Target recycling hybridization chain reaction

Abstract

The present invention relates to compositions and methods for detecting targets, and in particular to hybridization chain reactions for detecting nucleic acid targets. In some examples, the methods described herein include a looped hybridization reaction that induces modification of a polynucleotide linked to a detection moiety, enabling the detection moiety to generate a detectable signal.

Description

Invention Field

[0001] This invention relates to compositions and methods for detecting targets, and more particularly to hybridization chain reactions for detecting nucleic acid targets. By referencing the merging

[0002] This application claims priority to Australian Provisional Application No. 2023903269, the entire contents of which are incorporated herein by reference. background

[0003] Any discussion of prior art throughout the specification should not be construed as an admission that the prior art is widely known or forms part of common knowledge in the art.

[0004] In medicine, the presence of specific nucleic acids can indicate health status or infection. The ability to detect specific nucleic acids can be used for diagnosis, infection tracking, disease control, drug discovery, and cancer treatment. Furthermore, viral DNA or RNA surveillance is a valuable tool for limiting the spread of infection during disease outbreaks.

[0005] Current nucleic acid detection technologies are limited by cost and time. Known methods for detecting the presence of DNA or RNA sequences include polymerase chain reaction (PCR), which requires multiple reaction cycles with heating and cooling phases to increase the relative concentration of the target sequence during a process called amplification. In PCR, amplification requires a significant amount of time and effort due to the need for multiple reaction cycles and careful control of temperature changes. The time and equipment required for this temperature cycling limit the potential applications of PCR. Furthermore, the enzymes used in PCR may be inhibited by compounds present in biological samples, making PCR unsuitable for certain types of tests.

[0006] One amplification method designed to overcome some limitations of PCR is loop-mediated isothermal amplification (LAMP). Other amplification methods aimed at making amplification cheaper and more convenient are under development. Beatriz B. Oliveira et al. provided a review in "Isothermal Amplification of Nucleic Acids: The Race for the Next “Gold Standard”" published in Frontiers in Sensors (Vol. 2) in 2021.

[0007] One such method is called hybridization chain reaction (HCR), described in US 7,632,641 B2. However, HCR implementation lacks the reliability and sensitivity of PCR and LAMP. Feng Xuan and I-Ming Hsing described a modified HCR in their 2014 article, "Triggering hairpin-free chain-branching growth of fluorescent DNA dendrimers for nonlinear hybridization chain reaction," published in the Journal of the American Chemical Society (Vol. 136). In this HCR method, polymerization occurs exponentially, resulting in a continuously growing dendritic structure. However, this method, along with others, still makes HCR less sensitive, accurate, and reliable than other amplification methods.

[0008] One object of the present invention is to overcome or improve at least one of the disadvantages of the prior art, or to provide a useful alternative. Summary of the Invention

[0009] This disclosure relates to the detection of nucleic acids in biological samples. In some instances, this disclosure contemplates the use of a complex comprising a circular first nucleic acid that hybridizes with a complementary region spaced between non-complementary loop regions to a portion of a second nucleic acid. In some instances, the complex additionally comprises a single-stranded region complementary to a target nucleic acid suspected of being present in the sample. In use, the complex of the invention is exposed to a polynucleotide, at least one of which comprises a detection portion. The target nucleic acid hybridizes with a single-stranded region in the complex, and the second nucleic acid of the complex is detached from the complex. The polynucleotide then hybridizes with a portion of the first nucleic acid exposed by the removal of the second nucleic acid, and the complex is detected to indicate the presence of the target nucleic acid. In some instances, the polynucleotide hybridizes with the exposed first nucleic acid in a circumferential hybridization reaction. In some cases, the circumferential hybridization reaction induces modifications in the polynucleotide, thereby enabling detection. In some instances described below, the first nucleic acid is referred to as a circular path and the second nucleic acid as a chaperone.

[0010] In a first aspect, this disclosure provides a method for detecting a nucleic acid target in a sample, the method comprising: (a) contacting the sample with a reaction mixture comprising: (i) a detector comprising a circular path containing a polynucleotide hybridization region hybridizing with a polynucleotide chaperone, the circular path comprising a plurality of loops not hybridizing with the chaperone and an exposed single-stranded initiation hybridization region not hybridizing with the chaperone, wherein the initiation hybridization region is complementary to a hybridization region of a target; and (ii) one or more polynucleotides, wherein at least one of said polynucleotides is linked to a detection moiety; (b) hybridizing the target with the initiation hybridization region; (c) dissociating the chaperone from the circular path, thereby exposing a plurality of hybridization regions on the circular path; (d) hybridizing the one or more polynucleotides with the plurality of exposed hybridization regions on the circular path in a circumferential hybridization reaction, wherein the circumferential hybridization reaction induces modification of at least one polynucleotide linked to the detection moiety, such that the detection moiety is capable of generating a detectable signal; and (e) measuring the presence or absence of a detectable signal, wherein the detectable signal indicates the presence of the target in the sample. It should be understood that both the circular path and the chaperone are nucleic acid molecules containing a plurality of complementary hybridization regions.

[0011] In some instances, the detection portion is a fluorophore that emits a detectable fluorescent signal after spatial separation from the quencher. In some instances, the at least one polynucleotide is linked to both the fluorophore and the quencher, and the modification includes spatial separation of the fluorophore from the quencher, such that the fluorophore emits a detectable signal. In some instances, the at least one polynucleotide is linked to both the fluorophore and the quencher and includes a double-stranded region, wherein the strand of the double-stranded region dissociates after hybridization with a circular pathway, such that the fluorophore is spatially separated from the quencher and emits a detectable signal. In some instances, the one or more polynucleotides comprise a plurality of polynucleotides, each linked to the detection portion. In some instances, each of the plurality of polynucleotides is linked to both the fluorophore and the quencher and includes a double-stranded region, wherein the strand of each double-stranded region dissociates after hybridization with a circular pathway, such that each fluorophore is spatially separated from the quencher and emits a detectable signal.

[0012] In some instances of the first aspect, the separation of the mate from the loop path is facilitated by opening the loop of the loop path.

[0013] In some instances of the first aspect, circumferential hybridization reactions separate the target from the circumferential pathway.

[0014] In some instances of the first aspect, hybridization of the target and the initiating hybridization region involves the replacement of the mate's hybridization region by a circumferential pathway.

[0015] In some examples of the first aspect, the reaction mixture includes a detector dissociator comprising multiple polynucleotide hybridization regions that hybridize with the chaperone, thereby promoting the dissociation of the chaperone from the circular pathway and causing one or more loops in the circular pathway to open. The detector dissociator is preferably a single-stranded polynucleotide.

[0016] In some instances of the first aspect, at least a portion of the substituted hybridization region of the chaperone hybridizes with a hybridization region on the detector dissociation ion, and multiple hybridization regions on the detector dissociation ion hybridize with hybridization regions on the chaperone, thereby promoting the separation of the chaperone from the cyclic pathway.

[0017] In some examples of the first aspect, at least one polynucleotide is an exponentiator comprising a double-stranded portion of a complementary pair of hybridization regions and a single-stranded toehold extending beyond the double-stranded portion, and wherein the circumferential hybridization reaction comprises: hybridizing the toehold with a hybridization region on a circumferential path; and at least one hybridization region adjacent to the toehold hybridizing with a complementary hybridization region on a circumferential path, thereby at least partially opening the double-stranded portion.

[0018] In some examples of the first aspect, the indexor comprises a first-strand polynucleotide and a second-strand polynucleotide hybridized through at least two complementary hybridization regions, the first strand extending beyond the second strand to form a dangling splint and comprising at least one loop that does not hybridize with the second strand, and wherein the circumferential hybridization reaction comprises: the dangling splint hybridizing with a hybridization region on the circumferential path; and at least one hybridization region on the first strand adjacent to the dangling splint hybridizing with a hybridization region on the circumferential path, thereby displacing the hybridization region on the second strand of the indexor and opening the loop on the first strand of the indexor.

[0019] In some instances of the first aspect, the flanking regions of the ring of the indexinton are complementary hybridization pairs, which hybridize the first strand of the indexinton with the second strand of the indexinton.

[0020] In some examples of the first aspect, the reaction mixture further comprises an exponentiator hybridizer containing at least two polynucleotide hybridization regions that hybridize with one strand of the exponentiator, thereby separating the first strand of the exponentiator from the second strand of the exponentiator. The exponentiator hybridizer is preferably a single-stranded polynucleotide.

[0021] In some examples of the first aspect, the circumferential hybridization reaction includes: hybridization of a pendant with a hybridization region on a circumferential path; hybridization of at least one hybridization region of a pendant adjacent to the first strand with a hybridization region on the circumferential path, thereby displacing a hybridization region on the second strand of the indexed substituent and opening a loop on the first strand of the indexed substituent; and hybridization of a hybridization region of the indexed substituent with at least a portion of the displaced hybridization region on the second strand of the indexed substituent and another hybridization region on the indexed substituent with a hybridization region on the second strand of the indexed substituent, thereby separating the first strand of the indexed substituent from the second strand of the indexed substituent, optionally wherein at least one hybridization region of the first strand of the indexed substituent hybridizes with a hybridization region on the circumferential path, and at least one other hybridization region of the first strand of the indexed substituent does not hybridize with the circumferential path.

[0022] In some instances of the first aspect, the first strand of the indexer contains a hybridization region capable of hybridizing with the initiation hybridization region of another cyclic path, thereby initiating the amplification of the method.

[0023] In some instances of the first aspect, the first chain of the indexer includes a first hybridization region capable of hybridizing with an initial hybridization region of another cyclic path and a second hybridization region capable of hybridizing with an adjacent hybridization region on that other cyclic path, thereby initiating the amplification of the method.

[0024] In some instances of the first aspect, at least one hybridization region of the first strand of the indexed substituent that does not hybridize with the circular pathway hybridizes with the initiation hybridization region of another detector, thereby initiating the amplification of the method.

[0025] The first strand of the indexer may include a hybridization region capable of hybridizing with the initiating hybridization region of a circular pathway, thereby initiating amplification of the method. In some instances, at least one hybridization region of the first strand of the indexer that does not hybridize with a circular pathway hybridizes with the initiating hybridization region of another detector, thereby initiating amplification of the method. In some instances, the first strand of the indexer includes a hybridization region that is identical to or has at least about 80%, at least about 85%, at least about 90%, or at least about 95% identity with the target hybridization region. In some instances, at least one hybridization region of the first strand of the indexer that does not hybridize with a circular pathway includes a hybridization region that is identical to or has at least about 80%, at least about 85%, at least about 90%, or at least about 95% identity with the target hybridization region.

[0026] In some instances, the first strand of the indexer includes a first hybridization region capable of hybridizing with the initiating hybridization region and a second hybridization region capable of hybridizing with adjacent hybridization regions on the circular path, thereby initiating amplification by the method. In some instances, the first strand of the indexer includes two hybridization regions, each having at least about 80% identity, or at least about 85% identity, or at least about 90% identity, or at least about 95% identity, or 100% identity with a corresponding hybridization region on the target. In some instances, at least one hybridization region of the first strand of the indexer that does not hybridize with the circular path includes two hybridization regions, each having at least about 80% identity, or at least about 85% identity, or at least about 90% identity, or at least about 95% identity, or 100% identity with a corresponding hybridization region on the target.

[0027] In some instances of the first aspect, the first strand of the indexer contains a hybridization region that is identical to or has at least about 90% sequence identity with the hybridization region of the target.

[0028] In some instances of the first aspect, at least one hybridization region of the first strand of the indexed substituent that does not hybridize with the circular pathway contains a hybridization region that is identical to or has at least about 90% sequence identity with the hybridization region of the target.

[0029] In some instances of the first aspect, the loop of the first strand of the indexed substituent contains a target hybridization region or a sequence that has at least about 90% sequence identity with the target hybridization region.

[0030] In some instances of the first aspect, each chain of the exponent is connected to the detection portion, and the separation of the first and second chains of the exponent enables the detection portion of the exponent to be spatially separated and generate a detectable signal.

[0031] In some instances of the first aspect, the reaction mixture contains at least two different types of exponents.

[0032] In some instances of the first aspect, each type of indexer hybridizes with different hybridization regions on the cyclic path.

[0033] In some instances of the first aspect, the reaction mixture contains multiple exponential hybrids.

[0034] In some examples of the first aspect, one or more polynucleotides comprise: at least one first indexing unit comprising a first-strand polynucleotide and a second-strand polynucleotide hybridized through at least two complementary hybridization regions, the first strand extending beyond the second strand to form a single-stranded overhang and comprising at least one loop not hybridizing with the second strand, wherein each strand is connected to a detection portion; and at least one second indexing unit comprising a first-strand polynucleotide and a second-strand polynucleotide hybridized through at least two complementary hybridization regions, the first strand extending beyond the second strand to form a single-stranded overhang and comprising at least one loop not hybridizing with the second strand, wherein each strand is connected to a detection portion, and wherein the reaction mixture comprises The first exponent hybridizer contains at least two polynucleotide hybridization regions; and the second exponent hybridizer contains at least two polynucleotide hybridization regions, wherein the circumferential hybridization reaction comprises: at least one dangling protrusion of the first exponent hybridizes with a hybridization region on the circumferential path and at least one adjacent hybridization region on the first strand of the first exponent hybridizes with a hybridization region on the circumferential path, thereby displacing a hybridization region on the second strand of the first exponent hybridizer and opening the loop on the first strand of the first exponent hybridizer; at least one dangling protrusion of the second exponent hybridizes with a complementary hybridization region on the circumferential path and at least one adjacent hybridization region on the first strand of the second exponent hybridizes with a hybridization region on the circumferential path. This displaces the hybridization region on the second strand of the second exponent and opens the loop on the first strand of the second exponent; the hybridization region of the first exponent hybridizes with at least a portion of the displaced hybridization region on the second strand of the first exponent, and another hybridization region of the first exponent hybridizes with a hybridization region on the second strand of the first exponent, thereby separating the first strand of the first exponent from the second strand of the first exponent, thus enabling the detection portion of the first exponent to be spatially separated and generate a detectable signal; optionally, at least one hybridization region of the first strand of the first exponent hybridizes with a hybridization region on the loop path, and the first strand of the first exponent... At least one other hybridization region does not hybridize with the cyclic pathway; the hybridization region of the second exponent hybridizes with at least a portion of the substituted hybridization region on the second strand of the second exponent, and another hybridization region of the second exponent hybridizes with the hybridization region on the second strand of the second exponent, thereby separating the first strand of the second exponent from the second strand of the second exponent, thereby enabling the detection portion of the second exponent to be spatially separated and generate a detectable signal; optionally, at least one hybridization region of the first strand of the second exponent hybridizes with the hybridization region on the cyclic pathway, and at least one other hybridization region on the first strand of the second exponent does not hybridize with the cyclic pathway.

[0035] In some instances of the first aspect, at least one polynucleotide is a recycler comprising at least two hybridization regions, wherein the hybridization region of the recycler hybridizes with a hybridization region adjacent to the initiating hybridization region on the circular pathway, and another hybridization region of the recycler hybridizes with the initiating hybridization region, thereby displacing the target from the circular pathway.

[0036] In some examples of the first aspect, the recycle includes a double-stranded portion of a complementary hybridization region pair and a single-stranded portion extending beyond the double-stranded portion, the double-stranded portion being interrupted by a loop in one of the strands, the loop containing a hybridization region, and wherein the circumferential hybridization reaction comprises: the single-stranded portion hybridizing with a hybridization region on the circumferential path; at least one hybridization region adjacent to the single-stranded portion hybridizing with a hybridization region on the circumferential path adjacent to the initiating hybridization region, thereby opening the loop of the recycle; and the hybridization region of the loop of the recycle hybridizing with at least a portion of the initiating hybridization region, thereby displacing the target from the circumferential path.

[0037] In some examples of the first aspect, the recirculator comprises a first-strand polynucleotide and a second-strand polynucleotide hybridized via at least two complementary hybridization regions, the first strand extending beyond the second strand to form a single-stranded portion and comprising at least one loop that does not hybridize with the second strand, and wherein the circumferential hybridization reaction comprises: hybridizing the single-stranded portion with a hybridization region on the circumferential pathway; hybridizing a hybridization region of the recirculator adjacent to the single-stranded portion with a hybridization region of the circumferential pathway adjacent to the initiating hybridization region, thereby opening the loop of the recirculator; and hybridizing a hybridization region on the opened loop of the recirculator with at least a portion of the initiating hybridization region, thereby displacing the target from the circumferential pathway.

[0038] In some examples of the first aspect, the reaction mixture contains a releaser comprising at least two polynucleotide hybridization regions that hybridize with one strand of the recycle, thereby separating the first strand of the recycle from the second strand of the recycle. The releaser is preferably a single-stranded polynucleotide.

[0039] In some examples of the first aspect, the circumferential hybridization reaction includes: a single-stranded portion of the recycle hybridizing with a hybridization region on a circumferential path; a hybridization region of a neighboring single-stranded portion of the recycle hybridizing with a hybridization region of a neighboring initiating hybridization region on the circumferential path, thereby displacing the hybridization region of the second strand of the recycle and opening the loop on the first strand of the recycle; a hybridization region in the opened loop of the recycle hybridizing with at least a portion of the initiating hybridization region, thereby displacing the hybridization region of the target from the circumferential path; at least a portion of the displaced hybridization region of the second strand of the recycle hybridizing with a hybridization region on a releaser, and a neighboring hybridization region on the releaser hybridizing with a hybridization region on the second strand of the recycle, thereby separating the first strand of the recycle from the second strand of the recycle; and a hybridization region of the first strand of the recycle hybridizing with the initiating hybridization region, thereby separating the target from the circumferential path.

[0040] In some instances of the first aspect, the first strand of the recirculator contains a hybrid region that is identical to or has at least about 80% sequence identity with the target hybrid region, such as at least about 90% or at least about 95% sequence identity with the target hybrid region.

[0041] In some instances of the first aspect, the loop of the first strand of the recirculator contains a target hybridization region or a sequence having at least about 80% sequence identity with the target hybridization region, such as at least about 90% or at least about 95% sequence identity.

[0042] In some instances of the first aspect, each chain of the recirculator is connected to the detection portion, and the separation of the first and second chains of the recirculator enables the detection portion of the recirculator to be spatially separated and generate a detectable signal.

[0043] In some instances of the first aspect, the cyclic path contains multiple loops, each containing a hybridization region.

[0044] In some instances of the first aspect, the dangling of the indexinton hybridizes with the hybridization region present in the open loop of the cyclic path.

[0045] In some instances of the first aspect, the released target hybridizes with the initiation hybridization region of another detector, thereby initiating the amplification of the method.

[0046] In some instances of the first aspect, the cyclic path contains four loops.

[0047] In some instances of the first aspect, the loop path is a closed loop path.

[0048] In some instances of the first aspect, the circumferential hybridization reaction is carried out under isothermal conditions.

[0049] The methods described herein do not require the thermal cycling required for PCR. In some instances, the methods of this disclosure are performed under isothermal conditions. In some instances, the methods are performed at approximately 15°C, or approximately 16°C, or approximately 17°C, or approximately 18°C, or approximately 19°C, or approximately 20°C, or approximately 21°C, or approximately 22°C, or approximately 23°C, or approximately 24°C, or approximately 25°C, or approximately 26°C, or approximately 27°C, or approximately 28°C, or approximately 29°C, or approximately 30°C, or approximately 31°C, or approximately 32°C, or approximately 33°C, or approximately 34°C, or approximately 35°C, or approximately 36°C, or approximately... The method is carried out at temperatures ranging from approximately 37°C, or about 38°C, or about 39°C, or about 40°C, or about 41°C, or about 42°C, or about 43°C, or about 44°C, or about 45°C, or about 46°C, or about 47°C, or about 48°C, or about 49°C, or about 50°C, or about 51°C, or about 52°C, or about 53°C, or about 54°C, or about 55°C, or about 56°C, or about 57°C, or about 58°C, or about 59°C, or about 60°C. In some instances, the method is carried out at temperatures fluctuating by no more than about 10°C, or about 9°C, or about 8°C, or about 7°C, or about 6°C, or about 5°C, or about 4°C, or about 3°C, or about 2°C, or about 1°C, or about 0.5°C. In some instances of the first aspect, the circumferential hybridization reaction is carried out at temperatures ranging from about 20°C to about 25°C.

[0050] In some instances of the first aspect, the reaction mixture does not contain enzymes.

[0051] In some instances of the first aspect, the reaction mixture does not contain nuclease.

[0052] In some instances of the first aspect, the reaction mixture does not contain polymerase.

[0053] In some instances of the first aspect, (i) the first strand of the indexinon is connected to the fluorophore and the second strand of the indexinon is connected to the quencher; or (ii) the first strand of the indexinon is connected to the quencher and the second strand of the indexinon is connected to the fluorophore.

[0054] In some instances of the first aspect, (i) the first chain of the recycler is connected to the fluorophore and the second chain of the recycler is connected to the quencher; or (ii) the first chain of the recycler is connected to the quencher and the second chain of the recycler is connected to the fluorophore.

[0055] In a second aspect, this disclosure provides a composition comprising a detector comprising a circular pathway of a polynucleotide hybridization region that hybridizes with a polynucleotide chaperone, the circular pathway comprising one or more loops that do not hybridize with the chaperone and an exposed initiating hybridization region that does not hybridize with the chaperone, wherein the initiating hybridization region is complementary to the target hybridization region.

[0056] In some instances of the second aspect, the exposed initiation hybridization region is single-stranded.

[0057] In some examples of the second aspect, the composition further comprises a detector dissociation ion containing a plurality of polynucleotide hybridization regions capable of hybridizing with hybridization regions on a chaperone and dissociating the chaperone from the cyclic pathway.

[0058] In some examples of the second aspect, the composition further comprises an indexer comprising a first-strand polynucleotide and a second-strand polynucleotide hybridized through at least two complementary hybridization regions, the first strand extending beyond the second strand to form a single-stranded overhang and comprising at least one loop that does not hybridize with the second strand.

[0059] In some instances of the second aspect, the loop of the first strand of the indexer contains a target hybridization region or a sequence having at least about 70% sequence identity with the target hybridization region, such as a sequence having at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95% identity with the target hybridization region.

[0060] In some instances of the second aspect, the flanking regions of the ring of the indexinton are complementary hybridization pairs, which hybridize the first strand of the indexinton with the second strand of the indexinton.

[0061] In some instances of the second aspect, each chain of the exponent is connected to the detection part.

[0062] In some examples of the second aspect, the composition further comprises an indexed hybridon containing at least two polynucleotide hybridization regions capable of hybridizing with a hybridization region on the second strand of the indexed hybridon.

[0063] In some examples of the second aspect, the composition further comprises a recycler comprising a first-strand polynucleotide and a second-strand polynucleotide hybridized through at least two complementary hybridization regions, the first strand extending beyond the second strand to form a single-stranded portion and comprising at least one loop that does not hybridize with the second strand.

[0064] In some instances of the second aspect, the flanking regions of the recycle ring are complementary hybridization pairs, which hybridize the first strand of the recycle with the second strand of the recycle.

[0065] In some instances of the second aspect, the loop of the recycler contains a hybridization region capable of hybridizing with the cyclic path.

[0066] In some instances of the second aspect, the ring of the recycle includes a hybridization region capable of hybridizing with the initiating hybridization region.

[0067] In some instances of the second aspect, each chain of the recirculator is connected to the detection section.

[0068] In some examples of the second aspect, the composition further comprises a releaser containing at least two polynucleotide hybridization regions capable of hybridizing with a hybridization region on the second strand of the recycler.

[0069] In some instances of the second aspect, the cyclic path contains multiple loops, each containing a hybridization region.

[0070] In some instances of the second aspect, the dangling of the indexinon can hybridize with the hybridization region present in the open loop of the cyclic path.

[0071] In some instances of the second aspect, the circular path contains four loops.

[0072] In some instances of the second aspect, the loop path is a closed loop path.

[0073] In some instances of the second aspect, the composition does not contain an enzyme.

[0074] In some instances of the second aspect, the composition does not contain nuclease.

[0075] In some instances of the second aspect, the composition does not contain polymerase.

[0076] In some instances of the second aspect, (i) the first strand of the indexinon is connected to the fluorophore and the second strand of the indexinon is connected to the quencher; or (ii) the first strand of the indexinon is connected to the quencher and the second strand of the indexinon is connected to the fluorophore.

[0077] In some instances of the second aspect, (i) the first chain of the recycler is connected to the fluorophore and the second chain of the recycler is connected to the quencher; or (ii) the first chain of the recycler is connected to the quencher and the second chain of the recycler is connected to the fluorophore.

[0078] In a third aspect, this disclosure provides a method for detecting a target in a sample, wherein the target comprises at least two polynucleotide target hybridization regions, the method comprising: (a) contacting the sample with a reaction mixture comprising: (i) a detector comprising a circular pathway containing a polynucleotide hybridization region hybridizing with a polynucleotide chaperone, the circular pathway comprising one or more loops not hybridizing with the chaperone and an exposed single-stranded initiation hybridization region not hybridizing with the chaperone, wherein the initiation hybridization region is complementary to one of the target hybridization regions; (ii) a detector dissociation ion comprising a plurality of polynucleotide hybridization regions capable of hybridizing with hybridization regions on the chaperone; and (iii) an indexer comprising a dissociation ion containing at least two complementary single-stranded hybridization regions. (iv) a hybridization region that hybridizes a first-strand polynucleotide and a second-strand polynucleotide, the first strand extending beyond the second strand to form a single-stranded overhang and containing at least one loop that does not hybridize with the second strand; (v) an indexed hybridization region that contains at least two polynucleotide hybridization regions; (v) a recirculatory hybridization region that contains a first-strand polynucleotide and a second-strand polynucleotide hybridized through at least two complementary hybridization regions, the first strand extending beyond the second strand to form a single-stranded portion and containing at least one loop that does not hybridize with the second strand; and (v) a releaser that contains at least two polynucleotide hybridization regions, wherein each strand of the indexed hybridization region is connected to the detection portion and / or each strand of the recirculatory hybridization region is connected to the detection portion.(b) The reaction mixture is treated such that: (i) a first target hybridization region hybridizes with an initial hybridization region and a second target hybridization region hybridizes with an adjacent hybridization region on the circular pathway, thereby displacing the hybridization region of the partner; (ii) at least a portion of the displaced hybridization region on the partner hybridizes with a hybridization region on the detector dissociation ion and multiple hybridization regions on the detector dissociation ion hybridize with hybridization regions on the partner, thereby separating the partner from the circular pathway and opening one or more loops on the circular pathway and exposing the recycled portion of the polynucleotide hybridization region on the circular pathway; (iii) the dangling splint of the indexin hybridizes with a hybridization region on the circular pathway and at least one adjacent hybridization region on the first strand of the indexin hybrid hybridizes with a hybridization region on the circular pathway, thereby displacing the hybridization region on the second strand of the indexin hybrid and opening the loop on the first strand of the indexin hybrid; (iv) the hybridization region of the indexin hybrid hybrid hybridizes with at least a portion of the displaced hybridization region on the second strand of the indexin hybrid hybrid and another hybridization region on the indexin hybrid hybrid hybrid hybridizes with a hybridization region on the second strand of the indexin hybrid, thereby separating the first strand of the indexin hybrid from the second strand of the indexin hybrid, wherein at least one hybridization region on the first strand of the indexin hybrid hybrid hybridizes with the hybridization region on the circular pathway. (v) The single-stranded portion of the recirculator hybridizes with the hybridization region of the recirculating portion of the circular path, and the hybridization region of the adjacent single-stranded portion hybridizes with the second hybridization region on the recirculating portion, thereby displacing the hybridization region of the second strand of the recirculator and opening the loop on the first strand of the recirculator; (vi) At least a portion of the displaced hybridization region of the second strand of the recirculator hybridizes with the hybridization region on the releaser, and the adjacent hybridization region on the releaser hybridizes with the hybridization region on the second strand of the recirculator, thereby recirculating. (vii) The first strand of the indexing unit separates from the second strand of the recycle unit; (vii) the hybridization region of the first strand of the recycle unit hybridizes with the initiation hybridization region, thereby separating the target from the circular path, wherein the separation of the first strand of the indexing unit from the second strand of the indexing unit enables the detection portion of the indexing unit to be spatially separated and generate a detectable signal; and / or wherein the separation of the first strand of the recycle unit from the second strand of the recycle unit enables the detection portion of the recycle unit to be spatially separated and generate a detectable signal; (c) the presence or absence of the detectable signal is measured, wherein the detectable signal indicates the presence of the target in the sample.

[0079] In some instances of the third aspect, the flanking regions of the recycle ring are complementary hybridization pairs, which hybridize the first strand of the recycle with the second strand of the recycle.

[0080] In some instances of the third aspect, the loop of the recycler contains a hybridization region that hybridizes with the cyclic path.

[0081] In some instances of the third aspect, the ring of the recycle includes a hybridization region that hybridizes with the initial hybridization region.

[0082] In some instances of the third aspect, the cyclic path contains multiple loops, each containing a hybridization region.

[0083] In some instances of the third aspect, the dangling of the indexinon hybridizes with the hybridization region present in the open loop of the cyclic path.

[0084] In some instances of the third aspect, the released target hybridizes with the initiation hybridization region of another detector, thereby initiating the amplification of the method.

[0085] In some instances of the third aspect, at least one hybridization region of the first strand of the indexed substituent that does not hybridize with the circular pathway hybridizes with the initiation hybridization region of another detector, thereby initiating the amplification of the method.

[0086] In some instances of the third aspect, the first strand of the indexer contains a target hybridization region or a sequence that has at least about 90% identity with the target hybridization region.

[0087] In some instances of the third aspect, at least one hybridization region of the first strand of the indexed substituent that does not hybridize with the cyclic pathway contains a target hybridization region or a sequence that has at least about 90% identity with the target hybridization region.

[0088] In some instances of the third aspect, the loop of the first strand of the indexed substituent contains one of the target hybridization regions or a sequence that has at least about 90% identity with one of the target hybridization regions.

[0089] In some instances of the third aspect, the flanking regions of the ring of the indexinte are complementary hybridization pairs, which hybridize the first strand of the indexinte with the second strand of the indexinte.

[0090] In some instances of the third aspect, the reaction mixture contains multiple types of exponents.

[0091] In some instances of the third aspect, the circular path contains four loops.

[0092] In some instances of the third aspect, the first strand of the recycle contains the target hybridization region or a sequence that has at least about 90% identity with the target hybridization region.

[0093] In some instances of the third aspect, the loop of the recirculator contains one of the target hybridization regions or a sequence that has at least about 90% identity with one of the target hybridization regions.

[0094] In some instances of the third aspect, the recycling portion is adjacent to the initiation hybridization region.

[0095] In some instances of the third aspect, the loop path is a closed loop path.

[0096] In some instances of the third aspect, the treatment reaction mixture is carried out under isothermal conditions.

[0097] In some examples of the third aspect, the treatment reaction mixture is carried out at a temperature of about 20°C to about 25°C.

[0098] In some instances of the third aspect, the reaction mixture does not contain enzymes.

[0099] In some instances of the third aspect, the reaction mixture does not contain nuclease.

[0100] In some instances of the third aspect, the reaction mixture does not contain polymerase.

[0101] In some instances of the third aspect, (i) the first strand of the indexinon is connected to the fluorophore and the second strand of the indexinon is connected to the quencher; or (ii) the first strand of the indexinon is connected to the quencher and the second strand of the indexinon is connected to the fluorophore.

[0102] In some instances of the third aspect, (i) the first chain of the recycler is connected to the fluorophore and the second chain of the recycler is connected to the quencher; or (ii) the first chain of the recycler is connected to the quencher and the second chain of the recycler is connected to the fluorophore.

[0103] In a fourth aspect, this disclosure provides a method for detecting a target in a sample, wherein the target comprises at least two polynucleotide target hybridization regions, the method comprising: (a) contacting the sample with a reaction mixture comprising: (i) a detector comprising a circular pathway comprising a polynucleotide hybridization region hybridizing with a polynucleotide chaperone, the circular pathway comprising one or more loops not hybridizing with the chaperone and an exposed single-stranded initiation hybridization region not hybridizing with the chaperone, wherein the initiation hybridization region is complementary to one of the target hybridization regions; (ii) a detector dissociation comprising a plurality of polynucleotide hybridization regions capable of hybridizing with hybridization regions on the chaperone; and (iii) at least one first indexer comprising a first-stranded polynucleotide hybridized via at least two complementary hybridization regions. (iv) a first-chain polynucleotide and a second-chain polynucleotide, the first chain extending beyond the second chain to form a single-chain overhang and containing at least one loop that does not hybridize with the second chain, wherein each chain is connected to a detection portion; (v) a first-chain polynucleotide and a second-chain polynucleotide hybridized through at least two complementary hybridization regions, the first chain extending beyond the second chain to form a single-chain overhang and containing at least one loop that does not hybridize with the second chain, wherein each chain is connected to a detection portion; (vi) a first-chain polynucleotide hybridized unit containing at least two polynucleotide hybridization regions; (vii) a second-chain polynucleotide hybridized unit containing at least two polynucleotide hybridization regions; and (vii) a recycle unit containing a first-chain polynucleotide hybridized through at least two complementary hybridization regions. (viii) A first-strand polynucleotide and a second-strand polynucleotide, the first strand extending beyond the second strand to form a single-stranded portion and comprising at least one loop not hybridizing with the second strand, wherein each strand is connected to a detection portion; (b) a release molecule comprising at least two polynucleotide hybridization regions; and (c) processing the reaction mixture such that: (i) the first target hybridization region hybridizes with the initiating hybridization region and the second target hybridization region hybridizes with an adjacent hybridization region on the circular path, displacing the hybridization region of the partner; and (ii) at least a portion of the displaced hybridization region of the partner hybridizes with a hybridization region on the detection dissociation ion and multiple hybridization regions on the detection dissociation ion hybridize with hybridization regions on the partner, thereby separating the partner from the circular path and knocking one or more loops on the circular path. (iii) Opening and exposing the recycling portion of the hybridization region on the cyclic path, and optionally forming a product containing a partner that hybridizes with the detection dissociation ion; (iv) at least one first exponent hybridizes with the hybridization region on the cyclic path and at least one adjacent hybridization region on the first chain of the first exponent hybridizes with the hybridization region on the cyclic path, thereby displacing the hybridization region on the second chain of the first exponent and opening the loop on the first chain of the first exponent; (v) at least one second exponent hybridizes with the hybridization region on the cyclic path and at least one adjacent hybridization region on the first chain of the second exponent hybridizes with the hybridization region on the cyclic path, thereby displacing the hybridization region on the second chain of the second exponent and opening the loop on the first chain of the second exponent.(v) The hybridization region of the first exponent hybridizes with at least a portion of the substituted hybridization region on the second strand of the first exponent hybrid, and another hybridization region on the first exponent hybridizes with a hybridization region on the second strand of the first exponent hybrid, thereby separating the first strand of the first exponent from the second strand of the first exponent, thus enabling the detection portion of the first exponent to be spatially separated and generate a detectable signal, wherein at least one hybridization region of the first strand of the first exponent hybridizes with a hybridization region on a circular path and at least one hybridization region of the first strand of the first exponent does not hybridize with a circular path, optionally, wherein the product is formed by hybridization of the first exponent hybrid with the second strand of the first exponent; (vi) The hybridization region of the second exponent hybridizes with at least a portion of the substituted hybridization region on the second strand of the second exponent hybrid, and another hybridization region on the second exponent hybridizes with a hybridization region on the second strand of the second exponent hybrid, thereby separating the first strand of the second exponent from the second strand of the second exponent, thus enabling the detection portion of the second exponent to be spatially separated and generate a detectable signal, wherein at least one hybridization region of the first strand of the second exponent hybridizes with a hybridization region on a circular path and at least one hybridization region of the first strand of the first exponent hybrid does not hybridize with a circular path, optionally, wherein the product is formed by hybridization of the first exponent hybrid with the second strand of the first exponent; (vi) The hybridization region of the second exponent hybridizes with at least a portion of the substituted hybridization region on the second strand of the second exponent hybrid, and another hybridization region on the second exponent hybridizes with a hybridization region on the second strand of the second exponent hybrid, thereby separating the first strand of the second exponent from the second strand of the second exponent, thus enabling the detection portion of the second exponent to be spatially separated and generate a A hybridization region hybridizes with a hybridization region on a circular path, and at least one hybridization region of the first strand of the second indexor does not hybridize with the circular path. Optionally, the product is formed by hybridization of the first indexor hybrid with the second strand of the first indexor. (vii) The single-stranded portion of the recycle hybridizes with the hybridization region of the recycle portion of the circular path, and the hybridization region adjacent to the single-stranded portion hybridizes with the second hybridization region on the recycle portion, thereby displacing the hybridization region of the second strand of the recycle and opening the loop on the first strand of the recycle. (viii) At least one of the displaced hybridization regions of the second strand of the recycle... Partial hybridization occurs between the releaser and the hybridization region on the first strand of the recycler, and the adjacent hybridization region on the releaser hybridizes with the hybridization region on the second strand of the recycler, thereby separating the first strand of the recycler from the second strand, thus enabling the detection portion of the recycler to be spatially separated and generate a detectable signal; (ix) the hybridization region of the first strand of the recycler hybridizes with the initiating hybridization region, thereby separating the target from the cyclic path; optionally, the secondary product is formed by hybridization of the releaser and the second strand of the recycler; (c) the presence or absence of a detectable signal is measured, wherein the detectable signal indicates the presence of the target in the sample.

[0104] In some instances of the fourth aspect, the flanking regions of the recycle ring are complementary hybridization pairs, which hybridize the first strand of the recycle with the second strand of the recycle.

[0105] In some instances of the fourth aspect, the loop of the recycler contains a hybridization region that hybridizes with the cyclic path.

[0106] In some instances of the fourth aspect, the ring of the recycle includes a hybridization region that hybridizes with the initial hybridization region.

[0107] In some instances of the fourth aspect, the cyclic path contains multiple loops, each containing a hybridization region.

[0108] In some instances of the fourth aspect, the dangling protrusion of the first indexin hybridizes with the hybridization region present in the open loop of the cyclic path.

[0109] In some instances of the fourth aspect, the dangling protrusion of the second exponent hybridizes with the hybridization region present in the open loop of the cyclic path.

[0110] In some instances of the fourth aspect, the released target hybridizes with the initiation hybridization region of another detector, thereby initiating the amplification of the method.

[0111] In some instances of the fourth aspect, at least one hybridization region of the first strand of the first indexer that does not hybridize with the circular pathway hybridizes with the initiation hybridization region of another detector, thereby initiating the amplification of the method.

[0112] In some instances of the fourth aspect, the first strand of the first indexer contains a target hybridization region or a sequence that has at least about 90% identity with the target hybridization region.

[0113] In some instances of the fourth aspect, at least one hybridization region of the first strand of the first indexed substituent that does not hybridize with the cyclic pathway contains the target hybridization region or a sequence that has at least about 90% identity with the target hybridization region.

[0114] In some instances of the fourth aspect, the loop of the first strand of the first indexer contains one of the target hybridization regions or a sequence that has at least about 90% identity with one of the target hybridization regions.

[0115] In some instances of the fourth aspect, at least one hybridization region of the first strand of the second exponent that does not hybridize with the circular pathway hybridizes with the initiation hybridization region of another detector, thereby initiating the amplification of the method.

[0116] In some instances of the fourth aspect, the first strand of the second indexer contains a target hybridization region or a sequence that has at least about 90% identity with the target hybridization region.

[0117] In some instances of the fourth aspect, at least one hybridization region of the first strand of the second exponentiated substituent that does not hybridize with the cyclic pathway contains the target hybridization region or a sequence that has at least about 90% identity with the target hybridization region.

[0118] In some instances of the fourth aspect, the loop of the first strand of the second indexer contains one of the target hybridization regions or a sequence that has at least about 90% identity with one of the target hybridization regions.

[0119] In some instances of the fourth aspect, the flanks of the loop of the first exponent are complementary hybridization pairs, which hybridize the first strand of the first exponent with the second strand of the first exponent.

[0120] In some instances of the fourth aspect, the flanks of the ring of the second exponent are complementary hybridization pairs, which hybridize the first strand of the second exponent with the second strand of the second exponent.

[0121] In some instances of the fourth aspect, the complementary hybridization region pair of the first exponent contains a sequence different from the complementary hybridization region pair of the second exponent.

[0122] In some instances of the fourth aspect, the dangling of the first exponent contains a sequence different from that of the dangling of the second exponent.

[0123] In some instances of the fourth aspect, the reaction mixture contains two first exponents and one second exponent.

[0124] In some instances of the fourth aspect, the circular path contains four loops.

[0125] In some instances of the fourth aspect, the first strand of the recycle contains the target hybridization region or a sequence that has at least about 90% identity with the target hybridization region.

[0126] In some instances of the fourth aspect, the loop of the recirculator contains one of the target hybridization regions or a sequence that has at least about 90% identity with one of the target hybridization regions.

[0127] In some instances of the fourth aspect, the recycling portion is adjacent to the initiation hybridization region.

[0128] In some instances of the fourth aspect, the loop path is a closed loop path.

[0129] In some instances of the fourth aspect, the treatment reaction mixture is carried out under isothermal conditions.

[0130] In some examples of the fourth aspect, the treatment reaction mixture is carried out at a temperature of about 20°C to about 25°C.

[0131] In some instances of the fourth aspect, the reaction mixture does not contain enzymes.

[0132] In some instances of the fourth aspect, the reaction mixture does not contain nuclease.

[0133] In some instances of the fourth aspect, the reaction mixture does not contain polymerase.

[0134] In some instances of the fourth aspect, (i) the first strand of the first exponent is connected to the fluorophore and the second strand of the first exponent is connected to the quencher; or (ii) the first strand of the first exponent is connected to the quencher and the second strand of the first exponent is connected to the fluorophore.

[0135] In some instances of the fourth aspect, (i) the first strand of the second exponent is connected to the fluorophore and the second strand of the second exponent is connected to the quencher; or (ii) the first strand of the second exponent is connected to the quencher and the second strand of the second exponent is connected to the fluorophore.

[0136] In some instances of the fourth aspect, (i) the first chain of the recycler is connected to the fluorophore and the second chain of the recycler is connected to the quencher; or (ii) the first chain of the recycler is connected to the quencher and the second chain of the recycler is connected to the fluorophore.

[0137] In a fifth aspect, this disclosure provides a method for detecting a nucleic acid target in a sample, the method comprising: (a) contacting the sample with a reaction mixture comprising: (i) a detector comprising a circular pathway of a polynucleotide hybridizing with a polynucleotide chaperone, the circular pathway comprising a plurality of loops not hybridizing with the chaperone and an exposed single-stranded initiation region not hybridizing with the chaperone, wherein the initiation region is complementary to the target; and (ii) one or more polynucleotides, wherein at least one of the polynucleotides is linked to a detection portion; (b) hybridizing the target with the initiation region; (c) dissociating the chaperone from the circular pathway, thereby opening the loops of the circular pathway; (d) hybridizing one or more polynucleotides with the circular pathway in a circumferential hybridization reaction, wherein the circumferential hybridization reaction induces modification of at least one labeled polynucleotide such that the detection portion is capable of generating a detectable signal; and (e) measuring the presence or absence of the detectable signal, wherein the detectable signal indicates the presence of the target in the sample.

[0138] In some examples of the fifth aspect, circumferential hybridization reactions separate the target from the circumferential pathway.

[0139] In some instances of the fifth aspect, hybridization between the target and the initiation region partially replaces the mate from the circumferential pathway.

[0140] In some instances of the fifth aspect, the reaction mixture contains a detector dissociation ion that hybridizes with a chaperone, thereby dissociating the chaperone from the cyclic pathway and causing one or more loops of the cyclic pathway to open.

[0141] In some instances of the fifth aspect, the partially substituted partner hybridizes with the detector dissociative ion.

[0142] In some examples of the fifth aspect, at least one of the polynucleotides is an indexer comprising a double-stranded portion and a single-stranded overhang extending beyond the double-stranded portion, and said circumferential hybridization reaction comprises: (i) hybridization of the overhang with a circumferential pathway; and (ii) hybridization of at least one hybridization region adjacent to the overhang with a circumferential pathway, thereby at least partially opening the double-stranded portion.

[0143] In some instances of the fifth aspect, the indexinon comprises a hybridized first-strand polynucleotide and a second-strand polynucleotide, the first strand extending beyond the second strand to form a dangling protrusion and comprising at least one loop that does not hybridize with the second strand, and wherein the circumferential hybridization reaction comprises hybridizing the first strand of the indexinon with a circumferential pathway, thereby opening the loop on the first strand of the indexinon.

[0144] In some instances of the fifth aspect, the reaction mixture contains an indexinverter hybrid that hybridizes with one strand of the indexinverter, thereby separating the first strand of the indexinverter from the second strand of the indexinverter.

[0145] In some instances of the fifth aspect, the circumferential hybridization reaction includes: (i) hybridization of the first strand of the indexinteger with the circumferential pathway, thereby opening the loop on the first strand of the indexinteger; and (ii) hybridization of the indexinteger hybrid with the second strand of the indexinteger, thereby separating the first strand of the indexinteger from the second strand of the indexinteger.

[0146] In some instances of the fifth aspect, the first strand of the exponentiator can hybridize with the initiation region of another circular path, thereby initiating the amplification of the method.

[0147] In some instances of the fifth aspect, the first strand of the indexer contains a hybridization region that is identical to or has at least about 90% sequence identity with the hybridization region of the target.

[0148] In some instances of the fifth aspect, the loop of the first strand of the indexed substituent contains a hybridization region that is identical to or has at least about 90% sequence identity with the target hybridization region.

[0149] In some instances of the fifth aspect, each chain of the exponent is connected to the detection part, and the separation of the first and second chains of the exponent enables the detection part of the exponent to be spatially separated and generate a detectable signal.

[0150] In some instances of the fifth aspect, the reaction mixture contains multiple exponents.

[0151] In some instances of the fifth aspect, the reaction mixture contains multiple exponential hybrids.

[0152] In some instances of the fifth aspect, (a) one or more polynucleotides comprise: (i) at least one first exponentiated polynucleotide comprising a hybridized first-strand polynucleotide and a hybridized second-strand polynucleotide, the first strand extending beyond the second strand to form a single-stranded overhang and comprising at least one loop not hybridizing with the second strand, wherein each strand is connected to a detection moiety; and (ii) at least one second exponentiated polynucleotide comprising a hybridized first-strand polynucleotide and a hybridized second-strand polynucleotide, the first strand extending beyond the second strand to form a single-stranded overhang and comprising at least one loop not hybridizing with the second strand, wherein each strand is connected to a detection moiety; (b) the reaction mixture comprises: (i) a first exponentiated hybrid; and (ii) a second exponentiated hybrid; and (c) a circumferential hybridization reaction. This includes: (i) hybridization of the first strand of the first exponent with a cyclic path, thereby opening the loop on the first strand of the first exponent; (ii) hybridization of the first strand of the second exponent with a cyclic path, thereby opening the loop on the first strand of the second exponent; (iii) hybridization of the first exponent hybrid with the second strand of the first exponent, separating the first strand of the first exponent from the second strand of the first exponent, thereby enabling the detection portion of the first exponent to be spatially separated and generate a detectable signal; and (iv) hybridization of the second exponent hybrid with the second strand of the second exponent, separating the first strand of the second exponent from the second strand of the second exponent, thereby enabling the detection portion of the second exponent to be spatially separated and generate a detectable signal.

[0153] In some instances of the fifth aspect, the first and second exponents hybridize with different sequences on the circular path.

[0154] In some instances of the fifth aspect, at least one of the polynucleotides is a recycler that hybridizes with the initiation region of the cyclic pathway, thereby displacing the target from the cyclic pathway.

[0155] In some instances of the fifth aspect, the recirculator comprises a double-stranded portion interrupted by a loop in one strand; and a single-stranded portion extending beyond the double-stranded portion, and wherein the circumferential hybridization reaction comprises: (i) hybridization of the single-stranded portion with the circumferential path; (ii) hybridization of at least one hybridization region adjacent to the single-stranded portion with a hybridization region adjacent to the starting region on the circumferential path, thereby opening the loop of the recirculator; and (iii) hybridization of the opened loop of the recirculator with the starting region, thereby displacing the target from the circumferential path.

[0156] In some examples of the fifth aspect, the recirculator comprises a first-strand polynucleotide and a second-strand polynucleotide that are hybridized, the first strand extending beyond the second strand to form a single-stranded portion and comprising at least one loop that does not hybridize with the second strand, and wherein the circumferential hybridization reaction comprises: (i) the first strand of the recirculator hybridizing with a hybridization region adjacent to the initiation region of the circumferential pathway, thereby opening the loop of the recirculator; and (ii) the opened loop of the recirculator hybridizing with the initiation region, thereby displacing the target from the circumferential pathway.

[0157] In some instances of the fifth aspect, the reaction mixture contains a releaser that hybridizes with one strand of the recycler, thereby separating the first strand of the recycler from the second strand of the recycler.

[0158] In some examples of the fifth aspect, the circumferential hybridization reaction includes: (i) hybridization of the first strand of the recirculator with a region adjacent to the starting region of the circumferential path, thereby opening the loop on the first strand of the recirculator; (ii) hybridization of the opened loop of the recirculator with at least a portion of the starting region, thereby partially displacing the target from the circumferential path; (iii) hybridization of the second strand of the recirculator with the releaser, thereby separating the first strand of the recirculator from the second strand of the recirculator; and (iv) hybridization of the first strand of the recirculator with the starting region, separating the target from the circumferential path.

[0159] In some instances of the fifth aspect, the first strand of the recirculator contains a hybridization region that is identical to or has at least about 90% sequence identity with the target.

[0160] In some instances of the fifth aspect, the loop of the first strand of the recycle contains a sequence that is identical to or has at least about 90% identity with the target hybridization region.

[0161] In some instances of the fifth aspect, each chain of the recirculator is connected to the detection part, and the separation of the first and second chains of the recirculator enables the detection part of the recirculator to be spatially separated and generate a detectable signal.

[0162] In some instances of the fifth aspect, the cyclic path contains multiple loops.

[0163] In some instances of the fifth aspect, the dangling of the indexinton hybridizes with the open loop of the cyclic path.

[0164] In some instances of the fifth aspect, the released target hybridizes with the start region of another detector, thereby initiating the amplification of the method.

[0165] In some instances of the fifth aspect, the circular path contains four loops.

[0166] In some instances of the fifth aspect, the loop path is a closed loop path.

[0167] In some examples of the fifth aspect, the circumferential hybridization reaction is carried out under isothermal conditions.

[0168] In some examples of the fifth aspect, the circumferential hybridization reaction is carried out at a temperature of about 20°C to about 25°C.

[0169] In some instances of the fifth aspect, the reaction mixture does not contain enzymes.

[0170] In some instances of the fifth aspect, the reaction mixture does not contain nuclease.

[0171] In some instances of the fifth aspect, the reaction mixture does not contain polymerase. In some instances of the fifth aspect, (i) the first strand of the indexinon is linked to the fluorophore and the second strand of the indexinon is linked to the quencher; or (ii) the first strand of the indexinon is linked to the quencher and the second strand of the indexinon is linked to the fluorophore.

[0172] In some instances of the fifth aspect, (i) the first chain of the recycler is connected to the fluorophore and the second chain of the recycler is connected to the quencher; or (ii) the first chain of the recycler is connected to the quencher and the second chain of the recycler is connected to the fluorophore.

[0173] In a sixth aspect, this disclosure provides a method for forming a structure containing a circular pathway comprising hybrid monomers, the method comprising initiating a hybridization chain reaction by providing a target comprising a starting sequence to a closed pathway of a hybridization region; hybridizing the starting sequence with the circular pathway; exposing a recirculating portion of the circular pathway in response to a circumferential process of the hybridization chain reaction; and releasing the target from the circular pathway in response to the exposed recirculating portion.

[0174] In some instances of the sixth aspect, the hybridization chain reaction can be initiated by a target previously released by the execution method.

[0175] In some instances of the sixth aspect, the method includes activating at least one propagator on a circular pathway in response to a hybridization chain reaction, each activated propagator containing a starting sequence.

[0176] In some instances of the sixth aspect, the hybridization chain reaction can be initiated by a target that is a proliferator activated on another cyclic pathway by executing a method.

[0177] In some examples of the sixth aspect, the method includes detecting the products of the hybridization chain reaction by detecting the fluorescence response.

[0178] In a seventh aspect, this disclosure provides a hybridization chain reaction comprising initiating a hybridization chain by providing a target to a circular path of a hybridization region, the target containing a starting sequence; hybridizing the starting sequence with the circular path; exposing a recirculating portion of the circular path in response to a circumferential hybridization process on the circular path; and releasing an initiator from the circular path in response to the exposed recirculating portion.

[0179] In some instances of the seventh aspect, the hybridization chain reaction also includes providing a recycler to the exposed recycling portion, such that the target is released from the cyclic pathway in response to the reaction between the recycler and the exposed recycling portion. Brief description of the attached diagram

[0180] Figure 1 A system for analyzing samples containing a target and a reaction mixture.

[0181] Figure 2 . Figure 1 The components in the reaction mixture shown include a detector, a detector dissociation ion, a first indexed ion, a second indexed ion, a first indexed ion hybrid, a second indexed ion hybrid, a recycle ion, and a release ion.

[0182] Figure 3 . Figure 1 The target and Figure 2 The detectors shown are in which letters a, b, c, etc., represent hybridization regions, and the letters marked with * are complementary to the unmarked letters.

[0183] Figure 4 . Figure 2 The detectors shown are decomposed ions and Figure 3 The hybridization between the detector and the target shown produces a modified detector.

[0184] Figure 5 . Figure 4 The reaction results between the components are shown, including the detection of dissociation.

[0185] Figure 6 . Figure 5 The dissociation detectors shown and Figure 2 The exponentiation shown.

[0186] Figure 7 . Figure 6 The hybridization results between the components shown include the modified dissociated detector and Figure 2 The exponential hybrids shown are shown.

[0187] Figure 8 . Figure 7 The hybridization results shown are between the modified dissociated detector and the indexed hybrid, including the activated detector.

[0188] Figure 9 . Figure 8 The activation detector and Figure 2 The recycle sub-sub ...

[0189] Figure 10 . Figure 9 The hybridization results between the components shown and the releasers include the modified activated detectors.

[0190] Figure 11 Release the child and Figure 10 The hybridization results between the modified activated detectors shown include Figure 1 The target shown is attached to the modified activated detector.

[0191] Figure 12 . Figure 11 The target shown is released from the modified activated detector and three proliferators are attached to the detector.

[0192] Figure 13 . Figure 12 The types of proliferators shown act as Figure 3 The target of the type of detector shown generates Figure 4 Modified detectors of the types shown.

[0193] Figure 14 . Figure 12 The types of proliferators shown act as Figure 3 The target of the type of detector shown generates Figure 5 The types of dissociation detectors shown.

[0194] Figure 15 . Figure 13 and 14 The detailed reaction is in Figure 1 The following events occur simultaneously in the reaction mixture shown.

[0195] Figure 16 . Figure 1 Summary of the steps performed in the reaction mixture shown.

[0196] Figure 17 . Figure 1 The sequence of the target shown and Figure 2 The components shown.

[0197] Figure 18 An exponential target cyclic hybridization chain reaction with an open cyclic pathway. Fluorescence is expressed in arbitrary units (au). The legend shows the concentration of the target in the reaction. The no-detector-complex control omits both the detector-complex and the target. The temperatures shown above the figure are the incubation temperatures for the exponential target cyclic hybridization chain reaction within the time indicated in parentheses.

[0198] Figure 19 An exponential target-recycled hybridization chain reaction with an open circular pathway and a target added before the detector. The legend shows the target concentration during the reaction. The gray line is not the original result, but rather the result at the same time point. 1 fM Target results and 0 fM The calculated difference between target results. Fluorescence is expressed in arbitrary units (au).

[0199] Figure 20 An exponentially targeted recirculating hybridization chain reaction with an open circular pathway and a nuclease for removing residual unhybridized oligonucleotides during the preparation of the detection subcomplex. Fluorescence is expressed in arbitrary units (au).

[0200] Figure 21 An exponential target cyclic hybridization chain reaction with a closed loop pathway. Fluorescence is expressed in arbitrary units (au).

[0201] Figure 22 An exponentially targeted recirculating hybridization chain reaction with a closed circular pathway and a nuclease for removing residual unhybridized oligonucleotides during the preparation of the detection subcomplex. Fluorescence is expressed in arbitrary units (au).

[0202] Figure 23 Exponential target recycling hybridization chain reaction with a nonspecific (pseudo)target. The exponential target recycling hybridization chain reaction was performed using the target sequence (initiator; SEQ ID NO: 1) and pseudo-initiator-2 (a nonspecific sequence from the SARS-CoV-2 genome; SEQ ID NO: 14). Fluorescence is expressed in arbitrary units (au).

[0203] Figure 24 Target dependence of the exponential target-recycled hybridization chain reaction. The fluorescence of the reaction (expressed in arbitrary units (au)) depends on the target concentration (shown in the legend). Error bars represent the maximum and minimum values ​​for repeated samples.

[0204] Figure 25 An exponential target recycling hybridization chain reaction using a target within a larger sequence was employed. Initiators 2, 3 & 4 (SEQ ID NO: 19, 20 & 21, respectively) had target sequences flanked by non-target sequences. Fluorescence is expressed in arbitrary units (au). Error bars represent the maximum and minimum values ​​of replicate samples.

[0205] Figure 26Specificity of exponential target recycling hybridization chain reactions. Exponential target recycling hybridization chain reactions were performed using target sequences (initiators), non-specific sequences from the genomes of humans (pseudo-initiator 1; SEQ ID NO: 22), SARS-CoV-2 (pseudo-initiator 2; SEQ ID NO: 14), and Plasmodium (pseudo-initiator 3; SEQ ID NO: 23), and modified target sequences with 52-96% sequence identity to the target sequences, i.e., mutant initiators 1-6 (SEQ ID NO: 24-29). Fluorescence is expressed in arbitrary units (au). The reaction rate for each initiator is shown in Table 2.

[0206] Figure 27 Exponential target cyclic hybridization chain reactions at different temperatures. The relative reaction rate at each temperature was determined by dividing the result with the initiator by the control without the initiator. The maximum relative reaction rate was measured at 26 °C and the result is expressed as a percentage of the maximum reaction rate.

[0207] Figure 28. Exponential target recycle hybridization chain reaction in different buffers. A: The reaction was carried out in 2xSSC buffer containing 0.1% Tween 20 and 0 mM, 5 mM, and 10 mM MgCl2 (SSCT, SSCM / 2T, and SSCMT, respectively). B: The reaction was carried out in 10 mM MgCl2 buffer. 2, Fluorescence was measured in 2xSSC buffer (SSCMT) with 0.1% Tween 20 and pH 7, 1xTAE buffer (TAEM) with 12.5 mM MgCl2 and pH 8.3, and 1xHEPES buffer (KAH) with 100 mM potassium acetate and pH 7.5. Fluorescence is expressed in arbitrary units (au). Error bars indicate the maximum and minimum values ​​of replicate samples.

[0208] Figure 29 Following the closure of the circular pathway, ethanol precipitation was used to clean the exponential target recycling hybridization chain reaction of the detection complex. The exponential target recycling hybridization chain reaction was performed using the target sequence (initiator-3; SEQ ID NO: 20) and pseudo-initiator-2 (a non-specific sequence from the SARS-CoV-2 genome; SEQ ID NO: 14). Fluorescence is expressed in arbitrary units (au). Error bars represent the maximum and minimum values ​​of replicate samples.

[0209] Figure 30 After closing the circular pathway, single-stranded DNA nuclease and ethanol precipitation were used to clean the exponential target recirculating hybridization chain reaction of the detection subcomplex. Fluorescence is expressed in arbitrary units (au). Error bars represent the maximum and minimum values ​​of repeated samples.

[0210] Figure 31 The exponential target recycling hybridization chain reaction was purified using a silica gel column. The silica gel column was used to clean the detection complex after closing the loop pathway, and to clean the first exponent, second exponent, and recycling complexes after hybridization of the first and second chains. Fluorescence is expressed in arbitrary units (au). Error bars represent the maximum and minimum values ​​for replicate samples.

[0211] Figure 32 This is an exponentially targeted recycle hybridization chain reaction without the detection of dissociative ions. The reaction can proceed at a slower rate without the detection of dissociative ions. Background signal is also low when the detection of dissociative ions is omitted. Fluorescence is expressed in arbitrary units (au). Error bars represent the maximum and minimum values ​​of repeated samples.

[0212] Figure 33 Exponential target cyclic hybridization chain reactions were performed using different reagent concentrations. All reagents, except the initiator, were reacted at standard concentrations and half the standard concentrations. Fluorescence is expressed in arbitrary units (au). Error bars represent the maximum and minimum values ​​of replicate samples.

[0213] Figure 34 A chain reaction of exponentially targeted recycling hybridization with a second strand containing a reduced concentration of the indexiton and recyclon complex, and a hybridon / releaser. The reaction was carried out with a first strand containing a standard concentration of the indexiton and recyclon complex; however, the molar ratio of the second strand and the hybridon / releaser to these first strands was altered. The tested molar ratios are shown in the legend. Fluorescence is expressed in arbitrary units (au). Error bars represent the maximum and minimum values ​​for repeated samples.

[0214] Figure 35 A chain reaction of exponential target recycling hybridization with reduced concentrations of the first or second exponentialized complex. The reaction is carried out using reagents at standard concentrations or only half the standard concentrations of the first or second exponentialized complex. Fluorescence is expressed in arbitrary units (au). Error bars represent the maximum and minimum values ​​of replicate samples.

[0215] Figure 36 An exponential target-recycling hybridization chain reaction with a large dangling splint. The reaction was carried out with a modified chaperone, chaperone-77 (SEQ ID NO: 30), which resulted in a detector complex with a single-stranded region of 3 nucleotides longer for target dangling splint binding. Fluorescence is expressed in arbitrary units (au). Error bars represent the maximum and minimum values ​​for repeated samples.

[0216] Figure 37Background signal was reduced using modified indexer and / or recycler complexes. Different stem sequences in the cyclic pathway binding region on the indexer and / or recycler complex reduced spontaneous binding of hybrids and / or recyclers to the second strand of the indexer and / or recycler complex and background signal. Background fluorescence from the first indexer complex-8 was 46% of the level of the first indexer complex, while background fluorescence from recycler complex-9 and recycler complex-2 were 70% and 25% of the level of the recycler complex, respectively.

[0217] Figure 38 A chain reaction of exponential target recirculating hybridization was performed using a modified first exponentiomer. These modifications employed a two-stage strategy to further shift the initiation region of the first exponentiomer away from its cyclic pathway binding region, with the initiation region located in a second stage containing a hairpin hybrid. Fluorescence is expressed in arbitrary units (au). Error bars represent the maximum and minimum values ​​for replicate samples.

[0218] Figure 39 A chain reaction of exponential target recirculating hybridization was performed using a modified first exponent. These modifications employed a two-stage strategy to further move the initiation region of the first exponent away from its cyclic pathway binding region, with the split initiation region located in the second stage. Fluorescence is expressed in arbitrary units (au). Error bars represent the maximum and minimum values ​​for replicate samples.

[0219] Figure 40 An exponential target recycling hybridization chain reaction was performed using modified initiation regions and modified cyclic pathway indexer binding regions. These modifications reduced homodimerization of the initiation regions and further distanced the initiation regions of each indexer from their cyclic pathway binding regions. Fluorescence is expressed in arbitrary units (au). Error bars represent the maximum and minimum values ​​for replicate samples. definition

[0220] In the context of this specification, the terms “a” and “an” are used herein to refer to the grammatical object of one or more articles (i.e., at least one). For example, “a child” means one or more children.

[0221] The term “about” is understood to mean a range of + / - 10%, preferably + / - 5% or + / - 1%, or more preferably + / - 0.1%.

[0222] As used herein, the terms “complementary,” “complementarity,” “matching,” and “matching” refer to the ability of nucleotides (e.g., deoxyribonucleotides, ribonucleotides, or combinations thereof) to hybridize with each other via Watson-Crick base pairing, non-classical base pairing (including swinging base pairing and Hoogsteen base pairing) (e.g., LNA, PNA, or BNA), or unnatural base pairing (UBP). A nucleotide referred to as “complementary” or “complementary” to each other is a nucleotide that has the ability to hybridize together via Watson-Crick base pairing, via non-classical base pairing (swinging base pairing, Hoogsteen base pairing), or via unnatural base pairing (UBP) between their respective bases.

[0223] The terms “comprise”, “comprises”, “comprised”, “comprising”, “including”, or “having” in this specification and claims are used in an inclusive sense, that is, to indicate the presence of the referred feature but not to exclude the presence of other or further features.

[0224] The term "identity" refers to the relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, determined by sequence alignment and comparison. When two sequences are optimally aligned, the percentage of identity between them is a function of the number of common positions (i.e., %homology = number of common positions / total number of positions × 100). Determining the optimal alignment takes into account the number of vacancies and the length of each vacancy, which are essential factors for optimal alignment of two sequences. Sequence comparison and the determination of the percentage of identity between two sequences can be achieved using mathematical algorithms. The percentage of identity between two nucleotide sequences can be determined using the GAP program in the GCG software package, using the NWSgapdna.CMP matrix with vacancy weights set to 40, 50, 60, 70, or 80 and length weights set to 1, 2, 3, 4, 5, or 6. The percentage of identity between two nucleotide or amino acid sequences can also be calculated using the algorithm of E. Meyers and W. Miller (CABIOS, 4: 11-17 (1989)) (which has been incorporated into the ALIGN program), using the PAM120 weighted residue table with a vacancy length penalty of 12 and a vacancy penalty of 4. Alternatively, the percentage of identity between two amino acid sequences can be calculated using Needleman and Wunsch (… J. Mol.Biol.The algorithm (48):444-453 (1970) (which has been incorporated into the GAP program in the GCG software package) uses a Blossum62 or PAM250 matrix, with empty space weights set to 16, 14, 12, 10, 8, 6, or 4 and length weights set to 1, 2, 3, 4, 5, or 6 to determine the length. Other computer programs that can be used in this regard include BLASTP, BLASTN, and FASTA (Altschul). et al ., J . Molec. Biol ., 1990:215:403). Another method for determining the percentage of identity between two peptides involves the Clustal W algorithm (Thompson, JD, Higgines, DG and Gibson TJ, 1994, . Nucleic AcidRes 22(22): 4673-4680) together with the BLOSUM 62 rating matrix (Henikoff S & Henikoff, JG, 1992, Proc.Natl.Acad.Sci.USA 89: 10915-10919), which uses a gap opening penalty of 10 and a gap extension penalty of 0.1 to obtain the highest level of matching between two sequences, where at least 50% of the total length of one of the sequences is involved in the alignment.

[0225] As used herein, the term "isolated" refers to a material that is substantially or substantially free of components that are normally present in its natural state. For example, isolated polynucleotides as used herein refer to polynucleotides that have been purified from their flanking sequences in their natural state, such as DNA fragments that have been removed from sequences normally adjacent to the fragment.

[0226] As used herein, the term "multiple" means more than one. In certain aspects or implementations, multiple can mean 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 or more, as well as any integer and any range that can be derived therefrom.

[0227] As used herein, the terms “polynucleotide” and “nucleic acid” are used interchangeably and refer to a single- or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases, or analogs, derivatives, variants, fragments or combinations thereof, including but not limited to DNA, methylated DNA, alkylated DNA, RNA, methylated RNA, microRNA, siRNA, shRNA, mRNA, tRNA, snoRNA, stRNA, smRNA, pre-microRNA and pri-microRNA, other non-coding RNA, ribosomal RNA, its derivatives, its amplicon or any combination thereof.

[0228] When using numerical ranges to describe certain embodiments of this disclosure, it should be understood that each range should be considered to encompass its sub-ranges. For example, a range described as 1 to 6 should be considered to include sub-ranges such as 1 to 5, 2 to 4, 2 to 6, etc. Similarly, a range described as 1 to 6 should be considered to include sub-ranges such as 2 to 5, 1 to 3, 3 to 6, etc. Detailed Implementation

[0229] The following detailed description provides sufficiently detailed exemplary embodiments of the invention to enable those skilled in the art to practice it. The features or limitations of the various embodiments described do not necessarily limit other embodiments of the invention or the invention as a whole. Therefore, the following detailed description does not limit the scope of the invention, which is defined only by the claims.

[0230] This disclosure provides compositions and methods for detecting targets (e.g., nucleic acids, proteins, analytes, compounds, molecules, etc.). The method described herein may be referred to as target recycling hybridization chain reaction (TRHCR). HCR detects a specific nucleic acid sequence and outputs a chain reaction involving hybridization and conformational changes between pre-existing nucleic acid components (in the reaction mixture). In some instances, TRHCR involves guiding individual HCRs around a cyclic pathway, such that they ultimately release the initiating target sequence and expose / activate new target sequences. This provides several new target sequences for each asynchronous cycle / HCR and maintains low molecular weight products. This provides an exponential reaction that is not limited by increased viscosity or steric hindrance due to high molecular weight or highly branched products. TRHCR can also be designed as a linear reaction if quantification is required. When used as an exponential reaction, the method may be referred to as exponential target recycling hybridization chain reaction (ETHyR).

[0231] The compositions and methods described herein can be used to detect a target and / or the response to a target. The compositions and methods can be used for molecular computation (Bi) et al . Chem Soc Rev(2017) 46, 4281). In some instances, this disclosure provides a method for detecting a nucleic acid target in a sample, the method comprising: (a) contacting the sample with a reaction mixture comprising: (i) a detector comprising a circular path containing a polynucleotide hybridization region hybridizing with a polynucleotide chaperone, the circular path comprising a plurality of loops not hybridizing with the chaperone and an exposed single-stranded initiation hybridization region not hybridizing with the chaperone, wherein the initiation hybridization region is complementary to a hybridization region of the target; and (ii) one or more polynucleotides; (b) hybridizing the target with the initiation hybridization region; (c) dissociating the chaperone from the circular path, thereby exposing a plurality of hybridization regions on the circular path; and (d) hybridizing the one or more polynucleotides with the plurality of exposed hybridization regions on the circular path in a circumferential hybridization reaction.

[0232] Non-restrictive description of the implementation plan This section on the specific implementation plan is for reference. Figures 1 to 17 Scheme implementations are described. References to the accompanying drawings and components in those drawings are provided for ease of understanding and are not intended to limit the scope of the invention.

[0233] Figure 1 Figure 1 A method for identifying viral infection in a patient is shown. A nasal / throat swab 101 is used to obtain a sample from the patient. Cells, viruses, and other materials on the swab 101 are transferred to a sample tube 102 containing a buffer solution that releases viral nucleic acids and optionally inactivates any nucleases. The mixture in the sample tube 102 is treated 103 using known methods such as heating and rapid cooling to denature the viral nucleic acids. However, it should be understood that in some instances, denaturation is not part of the method. For example, the target may be a single-stranded nucleic acid. The single-stranded nucleic acid includes a target 105, which includes a nucleotide initiation sequence indicating the presence of a specific viral infection. This target 105 will serve as an initiator for amplification and may be part of a longer nucleic acid chain. The target may bind to another molecule to be detected (e.g., an analyte or another nucleic acid), thereby serving as a proxy for the detection of that other molecule.

[0234] In some instances, the nucleic acid is DNA. In some instances, the nucleic acid is RNA. The following description of the implementation scheme will refer to the target DNA sequence 105. However, those skilled in the art will understand that the starting RNA sequence can be used directly in the same process.

[0235] Known methods for detecting the presence of target sequences include PCR. To detect RNA sequences, PCR requires an initial step where RNA is reverse transcribed to produce DNA. Subsequently, multiple reaction cycles combining heating and cooling phases are needed to increase the relative concentration of the starting sequence. In PCR, amplification requires significant time and effort due to the need for careful control of temperature changes and multiple reaction cycles. In contrast, hybridization reactions performed at roughly constant temperatures are often referred to as isothermal reactions and do not require sophisticated equipment. HCR methods can be performed under isothermal conditions and avoid the use of enzymes. However, known HCR methods lack the reliability and sensitivity of PCR.

[0236] Figure 1 An improved amplification process was demonstrated, which avoids the need for repeated temperature cycles and enzymes. It was carried out under isothermal conditions, for example, at a temperature of approximately 25°C.

[0237] A solution of eight components 106 to 113 is mixed 114 to provide a detection solution 115, wherein components 106 to 113 do not react with each other (a condition known as metastability). Components 106 to 113 are nucleic acid components such as DNA. The detection solution 115 is mixed with a sample containing target 105 to form a reaction mixture 116, wherein amplification occurs over time. In the example shown, target 105 includes the starting sequence shown in SEQ ID NO: 1. However, it should be understood that the method described herein is not limited to a specific target sequence. Stirring can be used before and / or during amplification to accelerate amplification. Target 105 derived from sample 102 can be kept at a temperature slightly above freezing before being added to reaction mixture 116. Light 117 can be used to irradiate reaction mixture 116. Over time, reaction mixture 116 begins to emit fluorescence 118 in response to irradiation 117. This fluorescence 118 and its rate of change can be detected electronically. Furthermore, changes in fluorescence can be recorded to provide further characterization information. Fluorescence 118 indicates the presence of the target in the sample.

[0238] In one instance, for ease of dispensing, components 106 to 113 are pre-mixed as a dry powder and stored. Closer to use, water or a suitable buffer is added to the powder, and solution 115 is then tested to ensure it can be refrigerated or frozen until needed.

[0239] In some instances, target 105 indicates the presence of a specific form of cancer, and sample processing 103 is adjusted accordingly. The ability to detect cancer rapidly and inexpensively offers significant advantages for patient management, including improved prognosis, personalized treatment, monitoring of treatment success, and monitoring for recurrence after remission. In some instances, this testing is performed on a large scale in vitro to facilitate drug discovery. In some instances, this in vitro drug discovery is performed on a large scale to feed training data into artificial neural networks, thereby accelerating the design of successful therapies for both critical and non-critical health conditions.

[0240] In some instances, sample processing 103 includes activating target 105 by utilizing the presence of a target sequence in sample 102. The aim of doing so is to enable the design of components 106 to 113 that can effectively and efficiently detect any DNA or RNA sequence in sample 102.

[0241] Figure 2 Figure 1 The test solution 115 shown is in Figure 2 The following is a schematic description. Solution 115 contains a detector 106, which includes a circular path 201 containing a polynucleotide hybridization region that hybridizes with a chaperone of a largely complementary polynucleotide hybridization region 202. It should be understood that the circular path is not necessarily perfectly circular, and it can exist in other forms, such as oval or elliptical. The circular path can also include folds and twists (see, for example, Figure 3 Circular pathways can be open, with their 5' and 3' ends closely adjacent to each other. The ends of an open circular pathway can remain closely adjacent by hybridizing with a polynucleotide chaperone that bridges the two ends. In other examples, such as... Figure 2 As shown, the circular path is a closed circular path containing a continuous nucleic acid molecule without ends. A closed circular path can be formed by connecting the ends of an open circular path (optionally using a chaperone and a nuclease). Loops 203, 204, 205, and 206 drive the hybridization chain reaction along the nucleotide sequence. Loops 203, 204, 205, and 206 are present at multiple locations on the circular path 201. The circular path 201 also contains a single-stranded initiation portion 207, which provides an initiation site for the hybridization chain reaction. In the example shown, the circular path 201 has the sequence shown in SEQ ID NO: 2. However, it should be understood that different sequences can be used alternatively. Similarly, in the example shown, the hybridization chaperone 202 of the detector 106 has the sequence shown in SEQ ID NO: 3, but different sequences can be used alternatively. The detector molecule 106 in Figure 2The shapes are shown schematically and should be understood to be not precisely circular. These details have been omitted for clarity. The term "hybridization region" refers to a polynucleotide sequence capable of specifically hybridizing with a complementary polynucleotide sequence (i.e., a complementary hybridization region).

[0242] The detection solution 115 also contains an optional single-stranded nucleic acid detector dissociation ion 107, the length of which may be similar to the perimeter of the detector 106. In the illustrated example, the dissociation ion 107 has the sequence shown in SEQ ID NO: 4. However, it should be understood that different sequences may be used alternatively. The detection solution 115 also contains a first indexer 108, which comprises two polynucleotide chains 208 and 209, and a single-stranded overhang 210 and a loop 211. In the illustrated example, the first chain 208 has the sequence shown in SEQ ID NO: 5, and the second chain 209 has the sequence shown in SEQ ID NO: 6. However, it should be understood that different sequences may be used alternatively. The first chain 208 and the second chain 209 are labeled with a detection portion. In the illustrated example, the first chain 208 is labeled with a quencher molecule 212, which is in close proximity to the fluorophore 213 on the second chain 209, thereby suppressing the fluorescence of the fluorophore 213. It should be understood that the first chain can be labeled with a fluorophore, while the second chain can be labeled with a quencher.

[0243] The second exponent 109 has the same general structure as the first exponent 108, but has a different hybridization region sequence. The second exponent has a first strand 214, which in the illustrated example has the sequence shown in SEQ ID NO: 7; and a second strand 215, which in the illustrated example has the sequence shown in SEQ ID NO: 8. However, it should be understood that different sequences may be used alternatively. It also includes a detection portion in the form of a quencher 216 and a fluorophore 217. It should be understood that the quencher and fluorophore may be attached to the strand opposite to the strand shown in the figure. The detection solution 115 also includes a first exponent hybrid 110, which in the illustrated example has the sequence shown in SEQ ID NO: 9; and a second exponent hybrid 111, which in the illustrated example has the sequence shown in SEQ ID NO: 10. However, it should be understood that different sequences may be used alternatively.

[0244] The detection solution 115 also contains a recycler 112 structurally similar to 108 and 109. Each recycler comprises a first chain 218 and a second chain 219, wherein in the illustrated example, the first chain 218 has the sequence shown in SEQ ID NO: 11, and in the illustrated example, the second chain 219 has the sequence shown in SEQ ID NO: 12. However, it should be understood that different sequences may be used alternatively. Each recycler 112 is labeled with a detection portion in the form of a quencher 220 and a fluorophore 221, which may be attached to the chain opposite to the chain shown in the figure. The detection solution 115 also contains a releaser 113. In the illustrated example, each releaser 113 has the sequence shown in SEQ ID NO: 13. However, it should be understood that different sequences may be used alternatively.

[0245] It should be understood that the nucleic acid sequences of each component 106 to 113 in the detection solution 115 are designed such that they do not react with each other or with themselves in the absence of the target 105, and even so, under conditions commonly referred to as metastability, they only hybridize with their intended sequences.

[0246] Figure 3 Figure 1 Part of the contents of the reaction mixture 116 shown are in Figure 3 Detailed description is provided below. The reaction mixture 116 comprises at least one target 105, which includes a hybridization region a * 301 and hybrid region b * 302. Each detector 106 contains a circular path 201 of hybridization regions in the order labeled a, b, c, d, e, f, c, d, g, and h, wherein hybridization region a is exposed to its complementary sequence a in target 105. * Pairing. Hybridization regions b, d, f, d, and h are paired with their respective complementary sequences b. * d * f * d * and h * Hybridization. Figure 3 Other shapes of the detector 106 that may be naturally present in the reaction mixture 116 are also shown.

[0247] In the solution of reaction mixture 116, mixing occurs through a diffusion process, and the hybridization region a of target 105... * The hybridization region a is provided to the circular path 201 of the detector. Then, the hybridization region b is replaced from the detector 106. * The opening of ring 203 in the first c hybridization region makes this irreversible.

[0248] Figure 4 Figure 1 The additional contents of the reaction mixture 116 shown (caused by providing the target 105 to the detector 106) are in Figure 4 Detailed description is provided below. Target 105 has been replaced by the hybridization region b from the detector 106. * This generates a modified detector 401. This illustrates the first stage of a hybridization chain reaction occurring around the cyclic pathway 201 of detector 106.

[0249] Hybridization region b after substitution * 402 is now exposed and can pair with the detector dissociative ion 107, whose polynucleotide sequence begins with a shortened form of the b base sequence (denoted as 'b'). The full-length sequence containing the hybridization region of the detector dissociative ion 107 is 'b', d, f, d, and h. This can be considered complementary to the chaperone 202 of the modified detector 401.

[0250] The detection of the ion 107 first interacts with b. * Hybridization region 402 is crossbred, and then with d * Hybridization occurs in hybridization region 403, which then opens ring 204, making the process irreversible. A hybridization chain reaction proceeds in a circumferential manner, with the detection dissociation ion completely removing chaperone 202 from detection 106, simultaneously opening hybridization region rings c 205 and g 206, making the removal of chaperone 202 irreversible. However, it should be understood that although the detection dissociation ion can promote the separation of the chaperone from the cyclic pathway, it is not essential for the separation to occur. For example, other oligonucleotides in the reaction mixture can compete with the chaperone for hybridization with the cyclic pathway (see, for example, Example 17).

[0251] Figure 5 The dissociation of detector 106 (which is caused by) Figure 4 The reaction of the components shown causes) in Figure 5 Detailed description is provided below. Target 105 maintains hybridization with a portion of the circular path 201. The circular path 201 now includes an exposed recycling portion 502, which contains the sequences of hybridization region g and hybridization region h.

[0252] Furthermore, reaction mixture 116 contains product 503, which is the result of hybridization of detector dissociation ion 107 with chaperone 202 of modified detector 401. Product 503 contains double-stranded DNA, which is stable at room temperature relative to other components or products of reaction mixture 116.

[0253] Figure 6 Figure 5 The dissociation detector 501 shown, together with the first exponent 108 and the second exponent 109, is in Figure 6Detailed description is provided below. Each first indexer 108 has a hybridization region c. * The single-stranded dangling region 210. It can be observed that the dissociated detector 501 contains two exposed c-hybridization regions 203 and 205. The first indexer 108 is attached to the first exposed c-hybridization region 203, and hybridization continues with the substitution of the d-hybridization region 601, resulting in a * The opening of ring 211 makes the reaction irreversible. However, a * Once hybridization region 211 is opened, it cannot pair with hybridization region e 204, leaving the indexifier 108 partially paired with the dissociated detector 501. The same reaction occurs at two positions 203 and 205 on the dissociated detector 501, exhausting two type I indexifiers 108.

[0254] Starting at the e-hybridization region 204 on the hybridization detector 501, a similar reaction occurs between the second type of indexers 109, where the e-hybridization region 204 interacts with the e-indexers 109. * Complementary sequence 602 pairing.

[0255] The two types of indexers 108 and 109 contain detection portions in the form of fluorophores 213 and 217 and quenchers 212 and 216, respectively. The fluorophores and their respective quenchers remain intact, ensuring that fluorescence above the background level does not occur at this stage.

[0256] Figure 7 Figure 6 The reaction results of the dissociation detector 501 shown are compared with those of other components. Figure 7 Detailed description is provided below. The modified dissociated detector 701 now has indexers 108 and 109 attached thereto. The reaction mixture 116 contains a first indexer hybrid 110 and a second indexer hybrid 111. The first indexer hybrid 110 contains d * The shortened variant of the hybridization region (denoted as 'd*) preferentially hybridizes with the d hybridization region exposed on the first exponentiation 108 now suspended on the modified detector 701. Similarly, the second exponentiation hybrid 111 contains 'f'. * The hybridization region preferentially pairs with the f-hybridization region now exposed on the second indexer 109. These reactions outweigh other reactions that may occur at this stage, resulting in the activation of indexers 108 and 109 attached to the modified dissociated detector 701.

[0257] The reaction between the indexed hybrids 110, 111 and their respective indexed hybrids 108, 109 results in the activation of the chains of indexed hybrids 108, 109, which optionally become proliferators.

[0258] Figure 8 Figure 7 The activation results of the attached exponents 108 and 109 shown are in Figure 8 Detailed description is provided below. The now activated detector 801 has activated proliferators 802, 803, and 804 attached to it. Proliferators 802, 803, and 804 are single-stranded polynucleotides, each optionally containing α... * and b * Hybridization regions. These can function as target 105 in subsequent detection iterations.

[0259] Furthermore, proliferators 802, 803, and 804 retain quenchers 212 and 216, while fluorophores 213 and 217 attach to double-stranded products 805, 806, and 807. The separation of fluorophores 213 and 217 from their respective quenchers 212 and 216 causes them to emit fluorescence 118 in mixture 116.

[0260] Figure 9 In addition to recycler 112, which is one of the two components 112 and 113 that promote target recycling, Figure 8 The activated detector 801 shown in Figure 9 The process is described in further detail. Recycler 112 is provided in reaction mixture 116 for reacting with the exposed recycling portion 502 of the cyclic path 201 of the activated detector 801 or the dissociated detector 501. Recycler 112 optionally contains a starting sequence a. * b * 901. Recycled component 112 also contains a single-chain g. * Hybridization region 902, which can bind to its complementary sequence g in the exposed recycling portion 502. The recycler h... * Hybridization region 903 also hybridizes with its complementary sequence h in the exposed recycling section 502. This is achieved by opening the a in the recycling section 112. * The reaction becomes irreversible when the ring is removed. However, this is only possible by removing the b hybridization region of recycler 112. * Before free hybridization occurs in the hybridization region, the hybridization of recirculator 112 with activated detector 801 is unlikely to proceed significantly.

[0261] It should be understood that recycler 112 can be located in the cyclic pathway 201 of the hybridization region, as shown in the example. Figure 5The recycler 112 may bind to the exposed recycling portion 502 at any time after being dissociated. For example, the recycler 112 may bind to the cyclic pathway 201 before any of the exponents 108 and 109. This binding may occur in any order and may occur simultaneously in multiple different orders in the reaction mixture 116. It should also be understood that the positions of the hybridization regions on the cyclic pathway with which the exponents and recyclers hybridize may vary.

[0262] Figure 10 The hybridization results of recycle 112 and exposed recycle portion 502 are as follows: Figure 10 The details are described below. At this stage, further changes are made from the release of target 105 from the detector 1001, which is facilitated by the releaser 113.

[0263] Release sub 113 contains part h * The hybridization region sequence is denoted as 'h'. * 1002. The shortened ends minimize the likelihood of erroneous initiation due to DNA respiration at the 3' end of 221 during hybridization with 220. Release 113 preferentially interacts with DNA from... Figure 9 The h chain 1003 of the recycler 112 shown binds. Hybridization continues to replace the b chain 1005 from the remainder of the recycler 112. This causes b... * Hybridization region 1006 dissociates to provide a recycling chain, which will preferentially replace and release target 105 from detector 1001.

[0264] Release child 113's 'h * Part of it is complementary to a portion of the exposed recirculating portion 502 of the circular pathway 201. However, this short sequence may only be present at levels below approximately 16. o Temperature C is stable, and the reaction is carried out at the expected temperature of 25°C. o C may be unstable. Furthermore, longer matching chains will preferentially include, for example, recycle sub-112's g. * h * The sequence is combined with the h sequence, where 'h' * Some hybridizations may occur briefly. This similarly applies to the shortened sequences on the exponent hybrids 110 and 111.

[0265] Figure 11 Figure 10 The hybridization results of recycler 112 and releaser 113 shown are in Figure 11 Detailed description in the text. Dissociation of b * The hybridization region provides a recycling chain 1006, which preferentially replaces the remaining portion of the target 105 that is still hybridized with the circular pathway 201. This will cause the target 105 to be released from the detector 1101. Due to the hybridization region sequence g...* h * a * b * The length and only containing subset a * b * The target 105 is longer than that, and the release is irreversible.

[0266] The result of hybridization of releaser 113 is the separation of recycler quencher 220 from its corresponding fluorophore 221. Secondary product 1102 carries away fluorophore 221, promoting... Figure 1 The fluorescence shown is 118.

[0267] Figure 12 Figure 11 The result of the target 105 being released from the detector 1101 is shown in Figure 12 The target 105 is now free to move freely in the solution of the reaction mixture 116. Furthermore, the detector 1201 is now free to exit the target 105.

[0268] This recycling of target 105 differs from the amplification process. Firstly, the same target 105 can trigger... Figures 3 to 12 The type of multiple hybridization chain reaction shown increases the overall sensitivity of the system. Second, due to the lack of dendritic polymerization, the reaction mixture 116 and its products maintain similar molecular weights, allowing diffusion to continue without reducing the reaction rate. The lack of dendritic polymerization also minimizes steric hindrance. The subsequent sustained diffusion of the reaction products throughout the reaction mixture 116 ensures that the amplification rate is maintained. This contrasts with known HCR methods, where linear or exponential amplification is achieved by generating structures with accumulated molecular weights, reducing the diffusion rate and potentially increasing steric hindrance.

[0269] It should be understood that the amplification shown in the previous figure is exponential, as four initiators, including one target and three proliferators, can be used to initiate the reaction. Furthermore, four fluorophores are released at each stage. Therefore, for a count of n detection iterations, the number of fluorophores released is 4 to the power of (n+1). In some instances, a linear system can be provided where proliferators 802, 803, and 804 are not generated, and detection is performed because the same target 105 can be attached, transformed, and then released from any number of detectors 106; the only limitations are the number of detectors 106 in the detection mixture 116 and the time required for hybridization. In both linear and exponential implementations, this target recycling significantly improves the sensitivity, rate, and reliability of the detection.

[0270] Regardless of the reaction progress or the proportion of detector 106 used in reaction mixture 116, exponential sensitivity to target 105 can be obtained without steric interference because the molecular weights of activated detector 1201 and other reaction products are strictly limited. The unrestricted growth of dendritic polymer chains is a known consequence of exponential HCR methods. In contrast, the lack of this unrestricted increase in dendritic structure allows exponential sensitivity to be maintained longer as the reaction proceeds. It should be understood that the molecular weights of the components in reaction mixture 116 fluctuate with amplification, and the exponential sensitivity will decrease as the concentrations of components 106 to 113 decrease due to amplification.

[0271] Figure 13 The first instance initiated by a proliferator Figure 13 As shown in the figure. The fully hybridized detector 1201 has aprogenitor 803, which acts as Figure 3 The target 105 of the type of detector 106 shown has become, due to the chain reaction initiated by the proliferator 803 acting as the target 105, that detector 106 has become Figure 4 The modified detector 401 of the shown species. Proliferator 803 is hybridized with the modified detector 401. Figure 13 In the process, the two detectors 1201 and 401 are close together. However, when the proliferator 803, which acts as target 105, is released, the two detectors will freely separate, and proliferator 803 will be released to act as target 105 for other detectors 106 again. Such reactions may occur simultaneously for multiple proliferators 802, 803, and 804 of each detector 1201.

[0272] Figure 14 The second instance initiated by a proliferator Figure 14 As shown in the figure. The fully hybridized detector 1201 has aprogenitor 803, which acts as Figure 3 The target 105 of the type of detector 106 shown has become, due to the chain reaction initiated by the proliferator 803 acting as the target 105, that detector 106 has become Figure 5 The dissociated detector 501 is shown. A proliferator 803 hybridizes with the dissociated detector 501. Subsequent release of the proliferator 803, acting as target 105, will cause the two detectors to move away from each other. The proliferator 803 will then be released to again act as target 105 for other detectors 106.

[0273] Figure 15 The detection of multiple stages of different reactions occurring in mixture 116 can occur simultaneously with any other stage. Furthermore, the reactions can occur in various different sequences; for example, the order of generation of proliferators 802, 803, and 804 and / or release of target 105 can differ. Multi-stage reactions occurring simultaneously in the detection mixture, such as... Figure 15 As shown.

[0274] Over time, most of the detectors release each other during the exponential amplification process. Each iteration of the reaction results in the separation of multiple fluorophores from their respective quenchers, increasing the fluorescence 118 of the detection mixture 116.

[0275] By enabling an improved, robust amplification process that can be performed at room temperature, the method of releasing the detector helps to test the target of sample 103.

[0276] In this embodiment, the fluorophore and quencher pair are replaced by a guanine complex, thereby forming a guanine quadruplex on reaction products 805, 806, 807, and 1202. Such a method will be readily implemented by those skilled in the art. Detection of the presence of this reaction product is by no means limited to these and other known methods. In some instances, as a result of detection, reduced nicotinamide adenine dinucleotide (NADH) is oxidized to NAD, thereby enabling monitoring of the amplification results using inexpensive and widely available blood glucose meters.

[0277] Figure 16 exist Figure 1 The reaction mixture 116 shown in the figure is summarized in Figure 16 The recursive flowchart is shown. Hybridization chain reaction 1600 begins in step 1601 by providing a target 105 to the cyclic pathway 201 of the hybridization region in detector 106. In step 1602, the target 105 hybridizes with the cyclic pathway 201. In step 1603, an optional detector dissociation ion 107 is provided to detector 106, which helps remove the chaperone 202 of detector 106.

[0278] Next is the circumferential hybridization 1604. This begins in step 1605 with the exposure of the hybridization region ring c 203. Once the hybridization region ring e 203 is exposed, the exponentiation 108, as... Figure 7 The attachment process is shown. This ultimately leads to step 1607, in which proliferator 802 is activated by its dissociation from exponentiator 108. Subsequently, using proliferator 802 as a target / initiator 105, a recursive reaction 1600 occurs.

[0279] Following step 1605, hybridization region ring e 204 is exposed in step 1608, followed by attachment of exponent 109 in step 1609. In step 1610, proliferator 803 is activated by dissociation from exponent 109, and subsequently, a recursive hybridization chain reaction 1600 occurs using proliferator 803 as target / initiator 105. Following step 1608, hybridization region ring c 205 is exposed in step 1611, followed by attachment of exponent 109 in step 1612. In step 1613, proliferator 804 is activated by its dissociation from exponent 108. Further recursive hybridization chain reactions 1600 can then occur, with proliferator 804 acting as target / initiator 105.

[0280] In the final part of the circumferential hybridization 1604, the recycling portion 502 is exposed in step 1614. In step 1615, the recycling portion 502 hybridizes with the recycling member 112, as from... Figures 9 to 10 The progress is shown. In step 1616, the initiator 105 is released using the releaser 113, as shown from... Figures 10 to 11 and with Figure 12 The progress at the end is shown. In step 1616, target 105 is released so that it can be recycled for use as target 105 in recursive reaction 1600.

[0281] Steps 1605, 1606, and 1607 occur sequentially, as do steps 1605, 1608, 1611, and 1614. However, step 1609 may occur before step 1606 or step 1607. For example, the vertical overlap of steps 1607 and 1616 does not imply simultaneous timing. Step 1607 is not necessary at all. Steps 1608 through 1616 can proceed regardless of whether step 1606 or step 1607 is completed. Similarly, the other steps in the circumferential hybridization 1604 can be performed sequentially. Figure 16 The causal process can occur in any order supported by the causal flow. In other embodiments, other causal flows are also possible. In such an embodiment, target recycling can be facilitated by reaching the target 105 that triggers detection in step 1601 and releasing the target 105 in step 1616 via a circumferential hybridization 1604.

[0282] Figure 17 Figure 1 The target 105 shown is Figure 2 The components 106 to 113 shown for detection are in Figure 17 The following is a detailed description in illustrative form. Target 105 comprises two hybridization regions a having SEQ ID NO: 1. * and b * .exist Figure 17 In the schematic diagram, hybridization region a * and b * Separated by a hyphen. This is merely for readability. The sequence itself is a continuous single strand of nucleic acid bases.

[0283] The detector 106 comprises a circular path 201 having SEQ ID NO: 2 and a hybridization chaperone 202 having SEQ ID NO: 3. The detector 106 is shown schematically. Figure 17 In this configuration, the rapid identification of sequences and their complementary sequences is facilitated, which is useful for understanding their role in detection. This includes displaying a circular path 201 and a companion 202 with large dashes between its constituent hybridization regions, aligning these hybridization regions with their complementary sequences for easier understanding. The circular path 201 can be constructed as follows: Figure 17 The linear form shown is optionally covalently closed by connecting its two ends, as part of the preparation of the detection molecule 106. Cycloning can be performed according to the procedure described in detail in "The C-CircleAssay for alternative-lengthening-of-telomeres activity" published in Methods (Vol. 114) in 2016.

[0284] The detector dissociation ion 107 has SEQ ID NO: 4. The first exponentiation ion 108 comprises two DNA strands having SEQ ID NO: 5 and SEQ ID NO: 6. The quencher molecule is not shown, and suitable amplification detection methods using fluorophores or other mechanisms can be readily understood by those skilled in the art.

[0285] The second exponent 109 has a first strand of SEQ ID NO: 7 and a second strand of SEQ ID NO: 8. The first exponent hybrid 110 has SEQ ID NO: 9 and the second exponent hybrid 111 has SEQ ID NO: 10. The recycling unit 112 comprises a first strand of SEQ ID NO: 11 and a second strand of SEQ ID NO: 12. The release unit 113 has SEQ ID NO: 13.

[0286] Figure 2 As shown and Figure 17The components described in detail herein can be prepared using methods known in the art, or they can be purchased from commercial suppliers such as Integrated DNA Technologies Pte. Ltd., 41 SciencePark Road, #01-28 The Gemini, Singapore Science Park II, Singapore 117610, Republic of Singapore.

[0287] In some instances, deliberate mismatches in the hybridization region can be used to optimize hybridization and release of reactants. In some instances, target 105 is double-stranded, and the peptide nucleic acid is used for detector 106, allowing direct detection of double-stranded DNA or double-stranded RNA without conversion to single-stranded form. In such embodiments, a triple-stranded helix is ​​formed in detector 106, while other aspects are as follows. Figure 16 The procedure is shown. In the implementation scheme, other types of nucleic acids are used for detection.

[0288] Target and detection section The methods and compositions described herein can be used to detect targets, preferably nucleic acids, such as RNA or DNA. A target can indicate the presence of a specific substance (e.g., a virus or microorganism) in a sample. In this case, the methods described herein can detect viral or microbial nucleic acids. In some instances, the target indicates a disease or condition, such as a specific type of cancer. In some instances, the target is fetal nucleic acid present in the blood of a pregnant woman.

[0289] This disclosure provides a polynucleotide linked to a detection moiety. The polynucleotide may be indirectly linked to the detection moiety, or it may be directly labeled with the detection moiety.

[0290] In some instances, the methods described herein are used to detect analytes bound to nucleic acids. In such instances, the nucleic acid itself can be the target, capable of hybridizing with a circular pathway after binding to the analyte, or it can be a nucleic acid modified after binding to the analyte such that the target can hybridize with a circular pathway. In such instances, the detection of the target indicates the presence of the analyte in the sample. In such instances, the nucleic acid can be an aptamer or aptamyme with specific affinity for the analyte. The methods described herein can be used, for example, to detect analytes, ions, proteins, or peptides.

[0291] In some instances, the target may contain oligonucleotide pairs (such as a retractor), which form a complex in the presence of the nucleic acid of interest. In such instances, the retractor can hybridize with the circular pathway only in the presence of the nucleic acid of interest. For example, the nucleic acid of interest may be a pathogenic nucleic acid containing sequences complementary to the two components of the retractor. Hybridization of the pathogenic nucleic acid with the retractor aggregates the components, resulting in the formation of a hybridization region in the retractor that can hybridize with the initiation portion of the circular pathway. In such instances, the retractor is designed to hybridize with a specific nucleic acid of interest, while the remaining reaction components (e.g., detectors, indexers, etc.) can be generic for detecting any nucleic acid of interest.

[0292] A detectable signal can indicate the presence of a target. The term "detectable signal" refers to a signal generated by a detection portion attached to or otherwise associated with the nucleic acid (e.g., an indexer or recycler) of the present invention, typically generated when the nucleic acid is modified to alter its conformation, structure, orientation, position relative to other entities, etc. Modification can be induced, for example, by the presence of a target that causes a structural or conformational change in the nucleic acid attached to the detection portion. Non-limiting examples of such modification include the separation of the double-stranded portion of the nucleic acid. The detectable signal can be detected by a variety of methods, including fluorescence spectroscopy, surface plasmon resonance (SPR), mass spectrometry, NMR, electron spin resonance, colorimetry, blood glucose meter readings, polarized fluorescence spectroscopy, circular dichroism, immunoassay, chromatography, radiometry, photometry, scintillation scanning, electronic methods, electrochemical methods, UV, visible or infrared spectroscopy, enzymatic methods, or any combination thereof. The detection portion can be a fluorophore or a quencher. Quenchers include any molecule that, when in close proximity to a fluorophore, absorbs the emission energy generated by the fluorophore and dissipates the energy as heat or emits light with a wavelength longer than that of the fluorophore. Non-limiting examples of quenchers include Dabcyl, TAMRA, graphene, the FRET fluorophore, ZEN quencher, ATTO quencher, black hole quencher (BHQ), Iowa Black Dark quencher, and blackberry quencher (BBQ). Therefore, the spatial separation of the fluorophore from the quencher allows the fluorophore to emit a detectable signal in the form of fluorescence.

[0293] This disclosure provides an indexing nucleotide comprising a hybridized first-strand polynucleotide and a hybridized second-strand polynucleotide, the first strand extending beyond the second strand to form a single-stranded overhang and comprising at least one loop that does not hybridize with the second strand, wherein each strand is labeled with a detection portion. In some instances, the detection portion is a fluorophore and a quencher pair. For example, the first strand of the indexing nucleotide may be labeled with a fluorophore, and the second strand of the indexing nucleotide may be labeled with a quencher. In other instances, the first strand of the indexing nucleotide may be labeled with a quencher, and the second strand of the indexing nucleotide may be labeled with a fluorophore. Separation of the first and second strands allows the fluorophore to emit a detectable signal.

[0294] This disclosure also provides a recycler comprising a hybridized first-strand polynucleotide and a hybridized second-strand polynucleotide, the first strand extending beyond the second strand to form a single-stranded portion and comprising at least one loop that does not hybridize with the second strand, wherein each strand is labeled with a detection portion. In some instances, the first strand of the recycler may be labeled with a fluorophore, and the second strand of the recycler may be labeled with a quencher. In other instances, the first strand of the recycler may be labeled with a quencher, and the second strand of the recycler may be labeled with a fluorophore.

[0295] Other suitable detection components for use in this disclosure may include nanoparticles for colorimetric or SPR detection, reaction components for chemiluminescent detection (e.g., alkaline phosphatase or peroxidase), or electroactive substances for electrochemical detection (e.g., ferrocene, methylene blue, or peroxidase). For example: gold or silver nanoparticles on both strands of a nucleic acid for colorimetric detection; immobilizing one strand to a gold surface and gold nanoparticles on the opposing strand for SPR detection; and immobilizing one strand to an electrode surface and methylene blue molecules on the opposing strand for electrochemical detection.

[0296] Diagnostic uses The compositions and methods described herein can be used for diagnostic or prognostic methods. Diagnostic and / or prognostic methods can be performed in vitro or ex vivo.

[0297] In some instances, the methods described herein can be used to diagnose infections in subjects. For example, the methods can be used to diagnose bacterial, viral, fungal / yeast, protozoan, and / or nematode infections in subjects. In some instances, the virus is a coronavirus. Subjects can be humans or non-human animals.

[0298] The methods disclosed herein can be performed on a sample. The sample can be derived from any source. For example, the sample can be obtained from environmental sources, food sources, industrial sources, or through chemical synthesis. In some instances, the sample is a biological sample. Biological samples can be taken from a subject. Stored biological samples can also be used. Non-limiting examples of suitable biological samples include whole blood or its components (e.g., blood cells, plasma, serum), urine, cervical-vaginal mucus, feces, saliva, lymph, bile, sputum, tears, cerebrospinal fluid, bronchoalveolar lavage fluid, synovial fluid, semen, ascites tumor fluid, breast milk, and pus.

[0299] Reagent test kit This disclosure also provides a kit containing one or more reagents for carrying out the methods of the present invention. Typically, a kit for carrying out the methods of the present invention contains all the reagents required to carry out the method.

[0300] In some instances, the kit contains: - Circular pathways in polynucleotide hybridization regions; - A polynucleotide chaperone that is capable of hybridizing with a circular pathway, such that the circular pathway contains one or more loops that do not hybridize with the chaperone and an exposed single-stranded initiating polynucleotide hybridization region that does not hybridize with the chaperone, wherein the initiating hybridization region is complementary to one of the target hybridization regions; - Detection of a dissociative ion containing multiple polynucleotide hybridization regions capable of hybridizing with complementary hybridization regions on a chaperone; - A first indexer comprising a first-strand polynucleotide and a second-strand polynucleotide hybridized through at least two complementary hybridization regions, the first strand extending beyond the second strand to form a single-stranded overhang and comprising at least one loop that does not hybridize with the second strand, wherein each strand is labeled with a detection portion; - A second indexer comprising a first-strand polynucleotide and a second-strand polynucleotide hybridized through at least two complementary hybridization regions, the first strand extending beyond the second strand to form a single-stranded overhang and comprising at least one loop that does not hybridize with the second strand, wherein each strand is labeled with a detection portion; - A first exponent hybrid, comprising at least two polynucleotide hybridization regions capable of hybridizing with the second strand of the first exponent hybrid; - A second exponent hybrid, comprising at least two polynucleotide hybridization regions capable of hybridizing with the second strand of the second exponent hybrid; - A recycler comprising a first-strand polynucleotide and a second-strand polynucleotide hybridized via at least two complementary hybridization regions, the first strand extending beyond the second strand to form a single-stranded portion and comprising at least one loop not hybridizing with the second strand, wherein each strand is labeled with a detection portion; and / or - A releaser that contains at least two polynucleotide hybridization regions capable of hybridizing with the second strand of a recirculator.

[0301] Typically, the kits of this invention will also contain other reagents that can be used to perform the methods described herein. Other reagents may include ligases, nucleases, reaction buffers, storage buffers, and / or lysis buffers.

[0302] The kit can be either aliquot kits or combination kits as described herein. Aliquot kits contain reagents housed in individual containers and may include small glass containers, plastic containers, or plastic or paper strips. Such containers allow for efficient transfer of reagents from one compartment to another while avoiding cross-contamination of samples and reagents, and quantitatively add reagents or solutions from one compartment to another. Combination kits contain all components of the reaction assay in a single container (e.g., in a single cartridge containing each desired component).

[0303] The kit of the present invention may also include instructions for using the kit components in a suitable manner. The kit and method of the present invention can be used in conjunction with automated analytical equipment and systems.

[0304] This disclosure includes items and entries. The following describes the non-restrictive items and entries of this disclosure.

[0305] Project 1. A method for detecting nucleic acid targets in a sample, the method comprising: (a) Contact the sample with the reaction mixture, the reaction mixture comprising: - A detector comprising a circular pathway containing a polynucleotide hybridization region that hybridizes with a polynucleotide chaperone, the circular pathway comprising multiple loops that do not hybridize with the chaperone and an exposed single-stranded initiation hybridization region that does not hybridize with the chaperone, wherein the initiation hybridization region is complementary to the hybridization region of the target, and - One or more polynucleotides, wherein at least one of the polynucleotides is linked to the detection portion; (b) Hybridize the target with the initial hybridization region; (c) Separate the mate from the circumferential pathway, thereby exposing multiple hybridization regions on the circumferential pathway; (d) In a circumferential hybridization reaction, one or more polynucleotides are hybridized to multiple exposed hybridization regions along a circumferential pathway, wherein the circumferential hybridization reaction induces modification of at least one polynucleotide linked to a detection moiety, such that the detection moiety generates a detectable signal; and (e) Measure the presence or absence of a detectable signal, wherein the detectable signal indicates the presence of a target in the sample.

[0306] Project 2. The method of Project 1, wherein the detection component is a fluorophore, which emits a detectable fluorescent signal after being spatially separated from the quencher.

[0307] Project 3. The method of Project 1 or Project 2, wherein the at least one polynucleotide is linked to a fluorophore and a quencher, and wherein the modification includes spatial separation of the fluorophore from the quencher, such that the fluorophore emits a detectable signal.

[0308] Project 4. The method of any one of Projects 1 to 3, wherein the at least one polynucleotide is linked to a fluorophore and a quencher and comprises a double-stranded region, and wherein the strand of the double-stranded region dissociates after the at least one polynucleotide hybridizes with a circular pathway, such that the fluorophore is spatially separated from the quencher and emits a detectable signal.

[0309] Project 5. The method of any one of Projects 1 to 4, wherein the one or more polynucleotides comprises a plurality of polynucleotides, each polynucleotide being linked to a detection portion.

[0310] Project 6. The method of Project 5, wherein each of the plurality of polynucleotides is linked to a fluorophore and a quencher and comprises a double-stranded region, and wherein the strand of each double-stranded region is dissociated after hybridization with a circular pathway, such that each fluorophore is spatially separated from the quencher and emits a detectable signal.

[0311] Item 7. The method of any one of Items 1 to 6, wherein the separation of the companion from the circumferential path is facilitated by opening a loop in the circumferential path.

[0312] Project 8. The method of any one of Projects 1 to 7, wherein a circumferential hybridization reaction separates the target from the circumferential pathway.

[0313] Project 9. The method of any one of Projects 1 to 8, wherein the hybridization of the target and the initiating hybridization region replaces the hybridization region of the mate via a circumferential pathway.

[0314] Project 10. The method of Project 9, wherein the reaction mixture further comprises a detector dissociation ion, said detector dissociation ion comprising a plurality of polynucleotide hybridization regions that hybridize with the chaperone, thereby promoting the separation of the chaperone from the circular pathway.

[0315] Project 11. The method of Project 10, wherein at least a portion of the substituted hybridization region of the chaperone hybridizes with a hybridization region on the detection dissociation ion, and multiple hybridization regions on the detection dissociation ion hybridize with hybridization regions on the chaperone, thereby promoting the separation of the chaperone from the cyclic pathway.

[0316] Project 12. The method of any one of Projects 1 to 11, wherein at least one of the polynucleotides is an indexer, said indexer comprising a double-stranded portion of a complementary hybridization region pair and a single-stranded overhang extending beyond the double-stranded portion, and wherein said circumferential hybridization reaction comprises: - Hybridization between overhangs and hybridization zones on circumferential paths; and - At least one hybridization region adjacent to the overhang hybridizes with a complementary hybridization region on the circumferential path, thereby at least partially opening the double-stranded portion.

[0317] Project 13. The method of Project 12, wherein the indexer comprises a first-strand polynucleotide and a second-strand polynucleotide hybridized through at least two complementary hybridization regions, the first strand extending beyond the second strand to form a dangling overhang and comprising at least one loop that does not hybridize with the second strand, and wherein said circumferential hybridization reaction comprises: - Hybridization between overhangs and hybridization zones on circumferential paths; and - At least one hybridization region on the first chain adjacent to the overhang hybridization region on the loop path hybridizes with the hybridization region on the loop path, thereby displacing the hybridization region on the second chain of the exponent and opening the loop on the first chain of the exponent.

[0318] Project 14. The method of Project 13, wherein the flanking sides of the ring of the indexer are complementary hybridization pairs, which hybridize the first strand of the indexer with the second strand of the indexer.

[0319] Item 15. The method of Item 13 or Item 14, wherein the reaction mixture further comprises an indexintegrator hybridizer, said indexintegrator hybridizer comprising at least two polynucleotide hybridization regions hybridizing with one strand of the indexintegrator, thereby separating the first strand of the indexintegrator from the second strand of the indexintegrator.

[0320] Project 16. The method of Project 15, wherein the circumferential hybridization reaction includes: - Hybridization between overhangs and hybridization zones on the circular path; - At least one hybridization region of a neighboring overhang of the first strand hybridizes with a hybridization region on the loop path, thereby displacing the hybridization region on the second strand of the exponent and opening the loop on the first strand of the exponent; and - The hybridization region of the indexed hybrid hybrid crosses with at least a portion of the substituted hybridization region on the second strand of the indexed hybrid, and another hybridization region of the indexed hybrid hybrid crosses with the hybridization region on the second strand of the indexed hybrid, thereby separating the first strand of the indexed hybrid from the second strand of the indexed hybrid. Optionally, at least one hybridization region of the first strand of the indexed substituent hybridizes with a hybridization region on the cyclic pathway, and at least one hybridization region of the first strand of the indexed substituent does not hybridize with the cyclic pathway.

[0321] Project 17. The method of any one of Projects 13 to 16, wherein the first strand of the indexer contains a hybridization region capable of hybridizing with an initiating hybridization region of another circular path, thereby initiating the amplification of the method.

[0322] Project 18. The method of any one of Projects 13 to 17, wherein the first chain of the indexer includes a first hybridization region capable of hybridizing with an initiating hybridization region on an additional cyclic path and a second hybridization region capable of hybridizing with an adjacent hybridization region on the additional cyclic path, thereby initiating the amplification of the method.

[0323] Project 19. The method of Project 16, wherein at least one hybridization region of the first strand of the indexed molecule that does not hybridize with the circular pathway hybridizes with the initiation hybridization region of another detector, thereby initiating the amplification of the method.

[0324] Project 20. The method of any one of Projects 13 to 19, wherein the first strand of the indexer contains a hybridization region that is identical to or has at least about 90% sequence identity with the hybridization region of the target.

[0325] Project 21. The method of Project 16 or Project 19, wherein at least one hybridization region of the first strand of the indexed substituent that does not hybridize with the circular pathway contains a hybridization region that is identical to or has at least about 90% sequence identity with the hybridization region of the target.

[0326] Item 22. The method of any one of Items 13 to 21, wherein the loop of the first strand of the indexed substituent contains a target hybridization region or a sequence having at least about 90% sequence identity with the target hybridization region.

[0327] Project 23. The method of any one of Projects 13 to 22, wherein each chain of the exponent is connected to the detection portion, and wherein the separation of the first chain and the second chain of the exponent enables the detection portion of the exponent to be spatially separated and generate a detectable signal.

[0328] Item 24. The method of any one of Items 12 to 23, wherein the reaction mixture contains at least two different types of indexants.

[0329] Project 25. The method of Project 24, wherein each type of indexer hybridizes with different hybridization regions on a circular path.

[0330] Item 26. The method of any one of Items 15 to 25, wherein the reaction mixture contains a plurality of indexed hybrids.

[0331] Item 27. A method of any one of Items 1 to 11, wherein one or more polynucleotides comprise: - At least one first indexer comprising a first-strand polynucleotide and a second-strand polynucleotide hybridized through at least two complementary hybridization regions, the first strand extending beyond the second strand to form a single-stranded overhang and comprising at least one loop not hybridizing with the second strand, wherein each strand is connected to the detection portion; and - At least one second exponentiator comprising a first-strand polynucleotide and a second-strand polynucleotide hybridized through at least two complementary hybridization regions, the first strand extending beyond the second strand to form a single-stranded overhang and comprising at least one loop that does not hybridize with the second strand, wherein each strand is connected to the detection portion. And the reaction mixture contains: - The first exponential hybrid, which contains at least two polynucleotide hybridization regions; and - The second exponential hybrid, which contains at least two polynucleotide hybridization regions. And the circumferential hybridization reaction includes: - At least one dangling protrusion of the first indexer hybridizes with a hybridization region on the loop path, and at least one adjacent hybridization region on the first chain of the first indexer hybridizes with a hybridization region on the loop path, thereby displacing the hybridization region on the second chain of the first indexer and opening the loop on the first chain of the first indexer. - At least one dangling protrusion of the second exponent hybridizes with a complementary hybridization region on the loop path, and at least one adjacent hybridization region on the first strand of the second exponent hybridizes with a hybridization region on the loop path, thereby displacing the hybridization region on the second strand of the second exponent and opening the loop on the first strand of the second exponent. - The hybridization region of the first exponent hybridizes with at least a portion of the substituted hybridization region on the second strand of the first exponent, and another hybridization region on the first exponent hybridizes with the hybridization region on the second strand of the first exponent, thereby separating the first strand of the first exponent from the second strand of the first exponent, thus enabling the detection portion of the first exponent to be spatially separated and generate a detectable signal. Optionally, at least one hybridization region of the first strand of the first exponent hybridizes with a hybridization region on the cyclic pathway, and at least one hybridization region on the first strand of the first exponent does not hybridize with the cyclic pathway; and - The hybridization region of the second exponent hybridizes with at least a portion of the substituted hybridization region on the second strand of the second exponent, and another hybridization region on the second exponent hybridizes with the hybridization region on the second strand of the second exponent, thereby separating the first strand and the second strand of the second exponent, thus enabling the detection portion of the second exponent to be spatially separated and generate a detectable signal. Optionally, at least one hybridization region of the first strand of the second exponent hybridizes with a hybridization region on the cyclic pathway, and at least one hybridization region on the first strand of the second exponent hybridizes without hybridizing with the cyclic pathway.

[0332] Project 28. The method of any one of Projects 1 to 27, wherein at least one polynucleotide is a recycler comprising at least two hybridization regions, wherein the hybridization region of the recycler hybridizes with a hybridization region adjacent to the initiating hybridization region on the circular pathway, and the other hybridization region of the recycler hybridizes with the initiating hybridization region, thereby displacing the target from the circular pathway.

[0333] Project 29. The method of Project 28, where the recycle subcontract contains: The double-stranded portion of the complementary hybridization region pair is interrupted by a loop in one strand, the loop containing the hybridization region; and Extending beyond the double-chain portion, And the circumferential hybridization reaction mentioned above includes: - The single-stranded portion hybridizes with the hybridization region on the circular pathway; - At least one hybridization region adjacent to the single-stranded portion hybridizes with a hybridization region adjacent to the initiating hybridization region on the loop path, thereby opening the loop on the recycle; and - The hybridization region of the recycled ring hybridizes with at least a portion of the initiating hybridization region, thereby displacing the target from the ring path.

[0334] Item 30. The method of Item 28 or Item 29, wherein the recycle comprises a first-strand polynucleotide and a second-strand polynucleotide hybridized through at least two complementary hybridization regions, the first strand extending beyond the second strand to form a single-stranded portion and comprising at least one loop that does not hybridize with the second strand, and wherein said circumferential hybridization reaction comprises: - The single-stranded portion hybridizes with the hybridization region on the circular pathway; - The hybridization region of the recycler's adjacent single-stranded portion hybridizes with the hybridization region of the adjacent initiation hybridization region of the cyclic path, thereby opening the loop of the recycler; and - The hybridization region on the open loop of the recirculator hybridizes with at least a portion of the initial hybridization region, thereby displacing the target from the loop path.

[0335] Item 31. The method of Item 30, wherein the reaction mixture comprises a releaser containing at least two polynucleotide hybridization regions that hybridize with one strand of a recycle, thereby separating the first strand of the recycle from the second strand of the recycle.

[0336] Project 32. The method of Project 31, wherein the circumferential hybridization reaction includes: - The single-stranded portion of the recycler hybridizes with the hybridization region on the circular path; - The hybridization region of the adjacent single strand of the recycle hybridizes with the hybridization region of the adjacent starting hybridization region of the loop path, thereby displacing the hybridization region of the second strand of the recycle and opening the loop on the first strand of the recycle. - The hybridization region in the open loop of the recirculator hybridizes with at least a portion of the initial hybridization region, thereby displacing the hybridization region of the target from the loop path; - At least a portion of the substituted hybridization region of the second strand of the recirculator hybridizes with the hybridization region on the releaser, and an adjacent hybridization region on the releaser hybridizes with the hybridization region on the second strand of the recirculator, thereby separating the first strand of the recirculator from the second strand of the recirculator; and - The hybridization region of the first strand of the recycle hybridizes with the initiation hybridization region, thereby separating the target from the circular pathway.

[0337] Item 33. The method of any one of Items 30 to 32, wherein the first strand of the recirculator contains a hybrid region that is identical to or has at least about 90% sequence identity with the hybrid region of the target.

[0338] Item 34. The method of any one of Items 30 to 33, wherein the loop of the first strand of the recirculator contains a target hybridization region or a sequence having at least about 90% sequence identity with the target hybridization region.

[0339] Item 35. The method of any one of Items 30 to 34, wherein each chain of the recirculator is connected to the detection portion, and wherein the separation of the first chain and the second chain of the recirculator enables the detection portion of the recirculator to be spatially separated and generate a detectable signal.

[0340] Project 36. The method of any one of Projects 1 to 35, wherein the cyclic path contains multiple loops, each loop containing a hybridization region.

[0341] Project 37. The method of any one of Projects 12 to 27, wherein the dangling of an indexon hybridizes with a hybridization region present in an open loop of a cyclic path.

[0342] Project 38. The method of any one of Projects 8 to 37, wherein the released target hybridizes with the initiation hybridization region of another detector, thereby initiating the amplification of the method.

[0343] Project 39. A method from any of Projects 1 to 38, wherein the cyclic path contains four loops.

[0344] Project 40. A method from any of Projects 1 to 39, wherein the loop path is a closed loop path.

[0345] Item 41. The method of any one of Items 1 to 40, wherein the circumferential hybridization reaction is carried out under isothermal conditions.

[0346] Item 42. The method of Item 41, wherein the circumferential hybridization reaction is carried out at a temperature of about 20°C to about 25°C.

[0347] Item 43. The method of any one of Items 1 to 42, wherein the reaction mixture does not contain a nuclease.

[0348] Item 44. The method of any one of Items 1 to 43, wherein the reaction mixture does not contain polymerase.

[0349] Item 45. The method of any one of Items 1 to 44, wherein the reaction mixture does not contain an enzyme.

[0350] Project 46. The method of any one of Projects 13 to 45, wherein: (i) The first strand of the indexer is attached to the fluorophore and the second strand of the indexer is attached to the quencher; or (ii) The first strand of the exponent is connected to the quencher and the second strand of the exponent is connected to the fluorophore.

[0351] Project 47. The method of any one of Projects 30 to 46, wherein: (i) The first strand of the recycler is attached to the fluorophore and the second strand of the recycler is attached to the quencher; or (ii) The first strand of the recycler is attached to the quencher and the second strand of the recycler is attached to the fluorophore.

[0352] Item 48. A method for detecting nucleic acid targets in a sample, the method comprising: Contact the sample with the complex, which contains A cyclic polynucleotide that partially hybridizes with a polynucleotide chaperone, wherein the complex contains multiple unhybridized loops and a single-stranded initiation region complementary to a target nucleic acid suspected of being present in the sample; Introducing one or more polynucleotides, wherein at least one of the polynucleotides contains a detection moiety; After the target binds to the initiation region, the chaperone is separated from the cyclic polynucleotide; A circumferential hybridization reaction is performed to hybridize multiple polynucleotides with a loop region, wherein the circumferential hybridization reaction induces modification of at least one polynucleotide to generate a detectable signal; and The detection signal serves as an indication of the presence of the target in the sample.

[0353] Item 49. A composition comprising a detector comprising a circular pathway of a polynucleotide hybridization region that hybridizes with a polynucleotide chaperone, the circular pathway comprising one or more loops that do not hybridize with the chaperone and an exposed initiating hybridization region that does not hybridize with the chaperone, wherein the initiating hybridization region is complementary to the target hybridization region.

[0354] Compositions of Item 50 and Item 49, wherein the exposed initiation hybridization region is single-stranded.

[0355] Item 51. A composition of Item 49 or Item 50, the composition further comprising a detector dissociation ion comprising a plurality of polynucleotide hybridization regions capable of hybridizing with hybridization regions on a chaperone and dissociating the chaperone from the cyclic pathway.

[0356] Item 52. A composition of any one of items 49 to 51, the composition further comprising an indexer comprising a first-strand polynucleotide and a second-strand polynucleotide hybridized through at least two complementary hybridization regions, the first strand extending beyond the second strand to form a single-stranded overhang and comprising at least one loop that does not hybridize with the second strand.

[0357] Item 53. The composition of Item 52, wherein the loop of the first strand of the indexer contains a target hybridization region or a sequence having at least about 90% sequence identity with the target hybridization region.

[0358] Item 54. A composition of Item 52 or Item 53, wherein the flanking sides of the ring of the indexer are complementary hybridization pairs that hybridize the first strand of the indexer with the second strand of the indexer.

[0359] Item 55. A composition of any one of items 52 to 54, wherein each chain of the indexer is connected to the detection portion.

[0360] Item 56. A composition of any one of items 52 to 55, the composition further comprising an indexed hybridon comprising at least two polynucleotide hybridization regions capable of hybridizing with a hybridization region on the second strand of the indexed hybridon.

[0361] Item 57. A composition of any one of items 49 to 56, the composition further comprising a recycler comprising a first-strand polynucleotide and a second-strand polynucleotide hybridized through at least two complementary hybridization regions, the first strand extending beyond the second strand to form a single-stranded portion and comprising at least one loop that does not hybridize with the second strand.

[0362] Item 58. The composition of Item 57, wherein the flanking sides of the loop of the recycler are complementary hybridization pairs, which hybridize the first strand of the recycler with the second strand of the recycler.

[0363] Item 59. The composition of Item 57 or Item 58, wherein the ring of the recycler includes a hybridization region capable of hybridizing with a cyclic pathway.

[0364] Item 60. A composition of any one of Items 57 to 59, wherein the ring of the recycler includes a hybridization region capable of hybridizing with the initiating hybridization region.

[0365] Item 61. A composition of any one of Items 57 to 60, wherein each chain of the recycler is connected to the detection portion.

[0366] Item 62. A composition of any one of items 57 to 61, the composition further comprising a releaser comprising at least two polynucleotide hybridization regions capable of hybridizing with a hybridization region on the second strand of a recycler.

[0367] Item 63. A composition of any one of items 49 to 62, wherein the cyclic path comprises a plurality of rings, each ring comprising a hybridization region.

[0368] Item 64. A composition of any one of Items 52 to 63, wherein the dangling of the indexed one is capable of hybridizing with a hybridization region present in an open ring of a cyclic path.

[0369] Item 65. A composition of any one of items 49 to 64, wherein the cyclic path comprises four rings.

[0370] Item 66. A composition of any one of items 49 to 65, wherein the annular path is a closed annular path.

[0371] Item 67. A composition of any one of items 49 to 66, wherein the composition does not contain a nuclease.

[0372] Item 68. A composition of any one of items 49 to 67, wherein the composition does not contain polymerase.

[0373] Item 69. A composition of any one of items 49 to 68, wherein the composition does not contain an enzyme.

[0374] Item 70. A composition of any one of items 52 to 69, wherein: (i) The first strand of the indexer is attached to the fluorophore and the second strand of the indexer is attached to the quencher; or (ii) The first strand of the exponent is connected to the quencher and the second strand of the exponent is connected to the fluorophore.

[0375] Item 71. A composition of any one of items 57 to 70, wherein: (i) The first strand of the recycler is attached to the fluorophore and the second strand of the recycler is attached to the quencher; or (ii) The first strand of the recycler is attached to the quencher and the second strand of the recycler is attached to the fluorophore.

[0376] Item 1. A method for detecting a nucleic acid target in a sample, the method comprising: Contact the sample with the complex, which contains A cyclic polynucleotide that partially hybridizes with a polynucleotide chaperone, wherein the complex contains multiple unhybridized loops and a single-stranded initiation region complementary to a target nucleic acid suspected of being present in the sample; Introducing one or more polynucleotides, wherein at least one of the polynucleotides contains a detection moiety; After the target binds to the initiation region, the chaperone is separated from the cyclic polynucleotide; A circumferential hybridization reaction is performed to hybridize multiple polynucleotides with a loop region, wherein the circumferential hybridization reaction induces modification of at least one polynucleotide to generate a detectable signal; and The detection signal serves as an indication of the presence of the target in the sample.

[0377] Item 2. The method of Item 1, wherein the detection component is a fluorophore that emits a detectable fluorescent signal after being spatially separated from the quencher.

[0378] Item 3. The method of Item 1 or Item 2, wherein the at least one polynucleotide is linked to a fluorophore and a quencher, and wherein the modification comprises spatial separation of the fluorophore from the quencher, such that the fluorophore emits a detectable signal.

[0379] Item 4. The method of any one of Items 1 to 3, wherein the at least one polynucleotide is linked to a fluorophore and a quencher and comprises a double-stranded region, and wherein the strand of the double-stranded region dissociates after the at least one polynucleotide hybridizes with a circular pathway, such that the fluorophore is spatially separated from the quencher and emits a detectable signal.

[0380] Item 5. The method of any one of Items 1 to 4, wherein the one or more polynucleotides comprises a plurality of polynucleotides, each polynucleotide being linked to a detection portion.

[0381] Item 6. The method of Item 5, wherein each of the plurality of polynucleotides is linked to a fluorophore and a quencher and comprises a double-stranded region, and wherein the strand of each double-stranded region is dissociated after hybridization with a circular pathway, such that each fluorophore is spatially separated from the quencher and emits a detectable signal.

[0382] Item 7. The method of any one of Items 1 to 6, wherein the separation step includes opening the loop region.

[0383] Item 8. The method of any one of items 1 to 7, wherein a circumferential hybridization reaction separates the target from the cyclic polynucleotide.

[0384] Item 9. The method of any of Items 1 to 8, wherein hybridization of the target and the starting region is performed from the hybridization region of the cyclic polynucleotide replacement partner.

[0385] Item 10. The method of Item 9, wherein the complex further comprises a detector dissociation ion, said detector dissociation ion comprising a plurality of polynucleotide hybridization regions that hybridize with the chaperone, thereby facilitating the separation of the chaperone from the cyclic polynucleotide.

[0386] Item 11. The method of Item 10, wherein at least a portion of the substituted hybridization region of the chaperone hybridizes with a hybridization region on the detection dissociation ion, and multiple hybridization regions on the detection dissociation ion hybridize with hybridization regions on the chaperone, thereby facilitating the separation of the chaperone from the cyclic polynucleotide.

[0387] Item 12. The method of any one of items 1 to 11, wherein at least one of the polynucleotides is an indexer, said indexer comprising a double-stranded portion of a complementary hybridization region pair and a single-stranded overhang extending beyond the double-stranded portion, and wherein said circumferential hybridization reaction comprises: - Hybridization between the pendulous process and the hybridization region on the cyclic polynucleotide; and - At least one hybridization region adjacent to the overhang hybridizes with a complementary hybridization region on the cyclic polynucleotide, thereby at least partially opening the double-stranded portion.

[0388] Item 13. The method of Item 12, wherein the indexer comprises a first-strand polynucleotide and a second-strand polynucleotide hybridized through at least two complementary hybridization regions, the first strand extending beyond the second strand to form a dangling overhang and comprising at least one loop that does not hybridize with the second strand, and wherein said circumferential hybridization reaction comprises: - Hybridization between the pendulous process and the hybridization region on the cyclic polynucleotide; and - At least one hybridization region on the first strand adjacent to the dangling protrusion hybridizes with a hybridization region on the cyclic polynucleotide, thereby displacing the hybridization region on the second strand of the indexer and opening the loop on the first strand of the indexer.

[0389] Item 14. The method of Item 13, wherein the flanking sides of the ring of the indexer are complementary hybridization pairs, which hybridize the first strand of the indexer with the second strand of the indexer.

[0390] Item 15. The method of Item 13 or Item 14 further comprises an indexintegrator hybrid, said indexintegrator hybrid having at least two polynucleotide hybridization regions that hybridize with one strand of the indexintegrator, thereby separating the first strand of the indexintegrator from the second strand of the indexintegrator.

[0391] Item 16. The method of Item 15, wherein the circumferential hybridization reaction comprises: - Hybridization between the pendulous process and the hybridization region on the cyclic polynucleotide; - At least one hybridization region of a neighboring overhang of the first strand hybridizes with a hybridization region on a cyclic polynucleotide, thereby displacing the hybridization region on the second strand of the indexer and opening the loop on the first strand of the indexer; and - The hybridization region of the indexed hybrid crosses with at least a portion of the substituted hybridization region on the second strand of the indexed hybrid, and another hybridization region of the indexed hybrid crosses with the hybridization region on the second strand of the indexed hybrid, thereby separating the first strand of the indexed hybrid from the second strand of the indexed hybrid. Optionally, at least one hybridization region of the first strand of the indexed nucleotide hybridizes with a hybridization region on the cyclic polynucleotide, and at least one hybridization region of the first strand of the indexed nucleotide does not hybridize with the cyclic polynucleotide.

[0392] Item 17. The method of any one of Items 13 to 16, wherein the first strand of the indexinte contains a hybridization region capable of hybridizing with the initiation region of another cyclic polynucleotide, thereby initiating the amplification of the method.

[0393] Item 18. The method of any one of Items 13 to 17, wherein the first strand of the indexinteger comprises a first hybridization region capable of hybridizing with a start region on another cyclic polynucleotide and a second hybridization region capable of hybridizing with an adjacent hybridization region on the other cyclic polynucleotide, thereby initiating the amplification of the method.

[0394] Item 19. The method of Item 16, wherein at least one hybridization region of the first strand of an indexed nucleotide that does not hybridize with a cyclic polynucleotide hybridizes with the initiation region of another complex, thereby initiating the amplification of the method.

[0395] Item 20. The method of any one of Items 13 to 19, wherein the first strand of the indexer contains a hybridization region that is identical to or has at least about 90% sequence identity with the hybridization region of the target.

[0396] Item 21. The method of Item 16 or Item 19, wherein at least one hybridization region of the first strand of the indexed nucleotide that does not hybridize with a cyclic polynucleotide contains a hybridization region that is identical to or has at least about 90% sequence identity with the hybridization region of the target.

[0397] Item 22. The method of any one of Items 13 to 21, wherein the loop of the first strand of the indexed substituent contains a target hybridization region or a sequence having at least about 90% sequence identity with the target hybridization region.

[0398] Item 23. The method of any one of items 13 to 22, wherein each chain of the exponent is connected to the detection portion, and wherein the separation of the first chain and the second chain of the exponent enables the detection portion of the exponent to be spatially separated and generate a detectable signal.

[0399] Item 24. The method of any one of Items 12 to 23, wherein the reaction mixture contains at least two different types of indexants.

[0400] Item 25. The method of Item 24, wherein each type of indexation hybridizes with a different hybridization region on a cyclic polynucleotide.

[0401] Item 26. The method of any one of Items 15 to 25, wherein the reaction mixture comprises a plurality of indexed hybrids.

[0402] Item 27. The method of any one of items 1 to 11, wherein one or more polynucleotides comprise: - At least one first indexer comprising a first-strand polynucleotide and a second-strand polynucleotide hybridized through at least two complementary hybridization regions, the first strand extending beyond the second strand to form a single-stranded overhang and comprising at least one loop not hybridizing with the second strand, wherein each strand is connected to the detection portion; and - At least one second exponentiator comprising a first-strand polynucleotide and a second-strand polynucleotide hybridized through at least two complementary hybridization regions, the first strand extending beyond the second strand to form a single-stranded overhang and comprising at least one loop that does not hybridize with the second strand, wherein each strand is connected to the detection portion. And the reactants include: - The first exponential hybrid, which contains at least two polynucleotide hybridization regions; and - The second exponential hybrid, which contains at least two polynucleotide hybridization regions. And the circumferential hybridization reaction includes: - At least one dangling protrusion of the first indexer hybridizes with a hybridization region on a cyclic polynucleotide, and at least one adjacent hybridization region on the first strand of the first indexer hybridizes with a hybridization region on a cyclic polynucleotide, thereby displacing the hybridization region on the second strand of the first indexer and opening the loop on the first strand of the first indexer. - At least one dangling protrusion of the second indexer hybridizes with a complementary hybridization region on a cyclic polynucleotide, and at least one adjacent hybridization region on the first strand of the second indexer hybridizes with a hybridization region on a cyclic polynucleotide, thereby displacing the hybridization region on the second strand of the second indexer and opening the loop on the first strand of the second indexer. - The hybridization region of the first exponent hybridizes with at least a portion of the substituted hybridization region on the second strand of the first exponent, and another hybridization region on the first exponent hybridizes with the hybridization region on the second strand of the first exponent, thereby separating the first strand of the first exponent from the second strand of the first exponent, thus enabling the detection portion of the first exponent to be spatially separated and generate a detectable signal. Optionally, at least one hybridization region of the first strand of the first exponent hybridizes with a hybridization region on the cyclic polynucleotide, and at least one hybridization region on the first strand of the first exponent does not hybridize with the cyclic polynucleotide; and - The hybridization region of the second exponent hybridizes with at least a portion of the substituted hybridization region on the second strand of the second exponent, and another hybridization region on the second exponent hybridizes with the hybridization region on the second strand of the second exponent, thereby separating the first strand and the second strand of the second exponent, thus enabling the detection portion of the second exponent to be spatially separated and generate a detectable signal. Optionally, at least one hybridization region of the first strand of the second exponent hybridizes with a hybridization region on the cyclic polynucleotide, and at least one hybridization region on the first strand of the second exponent does not hybridize with the cyclic polynucleotide.

[0403] Item 28. The method of any one of items 1 to 27, wherein at least one polynucleotide is a recirculator comprising at least two hybridization regions, wherein the hybridization region of the recirculator hybridizes with a hybridization region adjacent to the initiation region on the cyclic polynucleotide, and the other hybridization region of the recirculator hybridizes with the initiation region, thereby replacing the target from the cyclic polynucleotide.

[0404] Item 29. The method of Item 28, wherein the recycle subcontains: The double-stranded portion of the complementary hybridization region pair is interrupted by a loop in one strand, the loop containing the hybridization region; and Extending beyond the double-chain portion, And the circumferential hybridization reaction mentioned above includes: - The single-stranded portion hybridizes with the hybridization region on the cyclic polynucleotide; - At least one hybridization region adjacent to the single-stranded portion hybridizes with a hybridization region adjacent to the initiation region on the cyclic polynucleotide, thereby opening the loop of the recirculator; and - The hybridization region of the recycled nucleotide hybridizes with at least a portion of the starting region, thereby replacing the target from the cyclic polynucleotide.

[0405] Item 30. The method of Item 28 or Item 29, wherein the recycle comprises a first-strand polynucleotide and a second-strand polynucleotide hybridized through at least two complementary hybridization regions, the first strand extending beyond the second strand to form a single-stranded portion and comprising at least one loop that does not hybridize with the second strand, and wherein said circumferential hybridization reaction comprises: - The single-stranded portion hybridizes with the hybridization region on the cyclic polynucleotide; - The hybridization region of the recirculator adjacent to the single-stranded portion hybridizes with the hybridization region of the cyclic polynucleotide adjacent to the initiation region, thereby opening the ring of the recirculator; and - The hybridization region on the opened loop of the recirculator hybridizes with at least a portion of the starting region, thereby replacing the target from the cyclic polynucleotide.

[0406] Item 31. The method of Item 30, wherein the reaction mixture comprises a releaser containing at least two polynucleotide hybridization regions that hybridize with one strand of the recycle, thereby separating the first strand of the recycle from the second strand of the recycle.

[0407] Item 32. The method of Item 31, wherein the circumferential hybridization reaction comprises: - The single-stranded portion of the recyclon hybridizes with the hybridization region on the cyclic polynucleotide; - The hybridization region of the adjacent single-stranded portion of the recycle hybridizes with the hybridization region of the adjacent initiation region of the cyclic polynucleotide, thereby displacing the hybridization region of the second strand of the recycle and opening the loop on the first strand of the recycle. - The hybridization region in the opened loop of the recirculator hybridizes with at least a portion of the starting region, thereby replacing the hybridization region of the target from the cyclic polynucleotide; - At least a portion of the substituted hybridization region of the second strand of the recirculator hybridizes with the hybridization region on the releaser, and an adjacent hybridization region on the releaser hybridizes with the hybridization region on the second strand of the recirculator, thereby separating the first strand of the recirculator from the second strand of the recirculator; and - The hybridization region of the first strand of the recycle hybridizes with the initiation region, thereby separating the target from the cyclic polynucleotide.

[0408] Item 33. The method of any one of items 30 to 32, wherein the first strand of the recirculator contains a hybrid region that is identical to or has at least about 90% sequence identity with the hybrid region of the target.

[0409] Item 34. The method of any one of items 30 to 33, wherein the loop of the first strand of the recirculator contains a target hybridization region or a sequence having at least about 90% sequence identity with the target hybridization region.

[0410] Item 35. The method of any one of items 30 to 34, wherein each chain of the recirculator is connected to the detection portion, and wherein the separation of the first chain and the second chain of the recirculator enables the detection portion of the recirculator to be spatially separated and generate a detectable signal.

[0411] Item 36. The method of any one of items 1 to 35, wherein the cyclic polynucleotide comprises a plurality of rings, each ring comprising a hybridization region.

[0412] Item 37. The method of any one of items 12 to 27, wherein the dangling of an indexinon hybridizes with a hybridization region present in the open ring of a cyclic polynucleotide.

[0413] Item 38. The method of any one of items 8 to 37, wherein the released target hybridizes with the initiation region of another complex, thereby initiating the amplification of the method.

[0414] Item 39. The method of any one of items 1 to 38, wherein the cyclic polynucleotide comprises four rings.

[0415] Item 40. The method of any one of items 1 to 39, wherein the cyclic polynucleotide is a closed cyclic polynucleotide.

[0416] Item 41. The method of any one of items 1 to 40, wherein the circumferential hybridization reaction is carried out under isothermal conditions.

[0417] Item 42. The method of Item 41, wherein the circumferential hybridization reaction is carried out at a temperature of about 20°C to about 25°C.

[0418] Item 43. The method of any one of items 1 to 42, wherein the reactants do not contain nucleases.

[0419] Item 44. The method of any one of items 1 to 43, wherein the reactants do not contain polymerase.

[0420] Item 45. The method of any of items 1 to 44, wherein the reactants do not contain enzymes.

[0421] Item 46. The method of any one of items 13 to 45, wherein: (i) The first strand of the indexer is attached to the fluorophore and the second strand of the indexer is attached to the quencher; or (ii) The first strand of the exponent is connected to the quencher and the second strand of the exponent is connected to the fluorophore.

[0422] Item 47. The method of any one of items 30 to 46, wherein: (i) The first strand of the recycler is attached to the fluorophore and the second strand of the recycler is attached to the quencher; or (ii) The first strand of the recycler is attached to the quencher and the second strand of the recycler is attached to the fluorophore.

[0423] sequence The sequences involved in this disclosure are listed in Table 1.

[0424] Table 1

[0425]

[0426]

[0427]

[0428]

[0429]

[0430]

[0431]

[0432]

[0433] Example Example 1: Open Loop Path The lyophilized polynucleotides listed in Table 1 were purchased from IDT, dissolved in TE (10 mM tris(hydroxymethyl)aminomethane-HCl, 0.1 mM disodium ethylenediaminetetraacetate; pH 7.6) at a concentration of 100 µM, and stored at -80 °C; working aliquots were stored at -25 °C. ° C.

[0434] Detection complex The double-stranded detector molecule was constructed by hybridizing two oligonucleotides, a circular pathway (SEQ ID NO: 2) and a chaperone (SEQ ID NO: 3). The circular pathway and chaperone were added to 100 µL of hybridization buffer (0.3 M sodium chloride, 0.03 M sodium citrate, 10 mM MgCl2; pH 7) at final concentrations of 1 µM and 2 µM, respectively. The mixture was heated to 94°C for 2 min to remove secondary structures and then hybridized at 25°C for 60 min. A detector dissociation ion (SEQ ID NO: 4) (which had been heated to 70°C for 2 min to remove secondary structures) was added to a final concentration of 2.5 µM, and the mixture was incubated at 25°C for 120 min to remove excess / unhybridized chaperone and provide free detector dissociation ions to the detector complex. The final detector complex was stored at -25°C. ° C.

[0435] First exponentiation, second exponentiation, and recirculator The first strand (SEQ ID NO: 5) and the second strand (SEQ ID NO: 6) of the first indexer were added to 100 µL of hybridization buffer (0.3 M sodium chloride, 0.03 M sodium citrate, 10 mM MgCl2; pH 7) at final concentrations of 1 µM and 2 µM, respectively. The mixture was heated to 94°C for 2 min to remove secondary structures and then hybridized at 25°C for 60 min. The first indexer hybrid (SEQ ID NO: 7) (which had been heated to 70°C for 2 min to remove secondary structures) was added to a final concentration of 2.5 µM, and the mixture was incubated at 25°C for 120 min to remove excess / unhybridized second strands of the first indexer and to provide free first indexer hybrids to the first indexer. The final first indexer complex was stored at -25°C. ° C. The second indexer complex, the second indexer hybridon, the recirculator complex, and the releaser are generated in a similar manner.

[0436] Exponential target recycling hybridization chain reaction The detecton, first indexer, second indexer, and recycling complex were warmed to 22°C. The target (SEQ ID NO: 1) was diluted in TE buffer and kept separate from other reagents until use. The target was incubated at 70°C for 2 minutes to remove secondary structures and then cooled to 22°C before use. The following substances were added to a new tube: 12 µL of the first indexer complex, 6 µL of the second indexer complex, and 6 µL of the recycling complex. The solutions were then mixed and centrifuged, and 3 µL of the detecton complex was added, followed by 3 µL of the target. The solutions were then mixed and centrifuged, incubated at 22–25°C, and fluorescence was measured on a fluorometer (FLUOstar Omega Plate Reader; BMG Labtech). The final concentrations of the circular pathway (Sequence 1), the first strand of the first indexer (Sequence 4), the first strand of the second indexer (Sequence 7), and the first strand of the recycling complex (Sequence 10) were 100 nM, 400 nM, 200 nM, and 200 nM, respectively. The result is subtracted from the background of the voids, and then normalized relative to the background of the voids. Figure 18 ).

[0437] Example 2: Open circular pathway, adding a target before detecting the subcomplex. The lyophilized polynucleotides listed in Table 1 were purchased from IDT, dissolved in TE (10 mM tris(hydroxymethyl)aminomethane-HCl, 0.1 mM disodium ethylenediaminetetraacetate; pH 7.6) at a concentration of 100 µM, and stored at -80 °C; working aliquots were stored at -25 °C. ° C.

[0438] Detection complex The cyclic pathway (SEQ ID NO: 2) and chaperone (SEQ ID NO: 3) were added to 100 µL of hybridization buffer (0.3 M sodium chloride, 0.03 M sodium citrate, 10 mM MgCl2; pH 7) at final concentrations of 1 µM and 2 µM, respectively. The mixture was heated to 94°C for 2 min to remove secondary structures and then hybridized at 25°C for 60 min. The detecton dissociation ion (SEQ ID NO: 4) (which had been heated to 70°C for 2 min to remove secondary structures) was added to a final concentration of 2.5 µM, and the mixture was incubated at 25°C for 120 min to remove excess / unhybridized chaperone and provide the complex with free detecton dissociation ions. The final detecton complex was stored at -25°C. ° C.

[0439] First exponentiation, second exponentiation, and recirculator The first strand (SEQ ID NO: 5) and the second strand (SEQ ID NO: 6) of the first indexer were added to 100 µL of hybridization buffer (0.3 M sodium chloride, 0.03 M sodium citrate, 10 mM MgCl2; pH 7) at final concentrations of 1 µM and 2 µM, respectively. The mixture was heated to 94°C for 2 min to remove secondary structures and then hybridized at 25°C for 60 min. The first indexer hybrid (SEQ ID NO: 7) (which had been heated to 70°C for 2 min to remove secondary structures) was added to a final concentration of 2.5 µM, and the mixture was incubated at 25°C for 120 min to remove excess / unhybridized second strands of the first indexer and to provide free first indexer hybrids to the first indexer complex. The final first indexer complex was stored at -25°C. ° C. The second indexer complex, the second indexer hybridon, the recirculator complex, and the releaser are generated in a similar manner.

[0440] Exponential target recycling hybridization chain reaction The complex of the detector, first indexer, second indexer, and recycler was warmed to 23°C. The target (SEQ ID NO: 1) was diluted in TE buffer and kept separate from other reagents until use. The target was incubated at 70°C for 2 minutes to remove secondary structures and cooled to 23°C before use. °C. Add the following substances to a new tube: 12 µL of the first indexer complex, 6 µL of the second indexer complex, and 6 µL of the recycling complex. The solutions are then mixed and centrifuged, and 3 µL of the target and then 3 µL of the detector complex are added. The solutions are then mixed and centrifuged, incubated at 23°C, and fluorescence is measured on a fluorometer (FLUOstar Omega Plate Reader; BMG Labtech). The final concentrations of the cyclic path (SEQ ID NO: 2), the first strand of the first indexer (SEQ ID NO: 5), the first strand of the second indexer (SEQ ID NO: 8), and the first strand of the recycling complex (SEQ ID NO: 11) are 100 nM, 400 nM, 200 nM, and 200 nM, respectively. The results are subtracted from the pore background and then normalized relative to the pore background. Figure 19 ).

[0441] Example 3: Exponential reaction, open circular pathway and nuclease cleanup The lyophilized polynucleotides listed in Table 1 were purchased from IDT, dissolved in TE (10 mM tris(hydroxymethyl)aminomethane-HCl, 0.1 mM disodium ethylenediaminetetraacetate; pH 7.6) at a concentration of 100 µM, and stored at -80 °C; working aliquots were stored at -25 °C. ° C.

[0442] Detection complex The circular pathway (SEQ ID NO: 2) and chaperone (SEQ ID NO: 3) were added to 100 µL of hybridization buffer (0.3 M sodium chloride, 0.03 M sodium citrate, 10 mM MgCl2; pH 7) at final concentrations of 1 µM and 1.5 µM, respectively. The mixture was heated to 94°C for 2 min to remove secondary structures, and then hybridized at 25°C for 30 min. The remaining, unhybridized single-stranded fraction was digested by adding T4 DNA ligase buffer to a final concentration of 1x, truncated exonuclease VIII (20 U; New England Biolabs [NEB]), and thermosensitive exonuclease I (100 U; NEB), incubated at 37°C for 30 min, and then heat-inactivated at 70°C for 30 min. After returning to the hybridization buffer via ethanol precipitation, the detector dissociation ion (SEQ ID NO:4) (which had been heated to 70°C for 2 min to remove secondary structure) was added to a final concentration of 2.0 µM, and the mixture was incubated at 25°C for 20 min to remove excess / unhybridized chaperones and provide the complex with free detector dissociation ions. The final detector complex was stored at -25°C. ° C.

[0443] First exponentiation, second exponentiation, and recirculator The first strand (SEQ ID NO: 5) and the second strand (SEQ ID NO: 6) of the first indexer were added to 100 µL of hybridization buffer (0.3 M sodium chloride, 0.03 M sodium citrate, 10 mM MgCl2; pH 7) at final concentrations of 1 µM and 1.5 µM, respectively. The mixture was heated to 94°C for 2 min to remove secondary structures and then hybridized at 25°C for 20 min. The first indexer hybrid (SEQ ID NO: 7) (which had been heated to 70°C for 2 min) was added to a final concentration of 2.0 µM, and the mixture was incubated at 25°C for 20 min to remove excess / unhybridized second strands of the first indexer and to provide free first indexer hybrids to the first indexer complex. The final first indexer complex was stored at -25°C. ° C. The second indexer complex, the second indexer hybridon, the recirculator complex, and the releaser are generated in a similar manner.

[0444] Exponential target recycling hybridization chain reaction The detection complex, first indexing complex, second indexing complex, and recycling complex were warmed to 25°C. The target (SEQ ID NO: 1) was diluted in TE buffer and kept separate from other reagents until use. The target was incubated at 70°C for 2 minutes to remove secondary structures and cooled to 23°C before use. ° C. Add the following substances to a new tube: 12 µL of the first indexer complex, 6 µL of the second indexer complex, and 6 µL of the recycling complex. The solutions are then mixed and centrifuged, and 3 µL of the detection complex and then 3 µL of the target are added. The solutions are then mixed and centrifuged, incubated at 25°C, and fluorescence is measured on a fluorometer (FLUOstar Omega Plate Reader; BMG Labtech). The final concentrations of the cyclic path (SEQ ID NO: 2), the first strand of the first indexer (SEQ ID NO: 5), the first strand of the second indexer (SEQ ID NO: 8), and the first strand of the recycling complex (SEQ ID NO: 11) are 100 nM, 400 nM, 200 nM, and 200 nM, respectively. The results are subtracted from the pore background and then normalized relative to the pore background. Figure 20 ).

[0445] Example 4: Closed loop path The lyophilized polynucleotides listed in Table 1 were purchased from IDT, dissolved in TE (10 mM tris(hydroxymethyl)aminomethane-HCl, 0.1 mM disodium ethylenediaminetetraacetate; pH 7.6) at a concentration of 100 µM, and stored at -80 °C; working aliquots were stored at -25 °C. ° C.

[0446] Detection complex The circular pathway (SEQ ID NO: 2) and chaperone (SEQ ID NO: 3) were added to 100 µL of hybridization buffer (0.3 M sodium chloride, 0.03 M sodium citrate, 10 mM MgCl2; pH 7) at final concentrations of 1 µM and 1.5 µM, respectively. The mixture was heated to 94°C for 2 min to remove secondary structures and then hybridized at 25°C for 30 min. The 5' and 3' nucleotides of the circular pathway oligonucleotides (which remain adjacent to each other through hybridization with the chaperone via the circular pathway) were ligated by adding 1x T4 DNA ligase buffer (NEB), 1 µg / mL recombinant albumin (NEB), and 800 U T4 DNA ligase (NEB), incubating overnight at 16°C, and then heat-inactivating at 65°C for 10 min. After returning to the hybridization buffer via ethanol precipitation, the detector dissociation ion (SEQ ID NO:4) (which had been heated to 70°C for 2 min to remove secondary structure) was added to a final concentration of 2.0 µM, and the mixture was incubated at 25°C for 20 min to remove excess / unhybridized chaperones and provide the complex with free detector dissociation ions. The final detector complex was stored at -25°C. ° C.

[0447] First exponentiation, second exponentiation, and recirculator The first strand (SEQ ID NO: 5) and the second strand (SEQ ID NO: 6) of the first indexer were added to 100 µL of hybridization buffer (0.3 M sodium chloride, 0.03 M sodium citrate, 10 mM MgCl2; pH 7) at final concentrations of 1 µmM and 1.5 µM, respectively. The mixture was heated to 94°C for 2 min to remove secondary structures and then hybridized at 25°C for 20 min. The first indexer hybrid (SEQ ID NO: 7) (which had been heated to 70°C for 2 min to remove secondary structures) was added to a final concentration of 2.0 µM, and the mixture was incubated at 25°C for 20 min to remove excess / unhybridized second strand of the first indexer and to provide free first indexer hybrid to the first indexer complex. The final first indexer complex was stored at -25°C. °C. The second indexer complex, the second indexer hybridon, the recirculator complex, and the releaser are generated in a similar manner.

[0448] Exponential target recycling hybridization chain reaction The detection subcomplex, first indexed subcomplex, second indexed subcomplex, and recycling subcomplex were warmed at 25°C. The target (SEQ ID NO: 1) was diluted in TE and kept separate from other reagents until use (to prevent contamination). The target was incubated at 70°C for 2 minutes to remove secondary structures and cooled to 23°C before use. ° C. Add the following substances to a new tube: 12 µL of the first indexer complex, 6 µL of the second indexer complex, and 6 µL of the recycling complex. Then mix and centrifuge the solutions, add 3 µL of the detection complex, and then add 3 µL of the target. Mix and centrifuge the solutions again, incubate at 25°C, and measure fluorescence on a fluorometer (FLUOstar Omega Plate Reader; BMG Labtech). The final concentrations of the cyclic path, the first strand of the first indexer, the first strand of the second indexer, and the first strand of the recycling complex were 100 nM, 400 nM, 200 nM, and 200 nM, respectively. The results were subtracted from the pore background and then normalized relative to the pore background. Figure 21 ).

[0449] Example 5: Closed circular pathway, nuclease cleanup The lyophilized polynucleotides listed in Table 1 were purchased from IDT, dissolved in TE (10 mM tris(hydroxymethyl)aminomethane-HCl, 0.1 mM disodium ethylenediaminetetraacetate; pH 7.6) at a concentration of 100 µM, and stored at -80 °C; working aliquots were stored at -25 °C. ° C.

[0450] Detection complex The circular pathway (SEQ ID NO: 2) and chaperone (SEQ ID NO: 3) were added to 100 µL of hybridization buffer (0.3 M sodium chloride, 0.03 M sodium citrate, 10 mM MgCl2; pH 7) at final concentrations of 1 µM and 1.5 µM, respectively. The mixture was heated to 94°C for 2 min to remove secondary structures and then hybridized at 25°C for 30 min. The 5' and 3' nucleotides of the circular pathway (which remain adjacent to each other through hybridization with the chaperone) were ligated by adding 1x T4 DNA ligase buffer (NEB), 1 µg / mL recombinant albumin (NEB), and 800 U T4 DNA ligase (NEB), incubating overnight at 16°C, and then heat-inactivating at 65°C for 10 min. The remaining, unhybridized single-stranded component was digested by adding truncated exonuclease VIII (20 U; NEB) and thermosensitive exonuclease I (100 U; NEB), incubating at 37°C for 30 min, and then heat-inactivating at 70°C for 30 min. After returning to the hybridization buffer via ethanol precipitation, the detector dissociation ion (SEQ ID NO:4) (which had been heated to 70°C for 2 min to remove secondary structure) was added to a final concentration of 2.0 µM, and the mixture was incubated at 25°C for 20 min to remove excess / unhybridized chaperones and provide free detector dissociation ions to the complex. The final detector complex was stored at -25°C. ° C.

[0451] First exponentiation, second exponentiation, and recirculator The first strand (SEQ ID NO: 5) and the second strand (SEQ ID NO: 6) of the first indexer were added to 100 µL of hybridization buffer (0.3 M sodium chloride, 0.03 M sodium citrate, 10 mM MgCl2; pH 7) at final concentrations of 1 µM and 1.5 µM, respectively. The mixture was heated to 94°C for 2 min to remove secondary structures and then hybridized at 25°C for 20 min. The first indexer hybrid (SEQ ID NO: 7), which had been heated to 70°C for 2 min to remove secondary structures, was added to a final concentration of 2.0 µM, and the mixture was incubated at 25°C for 20 min to remove excess / unhybridized second strands of the first indexer and to provide free first indexer hybrids to the complex. The final first indexer complex was stored at -25°C. The second indexer complex, second indexer hybrid, recycle complex, and release agent were generated in a similar manner.

[0452] Exponential target recycling hybridization chain reaction The detection subcomplex, first indexed subcomplex, second indexed subcomplex, and recycling subcomplex were warmed at 25°C. The target (SEQ ID NO: 1) was diluted in TE and kept separate from other reagents until use (to prevent contamination). The target was incubated at 70°C for 2 minutes to remove secondary structures and cooled to 23°C before use. ° C. Add the following substances to a new tube: 12 µL of the first indexer complex, 6 µL of the second indexer complex, and 6 µL of the recycling complex. Then mix and centrifuge the solutions, add 3 µL of the detection complex, and then add 3 µL of the target. Mix and centrifuge the solutions again, incubate at 25°C, and measure fluorescence on a fluorometer (FLUOstar Omega Plate Reader; BMG Labtech). The final concentrations of the cyclic path, the first strand of the first indexer, the first strand of the second indexer, and the first strand of the recycling complex were 100 nM, 400 nM, 200 nM, and 200 nM, respectively. The results were subtracted from the pore background and then normalized relative to the pore background. Figure 22 ).

[0453] Example 6: Non-specific target (dummy target) The lyophilized polynucleotides listed in Table 1 were purchased from IDT, dissolved in TE (10 mM tris(hydroxymethyl)aminomethane-HCl, 0.1 mM disodium ethylenediaminetetraacetate; pH 7.6) at a concentration of 100 µM, and stored at -80 °C; working aliquots were stored at -25 °C. ° C.

[0454] Detection complex The double-stranded detector molecule was constructed by hybridizing two oligonucleotides, a circular pathway (SEQ ID NO: 2) and a chaperone (SEQ ID NO: 3). The circular pathway and chaperone were added to 100 µL of hybridization buffer (0.3 M sodium chloride, 0.03 M sodium citrate, 10 mM MgCl2; pH 7) at final concentrations of 1 µM and 2 µM, respectively. The mixture was heated to 94°C for 2 min to remove secondary structures and then hybridized at 25°C for 60 min. A detector dissociation ion (SEQ ID NO: 4) (which had been heated to 70°C for 2 min to remove secondary structures) was added to a final concentration of 2.5 µM, and the mixture was incubated at 25°C for 120 min to remove excess / unhybridized chaperone and provide free detector dissociation ions to the detector complex. The final detector complex was stored at -25°C. ° C.

[0455] First exponentiation, second exponentiation, and recirculator The first strand (SEQ ID NO: 5) and the second strand (SEQ ID NO: 6) of the first indexer were added to 100 µL of hybridization buffer (0.3 M sodium chloride, 0.03 M sodium citrate, 10 mM MgCl2; pH 7) at final concentrations of 1 µM and 2 µM, respectively. The mixture was heated to 94°C for 2 min to remove secondary structures and then hybridized at 25°C for 60 min. The first indexer hybrid (SEQ ID NO: 7) (which had been heated to 70°C for 2 min to remove secondary structures) was added to a final concentration of 2.5 µM, and the mixture was incubated at 25°C for 120 min to remove excess / unhybridized second strands of the first indexer and to provide free first indexer hybrids to the first indexer. The final first indexer complex was stored at -25°C. ° C. The second indexer complex, the second indexer hybridon, the recirculator complex, and the releaser are generated in a similar manner.

[0456] Exponential target recycling hybridization chain reaction The detecton, first indexer, second indexer, and recycler complex were warmed to 23°C. The target (SEQ ID NO: 1) was diluted in TE buffer and kept separate from other reagents until use. The target was incubated with the sham target (PCR primers for 23 nucleotides of SARS-CoV-2, Centre of Disease Control, USA; SEQ ID NO: 14) at 70°C for 2 minutes to remove secondary structures and cooled to 23°C before use. ° C. Add the following substances to a new tube: 12 µL of the first indexer complex, 6 µL of the second indexer complex, and 6 µL of the recycling complex. The solutions are then mixed and centrifuged, and 3 µL of the detection complex and then 3 µL of the target or dummy target are added, each to a final concentration of 10 pM. The solutions are then mixed and centrifuged, incubated at 23°C, and fluorescence is measured on a fluorometer (FLUOstar Omega Plate Reader; BMG Labtech). The final concentrations of the cyclic path (SEQ ID NO: 2), the first strand of the first indexer (SEQ ID NO: 5), the first strand of the second indexer (SEQ ID NO: 8), and the first strand of the recycling complex (SEQ ID NO: 11) are 100 nM, 400 nM, 200 nM, and 200 nM, respectively. The results are subtracted from the pore background and then normalized relative to the pore background. Figure 23 ).

[0457] Example 7: Target-dependent reaction Lyophilized oligonucleotides (Table 1) were purchased from IDT and / or Bioneer, dissolved in 2xSSC (300 mM sodium chloride, 30 mM sodium citrate; pH 7.0) at a concentration of 100 µM and stored at -80 °C; working aliquots were stored at -25 °C.

[0458] Detection complex The double-stranded detection molecule was constructed by hybridizing two oligonucleotides, a circular pathway (SEQ ID NO: 2) and a chaperone (SEQ ID NO: 3). The circular pathway and the chaperone were added to 100 µL of 2x SSC (0.3 M sodium chloride, 0.03 M sodium citrate; pH 7) at final concentrations of 1 µM and 2 µM, respectively. The mixture was heated to 94°C for 2 min to remove secondary structures, and then hybridized at 25°C for 60 min, followed by hybridization at 16°C for 60 min. The 5' and 3' nucleotides of the circular pathway oligonucleotide (which remained adjacent to each other through hybridization of the circular pathway with the chaperone) were ligated by adding 1x T4 DNA ligase buffer (NEB), 1 µg / mL recombinant albumin (NEB), and T4 DNA ligase (800 U; NEB), incubating overnight at 16°C, and then heat-inactivating at 65°C for 10 min. Unhybridized cyclic pathways and chaperones were removed by purification on a silica membrane column (Minelute Reaction Cleanup Kit; QIAGEN) and eluted in 10 µL TE, which was then brought to 80 µL with SSCMT (0.3 M sodium chloride, 0.03 M sodium citrate, 10 mM MgCl2, 0.1% Tween 20; pH 7). Then, a detecton dissociation ion (SEQ ID NO: 15) (which had been heated to 70 °C for 2 min to remove secondary structures) was added to a final concentration of 2.5 µM, and the mixture was incubated at 25 °C for 10 min to remove any remaining unhybridized chaperones and provide the complex with free detecton dissociation ions. The final detecton complex was stored at -25 °C. ° C.

[0459] The first exponent, the second exponent, and the recycling complex (SEQ ID NO: 5, 6, 16, 8, respectively) 9、17、11、12、18) The first strand (SEQ ID NO: 5) and the second strand (SEQ ID NO: 6) of the first indexer were added to 100 µL of SSCMT at final concentrations of 1 µM and 2 µM, respectively. The mixture was heated to 94°C for 2 min to remove secondary structures and then hybridized at 25°C for 60 min. The first indexer hybrid (SEQ ID NO: 16) (which had been heated to 70°C for 2 min to remove secondary structures) was added to a final concentration of 2.5 µM, and the mixture was incubated at 25°C for 10 min to remove excess / unhybridized second strands of the first indexer and to provide free first indexer hybrids to the first indexer. The final first indexer complex was stored at -25°C. ° C. The second indexer complex, the second indexer hybridon, the recirculator complex, and the releaser are generated in a similar manner.

[0460] Exponential target recycling hybridization chain reaction The complex of the detector, first indexer, second indexer, and recycler was warmed to 23°C. The target (SEQ ID NO: 1) was diluted in 2xSSC and kept separate from other reagents until use (to prevent contamination). The target was incubated at 70°C for 2 minutes to remove secondary structures and cooled to 23°C before use. ° C. Add the following to a new tube: 4 µL of the first indexer complex, 2 µL of the second indexer complex, and 2 µL of the recycling complex. Mix the solutions and centrifuge, then add 1 µL of the detectant complex and then 1 µL (0-10 µM) of the initiator. Mix the solutions and centrifuge, incubate at 25°C, aliquot into duplicates, and measure fluorescence on a fluorometer (FLUOstar OmegaPlate Reader; BMG Labtech). The final concentrations of the cyclic pathway (SEQ ID NO: 2), the first strand of the first indexer (SEQ ID NO: 5), the first strand of the second indexer (SEQ ID NO: 8), and the first strand of the recycling complex (SEQ ID NO: 11) were 100 nM, 400 nM, 200 nM, and 200 nM, respectively. The results were subtracted from the pore background and then normalized relative to the pore background. Figure 24 Error bars represent the maximum and minimum values ​​of repeated samples.

[0461] Example 8: Target contained in a larger sequence Lyophilized oligonucleotides (Table 1) were purchased from IDT and / or Bioneer, dissolved in 2xSSC (300 mM sodium chloride, 30 mM sodium citrate; pH 7.0) at a concentration of 100 µM and stored at -80 °C; working aliquots were stored at -25 °C.

[0462] Detection complex The double-stranded detection molecule was constructed by hybridizing two oligonucleotides, a circular pathway (SEQ ID NO: 2) and a chaperone (SEQ ID NO: 3). The circular pathway and the chaperone were added to 100 µL of 2x SSC (0.3 M sodium chloride, 0.03 M sodium citrate; pH 7) at final concentrations of 1 µM and 2 µM, respectively. The mixture was heated to 94°C for 2 min to remove secondary structures, and then hybridized at 25°C for 60 min, followed by hybridization at 16°C for 60 min. The 5' and 3' nucleotides of the circular pathway oligonucleotide (which remained adjacent to each other through hybridization of the circular pathway with the chaperone) were ligated by adding 1x T4 DNA ligase buffer (NEB), 1 µg / mL recombinant albumin (NEB), and T4 DNA ligase (800 U; NEB), incubating overnight at 16°C, and then heat-inactivating at 65°C for 10 min. Unhybridized cyclic pathways and chaperones were removed by purification on a silica membrane column (Minelute Reaction Cleanup Kit; QIAGEN) and eluted in 10 µL TE, which was then brought to 80 µL with SSCMT (0.3 M sodium chloride, 0.03 M sodium citrate, 10 mM MgCl2, 0.1% Tween 20; pH 7). Then, a detecton dissociation ion (SEQ ID NO: 15) (which had been heated to 70 °C for 2 min to remove secondary structures) was added to a final concentration of 2.5 µM, and the mixture was incubated at 25 °C for 10 min to remove any remaining unhybridized chaperones and provide the complex with free detecton dissociation ions. The final detecton complex was stored at -25 °C. ° C.

[0463] The first exponent, the second exponent, and the recirculator (SEQ ID NO: 5, 6, 16, 8, 9, 17, respectively) 11、12、18) The first strand (SEQ ID NO: 5) and the second strand (SEQ ID NO: 6) of the first indexer were added to 100 µL of SSCMT at final concentrations of 1 µM and 2 µM, respectively. The mixture was heated to 94°C for 2 min to remove secondary structures and then hybridized at 25°C for 60 min. The first indexer hybrid (SEQ ID NO: 16) (which had been heated to 70°C for 2 min to remove secondary structures) was added to a final concentration of 2.5 µM, and the mixture was incubated at 25°C for 10 min to remove excess / unhybridized second strands of the first indexer and to provide free first indexer hybrids to the first indexer complex. The final first indexer complex was stored at -25°C. ° C. The second indexer complex, the second indexer hybridon, the recirculator complex, and the releaser are generated in a similar manner.

[0464] Exponential target recycling hybridization chain reaction The complex of the detector, first indexer, second indexer, and recycler was warmed to 23°C. The initiators (proposed targets; SEQ ID NO: 19, 20, and 21) were diluted in 2xSSC and kept separate from other reagents until use (to prevent contamination). The initiators were incubated at 70°C for 2 minutes to remove secondary structures and cooled to 23°C before use. ° C. Add the following to a new tube: 4 µL of the first indexing complex, 2 µL of the second indexing complex, and 2 µL of the recycling complex. Then mix and centrifuge the solution, add 1 µL of the detectant complex, and then add 1 µL of 2xSSC or 2 µL of 1 µM initiator-2-4. Then mix and centrifuge the solution, incubate at 25°C, aliquot twice, and measure fluorescence on a fluorometer (FLUOstar Omega Plate Reader; BMG Labtech). Subtract the pore background from the results and then normalize relative to the pore background. Error bars represent the maximum and minimum values ​​of replicate samples (…). Figure 25 ).

[0465] Example 9: Specificity Lyophilized oligonucleotides (Table 1) were purchased from IDT and / or Bioneer, dissolved in 2xSSC (300 mM sodium chloride, 30 mM sodium citrate; pH 7.0) at a concentration of 100 µM and stored at -80 °C; working aliquots were stored at -25 °C.

[0466] Detection complex The double-stranded detection molecule was constructed by hybridizing two oligonucleotides, a circular pathway (SEQ ID NO: 2) and a chaperone (SEQ ID NO: 3). The circular pathway and the chaperone were added to 100 µL of 2x SSC (0.3 M sodium chloride, 0.03 M sodium citrate; pH 7) at final concentrations of 1 µM and 2 µM, respectively. The mixture was heated to 94°C for 2 min to remove secondary structures, and then hybridized at 25°C for 60 min, followed by hybridization at 16°C for 60 min. The 5' and 3' nucleotides of the circular pathway oligonucleotide (which remained adjacent to each other through hybridization of the circular pathway with the chaperone) were ligated by adding 1x T4 DNA ligase buffer (NEB), 1 µg / mL recombinant albumin (NEB), and T4 DNA ligase (800 U; NEB), incubating overnight at 16°C, and then heat-inactivating at 65°C for 10 min. Unhybridized cyclic pathways and chaperones were removed by purification on a silica membrane column (Minelute Reaction Cleanup Kit; QIAGEN) and eluted in 10 µL TE, which was then brought to 80 µL with SSCMT (0.3 M sodium chloride, 0.03 M sodium citrate, 10 mM MgCl2, 0.1% Tween 20; pH 7). Then, a detecton dissociation ion (SEQ ID NO: 15) (which had been heated to 70 °C for 2 min to remove secondary structures) was added to a final concentration of 2.5 µM, and the mixture was incubated at 25 °C for 10 min to remove any remaining unhybridized chaperones and provide the complex with free detecton dissociation ions. The final detecton complex was stored at -25 °C. ° C.

[0467] The first exponent, the second exponent, and the recycling complex (SEQ ID NO: 5, 6, 16, 8, respectively) 9、17、11、12、18) The first strand (SEQ ID NO: 5) and the second strand (SEQ ID NO: 6) of the first indexer were added to 100 µL of SSCMT at final concentrations of 1 µM and 2 µM, respectively. The mixture was heated to 94°C for 2 min to remove secondary structures and then hybridized at 25°C for 60 min. The first indexer hybrid (SEQ ID NO: 16) (which had been heated to 70°C for 2 min to remove secondary structures) was added to a final concentration of 2.5 µM, and the mixture was incubated at 25°C for 10 min to remove excess / unhybridized second strands of the first indexer and to provide free first indexer hybrids to the first indexer complex. The final first indexer complex was stored at -25°C. ° C. The second indexer complex, the second indexer hybridon, the recirculator complex, and the releaser are generated in a similar manner.

[0468] Exponential target recycling hybridization chain reaction The complex of the detector, first indexer, second indexer, and recycler was warmed to 23°C. The initiator (SEQ ID NO: 1) was diluted in 2xSSC and kept separate from other reagents until use (to prevent contamination). The initiator was incubated at 70°C for 2 minutes to remove secondary structures and cooled to 23°C before use. ° C. Add the following to a new tube: 4 µL of the first indexer complex, 2 µL of the second indexer complex, and 2 µL of the recycling complex. Mix the solutions and centrifuge, then add 1 µL of the detector complex followed by 1 µL of 1 µM initiator or pseudo-initiators 1-3 (SEQ ID NO: 22, 14, and 23) or mutated initiators 1-6 (SEQ ID NO: 24-29). Mix the solutions and centrifuge, incubate at 25°C, aliquot twice, and measure fluorescence on a fluorometer (FLUOstar Omega Plate Reader; BMGLabtech). Subtract the pore background from the results and then normalize relative to the pore background. Figure 26 (Table 2).

[0469] Table 2

[0470] Example 10: Reaction Temperature Lyophilized oligonucleotides (Table 1) were purchased from IDT and / or Bioneer, dissolved in 2xSSC (300 mM sodium chloride, 30 mM sodium citrate; pH 7.0) at a concentration of 100 µM and stored at -80 °C; working aliquots were stored at -25 °C.

[0471] Detection complex The double-stranded detection molecule was constructed by hybridizing two oligonucleotides, a circular pathway (SEQ ID NO: 2) and a chaperone (SEQ ID NO: 3). The circular pathway and the chaperone were added to 100 µL of 2x SSC (0.3 M sodium chloride, 0.03 M sodium citrate; pH 7) at final concentrations of 1 µM and 2 µM, respectively. The mixture was heated to 94°C for 2 min to remove secondary structures, and then hybridized at 25°C for 60 min, followed by hybridization at 16°C for 60 min. The 5' and 3' nucleotides of the circular pathway oligonucleotide (which remained adjacent to each other through hybridization of the circular pathway with the chaperone) were ligated by adding 1x T4 DNA ligase buffer (NEB), 1 µg / mL recombinant albumin (NEB), and T4 DNA ligase (800 U; NEB), incubating overnight at 16°C, and then heat-inactivating at 65°C for 10 min. Unhybridized cyclic pathways and chaperones were removed by purification on a silica membrane column (Minelute Reaction Cleanup Kit; QIAGEN) and eluted in 10 µL TE, which was then brought to 80 µL with SSCMT (0.3 M sodium chloride, 0.03 M sodium citrate, 10 mM MgCl2, 0.1% Tween 20; pH 7). Then, a detecton dissociation ion (SEQ ID NO: 15) (which had been heated to 70 °C for 2 min to remove secondary structures) was added to a final concentration of 2.5 µM, and the mixture was incubated at 25 °C for 10 min to remove any remaining unhybridized chaperones and provide the complex with free detecton dissociation ions. The final detecton complex was stored at -25 °C. ° C.

[0472] The first exponent, the second exponent, and the recycling complex (SEQ ID NO: 5, 6, 16, 8, respectively) 9、17、11、12、18) The first strand (SEQ ID NO: 5) and the second strand (SEQ ID NO: 6) of the first indexer were added to 100 µL of SSCMT at final concentrations of 1 µM and 2 µM, respectively. The mixture was heated to 94°C for 2 min to remove secondary structures and then hybridized at 25°C for 60 min. The first indexer hybrid (SEQ ID NO: 16) (which had been heated to 70°C for 2 min to remove secondary structures) was added to a final concentration of 2.5 µM, and the mixture was incubated at 25°C for 10 min to remove excess / unhybridized second strands of the first indexer and to provide free first indexer hybrids to the first indexer complex. The final first indexer complex was stored at -25°C. ° C. The second indexer complex, the second indexer hybridon, the recirculator complex, and the releaser are generated in a similar manner.

[0473] Exponential target recycling hybridization chain reaction The complex of the detector, first indexer, second indexer, and recycler was warmed to 23°C. The initiator (SEQ ID NO: 1) was diluted in 2xSSC and kept separate from other reagents until use (to prevent contamination). The initiator was incubated at 70°C for 2 minutes to remove secondary structures and cooled to 23°C before use. ° C. Add the following to a new tube: 4 µL of the first indexing complex, 2 µL of the second indexing complex, and 2 µL of the recycling complex. Then mix and centrifuge the solution, add 1 µL of the detection complex, and then add 1 µL (0-10 µM) of the initiator. Mix and centrifuge the solution, aliquot into two portions, and store in a Thermomixer C. TM Incubate in Eppendorf at 22.3°C – 36°C and measure fluorescence every 5 minutes on a fluorometer (FLUOstar Omega Plate Reader; BMG Labtech) set to incubate at the same temperature; read every 5 minutes for one hour, and return the plate to the Thermomixer C between readings. TM The results were subtracted from the porosity background and then normalized relative to the porosity background. The relative reaction rate was determined by dividing the result with the initiator by the control without the initiator. Figure 27 ).

[0474] Example 11: Buffer Solution Lyophilized oligonucleotides (Table 1) were purchased from IDT and / or Bioneer, dissolved in 2xSSC (300 mM sodium chloride, 30 mM sodium citrate; pH 7.0) at a concentration of 100 µM and stored at -80 °C; working aliquots were stored at -25 °C.

[0475] Buffer solution used: SSCMT: 0.3 M sodium chloride, 0.03 M sodium citrate, 10 mM MgCl2, 0.1% Tween 20; pH 7.

[0476] SSCM / 2T: 0.3 M sodium chloride, 0.03 M sodium citrate, 5 mM MgCl2, 0.1% Tween 20; pH 7.

[0477] SSCT: 0.3 M sodium chloride, 0.03 M sodium citrate, 0.1% Tween 20; pH 7.

[0478] TAEM: 40 mM tris(hydroxymethyl)aminomethane acetate, 1 mM disodium ethylenediaminetetraacetate, 12.5 mM MgCl2; pH 8.3.

[0479] KAH: 100 mM potassium acetate, 30 mM 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES); pH 7.5.

[0480] Detection complex The double-stranded detection molecule was constructed by hybridizing two oligonucleotides, a circular pathway (SEQ ID NO: 2) and a chaperone (SEQ ID NO: 3). The circular pathway and the chaperone were added to 100 µL of 2x SSC (0.3 M sodium chloride, 0.03 M sodium citrate; pH 7) at final concentrations of 1 µM and 2 µM, respectively. The mixture was heated to 94°C for 2 min to remove secondary structures, and then hybridized at 25°C for 60 min, followed by hybridization at 16°C for 60 min. The 5' and 3' nucleotides of the circular pathway oligonucleotide (which remained adjacent to each other through hybridization of the circular pathway with the chaperone) were ligated by adding 1x T4 DNA ligase buffer (NEB), 1 µg / mL recombinant albumin (NEB), and T4 DNA ligase (800 U; NEB), incubating overnight at 16°C, and then heat-inactivating at 65°C for 10 min. Unhybridized cyclic pathways and chaperones were removed by purification on a silica membrane column (Minelute Reaction Cleanup Kit; QIAGEN) and eluted in 10 µL TE, which was then brought to 80 µL with SSCMT, SSCM / 2T, SSCT, TAEM, or KAH. Then, a detecton dissociation ion (SEQ ID NO: 15) (heated to 70 °C for 2 min to remove secondary structures) was added to a final concentration of 2.5 µM, and the mixture was incubated at 25 °C for 10 min to remove any remaining unhybridized chaperones and provide the complex with free detecton dissociation ions. The final detecton complex was stored at -25 °C. ° C.

[0481] The first exponent, the second exponent, and the recycling complex (SEQ ID NO: 5, 6, 16, 8, respectively) 9、17、11、12、18) The first strand (SEQ ID NO: 5) and the second strand (SEQ ID NO: 6) of the first indexer were added to 100 µL of SSCMT, SSCM / 2T, SSCT, TAEM, or KAH at final concentrations of 1 µM and 2 µM, respectively. The mixture was heated to 94°C for 2 min to remove secondary structures and then hybridized at 25°C for 60 min. The first indexer hybrid (SEQ ID NO: 16) (which had been heated to 70°C for 2 min to remove secondary structures) was added to a final concentration of 2.5 µM, and the mixture was incubated at 25°C for 10 min to remove excess / unhybridized second strands of the first indexer and to provide free first indexer hybrids to the first indexer complex. The final first indexer complex was stored at -25°C. ° C. The second indexer complex, the second indexer hybridon, the recirculator complex, and the releaser are generated in a similar manner.

[0482] Exponential target recycling hybridization chain reaction The complex of the detector, first indexer, second indexer, and recycler was warmed at 23°C. The initiator (target; SEQ ID NO: 1) or initiator 3 (target; SEQ ID NO: 20) was diluted in 2xSSC and kept separate from other reagents until use (to prevent contamination). The initiator was incubated at 70°C for 2 minutes to remove secondary structures and cooled to 23°C before use. ° C. Add the following to a new tube: 4 µL of the first indexing complex, 2 µL of the second indexing complex, and 2 µL of the recycling complex. Mix the solutions and centrifuge, then add 1 µL of the detectant complex followed by 1 µL of 2xSSC or 1 µM initiator or 1 µM initiator 3. Mix the solutions and centrifuge, incubate at 25°C, aliquot twice, and measure fluorescence on a fluorometer (FLUOstar Omega Plate Reader; BMG Labtech). Subtract the pore background from the results and then normalize relative to the pore background (Figure 28). Error bars represent the maximum and minimum values ​​for replicate samples.

[0483] Example 12: Cleaning of Ethanol Precipitation Lyophilized oligonucleotides (Table 1) were purchased from IDT and / or Bioneer, dissolved in 2xSSC (300 mM sodium chloride, 30 mM sodium citrate; pH 7.0) at a concentration of 100 µM and stored at -80 °C; working aliquots were stored at -25 °C.

[0484] Detection complex The double-stranded detection molecule was constructed by hybridizing two oligonucleotides, a circular pathway (SEQ ID NO: 2) and a chaperone (SEQ ID NO: 3). The circular pathway and the chaperone were added to 100 µL of 2x SSC (0.3 M sodium chloride, 0.03 M sodium citrate; pH 7) at final concentrations of 1 µM and 2 µM, respectively. The mixture was heated to 94°C for 2 min to remove secondary structures, and then hybridized at 25°C for 60 min, followed by hybridization at 16°C for 60 min. The 5' and 3' nucleotides of the circular pathway oligonucleotide (which remained adjacent to each other through hybridization with the chaperone via the circular pathway) were ligated by adding 1x T4 DNA ligase buffer (NEB), 1 µg / mL recombinant albumin (NEB), and T4 DNA ligase (800 U; NEB), incubating overnight at 16°C, and then heat-inactivating at 65°C for 10 min. After returning to SSCMT buffer (0.3 M sodium chloride, 0.03 M sodium citrate, 10 mM MgCl2, 0.1% Tween 20; pH 7) via ethanol precipitation, a detector dissociation ion (SEQ ID NO: 15) (which had been heated to 70°C for 2 min to remove secondary structure) was added to a final concentration of 2.5 µM, and the mixture was incubated at 25°C for 10 min to remove any remaining unhybridized chaperones and provide the complex with free detector dissociation ions. The final detector complex was stored at -25°C. ° C.

[0485] The first exponent, the second exponent, and the recycling complex (SEQ ID NO: 5, 6, 7, 8, 9, respectively) 10、11、12、13) The first strand (SEQ ID NO: 5) and the second strand (SEQ ID NO: 6) of the first indexer were added to 100 µL of SSCM (0.3 M sodium chloride, 0.03 M sodium citrate, 10 mM MgCl2; pH 7) at final concentrations of 1 µM and 2 µM, respectively. The mixture was heated to 94°C for 2 min to remove secondary structures and then hybridized at 23°C for 30 min. The first indexer hybrid -FL (SEQ ID NO: 7), which had been heated to 70°C for 2 min to remove secondary structures, was added to a final concentration of 2.5 µM, and the mixture was incubated at 23°C for 120 min to remove excess / unhybridized second strand of the first indexer and to provide free first indexer hybrid -FL to the first indexer complex. The final first indexer complex was stored at -25°C. ° C. The second indexer complex, the second indexer hybridon, the recirculator complex, and the releaser are generated in a similar manner.

[0486] Exponential target recycling hybridization chain reaction The complex of the detector, first indexer, second indexer, and recycler was warmed at 23°C. Initiator-3 (SEQ ID NO: 20) and pseudo-initiator-2 (SEQ ID NO: 14) were diluted in 2xSSC and kept separate from other reagents until use (to prevent contamination). Initiator-3 and pseudo-initiator-2 were incubated at 70°C for 2 minutes to remove secondary structures and cooled to 23°C before use. ° C. Add the following to a new tube: 4 µL of the first indexing complex, 2 µL of the second indexing complex, and 2 µL of the recycling complex. Mix the solutions and centrifuge, then add 1 µL of the detection complex followed by 1 µL of 2xSSC or 1 µM initiator-3 or 1 µM pseudo-initiator-2. Mix the solutions and centrifuge, incubate at 25°C, aliquot twice, and measure fluorescence on a fluorometer (FLUOstar Omega Plate Reader; BMGLabtech). Subtract the pore background from the results and then normalize relative to the pore background. Figure 29 Error bars represent the maximum and minimum values ​​of repeated samples.

[0487] Example 13: Cleaning of single-stranded DNA nuclease and ethanol precipitation Lyophilized oligonucleotides (Table 1) were purchased from IDT and / or Bioneer, dissolved in 2xSSC (300 mM sodium chloride, 30 mM sodium citrate; pH 7.0) at a concentration of 100 µM and stored at -80 °C; working aliquots were stored at -25 °C.

[0488] Detection complex The double-stranded detection molecule was constructed by hybridizing two oligonucleotides, a circular pathway (SEQ ID NO: 2) and a chaperone (SEQ ID NO: 3). The circular pathway and the chaperone were added to 100 µL of 2x SSC (0.3 M sodium chloride, 0.03 M sodium citrate; pH 7) at final concentrations of 1 µM and 2 µM, respectively. The mixture was heated to 94°C for 2 min to remove secondary structures, and then hybridized at 25°C for 60 min, followed by hybridization at 16°C for 60 min. The 5' and 3' nucleotides of the circular pathway oligonucleotide (which remained adjacent to each other through hybridization with the chaperone via the circular pathway) were ligated by adding 1x T4 DNA ligase buffer (NEB), 1 µg / mL recombinant albumin (NEB), and T4 DNA ligase (800 U; NEB), incubating overnight at 16°C, and then heat-inactivating at 65°C for 10 min. The remaining unhybridized single-stranded component was digested by adding truncated exonuclease VIII (20 U; NEB) and thermosensitive exonuclease I (100 U; NEB), incubating at 37°C for 30 min, and then heat-inactivating at 70°C for 30 min. After returning to SSCMT buffer (0.3 M sodium chloride, 0.03 M sodium citrate, 10 mM MgCl2, 0.1% Tween 20; pH 7) via ethanol precipitation, the detector dissociation ion (SEQ ID NO: 15) (which had been heated to 70°C for 2 min (to remove secondary structure)) was added to a final concentration of 2.5 µM, and the mixture was incubated at 25°C for 10 min to remove any remaining unhybridized partners and provide the complex with free detector dissociation ions. The final detector complex was stored at -25°C. ° C.

[0489] The first exponent, the second exponent, and the recirculator (SEQ ID NO: 5, 6, 16, 8, 9, 17, respectively) 11、12、18) The first strand (SEQ ID NO: 5) and the second strand (SEQ ID NO: 6) of the first indexer were added to 100 µL of SSCMT at final concentrations of 1 µM and 2 µM, respectively. The mixture was heated to 94°C for 2 min to remove secondary structures and then hybridized at 25°C for 60 min. The first indexer hybrid (SEQ ID NO: 16) (which had been heated to 70°C for 2 min to remove secondary structures) was added to a final concentration of 2.5 µM, and the mixture was incubated at 25°C for 10 min to remove excess / unhybridized second strands of the first indexer and to provide free first indexer hybrids to the first indexer complex. The final first indexer complex was stored at -25°C. °C. The second indexer complex, the second indexer hybridon, the recirculator complex, and the releaser are generated in a similar manner.

[0490] Exponential target recycling hybridization chain reaction The complex of the detector, first indexer, second indexer, and recycler was warmed to 23°C. Initiator-3 (SEQ ID NO: 20) was diluted in 2xSSC and kept separate from other reagents until use (to prevent contamination). Initiator-3 was incubated at 70°C for 2 minutes to remove secondary structures and cooled to 23°C before use. ° C. Add the following to a new tube: 4 µL of the first indexing complex, 2 µL of the second indexing complex, and 2 µL of the recycling complex. Mix the solutions and centrifuge, then add 1 µL of the detection complex followed by 1 µL of 2xSSC or 1 µM initiator-3. Mix the solutions and centrifuge, incubate at 25°C, aliquot twice, and measure fluorescence on a fluorometer (FLUOstar Omega PlateReader; BMG Labtech). Subtract the pore background from the results and then normalize relative to the pore background. Figure 30 Error bars represent the maximum and minimum values ​​of repeated samples.

[0491] Example 14: ETHyR reaction and silica membrane column purification of all complexes Lyophilized oligonucleotides (Table 1) were purchased from IDT and / or Bioneer, dissolved in 2xSSC (300 mM sodium chloride, 30 mM sodium citrate; pH 7.0) at a concentration of 100 µM and stored at -80 °C; working aliquots were stored at -25 °C.

[0492] Detection complex The double-stranded detection molecule was constructed by hybridizing two oligonucleotides, a circular pathway (SEQ ID NO: 2) and a chaperone (SEQ ID NO: 3). The circular pathway and the chaperone were added to 100 µL of 2x SSC (0.3 M sodium chloride, 0.03 M sodium citrate; pH 7) at final concentrations of 1 µM and 2 µM, respectively. The mixture was heated to 94°C for 2 min to remove secondary structures, and then hybridized at 25°C for 60 min, followed by hybridization at 16°C for 60 min. The 5' and 3' nucleotides of the circular pathway oligonucleotide (which remained adjacent to each other through hybridization of the circular pathway with the chaperone) were ligated by adding 1x T4 DNA ligase buffer (NEB), 1 µg / mL recombinant albumin (NEB), and T4 DNA ligase (800 U; NEB), incubating overnight at 16°C, and then heat-inactivating at 65°C for 10 min. Unhybridized cyclic pathways and chaperones were removed by purification on a silica membrane column (Minelute Reaction Cleanup Kit; QIAGEN) and eluted in 10 µL TE, which was then brought to 80 µL with SSCMT (0.3 M sodium chloride, 0.03 M sodium citrate, 10 mM MgCl2, 0.1% Tween 20; pH 7). Then, a detecton dissociation ion (SEQ ID NO: 15) (which had been heated to 70 °C for 2 min to remove secondary structures) was added to a final concentration of 2.5 µM, and the mixture was incubated at 25 °C for 10 min to remove any remaining unhybridized chaperones and provide the complex with free detecton dissociation ions. The final detecton complex was stored at -25 °C. ° C.

[0493] The first exponent, the second exponent, and the recirculator (SEQ ID NO: 5, 6, 16, 8, 9, 17, respectively) 11、12、18) The first strand (SEQ ID NO: 5) and the second strand (SEQ ID NO: 6) of the first indexer were added to 100 µL of 2xSSC at final concentrations of 1 µM and 2 µM, respectively. The mixture was heated to 94°C for 2 min to remove secondary structures, and then hybridized at 25°C for 60 min. The hybridized first and second strands of the first indexer were purified on a silica gel column (Nucleotide Removal Kit; QIAGEN) and eluted in 30 µL of TE, which was then brought to a final volume of 80 µL with SSCMT (0.3 M sodium chloride, 0.03 M sodium citrate, 10 mM MgCl2, 0.1% Tween 20; pH 7). The first indexer hybrid (SEQ ID NO: 16) (which had been heated to 70°C for 2 min to remove secondary structures) was added to a final concentration of 2.5 µM, and the mixture was incubated at 25°C for 10 min to remove the second strand of excess / unhybridized first indexers and to provide free first indexer hybrids to the first indexer complex. The final first indexer complex was stored at -25°C. ° C. The second indexer complex, the second indexer hybridon, the recirculator complex, and the releaser are generated in a similar manner.

[0494] Exponential target recycling hybridization chain reaction The complex of the detector, first indexer, second indexer, and recycler was warmed to 23°C. Initiator-3 (SEQ ID NO: 20) was diluted in 2xSSC and kept separate from other reagents until use (to prevent contamination). Initiator-3 was incubated at 70°C for 2 minutes to remove secondary structures and cooled to 23°C before use. ° C. Add the following to a new tube: 4 µL of the first indexing complex, 2 µL of the second indexing complex, and 2 µL of the recycling complex. Mix the solutions and centrifuge, then add 1 µL of the detection complex followed by 1 µL of 2xSSC or 1 µM initiator-3. Mix the solutions and centrifuge, incubate at 25°C, aliquot twice, and measure fluorescence on a fluorometer (FLUOstar Omega PlateReader; BMG Labtech). Subtract the pore background from the results and then normalize relative to the pore background. Figure 31 Error bars represent the maximum and minimum values ​​of repeated samples.

[0495] Example 15: ETHyR reaction without detection of dissociation ions Lyophilized oligonucleotides (Table 1) were purchased from IDT and / or Bioneer, dissolved in 2xSSC (300 mM sodium chloride, 30 mM sodium citrate; pH 7.0) at a concentration of 100 µM and stored at -80 °C; working aliquots were stored at -25 °C.

[0496] Detection of the subcomplexes (SEQ ID NO: 2 and 3) The double-stranded detection molecule was constructed by hybridizing two oligonucleotides, a circular pathway (SEQ ID NO: 2) and a chaperone (SEQ ID NO: 3). The circular pathway and the chaperone were added to 100 µL of 2x SSC (0.3 M sodium chloride, 0.03 M sodium citrate; pH 7) at final concentrations of 1 µM and 2 µM, respectively. The mixture was heated to 94°C for 2 min to remove secondary structures, and then hybridized at 25°C for 60 min, followed by hybridization at 16°C for 60 min. The 5' and 3' nucleotides of the circular pathway oligonucleotide (which remained adjacent to each other through hybridization with the chaperone via the circular pathway) were ligated by adding 1x T4 DNA ligase buffer (NEB), 1 µg / mL recombinant albumin (NEB), and T4 DNA ligase (800 U; NEB), incubating overnight at 16°C, and then heat-inactivating at 65°C for 10 min. The assay complex was purified by ethanol precipitation and returned to SSCMT buffer (0.3 M sodium chloride, 0.03 M sodium citrate, 10 mM MgCl2, 0.1% Tween 20; pH 7). The assay complex was stored at -25°C. ° C.

[0497] The first exponent, the second exponent, and the recirculator (SEQ ID NO: 5, 6, 7, 8, 9, 10, 11, respectively) 12、13) The first strand (SEQ ID NO: 5) and the second strand (SEQ ID NO: 6) of the first indexer were added to 100 µL of SSCM (0.3 M sodium chloride, 0.03 M sodium citrate, 10 mM MgCl2; pH 7) at final concentrations of 1 µM and 2 µM, respectively. The mixture was heated to 94°C for 2 min to remove secondary structures, and then hybridized at 23°C for 30 min. The first indexer hybrid -FL (SEQ ID NO: 7), which had been heated to 70°C for 2 min to remove secondary structures, was added to a final concentration of 2.5 µM, and the mixture was incubated at 23°C for 120 min to remove excess / unhybridized second strands of the first indexer and to provide free first indexer hybrid -FL to the first indexer complex. The final first indexer complex was stored at -25°C. °C. The second indexer complex, the second indexer hybridon, the recirculator complex, and the releaser are generated in a similar manner.

[0498] Exponential target recycling hybridization chain reaction The complex of the detector, first indexer, second indexer, and recycler was warmed to 23°C. Initiator-3 (SEQ ID NO: 20) was diluted in 2xSSC and kept separate from other reagents until use (to prevent contamination). Initiator-3 was incubated at 70°C for 2 minutes to remove secondary structures and cooled to 23°C before use. ° C. Add the following to a new tube: 4 µL of the first indexing complex, 2 µL of the second indexing complex, and 2 µL of the recycling complex. Mix the solutions and centrifuge, then add 1 µL of the detection complex, followed by 1 µL of 2xSSC or 1 µM initiator-3. Mix the solutions and centrifuge, incubate at 25°C, aliquot twice, and measure fluorescence on a fluorometer (FLUOstar Omega PlateReader; BMG Labtech). Subtract the pore background from the results and then normalize relative to the pore background. Figure 32 Error bars represent the maximum and minimum values ​​of repeated samples.

[0499] Example 16: Reducing the reagent concentration Lyophilized oligonucleotides (Table 1) were purchased from IDT and / or Bioneer, dissolved in 2xSSC (300 mM sodium chloride, 30 mM sodium citrate; pH 7.0) at a concentration of 100 µM and stored at -80 °C; working aliquots were stored at -25 °C.

[0500] Detection complex The double-stranded detection molecule was constructed by hybridizing two oligonucleotides, a circular pathway (SEQ ID NO: 2) and a chaperone (SEQ ID NO: 3). The circular pathway and the chaperone were added to 100 µL of 2x SSC (0.3 M sodium chloride, 0.03 M sodium citrate; pH 7) at final concentrations of 1 µM and 2 µM, respectively. The mixture was heated to 94°C for 2 min to remove secondary structures, then hybridized at 25°C for 60 min, followed by hybridization at 16°C for 60 min. The 5' and 3' nucleotides of the circular pathway oligonucleotide (which remained adjacent to each other through hybridization with the chaperone via the circular pathway) were ligated by adding 1x T4 DNA ligase buffer (NEB), 1 µg / mL recombinant albumin (NEB), and T4 DNA ligase (800 U; NEB), incubating overnight at 16°C, and then heat-inactivating at 65°C for 10 min. Unhybridized cyclic pathways and chaperones were removed by purification on a silica membrane column (MineluteReaction Cleanup Kit; QIAGEN) and eluted in 10 µL TE, which was then brought to 80 µL with SSCMT (0.3 M sodium chloride, 0.03 M sodium citrate, 10 mM MgCl2, 0.1% Tween 20; pH 7). Then, a detecton dissociation ion (SEQ ID NO: 15) (which had been heated to 70 °C for 2 min to remove secondary structures) was added to a final concentration of 2.5 µM, and the mixture was incubated at 25 °C for 10 min to remove any remaining unhybridized chaperones and provide the complex with free detecton dissociation ions. The final detecton complex was stored at -25 °C. ° C.

[0501] The first exponent, the second exponent, and the recirculator (SEQ ID NO: 5, 6, 16, 8, 9, 17, respectively) 11、12、18) The first strand (SEQ ID NO: 5) and the second strand (SEQ ID NO: 6) of the first indexer were added to 100 µL of SSCMT at final concentrations of 1 µM and 2 µM, respectively. The mixture was heated to 94°C for 2 min to remove secondary structures and then hybridized at 25°C for 60 min. The first indexer hybrid (SEQ ID NO: 16) (which had been heated to 70°C for 2 min to remove secondary structures) was added to a final concentration of 2.5 µM, and the mixture was incubated at 25°C for 10 min to remove excess / unhybridized second strands of the first indexer and to provide free first indexer hybrids to the first indexer complex. The final first indexer complex was stored at -25°C. ° C. The second indexer complex, the second indexer hybridon, the recirculator complex, and the releaser are generated in a similar manner.

[0502] Exponential target recycling hybridization chain reaction The complex of the detector, first indexer, second indexer, and recycler was warmed to 23°C and diluted 2-fold in SSCMT buffer as needed. The initiator (SEQ ID NO: 1) was diluted in 2xSSC and kept separate from other reagents until use (to prevent contamination). The initiator was incubated at 70°C for 2 minutes to remove secondary structures and cooled to 23°C before use. ° C. Add the following to a new tube: 4 µL of the first indexing complex, 2 µL of the second indexing complex, and 2 µL of the recycling complex. Mix the solutions and centrifuge, then add 1 µL of the detection complex followed by 1 µL of 2xSSC or 1 µM initiator. Mix the solutions and centrifuge, incubate at 25°C, aliquot twice, and measure fluorescence on a fluorometer (FLUOstar Omega Plate Reader; BMG Labtech). Subtract the pore background from the results and then normalize relative to the pore background. Figure 33 Error bars represent the maximum and minimum values ​​of repeated samples.

[0503] Example 17: Second strand with reduced concentration of exponentiation and recycling complex and hybrid / release ETHyR reaction of the release Lyophilized oligonucleotides (Table 1) were purchased from IDT and / or Bioneer, dissolved in 2xSSC (300 mM sodium chloride, 30 mM sodium citrate; pH 7.0) at a concentration of 100 µM and stored at -80 °C; working aliquots were stored at -25 °C.

[0504] Detection complex The double-stranded detection molecule was constructed by hybridizing two oligonucleotides, a circular pathway (SEQ ID NO: 2) and a chaperone (SEQ ID NO: 3). The circular pathway and the chaperone were added to 100 µL of 2x SSC (0.3 M sodium chloride, 0.03 M sodium citrate; pH 7) at final concentrations of 1 µM and 2 µM, respectively. The mixture was heated to 94°C for 2 min to remove secondary structures, and then hybridized at 25°C for 60 min, followed by hybridization at 16°C for 60 min. The 5' and 3' nucleotides of the circular pathway oligonucleotide (which remained adjacent to each other through hybridization of the circular pathway with the chaperone) were ligated by adding 1x T4 DNA ligase buffer (NEB), 1 µg / mL recombinant albumin (NEB), and T4 DNA ligase (800 U; NEB), incubating overnight at 16°C, and then heat-inactivating at 65°C for 10 min. Unhybridized cyclic pathways and chaperones were removed by purification on a silica membrane column (Minelute Reaction Cleanup Kit; QIAGEN) and eluted in 10 µL TE, which was then brought to 80 µL with SSCMT (0.3 M sodium chloride, 0.03 M sodium citrate, 10 mM MgCl2, 0.1% Tween 20; pH 7). Then, a detecton dissociation ion (SEQ ID NO: 15) (which had been heated to 70 °C for 2 min to remove secondary structures) was added to a final concentration of 2.5 µM, and the mixture was incubated at 25 °C for 10 min to remove any remaining unhybridized chaperones and provide the complex with free detecton dissociation ions. The final detecton complex was stored at -25 °C. ° C.

[0505] The first exponent, the second exponent, and the recirculator (SEQ ID NO: 5, 6, 16, 8, 9, 17, respectively) 11、12、18) The first strand (SEQ ID NO: 5) and the second strand (SEQ ID NO: 6) of the first indexer were added to 100 µL of SSCMT at final concentrations of 1 µM and 1.3 to 2 µM, respectively. The mixture was heated to 94°C for 2 min to remove secondary structures and then hybridized at 25°C for 60 min. The first indexer hybrid (SEQ ID NO: 16) (which had been heated to 70°C for 2 min to remove secondary structures) was added to a final concentration of 2.0 to 2.5 µM, and the mixture was incubated at 25°C for 10 min to remove excess / unhybridized second strands of the first indexer and to provide free first indexer hybrids to the first indexer complex. The final first indexer complex was stored at -25°C. °C. The second indexer complex, the second indexer hybridon, the recirculator complex, and the releaser are generated in a similar manner.

[0506] Exponential target recycling hybridization chain reaction The complex of the detector, first indexer, second indexer, and recycler was warmed to 23°C. The initiator (SEQ ID NO: 1) was diluted in 2xSSC and kept separate from other reagents until use (to prevent contamination). The initiator was incubated at 70°C for 2 minutes to remove secondary structures and cooled to 23°C before use. ° C. Add the following to a new tube: 4 µL of the first indexing complex, 2 µL of the second indexing complex, and 2 µL of the recycling complex. Mix and centrifuge the solutions, add 1 µL of the detection complex, and then add 1 µL of 2xSSC or 1 µM initiator. Mix and centrifuge the solutions, incubate at 25°C, aliquot twice, and measure fluorescence on a fluorometer (FLUOstar Omega Plate Reader; BMGLabtech). Subtract the pore background from the results and then normalize relative to the pore background. Figure 34 Error bars represent the maximum and minimum values ​​of repeated samples.

[0507] Example 18: ETHyR reaction with reduced concentration of the first or second exponential complex Lyophilized oligonucleotides (Table 1) were purchased from IDT and / or Bioneer, dissolved in 2xSSC (300 mM sodium chloride, 30 mM sodium citrate; pH 7.0) at a concentration of 100 µM and stored at -80 °C; working aliquots were stored at -25 °C.

[0508] Detection complex The double-stranded detection molecule was constructed by hybridizing two oligonucleotides, a circular pathway (SEQ ID NO: 2) and a chaperone (SEQ ID NO: 3). The circular pathway and the chaperone were added to 100 µL of 2x SSC (0.3 M sodium chloride, 0.03 M sodium citrate; pH 7) at final concentrations of 1 µM and 2 µM, respectively. The mixture was heated to 94°C for 2 min to remove secondary structures, and then hybridized at 25°C for 60 min, followed by hybridization at 16°C for 60 min. The 5' and 3' nucleotides of the circular pathway oligonucleotide (which remained adjacent to each other through hybridization of the circular pathway with the chaperone) were ligated by adding 1x T4 DNA ligase buffer (NEB), 1 µg / mL recombinant albumin (NEB), and T4 DNA ligase (800 U; NEB), incubating overnight at 16°C, and then heat-inactivating at 65°C for 10 min. Unhybridized cyclic pathways and chaperones were removed by purification on a silica membrane column (Minelute Reaction Cleanup Kit; QIAGEN) and eluted in 10 µL TE, which was then brought to 80 µL with SSCMT (0.3 M sodium chloride, 0.03 M sodium citrate, 10 mM MgCl2, 0.1% Tween 20; pH 7). Then, a detecton dissociation ion (SEQ ID NO: 15) (which had been heated to 70 °C for 2 min to remove secondary structures) was added to a final concentration of 2.5 µM, and the mixture was incubated at 25 °C for 10 min to remove any remaining unhybridized chaperones and provide the complex with free detecton dissociation ions. The final detecton complex was stored at -25 °C. ° C.

[0509] The first exponent, the second exponent, and the recirculator (SEQ ID NO: 5, 6, 16, 8, 9, 17, respectively) 11、12、18) The first strand (SEQ ID NO: 5) and the second strand (SEQ ID NO: 6) of the first indexer were added to 100 µL of SSCMT at final concentrations of 1 µM and 2 µM, respectively. The mixture was heated to 94°C for 2 min to remove secondary structures and then hybridized at 25°C for 60 min. The first indexer hybrid (SEQ ID NO: 16) (which had been heated to 70°C for 2 min to remove secondary structures) was added to a final concentration of 2.5 µM, and the mixture was incubated at 25°C for 10 min to remove excess / unhybridized second strands of the first indexer and to provide free first indexer hybrids to the first indexer complex. The final first indexer complex was stored at -25°C. ° C. The second indexer complex, the second indexer hybridon, the recirculator complex, and the releaser are generated in a similar manner.

[0510] Exponential target recycling hybridization chain reaction Warm the detecton, first indexer, second indexer, and recycler complex to 23°C, and dilute either the first or second indexer complex 2-fold in SSCMT buffer as needed. Dilute the initiator (SEQ ID NO: 1) in 2xSSC and keep it separate from other reagents until use (to prevent contamination). Incubate the initiator at 70°C for 2 minutes to remove secondary structures and cool to 23°C before use. ° C. Add the following to a new tube: 4 µL of the first indexing complex, 2 µL of the second indexing complex, and 2 µL of the recycling complex. Mix the solutions and centrifuge, then add 1 µL of the detection complex and subsequently 1 µL of 2xSSC or 1 µM initiator. Mix the solutions and centrifuge, incubate at 25°C, aliquot twice, and measure fluorescence on a fluorometer (FLUOstar Omega Plate Reader; BMGLabtech). Subtract the pore background from the results and then normalize relative to the pore background. Figure 35 ).

[0511] Example 19: Larger overhang Lyophilized oligonucleotides (Table 1) were purchased from IDT and / or Bioneer, dissolved in 2xSSC (300 mM sodium chloride, 30 mM sodium citrate; pH 7.0) at a concentration of 100 µM and stored at -80 °C; working aliquots were stored at -25 °C.

[0512] Detection of subcomplexes (SEQ ID NO: 2, 30 and 15) A double-stranded detector molecule with a single-stranded target-binding region of 3 nucleotides (10 nucleotides in length instead of 7 nucleotides) was constructed by hybridizing two oligonucleotides, a circular pathway (SEQ ID NO: 2) and chaperone-77 (SEQ ID NO: 30). The circular pathway and chaperone-77 were added to 100 µL of 2xSSC (0.3 M sodium chloride, 0.03 M sodium citrate; pH 7) at final concentrations of 1 µM and 2 µM, respectively. Heating to 94°C for 2 min was performed to remove secondary structures, followed by hybridization at 25°C for 60 min and then at 16°C for 60 min. The 5' and 3' nucleotides of the circular pathway oligonucleotides (which remain adjacent to each other through hybridization with chaperone-77 via the circular pathway) were ligated as follows: T4 DNA ligase buffer (NEB) to a final concentration of 1x, recombinant albumin (NEB) to a final concentration of 1 µg / mL, and T4 DNA ligase (800 U; NEB) were added, incubated overnight at 16°C, and then heat-inactivated at 65°C for 10 min. The detecton complex was purified by ethanol precipitation and returned to SSCMT buffer (0.3 M sodium chloride, 0.03 M sodium citrate, 10 mM MgCl2, 0.1% Tween 20; pH 7). Then, the detecton dissociation ion (SEQ ID NO: 15) (which had been heated to 70°C for 2 min (to remove secondary structure)) was added to a final concentration of 2.5 µM, and the mixture was incubated at 25°C for 10 min to remove any remaining unhybridized chaperone-77 and provide the complex with free detecton dissociation ions. The final detection complex was stored at -25°C. ° C.

[0513] The first exponent, the second exponent, and the recirculator (SEQ ID NO: 5, 6, 16, 8, 9, 17, respectively) 11、12、18) The first strand (SEQ ID NO: 5) and the second strand (SEQ ID NO: 6) of the first indexer were added to 100 µL of SSCM (0.3 M sodium chloride, 0.03 M sodium citrate, 10 mM MgCl2; pH 7) at final concentrations of 1 µM and 2 µM, respectively. The mixture was heated to 94°C for 2 min to remove secondary structures and then hybridized at 23°C for 30 min. The first indexer hybrid (SEQ ID NO: 16) (which had been heated to 70°C for 2 min to remove secondary structures) was added to a final concentration of 2.5 µM, and the mixture was incubated at 23°C for 120 min to remove excess / unhybridized second strands of the first indexer and to provide free first indexer hybrids to the first indexer complex. The final first indexer complex was stored at -25°C. °C. The second indexer complex, the second indexer hybridon, the recirculator complex, and the releaser are generated in a similar manner.

[0514] Exponential target recycling hybridization chain reaction The complex of the detector, first indexer, second indexer, and recycler was warmed to 23°C. Initiator-3 (SEQ ID NO: 20) was diluted in 2xSSC and kept separate from other reagents until use (to prevent contamination). Initiator-3 was incubated at 70°C for 2 minutes to remove secondary structures and cooled to 23°C before use. ° C. Add the following to a new tube: 4 µL of the first indexing complex, 2 µL of the second indexing complex, and 2 µL of the recycling complex. Mix the solutions and centrifuge, then add 1 µL of the detection complex followed by 1 µL of 2xSSC or 1 µM initiator-3. Mix the solutions and centrifuge, incubate at 25°C, and measure fluorescence on a fluorometer (FLUOstar Omega Plate Reader; BMGLabtech). Subtract the pore background from the results and then normalize relative to the pore background. Figure 36 ).

[0515] Example 20: Different stem sequences to reduce hybrids and / or recycles compared to exponents and / or recycles Spontaneous binding of the second chain of the complex Lyophilized oligonucleotides (Table 1) were purchased from IDT and / or Bioneer, dissolved in 2xSSC (300 mM sodium chloride, 30 mM sodium citrate; pH 7.0) at a concentration of 100 µM and stored at -80 °C; working aliquots were stored at -25 °C.

[0516] The sequence of the first exponentiation complex was modified by replacing some AT bonds with GC bonds to increase the binding strength between the first and second strands of the first exponentiation complex-8. The sequence of the recycling complex was modified by replacing some AT bonds with GC bonds to increase the binding strength between the first and second strands of the recycling complex-9. The sequence of the recycling complex was also modified separately by replacing some GC bonds with AT bonds to decrease the binding strength between the second strand of the recycling complex-2 and the releaser.

[0517] First exponent, first exponent complex-8, recycler, recycler complex-9 and recycler Complex-2 (SEQ ID NOs: 5, 6, 16, 31, 32 and 16; 11, 12, 18; 33, 34 and 18; 35, 36, 37, respectively) The first strand (SEQ ID NO: 31) and the second strand (SEQ ID NO: 32) of the first indexer were added to 100 µL of SSCM (0.3 M sodium chloride, 0.03 M sodium citrate, 10 mM MgCl2; pH 7) at final concentrations of 1 µM and 2 µM, respectively. The mixture was heated to 94°C for 2 min to remove secondary structures and then hybridized at 23°C for 30 min. The first indexer hybrid (SEQ ID NO: 16) (which had been heated to 70°C for 2 min to remove secondary structures) was added to a final concentration of 2.5 µM, and the mixture was incubated at 23°C for 120 min to remove excess / unhybridized second strands of the first indexer and to provide free first indexer hybrids to the first indexer complex. The final first indexer complex was stored at -25°C. ° C. The first indexing subcomplex-8, the recycling subcomplex, the recycling subcomplex-9, and the recycling subcomplex-2 are all generated in a similar manner from SEQ ID NO:31, 32, 16; 11, 12, 18; 33, 34 and 18; 35, 36, 37, respectively.

[0518] From the first exponent, the first exponent complex-8, the first exponent complex-2, and the recycler. Background signals of recycle subcomplex-9 and recycle subcomplex-2 The complex was warmed at 25°C, mixed, and centrifuged, incubated at 25°C, and fluorescence was measured on a fluorometer (FLUOstar OmegaPlate Reader; BMG Labtech). The results were subtracted from the pore background and then normalized relative to the pore background.

[0519] The background fluorescence from the first indexing complex-8 was 46% of the level of the first indexing complex, while the background fluorescence from the recycling complex-9 and recycling complex-2 was 70% and 25% of the level of the recycling complex, respectively. Figure 37 ).

[0520] Example 21: Different exponentiation subsequences to further shift the starting region of the exponentiation away from the circular path. district Various methods exist for modifying exponents to alter the position of the hybridization region, which can hybridize with the initiating hybridization region of another cyclic path. This embodiment uses a two-step strategy to further move the initiating hybridization region of the first exponent away from the cyclic path, wherein the initiating region is located in a second segment containing hairpin hybrids. Similar modifications can be made to the second exponent.

[0521] Lyophilized oligonucleotides (Table 1) were purchased from IDT and / or Bioneer, dissolved in 2xSSC (300 mM sodium chloride, 30 mM sodium citrate; pH 7.0) at a concentration of 100 µM and stored at -80 °C; working aliquots were stored at -25 °C.

[0522] Detection complex The double-stranded detection molecule was constructed by hybridizing two oligonucleotides, a circular pathway (SEQ ID NO: 2) and a chaperone (SEQ ID NO: 3). The circular pathway and the chaperone were added to 100 µL of 2x SSC (0.3 M sodium chloride, 0.03 M sodium citrate; pH 7) at final concentrations of 1 µM and 2 µM, respectively. The mixture was heated to 94°C for 2 min to remove secondary structures, and then hybridized at 25°C for 60 min, followed by hybridization at 16°C for 60 min. The 5' and 3' nucleotides of the circular pathway oligonucleotide (which remained adjacent to each other through hybridization of the circular pathway with the chaperone) were ligated by adding 1x T4 DNA ligase buffer (NEB), 1 µg / mL recombinant albumin (NEB), and T4 DNA ligase (800 U; NEB), incubating overnight at 16°C, and then heat-inactivating at 65°C for 10 min. Unhybridized cyclic pathways and chaperones were removed by purification on a silica membrane column (Minelute Reaction Cleanup Kit; QIAGEN) and eluted in 10 µL TE, which was then brought to 80 µL with SSCMT (0.3 M sodium chloride, 0.03 M sodium citrate, 10 mM MgCl2, 0.1% Tween 20; pH 7). Then, a detecton dissociation ion (SEQ ID NO: 15) (which had been heated to 70 °C for 2 min to remove secondary structures) was added to a final concentration of 2.5 µM, and the mixture was incubated at 25 °C for 10 min to remove any remaining unhybridized chaperones and provide the complex with free detecton dissociation ions. The final detecton complex was stored at -25 °C. ° C.

[0523] First exponent -H (SEQ ID NO: 38, 39, 40, 41, 42, 43) The first strand (SEQ ID NO: 38) and the second strand (SEQ ID NO: 39) of the first indexer-H-segment-1 were added to 100 µL of SSCMT at final concentrations of 2 µM and 4 µM, respectively. The mixture was heated to 94°C for 2 min to remove secondary structures and then hybridized at 25°C for 60 min. The first indexer hybridizer-H-segment-1 (SEQ ID NO: 40), which had been heated to 70°C for 2 min to remove secondary structures, was added to the first indexer-H-segment-1 complex at a final concentration of 5 µM and incubated at 25°C for 10 min to remove excess / unhybridized second strand of the first indexer-H-segment-1 and to provide free first indexer hybridizer-H-segment-1 to the first indexer-H-segment-1 complex. Segment-2 of the first indexer-H complex was generated in a similar manner. Equal volumes of segments-1 and segment-2 of the first indexer-H complex were then mixed and incubated at 25°C for 10 min. The final first exponentiation-H complex is stored at -25°C. ° C.

[0524] The second exponent and the recirculator (SEQ ID NO: 8, 9, 17 and 11, 12, 18, respectively) The first strand of the second indexer (SEQ ID NO: 8) and the second strand of the second indexer (SEQ ID NO: 9) were added to 100 µL of SSCMT at final concentrations of 1 µM and 2 µM, respectively. The mixture was heated to 94°C for 2 min to remove secondary structures and then hybridized at 25°C for 60 min. The second indexer hybrid (SEQ ID NO: 17), which had been heated to 70°C for 2 min to remove secondary structures, was added to a final concentration of 2.5 µM, and the mixture was incubated at 25°C for 10 min to remove excess / unhybridized second strands of the second indexer and to provide free second indexer hybrids to the second indexer complex. The final second indexer complex was stored at -25°C. ° C. The recycling subcomplex is generated in a similar manner.

[0525] Exponential target recycling hybridization chain reaction The detecton, first indexer-H, second indexer, and recycler complex were warmed to 23°C. Initiator-3 (SEQ ID NO: 20) was diluted in 2xSSC and kept separate from other reagents until use (to prevent contamination). The initiator was incubated at 70°C for 2 minutes to remove secondary structures and cooled to 23°C before use. °C. Add the following to a new tube: 4 µL of the first indexer-H complex, 2 µL of the second indexer complex, and 2 µL of the recycling complex. Mix the solutions and centrifuge, then add 1 µL of the detectant complex followed by 1 µL of 2xSSC or 1 µL of 1 µM initiator-3. Mix the solutions and centrifuge, incubate at 25°C, and measure fluorescence on a fluorometer (FLUOstar Omega Plate Reader; BMG Labtech). Subtract the pore background from the results and then normalize relative to the pore background. Figure 38 Error bars represent the maximum and minimum values ​​of repeated samples.

[0526] Example 22: Different exponentiation subsequences to further shift the starting region of the exponentiation away from the circular path. district Various methods exist for modifying exponents to optimize the location of the hybridization region, which can hybridize with the initiation hybridization region of another cyclic path. This embodiment uses a two-step strategy to further move the initiation hybridization region of the first exponent away from the cyclic path, where the split initiation region is located in the second segment. Similar modifications can be made to the second exponent.

[0527] Lyophilized oligonucleotides (Table 1) were purchased from IDT and / or Bioneer, dissolved in 2xSSC (300 mM sodium chloride, 30 mM sodium citrate; pH 7.0) at a concentration of 100 µM and stored at -80 °C; working aliquots were stored at -25 °C.

[0528] Detection complex The double-stranded detection molecule was constructed by hybridizing two oligonucleotides, a circular pathway (SEQ ID NO: 2) and a chaperone (SEQ ID NO: 3). The circular pathway and the chaperone were added to 100 µL of 2x SSC (0.3 M sodium chloride, 0.03 M sodium citrate; pH 7) at final concentrations of 1 µM and 2 µM, respectively. The mixture was heated to 94°C for 2 min to remove secondary structures, and then hybridized at 25°C for 60 min, followed by hybridization at 16°C for 60 min. The 5' and 3' nucleotides of the circular pathway oligonucleotide (which remained adjacent to each other through hybridization of the circular pathway with the chaperone) were ligated by adding 1x T4 DNA ligase buffer (NEB), 1 µg / mL recombinant albumin (NEB), and T4 DNA ligase (800 U; NEB), incubating overnight at 16°C, and then heat-inactivating at 65°C for 10 min. Unhybridized cyclic pathways and chaperones were removed by purification on a silica membrane column (Minelute Reaction Cleanup Kit; QIAGEN) and eluted in 10 µL TE, which was then brought to 80 µL with SSCMT (0.3 M sodium chloride, 0.03 M sodium citrate, 10 mM MgCl2, 0.1% Tween 20; pH 7). Then, a detecton dissociation ion (SEQ ID NO: 15) (which had been heated to 70 °C for 2 min to remove secondary structures) was added to a final concentration of 2.5 µM, and the mixture was incubated at 25 °C for 10 min to remove any remaining unhybridized chaperones and provide the complex with free detecton dissociation ions. The final detecton complex was stored at -25 °C. ° C.

[0529] First exponent-S complex (SEQ ID NO: 44-48) The first strand (SEQ ID NO: 44) and the second strand (SEQ ID NO: 45) of the first indexer-S-segment-1 were added to 100 µL of SSCMT at final concentrations of 1 µM and 2 µM, respectively. The mixture was heated to 94°C for 2 min to remove secondary structures and then hybridized at 25°C for 60 min. The first indexer hybridizer-S-segment-1 (SEQ ID NO: 46) (which had been heated to 70°C for 2 min to remove secondary structures) was added to the mixture at a final concentration of 2.5 µM, and the mixture was incubated at 25°C for 10 min to remove excess / unhybridized second strand of the first indexer-S-segment-1 and to provide free first indexer hybridizer-S-segment-1 to the first indexer-S-segment-1 complex. The first half of the first exponent hybrid -S-segment-2 (SEQ ID NO: 47) and the second half of the first exponent hybrid -S-segment-2 (SEQ ID NO: 48) (which had been heated to 70°C for 2 min to remove secondary structure) were each added at a final concentration of 2.0 µM, and the mixture was incubated at 25°C for 10 min. The final first exponent hybrid -S complex was stored at -25°C. ° C.

[0530] The second exponent and the recirculator (SEQ ID NO: 8, 9, 17 and 11, 12, 18, respectively) The first strand of the second exponent (SEQ ID NO: 8) and the second strand of the second exponent (SEQ ID NO: 9) were added to 100 µL of SSCMT at final concentrations of 1 µM and 2 µM, respectively. The mixture was heated to 94°C for 2 min to remove secondary structures and then hybridized at 25°C for 60 min. The second exponent hybrid (SEQ ID NO: 17), which had been heated to 70°C for 2 min to remove secondary structures, was added to the mixture at a final concentration of 2.5 µM, and the mixture was incubated at 25°C for 10 min to remove excess / unhybridized second strands of the second exponent and to provide free second exponent hybrids to the second exponent complex. The final second exponent complex was stored at -25°C. The recycled complex was produced in a similar manner.

[0531] Exponential target recycling hybridization chain reaction The complex of the detector, first indexer-S, second indexer, and recycler was warmed to 23°C. Initiator-3 (SEQ ID NO: 20) was diluted in 2xSSC and kept separate from other reagents until use (to prevent contamination). The initiator was incubated at 70°C for 2 minutes to remove secondary structures and cooled to 23°C before use. °C. Add the following to a new tube: 4 µL of the first indexer-S complex, 2 µL of the second indexer complex, and 2 µL of the recycling complex. Mix the solutions and centrifuge, then add 1 µL of the detectant complex followed by 1 µL of 2xSSC or 1 µL of 1 µM initiator-3. Mix the solutions and centrifuge, incubate at 25°C, and measure fluorescence on a fluorometer (FLUOstar Omega PlateReader; BMG Labtech). Subtract the pore background from the results and then normalize relative to the pore background. Figure 39 Error bars represent the maximum and minimum values ​​of repeated samples.

[0532] Example 23: Reduced initiator regions are dimerized and the initiator regions of exponents are further moved away from the loop path. Integration Zone The function of the initiation region can be improved by minimizing homodimerization between initiation regions on different molecules and / or by further shifting the initiation regions on the first strand of the exponents away from their respective circular pathway binding regions. This embodiment modifies the initiation region sequence to reduce homodimerization and further shift the initiation regions on the first strand of the exponents away from their respective circular pathway binding regions. This is achieved by using a four-nucleotide-shortened exponent binding region on the circular pathway and adding a 4-nucleotide linker sequence between the circular pathway binding region of each exponent and the initiation region binding domain of each exponent.

[0533] Lyophilized oligonucleotides (Table 1) were purchased from IDT and / or Bioneer, dissolved in 2xSSC (300 mM sodium chloride, 30 mM sodium citrate; pH 7.0) at a concentration of 100 µM and stored at -80 °C; working aliquots were stored at -25 °C.

[0534] Detection complex The double-stranded detector molecule was constructed by hybridizing two oligonucleotides, a circular pathway (SEQ ID NO: 49) and a chaperone (SEQ ID NO: 50). The circular pathway and the chaperone were added to 100 µL of 2x SSC (0.3 M sodium chloride, 0.03 M sodium citrate; pH 7) at final concentrations of 1 µM and 2 µM, respectively. The mixture was heated to 94°C for 2 min to remove secondary structures, and then hybridized at 25°C for 60 min, followed by hybridization at 16°C for 60 min. The 5' and 3' nucleotides of the circular pathway oligonucleotide (which remained adjacent to each other through hybridization of the circular pathway with the chaperone) were ligated by adding 1x T4 DNA ligase buffer (NEB), 1 µg / mL recombinant albumin (NEB), and T4 DNA ligase (800 U; NEB), incubating overnight at 16°C, and then heat-inactivating at 65°C for 10 min. Unhybridized cyclic pathways and chaperones were removed by purification on a silica membrane column (Minelute Reaction Cleanup Kit; QIAGEN) and eluted in 10 µL TE, which was then brought to 80 µL with SSCMT (0.3 M sodium chloride, 0.03 M sodium citrate, 10 mM MgCl2, 0.1% Tween 20; pH 7). Then, a detecton dissociation ion (SEQ ID NO: 51) (which had been heated to 70 °C for 2 min to remove secondary structures) was added to a final concentration of 2.5 µM, and the mixture was incubated at 25 °C for 10 min to remove any remaining unhybridized chaperones and provide the complex with free detecton dissociation ions. The final detecton complex was stored at -25 °C. ° C.

[0535] First exponentiation, second exponentiation, and recirculator The first strand (SEQ ID NO: 52) and the second strand (SEQ ID NO: 53) of the first indexer were added to 100 µL of SSCMT at final concentrations of 1 µM and 2 µM, respectively. The mixture was heated to 94°C for 2 min to remove secondary structures and then hybridized at 25°C for 60 min. The first indexer hybrid (SEQ ID NO: 54) (which had been heated to 70°C for 2 min to remove secondary structures) was added to a final concentration of 2.5 µM, and the mixture was incubated at 25°C for 10 min to remove excess / unhybridized second strands of the first indexer and to provide free first indexer hybrids to the first indexer complex. The final first indexer complex was stored at -25°C. ° C. The second indexer complex, the second indexer hybridon, the recirculator complex, and the releaser are generated in a similar manner.

[0536] Exponential target recycling hybridization chain reaction The complex of the detector, first indexer-S, second indexer, and recycler was warmed to 23°C. The initiator (SEQ ID NO: 28) was diluted in 2xSSC and kept separate from other reagents until use (to prevent contamination). The initiator was incubated at 70°C for 2 minutes to remove secondary structures and cooled to 23°C before use. ° C. Add the following to a new tube: 12 µL of the first indexing complex, 6 µL of the second indexing complex, and 6 µL of the recycling complex. Mix the solutions and centrifuge, then add 3 µL of the detection complex followed by 2 µL of 2xSSC, 2 µL of 0.1 µM, or 2 µL of 1 µM initiator. Mix the solutions and centrifuge, incubate at 25°C, and measure fluorescence on a fluorometer (FLUOstar Omega PlateReader; BMG Labtech). Subtract the pore background from the results and then normalize relative to the pore background. Figure 40 Error bars represent the maximum and minimum values ​​of repeated samples.

[0537] Those skilled in the art will understand that this disclosure may be implemented in many other forms.

Claims

1. A method for detecting nucleic acid targets in a sample, the method comprising: (a) Contacting the sample with a reaction mixture, the reaction mixture comprising: - A detector comprising a circular pathway containing a polynucleotide hybridization region that hybridizes with a polynucleotide chaperone, the circular pathway including multiple loops that do not hybridize with the chaperone and an exposed single-stranded initiation hybridization region that does not hybridize with the chaperone, wherein the initiation hybridization region is complementary to the hybridization region of the target. - One or more polynucleotides, wherein at least one of the polynucleotides is linked to the detection portion; (b) Hybridize the target with the initial hybridization region; (c) Separating the partner from the circumferential path to expose multiple hybridization regions on the circumferential path; (d) In a circumferential hybridization reaction, the one or more polynucleotides are hybridized to a plurality of exposed hybridization regions along the circumferential pathway, wherein the circumferential hybridization reaction induces modification of at least one polynucleotide linked to a detection portion, such that the detection portion is capable of generating a detectable signal; and (e) Measure the presence or absence of the detectable signal, wherein the detectable signal indicates the presence of a target in the sample.

2. The method according to claim 1, wherein the detection portion is a fluorophore, which emits a detectable fluorescent signal after being separated from the quencher space.

3. The method according to claim 1 or claim 2, wherein the at least one polynucleotide is linked to a fluorophore and a quencher, and wherein the modification includes spatial separation of the fluorophore from the quencher, such that the fluorophore emits a detectable signal.

4. The method according to any one of claims 1 to 3, wherein the at least one polynucleotide is linked to a fluorophore and a quencher and comprises a double-stranded region, and wherein the strand of the double-stranded region dissociates after the at least one polynucleotide hybridizes with the circular pathway, such that the fluorophore is spatially separated from the quencher and emits a detectable signal.

5. The method according to any one of claims 1 to 4, wherein the one or more polynucleotides comprise a plurality of polynucleotides, each polynucleotide being linked to a detection portion.

6. The method of claim 5, wherein each of the plurality of polynucleotides is linked to a fluorophore and a quencher and comprises a double-stranded region, and wherein the strands of each double-stranded region dissociate after hybridization with the cyclic pathway, such that each fluorophore is spatially separated from the quencher and emits a detectable signal.

7. The method according to any one of claims 1 to 6, wherein the separation of the companion from the loop path is facilitated by opening a loop in the loop path.

8. The method according to any one of claims 1 to 7, wherein the circumferential hybridization reaction separates the target from the circumferential path.

9. The method according to any one of claims 1 to 8, wherein the hybridization of the target with the initial hybridization region replaces the hybridization region of the partner from the circumferential path.

10. The method of claim 9, wherein the reaction mixture further comprises a detection dissociation ion comprising a plurality of polynucleotide hybridization regions that hybridize with the chaperone, thereby promoting the dissociation of the chaperone from the cyclic pathway.

11. The method of claim 10, wherein at least a portion of the substituted hybridization region of the mate hybridizes with a hybridization region on the detector dissociation ion, and a plurality of hybridization regions on the detector dissociation ion hybridize with hybridization regions on the mate, thereby promoting separation of the mate from the circumferential path.

12. The method according to any one of claims 1 to 11, wherein at least one of the polynucleotides is an indexer, the indexer comprising a double-stranded portion of a complementary hybridization region pair and a single-stranded overhang extending beyond the double-stranded portion, and wherein the circumferential hybridization reaction comprises: - The overhang hybridizes with the hybridization region on the annular path; and - At least one hybridization region adjacent to the overhang hybridizes with a complementary hybridization region on the circumferential path, thereby at least partially opening the double-stranded portion.

13. The method of claim 12, wherein the indexer comprises a first-strand polynucleotide and a second-strand polynucleotide hybridized through at least two complementary hybridization regions, the first strand extending beyond the second strand to form a dangling overhang and comprising at least one loop that does not hybridize with the second strand, and wherein the circumferential hybridization reaction comprises: - The overhang hybridizes with the hybridization region on the annular path; and - At least one hybridization region on the first chain adjacent to the overhang hybridizes with a hybridization region on the loop path, thereby replacing the hybridization region on the second chain of the exponent and opening the loop on the first chain of the exponent.

14. The method of claim 13, wherein the flanks of the ring of the indexinducer are complementary hybridization pairs that allow the first strand of the indexinducer to hybridize with the second strand of the indexinducer.

15. The method of claim 13 or claim 14, wherein the reaction mixture further comprises an indexintegrator hybridizer comprising at least two polynucleotide hybridization regions that hybridize with one strand of the indexintegrator, thereby separating the first strand of the indexintegrator from the second strand of the indexintegrator.

16. The method of claim 15, wherein the circumferential hybridization reaction comprises: - The overhang hybridizes with the hybridization region on the annular path; - At least one hybridization region of a neighboring overhang of the first chain hybridizes with a hybridization region on the loop path, thereby displacing the hybridization region on the second chain of the exponentiated subchain and opening the loop on the first chain of the exponentiated subchain; and - The hybridization region of the indexed hybrid hybrid crosses with at least a portion of the substituted hybridization region on the second strand of the indexed hybrid hybrid, and another hybridization region of the indexed hybrid hybrid crosses with a hybridization region on the second strand of the indexed hybrid hybrid, thereby separating the first strand of the indexed hybrid hybrid from the second strand of the indexed hybrid hybrid. Optionally, at least one hybridization region of the first chain of the indexed subchain hybridizes with a hybridization region on the cyclic path, and at least one hybridization region of the first chain of the indexed subchain does not hybridize with the cyclic path.

17. The method according to any one of claims 13 to 16, wherein the first chain of the indexer includes a hybridization region capable of hybridizing with an initial hybridization region of an additional cyclic path, thereby initiating the amplification of the method.

18. The method according to any one of claims 13 to 17, wherein the first chain of the indexer comprises a first hybridization region capable of hybridizing with an initial hybridization region on an additional cyclic path and a second hybridization region capable of hybridizing with an adjacent hybridization region on the additional cyclic path, thereby initiating the amplification of the method.

19. The method of claim 16, wherein at least one hybridization region of the first strand of the indexed substituent that does not hybridize with the circular pathway hybridizes with the initiation hybridization region of another detector, thereby initiating the amplification of the method.

20. The method according to any one of claims 13 to 19, wherein the first chain of the indexer comprises a hybridization region that is identical to or has at least about 90% sequence identity with the hybridization region of the target.

21. The method of claim 16 or claim 19, wherein at least one hybridization region of the first strand of the indexed substituent that does not hybridize with the cyclic pathway includes a hybridization region identical to the hybridization region of the target or a hybridization region having at least about 90% sequence identity with the hybridization region of the target.

22. The method according to any one of claims 13 to 21, wherein the loop of the first strand of the indexed subchain comprises a target hybridization region or a sequence having at least about 90% sequence identity with the target hybridization region.

23. The method according to any one of claims 13 to 22, wherein each chain of the exponent is connected to the detection portion, and wherein the separation of the first chain and the second chain of the exponent enables the detection portion of the exponent to be spatially separated and generate a detectable signal.

24. The method according to any one of claims 12 to 23, wherein the reaction mixture comprises at least two different types of exponents.

25. The method of claim 24, wherein each type of indexer hybridizes with a different hybridization region on the cyclic path.

26. The method according to any one of claims 15 to 25, wherein the reaction mixture comprises a plurality of exponential hybrids.

27. The method according to any one of claims 1 to 11, wherein the one or more polynucleotides comprise: - At least one first indexer comprising a first-strand polynucleotide and a second-strand polynucleotide hybridized through at least two complementary hybridization regions, the first strand extending beyond the second strand to form a single-stranded overhang and comprising at least one loop not hybridizing with the second strand, wherein each strand is connected to a detection portion; and - At least one second exponentiator, the second exponentiator comprising a first-strand polynucleotide and a second-strand polynucleotide hybridized through at least two complementary hybridization regions, the first strand extending beyond the second strand to form a single-stranded overhang and comprising at least one loop that does not hybridize with the second strand, wherein each strand is connected to the detection portion. And said reaction mixture comprises: - The first exponential hybrid, which contains at least two polynucleotide hybridization regions; and - The second exponential hybrid, which contains at least two polynucleotide hybridization regions. And the circumferential hybridization reaction mentioned above includes: - At least one dangling protrusion of the first indexer hybridizes with a hybridization region on the loop path, and at least one adjacent hybridization region on the first chain of the first indexer hybridizes with a hybridization region on the loop path, thereby displacing the hybridization region on the second chain of the first indexer and opening the loop on the first chain of the first indexer; - At least one dangling protrusion of the second indexer hybridizes with a complementary hybridization region on the loop path, and at least one adjacent hybridization region on the first chain of the second indexer hybridizes with a hybridization region on the loop path, thereby displacing the hybridization region on the second chain of the second indexer and opening the loop on the first chain of the second indexer; - The hybridization region of the first exponent hybridizes with at least a portion of the substituted hybridization region on the second strand of the first exponent, and another hybridization region on the first exponent hybridizes with a hybridization region on the second strand of the first exponent, thereby separating the first strand of the first exponent from the second strand of the first exponent, thus enabling the detection portion of the first exponent to be spatially separated and generate a detectable signal. Optionally, at least one hybridization region of the first exponentiated subchain hybridizes with a hybridization region on the cyclic path, and at least one hybridization region on the first exponentiated subchain does not hybridize with the cyclic path; and - The hybridization region of the second exponent hybridizes with at least a portion of the substituted hybridization region on the second strand of the second exponent, and another hybridization region on the second exponent hybridizes with the hybridization region on the second strand of the second exponent, thereby separating the first strand of the second exponent from the second strand of the second exponent, thus enabling the detection portion of the second exponent to be spatially separated and generate a detectable signal. Optionally, at least one hybridization region of the first chain of the second exponent hybridizes with a hybridization region on the cyclic path, and at least one hybridization region on the first chain of the second exponent hybridizes not with the cyclic path.

28. The method according to any one of claims 1 to 27, wherein at least one of the polynucleotides is a recycler comprising at least two hybridization regions, wherein the hybridization region of the recycler hybridizes with a hybridization region adjacent to the initiating hybridization region on the cyclic path, and the other hybridization region of the recycler hybridizes with the initiating hybridization region, thereby displacing the target from the cyclic path.

29. The method of claim 28, wherein the recycle subcomponent comprises: The double-stranded portion of the complementary hybridization region pair is interrupted by a loop in one strand, the loop containing the hybridization region; and The single-chain portion extends beyond the double-chain portion. And the circumferential hybridization reaction mentioned above includes: - The single-stranded portion hybridizes with the hybridization region on the circular path; - At least one hybridization region adjacent to the single-stranded portion hybridizes with a hybridization region adjacent to the initiating hybridization region on the cyclic path, thereby opening the loop of the recycler; and - The hybridization region of the loop of the recycler hybridizes with at least a portion of the initial hybridization region, thereby displacing the target from the loop path.

30. The method of claim 28 or claim 29, wherein the recycler comprises a first-strand polynucleotide and a second-strand polynucleotide hybridized through at least two complementary hybridization regions, the first strand extending beyond the second strand to form a single-stranded portion and comprising at least one loop that does not hybridize with the second strand, and wherein the circumferential hybridization reaction comprises: - The single-chain portion hybridizes with the hybridization region on the circumferential path; - The hybridization region of the recycle's adjacent single-stranded portion hybridizes with the hybridization region of the cyclic path adjacent to the initial hybridization region, thereby opening the loop of the recycle; and - The hybridization region on the opened loop of the recirculator hybridizes with at least a portion of the initial hybridization region, thereby displacing the target from the loop path.

31. The method of claim 30, wherein the reaction mixture comprises a releaser having at least two polynucleotide hybridization regions that hybridize with one strand of the recycle, thereby separating the first strand of the recycle from the second strand of the recycle.

32. The method of claim 31, wherein the circumferential hybridization reaction comprises: - The single-stranded portion of the recycler hybridizes with the hybridization region on the cyclic path; - The hybridization region of the adjacent single-strand portion of the recycle hybridizes with the hybridization region of the adjacent starting hybridization region of the loop path, thereby displacing the hybridization region of the second strand of the recycle and opening the loop on the first strand of the recycle; - The hybridization region in the opened loop of the recirculator hybridizes with at least a portion of the initial hybridization region, thereby displacing the hybridization region of the target from the cyclic path; - At least a portion of the substituted hybridization region of the second strand of the recycle hybridizes with the hybridization region on the releaser, and the adjacent hybridization region on the releaser hybridizes with the hybridization region on the second strand of the recycle, thereby separating the first strand of the recycle from the second strand of the recycle; and - The hybridization region of the first chain of the recycle subhybridizes with the initiation hybridization region, thereby separating the target from the cyclic path.

33. The method according to any one of claims 30 to 32, wherein the first chain of the recycler comprises a hybridization region that is identical to or has at least about 90% sequence identity with the hybridization region of the target.

34. The method according to any one of claims 30 to 33, wherein the loop of the first strand of the recycle subcontinent comprises a target hybridization region or a sequence having at least about 90% sequence identity with the target hybridization region.

35. The method according to any one of claims 30 to 34, wherein each chain of the recirculator is connected to the detection portion, and wherein the separation of the first chain and the second chain of the recirculator enables the detection portion of the recirculator to be spatially separated and generate a detectable signal.

36. The method according to any one of claims 1 to 35, wherein the cyclic path comprises a plurality of loops, each loop comprising a hybridization region.

37. The method according to any one of claims 12 to 27, wherein the dangling protrusion of the indexintegrator hybridizes with a hybridization region present in the open loop of the circumferential path.

38. The method according to any one of claims 8 to 37, wherein the released target hybridizes with the initiation hybridization region of another detector, thereby initiating the amplification of the method.

39. The method according to any one of claims 1 to 38, wherein the loop path comprises four loops.

40. The method according to any one of claims 1 to 39, wherein the loop path is a closed loop path.

41. The method according to any one of claims 1 to 40, wherein the circumferential hybridization reaction is carried out under isothermal conditions.

42. The method of claim 41, wherein the circumferential hybridization reaction is carried out at a temperature of about 20°C to about 25°C.

43. The method according to any one of claims 1 to 42, wherein the reaction mixture does not contain a nuclease.

44. The method according to any one of claims 1 to 43, wherein the reaction mixture does not contain polymerase.

45. The method according to any one of claims 1 to 44, wherein the reaction mixture does not contain an enzyme.

46. ​​The method according to any one of claims 13 to 45, wherein: (i) The first strand of the indexer is connected to a fluorophore and the second strand of the indexer is connected to a quencher; or (ii) The first chain of the indexer is connected to a quencher and the second chain of the indexer is connected to a fluorophore.

47. The method according to any one of claims 30 to 46, wherein: (i) The first chain of the recycler is connected to a fluorophore and the second chain of the recycler is connected to a quencher; or (ii) The first chain of the recycler is connected to a quencher and the second chain of the recycler is connected to a fluorophore.

48. A method for detecting a nucleic acid target in a sample, the method comprising: Contact the sample with the complex, the complex comprising A cyclic polynucleotide that partially hybridizes with a polynucleotide chaperone, wherein the complex comprises a plurality of unhybridized loops and a single-stranded initiation region complementary to a target nucleic acid suspected of being present in the sample; Introducing one or more polynucleotides, wherein at least one of the polynucleotides contains a detection moiety; After the target binds to the initiation region, the chaperone is separated from the cyclic polynucleotide; A circumferential hybridization reaction is performed to hybridize a plurality of the polynucleotides to a loop region, wherein the circumferential hybridization reaction induces modification of at least one polynucleotide to generate a detectable signal; and The signal is detected as an indication of the presence of a target in the sample.

49. A composition comprising a detector comprising a circular pathway of a polynucleotide hybridization region that hybridizes with a polynucleotide chaperone, the circular pathway comprising one or more loops that do not hybridize with the chaperone and an exposed initiating hybridization region that does not hybridize with the chaperone, wherein the initiating hybridization region is complementary to a target hybridization region.

50. The composition of claim 49, wherein the exposed initiation hybridization region is single-stranded.

51. The composition of claim 49 or claim 50, further comprising a detector dissociation ion comprising a plurality of polynucleotide hybridization regions capable of hybridizing with hybridization regions on the chaperone and dissociating the chaperone from the cyclic pathway.

52. The composition according to any one of claims 49 to 51, the composition further comprising an indexer comprising a first-strand polynucleotide and a second-strand polynucleotide hybridized through at least two complementary hybridization regions, the first strand extending beyond the second strand to form a single-stranded overhang and comprising at least one loop that does not hybridize with the second strand.

53. The composition of claim 52, wherein the loop of the first chain of the indexed substituent comprises a target hybridization region or a sequence having at least about 90% sequence identity with the target hybridization region.

54. The composition according to claim 52 or claim 53, wherein the flanks of the ring of the indexinducer are complementary hybridization pairs that hybridize the first strand of the indexinducer with the second strand of the indexinducer.

55. The composition according to any one of claims 52 to 54, wherein each chain of the indexer is connected to the detection portion.

56. The composition according to any one of claims 52 to 55, the composition further comprising an indexed hybridon, the indexed hybridon comprising at least two polynucleotide hybridization regions capable of hybridizing with a hybridization region on the second strand of the indexed hybridon.

57. The composition according to any one of claims 49 to 56, the composition further comprising a recycler comprising a first-strand polynucleotide and a second-strand polynucleotide hybridized through at least two complementary hybridization regions, the first strand extending beyond the second strand to form a single-stranded portion and comprising at least one loop that does not hybridize with the second strand.

58. The composition of claim 57, wherein the flanks of the ring of the recycler are complementary hybridization pairs that hybridize the first strand of the recycler with the second strand of the recycler.

59. The composition of claim 57 or claim 58, wherein the ring of the recycler includes a hybridization region capable of hybridizing with the cyclic path.

60. The composition according to any one of claims 57 to 59, wherein the ring of the recycler comprises a hybridization region capable of hybridizing with the initiating hybridization region.

61. The composition according to any one of claims 57 to 60, wherein each chain of the recycler is connected to the detection portion.

62. The composition according to any one of claims 57 to 61, the composition further comprising a releaser comprising at least two polynucleotide hybridization regions capable of hybridizing with a hybridization region on the second strand of the recycler.

63. The composition according to any one of claims 49 to 62, wherein the cyclic path comprises a plurality of loops, each loop comprising a hybridization region.

64. The composition according to any one of claims 52 to 63, wherein the dangling protrusion of the indexed ion is capable of hybridizing with a hybridization region present in the open loop of the circumferential path.

65. The composition according to any one of claims 49 to 64, wherein the cyclic path comprises four loops.

66. The composition according to any one of claims 49 to 65, wherein the loop path is a closed loop path.

67. The composition according to any one of claims 49 to 66, wherein the composition does not contain a nuclease.

68. The composition according to any one of claims 49 to 67, wherein the composition does not contain polymerase.

69. The composition according to any one of claims 49 to 68, wherein the composition does not contain an enzyme.

70. The composition according to any one of claims 52 to 69, wherein: (i) The first strand of the indexer is connected to a fluorophore and the second strand of the indexer is connected to a quencher; or (ii) The first chain of the indexer is connected to a quencher and the second chain of the indexer is connected to a fluorophore.

71. The composition according to any one of claims 57 to 70, wherein: (i) The first chain of the recycler is connected to a fluorophore and the second chain of the recycler is connected to a quencher; or (ii) The first chain of the recycler is connected to a quencher and the second chain of the recycler is connected to a fluorophore.

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