Compositions and methods relating to nucleic acid sensors

Abstract

The present disclosure provides compositions and methods related to nucleic acid sensors. In particular, the present disclosure provides transmembrane nucleic acid sensors, signal transducers, and molecular amplifiers for lysis-free detection of internal nucleic acids.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 317,424, filed March 7, 2022, which is incorporated by reference in its entirety for all purposes.

[0002] Sequence Listing The entire text of the computer-readable sequence listing submitted herewith, filed under the file name "SKYSG-40581-601_SEQUENCE_LISTING", created on March 7, 2023, with a file size of 12,686 bytes, is incorporated herein by reference.

[0003] Federal Grant Statement This invention was made with Government support under Grant No. AI144247 awarded by the National Institutes of Health. The United States Government has certain rights in this invention.

[0004] The present disclosure provides compositions and methods related to nucleic acid sensors. In particular, the present disclosure provides transmembrane nucleic acid sensors, signal transducers, and molecular amplifiers for lysis-free detection of internal nucleic acids. [Background technology]

[0005] Non-invasive detection of nucleic acid targets inside lipid bilayer membrane-encapsulated vesicles such as exosomes and cells has attracted great interest as it could lead to many applications in diverse fields of medicine and biomedical science. Many of these applications can exploit various features of nucleic acids. For example, naturally occurring stem-loop or hairpin structures of DNA or RNA are crucial as they form components of RNA secondary structure that act as recognition sites for proteins and as nucleation sites for RNA folding. Different lengths and sequences of loops and stems, as well as salt concentrations in the solution, affect the structural stability of hairpins as well as the thermodynamics and kinetics of conformational changes in folded and unfolded states. Moreover, this simple and powerful structure has been applied in, for example, molecular beacons, molecular computing, and hairpin chain reaction (HCR) amplification, among other applications.

[0006] Moreover, toehold-mediated strand displacement (TMSD) refers to a process in which one DNA strand in a DNA helical structure, called the protector strand, can be replaced by an invader strand that is complementary to the other strand in the original helical structure. The other strand in the original helical structure is called the original strand and has an overhang called the "toehold" that helps the invader strand to disengage and replace the protector strand. The TMSD process has many applications in DNA molecular machines, DNA computing, DNA sensing, and programmable DNA nanostructures, among others. Furthermore, hairpin chain reaction (HCR) is a powerful enzyme-free isothermal amplification method based on two (or more) metastable monomer hairpins. An initiator strand is introduced to trigger the polymerization of the monomers. The programmability of HCR has been exploited in many applications in DNA and RNA detection, in addition to RNA imaging in fixed cells. Summary of the Invention

[0007] Embodiments of the present disclosure include a nucleic acid sensor comprising a double-stranded stem domain comprising at least one hydrophobic tag, at least one toehold domain disposed at one end of the double-stranded stem domain, and optionally a hairpin domain disposed at an end of the double-stranded stem domain opposite the end at which the toehold domain is disposed.

[0008] In some embodiments, the sensor comprises two toehold domains disposed at either end of a double-stranded stem domain.

[0009] In some embodiments, the sensor does not include a hairpin domain.

[0010] In some embodiments, the sensor comprises two separate nucleic acid molecules having complementary sequences that form a double-stranded stem domain, and each of the separate nucleic acid molecules comprises a toehold domain.

[0011] In some embodiments, the sensor comprises a toehold domain at one end and a hairpin domain at the other end of a double-stranded stem domain.

[0012] In some embodiments, the sensor comprises a single nucleic acid molecule, the single nucleic acid molecule comprising internal complementary sequences that form a double-stranded stem domain.

[0013] In some embodiments, the nucleic acid sensor is a DNA molecule. In some embodiments, the nucleic acid sensor is an LNA molecule. In some embodiments, the nucleic acid sensor is an RNA molecule.

[0014] In some embodiments, at least one toehold domain is complementary to a target nucleic acid sequence.

[0015] In some embodiments, the target nucleic acid sequence is a DNA molecule or an RNA molecule.

[0016] In some embodiments, at least one toehold domain is from about 5 to about 20 nucleotides.

[0017] In some embodiments, the stem domain is about 10 to about 30 nucleotides.

[0018] In some embodiments, the hairpin domain is about 5 to about 20 nucleotides.

[0019] In some embodiments, the sensor comprises at least two hydrophobic tags. In some embodiments, the at least two hydrophobic tags are positioned about 120° to about 180° away from each other. In some embodiments, the at least two hydrophobic tags are positioned about 4 to about 6 nucleotides away from each other.

[0020] In some embodiments, the sensor comprises at least three hydrophobic tags. In some embodiments, the at least three hydrophobic tags are spaced about 90° to about 120° apart from one another. In some embodiments, the at least three hydrophobic tags are spaced about 2 to about 4 nucleotides apart from one another.

[0021] In some embodiments, the at least one hydrophobic tag is spaced across from about 1.5 nm to about 3.0 nm.

[0022] In some embodiments, the sensor comprises a nucleic acid sequence that is at least 90% identical to SEQ ID NO: 1 or 2. In some embodiments, the sensor comprises a nucleic acid sequence that is at least 90% identical to SEQ ID NO: 5, 8, or 11.

[0023] Embodiments of the present disclosure also include compositions comprising any of the nucleic acid sensors described herein.

[0024] In some embodiments, the composition comprises at least one reporter nucleic acid. In some embodiments, the at least one reporter nucleic acid comprises a sequence complementary to at least a portion of the optional hairpin domain. In some embodiments, the at least one reporter nucleic acid comprises a sequence capable of initiating at least one of: (i) toehold-mediated strand displacement (TMSD), (ii) loop-mediated isothermal amplification (LAMP), and / or (iii) hairpin chain reaction (HCR).

[0025] Embodiments of the present disclosure also include methods of detecting a target nucleic acid using any of the sensors described herein.

[0026] In some embodiments, the target nucleic acid is located in a membrane vesicle or within a cell. In some embodiments, the method includes detecting the target nucleic acid without lysing the membrane vesicle or cell.

[0027] In some embodiments, the target nucleic acid is DNA or RNA.

[0028] In some embodiments, detecting the target nucleic acid comprises an amplification step, hi some embodiments, the amplification step comprises at least one of toehold-mediated strand displacement (TMSD), loop-mediated isothermal amplification (LAMP), and / or hairpin chain reaction (HCR).

[0029] In some embodiments, the method includes a target detection step.

[0030] In some embodiments, the method includes sequencing the target nucleic acid.

[0031] Embodiments of the present disclosure also include kits that include any of the sensors described herein and instructions for detecting a target nucleic acid.

[0032] In some embodiments, the kit further comprises a standard or control. In some embodiments, the standard or control comprises a detection moiety. In some embodiments, the standard or control comprises a nucleic acid sequence that is at least 90% identical to SEQ ID NO: 3, 4, 6, 7, 9, 10, 12, or 13. [Brief description of the drawings]

[0033] [Figure 1] 1A and 1B are representative schematic diagrams of a DNA sensor according to one embodiment of the present disclosure. [Diagram 2] 1A and 1B are representative schematic diagrams of a method of using a double-stranded DNA sensor for nucleic acid detection according to one embodiment of the present disclosure. [Diagram 3] Representative confocal images showing insertion of the dsDNA sensor through the membrane, showing a green ring on the GUV in the right panel, and detection of the target strand forming a red ring on the GUV in the middle panel. [Figure 4] 1A and 1B are representative schematic diagrams of a method of using a hairpin DNA sensor for nucleic acid detection according to one embodiment of the present disclosure. [Diagram 5] 1 shows representative experimental results of a negative control experiment of the DNA sensor of the present disclosure. [Figure 6] 1A-1D are representative confocal images showing detection of internal nucleic acid targets in vesicles with several composition ratios of POPC and cholesterol. [Figure 7] Representative experimental results showing that the effect of the duplex sensor on unrelated molecules present in the GUV is minimal, causing only approximately 8.4% leakage on average across all three experiments. [Figure 8] A and B are representative confocal images showing insertion of a hairpin DNA sensor through the membrane, showing a green ring on the GUV in the left panel due to target binding to the sensor, and a negative control in the right panel with scrambled DNA inside the GUV that does not result in a green ring on the GUV. [Figure 9]A and B are schematics of the TMSD of the hairpin DNA sensor after the sensor opens to allow target binding and reporter strand binding within the vesicle in the top panel, and representative confocal images showing detection of the target forming a green ring on the GUV in the bottom left panel, and binding of the reporter strand to the open stem portion of the sensor forming a red ring on the GUV in the bottom right panel. [Figure 10] TMSD schematic of the double-stranded DNA sensor after intravesicular target binding allowing a portion of the target to bind to a reporter added to the outside of the GUV in the top panel, and representative confocal images showing target detection showing the formation of a red ring upon target binding to the hairpin sensor in the bottom left panel and binding of the reporter strand to a portion of the target protruding outward from the GUV membrane by TMSD forming a blue ring in the right panel. [Figure 11] 1 shows representative experimental results of a TMSD negative control experiment for a double-stranded DNA sensor that shows no reporter binding indicating no TMSD across the lipid membrane in the case of a DNA target in a scrambled vesicle of the present disclosure. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0034] The embodiments of the present disclosure provide a nanometer-sized biosensor made of DNA that penetrates lipid bilayer membranes, detects internal nucleic acid targets present in vesicles, and amplifies the signal from the detection event to allow detection of low concentrations of targets. The subject of the present disclosure is based on several phenomena and techniques closely related to DNA nanotechnology and molecular biology, including DNA hairpin structure, toehold-mediated strand displacement, DNA hybridization design and free energy calculation, molecular dynamics simulation, hairpin chain reaction, and bioconjugation. The embodiments of the present disclosure utilize the above multidimensional developments to create single-stranded and double-stranded DNA nanostructures with sequences, structures, and hydrophobic modifications designed to be fixed to and inserted across lipid bilayer membranes and detect internal nucleic acids of interest while transmitting information across the membrane by TMSD and amplifying information by isothermal amplification such as HCR. The use of TMSD and HCR or other isothermal amplification methods will have a wide range of applications in the DNA sensors of the present disclosure and their associated components.

[0035] Several types of nucleic acid sensors are described herein, each designed and tested to accommodate efficient insertion and fixation across the lipid bilayer membrane of vesicles for the detection of internal nucleic acid targets without dissolution. The transmission of the signal from the detection event across the membrane and the amplification of the information inside the vesicle are performed using toehold-mediated strand displacement (TMSD), isothermal amplification (e.g., hairpin chain reaction (HCR)), and other possible amplification methods. The simple approach of single-stranded and double-stranded DNA prevents stoichiometric problems that often arise in DNA biosensor synthesis. The simple application of TMSD in combination with isothermal amplification provides a simple means of transmitting and amplifying information across membranes using DNA biosensors to detect low concentrations of internal nucleic acids. Detection of biomarkers present in exosomes and cells can be performed. Furthermore, non-invasive detection methods with DNA nanosensors can also be used to diagnose diseases, determine the genotype of cells, and target therapeutic agents.

[0036] The section headings used in this section and throughout this disclosure are for organizational purposes only and are not intended to be limiting.

[0037] 1.Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. In case of conflict, this document, including definitions, will control. Although preferred methods and materials are described below, methods and materials similar or equivalent to those described herein can also be used in the practice or testing of this 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 are not intended to be limiting.

[0038] As used herein, the terms "comprise(s)", "include(s)", "having", "has", "can", "contain(s)", and variations thereof are intended to be open-ended transitional phrases, terms, or words that do not exclude additional acts or structures. The singular forms "a", "and", and "the" include plural referents unless the context clearly indicates otherwise. The present disclosure also contemplates other embodiments that "comprising", "consisting of", and "consisting essentially of" the embodiments or elements set forth herein, whether or not expressly stated.

[0039] In the description of numerical ranges herein, each intervening numerical value of the same degree of precision is expressly contemplated, for example, in the range 6 to 9, the numerical values ​​7 and 8 are contemplated in addition to 6 and 9, and in the range 6.0 to 7.0, the numerical values ​​6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are expressly contemplated.

[0040] As used herein, "correlated with" refers to in comparison to.

[0041] The term "single-stranded" oligonucleotide generally refers to an oligonucleotide that comprises a single string of covalently linked nucleotide residues.

[0042] The term "oligomer" or "oligonucleotide" includes RNA or DNA sequences of multiple nucleotides in either single-stranded or double-stranded form, specifically including short sequences such as dimers and trimers in either single-stranded or double-stranded form that may be intermediates in the production of specific binding oligonucleotides. The "modified" forms used in the candidate pool contain at least one non-natural residue. "Oligonucleotide" or "oligomer" is a general term for polydeoxyribonucleotides such as DNA (including 2'-deoxy-D-ribose or modified forms thereof), polyribonucleotides such as RNA (including D-ribose or modified forms thereof), and any other type of polynucleotide that is an N- or C-glycoside of a purine or pyrimidine base, or a modified purine or pyrimidine base, or an abasic nucleotide. "Oligonucleotide" or "oligomer" may also be used to describe artificially synthesized polymers similar to RNA and DNA molecules, including, but not limited to, peptide nucleic acid (PNA) and locked nucleic acid (LNA) oligos, or DNA and RNA molecules with modified backbones and nucleosides.

[0043] The term "RNA analog" or "RNA derivative" or "modified RNA" generally refers to a polymeric molecule that contains ribonucleosides as its units and also contains at least one of the following: 2'-deoxy, 2'-halo (including 2'-fluoro), 2'-amino (preferably unsubstituted or mono- or di-substituted), 2'-mono-, di- or tri-halomethyl, 2'-O-alkyl, 2'-O-halo-substituted alkyl, 2'-alkyl, azido, phosphorothioate, sulfhydryl, methylphosphonate, fluorescein, rhodamine, pyrene, biotin, xanthine, hypoxanthine, 2,6-diaminopurine, 2-hydroxy-6-mercaptopurine, and a 5-amino group having a sulfur at the 6-position or a halo or C at the 5-position. 1~5 Pyrimidine bases substituted with alkyl groups, basic linkers, 3'-deoxy-adenosine, and other available "chain terminator" or "non-extendable" analogs (at the 3' end of the RNA), or 32 P, 33 Labels such as P. Any of the above can be incorporated into RNA using standard synthetic techniques as disclosed herein.

[0044] The terms "binding activity" and "binding affinity" generally refer to the tendency of a ligand molecule to bind or not bind to a target. The energetics of these interactions are important in "binding activity" and "binding affinity" because they can include definitions of the concentrations of interaction partners, the rate at which these partners can associate, and the relative concentrations of bound and free molecules in solution.

[0045] "Complementary" refers to the property of two or more structural elements (e.g., peptides, polypeptides, nucleic acids, small molecules, etc.) that can hybridize with each other, dimerize, or otherwise form a complex together. For example, "complementary peptides and polypeptides" can form a complex together. Complementary elements may require assistance in forming the complex (e.g., by each interacting element), for example, to place each element in the proper conformation for complementarity, to colocalize each complementary element, and to lower the interaction energy of complementarity.

[0046] As used herein, the term "nucleotide sequence identity" or "nucleic acid sequence identity" refers to the presence of identical nucleotides at corresponding positions in two polynucleotides. Two polynucleotides have "identical" sequences if the sequences of nucleotides in the two polynucleotides are the same when aligned for maximum matching (e.g., within a comparison window). Sequence comparison between two or more polynucleotides is generally performed by comparing portions of the two sequences over a comparison window to identify and compare local regions of sequence similarity. The comparison window is generally about 20 to 200 contiguous nucleotides. The "percentage (%) of sequence identity" between polynucleotides, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 98, 99, or 100 percent sequence identity, can be determined by comparing two optimally aligned sequences over a comparison window, and the portion of the polynucleotide sequence within the comparison window can include additions or deletions (i.e., gaps) when compared to the reference sequence for optimal alignment of the two sequences. In some embodiments, the percentage is calculated by (a) determining the number of positions at which identical nucleobases occur in both sequences, (b) dividing the number of matched positions by the total number of positions within the comparison window, and (c) multiplying the result by 100. Optimal alignment of the sequences to be compared can also be performed by computerized implementation of known algorithms or by visual inspection. Readily available sequence comparison and multiple sequence alignment algorithms include the Basic Local Alignment Search Tool (BLAST) and the ClustalW / ClustalW2 / Clustal Omega programs available on the Internet (eg, at the EMBL-EBI website), respectively.Other suitable programs include, but are not limited to, GAP, BestFit, Plot Similarity, and FASTA, which are part of the Accelrys GCG package sold by Accelrys, Inc., San Diego, Calif. See also Smith & Waterman, 1981; Needleman & Wunsch, 1970; Pearson & Lipman, 1988; Ausubel et al., 1988; and Sambrook & Russell, 2001.

[0047] 2. Nucleic Acid Sensors Embodiments of the present disclosure include compositions and methods related to nucleic acid sensors. In particular, the present disclosure provides transmembrane nucleic acid sensors, signal transducers, and molecular amplifiers for lysis-free detection of internal nucleic acids.

[0048] According to these embodiments, the disclosure provides a nucleic acid sensor comprising a double-stranded stem domain comprising at least one hydrophobic tag, at least one toehold domain disposed at one end of the double-stranded stem domain, and optionally a hairpin domain disposed at an end of the double-stranded stem domain opposite the end at which the toehold domain is disposed.

[0049] In some embodiments, the sensor comprises two toehold domains disposed at both ends of the double-stranded stem domain. In some embodiments, the sensor does not comprise a hairpin domain (e.g., FIG. 1A). In some embodiments, the sensor comprises two separate nucleic acid molecules having complementary sequences that form the double-stranded stem domain. In some embodiments, each of the separate nucleic acid molecules comprises a toehold domain (e.g., FIG. 1A). According to these embodiments, the toehold domain may be open at one end (i.e., the 5' or 3' end of the nucleic acid molecule is not bound). In some embodiments, the 5' or 3' end of the toehold domain is bound to a detection moiety.

[0050] In some embodiments, the sensor comprises a toehold domain at one end of a double-stranded stem domain and a hairpin domain at the other end. In some embodiments, the sensor comprises a single nucleic acid molecule (e.g., FIG. IB). In some embodiments, the single nucleic acid molecule comprises internal complementary sequences that form the double-stranded stem domain (e.g., FIG. IB). According to these embodiments, the hairpin domain is closed (i.e., comprises a continuous sequence of nucleic acid).

[0051] In some embodiments, the nucleic acid sensor is a DNA molecule. In some embodiments, the nucleic acid sensor is an RNA molecule. In some embodiments, at least one toehold domain is complementary to a target nucleic acid sequence. In some embodiments, the target nucleic acid sequence is a DNA molecule or an RNA molecule.

[0052] In some embodiments, at least one toehold domain is about 5 to about 20 nucleotides. In some embodiments, at least one toehold domain is about 10 to about 20 nucleotides. In some embodiments, at least one toehold domain is about 15 to about 20 nucleotides. In some embodiments, at least one toehold domain is about 5 to about 15 nucleotides. In some embodiments, at least one toehold domain is about 5 to about 10 nucleotides. In some embodiments, at least one toehold domain comprises 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides.

[0053] In some embodiments, the stem domain is about 5 to about 30 nucleotides. In some embodiments, the stem domain is about 10 to about 30 nucleotides. In some embodiments, the stem domain is about 15 to about 30 nucleotides. In some embodiments, the stem domain is about 20 to about 30 nucleotides. In some embodiments, the stem domain is about 25 to about 30 nucleotides. In some embodiments, the stem domain is about 5 to about 25 nucleotides. In some embodiments, the stem domain is about 5 to about 20 nucleotides. In some embodiments, the stem domain is about 5 to about 15 nucleotides. In some embodiments, the stem domain comprises 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, or 20 nucleotides.

[0054] In some embodiments, the hairpin domain is about 5 to about 20 nucleotides. In some embodiments, the hairpin domain is about 10 to about 20 nucleotides. In some embodiments, the hairpin domain is about 15 to about 20 nucleotides. In some embodiments, the hairpin domain is about 5 to about 15 nucleotides. In some embodiments, the hairpin domain is about 5 to about 10 nucleotides. In some embodiments, the hairpin domain comprises 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides.

[0055] In some embodiments, the sensor comprises at least two hydrophobic tags. In some embodiments, the at least two hydrophobic tags are positioned about 120° to about 180° away from each other. In some embodiments, the at least two hydrophobic tags are positioned about 180° away from each other. In some embodiments, the at least two hydrophobic tags are positioned about 4 to about 6 nucleotides away from each other. In some embodiments, the at least two hydrophobic tags are positioned 2, 3, 4, 5, or 6 nucleotides away from each other.

[0056] In some embodiments, the sensor comprises at least three hydrophobic tags. In some embodiments, the at least three hydrophobic tags are positioned about 90° to about 120° away from each other. In some embodiments, the at least three hydrophobic tags are positioned about 120° away from each other. In some embodiments, the at least three hydrophobic tags are positioned about 2 to about 4 nucleotides away from each other. In some embodiments, the at least three hydrophobic tags are positioned about 1, 2, 3, or 4 nucleotides away from each other.

[0057] In some embodiments, the at least one hydrophobic tag is arranged to span from about 1.0 nm to about 3.0 nm. In some embodiments, the at least one hydrophobic tag is arranged to span from about 1.5 nm to about 3.0 nm. In some embodiments, the at least one hydrophobic tag is arranged to span from about 2.0 nm to about 3.0 nm. In some embodiments, the at least one hydrophobic tag is arranged to span from about 2.5 nm to about 3.0 nm. In some embodiments, the at least one hydrophobic tag is arranged to span from about 1.0 nm to about 2.5 nm. In some embodiments, the at least one hydrophobic tag is arranged to span from about 1.0 nm to about 2.0 nm. In some embodiments, the at least one hydrophobic tag is arranged to span from about 1.0 nm to about 1.5 nm.

[0058] In some embodiments, the sensor comprises a nucleic acid sequence that is at least 90% identical to SEQ ID NO:1 or 2. In some embodiments, the sensor comprises a nucleic acid sequence that is at least 91% identical to SEQ ID NO:1 or 2. In some embodiments, the sensor comprises a nucleic acid sequence that is at least 92% identical to SEQ ID NO:1 or 2. In some embodiments, the sensor comprises a nucleic acid sequence that is at least 93% identical to SEQ ID NO:1 or 2. In some embodiments, the sensor comprises a nucleic acid sequence that is at least 94% identical to SEQ ID NO:1 or 2. In some embodiments, the sensor comprises a nucleic acid sequence that is at least 95% identical to SEQ ID NO:1 or 2. In some embodiments, the sensor comprises a nucleic acid sequence that is at least 96% identical to SEQ ID NO:1 or 2. In some embodiments, the sensor comprises a nucleic acid sequence that is at least 97% identical to SEQ ID NO:1 or 2. In some embodiments, the sensor comprises a nucleic acid sequence that is at least 98% identical to SEQ ID NO:1 or 2. In some embodiments, the sensor comprises a nucleic acid sequence that is at least 99% identical to SEQ ID NO:1 or 2.

[0059] In some embodiments, the sensor comprises a nucleic acid sequence that is at least 90% identical to SEQ ID NO:5, 8, or 11. In some embodiments, the sensor comprises a nucleic acid sequence that is at least 91% identical to SEQ ID NO:5, 8, or 11. In some embodiments, the sensor comprises a nucleic acid sequence that is at least 92% identical to SEQ ID NO:5, 8, or 11. In some embodiments, the sensor comprises a nucleic acid sequence that is at least 93% identical to SEQ ID NO:5, 8, or 11. In some embodiments, the sensor comprises a nucleic acid sequence that is at least 94% identical to SEQ ID NO:5, 8, or 11. In some embodiments, the sensor comprises a nucleic acid sequence that is at least 95% identical to SEQ ID NO:5, 8, or 11. In some embodiments, the sensor comprises a nucleic acid sequence that is at least 96% identical to SEQ ID NO:5, 8, or 11. In some embodiments, the sensor comprises a nucleic acid sequence that is at least 97% identical to SEQ ID NO:5, 8, or 11. In some embodiments, the sensor comprises a nucleic acid sequence that is at least 98% identical to SEQ ID NO:5, 8, or 11. In some embodiments, the sensor comprises a nucleic acid sequence that is at least 99% identical to SEQ ID NO:5, 8, or 11.

[0060] The present disclosure also includes a method of detecting a target nucleic acid using any of the sensors described herein. In some embodiments, the target nucleic acid is located in a membrane vesicle or inside a cell. In some embodiments, the method includes detecting the target nucleic acid without lysing the membrane vesicle or the cell. In some embodiments, the target nucleic acid is DNA or RNA.

[0061] In some embodiments, detecting the target nucleic acid comprises an amplification step. In some embodiments, the amplification step comprises at least one of toehold-mediated strand displacement (TMSD), loop-mediated isothermal amplification (LAMP), and / or hairpin chain reaction (HCR). In some embodiments, the method comprises a target detection step. For example, in some embodiments, nucleic acid detection comprises the use of a fluorophore, a chromophore, a fluorophore pair, a fluorophore / quencher pair, or other detection moieties known in the art.

[0062] In some embodiments, the method comprises sequencing the target nucleic acid. For example, in some embodiments, detecting the target nucleic acid comprises sequencing using Sanger sequencing, next generation sequencing (NGS), or any other sequencing method known in the art.

[0063] The present disclosure also includes compositions comprising any of the nucleic acid sensors described herein. In some embodiments, the compositions include at least one reporter nucleic acid. In some embodiments, the at least one reporter nucleic acid comprises a sequence complementary to at least a portion of the optional hairpin domain. In some embodiments, the at least one reporter nucleic acid comprises a sequence capable of initiating at least one of (i) toehold-mediated strand displacement (TMSD), (ii) loop-mediated isothermal amplification (LAMP), and / or (iii) hairpin chain reaction (HCR).

[0064] The present disclosure also includes a kit comprising any of the sensors described herein and instructions for detecting a target nucleic acid. In some embodiments, the kit further comprises a standard or control. In some embodiments, the standard or control comprises a detection moiety. In some embodiments, the standard or control comprises a nucleic acid sequence that is at least 90% identical to SEQ ID NO: 3, 4, 6, 7, 9, 10, 12, or 13. In some embodiments, the standard or control comprises a nucleic acid sequence that is at least 91% identical to SEQ ID NO: 3, 4, 6, 7, 9, 10, 12, or 13. In some embodiments, the standard or control comprises a nucleic acid sequence that is at least 92% identical to SEQ ID NO: 3, 4, 6, 7, 9, 10, 12, or 13. In some embodiments, the standard or control comprises a nucleic acid sequence that is at least 93% identical to SEQ ID NO: 3, 4, 6, 7, 9, 10, 12, or 13. In some embodiments, the standard or control comprises a nucleic acid sequence that is at least 94% identical to SEQ ID NO: 3, 4, 6, 7, 9, 10, 12, or 13. In some embodiments, the standard or control comprises a nucleic acid sequence that is at least 95% identical to SEQ ID NO: 3, 4, 6, 7, 9, 10, 12, or 13. In some embodiments, the standard or control comprises a nucleic acid sequence that is at least 96% identical to SEQ ID NO: 3, 4, 6, 7, 9, 10, 12, or 13. In some embodiments, the standard or control comprises a nucleic acid sequence that is at least 97% identical to SEQ ID NO: 3, 4, 6, 7, 9, 10, 12, or 13. In some embodiments, the standard or control comprises a nucleic acid sequence that is at least 98% identical to SEQ ID NO: 3, 4, 6, 7, 9, 10, 12, or 13. In some embodiments, the standard or control comprises a nucleic acid sequence that is at least 99% identical to SEQ ID NO: 3, 4, 6, 7, 9, 10, 12, or 13.

[0065] 3. Arrays Various nucleic acid sequences (ie, SEQ ID NOs) referenced herein are set forth below.

[0066] [Table 1]

[0067] [Table 2]

[0068] Other suitable modifications will be readily apparent to those skilled in the art.

[0069] It should be understood that the above detailed description and accompanying examples are merely illustrative and should not be construed as limitations on the scope of the present disclosure, which is defined solely by the appended claims and their equivalents.

[0070] Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications, including but not limited to those related to the chemical structures, substituents, derivatives, intermediates, synthetic methods, compositions, formulations, or methods of use of the present disclosure, can be made without departing from the spirit and scope thereof. EXAMPLES

[0071] 4. Working Example It will be appreciated by those skilled in the art that other suitable modifications and adaptations of the disclosed methods described herein are readily applicable and recognizable, and may be made using suitable equivalents without departing from the scope of the disclosure or the aspects and embodiments disclosed herein. Having described the disclosure in detail, the disclosure will be more clearly understood by reference to the following examples, which are intended merely to illustrate some aspects and embodiments of the disclosure, and should not be considered as limiting the scope of the disclosure. The disclosures of all journal references, U.S. patents, and publications referenced herein are incorporated herein by reference in their entirety.

[0072] The present disclosure has multiple aspects, which are illustrated by the following non-limiting examples.

[0073] Example 1 Sensor design, structure and working principle. In one embodiment, the single-stranded DNA sensor is composed of a toehold portion, a stem portion and a hairpin portion. The double-stranded DNA has two toeholds at each end connected by a stem portion. This embodiment requires one or two strands, which is very powerful in overcoming the stoichiometric problem in the formation of the sensor, which is often a challenge in DNA nanotechnology. Moreover, this simple approach is advantageous in terms of insertion kinetics and the number of hydrophobic tags required.

[0074] Based on the length of the target, the toehold can be about 6-13 nucleotides that are complementary to the portion of the internal nucleic acid target to be detected. The stem is designed to provide structural integrity to the hairpin and duplex structure. The toehold mediates the displacement of the stem portion, allowing the signal to be transferred from the inside to the outside. The stem has about 14-15 base pairs depending on the length of the target, which are the same or complementary to the target sequence. The hairpin in the single-stranded sensor is designed to trigger the amplification of isothermal amplification such as HCR or other methods such as rolling circle amplification. The hairpin is selected to be the same length as the toehold to maintain a balance of polar groups on both sides of the stem. Hydrophobic modifications are strategically placed on one side of the stem. As a result, the other side of the stem can be separated from the membrane once TMSD is completed. Figures 1A-1B show a schematic of the DNA sensor design.

[0075] For the DNA sensor to cover the hydrophobic liquid efficiently, the spatial arrangement of the hydrophobic tags (either 2 or 3 tags) is carefully designed such that, when projected onto the cross section of the stem, the spacing between the hydrophobic tags is 180 degrees for 2 tags and 120 degrees for 3 tags (see Figures 1A and 1B, center panel). Thus, the angular spacing between two tags is given by 5 nucleotides for 2 tags and 3 and 4 nucleotides for 3 tags. This can be seen in the left panels of Figures 1A and 1B, where the hydrophobic tags are shown in green and the spacing between them is also shown. As a result, the stem is fixed perpendicularly to the membrane surface inside the lipid bilayer membrane. Based on the spacing of 5 nucleotides and 3-4 nucleotides, the distance covered by the hydrophobic tags is about 1.8-2.5 nm, which is smaller than the known lipid bilayer thickness of 3.5-4.0 nm, which allows the stem to be located perpendicularly in the membrane, with the hairpin and toehold free on opposite sides of each other.

[0076] The hydrophobic tags are asymmetrically positioned along the stem to promote the insertion of the toehold into the vesicle for better internal nucleic acid detection. As a result, the hydrophobic groups are located close to the toehold region, imparting less polar nucleic acid to the toehold stem side. Assuming that the toehold and hairpin are of the same length, the orientation upon insertion is determined solely by the position of the hydrophobic groups on the stem, which is versatile from a design point of view. The arrangement of the hydrophobic tags is shown in the left panel of Figures 1A and 1B.

[0077] The introduction of two mismatches into the stem, 14-15 base pairs in length, confers faster TMSD kinetics with an optimal toehold length of approximately 6-10 nt (Zhang and Winfree, 2009). The mismatches are carefully selected and positioned to not affect the integrity of the stem structure but only promote the kinetics of TMSD. These mismatches are located in the vicinity of hydrophobic groups. Nupak shows a high equilibrium probability around the mismatches, thus confirming the integrity of the stem, but reducing the stability of this region.

[0078] Once TMSD is complete, the signal is transmitted across the membrane. As a result of being displaced by the target strand, one of the single-stranded units of the stem region is released from the membrane constraint and becomes outside the vesicle. A hairpin chain reaction is triggered as the open single-stranded hairpin portion is displaced by the single-stranded unit of the stem. The right panel of Figure 1B shows two monomer hairpins of HCR for the single-stranded DNA sensor. These are specifically designed in such a way that the reaction is triggered only if the internal target detection and TMSD event across the lipid bilayer membrane is successful. For the double-stranded sensor, the internal target strand is designed to have a portion that serves as a target for the signal strand, with a fluorophore to bind and detect TMSD. HCR cannot be performed in double-stranded DNA sensors due to the target length limit, and Figure 1A shows the internal target and signal strand in a double-stranded DNA sensor.

[0079] In the case of double-stranded DNA, the signal strand binds to part c shown in FIG. 1A. In the hairpin sensor, HCR amplification occurs when the hairpin is opened. Binding of the signal strand or monomer of the HCR allows detection of a fluorophore signal, which can be detected by microscopy or gel assay. In microscopy, the signal strand and one of the hairpin monomers can be bound to a fluorophore to detect the polymer formed from the HCR. It is also possible to monitor detection on a plate reader using monomer hairpins with emitter / quencher or donor / acceptor pairs, or using fluorescence dequenching detection or FRET.

[0080] Using the design principles described above, we then use Nuack to design and generate optimized sequences for single-stranded and double-stranded DNA sensors. Among the initial targets is a sequence from the microRNA miR23b that is adapted for practical applications. The DNA sensors are shown in Tables 1 and 2 along with their targets, the signal strand for the double-stranded sensor, and the hairpin monomer for the single-stranded sensor.

[0081] A proof-of-concept experiment was carried out using giant unilamellar vesicles (GUVs), which contain phospholipids and some cholesterol in their membrane. The GUVs are synthesized with target DNA encapsulated inside them. Using fluorescence confocal microscopy, both the DNA target and the DNA sensor can be labeled with fluorescent dyes, allowing the analysis of the interaction of the DNA target with the GUVs.

[0082] Example 2 Insertion of double-stranded and hairpin DNA sensors across a membrane as a proof of concept for DNA sensor insertion across a membrane, and detection of single-stranded DNA within a GUV. Experiments were performed to demonstrate that a DNA sensor can be inserted into a lipid bilayer membrane and sense an internal target inside the vesicle. To demonstrate how insertion and detection works, very simple vesicles were synthesized from a minimal lipid composition of POPC and cholesterol. The left panels of Figures 2A and 4A show a schematic of the process after the target binds to the sensor containing double-stranded and single-stranded DNA. Figure 8A shows successful intravesicular target detection with a hairpin DNA sensor forming a green ring on the GUV, and Figure 8B is a negative control by having a scrambled intravesicular DNA target, where no green ring is observed.

[0083] The detection of an internal nucleic acid target is demonstrated by using a test sample of GUVs encapsulating a specific target DNA that is complementary to the toehold of the single-stranded and double-stranded DNA sensors. The GUVs are synthesized by the inverse emulsion or cDICE method, in which POPC phospholipids and cholesterol are present in a ratio of 70%:30%, respectively. The inner solution contains a specific concentration of sucrose, as well as 250 mM KCl, and a target concentration of 100-1000 nM. There is an outer solution of a specific concentration of glucose and 250 mM KCl. The concentrations of sucrose and glucose are adjusted to ensure equal osmolarity of the inner and outer solutions. The DNA sensor is then mixed with the test sample and incubated for approximately 1.5 hours before being examined under a confocal microscope. In Figure 1A-1B, the DNA sensor is labeled with sybr gold and the internal target DNA is tagged with a Cy5 fluorophore, referred to as fluorophore 1. Figure 3 shows confocal images demonstrating the insertion of a double-stranded DNA sensor through the membrane showing a green ring on the GUV in the right panel, and detection of the target strand forming a red ring on the GUV in the middle panel. Fluorescence confocal microscopy experiments are used to confirm insertion and detection.

[0084] Example 3 Toehold-Mediated Strand Displacement Across a GUV Membrane. Toehold-Mediated Strand Displacement (TMSD) occurs in solution in the absence of a constraining membrane surrounding the DNA (Yurke et al, 2000). In this embodiment, the DNA sensor undergoes TMSD under the constraint of a lipid bilayer membrane. In the design section, it was noted that the sensor has a hydrophobic tag on one side of the double-stranded stem, and the other side is released to the exterior of the vesicle upon completion of TMSD across the membrane. TMSD across the membrane has a total Gibbs free energy difference of zero from a single base pair perspective. The reaction rate of TMS is expected to be slower than in solution only in the absence of a constraining membrane. The right panels of Figures 2A and 4A show schematics of single-stranded and double-stranded DNA sensors.

[0085] The GUV test sample described in section II is used. It is tagged with a signal strand with a different fluorophore than the target. For the single-stranded DNA sensor, the signal strand is designed in Nupac (nuack.org) so that leakage does not occur if TMSD across the membrane does not occur. Furthermore, the assays shown in Figures 2A and 4A are carefully designed so that false positives are not induced. Once the detection of TMSD across the membrane is completed, the detection of the internal target information is transmitted to the outside, and the signal strand is expected to bind to the part of the sensor that has been released to the outside but is still fixed to the membrane by the sensor, emitting a fluorescent signal from the sensor surface, as shown in Figure 2B for the double-stranded DNA sensor. This event was examined using fluorescence confocal microscopy.

[0086] Example 4 HCR amplification of internal target detection in hairpin sensor. Amplification of the detection event is demonstrated using HCR amplification. Hairpin monomers were designed using Nuack (nuack.org). The hairpins and assays are checked for leakage and false positive signals. A schematic of the experiment is shown in Figure 4B. One of the hairpins is labeled with a different fluorophore than the target. As a result of HCR amplification, the sensor should be able to detect low concentrations of DNA target. The same GUV test sample is mixed with the sensor and both monomer hairpins. Amplification is observed using fluorescence confocal microscopy.

[0087] Example 5 Several GUV composition ratios are tested in the double-stranded sensor. Experiments were carried out to confirm the ability of the double-stranded sensor to insert and detect internal nucleic acid targets in vesicles with several composition ratios of POPC and cholesterol. These experiments are to test the ability of the double-stranded sensor to detect targets encapsulated in different lipid bilayer membrane compositions. Figure 6 shows the red ring as a result of internal nucleic acid detection, proving that the double-stranded sensor still works very well under different synthetic lipid bilayer compositions.

[0088] Characterization of leakage assay of dye small molecules and invasiveness of double-stranded sensor. The invasiveness of the double-stranded sensor is also confirmed by performing a dye small molecule leakage assay. Confocal images showed that the sensor did not cause any visible damage to the GUV. However, the leakage assay was performed to test how the sensor only affects the extremely small dye small molecules and binds to a specific length of single-stranded DNA inside the GUV that is unrelated to the sensor. The leakage of ATTO488 dye molecules, ATTO488-15nt single-stranded DNA, and ATTO488-22nt single-stranded DNA was tested. Figure 7 shows the effect of the sensor on the dye small molecules. From this result, it was concluded that the double-stranded sensor has minimal effect on unrelated molecules present in the GUV, causing leakage of about 8.4% on average in all three experiments.

[0089] Example 6 Possibility of TMSD across lipid bilayer membranes in double-stranded and hairpin DNA sensors. Experiments were performed to confirm the possibility of TMSD across lipid membranes in double-stranded and hairpin DNA sensors. The corresponding DNA targets of double-stranded and hairpin DNA sensors are encapsulated inside the GUVs. Then, each sensor is added and incubated for several hours to allow TMSD to proceed. Finally, a reporter specific to the sensor is added to examine the occurrence of TMSD.

[0090] FIG. 9A shows a schematic of the experiment and process for the hairpin DNA sensor. FIG. 9B shows the detection of the intravesicular target forming a green ring on the GUV in the left panel, and the binding of the reporter strand from outside the GUV to the open stem portion of the sensor forming a red ring on the GUV. For the double-stranded DNA sensor, the top panel of FIG. 10 shows a schematic of the process of TMSD crossing the membrane and penetrating some portions of the target to allow the reporter to bind to them from outside the GUV, and the bottom panel shows representative confocal images, where target detection by the double-stranded sensor forms a red ring on the GUV in the bottom left panel, and reporter strand binding to the sensor forms a blue ring on the GUV in the bottom right panel. A negative control experiment with a scrambled intravesicular DNA target and a double-stranded DNA sensor shows no formation of a blue ring on the GUV, as shown in FIG. 11.

[0091] It should be understood that the above detailed description and accompanying examples are merely illustrative and should not be construed as limitations on the scope of the disclosed embodiments, which is defined only by the appended claims and their equivalents. Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications, including but not limited to those related to the chemical structures, substituents, derivatives, intermediates, synthetic methods, compositions, formulations, or methods of use of the disclosed embodiments, can be made without departing from the spirit and scope thereof.

[0092] For completeness, various aspects of embodiments of the disclosure are set out in the following numbered clauses.

[0093] Clause 1. A nucleic acid sensor comprising: a double-stranded stem domain comprising at least one hydrophobic tag; at least one toehold domain disposed at one end of said double-stranded stem domain; and optionally, a hairpin domain disposed at an end of said double-stranded stem domain opposite to the end at which the toehold domain is disposed.

[0094] Clause 2. The sensor of clause 1, comprising two toehold domains disposed at either end of the double-stranded stem domain.

[0095] Clause 3. The sensor of clause 1 or clause 2, which does not comprise a hairpin domain.

[0096] Clause 4. The sensor of any of clauses 1-3, comprising two separate nucleic acid molecules having complementary sequences that form the double-stranded stem domain, each of the separate nucleic acid molecules comprising a toehold domain.

[0097] Clause 5. The sensor of clause 1, comprising a toehold domain at one end of the double-stranded stem domain and a hairpin domain at the other end.

[0098] Clause 6. The sensor of clause 5, comprising a single nucleic acid molecule, said single nucleic acid molecule comprising internal complementary sequences that form said double-stranded stem domain.

[0099] Clause 7. The sensor of any of clauses 1 to 6, which is a DNA molecule, an LNA molecule, or a combination thereof.

[0100] Clause 8. The sensor according to any one of clauses 1 to 6, which is an RNA molecule.

[0101] Clause 9. The sensor of any of clauses 1-8, wherein said at least one toehold domain is complementary to a target nucleic acid sequence.

[0102] Clause 10. The sensor of clause 10, wherein the target nucleic acid sequence is a DNA molecule or an RNA molecule.

[0103] Clause 11. The sensor of any of clauses 1-10, wherein said at least one toehold domain is from about 5 to about 20 nucleotides.

[0104] Clause 12. The sensor of any one of clauses 1 to 11, wherein the stem domain is from about 5 to about 30 nucleotides.

[0105] Clause 13. The sensor of any of clauses 1 to 12, wherein the hairpin domain is from about 5 to about 20 nucleotides.

[0106] Clause 14. A sensor according to any one of clauses 1 to 13, comprising at least two hydrophobic tags.

[0107] Clause 15. The sensor of clause 14, wherein the at least two hydrophobic tags are positioned about 120° to about 180° apart from each other.

[0108] Clause 16. The sensor of clause 14 or clause 15, wherein the at least two hydrophobic tags are positioned about 4 to about 6 nucleotides apart from each other.

[0109] Clause 17. A sensor according to any one of clauses 1 to 13, comprising at least three hydrophobic tags.

[0110] Clause 18. The sensor of clause 17, wherein the at least three hydrophobic tags are positioned at about 90° to about 120° apart from each other.

[0111] Clause 19. The sensor of clause 17 or clause 18, wherein the at least three hydrophobic tags are spaced from about 2 to about 4 nucleotides apart from one another.

[0112] Clause 20. The sensor of any of clauses 1-19, wherein the at least one hydrophobic tag is disposed to span from about 1.0 nm to about 3.0 nm.

[0113] Clause 21. A sensor according to any one of clauses 1 to 20, comprising a nucleic acid sequence which is at least 90% identical to SEQ ID NO: 1 or 2.

[0114] Clause 22. A sensor according to any one of clauses 1 to 20, comprising a nucleic acid sequence which is at least 90% identical to SEQ ID NO: 5, 8, or 11.

[0115] Clause 23. A composition comprising the nucleic acid sensor according to any one of clauses 1 to 22.

[0116] Clause 24. The composition of clause 23, further comprising at least one reporter nucleic acid, said at least one reporter nucleic acid comprising a sequence complementary to at least a portion of said optional hairpin domain.

[0117] Clause 25. The composition of clause 23, further comprising at least one reporter nucleic acid, wherein the at least one reporter nucleic acid comprises a sequence capable of avoiding at least one of: (i) toehold-mediated strand displacement (TMSD); (ii) loop-mediated isothermal amplification (LAMP); and / or (iii) hairpin chain reaction (HCR).

[0118] Clause 26. A method for detecting a target nucleic acid using any of the sensors described in clauses 1 to 22 or the compositions described in clauses 23 to 25.

[0119] Clause 27. The method according to clause 26, wherein the target nucleic acid is located in a membrane vesicle or intracellularly.

[0120] Clause 28. The method of clause 27, comprising detecting the target nucleic acid without lysing the membrane vesicle or cell.

[0121] Clause 29. The method of any one of clauses 26 to 28, wherein the target nucleic acid is DNA or RNA.

[0122] Clause 30. The method of any one of clauses 26 to 28, wherein detecting the target nucleic acid comprises an amplification step.

[0123] Clause 31. The method of clause 30, wherein the amplifying step comprises at least one of toehold-mediated strand displacement (TMSD), loop-mediated isothermal amplification (LAMP), and / or hairpin chain reaction (HCR).

[0124] Clause 32. A method according to any one of clauses 26 to 31, comprising a target detection step.

[0125] Clause 33. A method according to any one of clauses 26 to 32, comprising sequencing the target nucleic acid.

[0126] Clause 34. A kit comprising any of the sensors described in clauses 1 to 22 and instructions for detecting a target nucleic acid.

[0127] Clause 35. The kit of clause 34, further comprising a standard or control.

[0128] Clause 36. The kit of clause 35, wherein the standard or control comprises a detection moiety.

[0129] Clause 37. The kit of clause 35, wherein the standard or control comprises a nucleic acid sequence that is at least 90% identical to SEQ ID NO: 3, 4, 6, 7, 9, 10, 12, or 13.

Claims

1. A double-stranded stem domain containing at least one hydrophobic tag, At least one toehold domain located at one end of the double-stranded stem domain, A nucleic acid sensor comprising, optionally, a hairpin domain located at the end opposite to the end of the double-stranded stem domain in which the toehold domain is located.

2. The nucleic acid sensor according to claim 1, wherein the nucleic acid sensor comprises two toehold domains located at both ends of the double-stranded stem domain, and / or the nucleic acid sensor does not contain a hairpin domain.

3. Furthermore, (i) The nucleic acid sensor comprises two separate nucleic acid molecules having complementary sequences that form the double-stranded stem domain, each of the separate nucleic acid molecules comprising a toehold domain. (ii) The nucleic acid sensor comprises one toehold domain and one hairpin domain at the terminal opposite to the double-stranded stem domain, preferably the nucleic acid sensor comprises a single nucleic acid molecule, the single nucleic acid molecule comprising an internal complementary sequence that forms the double-stranded stem domain; (iii) The nucleic acid sensor is a DNA molecule, an LNA molecule, or a combination thereof; (iv) The nucleic acid sensor is an RNA molecule; and (v) The at least one toehold domain is complementary to the target nucleic acid sequence, preferably the target nucleic acid sequence is a DNA molecule or an RNA molecule; A nucleic acid sensor according to claim 1, defined by one of the following.

4. Furthermore, (i) The at least one toehold domain is approximately 5 to approximately 20 nucleotides; (ii) The stem domain is approximately 5 to approximately 30 nucleotides; and (iii) The hairpin domain is approximately 5 to approximately 20 nucleotides. A nucleic acid sensor according to claim 1, defined by one or more of the following:

5. The nucleic acid sensor according to claim 1, comprising at least two hydrophobic tags, preferably the at least two hydrophobic tags being positioned about 120° to about 180° apart from each other, or about 4 to about 6 nucleotides apart from each other.

6. The nucleic acid sensor according to claim 1, comprising at least three hydrophobic tags, preferably the at least three hydrophobic tags being arranged at a distance of about 90° to about 120° from each other, or at a distance of about 2 to about 4 nucleotides from each other.

7. The nucleic acid sensor according to claim 1, wherein the at least one hydrophobic tag is arranged to extend over a range of about 1.0 nm to about 3.0 nm.

8. The nucleic acid sensor according to claim 1, comprising a nucleic acid sequence that is at least 90% identical to sequence number 1 or 2.

9. The nucleic acid sensor according to claim 1, comprising a nucleic acid sequence that is at least 90% identical to sequence number 5, 8, or 11.

10. A composition comprising a nucleic acid sensor according to any one of claims 1 to 9.

11. It further contains at least one reporter nucleic acid, (a) The at least one reporter nucleic acid contains a sequence complementary to at least a portion of the optional hairpin domain, or (b) The composition according to claim 10, wherein the at least one reporter nucleic acid comprises a sequence capable of initiating at least one of (i) toehold-mediated strand substitution (TMSD), (ii) loop-mediated isothermal amplification (LAMP), and / or (iii) hairpin chain reaction (HCR).

12. A method for detecting a target nucleic acid using a nucleic acid sensor according to any one of claims 1 to 9.

13. Furthermore, (i) The target nucleic acid is located in a membrane vesicle or cell, and the method further preferably includes detecting the target nucleic acid without lysing the membrane vesicle or cell; (ii) The target nucleic acid is DNA or RNA; (iii) Detection of the target nucleic acid includes an amplification step, preferably the amplification step includes at least one of toehold-mediated strand substitution (TMSD), loop-mediated isothermal amplification (LAMP), and / or hairpin chain reaction (HCR); (iv) The method includes a target detection step; and (v) The method includes sequencing the target nucleic acid, The method according to claim 12, defined by one or more of the following.

14. A kit comprising a nucleic acid sensor according to any one of claims 1 to 9, and instructions for detecting a target nucleic acid.

15. The kit according to claim 14, further comprising a standard substance or control, preferably the standard substance or control comprising a detection portion or a nucleic acid sequence that is at least 90% identical to SEQ ID NOs: 3, 4, 6, 7, 9, 10, 12, or 13.

16. A double-stranded stem domain, At least one toehold domain located at one end of the double-stranded stem domain, A nucleic acid sensor comprising, optionally, a hairpin domain located at the end opposite to the end of the double-stranded stem domain in which the toehold domain is located.

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