Compositions and methods related 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 cleavage-free detection of internal nucleic acids.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 317,424, filed on March 7, 2022, which is incorporated herein by reference in its entirety for all purposes.

[0003] Sequence Listing

[0004] The text name of the computer-readable sequence listing submitted herewith is "SKYSG-40581-601", was created on March 7, 2023, has a file size of 12,686 bytes, and the text is hereby incorporated by reference in its entirety.

[0005] Statement Regarding Federal Funding

[0006] This invention was made with government support under Grant No. AI 144247 awarded by the National Institutes of Health. The government has certain rights in this invention. Technical Field

[0007] The present disclosure provides compositions and methods related to nucleic acid sensors. Specifically, the present disclosure provides transmembrane nucleic acid sensors, signal transducers, and molecular amplifiers for non-lytic detection of internal nucleic acids. Background Art

[0008] Non-invasive detection of nucleic acid targets in vesicles (such as exosomes and cells) surrounded by lipid bilayer membranes is very interesting because it can be used in many branches of medicine and biomedical science. Many applications in these applications can utilize various features of nucleic acids. For example, the stem-loop structure or hairpin structure of DNA or RNA is naturally occurring, and this structure is very important because it forms a building block of RNA secondary structure that serves as a recognition site for protein and a nucleation site for RNA folding. In addition to the salt concentration in the solution, the different lengths and sequences of the loop and stem also affect the structural stability of the hairpin, as well as the thermodynamics and kinetics of conformational changes under folding and unfolding states. In addition, simple and powerful structures have been applied to, for example, molecular beacons, molecular computing, and hairpin chain reaction (HCR) amplification, as well as other uses.

[0009] In addition, toehold mediated strand displacement (TMSD) refers to a process in which a DNA strand called a protective strand in a DNA helix structure can be displaced and replaced by an invading strand that is complementary to another strand in the original helix structure. The other strand in the original helix structure is called the original strand, which has a protruding end called a "toehold", which helps the invading strand move and replace the protective strand. The TMSD process has many applications in DNA molecular machines, DNA computing, DNA sensing, and programmable DNA nanostructures. Moreover, the hairpin chain reaction (HCR) is a powerful enzyme-free isothermal amplification method based on two (or more) metastable monomer hairpins. In order to trigger the polymerization of the monomer, an initiator strand is introduced. In addition to RNA imaging in fixed cells, HCR programmability has been developed for many applications in DNA and RNA detection. Summary of the invention

[0010] 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 located at the end of the double-stranded stem domain; and optionally, a hairpin domain located at the end of the double-stranded stem domain opposite to the toehold domain.

[0011] In some embodiments, the sensor comprises two toehold domains located at either end of the double-stranded stem domain.

[0012] In some embodiments, the sensor does not comprise a hairpin domain.

[0013] In some embodiments, the sensor comprises two separate nucleic acid molecules having complementary sequences that form the double-stranded stem domain, and wherein each of the separate nucleic acid molecules comprises a toehold domain.

[0014] In some embodiments, the sensor comprises a toehold domain and a hairpin domain at opposite ends of the double-stranded stem domain.

[0015] In some embodiments, the sensor comprises a single nucleic acid molecule, and wherein the single nucleic acid molecule comprises an internal complementary sequence that forms the double-stranded stem domain.

[0016] 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.

[0017] In some embodiments, the at least one toehold domain is complementary to a target nucleic acid sequence.

[0018] In some embodiments, the target nucleic acid sequence is a DNA molecule or an RNA molecule.

[0019] In some embodiments, the at least one toehold domain is about 5 to about 20 nucleotides.

[0020] In some embodiments, the stem domain is about 10 to about 30 nucleotides.

[0021] In some embodiments, the hairpin domain is about 5 to about 20 nucleotides.

[0022] 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° from each other. In some embodiments, the at least two hydrophobic tags are positioned about 4 to about 6 nucleotides from each other.

[0023] 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° from each other. In some embodiments, the at least three hydrophobic tags are positioned about 2 to about 4 nucleotides from each other.

[0024] In some embodiments, the at least one hydrophobic tag is positioned such that the at least one hydrophobic tag spans from about 1.5 nm to about 3.0 nm.

[0025] 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.

[0026] Embodiments of the present disclosure also include compositions comprising any of the nucleic acid sensors described herein.

[0027] In some embodiments, the composition comprises at least one reporter gene nucleic acid. In some embodiments, the at least one reporter gene nucleic acid comprises a sequence complementary to at least a portion of an optional hairpin domain. In some embodiments, the at least one reporter gene nucleic acid comprises a sequence capable of inducing at least one of: (i) toehold-mediated strand displacement (TMSD); (ii) loop-mediated isothermal amplification (LAMP); and / or (iii) hairpin chain reaction (HCR).

[0028] Embodiments of the present disclosure also include methods of detecting a target nucleic acid using any of the sensors described herein.

[0029] In some embodiments, the target nucleic acid is located within a membrane vesicle or a cell. In some embodiments, the method comprises detecting the target nucleic acid without lysing the membrane vesicle or the cell.

[0030] In some embodiments, the target nucleic acid is DNA or RNA.

[0031] 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).

[0032] In some embodiments, the method includes a target detection step.

[0033] In some embodiments, the method comprises sequencing the target nucleic acid.

[0034] Embodiments of the present disclosure also include kits comprising any of the sensors described herein and instructions for detecting a target nucleic acid.

[0035] In some embodiments, the kit further comprises a calibrator or control. In some embodiments, the calibrator or control comprises a detection portion. In some embodiments, the calibrator 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

[0036] Figure 1A-1B : A representative schematic diagram of a DNA sensor according to one embodiment of the present disclosure.

[0037] Figure 2A-2B : A representative schematic diagram of a method for nucleic acid detection using a double-stranded DNA sensor according to one embodiment of the present disclosure.

[0038] Figure 3 : Representative confocal images showing the insertion of the double-stranded DNA sensor across the membrane, showing a green ring on the GUV in the right image and the detection of the target strand forming a red ring on the GUV in the middle image.

[0039] Figures 4A-4B : A representative schematic diagram of a method for nucleic acid detection using a hairpin DNA sensor according to one embodiment of the present disclosure.

[0040] FIG. 5 : Representative experimental results of negative control experiments of the DNA sensor of the present disclosure.

[0041] Figure 6: Representative confocal images showing the detection of internal nucleic acid targets in vesicles with several POPC and cholesterol composition ratios.

[0042] Figure 7 : Representative experimental results, demonstrating that the double-chain sensor has minimal effect on irrelevant molecules present in GUVs, causing only an average of about 8.4% leakage in all three experiments.

[0043] Figures 8A-8B : Representative confocal images showing the insertion of the hairpin DNA sensor across the membrane, showing the green ring formed on the GUV due to the binding of the target to the sensor in the left image, and the negative control in which the scrambled DNA inside the GUV did not produce a green ring on the GUV in the right image.

[0044] Figures 9A-9B : Schematic diagram of the TMSD of the hairpin DNA sensor after target binding and opening of the sensor within the vesicle to enable binding of the reporter strand is shown in the upper panel, and representative confocal images of detection of the target forming a green ring on the GUV are shown in the lower left panel and binding of the reporter strand on the open stem portion of the sensor forming a red ring on the GUV are shown in the lower right panel.

[0045] Fig.10 : Schematic diagram of the TMSD of the double-stranded DNA sensor after target binding inside the vesicle allows a certain part of the target to bind to the reporter gene added outside the GUV, and representative confocal images of target detection showing the formation of a red ring due to target binding to the hairpin sensor and the binding of the reporter gene strand to the part of the target protruding outward from the GUV membrane due to the TMSD formation of a blue ring on the right.

[0046] Fig.11 : Representative experimental results of TMSD negative control experiments of double-stranded DNA sensors, which show no reporter gene binding, indicating the absence of TMSD across lipid membranes in the case of hybrid intra-vesicle DNA targets of the present disclosure. DETAILED DESCRIPTION

[0047] Embodiments of the present disclosure provide nanometer-sized biosensors made of DNA that can span lipid bilayer membranes, detect internal nucleic acid targets present in vesicles, and amplify signals from detection events to enable detection of low-concentration targets. The inventive subject matter of the present disclosure is based on several phenomena and technologies closely related to DNA nanotechnology and molecular biology, including DNA hairpin structures, toehold-mediated strand displacement, DNA hybridization design, free energy calculations, molecular dynamics simulations, hairpin chain reactions, and bioconjugation. Embodiments of the present disclosure utilize the multidisciplinary developments mentioned above to create single-stranded and double-stranded DNA nanostructures with sequences, structures, and hydrophobic modifications designed in such a way that they can be anchored, inserted into lipid bilayer membranes, and detect internal nucleic acids of interest, while allowing information to be transduced across the membrane via TMSD and amplified via isothermal amplification such as HCR. The use of TMSD and HCR or other isothermal amplification methods will be widely used in DNA sensors and related components of the present disclosure.

[0048] Several types of nucleic acid sensors are described herein, and each type has been designed and tested to adapt to the effective insertion and anchoring of the lipid bilayer membrane across the vesicle for detection of internal nucleic acid targets without cleavage. The signal transduction from the transmembrane detection event and the amplification of the internal information of the vesicle are carried out using toehold-mediated strand displacement (TMSD), isothermal amplification (e.g., hairpin chain reaction (HCR)) and other possible amplification methods. Simple methods for single-stranded DNA and double-stranded DNA will prevent the stoichiometric problems often encountered in the synthesis of DNA biosensors. The simple application of TMSD together with isothermal amplification provides a simple method for using DNA biosensors to transfer and amplify information across membranes to detect low-concentration internal nucleic acids. Detection of biomarkers present in exosomes and cells can be performed. In addition, the non-invasive detection method of DNA nanosensors can also be used to diagnose diseases and genotype cells, as well as targeted therapy.

[0049] The section headings as used in this section and the entire disclosure herein are for organizational purposes only and are not intended to be limiting.

[0050] 1. Definition

[0051] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art. In the event of a conflict, this document (including definitions) shall prevail. Although methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present disclosure, preferred methods and materials are described below. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods and embodiments disclosed herein are illustrative only and are not intended to be restrictive.

[0052] As used herein, the terms "comprise," "include," "having," "has," "can," "contain," and variations thereof are intended to be open-ended conjunctions, terms, or words that do not exclude the possibility of additional actions or structures. Unless the context clearly dictates otherwise, the singular forms "a," "an," and "the" include plural referents. The present disclosure also contemplates other embodiments "comprising," "consisting of," and "consisting essentially of," whether or not explicitly stated.

[0053] For the recitation of numerical ranges herein, each intermediate number with the same degree of precision is expressly contemplated. For example, for a range of 6 to 9, the numbers 7 and 8 are included in addition to 6 and 9, and for a range of 6.0 to 7.0, the numbers 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 included.

[0054] As used herein, "correlating to" means compared to.

[0055] The term "single-stranded" oligonucleotide generally refers to those oligonucleotides containing a series of covalently linked nucleotide residues.

[0056] The term "oligomer" or "oligonucleotide" includes RNA or DNA sequences of more than one nucleotide in single-stranded or duplex form, specifically including short sequences in single-stranded or duplex form, such as dimers and trimers, which can be intermediates for producing specific binding oligonucleotides. The "modified" form used in the candidate library contains at least one non-natural residue. "Oligonucleotide" or "oligomer" generally refers to polydeoxyribonucleotides (containing 2'-deoxy-D-ribose or its modified form), such as DNA, polyribonucleotides (containing D-ribose or its modified form), such as RNA, and any other type of polynucleotide, which is an N-glycoside or C-glycoside of a purine or pyrimidine base, or a modified purine or pyrimidine base or abasic nucleotide. "Oligonucleotide" or "oligomer" can also be used to describe artificially synthesized polymers, which are similar to RNA and DNA or DNA and RNA molecules with modified backbones and nucleosides, including but not limited to oligomers of peptide nucleic acids (PNA) and locked nucleic acids (LNA).

[0057] The term "RNA analogue" or "RNA derivative" or "modified RNA" generally refers to a polymeric molecule which, in addition to ribonucleosides, contains as its units at least one of the following: 2'-deoxy, 2'-halo (including 2'-fluoro), 2'-amino (preferably unsubstituted or mono- or di-substituted), 2'-monohalo, dihalo or trihalomethyl, 2'-O-alkyl, 2'-O-haloalkyl, 2'-alkyl, azido, phosphorothioate, thiol, methylphosphonate, fluorescein, rhodamine, pyrene, biotin, xanthine, hypoxanthine, 2,6-diaminopurine, 2-hydroxy-6-mercaptopurine and 2'-sulfur at the 6-position or halogen or C at the 5-position. 1-5 Alkyl-substituted pyrimidine bases, basic linkers, 3′-deoxyadenosine, and other available “chain terminators” or “non-extendable” analogs (at the 3′-end of the RNA), or labels such as 32 P. 33 All of the foregoing can be incorporated into RNA using standard synthetic techniques disclosed herein.

[0058] 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 significant in "binding activity" and "binding affinity" because they can include definitions of the concentrations of the interaction partners, the rates at which these partners are able to associate, and the relative concentrations of bound and free molecules in solution.

[0059] "Complementarity" refers to the characteristic of two or more structural elements (e.g., peptides, polypeptides, nucleic acids, small molecules, etc.) that are able to hybridize, dimerize, or otherwise form a complex with each other. For example, "complementary peptides and polypeptides" are able to come together to form a complex. Complementary elements may require assistance to form a complex (e.g., from an interacting element), for example, placing the elements in the correct conformation for complementation, co-localizing complementary elements, reducing the complementary interaction energy, etc.

[0060] As used herein, the term "nucleotide sequence identity" or "nucleic acid sequence identity" refers to the presence of identical nucleotides in the corresponding positions of two polynucleotides. If the nucleotide sequences in two polynucleotides are identical when the maximum correspondence is compared (e.g., in a comparison window), the polynucleotides have an "identical" sequence. The sequence comparison between two or more polynucleotides is usually performed by comparing the parts of the two sequences on a comparison window to identify and compare local regions with sequence similarity. The comparison window is generally about 20 to 200 continuous nucleotides. The "percentage of sequence identity" of a polynucleotide, such as about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity, can be determined by comparing two optimally aligned sequences over a comparison window, wherein for optimal alignment of the two sequences, the portion of the polynucleotide sequence in the comparison window may include additions or deletions (i.e., gaps) compared to the reference sequence. In some embodiments, the percentage is calculated by: (a) determining the number of positions where the identical nucleic acid base occurs in the two sequences; (b) dividing the number of matched positions by the total number of positions in the comparison window; and (c) multiplying the result by 100. Optimal alignment of sequences for comparison can also be performed by computerized implementation of known algorithms or by visual inspection. Readily available sequence comparison and multiple sequence alignment algorithms are the Basic Local Alignment Search Tool (BLAST) and ClustalW / ClustalW2 / Clustal Omega programs, respectively, available on the Internet (e.g., the EMBL-EBI website). Other suitable programs include, but are not limited to, GAP, BestFit, Plot Similarity, and FASTA, which are part of the Accelrys GCG software package available from Accelrys, Inc., San Diego, California, USA. See also Smith and Waterman, 1981; Needleman and Wunsch, 1970; Pearson and Lipman, 1988; Ausubel et al., 1988; and Sambrook and Russell, 2001.

[0061] 2. Nucleic acid sensors

[0062] Embodiments of the present disclosure include compositions and methods related to nucleic acid sensors. Specifically, the present disclosure provides transmembrane nucleic acid sensors, signal transducers, and molecular amplifiers for non-lytic detection of internal nucleic acids.

[0063] According to these embodiments, the present disclosure provides a nucleic acid sensor comprising: a double-stranded stem domain comprising at least one hydrophobic tag, at least one toehold domain located at the end of the double-stranded stem domain; and optionally, a hairpin domain located at the end of the double-stranded stem domain opposite to the toehold domain.

[0064] In some embodiments, the sensor comprises two foothold domains located 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 forming a double-stranded stem domain. In some embodiments, each of the separate nucleic acid molecules comprises a foothold domain (e.g., FIG. 1A ). According to these embodiments, the foothold domain can be open at one end (i.e., the 5' or 3' end of the nucleic acid molecule is non-conjugated). In some embodiments, the 5' or 3' end of the foothold domain is conjugated to a detection moiety.

[0065] In some embodiments, the sensor comprises a toehold domain and a hairpin domain at opposite ends of the double-stranded stem domain. In some embodiments, the sensor comprises a single nucleic acid molecule (e.g., FIG. 1B ). In some embodiments, the single nucleic acid molecule comprises an internal complementary sequence that forms a double-stranded stem domain (e.g., FIG. 1B ). According to these embodiments, the hairpin domain is closed (i.e., it comprises a continuous nucleic acid sequence).

[0066] 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, the 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.

[0067] In some embodiments, the at least one toehold domain is about 5 to about 20 nucleotides. In some embodiments, the at least one toehold domain is about 10 to about 20 nucleotides. In some embodiments, the at least one toehold domain is about 15 to about 20 nucleotides. In some embodiments, the at least one toehold domain is about 5 to about 15 nucleotides. In some embodiments, the at least one toehold domain is about 5 to about 10 nucleotides. In some embodiments, the at least one toehold domain includes 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides.

[0068] 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 includes 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.

[0069] 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 includes 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides.

[0070] 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° from each other. In some embodiments, the at least two hydrophobic tags are positioned about 180° from each other. In some embodiments, the at least two hydrophobic tags are positioned about 4 to about 6 nucleotides from each other. In some embodiments, the at least two hydrophobic tags are positioned 2, 3, 4, 5, or 6 nucleotides from each other.

[0071] 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° from each other. In some embodiments, the at least three hydrophobic tags are positioned about 120° from each other. In some embodiments, the at least three hydrophobic tags are positioned about 2 to about 4 nucleotides from each other. In some embodiments, the at least three hydrophobic tags are positioned about 1, 2, 3, or 4 nucleotides from each other.

[0072] In some embodiments, the at least one hydrophobic tag is positioned so that the at least one hydrophobic tag spans about 1.0nm to about 3.0nm. In some embodiments, the at least one hydrophobic tag is positioned so that the at least one hydrophobic tag spans about 1.5nm to about 3.0nm. In some embodiments, the at least one hydrophobic tag is positioned so that the at least one hydrophobic tag spans about 2.0nm to about 3.0nm. In some embodiments, the at least one hydrophobic tag is positioned so that the at least one hydrophobic tag spans about 2.5nm to about 3.0nm. In some embodiments, the at least one hydrophobic tag is positioned so that the at least one hydrophobic tag spans about 1.0nm to about 2.5nm. In some embodiments, the at least one hydrophobic tag is positioned so that the at least one hydrophobic tag spans about 1.0nm to about 2.0nm. In some embodiments, the at least one hydrophobic tag is positioned so that the at least one hydrophobic tag spans about 1.0nm to about 1.5nm.

[0073] 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 NOs: 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.

[0074] 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.

[0075] Embodiments of the present disclosure also include methods for detecting a target nucleic acid using any sensor described herein. In some embodiments, the target nucleic acid is located in a membrane vesicle or cell. In some embodiments, the method includes detecting the target nucleic acid without lysing the membrane vesicle or cell. In some embodiments, the target nucleic acid is DNA or RNA.

[0076] In some embodiments, detecting the target nucleic acid includes an amplification step. In some embodiments, the amplification step includes 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 includes a target detection step. For example, in some embodiments, nucleic acid detection includes the use of fluorophores, chromophores, fluorophore pairs, fluorophore-quencher pairs, or other detection moieties known in the art.

[0077] In some embodiments, the method includes sequencing the target nucleic acid.For example, in some embodiments, the detection of the target nucleic acid includes sequencing using Sanger sequencing, next generation sequencing (NGS) or any other sequencing method known in the art.

[0078] Embodiments of the present disclosure also include compositions comprising any nucleic acid sensor described herein. In some embodiments, the composition comprises at least one reporter gene nucleic acid. In some embodiments, the at least one reporter gene nucleic acid comprises a sequence complementary to at least a portion of an optional hairpin domain. In some embodiments, the at least one reporter gene nucleic acid comprises a sequence capable of inducing at least one of the following: (i) toehold-mediated strand displacement (TMSD); (ii) loop-mediated isothermal amplification (LAMP); and / or (iii) hairpin chain reaction (HCR).

[0079] Embodiments of the present disclosure also include kits comprising any sensor described herein and instructions for detecting a target nucleic acid. In some embodiments, the kit further comprises a calibrator or control. In some embodiments, the calibrator or control comprises a detection portion. In some embodiments, the calibrator or control comprises a nucleic acid sequence at least 90% identical to SEQ ID NO: 3, 4, 6, 7, 9, 10, 12 or 13. In some embodiments, the calibrator or control comprises a nucleic acid sequence at least 91% identical to SEQ ID NO: 3, 4, 6, 7, 9, 10, 12 or 13. In some embodiments, the calibrator or control comprises a nucleic acid sequence at least 92% identical to SEQ ID NO: 3, 4, 6, 7, 9, 10, 12 or 13. In some embodiments, the calibrator or control comprises a nucleic acid sequence at least 93% identical to SEQ ID NO: 3, 4, 6, 7, 9, 10, 12 or 13. In some embodiments, the calibrator 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 calibrator 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 calibrator 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 calibrator 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 calibrator 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 calibrator 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.

[0080] 3. Sequence

[0081] Provided below are various nucleic acid sequences (ie, SEQ ID NOs) referenced herein.

[0082] Table 1:

[0083]

[0084] Table 2:

[0085]

[0086]

[0087] Other suitable modifications will be apparent to those skilled in the art.

[0088] It should be understood that the foregoing detailed description and the accompanying examples are merely illustrative and should not be taken as limiting the scope of the present disclosure, which is defined solely by the appended claims and their equivalents.

[0089] Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications may be made without departing from the spirit and scope of the invention, including but not limited to those changes and modifications related to the chemical structures, substituents, derivatives, intermediates, syntheses, compositions, formulations or methods of use of the present invention.

[0090] 4. Examples

[0091] It will be apparent to those skilled in the art that other suitable modifications and adaptations of the disclosed methods described herein are readily applicable and understandable, and can be made using suitable equivalents without departing from the scope of the disclosure or the aspects and embodiments disclosed herein. Having now described the disclosure in detail, it will be more clearly understood by reference to the following examples, which are intended only to illustrate some aspects and embodiments of the disclosure and should not be construed as limiting the scope of the disclosure. All journal references, U.S. patents, and published disclosures cited herein are hereby incorporated by reference in their entirety.

[0092] The present disclosure has several aspects, illustrated by the following non-limiting examples.

[0093] Example 1

[0094] Design, structure and working principle of the sensor. In one embodiment, the single-stranded DNA sensor consists of a foothold, a stem and a hairpin segment. The duplex DNA has two footholds at each end, connected by a stem segment. This embodiment requires one or two chains, which is very powerful in overcoming the stoichiometric problem during sensor formation, which is generally a challenge for DNA nanotechnology. In addition, this simple method will facilitate insertion kinetics and the number of hydrophobic tags required.

[0095] Based on the target length, the foothold can be about 6-13 nucleotides, which are complementary to the part to be detected of the internal nucleic acid target. The stem is designed to provide structural integrity for hairpin and duplex structure. The foothold mediates the displacement of the stem segment, allowing the signal to be transduced from the inside to the outside. The stem has about 14-15 base pairs, which are identical or complementary to the target sequence depending on the target length. 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). In order to keep the polar group balance on both sides of the stem, the hairpin is selected to have the same length as the foothold. Hydrophobic modification is strategically placed on one side of the stem. Therefore, once TMSD is completed, the other side of the stem can be separated from the film. Figure 1A-1B A schematic diagram of the DNA sensor design is shown.

[0096] In order to effectively cover the hydrophobic liquid with the DNA sensor, the spatial arrangement of the hydrophobic tags (two or three tags) is carefully designed in such a way that when projected onto the cross section of the stem, the distance between them is 180 degrees for two hydrophobic tags, or 120 degrees for three hydrophobic tags (see the middle figure of Figures 1A and 1B). Therefore, the angular spacing between the two tags is 5 nucleotides in the case of two tags, and 3 and 4 nucleotides in the case of three tags. This can be seen in the left figure of Figures 1A and 1B, where the hydrophobic tags are shown in green and the spacing between them is also shown. Therefore, the stem will be anchored perpendicular 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 to 2.5nm, which is less than the known thickness of the lipid bilayer of 3.5-4.0nm, which ensures that the stem is located vertically in the membrane and the hairpin and foothold are free on the opposite side.

[0097] The hydrophobic tag is placed asymmetrically along the stem to facilitate the insertion of the toehold into the vesicle for successful internal nucleic acid detection. Therefore, the hydrophobic group is positioned closer to the toehold region, allowing it to produce less polar nucleic acids on the stem side of the toehold. Considering that the toehold and the hairpin are the same length, the orientation during insertion depends only on the position of the hydrophobic group on the stem, which is very versatile in terms of design. The placement of the hydrophobic tag is shown in Figure 1A and the left figure of Figure 1B.

[0098] Two mismatches were introduced in the 14-15 base pair long stem to generate faster TMSD kinetics and an optimal toehold length of around 6-10 nucleotides (Zhang and Winfree, 2009). The mismatches were carefully chosen and placed so that they would not affect the integrity of the stem structure but would only contribute to the TMSD kinetics. The mismatches were located near hydrophobic groups. Nupack showed a high equilibrium probability around the mismatches, thus confirming the integrity of the stem but reducing the stability of this region.

[0099] Once TMSD is completed, the signal is transduced across the membrane. Due to displacement by the target chain, one of the single-stranded units in the stem region will be released from the membrane restriction to the outside of the vesicle. When the opened single-stranded hairpin segment is replaced by the single-stranded unit of the stem, the hairpin chain reaction is triggered. The right figure of Figure 1B shows two monomer hairpins of HCR in the case of a single-stranded DNA sensor. They are specially designed in such a way that the reaction will be triggered only when there is an internal target detection and a successful TMSD event across the lipid bilayer membrane. For double-stranded sensors, the internal target chain is designed to have a target that acts as a signal chain with a fluorophore to bind and detect a segment of TMSD. Due to the length limit of the target, HCR cannot be performed on double-stranded DNA sensors, and Figure 1A shows the internal target and signal chain of double-stranded DNA sensors.

[0100] In the case of double-stranded DNA, the signal strand will be bound to the portion c shown in Figure 1A. For hairpin sensors, once the hairpin is opened, HCR amplification will occur. The combination of the signal strand or HCR monomer allows the detection of a fluorophore signal, which can be detected by microscopy or gel assay. In microscopy, one of the signal strand and the hairpin monomer can be conjugated with a fluorophore to detect the polymer formed by HCR. Monomer hairpins with emitter-quencher pairs or donor-acceptor pairs can also be used, or by using fluorescence dequenching detection or FRET, monitoring detection on a microplate reader.

[0101] Using the above design principles, Nupack can be used to design and generate optimized sequences for single-stranded and double-stranded DNA sensors. One of the initial targets was a sequence from the microRNA miR23b, which is an adaptation for practical applications. Tables 1 and 2 list the DNA sensors, their targets, the signal strands for double-stranded sensors, and the hairpin monomers for single-stranded sensors.

[0102] Giant unilamellar vesicles (GUVs) containing phospholipids and some cholesterol in their membranes were used to carry out the proof-of-concept experiments. GUVs were synthesized with the target DNA encapsulated within them. Using fluorescence confocal microscopy, both the DNA target and the DNA sensor would be labeled with a fluorescent dye to be able to analyze the interaction of the DNA target with the GUV.

[0103] Example 2

[0104] Double-stranded and hairpin DNA sensor transmembrane insertion and detection of single-stranded DNA within GUVs as a proof-of-concept for DNA sensor transmembrane insertion. Experiments were performed to demonstrate that the DNA sensor can insert into the lipid bilayer membrane and sense internal targets within the vesicles. To demonstrate how the insertion and detection work, 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 once the target binds to the sensor containing double-stranded and single-stranded DNA. Figure 8A shows successful intravesicular target detection by a hairpin DNA sensor that forms a green ring on the GUV, and Figure 8B is a negative control by a scrambled intravesicular DNA target with no green ring observed.

[0105] Detection of internal nucleic acid targets was demonstrated by using GUV test samples encapsulating specific target DNA complementary to the toeholds of single-stranded and double-stranded DNA sensors. Such GUVs were synthesized by reverse emulsion or cDICE method with POPC phospholipid and cholesterol in a ratio of 70%:30%, respectively. The internal solution contained a certain concentration of sucrose, as well as 250mM of KCl and 100-1000nM concentration of the target. An external solution with a certain concentration of glucose and 250mM of KCl was present. The concentrations of sucrose and glucose were adjusted to balance the osmotic pressure of the internal and external solutions. The DNA sensor was then mixed with the test sample and incubated for about 1.5 hours before being examined under a confocal microscope. Figure 1A-1B In the assay, the DNA sensor was labeled with SYBR gold, while the internal target DNA was labeled with a Cy5 fluorophore (referred to as fluorophore 1). Figure 3 Confocal images showing the insertion of a double-stranded DNA sensor across a membrane, a green ring on the GUV in the right image and detection of the target strand forming a red ring on the GUV in the middle image are shown. Insertion and detection were experimentally examined using fluorescence confocal microscopy.

[0106] Example 3

[0107] Toehold-mediated strand displacement across GUV membranes. Toehold-mediated strand displacement (TMSD) occurs in solution when there is no limiting membrane around the DNA (Yurke et al., 2000). In this embodiment, the DNA sensor undergoes TMSD under lipid bilayer membrane constraints. In the design section, it is noted that the sensor has a hydrophobic tag on one side of the double-stranded stem, allowing the other side to be released to the outside of the vesicle once TMSD is completed across the membrane. From the perspective of a single base pair, the difference in total Gibbs free energy for transmembrane TMSD will be zero. It is expected that the kinetics of TMS will be slower than in solution only in the absence of a limiting membrane. The right figure of Figure 2A and Figure 4A shows a schematic diagram of a single-stranded and double-stranded DNA sensor.

[0108] The GUV test sample described in Section II was used. It was labeled with a signal chain having a different fluorophore than the target. In the case of the single-stranded DNA sensor, the signal chain was designed with Nupack (nupack.org) to ensure that leakage would not occur if transmembrane TMSD did not occur. In addition, the assays shown in Figures 2A and 4A were carefully designed so that false positives would not be triggered. After the transmembrane TMSD is completed, the detection of the internal target information is transduced to the outside and the signal chain is expected to bind to the sensor portion that is released to the outside but still anchored to the membrane by the sensor, so as to emit a fluorescent signal from the sensor surface, as shown in Figure 2B for the double-stranded DNA sensor. The event was investigated using fluorescent confocal microscopy.

[0109] Example 4

[0110] HCR amplification of internal target detection in hairpin sensor. HCR amplification is used to demonstrate amplification of detection events. Nupack (nupack.org) is used to design hairpin monomers. Leakage and false positive signals of hairpins and assays are checked. A schematic diagram 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 targets. The same GUV test sample was mixed with the sensor and two monomer hairpins. Amplification was observed using fluorescence confocal microscopy.

[0111] Example 5

[0112] Testing of several GUV composition ratios of the double-stranded sensor. Experiments were performed to examine the ability of the double-stranded sensor to insert and detect internal nucleic acid targets in vesicles with several POPC and cholesterol composition ratios. These experiments were 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, demonstrating that the double-stranded sensor still functions well under different synthetic lipid bilayer compositions.

[0113] Small dye molecule leakage assay and invasive characterization of the double-stranded sensor. The invasiveness of the double-stranded sensor was also checked by performing a small dye molecule leakage assay. Confocal images indicated that the sensor did not cause any visible damage to the GUV. However, a leakage assay was performed to test how it affects only very small dye molecules and conjugated to a certain length of single-stranded DNA within the GUV that is not associated with the sensor. The leakage of ATTO488 dye molecules, ATTO488-15nt single-stranded DNA, and ATTO488-22nt single-stranded DNA was tested. Figure 7 Its effect on a small dye molecule is depicted. From the results, it can be concluded that the double-stranded sensor has minimal effect on irrelevant molecules present in the GUV, causing only an average of about 8.4% leakage across all three experiments.

[0114] Example 6

[0115] Potential for TMSD across lipid bilayer membranes on double-stranded and hairpin DNA sensors. Experiments were performed to examine the potential for TMSD across lipid membranes on double-stranded and hairpin DNA sensors. The corresponding DNA targets for the double-stranded and hairpin DNA sensors were encapsulated inside the GUVs. The sensors were then added and incubated for several hours to allow TMSD to proceed. Finally, a reporter gene specific for the sensor was added to probe the occurrence of TMSD.

[0116] Figure 9A shows a schematic diagram of the experiment and process of the hairpin DNA sensor. Figure 9B shows the detection of the target inside the vesicle forming a green ring on the GUV on the left, and the binding of the reporter gene strand from outside the GUV to the open stem part of the sensor forming a red ring on the GUV. In the case of the double-stranded DNA sensor, Fig.10 The upper panel shows a schematic diagram of the process of penetration of a certain portion of the transmembrane TMSD and its use for reporter binding of the target from outside the GUV, and the lower panel shows a representative confocal image, where in the lower left panel the detection of the target by the double-stranded sensor forms a red ring on the GUV, and in the lower right panel a blue ring forms on the GUV due to the binding of the reporter strand to the sensor. Fig.11 As shown, negative control experiments with promiscuous intravesicular DNA targets and using double-stranded DNA sensors showed that no blue rings were formed on the GUVs.

[0117] It should be understood that the foregoing detailed description and the accompanying examples are merely illustrative and should not be considered as limiting the scope of the embodiments of the present disclosure, which is limited 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 may be made without departing from the spirit and scope of the present invention, including but not limited to those changes and modifications related to the chemical structures, substituents, derivatives, intermediates, syntheses, compositions, formulations, or methods of use of the embodiments of the present disclosure.

[0118] For reasons of completeness, various aspects of embodiments of the present disclosure are set forth in the following numbered clauses:

[0119] Item 1. A nucleic acid sensor comprising: a double-stranded stem domain comprising at least one hydrophobic tag; at least one toehold domain located at the end of the double-stranded stem domain; and optionally, a hairpin domain located at the end of the double-stranded stem domain opposite to the toehold domain.

[0120] Clause 2. The sensor of Clause 1, wherein the sensor comprises two toehold domains located at both ends of the double-stranded stem domain.

[0121] Clause 3. The sensor of Clause 1 or Clause 2, wherein the sensor does not comprise a hairpin domain.

[0122] Clause 4. The sensor of any one of clauses 1 to 3, wherein the sensor comprises two separate nucleic acid molecules having complementary sequences forming the double-stranded stem domain, and wherein each of the separate nucleic acid molecules comprises a toehold domain.

[0123] Item 5. The sensor of Item 1, wherein the sensor comprises a toehold domain and a hairpin domain at opposite ends of the double-stranded stem domain.

[0124] Clause 6. The sensor of Clause 5, wherein the sensor comprises a single nucleic acid molecule, and wherein the single nucleic acid molecule comprises an internal complementary sequence that forms the double-stranded stem domain.

[0125] Clause 7. The sensor of any one of Clauses 1 to 6, wherein the nucleic acid sensor is a DNA molecule, an LNA molecule, or a combination thereof.

[0126] Clause 8. The sensor of any one of Clauses 1 to 6, wherein the nucleic acid sensor is an RNA molecule.

[0127] Item 9. The sensor of any one of Items 1 to 8, wherein the at least one toehold domain is complementary to a target nucleic acid sequence.

[0128] Item 10. The sensor of Item 10, wherein the target nucleic acid sequence is a DNA molecule or an RNA molecule.

[0129] Item 11. The sensor of any one of Items 1 to 10, wherein the at least one toehold domain is about 5 to about 20 nucleotides.

[0130] Item 12. The sensor of any one of Items 1 to 11, wherein the stem domain is about 5 to about 30 nucleotides.

[0131] Item 13. The sensor of any one of Items 1 to 12, wherein the hairpin domain is about 5 to about 20 nucleotides.

[0132] Clause 14. The sensor of any one of Clauses 1 to 13, wherein the sensor comprises at least two hydrophobic tags.

[0133] Clause 15. The sensor of Clause 14, wherein the at least two hydrophobic tags are positioned about 120° to about 180° from each other.

[0134] Item 16. The sensor of Item 14 or Item 15, wherein the at least two hydrophobic tags are positioned about 4 to about 6 nucleotides away from each other.

[0135] Clause 17. The sensor of any one of Clauses 1 to 13, wherein the sensor comprises at least three hydrophobic tags.

[0136] Clause 18. The sensor of Clause 17, wherein the at least three hydrophobic tags are positioned about 90° to about 120° from each other.

[0137] Item 19. The sensor of Item 17 or Item 18, wherein the at least three hydrophobic tags are positioned about 2 to about 4 nucleotides away from each other.

[0138] Clause 20. The sensor of any one of Clauses 1 to 19, wherein the at least one hydrophobic tag is positioned such that the at least one hydrophobic tag spans from about 1.0 nm to about 3.0 nm.

[0139] Clause 21. The sensor of any one of Clauses 1 to 20, wherein the sensor comprises a nucleic acid sequence that is at least 90% identical to SEQ ID NO: 1 or 2.

[0140] Clause 22. The sensor of any one of Clauses 1 to 20, wherein the sensor comprises a nucleic acid sequence that is at least 90% identical to SEQ ID NO: 5, 8 or 11.

[0141] Item 23. A composition comprising the nucleic acid sensor according to any one of Items 1 to 22.

[0142] Clause 24. The composition of Clause 23, wherein the composition further comprises at least one reporter nucleic acid, wherein the at least one reporter nucleic acid comprises a sequence complementary to at least a portion of the optional hairpin domain.

[0143] Item 25. A composition as described in Item 23, wherein the composition further comprises at least one reporter gene nucleic acid, wherein the at least one reporter gene nucleic acid comprises a sequence capable of inducing at least one of the following: (i) toehold-mediated strand displacement (TMSD); (ii) loop-mediated isothermal amplification (LAMP); and / or (iii) hairpin chain reaction (HCR).

[0144] Clause 26. A method of detecting a target nucleic acid using any sensor according to clauses 1 to 22 or a composition according to clauses 23 to 25.

[0145] Item 27. The method of Item 26, wherein the target nucleic acid is located in a membrane vesicle or a cell.

[0146] Clause 28. The method of Clause 27, wherein the method comprises detecting the target nucleic acid without lysing the membrane vesicle or the cell.

[0147] Clause 29. The method of any one of Clauses 26 to 28, wherein the target nucleic acid is DNA or RNA.

[0148] Clause 30. The method of any one of Clauses 26 to 28, wherein detecting the target nucleic acid comprises an amplification step.

[0149] Clause 31. The method of clause 30, wherein the amplification step comprises at least one of toehold-mediated strand displacement (TMSD), loop-mediated isothermal amplification (LAMP) and / or hairpin chain reaction (HCR).

[0150] Clause 32. The method of any one of clauses 26 to 31, wherein the method comprises a target detection step.

[0151] Clause 33. The method of any one of Clauses 26 to 32, wherein the method comprises sequencing the target nucleic acid.

[0152] Clause 34. A kit comprising any sensor as described in Clauses 1 to 22 and instructions for detecting a target nucleic acid.

[0153] Clause 35. The kit of Clause 34, further comprising a calibrator or a control.

[0154] Clause 36. The kit of Clause 35, wherein the calibrator or control comprises a detection moiety.

[0155] Clause 37. The kit of Clause 35, wherein the calibrator 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 nucleic acid sensor, comprising: a double-stranded stem domain comprising at least one hydrophobic tag; at least one toehold domain located at a terminus of the double-stranded stem domain; and Optionally, a hairpin domain is located at the end of the double-stranded stem domain opposite the toehold domain.

2. The sensor of claim 1, wherein the sensor comprises two toehold domains located at both ends of the double-stranded stem domain.

3. A sensor as claimed in claim 1 or claim 2, wherein the sensor does not comprise a hairpin domain.

4. The sensor of claim 1, wherein the sensor comprises two separate nucleic acid molecules having complementary sequences that form the double-stranded stem domain, and wherein each of the separate nucleic acid molecules comprises a toehold domain.

5. The sensor of claim 1, wherein the sensor comprises a toehold domain and a hairpin domain at opposite ends of the double-stranded stem domain.

6. The sensor of claim 5, wherein the sensor comprises a single nucleic acid molecule, and wherein the single nucleic acid molecule comprises an internal complementary sequence that forms the double-stranded stem domain. The sensor of claim 1 , wherein the nucleic acid sensor is a DNA molecule, an LNA molecule, or a combination thereof.

8. The sensor of claim 1, wherein the nucleic acid sensor is an RNA molecule.

9. The sensor of claim 1, wherein the at least one toehold domain is complementary to a target nucleic acid sequence.

10. The sensor of claim 10, wherein the target nucleic acid sequence is a DNA molecule or an RNA molecule.

11. The sensor of claim 1, wherein the at least one toehold domain is about 5 to about 20 nucleotides.

12. The sensor of claim 1, wherein the stem domain is about 5 to about 30 nucleotides.

13. The sensor of claim 1, wherein the hairpin domain is about 5 to about 20 nucleotides.

14. The sensor of claim 1, wherein the sensor comprises at least two hydrophobic tags.

15. The sensor of claim 14, wherein the at least two hydrophobic tags are positioned about 120° to about 180° from each other.

16. The sensor of claim 14, wherein the at least two hydrophobic tags are positioned about 4 to about 6 nucleotides away from each other.

17. The sensor of claim 1, wherein the sensor comprises at least three hydrophobic tags.

18. The sensor of claim 17, wherein the at least three hydrophobic tags are positioned about 90° to about 120° from each other.

19. The sensor of claim 17, wherein the at least three hydrophobic tags are positioned about 2 to about 4 nucleotides away from each other.

20. The sensor of claim 1, wherein the at least one hydrophobic tag is positioned such that the at least one hydrophobic tag spans from about 1.0 nm to about 3.0 nm.

21. The sensor of claim 1, wherein the sensor comprises a nucleic acid sequence that is at least 90% identical to SEQ ID NO: 1 or 2.

22. The sensor of claim 1, wherein the sensor comprises a nucleic acid sequence that is at least 90% identical to SEQ ID NO: 5, 8, or 11.

23. A composition comprising the nucleic acid sensor according to claim 1.

24. The composition of claim 23, wherein the composition further comprises at least one reporter nucleic acid, wherein the at least one reporter nucleic acid comprises a sequence complementary to at least a portion of the optional hairpin domain.

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

26. A method for detecting a target nucleic acid using any sensor according to claim 1 or the composition according to claim 23.

27. The method of claim 26, wherein the target nucleic acid is located within a membrane vesicle or a cell.

28. The method of claim 27, wherein the method comprises detecting the target nucleic acid without lysing the membrane vesicle or cell.

29. The method of claim 26, wherein the target nucleic acid is DNA or RNA.

30. The method of claim 26, wherein detecting the target nucleic acid comprises an amplification step.

31. The method of claim 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).

32. The method of claim 26, wherein the method comprises a target detection step.

33. The method of claim 26, wherein the method comprises sequencing the target nucleic acid.

34. A kit comprising any sensor of claim 1 and instructions for detecting a target nucleic acid.

35. The kit of claim 34, further comprising a calibrator or control.

36. The kit of claim 35, wherein the calibrator or control comprises a detection moiety.

37. The kit of claim 35, wherein the calibrator 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.

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