-length proteins through an engineered nanopore under opposing electrophoretic force. Nat Biotechnol. 2024 Aug;42(8):1275-1281. doi: 10.1038 / s41587-023-01954-x. Epub 2023 Sep 18. PMID: 37723268.6. Yu L, Kang X, Li F, Mehrafrooz B, Makhamreh A, Fallahi A, Foster JC, Aksimentiev A, Chen M, Wanunu M. Unidirectional single-file transport of full-length proteins through a nanopore. Nat Biotechnol. 2023 Aug;41(8): 1130-1139. doi:10.1038 / s41587-022-01598-3. Epub 2023 Jan 9. Erratum in: Nat Biotechnol. 2023 Oct;41(10):1483. doi: 10.1038 / s41587-023-01995-2. PMID: 36624148; PMCID: PMC 10329728.7. Sauciuc A, Whittaker J, Tadema M, Tych K, Guskov A, Maglia G. Blobs form during the single-file transport of proteins across nanopores. Proc Natl Acad Sci U S A. 2024 Sep 17;121(38):e2405018121. doi: 10.1073 / pnas.240501812L Epub 2024 Sep 12.- 37 - 5001986. vl5200.2441001Erratum in: Proc Natl Acad Sci U S A. 2025 Mar 25;122(12):e2503397122. doi: 10.1073 / pnas.2503397122. PMID: 39264741; PMCID: PMC11420176.8. Soni N, Freundlich N, Ohayon S, Huttner D, Meller A. Single-File Translocation Dynamics of SDS-Denatured, Whole Proteins through Sub-5 nm Solid-State Nanopores. ACS Nano. 2022 Jul 26; 16(7): 11405-11414. doi:10.1021 / acsnano.2c05391. Epub 2022 Jul 3. PMID: 35785960; PMCID:PMC7613183.9. Martin-Baniandres, P., Lan, WH., Board, S. et al. Enzyme-less nanopore detection of post-translational modifications within long polypeptides. Nat. Nanotechnol. 18, 1335-1340 (2023).10. Choudhary C, Weinert BT, Nishida Y, Verdin E, Mann M. The growing landscape of lysine acetylation links metabolism and cell signalling. Nat Rev Mol Cell Biol. 2014 Aug;15(8):536-50. doi: 10.1038 / nrm3841. PMID: 25053359.11. Melchior WB, Fahrney D. Ethoxyformylation of proteins. Reaction of ethoxyformic anhydride with .alpha. -chymotrypsin, pepsin, and pancreatic ribonuclease at pH 4. Biochemistry 19709 (2), 251-258. DOI: 10.1021 / bi00804a010.12. Narumi R, Yamamoto T, Inoue A, Arata T. Substrate-induced conformational changes in sarcoplasmic reticulum Ca2+-ATPase probed by surface modification using diethylpyrocarbonate with mass spectrometry. FEBS Lett. 2012 Sep 21;586(19):3172-8. doi: 10.1016 / j.febslet.2012.06.029. Epub 2012 Jul 5. PMID: 22771786.13. Fatemeh Jahani, Mahmood Tajbakhsh, Hamid Golchoubian, Samad Khaksar.Guanidine hydrochloride as an organocatalyst for N-Boc protection of amino groups. Tetrahedron Letters. Volume 52, Issue 12, 2011, Pages 1260-1264, ISSN 0040-4039.- 38 - 5001986. vl

Claims

5200.2441001CLAIMSWhat is claimed is:

1. A method of translocating an analyte through a nanopore in a structure defining an interior cavity and a barrier dividing the interior cavity into a first chamber and a second chamber, the barrier comprising a channel structure defining the nanopore therethrough fluidically coupling the first and second chambers, the first chamber comprising the analyte, the method comprising:modifying at least one side group of the analyte, said modifying changing at least one of an electrical property or a chemical property of the analyte; and applying a voltage gradient between the first chamber and the second chamber via the nanopore, said applying causing a translocation of the analyte through the nanopore from the first chamber to the second chamber, said modifying the at least one side group causing a corresponding change in the translocation of the analyte in the presence of the voltage gradient relative to translocation absent the modifying of the at least one side group of the analyte in the presence of the voltage gradient.

2. The method of Claim 1, wherein modifying the at least one side groups of the analyte comprises introducing a reagent to the analyte, the reagent reacting selectively with the at least one side groups.

3. The method of Claim 2, wherein the reagent reacts selectively with at least one of:a charged side group of the at least one side chains; anda thiol side group of the at least one side chains.

4. The method of Claim 2, wherein the reagent comprises at least one of:an anhydride, wherein the anhydride comprises a maleic anhydride or a carbonic anhydride, or a precursor or derivative thereof;an activated ester, wherein the activated ester comprises an N- hydroxysuccinimide ester, a pentafluorophenyl ester, a tetrafluorophenyl ester, a sulfo-tetrafluorophenyl ester, or a precursor of derivative thereof;an isocyanate;an acyl halide;- 39 - 5001986. vl5200.2441001an epoxide;a Michael acceptor, wherein the Michael acceptor comprises maleimide, acrylamide, methacrylmide, propiolamide, or a precursor or derivative thereof; and an alpha-halo carbonyl, wherein the alpha-halo carbonyl comprises iodoacetamide, bromoacetamide, or chloroacetamide.

5. The method of Claim 2, further comprising introducing a reducing agent to the analyte and the reagent, wherein the reducing agent comprises one or more of tris(2- carboxyethyl)phosphine, diothitreitol, or 2-mercaptoethanol.

6. The method of Claim 1, wherein modifying the at least one side groups of the analyte comprises tuning a pH of a solution of the first chamber or the second chamber to change a charge of the at least one side groups.

7. The method of Claim 1, wherein the analyte is a polypeptide or protein.

8. The method of Claim 1, further comprising:observing an electronic signature of the analyte produced by the analyte passing through the nanopore; anddetermining a property of the analyte based on the electronic signature observed.

9. The method of Claim 1, further comprising:controlling a rate of translocation of the analyte through the nanopore based on one or more of a salt concentration of the first or the second solution, a salt species of the first or the second solution, pH of the first or the second solution, or the voltage gradient applied.

10. A method of translocating an analyte through a nanopore in a structure defining an interior cavity and a barrier dividing the interior cavity into a first chamber and a second chamber, the barrier comprising a lipid bilayer disposed on a side of the barrier facing the second chamber and a channel structure defining the nanopore- 40 - 5001986. vl5200.2441001therethrough fluidically coupling the first and second chambers, the method comprising:introducing the analyte to the first chamber, wherein said first chamber contains a first solution, the second chamber contains a second solution, and wherein the first solution and the second solution are compositionally different; and applying a voltage gradient between the first chamber and the second chamber via the nanopore, the applying the voltage gradient causing a translocation of the analyte through the nanopore from the first chamber to the second chamber.

11. The method of Claim 10, wherein:the first solution comprises a denaturing agent and wherein the denaturing agent comprises at least one of guanidinium chloride, urea, or sodium dodecyl sulfate (SDS).

12. The method of Claim 11, wherein:the first solution comprises the denaturing agent at a concentration that does not disrupt a lipid bilayer or the channel structure.

13. The method of Claim 10, wherein:a pH of the first solution is sufficiently different from a pH of the second solution such that a charge of a side group of the analyte in the first solution is different to a charge of the side group in the second solution, and wherein the side group of the analyte undergoes a change in the charge of the side group as it passes through the nanopore.

14. The method of Claim 10, wherein:a concentration of an electrolyte in the first chamber is different from a concentration of the electrolyte in the second chamber and wherein the electrolyte comprises at least one of potassium (K+) ions or chloride (Cl’) ions.

15. The method of Claim 10, further comprising:inserting the channel structure into the barrier, the channel structure defining a first aperture and a second aperture of the nanopore, the first aperture having a smaller- 41 - 5001986. vl5200.2441001circumference than the second aperture, the channel structure being inserted such that the first aperture is in fluidic contact with one of the first or the second solution and the second aperture is in fluidic contact with the other of the first or the second solution.

16. The method of Claim 15, further comprising controlling an orientation of the channel structure in the barrier by containing, by the second solution, the channel structure, the channel structure being contained by the second solution causing the channel structure to be inserted such that the first aperture is in fluidic contact with the first solution and the second aperture is in fluidic contact with the second solution.

17. The method of Claim 15, wherein inserting the channel structure further includes positioning the channel structure such that a distance between the smaller aperture and the barrier is smaller than a distance between the larger aperture and the barrier.

18. The method of Claim 10, further comprising:observing an electronic signature of the analyte produced by the analyte passing through the nanopore; anddetermining a property of the analyte based on the electronic signature observed.

19. A system for translocating an analyte through a nanopore, the system comprising:a structure defining an interior cavity, the structure comprising a barrier dividing the interior cavity into a first chamber configured to contain a first solution and a second chamber configured to contain a second solution compositionally different from the first solution, wherein the barrier comprises a lipid bilayer disposed on a side of the barrier facing the second chamber and a channel structure defining the nanopore therethrough fluidically coupling the first and second chambers; and voltage source electrodes configured to apply a voltage gradient to the first and second chambers via the nanopore, the voltage gradient applied causing a translocation of the analyte of the first solution or the second solution through the nanopore.- 42 - 5001986. vl5200.244100120. The system for translocating an analyte of Claim 19, wherein:the channel structure defines a first aperture and a second aperture of the nanopore, the first aperture having a smaller circumference than the second aperture, and wherein the first aperture is in fluidic contact with the first solution and the second aperture is in fluidic contact with the second solution.5001986. vl

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