WO2026006174A1

WO2026006174A1 - Compositions and methods related to removal of transmembrane sensors

Info

Publication number: WO2026006174A1
Application number: PCT/US2025/034778
Authority: WO
Inventors: Rizal F. HARIADI; Ranjan SASMAL; Youssef HASSAN
Original assignee: Arizona State University ASU; Arizona State University Downtown Phoenix campus
Current assignee: Arizona State University ASU; Arizona State University Downtown Phoenix campus
Priority date: 2024-06-24
Filing date: 2025-06-23
Publication date: 2026-01-02
Anticipated expiration: 2026-12-24

Abstract

The present disclosure provides compositions and methods related to the removal of transmembrane sensors from cell membranes. Among other innovations, the present disclosure provides methods involving the use of cyclodextrin to remove transmembrane sensors comprising cholesterol from a cell membrane to prevent or reduce sensor-induced cytotoxicity.

Description

COMPOSITIONS AND METHODS RELATED TO REMOVAL OF TRANSMEMBRANE SENSORS

STATEMENT REGARDING RELATED APPLICATIONS

|0001] This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63/663,231, filed June 24, 2024, the entire contents of which are incorporated herein by reference for all purposes.

FIELD

[0002] The present disclosure provides compositions and methods related to the removal of transmembrane sensors from cell membranes. In particular, the present disclosure provides novel methods involving the use of cyclodextrin to quench transmembrane sensors containing cholesterol, effectively facilitating their removal from the cell membrane and reducing/preventing sensor-induced cytotoxicity.

BACKGROUND

[0003] Recent advances in the field of molecular sensor technology include the development of transmembrane sensors for detecting specific targets within cells without breaking the cells open. However, a significant challenge remains in mitigating the cytotoxicity of these sensors, as they can stress cells when they remain bound for extended periods. Embodiments of the present disclosure leverage the properties of cyclodextrin to remove sensors labeled with cholesterol from cell membranes, thus providing a practical solution to enhance the viability of sensor-based applications.

SUMMARY

[0004] Embodiments of the present disclosure include a system comprising a transmembrane nucleic acid-based sensor and cyclodextrin. In some embodiments, the cyclodextrin is a-cyclodextrin, [3-cyclodextrin, and/or y-cyclodextrin.

[0005] In some embodiments, the sensor is amphiphilic. In some embodiments, the sensor comprises a hydrophobic tag that enables integration of the sensor with cell membranes. The hydrophobic tag comprises cholesterol. In some embodiments, the sensor comprises a sensing domain complementary to a target intracellular nucleic acid. In some embodiments, the sensor comprises an indicator that produces a detectable shift in signal upon binding of the target intracellular nucleic acid to the sensor. In some embodiments, the indicator comprises a fluorophore. In some embodiments, the sensor comprises the fluorophore and a quencher, wherein the fluorophore and quencher are positioned on the sensor such that a signal from the fluorophore is quenched in the absence of the target nucleic acid, and wherein binding of the target nucleic acid to the sensor displaces the fluorophore from the quencher, thus producing the detectable shift in signal.

[0006] In some embodiments, the system further comprises one or more cells.

[0007] In some aspects, provided herein are methods for reducing sensor cytotoxicity. In some embodiments, the method comprises incubating a population of cells comprising transmembrane nucleic acid-based sensors with cyclodextrin. In some embodiments, the cyclodextrin is a-cyclodextrin, |3-cyclodextrin, and/or y-cyclodextrin. The sensors comprise a hydrophobic tag, namely at least one cholesterol moiety, that enables integration of a sensor with a cell membrane, and formation of a host-guest complex between the cyclodextrin and the hydrophobic tag (e.g. cholesterol) removes the sensor from the cell membrane.

[0008] In some embodiments, incubation with cyclodextrin removes at least 70% of the sensors from the cell membranes after 30 minutes or less of incubation with cyclodextrin. In some embodiments, incubation with cyclodextrin removes at least 80% of the sensors from the cell membranes. In some embodiments, incubation with cyclodextrin removes at least 90% of the sensors from the cell membranes.

[0009] In some embodiments, the sensor is amphiphilic. In some embodiments, the sensor comprises a hydrophobic tag, namely at least one cholesterol moiety, that enables integration of the sensor with cell membranes. In some embodiments, the sensor comprises a sensing domain complementary to a target intracellular nucleic acid. In some embodiments, the sensor comprises an indicator that produces a detectable shift in signal upon binding of the target intracellular nucleic acid to the sensor. In some embodiments, the indicator comprises a fluorophore. In some embodiments, the sensor comprises the fluorophore and a quencher, wherein the fluorophore and quencher are positioned on the sensor such that a signal from the fluorophore is quenched in the absence of the target nucleic acid, and wherein binding of the target nucleic acid to the sensor displaces the fluorophore from the quencher, thus producing the detectable shift in signal. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] FIG. 1 : Representative schematic diagrams showing different representations of 0- cyclodextrin. On the left is a chemical structure, while on the right are simplified 3D models.

0-cyclodextrin has a cup-like shape. Its height from top to bottom (primary to secondary hydroxyl face) is about 0.79 nm. The secondary hydroxyl face is slightly wider than the primary one. The cavity size varies among cyclodextrin types and is predicted at 0.5-1.5 nm. The arrows in the diagram indicate the positions of primary (open arrow) and secondary (closed arrow) hydroxyl groups on the 0 cyclodextrin structure.

[0011] FIG. 2: Representative schematic diagrams of chemical structures of different types of cyclodextrins. (FIG. 2A) a-Cyclodextrin (a-CD): A cyclic oligosaccharide composed of 6 glucopyranoside units; (FIG. 2B) 0-Cyclodextrin (0-CD): A cyclic oligosaccharide composed of 7 glucopyranoside units; and (FIG. 2C) y-Cyclodextrin (y-CD): A cyclic oligosaccharide composed of 8 glucopyranoside units. Each structure shows the arrangement of glucopyranoside units linked by a- 1,4 glycosidic bonds, forming a truncated cone shape with a hydrophobic interior cavity and hydrophilic exterior. The increasing number of units from a- CD to y-CD results in a progressively larger ring structure and cavity size.

[0012] FIG. 3: Representative diagrams showing formation of cholesterol-cyclodextrin guest-host complexes. (FIG. 3A) Monomer complex formation: A single cyclodextrin molecule (blue cylinder) interacts with one cholesterol molecule (red oval) to form a 1 :1 cyclodextrin monomer: cholesterol complex. The hydrophobic cholesterol molecule is partially enclosed within the cyclodextrin cavity. (FIG. 3B) Dimer complex formation: Two cyclodextrin molecules (blue cylinders) interact with one cholesterol molecule (red oval) to form a 1 :2 cyclodextrin dimer: cholesterol complex. The cholesterol molecule is more fully encapsulated between the cavities of two cyclodextrin units. In both cases, the process is reversible, as indicated by the bidirectional arrows. These complexes demonstrate the ability of cyclodextrins to form host-guest complexes with hydrophobic molecules like cholesterol, potentially enhancing their solubility and bioavailability.

[0013] FIG. 4: Representative schematic diagram showing the removal of cholesterol- labeled transmembrane sensors from a lipid membrane using cyclodextrin and its effect on a fluorescent biosensor. Left: A lipid membrane containing cholesterol-modified transmembrane sensors. Embedded in the membrane is a DNA-based biosensor (Cy5-Target) consisting of: a fluorophore, a quencher, a target binding site, and cholesterol anchors attaching the biosensor to the membrane. Right: Upon addition of cyclodextrin (cones), cyclodextrin: cholesterol complexes form and the cholesterol-modified transmembrane sensor is extracted from the membrane.

[0014] FIG. 5: Depletion of cholesterol-modified transmembrane sensors (labeled with Cy3; green) from lipid membranes using beta-cyclodextrin. (FIG. 5 A) The left panel shows fluorescence microscopy image of giant unilamellar vesicles (GUVs) composed of 10% LPC/EggPC, containing cholesterol-modified transmembrane sensors (labeled with Cy3; green) on the outer membrane surface of the vesicles. The right panel shows the same field of view after 30 minutes of treatment with 5 mM beta-cyclodextrin. (FIG. 5B) Fluorescence intensity profiles along GUV membranes before (solid lines) and after (dashed lines) beta- cyclodextrin treatment. Different colors represent measurements from multiple GUVs. The significant decrease in fluorescence intensity after beta-cyclodextrin treatment indicates approximately 90% depletion of cholesterol-modified transmembrane sensors (labeled with Cy3; green) Scale bars: 20 pm.

DETAILED DESCRIPTION

[0015] A significant challenge with the use of molecular sensors includes developing ways to mitigate the cytotoxicity of these sensors, as they can stress cells when they remain bound for extended periods. Embodiments of the present disclosure leverage the properties of cyclodextrin to remove sensors from cell membranes, thus providing a practical solution to enhance the viability of sensor-based applications. As described further herein, embodiments of the present disclosure provide compositions and methods related to the removal of transmembrane sensors containing cholesterol from membranes. In some embodiments, the present disclosure pertains to the removal of membrane-bound transmembrane sensors that comprise amphiphilic DNA designed to bind specific RNA biomarkers, thereby reducing toxicity in various downstream applications. The present disclosure provides a novel method for removing these sensors from cell membranes post-measurement to reduce cytotoxicity. Among other innovations, the present disclosure provides methods involving the use of cyclodextrin remove sensors from the cell membrane. In some embodiments, the methods herein achieve over 90% clearance of the sensors from cells.

[0016] Section headings as used in this section and the entire disclosure herein are merely for organizational purposes and are not intended to be limiting. 1. Definitions

[0017] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present disclosure. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

[0018] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “and” and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of’ and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.

[0019] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6- 9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.

10020] As used herein, the term “about” used in connection with a quantity is inclusive of the stated value and has the meaning dictated by the context (for example, it includes at least the degree of error associated with the measurement of the particular quantity). The term “about” should also be considered as disclosing the range defined by the absolute values of the two endpoints. For example, the expression “from about 2 to about 4” also discloses the range “from 2 to 4.” The term “about” may refer to ±10% of the indicated number. For example, “about 10%” may indicate a range of 9% to 11%, and “about 1” may mean from 0.9 - 1.1. Other meanings of “about” may be apparent from the context, such as rounding off; for example, “about 1” may also mean from 0.5 to 1.4.

[0021] The term “cytotoxicity” refers to damage to a cell. Damage to a cell may be evidenced by necrosis, apoptosis, autophagy, reduced or inhibited cell growth, reduced or inhibited cell division, and the like. [0022] As used herein, the term “nucleic acid” refers to any nucleic acid containing molecule, including but not limited to, DNA, RNA, and base analogs thereof. The term encompasses sequences that include base analogs of DNA and RNA including, but not limited to, 4-acetylcytosine, 8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine, 5-(carboxyhydroxylmethyl) uracil, 5 -fluorouracil, 5 -bromouracil, 5- carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil, inosine, N6-isopentenyladenine, 1 -methyladenine, 1 -methylpseudouracil, 1-methylguanine, 1 -methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine, 7-methylguanine, 5-methylaminomethyluracil, 5- methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,

5 ’ -methoxycarbonylmethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5 -oxy acetic acid methylester, uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5 -methyluracil, N- uracil-5 -oxy acetic acid methylester, uracil-5 -oxyacetic acid, pseudouracil, queosine, 2- thiocytosine, and 2,6-diaminopurine.

[0023] As used herein, a “double-stranded nucleic acid” may be a portion of a nucleic acid, a region of a longer nucleic acid, or an entire nucleic acid. A “double-stranded nucleic acid” may be, e.g., without limitation, a double- stranded DNA, a double-stranded RNA, a doublestranded DNA/RNA hybrid, etc. A single-stranded nucleic acid having secondary structure (e.g., base-paired secondary structure) and/or higher order structure comprises a “double- stranded nucleic acid”. For example, triplex structures are considered to be “double-stranded.” In some embodiments, any base-paired nucleic acid is a “double- stranded nucleic acid.” [0024] The term “single-stranded” oligonucleotides generally refers to those oligonucleotides that contain a single covalently linked series of nucleotide residues.

[0025] “Complementary” refers to the characteristic of two or more structural elements (e.g., peptide, polypeptide, nucleic acid, small molecule, etc.) of being able to hybridize, dimerize, or otherwise form a complex with each other. For example, a “complementary peptide and polypeptide” are capable of coming together to form a complex. Complementary elements may require assistance to form a complex (e.g., from interaction elements), for example, to place the elements in the proper conformation for complementarity, to co-localize complementary elements, to lower interaction energy for complementation, etc.

[0026] When used in reference to polynucleotides (e.g., a sequence of nucleotides such as all or a portion of a nucleic acid molecule or a target nucleic acid), the terms “complementary” or “complementarity” are used in reference to polynucleotides related by the base-pairing rules. For example, the sequence “5’-A-G-T-3’“ is complementary to the sequence “3’-T-C-A-5’.” Complementarity may be “partial,” in which only some of the nucleic acids’ bases are matched according to the base pairing rules. Or, there may be “complete” or “total” complementarity between the nucleic acids. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. This is of particular importance in amplification reactions, as well as detection methods that depend upon binding between nucleic acids. Either term may also be used in reference to individual nucleotides, especially within the context of polynucleotides. For example, a particular nucleotide within an oligonucleotide may be noted for its complementarity, or lack thereof, to a nucleotide within another nucleic acid strand, in contrast or comparison to the complementarity between the rest of the oligonucleotide and the nucleic acid strand.

[0027] In some contexts, the term “complementarity” and related terms (e.g., “complementary,” “complement”) refers to the nucleotides of a nucleic acid sequence that can bind to another nucleic acid sequence through hydrogen bonds, e.g., nucleotides that are capable of base pairing, e.g., by Watson-Crick base pairing or other base pairing. As an example, nucleotides that can form base pairs, e.g., that are complementary to one another, include the pairs: cytosine and guanine, thymine and adenine, and adenine and uracil. The percentage complementarity need not be calculated over the entire length of a nucleic acid sequence. The percentage of complementarity may be limited to a specific region of which the nucleic acid sequences that are base-paired, e.g., starting from a first base-paired nucleotide and ending at a last base-paired nucleotide. The complement of a nucleic acid sequence as used herein refers to an oligonucleotide which, when aligned with the nucleic acid sequence such that the 5’ end of one sequence is paired with the 3’ end of the other, is in “antiparallel association.” Certain bases not commonly found in natural nucleic acids may be included in the nucleic acids of the present disclosure and include, for example, inosine and 7- deazaguanine. Complementarity need not be perfect; stable duplexes may contain mismatched base pairs or unmatched bases. Those skilled in the art of nucleic acid technology can determine duplex stability empirically considering a number of variables including, for example, the length of the oligonucleotide, base composition and sequence of the oligonucleotide, ionic strength and incidence of mismatched base pairs.

[0028] Thus, in some embodiments, “complementary” refers to a first nucleobase sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the complement of a second nucleobase sequence over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or more nucleobases, or that the two sequences hybridize under stringent hybridization conditions. “Fully complementary” means each nucleobase of a first nucleic acid is capable of pairing with each nucleobase at a corresponding position in a second nucleic acid. For example, in certain embodiments, an oligonucleotide wherein each nucleobase has complementarity to a nucleic acid has a nucleobase sequence that is identical to the complement of the nucleic acid over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or more nucleobases.

[0029] As used herein, “hybridization chain reaction (HCR)” is an enzyme-free isothermal nucleic acid amplifying process. It involves using two or more metastable monomer hairpins that react with an initiator strand to initiate the polymerization process (Dirks and Pierce, 2004; US Patent US20050260635A1; US Patent US20120190835A1). To trigger the polymerization of the monomers, the initiator strand is introduced. The potential for programmability of HCR has been utilized in numerous applications, including the detection of DNA and RNA, as well as RNA imaging in fixed cells (Choi et al, 2010; Molecular Instruments Inc.).

[0030] As used herein, “toehold-mediated strand displacement (TMSD)” refers to a process in which a DNA strand in a DNA helix structure called the protector strand can be displaced and replaced by an invader strand that is complementary to the other strand in the original helix structure. The other strand in the original helix structure is called the original strand, which has an overhang called a “toehold” that assists the invading strand in dislodging and replacing the protector strand. Yurke et al introduced the concept of TMSD to the field of DNA nanotechnology where they constructed a nanomolecular machine powered by DNA (Yurke et al, 2000). The TMSD process has many applications such as DNA nanotechnology, DNA molecular machines, DNA computing, DNA sensing, and programmable DNA nanostructures, among others.

[0031] The term “toehold domain” as used herein refers to a region of a nucleic acid molecule that can function as a toehold in a TMSD process. In some embodiments, the toehold domain is complementary relative to at least a portion of a specified other nucleic acid molecule (e.g., a target nucleic acid).

2. Systems and Methods

[0032] Embodiments of the present disclosure include systems and methods related to nucleic acid sensors. In particular, the present disclosure provides systems and methods that leverage the properties of cyclodextrin to remove sensors from cell membranes, thus providing a practical solution to enhance the viability of sensor-based applications.

[0033] In some aspects, provided herein are systems comprising a transmembrane nucleic acid-based sensor and cyclodextrin.

10034] In some embodiments, provided herein is a method of reducing sensor cytotoxicity, comprising incubating a population of cells comprising transmembrane nucleic acid-based sensors with cyclodextrin. The sensors comprise at least one cholesterol moiety (e.g. the hydrophobic tag) that enables integration of a sensor with a cell membrane, and formation of a host-guest complex between the cyclodextrin and the hydrophobic tag (e.g. cholesterol) removes the sensor from the cell membrane.

[0035] The term “cyclodextrin” refers to a cyclic oligosaccharide containing 6, 7, or 8 glucopyranoside units. In some embodiments, the cyclodextrin is a-Cyclodextrin (a-CD), a cyclic oligosaccharide composed of 6 glucopyranoside units, P-Cyclodextrin (|3-CD), a cyclic oligosaccharide composed of 7 glucopyranoside units; and/or y-Cyclodextrin (y-CD), a cyclic oligosaccharide composed of 8 glucopyranoside units. In some embodiments, the cyclodextrin a single type of cyclodextrin (e.g. one or more a-CD units, one or more (3-CD units, or one or more y-CD units). In some embodiments, the cyclodextrin comprises different types of cyclodextrins (e.g. a combination of one or more a-CD units, one or more (CCD units, and/or one or more y-CD units).

[0036] In the systems and methods herein, cyclodextrin forms a host-guest complex with a cholesterol in the transmembrane nucleic-acid based sensor, thereby facilitating removal of the sensor from the cell membrane. This is advantageous in that long-term incubation with such a sensor can be cytotoxic. Removal of the sensor thus improves viability of using sensors to detect intracellular targets while minimizing or preventing cytotoxicity to the cells. Thus, cells can be contacted with a sensor to detect an intracellular target then contacted with cyclodextrin to remove the sensor from the cell membrane and avoid or reduce cytotoxicity induced by the sensor itself, thus improving cell viability during further downstream evaluations of the same cells. For example, in some embodiments cells are contacted with a sensor to detect an intracellular target, subjected to downstream evaluations such as Fluorescence- Activated Cell Sorting (FACS) or Magnetic- Activated Cell Sorting (MACS), then contacted with cyclodextrin to remove the sensor from the cell membrane, and finally subjected to further downstream processing of the same cells with improved viability of the cells as a result of avoiding or reducing cytotoxicity induced by the sensor itself. [0037] In some embodiments, incubation with cyclodextrin removes at least 70% of the sensors from the cell membranes after 30 minutes or less of incubation with cyclodextrin. In some embodiments, incubation with cyclodextrin removes at least 80% of the sensors from the cell membranes after 30 minutes or less of incubation with cyclodextrin. In some embodiments, incubation with cyclodextrin removes at least 90% of the sensors from the cell membranes after 30 minutes or less of incubation with cyclodextrin. In some embodiments, cells are incubated with cyclodextrin for less than 2 hours, less than 1 hour, less than 45 minutes, or less than 30 minutes to remove sensors from cell membranes. In some embodiments, cells are incubated with cyclodextrin for about 5 minutes to about 2 hours, about 10 minutes to about 1 hour, about 15 minutes to about 45 minutes, about 20 minutes to about 40 minutes, or about 25 minutes to about 30 minutes.

[0038] In some embodiments, the concentration of cyclodextrin in the system or incubated with the cells in the methods herein is dependent on the number of sensors integrated into cell membranes and the desired clearance of sensors from the cell membranes. Cyclodextrin can for monomeric complexes with the hydrophobic moiety or dimeric complexes with the hydrophobic moiety (e.g. two cyclodextrin molecules bound to a single hydrophobic moiety). In some embodiments, the concentration of cyclodextrin in the system or incubated with cells in the method is such that the ratio of cyclodextrin molecules to sensor molecules is at least 0.1 :1, 0.2:1, 0.3:1, 0.4: 1, 0.5: 1, 0.6:1, 0.7: 1, 0.8: 1, 0.9:1, or 1:1. In some embodiments, the concentration of cyclodextrin in the system or incubated with cells in the method is such that the ratio of cyclodextrin molecules to sensor molecules about equal., e.g. about 1: 1. In some embodiments, the concentration of cyclodextrin in the system or incubated with cells in the method is such that the ratio of cyclodextrin molecules to sensor molecules greater than 1 : 1 (e.g. at least about 1:1, at least about 1.5: 1, at least about 2: 1, at least about 2.5: 1, at least about 3:1, at least about 3.5:1, at least about 4: 1, at least about 4.5:1, at least about 5: 1, etc.).

|0039] A “transmembrane nucleic acid-based sensor” refers to a sensor composed of nucleic acid that inserts across the cell membrane. The “transmembrane nucleic acid-based sensor” is referred to herein simply as a “sensor”. In some embodiments, a transmembrane sensor comprises an intracellular domain, a transmembrane domain, and an extracellular domain. An exemplary sensor (i.e. transmembrane nucleic acid-based sensor) is shown in FIG. 4. The sensor may comprise natural and/or modified nucleic acids (e.g., DNA, LNA, PNA, RNA, and the like) or analogs thereof. In some embodiments, the nucleic acid sensor is a DNA molecule. In some embodiments, the nucleic acid sensor is an RNA molecule. [0040] In some embodiments, the sensor is amphiphilic. The term “amphiphilic” indicates that the sensor has hydrophobic and hydrophilic regions, components, or properties. For example, in some embodiments the sensor is an amphiphilic DNA sensor. An amphiphilic DNA sensor may be chosen for its programmability and compatibility with the dimensions and properties of target RNA biomarkers.

[0041] The sensor may comprise one or a plurality of nucleic acid strands. For example, in some embodiments the sensor comprises a single nucleic acid strand (e.g. is a single-stranded sensor). In some embodiments the sensor comprises two nucleic acid strands. In some embodiments, the sensor is a double-stranded nucleic acid sensor. In some embodiments, the sensor comprises a stem-loop structure (also referred to as a “hairpin” structure). For example, in some embodiments the sensor comprises a stem-loop structure with a stem where two nucleic acid strands are at least partially complementary and one or more loops. In some embodiments, the sensor comprises an intracellular loop, an extracellular loop, or both. In some embodiments, the sensor comprises four nucleic acid strands that form a Holliday junction, and one or two loops positioned at one or opposing ends of the sensor. In some embodiments, the sensor comprises a toehold domain. In some embodiments, one of the loops (e.g. the intracellular loop) of a sensor serves as a toehold domain for the binding of target nucleic acids. Exemplary sensors are described in PCT/US2024/025661 and PCT/US2023/063860, the entire contents of each of which are incorporated herein by reference for all purposes.

[0042] The sensor comprises a hydrophobic tag that enables integration of the sensor with cell membranes (e.g. insertion into the cell membrane). The hydrophobic tag comprises cholesterol. In some embodiments, the sensor comprises two hydrophobic tags (e.g. two cholesterol moieties) to facilitate insertion of the sensor into the cell membrane. The hydrophobic tags (e.g. cholesterol moieties) may be positioned on the sensor in any suitable arrangement to facilitate insertion into the cell membrane. For example, the cholesterol moieties may be arranged symmetrically on the nucleic acid strands of the sensor. As another example, the cholesterol moieties may be arranged asymmetrically on the nucleic acid strands of the sensor. This asymmetric design may confer an advantage to the sensor by creating enough imbalance for the polar backbone of the sensor to eventually flip and insert through the membrane, allowing for the detection of targets within the cell/vesicle.

[0043] In some embodiments, the hydrophobic tag facilitates “flipping” of the sensor across the cell membrane to detect intracellular targets. In some embodiments, the target is fluorescently labeled and the sensor inserts and flips across the cell membrane to detect the intracellular target, producing a fluorescent ring referred to as a “halo” on the intracellular surface of the cell membrane. In some embodiments, the sensor comprises a fluorescently labeled “cargo” strand that forms a duplex with the sensor strand, this duplex flips across the cell membrane and binds to a non-fluorescent target which displaces the cargo strand, releasing the fluorescently labeled cargo strand into the intracellular space thereby producing a fluorescent signal that fills the cell. In some embodiments, multiple fluorescent moieties are used to create multiple fluorescent signals. For example, in some embodiments the sensor comprises a fluorescent cargo strand carrying first fluorescent label that forms a duplex that inserts and flips across the cell membrane, creating a ring in the color of the first fluorescent signal (e.g. the halo) on the intracellular surface of the cell membrane. A fluorescently labeled intracellular target labeled with a second fluorescent label binds to the sensor and displaces the cargo strand, filling the cell with the fluorescent signal from the first fluorescent label and producing a halo ring in the second fluorescent color along the inner surface of the cell membrane. For any of the above-described sensor embodiments, the sensor can be removed from the cell membrane by cyclodextrin, which forms a host-guest complex with the hydrophobic tag(s) of the sensor effectively facilitating removal of the sensor.

[0044] In some embodiments, after binding of a target to the sensing domain a conformational change occurs that facilitates the sensor “flopping” back across the cell membrane and transmission of the signal extracellularly. This is referred to as a “flip-flop” sensor. The signal transduced by the flip-flop sensors can be amplified exogenously using various amplification methods, including hybridization chain reaction, HCR, rolling circle amplifications, and other amplification reactions. Cyclodextrin can be used to from a hostguest complex with the hydrophobic tag(s) of a flip-flop sensor, thereby facilitating it’s removal from the cell membrane.

[0045] The sensor comprises a sensing domain complementary to a target nucleic acid. In some embodiments, the sensing domain is complementary to a target intracellular nucleic acid, for example, in some embodiments the sensor is a DNA sensor containing a sensing domain complementary to a target intracellular RNA. In some embodiments, the sensor contains a sensing domain for detection of RNA species including siRNA, miRNA or IncRNA. In some embodiments, the target intracellular nucleic acid is DNA. In some embodiments, the target nucleic acid is fluorescently labeled. In some embodiments, the target nucleic acid is not fluorescently labeled.

|0046] The length and sequence of the sensing domain depends on the target nucleic acid to be detected. In some embodiments, the sensing domain comprises 5 to 50 nucleotides (e.g. 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides). In some embodiments, the sensing domain comprises 5 to 50, 5 to 45, 5 to 40, 5 to 35, 5 to 30, 5 to 25, 5 to 20, or 5 to 15 nucleotides.

10047] In some embodiments, the strand of the sensor or each strand of the sensor is 20 to 100 nucleotides in length, including the sensing domain. In some embodiments, the strand or each strand of the sensor is 20 to 100, 30 to 100, 40 to 100, 50 to 100, 60 to 100, 70 to 100, 80 to 100, or 90 to 100 nucleotides in length.

[0048] In some embodiments, the sensor comprises an indicator that produces a detectable shift in signal upon binding of the target nucleic acid to the sensor. In some embodiments, the indicator comprises a fhiorophore. Any suitable fluorophore may be used. In some embodiments, the sensor comprises a fluorophore and a quencher, wherein the fluorophore and quencher are positioned such that a signal from the fluorophore is quenched in the absence of the target nucleic acid. In some embodiments, the fluorophore and the quencher are on the same nucleic acid strand. In some embodiments, the fluorophore and the quencher are on different nucleic acid strands. For example, in some embodiments one strand of the sensor comprises a fluorophore and another strand of the sensor comprises a quencher positioned such that the fluorophore is inhibited (quenched) in the absence of the target nucleic acid. Binding of the target nucleic acid to the sensor results in strand displacement, removing the strand containing the quencher from the strand containing the fluorophore thereby producing a detectable signal.

[0049] In some embodiments, the system comprises at least one reporter nucleic acid. In some embodiments, the at least one reporter nucleic acid comprises a sequence that is complementary to at least a portion of a nucleic acid molecule of the sensor. In some embodiments, the at least one reporter nucleic acid comprises a sequence capable of initiating at least one of: (i) loop-mediated isothermal amplification (LAMP) and/or (ii) hairpin chain reaction (HCR).

[0050] In some embodiments, the system comprises cells. For example, in some embodiments the system comprises cells, a nucleic-acid based transmembrane sensor, and cyclodextrin. In some embodiments, the nucleic-acid based transmembrane sensor is inserted within the cell membrane of the cells, and cyclodextrin is added to the cells to remove the sensor from the cell membrane.

|0051] In some embodiments, the system is a kit. In some embodiments, the system is a kit comprising the sensor and cyclodextrin. In some embodiments, the sensor and cyclodextrin are packaged in separate containers within the kit. In some embodiments, the sensor is designed to detection of a desired target. In some embodiments, the kit further comprises additional reagents for use in performing the methods herein, including cell culture medium, wash buffers, and the like. The kit may further comprise instructions for use, e.g. instructions for using the sensor to detect the target and instructions for removing the sensor using cyclodextrin.

[0052] The application of cyclodextrin to remove sensors from cell membranes can be used in a variety of different methods and compositions. The specific conditions can be optimized for sensor removal and to reduce cytotoxicity in an efficient manner. DNA-based transmembrane sensors with cholesterol modifications can be used for specific RNA detection while keeping the cells alive (e.g., without cell lysis). Currently, there is no other methods and compositions that facilitate the removal of cholesterol-labeled sensors from cell membranes.

|0053] Other benefits include of the various embodiments of the present disclosure include the simplicity of reduced cytotoxicity. For example, the method of the present disclosure minimizes cell stress by limiting sensor incubation time and ensuring rapid removal of sensors. Another benefit includes increase efficiency. For example, over 90% of the sensors can be removed from cell membranes, significantly improving the viability of sensor-based applications. Another benefit includes versatility. For example, the technology can be applied in various settings, including FACS and MACS, enhancing its commercial potential.

3. Examples

[0054] It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the methods of the present disclosure described herein are readily applicable and appreciable, and may be made using suitable equivalents without departing from the scope of the present disclosure or the aspects and embodiments disclosed herein. Having now described the present disclosure in detail, the same will be more clearly understood by reference to the following examples, which are merely intended only to illustrate some aspects and embodiments of the disclosure, and should not be viewed as limiting to the scope of the disclosure. The disclosures of all journal references, U.S. patents, and publications referred to herein are hereby incorporated by reference in their entireties.

[0055] The present disclosure has multiple aspects, illustrated by the following non-limiting examples. Example 1

[0056] Experiments were conducted to assess the functionality of the compositions and methods of the present disclosure.

[0057] The structure of various cyclodextrins is shown in FIG. 2. Cells can be incubated with a transmembrane nucleic-acid based sensor for detection of various intracellular targets. After detection of the intracellular target(s), sensors are removed using cyclodextrin which forms a host-guest complex with cholesterol present in the sensors, thereby facilitating their removal from the cell membrane. An exemplary sensor structure is shown in FIG. 4. However, various transmembrane nucleic acid-based sensors (e.g. including single-stranded sensors, double-stranded sensors, sensors containing four nucleic acid strands that form a Holliday junction, stem-loop sensors (e.g. hairpin sensors, etc.) including cholesterol may be used to detect the intracellular target and the subsequently removed using cyclodextrin which forms host-guest complexes with the cholesterol, facilitating removal of the sensor from the cell membrane.

[0058] As shown in FIG. 5 , cholesterol-modified transmembrane sensors (labeled with Cy 3 ; green) were depleted from lipid membranes using beta-cyclodextrin. Fluorescence microscopy images of giant unilamellar vesicles (GUVs) composed of 10% LPC/EggPC, containing cholesterol-modified transmembrane sensors (labeled with Cy3; green) on the outer membrane surface of the vesicles are shown in the left panel of FIG. 5A. The center panel of FIG. 5A shows the same field of view after 30 minutes of treatment with 5 mM beta-cyclodextrin. FIG. 5B shows shows intensity profiles along GUV membranes before (solid lines) and after (dashed lines) beta-cyclodextrin treatment. Different colors represent measurements from multiple GUVs. The significant decrease in fluorescence intensity after beta-cyclodextrin treatment indicates approximately 90% depletion of cholesterol-modified transmembrane sensors (labeled with Cy3; green) Scale bars: 20 pm. As such, cyclodextrin effectively cleared 90% of the sensors from the cell membranes after a 30 minute incubation period, demonstrating the simplicity and utility of the presently described systems and methods for removal of transmembrane sensors from cell membranes, e.g. after detection of an intracellular target. The systems and methods herein can be used to reduce or inhibit sensor-induced cytotoxicity and promote cell viability for additional downstream applications after intracellular detection of a target. In some embodiments, the sensor is removed after intracellular detection of the target and following FACS, MACS, etc. procedures. After intracellular detection of the target and optionally FACS/MACS analysis, cyclodextrin treatment removes sensors from the same population of cells in which the intracellular target was detected, enabling further downstream processing.

[0059] The disclosure is further described in the following clauses:

[0060] Clause 1: A system comprising a transmembrane nucleic acid-based sensor and cyclodextrin.

[0061] Clause 2: The system of clause 1 , wherein the sensor is amphiphilic.

[0062] Clause 3: The system of clause 1 or clause 2, wherein the sensor comprises a hydrophobic tag that enables integration of the sensor with a cell membrane and wherein the cyclodextrin forms a host-guest complex with the hydrophobic tag to facilitate removal of the sensor from the cell membrane, wherein the hydrophobic tag comprises cholesterol.

[0063] Clause 4: The system of any one of clauses 1-3, wherein the sensor comprises a sensing domain complementary to a target intracellular nucleic acid.

[0064] Clause 5: The system of any one of clauses 1-4, wherein the sensor comprises an indicator that produces a detectable shift in signal upon binding of the target intracellular nucleic acid to the sensor.

[0065] Clause 6: The system of clause 5, wherein the indicator comprises a fluorophore.

10066] Clause 7: The system of clause 6, wherein the sensor comprises the fluorophore and a quencher, wherein the fluorophore and quencher are positioned on the sensor such that a signal from the fluorophore is quenched in the absence of the target nucleic acid, and wherein binding of the target nucleic acid to the sensor displaces the fluorophore from the quencher, thus producing the detectable shift in signal.

[0067] Clause 8: The system of any one of causes 1-7, wherein the cyclodextrin is a- cyclodextrin, P-cyclodextrin, and/or y-cyclodextrin.

[0068] Clause 9: The system of any one of clauses 1 -8, further comprising one or more cells.

[0069] Clause 10: A method for reducing sensor cytotoxicity, the method comprising incubating a population of cells comprising transmembrane nucleic acid-based sensors with cyclodextrin, wherein the sensors comprise a hydrophobic tag that enables integration of a sensor with a cell membrane and wherein formation of a host-guest complex between the cyclodextrin and the hydrophobic tag removes the sensor from the cell membrane, wherein the hydrophobic tag comprises cholesterol.

[0070] Clause 11 : The method of clause 10, wherein incubation with cyclodextrin removes at least 70% of the sensors from the cell membranes after 30 minutes or less of incubation with cyclodextrin.

[0071] Clause 12: The method of clause 10, wherein incubation with cyclodextrin removes at least 80% of the sensors from the cell membranes.

[0072] Clause 13: The method of clause 10, wherein incubation with cyclodextrin removes at least 90% of the sensors from the cell membranes.

[0073] Clause 14: The method of any one of clauses 10-13, wherein the sensor comprises a sensing domain complementary to a target intracellular nucleic acid.

[0074] Clause 15: The method of any one of clause 10-14, wherein the sensor comprises an indicator that produces a detectable shift in signal upon binding of the target intracellular nucleic acid to the sensor.

[0075] Clause 16: The method of clause 15, wherein the indicator comprises a fluorophore.

[0076] Clause 17: The method of clause 16, wherein the sensor comprises the fluorophore and a quencher, wherein the fluorophore and quencher are positioned on the sensor such that a signal from the fluorophore is quenched in the absence of the target nucleic acid, and wherein binding of the target nucleic acid to the sensor displaces the fluorophore from the quencher, thus producing the detectable shift in signal.

[0077] Clause 18: The method of any one of clauses 10-17, wherein the cyclodextrin is a- cyclodextrin, P-cyclodextrin, and/or y-cyclodextrin.

[0078] Clause 19: The method of any one of clauses 10-18, wherein the ratio of cyclodextrin molecules added to the population of cells to sensor molecules present in the population of cells is about 0.1 :1 to about 5:1

[0079] Clause 20: The method of clause 19, wherein the ratio is about 0.5:1 to about 4:1.

Claims

CLAIMS What is claimed is:

1. A system comprising a transmembrane nucleic acid-based sensor and cyclodextrin.

2. The system of claim 1, wherein the sensor is amphiphilic.

3. The system of claim 1, wherein the sensor comprises a hydrophobic tag that enables integration of the sensor with a cell membrane and wherein the cyclodextrin forms a hostguest complex with the hydrophobic tag to facilitate removal of the sensor from the cell membrane, wherein the hydrophobic tag comprises cholesterol.

4. The system of claim 1 , wherein the sensor comprises a sensing domain complementary to a target intracellular nucleic acid.

5. The system of claim 4, wherein the sensor comprises an indicator that produces a detectable shift in signal upon binding of the target intracellular nucleic acid to the sensor.

6. The system of claim 5, wherein the indicator comprises a fluorophore.

7. The system of claim 6, wherein the sensor comprises the fluorophore and a quencher, wherein the fluorophore and quencher are positioned on the sensor such that a signal from the fluorophore is quenched in the absence of the target nucleic acid, and wherein binding of the target nucleic acid to the sensor displaces the fluorophore from the quencher, thus producing the detectable shift in signal.

8. The system of claim 1, wherein the cyclodextrin is a-cyclodextrin, |3-cyclodextrin, and/or y-cyclodextrin.

9. The system of claim 1 , further comprising one or more cells.

10. A method for reducing sensor cytotoxicity, the method comprising incubating a population of cells comprising transmembrane nucleic acid-based sensors with cyclodextrin, wherein the sensors comprise a hydrophobic tag that enables integration of a sensor with a cell membrane and wherein formation of a host-guest complex between the cyclodextrin and the hydrophobic tag removes the sensor from the cell membrane, wherein the hydrophobic tag comprises cholesterol.

11. The method of claim 10, wherein incubation with cyclodextrin removes at least 70% of the sensors from the cell membranes after 30 minutes or less of incubation with cyclodextrin.

12. The method of claim 10, wherein incubation with cyclodextrin removes at least 80% of the sensors from the cell membranes.

13. The method of claim 10, wherein incubation with cyclodextrin removes at least 90% of the sensors from the cell membranes.

14. The method of claim 10, wherein the sensor comprises a sensing domain complementary to a target intracellular nucleic acid.

15. The method of claim 10, wherein the sensor comprises an indicator that produces a detectable shift in signal upon binding of the target intracellular nucleic acid to the sensor.

16. The method of claim 15, wherein the indicator comprises a fluorophore.

17. The method of claim 16, wherein the sensor comprises the fluorophore and a quencher, wherein the fluorophore and quencher are positioned on the sensor such that a signal from the fluorophore is quenched in the absence of the target nucleic acid, and wherein binding of the target nucleic acid to the sensor displaces the fluorophore from the quencher, thus producing the detectable shift in signal.

18. The method of claim 10, wherein the cyclodextrin is a-cyclodextrin, [ -cyclodextrin, and/or y-cyclodextrin.

19. The method of claim 10, wherein the ratio of cyclodextrin molecules added to the population of cells to sensor molecules present in the population of cells is about 0. 1 : 1 to about 5:1.

20. The method of claim 19, wherein the ratio is about 0.5:1 to about 4: 1.