Methods for sensing an analyte and corresponding analytes and setups

Abstract

Methods and corresponding analytes and setups for sensing an analyte are disclosed. An example method comprises configuring an analyte to self-modulate translocation of the analyte through a channel. The configuring comprises coupling at least one chemical group to a corresponding one or more residues of the analyte. The method further comprises causing the translocation of the analyte with the at least one chemical group coupled thereto through the channel. Each chemical group is configured to cause a corresponding transient change in the translocation. The method further comprises sensing a property associated with the analyte translocating through the channel. the corresponding transient change caused by a given chemical group of the at least one chemical group modulates the sensing of a portion of the analyte. By modulating sensing of the analyte, embodiments described herein can, for non-limiting example, enhance the fidelity or quality of information acquired of the analyte.

Description

5200.2455001Methods for Sensing an Analyte and Corresponding Analytes and SetupsRELATED APPLICATION

[0001] This application claims the benefit of U.S. Provisional Application No.63 / 788,779, filed on April 14, 2025. The entire teachings of the above application are incorporated herein by reference.INCORPORATION BY REFERENCE OF MATERIAL IN XML

[0002] This application incorporates by reference the Sequence Listing contained in the following extensible Markup Language (XML) file being submitted concurrently herewith:

[0003] File name: 52002455001. xml; created April 14, 2026, 6,609 Bytes in size.GOVERNMENT SUPPORT

[0004] This invention was made with government support under Grant No.R01HG012553 awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUND

[0005] Sequence-related information associated with some analyte molecules (which may also be referred to herein as analytes), such as proteins or nucleic acids for non-limiting examples, may be determined based on translocating said analyte through nanopores. The translocation of such analytes can enable sensing of portions of an analyte, which may comprise, for example, amino acid or nucleotide residues, as the portions pass through the nanopores. In addition to successful translocation, the rate of translocation, including the ability to control or modulate said rate, may be an important factor in enabling high-fidelity and efficient recovery of desired sequence-related information of analytes.SUMMARY

[0006] Example embodiments of methods, systems, and analytes or compounds described herein may be directed to modulating translocation of analytes, which may include, for nonlimiting example, protein analytes, through a channel, e.g., a nanopore, for accessing sequence-related information of said analytes.- 1 - 5051544. vl5200.2455001

[0007] An example embodiment is directed to a method of sensing an analyte. The method comprises configuring an analyte to self-modulate translocation of the analyte through a channel. The configuring comprises coupling at least one chemical group to a corresponding one or more residues of the analyte. The method further comprises causing the translocation of the analyte with the at least one chemical group coupled thereto through the channel. Each chemical group of the at least one chemical group is configured to cause a corresponding transient change in the translocation of the analyte. The method further comprises sensing a property associated with the analyte translocating through the channel. The corresponding transient change caused by a given chemical group of the at least one chemical group modulates the sensing of a portion of the analyte associated with the given chemical group.

[0008] The at least one chemical group can comprise at least one stall er group and the corresponding transient change in the translocation of the analyte can comprise a transient delay of the translocation caused by the at least one staller group blocking the translocation of the analyte through the channel. The transient delay can enable sensing the portion of the analyte over an extended period of time relative to a different portion of the analyte that is not associated with the at least one staller group. The staller group can comprise one or more of the following: an organic dye or a precursor or derivative thereof, wherein the organic dye comprises one or more of (a) an Alexa Fluor dye, a cyanine dye, a 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) dye, a rhodamine dye, or a quencher dye; (b) an oligomeric structure, wherein the oligomeric structure comprises one or more nucleotides or nucleotide variants; (c) a structure comprising a peptide or peptide variants; (d) a hydrophilic flexible polymer chain, wherein the hydrophilic flexible polymer chain comprises one or more of poly(ethylene glycol), poly(vinyl alcohol), or polypropylene glycol); (e) a dendron; (f) an oligosaccharide; (g) an organometallic complex, wherein the organometallic complex comprises one or more of nickel, copper, osmium, ruthenium, rhodium, iridium, zinc, cobalt, cadmium, iron, silver, or manganese; (h) a metallo-organic framework (MOF), wherein the MOF comprises a polyoxometalate cluster; or (i) a compound having universal affinity to proteins, wherein the compound comprises one or more of tris(l,10-phenanthroline) ruthenium(II), Coomassie Blue, nitrolotri acetic acid and precursors or derivatives thereof, or 4,4'-bis(phenylamino)-[l,l'-binaphthalene]-5,5'-disulfonic acid (bis-ANS). The method can further comprise causing a stalling moiety of the staller group to undergo a conformational change from a first conformation to at least a second conformation. The first conformation- 2 - 5051544. vl5200.2455001and the at least the second conformation of the stalling moiety can have differing capacities to block the translocation of the analyte through the channel.

[0009] The at least one chemical group can be configured to undergo a conformational change based on a state of internal bonding within the at least one chemical group. The internal bonding can comprise covalent bonding or non-covalent interactions. The conformations of the chemical group can comprise one or more of origamis, hairpins, G-quadruplexes, structure-forming aptamers, or duplexes.

[0010] Coupling the at least one chemical group to the at least one corresponding residue can comprise coupling the at least one chemical group to a side chain or a post-translational modification of the corresponding residue. Coupling the at least one chemical group to the corresponding residue can comprise causing a formation of a covalent bond.

[0011] Coupling the at least one chemical group to the corresponding residue of the analyte can comprise chemoselectively coupling a chemical group of the at least one chemical group to the corresponding residue, the corresponding residue being of a given type of residues of the analyte. Coupling a chemical group of the at least one chemical group to the corresponding residue can further comprise one or more of: (a) coupling a linker group to the corresponding residue and coupling the chemical group to the linker molecule, e.g., wherein the linker group comprises a biorthogonal handle, or (b) coupling a linker moiety of the chemical group to the corresponding residue, wherein the linker moiety comprises N-succinimidyl ester and the corresponding residue is lysine.

[0012] The at least one chemical group can comprise at least a first type and at least a second type of chemical group. Each type of chemical group can be configured to couple with a corresponding residue type of the analyte.

[0013] Sensing the property associated with the analyte translocating through the channel can comprise detecting a current associated with the analyte translocating through the channel.

[0014] The corresponding transient change of the each chemical group can be associated with the each chemical group arriving at or passing through the channel.

[0015] Another example embodiment can be directed to a self-modulating analyte. The self-modulating analyte comprises an analyte comprising one or more residues disposed in sequential arrangement. The self-modulating analyte further comprises at least one chemical group coupled to the one or more residues. The at least one chemical group is configured to- 3 - 5051544. vl5200.2455001cause an associated transient change in translocation of the analyte through a channel configured for sequential translocation of the one or more residues of the analyte.

[0016] The at least one chemical group can comprise at least one stall er group and the corresponding transient change in the translocation of the analyte can comprise a transient delay of the translocation caused by the at least one staller group blocking the translocation of the analyte through the channel. The transient delay can enable sensing the portion of the analyte over an extended period of time relative to a different portion of the analyte that is not associated with the at least one staller group. The staller group can comprise one or more of the following: an organic dye or a precursor or derivative thereof, wherein the organic dye comprises one or more of (a) an Alexa Fluor dye, a cyanine dye, a 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) dye, a rhodamine dye, or a quencher dye; (b) an oligomeric structure, wherein the oligomeric structure comprises one or more nucleotides or nucleotide variants; (c) a structure comprising a peptide or peptide variants; (d) a hydrophilic flexible polymer chain, wherein the hydrophilic flexible polymer chain comprises one or more of poly(ethylene glycol), poly(vinyl alcohol), or polypropylene glycol); (e) a dendron; (f) an oligosaccharide; (g) an organometallic complex, wherein the organometallic complex comprises one or more of nickel, copper, osmium, ruthenium, rhodium, iridium, zinc, cobalt, cadmium, iron, silver, or manganese; (h) a metallo-organic framework (MOF), wherein the MOF comprises a polyoxometalate cluster; or (i) a compound having universal affinity to proteins, wherein the compound comprises one or more of tris(l,10-phenanthroline) ruthenium(II), Coomassie Blue, nitrolotri acetic acid and precursors or derivatives thereof, or 4,4'-bis(phenylamino)-[l,l'-binaphthalene]-5,5'-disulfonic acid (bis-ANS). The staller group can be configured to be rigid wherein, in a given conformation of the staller group and a corresponding residue coupled to the staller group, a length associated with the staller group and the corresponding residue is larger than the diameter of the nanopore.

[0017] The self-modulating analyte can further comprise a linker molecule coupling a chemical group of the at least one chemical group to a corresponding residue of the one or more residues.

[0018] The at least one chemical group can be chemoselectively coupled to a corresponding residue of the one or more residues, the corresponding residue being of a given type of residues of the analyte.- 4 - 5051544. vl5200.2455001

[0019] The at least one chemical group can comprise at least a first type and at least a second type of chemical group. Each type of chemical group can be configured to be coupled to a corresponding residue type of the analyte.

[0020] The at least one chemical group can be coupled to the one or more residues via one or more of (a) a linker group configured to couple the corresponding residue and the chemical group, wherein the linker group comprises a biorthogonal handle, or (b) a linker moiety of the chemical group to the corresponding residue, wherein the linker moiety comprises N-succinimidyl ester and the corresponding residue is lysine.

[0021] Another example embodiment can be directed to a setup for sensing a property of an analyte. The setup comprises means for causing translocation an analyte through a channel. The setup further comprises means for the analyte to self-modulate translocation of the analyte through the channel. The analyte is configured to self-modulate at one or more residues of the analyte and the self-modulating causes a corresponding transient change associated with the one or more residues during the translocation of the analyte. The setup further comprises means for sensing a property associated with the analyte translocating through the channel, the corresponding transient change caused by a given chemical group of the at least one chemical group modulating the sensing of a portion of the analyte associated with the given chemical group.BRIEF DESCRIPTION OF THE DRAWINGS

[0022] 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.

[0023] FIG. l is a block diagram of an example embodiment of a method of sensing an analyte.

[0024] FIG. 2 is a schematic illustration of an example embodiment of a device comprising a nanopore structure that may be used for sensing an analyte.

[0025] FIGS. 3 A-3D are schematic illustrations of example embodiments of a method of sensing an analyte, wherein the analyte is configured to self-modulate translocation.

[0026] FIG. 3E is a plot illustrating an example current trace that may be associated with translocation of the analyte of FIGS. 3A-3D.- 5 - 5051544. vl5200.2455001

[0027] FIGS. 4A-4B are schematic illustrations of example schemes for coupling a chemical group to an analyte, according to example embodiments.

[0028] FIGS. 5A and 5B are schematic illustrations of an example conjugation of a chemical group to an analyte, according to an example embodiment.

[0029] FIGS. 6A and 6B are schematic illustrations of an example conjugation of another chemical group to an analyte, according to an example embodiment.

[0030] FIGS. 7A-7C are schematic illustrations of an example conjugation of a chemical group to an analyte via a linker, according to an example embodiment.

[0031] FIGS. 8A and 8B are schematic illustrations of an example staller that may be a chemical group used in an example embodiment.

[0032] FIGS. 9A-9D are schematic illustrations of conformations and corresponding sizes of example embodiments of a self-modulating analyte comprising an analyte and one or more chemical groups conjugated thereto.

[0033] FIGS. 10 A- 10C are scatter plots illustrating dwell time versus current blockade for translocation of an unmodified analyte through a nanopore at applied voltages of 60 mV, 70 mV, and 80 mV, according to an example embodiment.

[0034] FIG. 11 A-l IF are histograms of dwell time (FIGS. 11 A-l 1C) and current blockade (FIGS. 1 ID-1 IF) based on the scatter plots of FIGS. 10A-10C.

[0035] FIGS. 12A and 12B are plots of dwell time and current blockade with respect to applied voltage based on the scatter plots of FIGS. 10A-10C.

[0036] FIG. 13 is a plot of an example current trace associated with sensing an unmodified analyte translocating through a nanopore.

[0037] FIGS. 14A-14L are plots illustrating example dwell time and current blockade results for an example embodiment of sensing an analyte, the analyte being a self-modulating analyte, during translocation at applied voltages of 80 mV, 90 mV, 100 mV, and 120 mV.

[0038] FIGS. 15A and 15B are plots illustrating dwell time and current blockade, respectively, versus applied voltage based on the example embodiment of FIGS. 14A-14L.

[0039] FIGS. 16A-16P are plots illustrate example characteristic translocation patterns associated with sensing an example embodiment of a self-modulating analyte translocating a through channel.

[0040] FIGS. 17-20 are plots of translocation events associated with example embodiments of sensing a self-modulating analyte.- 6 - 5051544. vl5200.2455001

[0041] FIG. 21 A illustrates an example model protein that may be used for evaluating an example embodiment of a method for sensing an analyte.

[0042] FIG. 2 IB is a plot of an example current trace associated with sensing of a selfmodulating analyte comprising the model protein of FIG. 21 A and AF647 modifications to lysine groups of the model protein.

[0043] FIGS. 22A-22C are plots illustrating example current traces associated with example embodiments of sensing an analyte, the analyte being a self-modulating analyte, through another example nanopore.

[0044] FIGS. 23 A and 23B are plots illustrating counts of proteins having or not having lysine residues (FIG. 23 A) and numbers of lysine residues within proteins (FIG. 23B).

[0045] FIGS. 24A-24E are chemical structures of example chemical groups that may be coupled to an analyte, according to an example embodiment.DETAILED DESCRIPTION

[0046] A description of example embodiments follows.

[0047] Translocation of analytes, which can comprise unfolded proteins or polypeptides as non-limiting examples, through a channel such as a nanopore can enable access to sequence-related information of said analytes [1, 2] (numbers enclosed in brackets refer to references cited at the end of the present Specification). Prior studies have demonstrated mechanisms of unfolding and translocating protein analytes, with non-limiting examples comprising incorporating enzymes that can unfold a protein (such as through integration of an unfoldase onto the nanopore[3]), engineering the nanopore to enhance driving forces (such as electroosmotic force) to mechanically pull apart folded protein structure, and / or unfolding the protein by denaturants such as urea and guanidine [4, 5], In addition to translocation of the analytes through a channel, regulation of said translocation may be useful for ensuring quality and fidelity of information acquired from said analytes.

[0048] In some cases, when compared with enzyme-based approaches, enzyme-free approaches (which may include, for non-limiting example, the denaturant-based techniques), may hold potential for more efficient capturing of analytes by a nanopore while avoiding complications such as those associated with a need to preserve activity of the enzyme, a need to incorporate specific peptide sequences for recognition, and stalling or backstepping of the enzyme on certain motifs. However, an enzyme may also serve as a molecular ratchet to regulate the speed of translocation and may generate steps that allow discrete reading of- 7 - 5051544. vl5200.2455001certain segments of the analyte protein. Without such a molecular control, enzyme-free systems may have a dilemma such that a higher driving force, which may be associated with high capture rates and pulling an analyte through a pore, may result in a faster translocation, which can compromise detailed reading of a current trace due to factors such as instrumental limits of bandwidth or signal -noise ratio (SNR). Besides, without a stepping mechanism, unregulated translocation of macromolecules may fit in a drift-diffusion model of motion, with a stochastic one-dimensional diffusion factor contributing to varied translocation speed and irreproducibility of an exact current trace [6],

[0049] Despite prior efforts to compensate for such issues, for example, with pooling multiple translocations and advanced signal analysis methods [7], obtaining comparable signal quality with stepped motion may remain elusive. Therefore, adding a stepping mechanism can still be beneficial.

[0050] Prior efforts to create enzyme-free stepping mechanisms have included (i) adding a cysteine track on an inner lumen of a nanopore and exploiting disulfide exchange events to hop cysteines on an analyte along the track [8], (ii) direct mechanical pulling with piezoelectric [9] or optical tweezer actuation

[0010] , or (iii) incorporating expansion structures into the sequence

[0011] , However, these approaches may not work seamlessly for single-file full-length protein translocation. For example, hopping an analyte by a track in a nanopore may be designed to work with short strands of nucleic acids so that an analyte can be effectively traversed with a single thiol group on the terminus of the analyte. However, such an approach may not be functional with lengths associated with full-length proteins. Direct mechanical pulling can be limited by instrumental precision and can be incompatible with desired high-throughput applications of nanopore sensors. Incorporating expansion structures into the sequence can be exclusive to nucleic acid analytes due to a dependence on basepairing. For full-length protein analytes, an effective enzyme-free stepping or speed regulation mechanism is not yet available.

[0051] Embodiments of systems, compositions, devices, and methods described herein can be directed to a process of chemically labeling an analyte, for example, an analyte protein, with bulky moieties on specific sidechains, which can be selected for using chemoselective conjugation as a non-limiting example. As used herein, chemoselective conjugation can refer to selective conjugation of chemical groups to one or more residues or monomers of an analyte, e.g., amino acids of proteins for non-limiting example, based on, for example, chemical properties of a portion of the one or more residues of the analyte.- 8 - 5051544. vl5200.2455001Chemoselective conjugation can comprise conjugating a chemical group to a given type or class of residue. With respect to the example of protein analytes, a type of residue can comprise, for non-limiting examples, a specific amino acid type (e.g., lysine, arginine, aspartic acid, etc.) or groups of residues based on chemical properties (e.g., positively charged, negatively charged, polar, non-polar, or other categorizations).

[0052] According to some embodiments, the bulky moieties can be configured to cause modulation, for non-limiting example, stalling or slowing, of translocation of the analyte at or in proximity to the labels. This modulation can be helpful for reading a segment of analyte proteins adjacent to the label. In some embodiments, reading of the segment of the analyte protein can be processed as a step with enhanced signal fidelity when compared to free translocation.

[0053] As used herein, an analyte can refer to compound for which sequence information is being acquired, for example, using a nanopore experiment. The analyte can comprise, for non-limiting examples, full-length proteins, partial proteins, polypeptides, nucleic acids, and other structures comprising sequential residues or structures. A self-modulating analyte can refer to an analyte that is configured to cause one or more transient changes to translocation of said analyte through a channel such as a nanopore. The self-modulating analyte may cause the one or more transient changes without modifying experimental conditions, e.g., an applied voltage or potential for causing translocation of an analyte in a nanopore device or a buffer composition of the nanopore device. For non-limiting example, at a constant applied potential, a self-modulating analyte may cause a slowing or stalling of translocation. As further described herein, the self-modulating analyte can comprise an analyte, e.g., a protein, and one or more chemical groups coupled to residues or post-translational modifications of the analyte. As used herein, post-translational modifications can refer to, for non-limiting example, chemical groups associated with one or more residues of an analyte resulting from chemical modification of the analyte during (e.g., to a portion of an analyte that has already been formed) or following translation.

[0054] As used herein, a channel can refer to an aperture through which an analyte can be translocated or passed through. The channel can comprise a nanopore, for non-limiting example, a solid-state or a biological nanopore, wherein a biological nanopore structure such as a channel protein defines a nanopore channel. According to some embodiments, the channel can be configured to fluidically couple two or more chambers, wherein each chamber can contain a solution and wherein one or more chambers can contain an analyte. The analyte- 9 - 5051544. vl5200.2455001can be translocated through the channel, for non-limiting example, by an applied voltage. In some embodiments, the channel can be used to sense the analyte or a property associated with the analyte. For non-limiting example, a device comprising the channel can be used to sense a current caused by an applied potential (e.g, wherein the potential is applied to the two or more chambers). The current flow through the channel may vary during translocation of an analyte and measurements of the current can reveal information about said analyte.

[0055] Embodiments described herein can comprise a process in which protein analytes intended for nanopore detection by single-file translocation are preprocessed. In some embodiments, preprocessing can comprise using bulky moieties (which can be referred to as ‘staller’ hereinafter) to chemically label certain sidechains of an analyte using chemoselective conjugation, which may be useful for creating intermittent stalling or slowing of translocation at the labels. Translocation of the labeled analytes may produce current traces comprising characteristic spikes or steps, which can be useful for enhancing accuracy of fingerprinting of analyte proteins.

[0056] According to some embodiments, a rationale and scheme for fingerprinting proteins using the method described hereinabove can be borne from data described hereinbelow with reference to FIGS. 23A and 23B. Out of 20,820 human proteins reviewed in the Uniprot database, a vast majority (20,644) contain at least one lysine residue, with the human proteins comprising a median lysine count of 21, and 20,199 proteins containing more than two lysines (FIG. 23 A). Considering the human proteins lacking any lysines, this population may comprise primarily of shorter proteins compared to the population with lysines, with median amino acid sequence lengths of 71 and 414, respectively (FIG. 23 A). According to some embodiments, by labeling locations of each lysine in proteins, a “barcode” unique to each protein may be created and read as a series of stalls separated by distinct time periods throughout the translocation of the analyte. A protein with a median number of lysines may contain 21 such stalls, which can be recorded in nanopore experiments as spikes or plateaus in a corresponding current trace, separated by a distribution of possible distances between consecutive lysines that can be mapped and matched against databases of known protein sequences (FIG. 23B). According to some embodiments, the barcode data may be useful as a standalone measurement or may be used in conjunction with sequencing data acquired from analytes. In other embodiments, such a methodology can be further extrapolated to include the labeling of other amino acids in addition to or as a substitution of lysines, which can alter the barcode and provide alternative readouts during- 10 - 5051544. vl5200.2455001nanopore experiments. While modifications to lysine are described herein, such modifications are not intended to be limiting and modification of residues of an analyte can be carried out chemoselectively for a residue type of choice.

[0057] FIG. 1 is a block diagram of an example embodiment of a method 101 of sensing an analyte. The method 101 can comprise configuring 103 an analyte to self-modulate translocation of the analyte through a channel. The configuring can comprise coupling at least one chemical group to a corresponding residue of the analyte. The method 101 can further comprise causing 105 the translocation of the analyte with the at least one chemical group coupled thereto through the channel. Each chemical group of the at least one chemical group can be configured to cause a corresponding transient change in the translocation of the analyte. The method 101 can further comprise sensing 107 a property associated with the analyte translocating through the channel. The corresponding transient change caused by a given chemical group of the at least one chemical group can modulate the sensing of a portion of the analyte associated with the given chemical group.

[0058] FIG. 2 is a schematic illustration of an example embodiment of a device 200 comprising a nanopore structure 202 that may be used for sensing an analyte. The device 200 can comprise a first chamber 204 and a second chamber 206, which may be separated by a surface or a membrane , e.g., the membrane 208. According to some embodiments, the first chamber 204 and the second chamber 206 can be a trans chamber and a cis chamber, respectively. Trans and cis chambers may be determined based on their relation to the nanopore structure. For example, the nanopore structure 202 can comprise a narrower portion 209 (e.g., a stem portion) and a wider portion 211 (e.g., a vestibule portion). The trans side or trans chamber 204 may be the chamber contacting the narrower or stem side 209 of the nanopore structure 202. Alternatively, the nanopore structure 202 may comprise a portion that extends away from the membrane 208. The trans chamber 204 can be the chamber or side in which an aperture of the nanopore structure 202 is disposed closer to a surface of the membrane 208.

[0059] The nanopore structure 202 can define a channel fluidically coupling the first chamber 204 to the second chamber 206. An analyte, e.g., a protein 210 can be translocated through the channel of the nanopore structure 202, for example, based on an applied voltage from a voltage source 212. As described hereinabove, alternative means may also be applied for translocating the analyte. The analyte may experience an electroosmotic force (EOF) 213, which may be due to electrolyte concentrations in buffer solutions of the first chamber 204- 11 - 5051544. vl5200.2455001and the second chamber 206, and an electrophoretic force (EPF) 215, which may be caused by electrical interactions between the the applied voltage and side groups of the analyte. The buffer solutions of the first chamber 204 and the second chamber 206 may be asymmetric, i.e., different with respect to each other. In particular, the buffer solutions, without consideration of the analyte, of the first chamber 204 and the second chamber 206 may be different from each other. For example, the first chamber 204 or trans chamber, which contains the analyte, can comprise a denaturing agent 214, e.g., guanidinium chloride (GdmCl) for non-limiting example, for unfolding a protein analyte.

[0060] According to some embodiments, electroosmotic flow (EOF) 213 and electrophoretic force (EPF) 215 may be the primary driving forces facilitating the passage of the linearized polypeptide through the pore. Additionally, in the presence of high concentrations of GdmCl 214 (e.g., at concentrations of greater than IM), Gdm+ ions may be sticky and reside in the nanopore lumen, which can lead to a large EOF of chloride counterions and may drive the passage, or translocation, of protein chains through a nanopore. The analyte protein can carry heterogeneous charges from various residues, which experience EPF either along or against the direction of translocation

[0061] FIGS. 3 A-3D are schematic illustrations of example embodiments of sensing an analyte, wherein the analyte is configured to self-modulate translocation. In particular, FIGS.3 A-3D illustrate an example device 300 comprising a nanopore structure 302 that is similar to the device 200 and the nanopore structure 202 of FIG. 2. The analyte 310 can comprise a protein analyte configured to self-modulate translocation, for example, through coupling of chemical groups, e.g., the chemical groups 316-1, 316-2, 316-3. During translocation of the analyte 310, for example, using an applied voltage from a voltage source 312, each chemical group, for example, the chemical group 316-1, can be configured to cause a corresponding transient change in the translocation of the analyte 310, which may modulate sensing of an analyte or a property thereof, for example, a current trace associated with translocation of the analyte 310 through the nanopore structure 302. FIGS. 3A-3C illustrate example corresponding stalling associated with chemical groups coupled to the analyte 310. As described hereinabove, the chemical groups may be configured to cause a transient change in the translocation of the analyte 310, but the analyte 310 can eventually fully translocate through the channel or nanopore, as illustrated in FIG. 3D.

[0062] According to some embodiments, an analyte, e.g., the analyte 310, may comprise partial or complete modification of target residues, e.g., the lysine residues described- 12 - 5051544. vl5200.2455001hereinabove. The analyte 310 can further comprise segments, e.g., the a segment 320-1, the b segment 320-2, and the c segment 320-3. The segments may be portions of the analyte 310 disposed proximally to a chemical group. In some embodiments, the segments may be of a length corresponding to a reading frame of a nanopore structure. The modulation of translocation of the analyte 310 through the nanopore structure 302, e.g., stalling of the analyte 310 in the nanopore structure 302, can enable modulation of sensing properties associated with the segments.

[0063] FIG. 3E is a plot 340 of an example current trace that may be associated with translocation of the analyte of FIGS. 3A-3D. The chemical groups of the analyte 310 can comprise staller groups, which may be configured to cause self-modulation of the analyte 310 translocating through the nanopore structure 302 by delaying or stalling said translocation. In a current trace acquired based on this translocation, signals associated with the segments may be, for example, extended, due to a greater dwell time of the segments in the nanopore structure 302. According to the example embodiment, the stalling of FIGS. 3A, 3B, and 3C may correspond to stepwise segments 1, 2, and 3, respectively, of the current trace.

[0064] Embodiments for modulating translocation of an analyte described herein may be applicable to both solid-state and biological nanopores. In some embodiments, stallers may include moieties that are large enough to transiently stall, or significantly slow the translocation of the analyte protein, but not so large as to cause a permanent stalling, or clogging, of the translocation. Chemoselective conjugation on certain sidechains can refer to conjugation on side chains by linkers on the staller that preferentially react with specific amino acids. For non-limiting examples, linkers can comprise N-Hydroxysuccinimide esters on lysine, maleimides on cysteine, 4-Phenyl-3H-l,2,4-triazoline-3,5(4H)-diones (PTADs) on tyrosine. Chemoselective conjugation can also include, but is not limited to, conjugation on side chains with a certain type of post-translational modification (PTM) by linkers on the staller that preferentially react with specific PTMs on one or more types of amino acids, such as zinc-dipicolylamine on phosphorylation, phenylboronic acid on glycosylation.

[0065] FIGS. 4A-4B are schematic illustrations of example schemes 422a, 422b for coupling a chemical group to an analyte, according to example embodiments. In the example embodiment, the chemical group can be a staller 424a, 424b configured to cause stalling of the analyte during translocation. The scheme 422a of FIG. 4A can comprise direct coupling or conjugation of a staller 424a to an analyte 410 (e.g., a protein) with a target moiety 425- 13 - 5051544. vl5200.2455001indicated by R. In the scheme 422a, a linker can be a part of a staller molecule, as indicated by the reactive staller 426a, and the staller may react with the side chain in one reaction.

[0066] The scheme 422b of FIG. 4B can comprise conjugation of the analyte 410 having the target moiety 425 indicated by R with a linker 428 first to form an analyte-linker complex 430. The analyte-linker complex 430 can optionally undergo purification 432 (as indicated by the parenthesis). A reactive staller 426b can be reacted to with the analyte-linker complex 430 to couple the staller 424b to the analyte 410.

[0067] According to some embodiments, the target moiety 425 on the protein can denote moieties of interest, including but not limited to sidechains of specific residues, specific PTMs, or specific motifs of an analyte. In some embodiments, such linkers can be embodied as separate molecules with an intermediate biorthogonal handle, such as an alkyne as a nonlimiting example, and can be reacted with the side chains first. Stallers with a complementary biorthogonal handle, such as an azide, can be added subsequently.

[0068] FIGS. 5A and 5B are schematic illustrations of an example conjugation of a chemical group to an analyte, according to an example embodiment. FIG. 5 A illustrates an example chemical reaction 522, wherein the chemical group can be a staller, for example, Alexa fluor 647 (AF647). The staller can be a reactive staller 526 with amine-N-hydroxysuccinimide (NHS) conjugated thereon and can be reacted with the analyte 510 in a single reaction targeting a target moiety 525 of a lysine residue of the analyte 510. The reactive staller 526 can be reacted with the analyte 510 in a single step to form an analyte-staller complex 534, which may be an example embodiment of a self-modulating analyte. FIG. 5B illustrates the example chemical group (in particular, a reactive staller 525) with the NHS conjugated thereon, AF647-NHS.

[0069] FIGS. 6A and 6B are schematic illustrations of an example conjugation of another chemical group to an analyte, according to an example embodiment. FIG. 6A illustrates an example chemical reaction 622, wherein the chemical group can be a staller, for example, Cyanine 5 (Cy5). The analyte 610 can be reacted with a reactive staller 626 comprising maleimide conjugated to a Sulfo Cy5 and can be reacted with the analyte 610 in a single reaction targeting a target moiety 625 of a cysteine residue of the analyte to form a selfmodulating analyte 634. FIG. 6B illustrates the example chemical group with the maleimide conjugated thereon, Sulfo Cy5-maleimide 626.

[0070] FIGS. 7A-7C are schematic illustrations of an example conjugation of a chemical group to an analyte via a linker, according to an example embodiment. FIG. 7A illustrates an- 14 - 5051544. vl5200.2455001example chemical reaction 722 for conjugating a chemical group, which can be a staller, e.g., Cy5, onto an analyte 710. In this reaction, the staller may be conjugated to a target moiety 725 lysine residues of the analyte 710 in a plurality of steps. A first step of the reaction 722 can comprise conjugating a linker 728, for example, endo-bicyclonon-4-yne (endoBCN)-NHS, to the lysine residue 725 of the analyte 710 to form an analyte-linker complex 730. As a second step, a reactive staller, e.g., sulfo Cy5-azide 726, can be conjugated to the linker of the analyte-linker complex 730, thereby conjugating the staller onto the analyte to form a self-modulating analyte 734. FIG. 7B illustrates the example linker, endoBCN-NHS 728, used in the reaction of FIG. 7A. FIG. 7C illustrates the example staller group, sulfo Cy5-azide 726, used in the reaction of FIG. 7 A.

[0071] FIGS. 8A and 8B are schematic illustrations of an example staller that may be a chemical group used in an example embodiment. FIG. 8A is a chemical structure of an example staller, Alexa Fluor 647, in a potential conformation of the staller. A fluorescence core with a resonant bond system of the AF647 may rigidify part of the molecule (which may be indicated by the bolded bonds in the drawing) so that a structure of the molecule may not change significantly according to general chemistry principles. FIG. 8B illustrates a 3-dimensional (3D) chemical structure of the AF647 illustrated in FIG. 8A. As indicated the AF647 may have a width, as indicated by the dotted line, of approximately 2.1 nm, which may be larger than a diameter of an example channel, e.g., a 1.2 nm reported diameter for a Mycobacterium smegmatis porin A (MspA) nanopore

[0012] , When conjugated to the analyte, the staller-analyte complex may be even larger.

[0072] FIGS. 9A-9D are schematic illustrations of conformations and corresponding sizes of example embodiments of a self-modulating analyte comprising an analyte and one or more chemical groups conjugated thereto. FIG. 9A illustrates a chemical structure an example embodiment of a self-modulating analyte 934 comprising an analyte 910 having a sequence GGGGGKGGGGG with a chemical group, in particular, a staller group 926, conjugated to the lysine of the analyte. FIG. 9B illustrates a chemical structure of an example conformation of the self-modulating analyte 934 of FIG. 9A, indicating that in the conformation, the selfmodulating analyte 934 may have a width of 2.56 nm, as measured from a backbone of the analyte 910 to a distal end of the staller 926 from the backbone. FIG. 9C illustrates a chemical structure of another example conformation of the analyte of FIG. 9 A. In the conformation of FIG. 9C, the width of the self-modulating analyte, from a backbone of the analyte to a distal portion of a staller, can be around 1.04 nm, which may be small enough to- 15 - 5051544. vl5200.2455001pass through a channel of a MspA nanopore. FIG. 9D illustrates another chemical structure of the example conformation of FIG. 9C.

[0073] According to some embodiments, selection of a chemical group serving as a staller can be based on hindrance of motion by approximating a stall-or-pass pattern. For example, in some embodiments, a staller can completely stall translocation when assuming certain conformations. The staller may be configured to or may be capable of turning or reconfiguring into another conformation that allows passage. In the conformation enabling passage of the staller, the staller may pass through a channel or pore at a steady speed within fluctuation. According to some embodiments, conformations can comprise, for non-limiting examples, one or more of the following structures: origamis, hairpins, G-quadruplexes, structure-forming aptamers, or duplexes.

[0074] In some embodiments, a bulkiness of or associated with a staller, which may be used to achieve such stall-or-pass action described hereinabove, may more precisely comprise: 1) there exists some conformation of the staller so that, the resultant conjugated structure including the peptide backbone has a width higher than the diameter of the constriction of the pore (for example, the staller illustrated in FIG. 9B), and / or the staller contains a rigid structure that is wider (width in the sense of the maximum distance among pairs of atoms in the staller) than the diameter of the constriction of the pore (for example, the staller of FIGS. 8A and 8B); 2) there also exists some conformation of the staller so that, the resultant conjugated structure including the peptide backbone has a width lower than the diameter of the constriction of the pore (for example, the stallers of FIGS. 9C and 9D). In some embodiments, a width of a conformation can be counted as a maximum distance to the peptide backbone, among atoms of the staller. According to some embodiments, the conformations may be discrete conformations and may instead operate, for non-limiting example, on a continuum. For example, a self-modulating analyte can comprise an analyte with a coupled chemical group having freedom to rotate around a given axis. The rotation of the chemical group may facilitate stalling or passing of the analyte through a channel.

[0075] According to some embodiments, it may be beneficial for complication of a current trace, which can be acquired during translocation of an analyte, associated with a flexible staller structure to be minimized (the complication can comprise, for non-limiting example, fluctuation of ion exclusive volume due to internal conformational fluctuation and ineffective stalling due to compressed staller structure squeezed through a pore, etc.). Thus, according to some embodiments, the stallers can be preferably selected to be rigid, with- 16 - 5051544. vl5200.2455001minimal internal degrees of bond rotation, with conformational variation necessary to switch between stall and pass accounted by, for non-limiting example, 1) a linkage between a staller and a sidechain of an analyte residue, and flexibility of the sidechain per se; and / or 2) a state change of internal bonding, including but not limited to hydrogen bonding, salt bridge, electrostatic attraction / ion pairing, chelation, and / or change of covalent bond; and / or 3) interactions with a pore, such as affinity to the pore lumen, or motifs on the pore vestibule.

[0076] In some embodiments, a flexible staller structure may be preferred. In such embodiments, the flexible structure may be in reference to internal flexibility of a chemical or staller group or at flexibility at a linking or coupling portion between a staller and an analyte. The rigid and flexible staller structures may be employed in conjunction with one another to provide differential modulation of translocation associated with different residues based on, for example, chemoselective conjugation.

[0077] Embodiments for sensing an analyte described hereinbelow may comprise selfmodulating analytes with chemical groups such as stallers, which may be described based on categories of a moiety of the staller, e.g., a bulky moiety, configured for causing stalling of the analyte. While example embodiments are provided, it should be understood that said examples are not intended to be limiting and that one of skill in the art may reasonably understand that derivatives, precursors, or analogs of compounds or compositions described may be used to achieve a desired effect of modulating translocation of an analyte through a channel.

[0078] As described hereinabove, embodiments can include but are not limited to derivatives that have linkers with conjugation handles. Such linkers can include, for nonlimiting example, one or more of chains with a contour length of less than about 10 nm, with a backbone of carbon-carbon bonds, amides, ethers, phosphoesters, etc., and a terminal functionalization for conjugation such as alkynes, azides, alkenes (including but not limited to strained alkenes such as trans-cyclooctene (TCO) derivatives, acrylamides, acrylates, maleimide), carbonyls (including but not limited to ketones and aldehydes), halides (including but not limited to acyl and sulfonyl halides, carbonyl-alpha halides), epoxides, amines, tetrazines, reactive esters (including but not limited to succinimidyl esters, sulfonate esters, pentafluorophenyl esters, tetrafluorophenyl esters, squarate monoesters), isocyanates, isothiocyanates, carboxyls (through activation with e.g. carbodiimides), PTADs, or other example conjugation handles described herein.- 17 - 5051544. vl5200.2455001

[0079] In some embodiments, choices of rigid stallers can comprise one or more organic dyes, which can typically comprise extended n conjugated systems as a backbone for resonance with photons of desired wavelength, which are rigid. One advantage of organic dyes is their wide commercial availability due to their broad range of applications. As used herein, dyes can be in reference to, for non-limiting example, organic substances, whether pure compound or mixtures, with significant optical (ultraviolet (UV) / visible / infrared (IK)) absorbance properties. In some embodiments, the dyes can comprise fluorescent properties, including but not limited to those commercially sold or generally referred to as “dye”, “pigment”, “quencher”, “fluorophore”, “chromophore”, “colorant”, “pigment”. It should be noted that, in some embodiments and with respect to staller groups, geometry of the dye or similar compound may be a primary factor for selecting the dye. As such, colorless / bleached states / derivatives of dyes that still retain backbone bulkiness can also be included as embodiments of dyes, such as leuco methylene blue or colorless state of phenolphthalein, and all substances that can generally be referred to as “leuco dyes.” In some embodiments, photon emitting dyes (e.g., colored dyes, infrared, or ultraviolet) can be used for, e.g., multiplexed sampling combining optical and channel-based sensing modalities.

[0080] In some embodiments, a choice of rigid stallers can comprise oligonucleotides with rigid motifs. Such stallers may undergo a state change based on, for non-limiting example, hydrogen bonding, such as unzipping of oligonucleotide hairpins, unfolding of oligonucleotide origamis, or dissociation of oligonucleotide double strands or other motifs. In some embodiments, rigid stallers can also include oligomers of similar functionality with other backbones such as peptide beta-hairpins, peptide nucleic acid motifs. One advantage of such motifs can comprise tunability of the stability of the stalling state by altering the sequence, which may be useful for altering stalling time.Example Embodiments

[0081] According to some embodiments, sensing an analyte, the analyte configured to modulate, including self-modulating, translocation of said analyte through a channel can be evaluated by modifying an example protein analyte with a bulky molecule. The bulky molecule may modulate, e.g., slow or stall, translocation of the protein analyte and enhance fingerprinting resolution. In an example embodiment, a designed repeat protein (RP) can be used as a model protein. Such a protein can comprise four repeating domains, each containing 4-5 lysines (as further described hereinbelow with respect to FIG. 21 A). Additionally, an- 18 - 5051544. vl5200.24550018*His tag may be incorporated for purification, while a 10-aspartic acid (DIO) tag can be incorporated to facilitate electrophoretic capture in the nanopore.

[0082] While example embodiments of experiments and methods of sensing an analyte via translocation of said analyte through a channel are described herein, it should be understood that the embodiments are not intended to be limiting. Embodiments of methods, systems, devices, or compositions for sensing an analyte can be carried out using conventional architectures, for example, conventional nanopore architectures. Alternatively, the embodiments may be carried out in modified channel or device architectures. Nonlimiting examples of such architectures can comprise one or more of: (i) trans-to-cis, cis-to-trans, or bidirectional translocation; (ii) solid-state nanopore devices, including pores formed in silicon nitride, graphene, or other synthetic membranes; (iii) hybrid systems comprising biological nanopores embedded in synthetic or polymer-supported membranes; (iv) arrays of nanopores operated in parallel for, e.g., multiplexed sensing; (v) microfluidic or nanofluidic devices configured to control analyte delivery, confinement, or residence time at the pore; (vi) systems employing surface-functionalized nanopores or membranes to modulate analytepore interactions; (vii) architectures incorporating gradients other than ionic concentration, including pH gradients, chemical gradients, or viscosity gradients across the channel; (viii) devices configured to apply time-dependent or spatially varying electric fields, including pulsed, alternating, or stepped voltage profiles; (ix) configurations incorporating pressure-driven or flow-assisted transport across the channel.

[0083] In the absence of lysine modification, RP can translocate through a channel, e.g., a biological nanopore such as an MspA nanopore, under asymmetric buffer conditions (cis: 1 M potassium chloride (KC1), 10 mM tri s(hydroxymethyl)aminom ethane (Tris), pH 7.5; trans: 0.6 M KC1, 3 M GdmCl, 10 mM Tris, pH 7.5). The translocation can be primarily driven by a strong electroosmotic flow (EOF), which may be generated by chloride ions, and an electrophoretic force (EPF), which can either align with or oppose the direction of translocation depending on residue charge. FIG. 2, as described hereinabove, illustrates an example embodiment of a nanopore device 200, comprising a nanopore structure 202, an analyte 210, and representations of the EOF and EPF that may act upon the analyte 210. Under these conditions, the RP may exhibit rapid translocation, with an average dwell time of approximately 1 ms and a current blockade of 82.5% at 60 mV.

[0084] FIGS. 10A-10C are scatter plots 1040a-c illustrating dwell time versus current blockade for translocation of an analyte through a nanopore at applied voltages of 60 mV- 19 - 5051544. vl5200.2455001(1040a), 70 mV (1040b), and 80 mV (1040c), according to an example embodiment. As illustrated, the dwell time can decrease with increasing voltage while the current blockade can remain constant. The analyte can be the RP described hereinabove. However, such brief translocation times may limit the information retrievable from current traces.

[0085] FIG. 11 A-l IF are histograms 1140a-f of dwell time (FIGS. 11 A-l 1C) and current blockade (FIGS. 1 ID-1 IF) based on the scatter plots of FIGS. 10A-10C. The histograms of FIGS. 11 A-l 1C illustrate log dwell time distribution for translocation events at 60 mV (1140a), 70 mV (1140b), and 80 mV (1140c), respectively. The histograms of FIGS. 11D-1 IF illustrate current blockade distribution for translocation events at 60 mV (1140d), 70 mV (1140e), and 80 mV (1140f), respectively.

[0086] FIGS. 12A and 12B are plots 1240a, 1240b of dwell time and current blockade with respect to applied voltage based on the scatter plots of FIGS. 10A-10C. As described hereinabove, higher voltage can result in faster translocation or, correspondingly, lower dwell times (1240a). The current blockade may remain relatively consistent with increasing voltage (1240b), which can indicate similar translocation events (e.g., with respect to types of residues or analyte being translocated).

[0087] FIG. 13 is a plot 1340 of an example current trace associated with sensing an analyte translocating through a nanopore. The current traces may be representative of translocation events (e.g., a trough can be associated with one or more translocation events), during translocation at 80 mV applied voltage. The example current trace is acquired using experimental conditions comprising asymmetric buffer conditions, with the cis side containing 1 M KC1 and 10 mM Tris (pH 7.5), and the trans side containing 0.6 M KC1, 3 M GdmCl, and 10 mM Tris (pH 7.5).

[0088] As described hereinabove, modulation, and self-modulation in particular, of translocation of analytes can be useful for addressing this limitation. In the example embodiment, Alexa Fluor 647 (AF647) can be used as a bulky molecular brake to slow or stall protein translocation. Af647 can be conjugated to lysine side chains via an NHS ester reaction, forming a stable amide bond, for example, as described hereinabove with respect to FIGS. 5A and 5B. In an example embodiment, AF647-modified RP may be prepared by incubating a protein (e.g., the RP) with an excess of AF647-NHS in the presence of dimethyl sulfoxide (DMSO). FIGS. 3A-3D illustrate an example embodiment of a modified protein analyte, which can be a self-modulating protein analyte comprising an analyte and one or more chemical groups coupled or conjugated there to., and translocation of said analyte- 20 - 5051544. vl5200.2455001through a channel for sensing the analyte. The modified protein analyte can be unfolded by guanidinium chloride (GdmCl), and translocating through a biological nanopore from the trans to the cis side. Due to the presence of four sulfonate groups in AF647, the modified protein can acquire a greater negative charge compared to its unmodified counterpart, which, in theory, may facilitate translocation, for example, due to increased EPF. However, experimental results may reveal an opposite effect. When RP’s lysine residues were sparsely labeled with AF647 at a stoichiometric ratio of 4:1 (lysine:dye), the translocation rate decreased approximately 50-fold relative to the unmodified protein (data not shown). Further increasing the modification density to a stoichiometric ratio of 1:5 (lysine:dye) can produce an even more pronounced slowdown in translocation.

[0089] FIGS. 14A-14L are plots 1440a-l illustrating example dwell time and current blockade results for an example embodiment of sensing an analyte, the analyte being a selfmodulating analyte, during translocation at applied voltages of 80 mV (1440a-c), 90 mV (1440d-f), 100 mV (1440g-i), and 120 mV (1440j -1). As described hereinabove, the analyte can be the RP protein analyte and the chemical groups, e.g., stallers, conjugated to the protein analyte can comprise AF647. The self-modulating analyte, comprising the RP protein analyte and the chemical groups can be translocated through a nanopore structure, for non-limiting example, a MspA nanopore structure.

[0090] The plot 1440a of FIG. 14A is a scatter plot of dwell time (Log td) versus current blockade (AZ / Zo) for translocating a self-modulating analyte at 80 mV applied voltage, which can be similar to the analyte 310 described herein with respect to FIGS. 3 A-3D, through a nanopore. The plots 14440b, 1440c of FIGS. 14B and 14C are histograms corresponding to the current blockade and the dwell time, respectively, of the plot 1440a.

[0091] The plot 1440d of FIG. 14D is a scatter plot of dwell time (Log td) versus current blockade (AZ / Zo) for translocating a self-modulating analyte at 90 mV applied voltage through a nanopore. The plots 1440e, 1440f of FIGS. 14E and 14F are histograms corresponding to the current blockade and the dwell time, respectively, of the plot 1440d.

[0092] The plot 1440g of FIG. 14G is a scatter plot of dwell time (Log td) versus current blockade (AZ / Zo) for translocating a self-modulating analyte at 100 mV applied voltage through a nanopore. The plots 1440h, 1440i of FIGS. 14H and 141 are histograms, corresponding to the current blockade and the dwell time, respectively, of the plot 1440g.

[0093] The plot 1440j of FIG. 14J is a scatter plot of dwell time (Log td versus current blockade (AZ / Zo) for translocating a self-modulating analyte at 120 mV applied voltage- 21 - 5051544. vl5200.2455001through a nanopore. The plots 1440k, 14401 of FIGS. 14K and 14L are histograms corresponding to the current blockade and the dwell time, respectively, of the plot 1440j.

[0094] According to the example embodiment described herein with respect to FIGS. 14A-14L, two distinct populations (for example, as indicated by dashed circles Pl and P2 of FIG. 14A) may be observed at 80 mV applied voltage for translocation. Pl and P2 had average dwell times of 4.5 ms and 737.9 ms, respectively, with corresponding current blockades of 85.4% and 90.2%. As voltage increased, the translocation time of both populations decreased. The current blockade of Pl decreased, whereas P2 increased. These results may suggest that both populations correspond to translocation events. The slightly longer translocation time of Pl compared to the unmodified RP may suggest that Pl events likely arise from incomplete modification. The increased translocation time and current blockade of P2 may indicate that P2 events represent translocations of RP close to full modification.

[0095] FIGS. 15A and 15B are plots 1540a, 1540b illustrating dwell time and current blockade, respectively, versus applied voltage based on the example embodiment of FIGS.14A-14L. As described hereinabove, overall dwell times for both populations may decrease with respect to increasing voltage (1540a). Current blockade may decrease for the Pl population with increasing voltage and increase for the P2 population with increasing voltage (1540b).

[0096] FIGS. 16A-16P are plots 1640a-p illustrating example characteristic translocation patterns associated with sensing an example embodiment of a self-modulating analyte translocating a through channel. The plot 1640a of FIG. 16A is a continuous current recording and may comprise four sequential translocation events of the self-modulating analyte, which can comprise the RP protein analyte described hereinabove with AF647 modifications to lysine residues. The current recording of the plot may be acquired during translocation with an applied potential of 80 mV. The plots 1640b-p of FIGS. 16B-16P represent zoomed-in views of individual translocation events, which may be similar to each of the four translocation events of the plot 1640a of FIG. 16A, of AF647-modified RP during translocation at 80 mV applied potential.

[0097] According to some embodiments, the plots 1640a-p may show example individual current traces of P2 events at 80 mV. Although translocation times may vary, all traces may exhibit a similar pattern comprising distinctive stepwise current changes. Based on the hypothesis presented herein, this stepwise behavior may arise from a two-phase translocation- 22 - 5051544. vl5200.2455001process: while the highly negatively charged AF647 is expected to translocate rapidly through the constriction region due to the electrophoretic force (EPF), it likely stalls at the nanopore entrance before entering the constriction due to its bulky structure. During this stalling phase, the peptide region preceding the AF647-modified lysine may be exposed to the sensing zone, potentially for an extended period of time, which may allow the peptide region to be sequentially read by the nanopore, which may contribute to the observed stepwise current blockade. Once the AF647-modified lysine reaches the constriction, the strong EPF and EOF at the sensing zone may drive it quickly through, which may cause a transition to a different blockade level.

[0098] FIGS. 17-20 are plots of translocation events associated with example embodiments of sensing an analyte. As described hereinabove, the analyte can be a selfmodulating analyte such as the RP protein analyte described hereinabove with AF647 conjugated to lysine groups of the RP protein analyte. The plots 1740a-f of FIG. 17 illustrate example current traces associated with translocation events of analytes under a 70 mV applied potential. The plots 1840a-f of FIG. 18 illustrate example current traces associated with translocation events of analytes under a 90 mV applied potential. The plots 1790a-f of FIG. 19 illustrate example current traces associated with translocation events of analytes under a 100 mV applied potential. The plots 2040a-f of FIG. 20 illustrate example current traces associated with translocation events of analytes under a 120 mV applied potential.

[0099] FIG. 21 A illustrates an example model protein that may be used for evaluating an example embodiment of a method for sensing an analyte. The model protein may correspond to the repeat protein (RP) described hereinabove and may comprise four repeating domains, as indicated by Fl, F2, F3, and F4. The RP may further include a poly-aspartic acid tail and a poly-histidine tail. The domains (Fl, F2, F3, F4) are derived from E. coli maltose binding protein (MDP) and the RP is an artificial sequence created by the four domains, as presented in an accompanying sequence listing. The poly-histidine tail, which can be added, e.g., to a N- or C-terminus of a protein, can comprise a polyhistidine-tag, best known by the trademarked name His-tag, which is an amino acid motif in proteins that typically consists of at least six histidine (His) residues, often at the N- or C-terminus of the protein. It is also known as a hexa histidine-tag, 6xHis-tag, or His6 tag.

[0100] FIG. 21B is a plot 2140 of an example current trace associated with sensing of a self-modulating analyte comprising the model protein of FIG. 21A and AF647 modifications to lysine groups of the model protein. The current trace may be a representative example of- 23 - 5051544. vl5200.2455001AF647-modified RP translocation at 70 mV. The translocation can begin with the polyaspartic acid (DIO) tail entering the pore, followed sequentially by domains F4, F3, F2, and Fl, as indicated in the plot.

[0101] According to some embodiments, mechanisms of translocation of modified analytes, including analytes having chemical groups conjugated to given side chains or residues, may be further investigated by analyzing a number of distinct current levels per translocation event. Events similar to the P2 events described hereinabove with reference to FIGS. 14A-14L may exhibit 10-12 discrete steps, as may be illustrated in FIGS. 16A-21B, which may suggest that a device comprising a nanopore can sequentially detect or sense segments of an analyte, e.g., a protein, before each AF647-modified lysine that enters a constriction of a channel or nanopore. These steps may indicate that the stepwise pattern may not be solely caused by the bulky fluorophore but also reflects sequence-dependent peptide interactions within the nanopore.

[0102] Additionally, voltage dependence of stepwise translocations can be examined. As illustrated in FIGS. 16A-18, as voltage increases from 70 mV to 90 mV, the dwell time of individual steps may decrease while an overall stepwise pattern may remain present and consistent. However, further increasing the voltage to 100 mV or higher can result in a reduction of stepwise current changes. This voltage-dependent behavior may be because a stronger electrophoretic force (EPF) can shorten stalling phases associated with AF647, accelerating its passage through the pore and thereby reducing the resolution of stepwise events. These results may support the idea that the protein initially undergoes a sequencedependent pausing phase at the pore entrance before AF647-driven rapid translocation through the constriction.

[0103] FIGS. 22A-22C are plots 2240a-c illustrating example current traces associated with example embodiments of sensing an analyte, the analyte being a self-modulating analyte, through another example nanopore. In the example embodiment, the nanopore can be a CytK nanopore while the self-modulating analyte can comprise the RP analyte with AF647 modifications or labeling to lysine groups of the RP analyte. Translocation of the RP analyte can be carried out under conditions comprising IM KC1, 2M GdmCl, 10 Mm Tris, and pH7.5 and in a trans-to-cis direction.

[0104] As indicated hereinabove at least with respect to FIGS. 21 A-22C, distinctive current traces may reveal a repeating pattern occurring in four sequential segments of the RP analyte. Similar current traces can be observed when the dye-modified RP translocated- 24 - 5051544. vl5200.2455001through another nanopore, for example, the CytK nanopore of FIGS. 22A-22C. This pattern may correspond with the four repeating domains of RP, with a translocation sequence order of F4, F3, F2, and Fl, as described with respect to FIG. 21 A. The consistency of these patterns across multiple events may suggest that the nanopore can be capable of detecting domain-level structural features, further supporting the hypothesis that stepwise current blockades may contain valuable peptide information beyond a signal associated with a chemical group or staller group, e.g., a fluorophore signal.

[0105] The findings described herein may demonstrate that chemical modifications to an analyte, for non-limiting example, AF647 labeling of side chains of said analyte, can introduce controlled stalling and stepwise translocation, which can be useful for enhancing the resolution of nanopore fingerprinting.

[0106] According to some embodiments, sensing analytes as described herein can further comprise, for non-limiting examples, systematic variation of modification sites and fluorophore properties, which may be optimized for detection of sequence-dependent interactions. Additionally, integration of this approach with computational modeling and machine learning could be useful for enabling extraction of even more detailed structural sequence information, which may pave the way for improved analyte characterization based on translocation of analytes through a channel such as a nanopore.

[0107] FIGS. 23 A and 23B are plots 2340a, 2340b illustrating counts of proteins having or not having lysine residues (2340a) and numbers of lysine residues within proteins (2340b). The data of the plots are derived from the Uniprot database and may reveal that a majority of proteins may contain one or more lysine residues whereas a majority of proteins having lysine residues comprise at least two or more.

[0108] FIGS. 24A-24E are chemical structures of example chemical groups that may be coupled to an analyte, according to an example embodiment. The chemical groups can be used as, for example, stalling groups configured to cause a transient change such as a delay during translocation of the analyte through a channel, e.g., a nanopore. Such chemical groups can comprise, for non-limiting example, AF647 (FIG. 24A), infrared 750 (IR750, FIG. 24B), Cyanine 3 (Cy3, FIG. 24C), Sulfo-Cyanine 5 (Sulfo-Cy5, FIG. 24D), or 6-carboxy-X-rhodamine (FIG. 24E).

[0109] Methods

[0110] An example embodiment of a method of sensing an analyte translocating through a channel is presented hereinbelow. The embodiment being provided for exemplification and- 25 - 5051544. vl5200.2455001is not intended to be limiting. Embodiments may utilize, for non-limiting example, alternative dyes, linking mechanisms between the dye and analyte, nanopore architecture or design, or experimental protocols.

[0111] 1. SPARSE LABELING OF PROTEIN WITH A CY5 DERIVATIVE

[0112] According to an example embodiment, an engineered model protein (RP) containing 19 lysines with 4 repeated domains excerpted from E. Coli maltose binding protein (MBP) can be developed and used to evaluate embodiments of methods, analytes, and systems described herein. An RP sample of 6.8 mg / mL stock in 40mM Tris, 6M GdmCl pH 7.5 buffer was buffer exchanged into phosphate buffered saline (PBS), 6M GdmCl buffer using Zeba desalting column 7K molecular-weight cutoff (MWCO), and a lysine-specific dye Alexa Fluor™ 647 NHS ester 1 mM stock in DMSO was mixed at a volume ratio of sample:dye=l:l, namely a stoichiometric ratio of lysine:dye=4:l, resulting in sparsely labelled lysine on the protein. The mixture was incubated at 37°C overnight, and buffer exchanged into lOmM Tris, 6M GdmCl pH 7.5 buffer Zeba desalting column 7K MWCO. The product is stored at 4°C until tested for nanopore translocation.

[0113] 2. DENSE LABELING OF RP WITH A CY5 DERIVATIVE

[0114] An RP sample of 6.8 mg / mL stock in 40mM Tris, 6M GdmCl pH 7.5 buffer was buffer exchanged into PBS, 6M GdmCl buffer using Zeba desalting column 7K MWCO (Thermo Scientific™), and a lysine-specific dye BP Fluor 647 NHS Ester (BroadPharm®) 5mM stock in DMSO was mixed at a volume ratio of sample:dye=l :4, namely a stoichiometric ratio of lysine:dye=l:5, resulting in dense (or, according to some embodiments, complete) labeling of lysines on the protein. The mixture was incubated at 37°C overnight, and a final concentration 12.5 mM ethanolamine, 6.2mM HC1 was added to quench excess NHS ester. The mixture was then buffer exchanged into lOmM Tris, 6M GdmCl pH 7.5 buffer Zeba desalting column 7K MWCO. The product was stored at 4°C until being tested for nanopore translocation.

[0115] 3. DENSE LABELING OF OTHER PROTEINS WITH A CY5 DERIVATIVE

[0116] According to another example embodiment, other model proteins can also be tested to evaluate embodiments of methods, analytes, and systems described herein, including recombinant human PLCZ1 (50 lysines), Escherichia coli biotin ligase (BirA) (18 lysines), and MBP (37 lysines). The proteins can all be engineered mutants with a (Asp)io tail and a (His)s His Tag. The stock concentrations were: hPLCZl 5.5mg / mL, BirA 2.4mg / mL, MBP 3.5mg / mL. The stocks were buffer exchanged to buffers of 40mM 4-(2-hydroxyethyl)-l-- 26 - 5051544. vl5200.2455001piperazineethanesulfonic acid (HEPES), 6M GdmCl, pH 7.0 with (for hPLCZl and BirA) or without (for MBP) lOmM Tris(2-carboxyethyl)phosphine (TCEP)-hydrochloride (HC1), lOmM sodium hydroxide (NaOH), using Zeba desalting column 7K MWCO. The lysinespecific dye BP Fluor 647 NHS Ester (BroadPharm®) 5mM stock in DMSO was mixed at a volume ratio of sample:dye=l:4, for a stoichiometric ratio of lysine:dye of l:5(hPLCZl), l:17(BirA), and 1:6(MBP), resulting in dense (or ideally, complete) labeling of lysines on the protein. The mixtures were incubated at 37°C overnight, then ethanolamine at a final concentration of 70mM in 35mM HC1 was added to quench excess NHS ester. The mixtures were then buffer exchanged into lOmM Tris, 6M GdmCl pH 7.5 buffer Zeba desalting column 7K MWCO. The products were stored at 4°C until tested for nanopore translocation.

[0117] 4. DENSE LABELING OF RP WITH A CY7 DERIVATIVE

[0118] An RP sample of 6.8 mg / mL stock was buffer exchanged to a buffer of 40mM HEPES, 6M GdmCl, pH 7.0 using Zeba desalting column 7K MWCO. The lysine-specific dye IR 750 NHS ester 5mM stock in DMSO was mixed at a volume ratio of sample:dye=l :4, for a stoichiometric ratio of lysine:dye=l:5, resulting in dense (or ideally, complete) labeling of lysines on the proteins. The mixture was incubated at 37°C overnight, then ethanolamine at a final concentration of 70mM in 35mM HC1 was added to quench excess NHS ester. The mixtures was then buffer exchanged into lOmM Tris, 6M GdmCl pH 7.5 buffer Zeba desalting column 7K MWCO. The product was stored at 4°C until tested for nanopore translocation.

[0119] 5. NANOPORE EXPERIMENT

[0120] According to an example embodiment, current recordings can be performed using a two-chamber flow cell separated by a 25 pm thick polytetrafluoroethylene membrane with a -100 pm aperture. The aperture can be pretreated with 5% hexadecane in pentane, followed by the addition of 500 pL of electrolyte buffer to each chamber. A drop of 10 mg / mL diphytanoylphosphatidylcholine (DPhPC) in pentane was applied to each side, and a planar lipid bilayer was formed, for example, using the Montal-Mueller technique by pipetting the buffer. Ag / AgCl electrodes connected to a patch-clamp amplifier can be immersed in the electrolyte buffer, with the ground electrode in the trans chamber and the reference electrode in the cis chamber. MspA nanopores can be added to the cis chamber to ensure the correct orientation, with the stem side facing the trans chamber. For trans-to-cis translocation, protein analytes can be added to the trans chamber, which may comprise 0.6 M KC1, 3 M- 27 - 5051544. vl5200.2455001guanidinium chloride (GdmCl), and 10 mM Tris-HCl at pH 7.5, while the cis chamber comprising 1 M KC1 and 10 mM Tris-HCl at pH 7.5.

[0121] 6. DATA ANALYSIS

[0122] The single-channel recording data was analyzed to extract dwell times and current blockades using a home-written software Pythion with a low pass filter of 10 kHz.Additional Example Embodiments

[0123] Example embodiments for modulation of translocation of proteins may be advantageous due to, for non-limiting example, one or more of the following reasons: use of off-the-shelf and readily available chemical groups for modulation of translocation, chemoselective conjugation of chemical groups to an analyte based on side chain / residue chemistry (or a derivative thereof, e.g., post-translational modification in a protein analyte), compatibility with both denaturing and non-denaturing workflows, as the conjugation of the tag to the analyte is not destroyed by common protein denaturants, making it possible to even switch between denaturing or non-denaturing conditions at different steps of the sample preparation.

[0124] Embodiments described herein may additionally access information about vicinal sequences around a target moiety to which a chemical group, e.g., a staller, is bound. Such embodiments may differ from existing systems using conjugation chemistry to test for existence of target moieties on an analyte, e.g., an analyte protein, by acquiring sequence information of the analyte associated with the target moiety. In an example embodiment, the associated sequence information may comprise, as described hereinabove, a sequence of amino acids (e.g., ranging from 1-25 amino acids as a non-limiting example) upstream or downstream from the target moiety to which the chemical group is bound. The sequence of amino acids may or may not be directly adjacent to the target moiety. Such techniques can also be combined with label-free fingerprinting of protein sequences by nanopores.

[0125] According to some embodiments, coupling or conjugating one or more chemical groups, e.g., stall groups, to an analyte can also be performed in non-denaturing conditions where an example analyte protein can assume certain native or functional folded conformation, with only surface or functionally relevant (e.g. activated serine in hydrolases conjugating to Phenylmethyl sulfonyl fluoride (PMSF)-derived stallers as a non-limiting example) conjugation. The surface or functionally relevant portions of the analytes can then- 28 - 5051544. vl5200.2455001be subsequently profiled and located together with information of vicinal sequences, as a way to probe a native or functional structure of the analyte.

[0126] While prior studies have reported targeted conjugation of residues of an analyte to modify a detectable ionic current blockade (e.g., spikes in a current trace) associated with the residue, example embodiments described herein may be advantageous due to the additional regulation of movement resulting in step-like signals reflecting vicinal sequences of a given residue with a chemical group coupled or conjugated thereto, which can enable a distinct detection mechanism. Furthermore, while binder proteins have previously been used for stalling translocation of nucleic acids in nanopore genomics, such techniques may rely on pair-based coupling that may not be easily altered or redesigned for protein sequency and the reliability of the binder in a protein-unfolding environment is not guaranteed. Embodiments described herein can utilize covalent attachment of a chemical group, e.g., a staller group, for robust and secure bonding while providing higher accuracy (to single residue) spatial resolution and information on the vicinal sequences which may not be disclosed for nucleic acid applications.

[0127] According to some embodiments, a sample comprising an analyte, e.g., a protein, can be prepared for nanopore-based analysis, which can comprise fingerprinting, sequencing, or other techniques. Analysis of the analyte can comprise coupling a bulky moiety (‘staller’) to side chains of the residues on the protein for the purpose of transiently stalling the translocation to yield discrete signal levels suitable for enzyme-free fingerprinting the analyte protein. The fingerprinting can comprise but is not limited to detection, discrimination and characterization of the analyte. For embodiments of analytes comprising protein analytes, protein sequence permutations that are transiently immobilized at the pore can be distinguishable because they are substantially different from one another. The protein sample can include full-length analyte proteins and / or truncated analyte proteins. The staller can be linked in a manner that is chemoselective to sidechains of specific amino acid residues, or to specific post-translational modifications on sidechains of amino acids. More than one type of amino acid can be labelled by stallers or other chemical groups, the staller or chemical groups for different types of amino acid being of the same or different types (e.g., specific compound of the staller or chemical group). The staller molecule can comprise a linker moiety that reacts directly with the targeted amino acid sidechain or the targeted modifications on the sidechain. A separate linker molecule comprising a biorthogonal handle can be added to the targeted sidechain, and the staller with a complementary handle can be added subsequently to- 29 - 5051544. vl5200.2455001the linker. The pair of biorthogonal handles can comprise an alkyne and an azide, exploiting azide-alkyne cycloaddition. The sidechain-targeting moiety can comprise N-succinimidyl ester, targeting lysine sidechains.

[0128] The staller can comprise an organic dye as a bulky moiety, with the organic dye comprising but not limited to Alexa Fluor series (e.g., AF647, 555, 594, 680), IR750, Cyanine dyes (e.g., Cy3, Cy5, Cy7), BODIPY dyes, rhodamine-based dyes, or quencher dyes (e.g. DABCYL, DusQ3), including precursors or derivatives thereof.

[0129] The staller can comprise a bulky moiety having variants capable of conformational switch by change of internal bonding, where the internal bonding can include covalent and / or noncovalent interactions. The staller can comprise an oligomer structure of nucleotides or nucleotide variants. The oligomer structure of nucleotides or nucleotide variants can be capable of conformational switch by change of state of hydrogen bonding, wherein conformations can include but are not limited to origamis, hairpins, or duplexes. The nucleotide variant can comprise peptide nucleic acid oligomers.

[0130] The staller can comprise a structure of peptide or peptide variants, wherein the structure of peptide or peptide variants can be capable of undergoing a conformational switch by change of state of hydrogen bonding. Example conformations can include but are not limited to beta hairpins.

[0131] The staller can comprise a hydrophilic flexible polymer chain such as, for example poly(ethylene glycol), poly(vinyl alcohol), or polypropylene glycol) for the purpose of adjusting spacing between the side chain and the bulky moiety.

[0132] The staller can be a hydrophilic flexible polymer and have a composition of one or more of: (i) greater than or equal to 4 monomers and (ii) less than or equal to 10 kDa molecular weight, for the purpose of serving as the bulky moiety itself. The hydrophilic flexible polymer comprises non-linear topology, including, but not limited to, circular or branched topologies.

[0133] The staller can comprise dendrons, oligosaccharides, or moieties with universal affinity to proteins, such as tris(l,10-phenanthroline) ruthenium(II), Coomassie Blue, and JUNTA where M = Cu, Ni, Co, or another transition metal, and bis- ANS (4,4'-bis(phenylamino)-[l,l'-binaphthalene]-5,5'-disulfonic acid). The staller can comprise organometallic complexes, wherein the staller binds to the target amino acid side chain or modification by covalent binding or by coordination. The staller can be an organic moiety containing a metal, including, but not limited to, one of nickel, copper, osmium, ruthenium,- 30 - 5051544. vl5200.2455001rhodium, iridium, zinc, cobalt, cadmium, iron, silver, or manganese. The staller can be a metallo-organic framework (MOF) such as a polyoxometalate (POM) cluster. As noted hereinabove, a chemical group, e.g., a staller, can comprise any of the structures described hereinabove and may be selected based on its ability to cause a transient stalling during translocation of an analyte through a channel, e.g., a nanopore.

[0134] The slowing down or stalling of translocation by the staller can be detected as signal current spikes or plateaus or a combination of signal spikes and / or plateaus within the current trace. The presence or absence of one or more stallers on specific target sites on the protein or peptide can be measured in the current trace as a difference in the observed signal, informing of the composition or variations in the sample such as the presence of a post-translational modification or an amino acid substitution.

[0135] The teachings of all patents, published applications and references cited herein are incorporated by reference in their entirety.

[0136] 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

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[0138] 2. Ritmejeris, J., Chen, X. & Dekker, C. Single-molecule protein sequencing with nanopores. NatRevBioeng 3, 303-316 (2025). https: / / doi.org / 10.1038 / s44222-024-00260-8

[0139] 3. Motone, K., Kontogiorgos-Heintz, D., Wee, J. et al. Multi-pass, singlemolecule nanopore reading of long protein strands. Nature 633, 662-669 (2024).

[0140] 4. Yu, L., Kang, X., Li, F. et al. Unidirectional single-file transport of full-length proteins through a nanopore. Nat Biotechnol 41, 1130-1139 (2023).

[0141] 5 Martin-Baniandres P, Lan WH, Board S, Romero-Ruiz M, Garcia-Manyes S, Qing Y, Bayley H. Enzyme-less nanopore detection of post-translational modifications within long polypeptides. NatNanotechnol. 2023 Nov;18(ll):1335-1340. doi: 10.1038 / s41565-023-01462-8. Epub 2023 Jul 27. PMID: 37500774; PMCID: PMC10656283.- 31 - 5051544. vl5200.2455001

[0142] 6. Sanacora G, Treccani G, Popoli M. Towards a glutamate hypothesis of depression: an emerging frontier of neuropsychopharmacology for mood disorders.Neuropharmacology. 2012 Jan;62(l):63-77. doi: 10.1016 / j.neuropharm.2011.07.036. Epub 2011 Aug 3. PMID: 21827775; PMCID: PMC3205453.

[0143] 7. Wen C, Dematties D, Zhang SL. A Guide to Signal Processing Algorithms for Nanopore Sensors. ACS Sens. 2021 Oct 22;6(10):3536-3555. doi:10.1021 / acssensors.lc01618. Epub 2021 Oct 4. PMID: 34601866; PMCID: PMC8546757.

[0144] 8. Qing Y, Bayley H. Enzymeless DNA Base Identification by Chemical Stepping in aNanopore. J Am Chem Soc. 2021 Nov 3; 143(43): 18181-18187. doi:10.1021 / jacs.lc07497. Epub 2021 Oct 20. PMID: 34669377.

[0145] 9. Leitao, S.M., Navikas, V., Miljkovic, H. etal. Spatially multiplexed singlemolecule translocations through a nanopore at controlled speeds. Nat. NanotechnoL 18, 1078-1084 (2023).

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Claims

5200.2455001CLAIMSWhat is claimed is:

1. A method of sensing an analyte, the method comprising:configuring an analyte to self-modulate translocation of the analyte through a channel, the configuring comprising coupling at least one chemical group to a corresponding one or more residues of the analyte;causing the translocation of the analyte with the at least one chemical group coupled thereto through the channel, each chemical group of the at least one chemical group configured to cause a corresponding transient change in the translocation of the analyte; andsensing a property associated with the analyte translocating through the channel, the corresponding transient change caused by a given chemical group of the at least one chemical group modulating the sensing of a portion of the analyte associated with the given chemical group.

2. The method of Claim 1, wherein the at least one chemical group comprises at least one staller group and wherein the corresponding transient change in the translocation of the analyte comprises a transient delay of the translocation caused by the at least one staller group blocking the translocation of the analyte through the channel, further wherein the transient delay enables sensing the portion of the analyte over an extended period of time relative to a different portion of the analyte that is not associated with the at least one staller group.

3. The method of Claim 2, wherein the staller group comprises one or more of:an organic dye or a precursor or derivative thereof, further wherein the organic dye comprises one or more of an Alexa Fluor dye, a cyanine dye, a 4,4-difluoro-4- bora-3a,4a-diaza-s-indacene (BODIPY) dye, a rhodamine dye, or a quencher dye.; an oligomeric structure, further wherein the oligomeric structure comprises one or more nucleotides or nucleotide variants;a structure comprising a peptide or peptide variants;- 33 - 5051544. vl5200.2455001a hydrophilic flexible polymer chain, further wherein the hydrophilic flexible polymer chain comprises one or more of poly(ethylene glycol), poly(vinyl alcohol), or polypropylene glycol);a dendron;an oligosaccharide;an organometallic complex, further wherein the organometallic complex comprises one or more of nickel, copper, osmium, ruthenium, rhodium, iridium, zinc, cobalt, cadmium, iron, silver, or manganese;a metallo-organic framework (MOF), further wherein the MOF comprises a polyoxometalate cluster; ora compound having universal affinity to proteins, further wherein the compound comprises one or more of tris(l,10-phenanthroline) ruthenium(II), Coomassie Blue, nitrolotri acetic acid and precursors or derivatives thereof, or 4,4'- bis(phenylamino)-[l,l'-binaphthalene]-5,5'-disulfonic acid (bis-ANS).

4. The method of Claim 2, further comprising causing a stalling moiety of the staller group to undergo a conformational change from a first conformation to at least a second conformation, the first conformation and the at least a second conformation of the stalling moiety having differing capacities to block the translocation of the analyte through the channel.

5. The method of Claim 1, wherein the at least one chemical group is configured to undergo a conformational change based on a state of internal bonding within the at least one chemical group, the internal bonding comprising covalent bonding or non- covalent interactions, and further wherein conformations of the chemical group comprise one or more of origamis, hairpins, G-quadruplexes, structure-forming aptamers, or duplexes.

6. The method of Claim 1, wherein coupling the at least one chemical group to the corresponding residue comprises coupling the at least one chemical group to a side chain or a post-translational modification of the corresponding residue, further wherein coupling the at least one chemical group to the corresponding residue comprises causing a formation of a covalent bond.- 34 - 5051544. vl5200.24550017. The method of Claim 1, wherein coupling the at least one chemical group to the corresponding residue of the analyte comprises chemoselectively coupling a chemical group of the at least one chemical group to the corresponding residue, the corresponding residue being of a given type of residues of the analyte.

8. The method of Claim 7, wherein coupling a chemical group of the at least one chemical group to the corresponding residue further comprises one or more of:coupling a linker group to the corresponding residue and coupling the chemical group to the linker molecule, further wherein the linker group comprises a biorthogonal handle; orcoupling a linker moiety of the chemical group to the corresponding residue, further wherein the linker moiety comprises N-succinimidyl ester and the corresponding residue is lysine.

9. The method of Claim 1, wherein the at least one chemical group comprises at least a first type and at least a second type of chemical group, wherein each type of chemical group is configured to couple with a corresponding residue type of the analyte.

10. The method of Claim 1, wherein sensing the property associated with the analyte translocating through the channel comprises detecting a current associated with the analyte translocating through the channel.

11. The method of Claim 1, wherein the corresponding transient change of the each chemical group is associated with the each chemical group arriving at or passing through the channel.

12. A self-modulating analyte comprising:an analyte comprising one or more residues disposed in sequential arrangement; andat least one chemical group coupled to the one or more residues, the at least one chemical group configured to cause an associated transient change in- 35 - 5051544. vl5200.2455001translocation of the analyte through a channel configured for sequential translocation of the one or more residues of the analyte.

13. The self-modulating analyte of Claim 12, wherein the at least one chemical group comprises at least one staller group and wherein the transient change in the translocation of the analyte comprises a transient delay of the translocation caused by a staller group blocking the translocation of the analyte through the channel.

14. The self-modulating analyte of Claim 13, wherein the staller group comprises one or more of:an organic dye or a precursor or derivative thereof, further wherein the organic dye comprises one or more of an Alexa Fluor dye, a cyanine dye, a 4,4-difluoro-4- bora-3a,4a-diaza-s-indacene (BODIPY) dye, a rhodamine dye, or a quencher dye.; an oligomeric structure, further wherein the oligomeric structure comprises one or more nucleotides or nucleotide variants;a structure comprising peptides or peptide variants;a hydrophilic flexible polymer chain, further wherein the hydrophilic flexible polymer chain comprises one or more of poly(ethylene glycol), poly(vinyl alcohol), or polypropylene glycol);a dendron;an oligosaccharide;an organometallic complex, further wherein the organometallic complex comprises one or more of nickel, copper, osmium, ruthenium, rhodium, iridium, zinc, cobalt, cadmium, iron, silver, or manganese;a metallo-organic framework (MOF), further wherein the MOF comprises a polyoxometalate cluster; ora compound having universal affinity to proteins, further wherein the compound comprises one or more of tris(l,10-phenanthroline) ruthenium(II), Coomassie Blue, nitrolotri acetic acid and precursors or derivatives thereof, or 4,4'- bis(phenylamino)-[l,l'-binaphthalene]-5,5'-disulfonic acid (bis- ANS).

15. The self-modulating analyte of Claim 13, wherein the staller group is configured to be rigid and wherein, in a given conformation of the staller group and a corresponding- 36 - 5051544. vl5200.2455001residue coupled to the staller group, a length associated with the staller group and the corresponding residue is larger than the diameter of the nanopore.

16. The self-modulating analyte of Claim 12, further comprising a linker molecule coupling a chemical group of the at least one chemical group to a corresponding residue of the one or more residues.

17. The self-modulating analyte of Claim 12, wherein the at least one chemical group is chemoselectively coupled to a corresponding residue of the one or more residues, the corresponding residue being of a given type of residues of the analyte.

18. The self-modulating analyte of Claim 12, wherein the at least one chemical group comprises at least a first type and at least a second type of chemical group, wherein each type of chemical group is configured to couple with a corresponding residue type of the analyte.

19. The self-modulating analyte of Claim 12, wherein the at least one chemical group is coupled to the one or more residues via one or more of:a linker group configured to couple the corresponding residue and the chemical group, further wherein the linker group comprises a biorthogonal handle; or a linker moiety of the chemical group to the corresponding residue, further wherein the linker moiety comprises N-succinimidyl ester and the corresponding residue is lysine.

20. A setup for sensing a property of an analyte, the setup comprising:means for causing translocation an analyte through a channel;means for the analyte to self-modulate translocation of the analyte through the channel, the analyte being configured to self-modulate the translocation at one or more residues of the analyte and the self-modulating causing a corresponding transient change associated with the one or more residues during the translocation of the analyte; andmeans for sensing a property associated with the analyte translocating through the channel, the corresponding transient change caused by a given chemical group of- 37 - 5051544. vl5200.2455001the at least one chemical group modulating the sensing of a portion of the analyte associated with the given chemical group.- 38 - 5051544. vl

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