Catalytically active nucleic acid nanostructures with polymerase responsiveness

Abstract

The present invention relates to catalytically active signal-generating nucleic acid nanostructures responsive to polymerase activity, methods for using the same, and devices and kits containing the same. More specifically, the present invention provides highly sensitive and catalytically active signal-generating nanostructures comprising DNAzymes / RNAzymes and stimulus-responsive elements, which can be used alone or as integrated circuits in combination with target-recognition nanostructures capable of converting molecular signals into polymerase activity.

Description

[Technical Field]

[0001] The present invention relates to catalytically active signal-generating nucleic acid nanostructures responsive to polymerase activity, methods for using the same, and devices and kits containing the same. More specifically, the present invention provides highly sensitive and catalytically active signal-generating nanostructures comprising DNAzymes / RNAzymes and stimulus-responsive elements, which can be used alone or as integrated circuits in combination with target-recognition nanostructures capable of converting molecular signals into polymerase activity. [Background technology]

[0002] Nucleic acid detection is widely used, for example, in diagnostics. Clinical laboratories are increasingly using nucleic acid technologies to provide previously unknown molecular information about infectious diseases [Niemz, A., Ferguson, TM & Boyle, DS Trends Biotechnol 29: 240-250 (2011); Nong, RY, et al., Expert Rev Proteomics 9: 21-32 (2012); Zumla, A. et al. Lancet Infect Dis 14: 1123-1135 (2014)].

[0003] Currently, pathogen nucleic acid detection is largely limited to large-scale, centralized clinical laboratories. This limited application is due in part to the complexity and cost of conventional technologies. Commercially available tests primarily utilize polymerase chain reaction (PCR) to amplify and detect specific DNA targets. These assays require large, specialized equipment for PCR thermal cycling and fluorescence measurement, as well as skilled personnel to perform these operations. While improved isothermal amplification tests have been developed to reduce the need for thermal cycling equipment, these tests have limitations. For example, loop-mediated isothermal amplification (LAMP) has stringent sequence requirements, making it difficult to generalize [Zhao, Y., et al., Chem Rev 115: 12491-12545 (2015)]. Furthermore, like other nucleic acid amplification methods, LAMP is prone to false positives (e.g., due to primer-dimer formation). Although it is possible to improve detection accuracy by using sequence-specific signal-generating probes (e.g., fluorescent Taqman reporters), such probes are expensive and complex to use [Gardner, SN, et al., J Clin Microbiol 41: 2417-2427 (2003)]. Furthermore, each DNA target requires a dedicated sequence-specific probe to bind to the DNA target and generate a signal during target amplification, making this method even more expensive and difficult to use for multiplexed analyses and complex computations [Juskowiak, B. Anal Bioanal Chem 399: 3157-3176 (2011)].

[0004] There is a need for improved molecular platforms that enable rapid and visual detection of nucleic acids and other target molecules in a modular manner. Summary of the Invention [Means for solving the problem]

[0005] The present invention relates to responsive and catalytically active nucleic acid nanostructures. These nanostructures can be made to respond to various stimuli and targets, including polymerase activity. The core of these nanostructures contains DNAzymes / RNAzymes, which are nucleic acids that catalyze specific chemical reactions, and elements that respond to various stimuli. As an example, the inventors have designed and developed nanostructures incorporating G-quadruplex hemin DNAzymes and polymerase response elements and demonstrated their compatibility and performance with a variety of assay formats. By incorporating independent response elements that can convert molecular signals into polymerase activity, these systems can be used to measure various molecular targets and / or their combinations, and they exhibit robust performance under a variety of environmental conditions. The present invention provides novel catalytically active nanostructures for signal generation with improved sensitivity, speed of results, and robustness.

[0006] In a first aspect, a catalytic nucleic acid nanostructure is provided that comprises a DNAzyme / RNAzyme and a stimulus-responsive element. It will be appreciated that there are several known DNAzymes / RNAzymes that can be used for signal generation in the present invention.

[0007] In some embodiments, the DNAzyme / RNAzyme comprises: a ribonuclease such as ribonuclease 8-17, ribonuclease 10-23 or Dz10-66 deoxyribozyme; deoxyribonucleases such as 10MD5 deoxyribozyme or 9NL27 deoxyribozyme; peroxidases such as G-quadruplex hemin; enzymes with ligation activity, such as E47 deoxyribozyme; phosphatases such as 14WM9 deoxyribozyme; Amide hydrolases, such as AmideAm1 deoxyribozymes; and RNA branching enzymes such as 9F7 deoxyribozyme or 7S11 deoxyribozyme The compound may be selected from the group comprising:

[0008] In a preferred embodiment, the DNAzyme / RNAzyme is a guanine (G)-quadruplex hemin DNAzyme, which preferably comprises the nucleotide sequence 5'-CTGGGAGGGAGGGAGGGA-3' (SEQ ID NO: 1).

[0009] In some embodiments, the stimulus response element comprises a polymerase response element that inhibits DNAzyme / RNAzyme activity in the presence of a polymerase.

[0010] In some embodiments, the polymerase response element does not have a hairpin structure. In a preferred embodiment, the polymerase response element comprises the nucleotide sequences set forth in SEQ ID NOs: 2 and 3: 5'-CTGGGAGGGAGGGAGGGAATGCTAACGCATTGTCGATAGC-3' (SEQ ID NO: 2) and 5'-GCTATCGACAATGCGTT-3' (SEQ ID NO: 3).

[0011] In some embodiments, the polymerase response element has an internal hairpin structure.

[0012] In some embodiments, the polymerase response element or self-priming portion of the signal-generating nanostructure comprises the nucleic acid sequence 5'-AACGCATTGTCGATAGCTCAGCTGTCTGAGCTATCGACAATGCGTT-3' (SEQ ID NO: 10).

[0013] In some embodiments, the catalytically active nucleic acid nanostructure of the invention comprises a G-quadruplex hemin DNAzyme, 5'-CTGGGAGGGAGGGAGGGAATGCTAACGCATTGTCGATAGCTCTGTCGCTATCGACAATGCGTT-3' (SEQ ID NO: 4); 5'-CTGGGAGGGAGGGAGGGAATGCTAACGCATTGTCGATAGCTCTGTCGCTATCGACAATGCGTTAGCAT-3' (SEQ ID NO: 5); 5'-CTGGGAGGGAGGGAGGGAATGCTAACGCATTGTCGATAGCTCTGTCGCTATCGACAATGCGTTAGCATCCC-3' (SEQ ID NO: 6); 5'-CTGGGAGGGAGGGAGGGAATGCTAACGCATTGTCGATAGCTCTGTCGCTATCGACAATGCGTTAGCATCCCTCCC-3' (SEQ ID NO: 7); 5'-CTGGGAGGGAGGGAGGGAATGCTAACGCATTGTCGATAGCTCTGTCGCTATCGACAATGCGTTAGCATCCCTCCCTCCC-3' (SEQ ID NO: 8); and 5'-CTGGGAGGGAGGGAGGGAATGCTAACGCATTGTCGATAGCTCTGTCGCTATCGACAATGCGTTAGCATCCCTCCCTCCCTCCCAG-3' (SEQ ID NO: 9) The nucleic acid sequence comprises a nucleic acid sequence selected from the group comprising:

[0014] It will be understood that intermediate (partial) sequences within SEQ ID NOs: 4 to 9 are also included within the scope of the present invention.

[0015] In some embodiments, when the internal hairpin structure is present, extension of the polymerase response element by a polymerase abolishes catalytic activity.

[0016] In some embodiments, the DNAzyme / RNAzyme activity is a peroxidase activity.

[0017] In some embodiments, the activity of the peroxidase substrate is detected by a variety of methods, including but not limited to, colorimetric, fluorescent, electrochemical, and luminescent methods.

[0018] In another aspect of the invention, there is provided a method for detecting polymerase activity in a test sample, comprising the steps of: (a) providing a test sample; (b) providing a composition comprising a catalytically active nucleic acid nanostructure according to any aspect of the present invention; (c) contacting the sample of a) with the composition of b) in the presence of a DNAzyme / RNAzyme substrate, optionally in the further presence of a signal-generating reagent; and (d) detecting the expression of a signal, thereby indicating the level of polymerase activity in the sample, which is inversely related to the signal intensity. A method is provided which includes:

[0019] In some embodiments, the test sample comprises a polymerase, such as a DNA polymerase.

[0020] In another aspect of the present invention, there is provided a method for detecting a target molecule in a sample, comprising the steps of: (a) providing a test sample; (b) providing a composition comprising at least one DNA polymerase enzyme and at least one recognition nanostructure, wherein the recognition nanostructure comprises a DNA aptamer specific to the DNA polymerase enzyme, and the DNA aptamer is configured to recognize a target molecule in the sample; or (c) providing a composition comprising at least one DNA polymerase enzyme and at least one recognition nanostructure, wherein the recognition nanostructure comprises a DNA aptamer specific to the DNA polymerase enzyme, the DNA aptamer having a conserved sequence region and a variable sequence region, the variable sequence region comprising an overhanging end portion of at least 10 nucleotides that forms a double strand complementary to a portion of an inverter oligonucleotide, and the inverter oligonucleotide is configured to recognize a target molecule in the sample with higher affinity than the double-stranded region of the variable sequence; (d) contacting the test sample with the composition of (b) or (c), (i) When the target molecule binds to the recognition sequence region of the aptamer (b), the formation of a stable aptamer-DNA polymerase enzyme complex is promoted, inhibiting the DNA polymerase enzyme activity; (ii) when the target molecule binds to the inverter oligonucleotide of (c), the inhibition of the DNA polymerase enzyme by the DNA aptamer is released by destabilizing the recognition nanostructure; (e) providing a catalytically active nucleic acid nanostructure according to any of the aspects of the present invention; (f) contacting the nanostructure with the product of step (b) or (c) in the presence of a DNAzyme / RNAzyme substrate, optionally in the further presence of a signal-generating reagent; and (g) by detecting signal expression; (i) when composition (b) is used, the signal intensity indicates the presence of the target molecule in the sample; (ii) when composition (c) is used, the signal intensity indicates the absence of the target molecule in the sample. A method is provided which includes:

[0021] Suitable recognition nanostructures are described and defined in PCT patent application PCT / SG2019 / 050328, published as WO2020 / 009660, the contents of which are incorporated herein by reference.

[0022] In some embodiments, the conserved sequence region of the DNA aptamer specific for the DNA polymerase enzyme in the recognition nanostructure comprises the nucleic acid sequence 5'-CAATGTACAGTATTG-3' (SEQ ID NO: 18).

[0023] In some embodiments, the inverter oligonucleotide is at least one nucleotide longer than the double-stranded region of the aptamer. Preferably, the inverter oligonucleotide is about twice as long as the double-stranded region of the aptamer.

[0024] In some embodiments, approximately half of the length of the inverter oligonucleotide forms an aptamer-inverter duplex, and the remaining approximately half forms a cohesive end portion.

[0025] In some embodiments, the method according to any aspect of the invention further comprises providing a second recognition nanostructure complementary to a target nucleic acid different from the target nucleic acid of the first recognition nanostructure in the sample for simultaneous detection of two species.

[0026] In some embodiments, mismatches are introduced into the double-stranded region of the variable sequence region to confer strong sequence specificity, which is useful for simultaneous detection of multiple closely related target nucleic acids, such as for determining viral subtypes.

[0027] In some embodiments, the target is at least one nucleic acid selected from the group including DNA, RNA, PNA and other nucleic acid analogs.

[0028] In some embodiments, the target is at least one nucleic acid associated with non-human or human disease, genetic variants, forensics, strain identification, environmental contamination, and / or food contamination.

[0029] In some embodiments, the target is a pathogen, hi some embodiments, the pathogen is a virus.

[0030] In some embodiments, the test sample comprises a target molecule selected from the group comprising DNA, RNA, PNA, protein, lipid, small molecule, and metabolites and modifications thereof.

[0031] In another aspect of the present invention, there is provided a method for detecting a target nucleic acid in a sample, comprising the steps of: (a) providing a sample containing nucleic acid; (b) providing a composition comprising at least one DNA polymerase enzyme and at least one recognition nanostructure, wherein the recognition nanostructure comprises a DNA aptamer specific to the DNA polymerase enzyme, the DNA aptamer having a conserved sequence region and a variable sequence region, the variable sequence region comprising an overhanging end portion of at least 10 nucleotides complementary to a target nucleic acid in the sample; or (c) providing a composition comprising at least one DNA polymerase enzyme and at least one recognition nanostructure, wherein the recognition nanostructure comprises a DNA aptamer specific to the DNA polymerase enzyme and an inverter oligonucleotide, the DNA aptamer having a conserved sequence region and a variable sequence region, the variable sequence region comprising an overhanging end portion of at least 10 nucleotides that forms a duplex complementary to a portion of the inverter oligonucleotide, the inverter oligonucleotide being at least one nucleotide longer than the aptamer-inverter duplex, and the inverter oligonucleotide having more than 10 nucleotides complementary to a target nucleic acid in the sample; (d) contacting the sample containing nucleic acid with the composition of (b) or (c), (i) when the target nucleic acid binds to the variable sequence region of the aptamer of (b), the formation of a stable aptamer-DNA polymerase enzyme complex is promoted, thereby inhibiting DNA polymerase enzyme activity; (ii) when the target nucleic acid binds to the inverter oligonucleotide of (c), the inhibition of the DNA polymerase enzyme by the DNA aptamer is released due to the destabilization of the recognition nanostructure; (e) providing a catalytically active nucleic acid nanostructure according to any of the aspects of the present invention; (f) contacting the nanostructure with the product of step (d) in the presence of a DNAzyme / RNAzyme substrate, optionally in the presence of a signal-expressing reagent; and (g) by detecting signal expression; (i) when composition (b) is used, the signal intensity indicates the presence of the target nucleic acid in the sample; (ii) if composition (c) is used, the signal intensity indicates the absence of target nucleic acid in the sample. A method is provided which includes:

[0032] In another aspect of the present invention, there is provided a device comprising a catalytically active nucleic acid nanostructure according to any of the aspects of the present invention immobilized on a surface.

[0033] In some embodiments, the device comprises: (i) comprising at a first location composition b) or composition c) of claim 10 comprising at least one DNA polymerase enzyme and at least one recognition nanostructure; (ii) a catalytically active nucleic acid nanostructure according to any embodiment of the present invention is attached to the second position; (iii) an intermediate stage configured to mix the detection nanostructure with the sample nucleic acid and deliver the activated enzyme to the second location; It is characterized by:

[0034] In some embodiments, the device is selected from the group comprising a microfluidic device and a lateral flow device.

[0035] In some embodiments, the device comprises an electrode.

[0036] In another aspect of the present invention, there is provided a nucleic acid detection kit, comprising: (a) a composition comprising at least one DNA polymerase enzyme and at least one recognition nanostructure, wherein the recognition nanostructure comprises a DNA aptamer specific for the DNA polymerase enzyme, the DNA aptamer having a conserved sequence region and a variable sequence region, the variable sequence region comprising an overhanging portion of at least 10 nucleotides complementary to a target nucleic acid; and / or (b) A composition comprising at least one DNA polymerase enzyme and at least one recognition nanostructure, wherein the recognition nanostructure comprises a DNA aptamer specific to the DNA polymerase enzyme and an inverter oligonucleotide, the DNA aptamer having a conserved sequence region and a variable sequence region, the variable sequence region comprising an overhanging end portion of at least 10 nucleotides that forms a duplex complementary to a portion of the inverter oligonucleotide, the inverter oligonucleotide being at least one nucleotide longer than the aptamer-inverter duplex, and the inverter oligonucleotide having more than 10 nucleotides complementary to a target nucleic acid. Including, optionally (c) further comprising a catalytically active nucleic acid nanostructure according to any aspect of the present invention; Optionally, (d) a DNAzyme / RNAzyme substrate may be further included; Optionally, (e) the signal generating reagent may further be included. A kit is provided.

[0037] In another aspect of the present invention, there is provided a molecular detection kit, comprising: (a) a composition comprising at least one DNA polymerase enzyme and at least one recognition nanostructure, wherein the recognition nanostructure comprises a DNA aptamer specific to the DNA polymerase enzyme, the DNA aptamer having a conserved sequence region and a variable sequence region, the variable sequence region comprising an overhanging end portion of at least 10 nucleotides that forms a double strand complementary to a portion of an inverter oligonucleotide, the inverter oligonucleotide being configured to recognize a target molecule in a sample with higher affinity than the double-stranded region of the variable sequence; and (b) a catalytically active nucleic acid nanostructure according to any one of the aspects of the present invention; Including, optionally further comprising (c) a DNAzyme / RNAzyme substrate; Optionally, (d) the signal generating reagent may further be included. A kit is provided. [Brief explanation of the drawings]

[0038] [Figure 1] Figure 1 shows the activity of DNAzyme nanostructures with various signal-generating elements. The activity of DNAzyme nanostructures is shown before incubation (a), after 30 minutes of incubation in the presence of DNA polymerase (b), and after 30 minutes of incubation in the absence of DNA polymerase (c). N=3 experiments were performed, and significance was calculated using t-test with Bonferroni correction. ns: corrected p-value > 0.05, **: corrected p-value < 0.005, ****: corrected p-value < 0.00005.

[0039] [Figure 2] 1 shows the responsiveness of a DNAzyme signal-generating nanostructure when specific nucleotides (SEQ ID NOS: 4 to 9) are elongated. The experiment was carried out with N=3.

[0040] [Figure 3] Figure 1 shows the types of measurement formats for signal-generating nanostructures. Each graph shows the signal generated by the substrate in the presence (black) and absence (white) of signal-generating nanostructures. The types of measurement formats are detailed in Table 2. Electrochemical measurements were performed using nanostructures immobilized on a surface, while other measurements were performed using nanostructures in solution. Experiments were performed in triplicate.

[0041] [Figure 4a-4b] These are the results of measurements using a signal-generating nanostructure under various polymerase assay conditions. a) Time course of signal measured in the presence of different dilutions of DNA polymerase (1x, 10x, 100x, no polymerase added). b) Signal from the signal-generating nanostructure after 30 minutes of incubation with DNA polymerase in the presence of various concentrations of an enzyme-inhibiting contaminant (HCl). N=3 experiments were performed.

[0042] [Figure 5] Conditions for constructing recognition nanostructures for various substrates. Construction of signal-generating nanostructures for different targets. Recognition of DNA / RNA (left and center) requires a target recognition element that hybridizes to complementary sequences by base pairing. Recognition of proteins and small molecules requires an aptamer that folds and binds to the target (right). The graph at the bottom shows the signal measured with varying amounts of target in an assay combined with a signal-generating nanostructure. Experiments were performed with N=3.

[0043] [Figure 6a-6b] Figures showing the sensitivity and speed of the measurement. a) Titration curves obtained using specific targets or scrambled sequences in assays combined with signal-generating nanostructures. b) Signal time course in experiments using 10 copies of specific or scrambled targets. Experiments were performed in triplicate.

[0044] [Figure 7] This figure shows that the functionality of immobilized nanostructures is maintained. Both the non-immobilized recognition nanostructure (left) and the surface-immobilized recognition nanostructures (the two in the middle, each with a different configuration) retain their specificity and sensitivity (left, no target = no signal; right, target = high signal). This property is not observed in the surface-only control (right) (high signal regardless of the presence or absence of target), indicating that this property is a property of the recognition nanostructure, not the surface. The experiment was performed with N=3. DETAILED DESCRIPTION OF THE INVENTION

[0045] For convenience, the references cited herein are provided in a list of references at the end of the examples. The contents of such references are incorporated herein by reference in their entirety.

[0046] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. For convenience, certain terms used in the specification, examples, and appended claims are collected below.

[0047] It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly indicates otherwise. Thus, for example, a reference to "a target sequence" includes a plurality of such target sequences, and a reference to "an enzyme" is a reference to one or more enzymes and equivalents thereof known to those skilled in the art.

[0048] In this specification, the term "aptamer" refers to a single-stranded DNA molecule or a single-stranded RNA molecule.Aptamer binds to various molecules such as DNA, protein, small molecule, etc. with high affinity and high specificity.For example, in this specification, aptamer binds strongly to polymerase in the absence of target DNA, target protein or target small molecule, and inhibits polymerase activity.

[0049] The term "nucleic acid" or "nucleic acid sequence" as used herein refers to an oligonucleotide, nucleotide, polynucleotide, or fragment thereof; genomic DNA, genomic RNA, synthetic DNA, or RNA, which may be single-stranded or double-stranded and may be the sense or antisense strand; peptide nucleic acid (PNA); or DNA-like or RNA-like material.

[0050] As used herein, the term "oligonucleotide" refers to a nucleic acid sequence consisting of at least about 6 to 60 nucleotides, preferably about 15 to 30 nucleotides, and most preferably about 20 to 25 nucleotides, and is usable for PCR amplification, hybridization assays, or microarrays. As used herein, the term "oligonucleotide" is essentially synonymous with "amplimer," "primer," "oligomer," and "probe," as these terms are generally defined in the art. As used herein, the recognition nanostructure may include an inverter oligonucleotide.

[0051] As used herein, the term "inverter sequence" or "inverter oligonucleotide" refers to an oligonucleotide complementary to a target nucleic acid sequence, with a portion forming a double strand and another portion forming a protruding end. It has been shown herein that sequences longer than 20 nucleotides, comprising a double-stranded sequence and a protruding end sequence, exert a strong inhibitory effect on DNA polymerase by stabilizing the aptamer that binds to the DNA polymerase enzyme. This inhibitory effect can be eliminated in the presence of a complementary target at ambient temperature. The presence or absence of the inverter sequence determines the functional state (e.g., on or off) of the recognition element. In the presence of the inverter sequence, the target can turn on polymerase activity, while in the absence of the inverter sequence, the target can turn off polymerase activity.

[0052] As used herein, the term "variable sequence region" refers to the region in a recognition nanostructure that determines the sequence specificity for a target sequence (i.e., the region that defines the target sequence that can be recognized). The inverter sequence and a portion of the aptamer sequence are included in this variable sequence region. This region can be altered to enable detection of new targets. When an inverter sequence is not used, the "variable sequence region" refers to the protruding end portion of the aptamer that is complementary to the target nucleic acid.

[0053] The term "sample" is used herein in its broadest definition. For example, biological samples suspected of containing genomic sequences of human papillomavirus (HPV), such as, but not limited to, HPV 6, 16, 18, 31, 33, and 5, include body fluids; cell extracts, chromosomes, organelles, or membranes isolated from cells; cells; genomic DNA, RNA, or cDNA (in solution or bound to a solid support); tissues; tissue prints, and the like.

[0054] It will be understood that the oligonucleotides used in the present invention may be structurally and / or chemically modified, for example, to prolong the activity of the oligonucleotides in samples that may contain nucleases when performing the methods of the present invention, or to extend the shelf life of the kits. Thus, the aptamers, inverters, signal-generating nanostructures, or any oligonucleotide primers or probes used in the present invention may be chemically modified. In some embodiments, the structural and / or chemical modifications include the addition of tags such as fluorescent tags, radioactive tags, biotin, or 5' tails during synthesis, the addition of phosphorothioate (PS) linkages, 2'-O-methylation, and / or the addition of phosphoramidite C3 spacers.

[0055] As used herein, the terms "comprising" or "including" are to be construed as specifying the presence of the feature, integer, step, or element described herein denoted by these terms, but do not exclude the presence or addition of one or more features, integers, steps, or elements or groups thereof. In the context of the present disclosure, the terms "comprising" or "including" also encompass the meaning "consisting of." Accordingly, variations of the term "comprising," such as "comprise" or "comprises," and variations of the term "including," such as "include" or "includes," are similarly broad in meaning. [Example]

[0056] Standard molecular biology techniques known in the art that are not specifically described were generally performed as described in Green and Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory, New York (2012).

[0057] Example 1 Design of responsive and catalytic nucleic acid nanostructures The present inventors have designed and developed catalytically active nucleic acid nanostructures and successfully imparted responsiveness to these catalytic activities by incorporating stimuli-responsive elements into these structures. Specifically, we first created a core structure containing DNAzymes / RNAzymes, which are nucleic acids that catalyze specific chemical reactions [Li, W. et al., Nucleic Acids Research 44: 7373-7384 (2016)], and then incorporated stimuli-responsive elements into this core structure. For example, the present inventors created a three-dimensional DNA structure containing a G-quadruplex hemin DNAzyme (SEQ ID NO: 1) with peroxidase activity.

[0058] The inventors then devised and optimized the incorporation of a polymerase activity response element into this nanostructure (SEQ ID NOS: 4-9). The nanostructure thus constructed contains both the binding site required for polymerase activity and the essential catalytic domain (SEQ ID NOS: 2 and 3, SEQ ID NOS: 4-9). It is configured so that peroxidase catalytic activity changes in response to polymerase activity. Specifically, the response element provides substrates for polymerase activity, leading to unfolding and loss of catalytic activity. Although secondary and higher-order DNA structures are known to inhibit polymerase activity [Nelms, BL and Labosky, PA Scientific Reports 1: 106 (2011)], we confirmed that the designed nanostructure does not inhibit polymerase activity even in the absence of the polymerase activity response element. Nanostructures incorporating the polymerase activity response element retain catalytic activity in the absence of polymerase activity, but lose that catalytic activity in the presence of polymerase activity (Figure 1). Among the several polymerase response elements tested, the best design was one with an internal hairpin structure. Based on this result, we hypothesized that the internal hairpin structure could improve the stability of the polymerase response element through self-priming (SEQ ID NO: 10) (Figure 1). We further optimized the design of the response element and its location relative to the catalytic domain. Specifically, we adjusted the length of the 5' overhang of the response element so that the nanostructure would be highly responsive to polymerase activity. In the optimized design (SEQ ID NO: 4), catalytic activity was completely abolished after extension of just a few bases by polymerase. If the signal decreases exponentially with the decline in catalytic activity, nanostructures with this design would be highly sensitive to polymerase activity (Figure 2). The nanostructure sequences and target sequences are listed in Table 1. [Table 1]

[0059] Example 2 Measurement of catalytic activity using various measurement methods The catalytic activity of the fabricated nanostructures can be measured using various substrates as signal-generating elements, allowing for rapid detection at room temperature, and can be adapted to various measurement methods, including, but not limited to, colorimetry, fluorometry, electrochemistry, and luminescence (Table 2, Figure 3).

[0060] [Table 2]

[0061] Colorimetric measurement has the advantage of being highly portable. By immobilizing the fabricated nanostructures on an electrode, highly sensitive and portable measurements are possible.

[0062] Example 3 Detection of polymerase amount and / or activity The catalytic activity of the designed nanostructure (SEQ ID NO: 4) can be used to directly measure the amount of polymerase. Solutions containing the nanostructures were mixed with different amounts of polymerase, and aliquots of the reaction mixture were sampled at regular intervals to measure DNAzyme activity. The lower the amount of polymerase in the reaction mixture, the more slowly DNAzyme activity decreased over time. This is because a small amount of enzyme is insufficient to disrupt the DNAzyme structure (Figure 4a). This can be used to detect the amount of DNA polymerase. For example, this can be used to determine the yield of purified recombinant enzymes. This system can also be used to examine polymerase activity (activation or inhibition) (Figure 4b). For example, different amounts of chemical additives can be added to examine the effect on the activity of a fixed amount of enzyme.

[0063] Example 4 Detection of various stimuli By combining the catalytic nanostructures described above with other responsive nanostructures or other mechanisms for controlling DNA polymerase availability and / or activation, systems can be constructed that respond to and specifically measure a variety of other stimuli and target molecules, including, but not limited to, DNA, RNA, proteins, lipids, small molecules, metabolites, and modifications, in a variety of different solution environments (e.g., cell lysates, chemical buffers, etc.). To demonstrate this, we constructed a system using recognition nanostructures (SEQ ID NOS: 11 and 12) specifically designed to activate / inactivate DNA polymerase in response to specific nucleic acid targets (SEQ ID NOS: 13 and 14, human actin β DNA and RNA) [PCT Patent Publication No. WO 2020 / 009660, incorporated herein by reference; Ho, NRY et al., Nature Communications 9: 3238 (2018)], positioned upstream of the catalytic nucleic acid nanostructure described above. This fusion system enabled the detection of DNA and RNA targets with comparable efficiency. This system can also be used to detect protein or small molecule targets by matching the recognition nanostructure with an aptamer for a specific protein target. We designed a recognition nanostructure that responds to the EPCAM protein by incorporating an aptamer for the protein into the inverter sequence (SEQ ID NOs: 16 and 17). We confirmed that polymerase activity increased with an increase in the number of cells expressing this protein (Figure 5). When the responsive DNAzyme nanostructure was immobilized on an electrode surface, electrons generated by DNAzyme activity rapidly shuttled across the electrode, generating a measurable current. This electrochemical measurement allowed us to detect minute changes in DNAzyme activity. By combining this with a recognition nanostructure, we were able to detect changes in DNAzyme activity as small as 10 1 The scrambled sequence (SEQ ID NO: 15) was able to detect 10 specific targets. 11 The presence of the copy did not produce a signal (Fig. 6a), which allowed discrimination from the scrambled target within minutes and reached saturation in just 20 minutes (Fig. 6b).

[0064] Example 5 Solid-phase nanostructures for functional control By immobilizing various nanostructures on surfaces, their functionality can be controlled under various conditions, and the permutation of functionality can be controlled. Specifically, the nanostructures described herein can be immobilized on a variety of surfaces, including gold, polystyrene, and silica. These surfaces can be functionalized using common bioconjugation reactions, such as carbodiimide crosslinking, succinimide crosslinking, dithiol crosslinking, gold-thiol reactions, and avidin-biotin reactions. Furthermore, the addition of linkers or surface treatment groups (e.g., poly(ethylene glycol) or poly(ethylene oxide)) allows for optimization of surface density and molecular configuration, allowing for control of functionality and surface patterning. For example, we immobilized the recognition nanostructures described in Example 4 (SEQ ID NOS: 11 and 12) that recognize the human β-actin gene to demonstrate that immobilization does not affect the recognition nanostructure's ability to bind and inhibit DNA polymerase. Polymerase inactivation was not observed on surfaces without nanostructures, confirming that polymerase inactivation is a property of the recognition nanostructures. Importantly, the immobilized recognition nanostructures bound to the programmed target nucleic acid sequence and activated the polymerase equally well as non-immobilized nanostructures (Figure 7). Furthermore, nanostructures can be immobilized in various molecular configurations (Figure 7), allowing for permutation of functionality (information flow). This immobilization allows for array-like patterning for the detection of various targets [Yeh, EC et al., Sci Adv 3: e1501645 (2017)] and high analytical performance even in various environments that would otherwise inhibit nanostructure functionality (e.g., lysis buffers, detergents, ethylenediaminetetraacetic acid, or components of biological samples such as IgG, hemoglobin, proteases, and heparin) [Zumla, A. et al., Lancet Infect Dis 14: 1123-1135 (2014)]. Therefore, such systems enable direct detection from samples without extensive purification steps.

[0065] summary The advantages of the present invention are as follows: 1. It is faster and more sensitive than non-catalytic nanostructures. 2. It can achieve high performance in various environments with various measurement methods and is highly portable. 3. High analytical performance can be achieved even in various environments where the functionality of nanostructures would otherwise be impaired.

[0066] References 1. Gardner, SN, Kuczmarski, TA, Vitalis, EA & Slezak, TR Limitations of TaqMan PCR for detecting divergent viral pathogens illustrated by hepatitis A, B, C, and E viruses and human immunodeficiency virus. J Clin Microbiol 41, 2417-2427 (2003). 2. Ho, NRY et al. Visual and modular detection of pathogenic acids with enzyme-DNA nucleic molecular complexes. Nature Communications 9, 3238 (2018). 3. Juskowiak, B. Nucleic acid-based fluorescent probes and their analytical potential. Anal Bioanal Chem 399, 3157-3176 (2011). 4. Li, W. et al. Insight into G-quadruplex-hemin DNAzyme / RNAzyme: adjacent adenine as the intramolecular species for remarkable enhancement of enzymatic activity. Nucleic acids research 44, 7373-7384 (2016). 5. Niemz, A., Ferguson, T. M. & Boyle, D. S. Point-of-care nucleic acid testing for infectious diseases. Trends Biotechnol 29, 240-250 (2011). 6. Nelms, B. L. & Labosky, P. A. A predicted hairpin cluster correlates with barriers to PCR, sequencing and possibly BAC recombineering. Scientific Reports 1, 106 (2011). 7. Nong, R. Y., Gu, J., Darmanis, S., Kamali-Moghaddam, M. & Landegren, U. DNA-assisted protein detection technologies. Expert Rev Proteomics 9, 21-32 (2012). 8. Yeh, E. C. et al. Self-powered integrated microfluidic point-of-care low-cost enabling (SIMPLE) chip. Sci Adv 3, e1501645 (2017). 9. Zhao, Y., Chen, F., Li, Q., Wang, L. & Fan, C. Isothermal Amplification of Nucleic Acids. Chem Rev 115, 12491-12545 (2015). 10. Zumla, A. et al. Rapid point of care diagnostic tests for viral and bacterial respiratory tract infections--needs, advances, and future prospects. Lancet Infect Dis 14, 1123-1135 (2014).

Claims

1. A catalytic nucleic acid nanostructure comprising a G-quadruplex hemin DNAzyme and a polymerase response element having an internal hairpin structure that is elongated in the presence of a polymerase, The catalytically active nucleic acid nanostructure, wherein the extension inhibits the catalytic activity of the G-quadruplex hemin DNAzyme.

2. A nucleic acid nanostructure with catalytic activity as described in claim 1, wherein the G-quadruplex hemin DNAzyme comprises the nucleic acid sequence shown in SEQ ID NO:

1.

3. A nucleic acid nanostructure with catalytic activity described in claim 1 or 2, wherein the polymerase response element comprises the nucleic acid sequence shown in SEQ ID NO:

10.

4. A nucleic acid nanostructure having catalytic activity as described in claim 3, comprising a nucleic acid sequence shown in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO:

9.

5. A catalytically active nucleic acid nanostructure described in any one of claims 1 to 4, wherein the activity of the G-quadruplex hemin DNAzyme substrate is detected by various methods including, but not limited to, colorimetric, fluorescent, electrochemical and luminescent methods.

6. 1. A method for detecting polymerase activity in a test sample, comprising: (a) providing a test sample; (b) providing a composition comprising the catalytically active nucleic acid nanostructure of any one of claims 1 to 5; (c) contacting the sample of a) with the composition of b) in the presence of a G-quadruplex hemin DNAzyme substrate; and (d) detecting the expression of a signal, thereby indicating the level of polymerase activity in the sample, which is inversely related to the signal intensity. A method comprising:

7. 1. A method for detecting a target molecule in a sample, comprising: (a) providing a test sample; (b) providing a composition comprising at least one DNA polymerase enzyme and at least one recognition nanostructure, wherein the recognition nanostructure comprises a DNA aptamer specific to the DNA polymerase enzyme, the DNA aptamer being configured to recognize a target molecule in the sample; or (c) providing a composition comprising at least one DNA polymerase enzyme and at least one recognition nanostructure, wherein the recognition nanostructure comprises a DNA aptamer specific to the DNA polymerase enzyme, the DNA aptamer having a conserved sequence region and a variable sequence region, the variable sequence region comprising an overhanging end portion of at least 10 nucleotides that forms a double strand complementary to a portion of an inverter oligonucleotide, and the inverter oligonucleotide is configured to recognize a target molecule in the sample with higher affinity than the double-stranded region of the variable sequence; (d) contacting the test sample with the composition of (b) or (c), (i) When a target molecule binds to the recognition sequence region of the aptamer (b), the formation of a stable aptamer-DNA polymerase enzyme complex is promoted, inhibiting the DNA polymerase enzyme activity; (ii) when the target molecule binds to the inverter oligonucleotide of (c), the inhibition of the DNA polymerase enzyme by the DNA aptamer is released due to the destabilization of the recognition nanostructure; (e) providing a catalytically active nucleic acid nanostructure according to any one of claims 1 to 5; (f) contacting the nanostructure with the product of step (b) or (c) in the presence of a G-quadruplex hemin DNAzyme substrate; and (g) by detecting signal expression; (i) when composition (b) is used, the signal intensity indicates the presence of the target molecule in the sample; (ii) when composition (c) is used, the signal intensity indicates the absence of the target molecule in the sample. A method comprising:

8. 1. A method for detecting a target nucleic acid in a sample, comprising: (a) providing a sample containing nucleic acid; (b) providing a composition comprising at least one DNA polymerase enzyme and at least one recognition nanostructure, wherein the recognition nanostructure comprises a DNA aptamer specific to the DNA polymerase enzyme, the DNA aptamer having a conserved sequence region and a variable sequence region, the variable sequence region comprising an overhanging end portion of at least 10 nucleotides complementary to a target nucleic acid in the sample; or (c) providing a composition comprising at least one DNA polymerase enzyme and at least one recognition nanostructure, wherein the recognition nanostructure comprises a DNA aptamer specific to the DNA polymerase enzyme and an inverter oligonucleotide, the DNA aptamer having a conserved sequence region and a variable sequence region, the variable sequence region comprising an overhanging end portion of at least 10 nucleotides that forms a duplex complementary to a portion of the inverter oligonucleotide, the inverter oligonucleotide being at least one nucleotide longer than the aptamer-inverter duplex, and the inverter oligonucleotide having more than 10 nucleotides complementary to a target nucleic acid in the sample; (d) contacting the sample containing nucleic acid with the composition of (b) or (c), (i) when the target nucleic acid binds to the variable sequence region of the aptamer of (b), the formation of a stable aptamer-DNA polymerase enzyme complex is promoted, thereby inhibiting DNA polymerase enzyme activity; (ii) when the target nucleic acid binds to the inverter oligonucleotide of (c), the inhibition of the DNA polymerase enzyme by the DNA aptamer is released due to the destabilization of the recognition nanostructure; (e) providing a catalytically active nucleic acid nanostructure according to any one of claims 1 to 5; (f) contacting the nanostructure with the product of step (d) in the presence of a G-quadruplex hemin DNAzyme substrate; and (g) by detecting signal expression; (i) when composition (b) is used, the signal intensity indicates the presence of the target nucleic acid in the sample; (ii) when composition (c) is used, the signal intensity indicates the absence of target nucleic acid in the sample. A method comprising:

9. A device comprising the catalytically active nucleic acid nanostructure of any one of claims 1 to 5 immobilized on a surface thereof.

10. (i) comprising at a first location the composition of (b) or the composition of (c) of claim 8, comprising at least one DNA polymerase enzyme and at least one recognition nanostructure; (ii) a catalytically active nucleic acid nanostructure according to any one of claims 1 to 5 attached to a second location; (iii) an intermediate stage configured to mix the catalytically active nucleic acid nanostructure with a sample nucleic acid and deliver the activated enzyme to the second location; 10. The device of claim 9.

11. 11. The device according to claim 9 or 10, selected from the group comprising microfluidic devices and lateral flow devices.

12. A device according to any one of claims 9 to 11, comprising an electrode.

13. A nucleic acid detection kit, comprising: (a) a composition comprising at least one DNA polymerase enzyme and at least one recognition nanostructure, wherein the recognition nanostructure comprises a DNA aptamer specific for the DNA polymerase enzyme, the DNA aptamer having a conserved sequence region and a variable sequence region, the variable sequence region comprising an overhanging portion of at least 10 nucleotides complementary to a target nucleic acid; and / or (b) A composition comprising at least one DNA polymerase enzyme and at least one recognition nanostructure, wherein the recognition nanostructure comprises a DNA aptamer specific to the DNA polymerase enzyme and an inverter oligonucleotide, the DNA aptamer having a conserved sequence region and a variable sequence region, the variable sequence region comprising an overhanging end portion of at least 10 nucleotides that forms a duplex complementary to a portion of the inverter oligonucleotide, the inverter oligonucleotide being at least one nucleotide longer than the aptamer-inverter duplex, and the inverter oligonucleotide having more than 10 nucleotides complementary to a target nucleic acid. Including, (c) A kit further comprising a catalytically active nucleic acid nanostructure according to any one of claims 1 to 5.

14. A molecular detection kit, comprising: (a) a composition comprising at least one DNA polymerase enzyme and at least one recognition nanostructure, wherein the recognition nanostructure comprises a DNA aptamer specific to the DNA polymerase enzyme, the DNA aptamer having a conserved sequence region and a variable sequence region, the variable sequence region comprising an overhanging end portion of at least 10 nucleotides that forms a double strand complementary to a portion of an inverter oligonucleotide, the inverter oligonucleotide being configured to recognize a target molecule in a sample with higher affinity than the double-stranded region of the variable sequence; and (b) A nucleic acid nanostructure having catalytic activity according to any one of claims 1 to 5. Includes a kit.

15. The method of claim 6, wherein step (c) is carried out in the presence of a G-quadruplex hemin DNAzyme substrate and a signal expression reagent.

16. The method described in claim 7 or 8, wherein step (f) is carried out in the presence of a G-quadruplex hemin DNAzyme substrate and a signal expression reagent.

17. The nucleic acid detection kit described in claim 13, further comprising (d) a G-quadruplex hemin DNAzyme substrate and / or (e) a signal expression reagent.

18. The molecular detection kit described in claim 14, further comprising (c) a G-quadruplex hemin DNAzyme substrate and / or (d) a signal expression reagent.

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