Cascade nucleic acid protocols for ultraspecific molecular detection, transduction, and amplification

Abstract

Disclosed herein is the design and implementation of a set of novel molecular mechanisms that exploit programmable competitive hybridization and strand displacement of nucleic acids to effectively suppress the generation of non-specific reaction products (i.e., false positives) in molecular detection, transduction, and isothermal amplification of DNA or RNA targets. Some of the mechanisms described herein can also be applied to improve the performance of thermal cycling-based amplification methods.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 315,635, filed March 2, 2022, which is incorporated by reference in its entirety. [Background technology]

[0002] Nucleic acid sequence amplification is a reaction in which a nucleic acid sequence is amplified in large amounts. An isothermal reaction is a reaction in which there is limited change in temperature. Numerous isothermal amplification techniques have been developed to allow rapid and sensitive detection of nucleic acid sequences without the need for precise temperature cycling as required by conventional polymerase chain reaction (PCR), enabling the implementation of low-cost molecular diagnostics for point-of-care and field-deployable applications. State-of-the-art isothermal amplification protocols include loop-mediated isothermal amplification (LAMP), which includes reverse transcription-LAMP (RT-LAMP) as a subset.

[0003] Additional "LAMP-like" isothermal amplification reactions include other variations of isothermal nucleic acid amplification protocols using, for example, dual priming, swarm priming, stem priming, hairpin primers, etc. In general, LAMP and LAMP-like isothermal amplification reactions are prone to false positives due in part to the simultaneous use of multiple primers for target recognition, which increases the likelihood of primer-primer interactions and formation of secondary primer structures, resulting in non-specific amplification in the absence of target sequences. 1

[0004] Nucleic acid hybridization is a technique in which a single-stranded nucleic acid sequence interacts with a complementary nucleic acid sequence to form a nucleic acid complex. In PCR and LAMP protocols, partial hybridization between primers and non-target sequences can also lead to false amplification. Despite careful primer design and in silico optimization attempts, there is still a lack of universal metrics to predict primer performance during actual amplification. 2 As a result, time-consuming experimental screening and manual adjustment of multiple candidate primer sets is usually unavoidable in practice, making the already sophisticated assay design and optimization process even more difficult and tedious.

[0005] To improve assay reliability, prior approaches have used chemical or enzymatic additives to slow or prevent the generation of non-specific amplicons. 3 However, these techniques not only increase the cost of the assay, but also alter and complicate the overall reaction conditions.Other techniques for improving the signal-to-noise ratio of isothermal amplification rely on the use of sequence-specific reporter probes or similar constructs to detect the target amplicons. 4~8 However, these techniques only distinguish between the target amplicon and the resulting pool of amplification products, and cannot prevent the exponential generation of non-specific amplicons starting from the early stages of the reaction, resulting in assay readouts that rely on target-specific detection (e.g., fluorescent quencher probes). Thus, such methods cannot enable the use of more convenient and potentially lower-cost methods that directly probe total DNA synthesis during amplification (e.g., pH-based colorimetric changes, turbidity, intercalating dyes). These drawbacks limit the general use of such enhanced techniques in point-of-care assays that require rapid visual interpretation of test results without relying on specialized readout equipment.

[0006] Therefore, there is a continuing need for simple, efficient, programmable approaches to improve assay specificity that can be generally applied to different molecular detection, transduction, amplification, and readout protocols. Summary of the Invention

[0007] Disclosed herein are compositions and methods directed to novel molecular mechanisms that utilize programmable competitive hybridization and strand displacement of nucleic acids to effectively suppress the generation of non-specific reaction products (i.e., false positives) in molecular detection, transduction, and isothermal amplification of DNA or RNA targets. Specifically, the disclosure proposes a general framework of several molecular mechanisms, referred to herein as "sheathing / desheathing," "thresholding / dethresholding," and "deactivation / reactivation." These molecular mechanisms utilize short single-stranded nucleic acid oligonucleotides (i.e., "oligos") or pre-annealed oligodimers with programmable interactions with primers, target sequences, or transduced strands of target sequences to limit or moderate the initiation and progression of further reactions in different molecular protocols.

[0008] Strand displacement is a reaction in a nucleic acid complex that exchanges one nucleic acid strand for another displaced strand. The proposed molecular mechanism can be designed, modularized, and implemented in vitro, based on well-characterized principles of nucleic acid hybridization and strand displacement. 9~12 By programming the thermodynamic and kinetic parameters of hybridization and strand displacement, these molecular mechanisms can be custom programmed and integrated into different molecular detection, transduction, amplification, and readout pathways to ensure improved specificity for detecting minute amounts of target DNA or RNA molecules without sacrificing overall reaction speed and sensitivity.

[0009] A reaction cascade is a series of reactions. Transduction of a nucleic acid strand is a reaction in which one input nucleic acid strand is displaced with another output nucleic acid strand. Threshold transduction is transduction that presupposes a threshold amount of input nucleic acid strand. Reactions such as amplification or transduction are armored when they cannot occur without proper initiation. The designs disclosed herein allow for the construction of effective nucleic acid reaction cascades, including but not limited to (1) armored amplification (Figures 1-3), (2) armored transduction plus amplification (Figures 4-6), (3) threshold transduction plus amplification (Figure 7), and (4) armored / threshold transduction plus reactivation amplification (Figure 8). Furthermore, the thermodynamic properties inherent to competitive hybridization and strand displacement allow for the elimination of single base mismatches during target recognition. 13~15 This also enables sensitive discrimination of mutations, which directly facilitates mutation exclusion and rule-out for multiplexed molecular diagnostic applications (Figure 9).

[0010] It should be understood that the molecular mechanisms disclosed herein (including sheathing, desheathing, thresholding, dethresholding, deactivation, reactivation) are not limited to the exemplary designs of the nucleic acid modules described herein. For example, the basic nucleic acid components of the disclosed reaction protocols (e.g., sheathing strand, threshold complex, deactivation strand, etc.) can be designed and optimized according to the requirements of the intended assay, for example, with flexible selection of hybridization sites, length, stability, mismatch base usage, toehold, and nucleic acid sequence variants or modifications. In some embodiments, for example, the nucleic acid module is constructed as a DNA hairpin instead of a basic duplex form of construct. 16 Some embodiments may be designed to utilize PNA and LNA instead of or in combination with DNA and / or RNA. 17~19 The use of nucleic acid analogues such as may be exploited to achieve desired thermodynamic properties of molecular mechanisms.

[0011] It should also be understood that the application of the disclosed molecular mechanisms is not limited to a particular method or implementation of a molecular detection, transduction, amplification, and / or readout system unless otherwise specified. For example, the disclosed molecular mechanisms can be utilized to improve detection of nucleic acid targets, including, but not limited to, DNA, dsDNA, ssDNA, RNA, mRNA, microRNA, tRNA, rRNA, sgRNA, siRNA, and analogs of the foregoing, such as PNA, LNA, TNA, HNA, GNA, etc.

[0012] In some embodiments, the disclosed molecular mechanisms can be leveraged to improve the specificity of various isothermal amplification techniques other than traditional LAMP and / or RT-LAMP, including, but not limited to, variations of LAMP-like amplification techniques based on dual priming, swarm priming, stem priming, hairpin primers, etc. 20~25 Some of the underlying mechanisms proposed herein (e.g., packaging and depackaging) can also be strategically exploited to improve the performance of traditional thermal cycling-based amplification methods such as PCR, RT-PCR, qPCR, and RT-qPCR.

[0013] This Summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This Summary is not intended to identify all key features or essential features of the claimed subject matter, nor is it intended to be used alone as an aid in determining the scope of the claimed subject matter.

[0014] Various objects, features, characteristics and advantages of the present invention will become apparent and become more readily understood from the following description of embodiments, taken in conjunction with the accompanying drawings and the appended claims, all of which form a part hereof. [Brief description of the drawings]

[0015] [Figure 1] AC illustrate exemplary schemes of armoured primers and amplification protocols. [Diagram 2] 1 illustrates components of an exemplary scheme for an exteriorized LAMP protocol. [Figure 3A] 1 illustrates the reaction pathway of an exemplary scheme of the armored LAMP protocol. [Figure 3B] 1 illustrates the reaction pathway of an exemplary scheme of the armored LAMP protocol. [Figure 4] 1 illustrates components of an exemplary schematic of an armored transduction protocol. [Diagram 5] 1 illustrates the reaction pathway of an exemplary scheme of an armored transduction protocol. [Figure 6A] 1 shows a universal LAMP protocol primed by a non-armored transduction chain. [Figure 6B] FIG. 1 illustrates a universal LAMP protocol primed with a non-armored transduction chain. [Figure 7] AC illustrate an exemplary scheme of the threshold transduction and amplification protocol. [Figure 8] AC illustrate exemplary schemes for reactivation and amplification of deactivated primers. [Figure 9] A and B illustrate the mechanism of ultraspecific target discrimination through the use of armed primers. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] overview In this disclosure, a nucleic acid-based molecular mechanism is described that can be utilized to address the limitations of non-specific amplification (false positives) associated with conventional isothermal amplification protocols such as LAMP and RT-LAMP. The underlying principles can also be custom programmed and broadly applied to improve the specificity and signal-to-noise ratio of other molecular detection, transduction, amplification, and readout protocols.

[0017] Exterior / De-exterior In some embodiments, armoring can be accomplished by the introduction of a short, single-stranded nucleic acid "armor strand" that contains a subsequence of sufficient length to be complementary to the 3' segment of a primer used in a nucleic acid detection, transduction, or amplification protocol. The 3' end of the armor strand is designed to inhibit polymerase extension (e.g., by adding a short overhang "toehold" that incorporates a 3' inverted dT, 3' ddC, 3' C3 spacer, 3' amino, or a modification such as a 3' phosphorylation that blocks extension by DNA polymerases that incorporate non-canonical nucleobases or G-quadruplexes).

[0018] During the assay reaction, the armor strand is present in solution at a moderately high concentration, such that its competitive binding to the primer creates a higher energy threshold for complete primer binding to a priming site on the target sequence to occur, thereby increasing the stringency for initiation of target-specific amplification. In some embodiments, a "moderately high concentration" for the armor strand can be 0.05x, 0.1x, 0.2x, 0.3x, 0.4x, 0.5x, 0.6x, 0.7x, 0.8x, 0.9x, 1.0x, 1.1x, 1.2x, 1.3x, 1.4x, 1.5x, 1.6x, 1.7x, 1.8x, 1.9x, or 2.0x the concentration of the corresponding primer to which it binds, or within a range using any combination of the foregoing as an endpoint.

[0019] With proper adjustment of the concentration and thermodynamic parameters of the armor strand, hybridization between the armor strand and the primer can serve to block or at least temporarily separate the 3' end of the primer, effectively inhibiting spurious primer-primer and primer-template interactions and reducing false positives. Furthermore, once a primer is armored (i.e., bound to its armor strand), it must undergo initial partial binding to the target priming site, followed by branch migration and strand displacement, to release the initially bound armor strand (i.e., de-armoring) before the primer can fully hybridize to its target sequence and initiate polymerase elongation.

[0020] In some embodiments, the binding sites and sequences of the primers and armored strands can be tailored and optimized to discriminate single base mutations on target DNA or RNA due to the high specificity driven by the thermodynamic properties of the nucleic acid strand displacement reaction. The degree of armoring can be controlled by adjusting the initial concentration of armored strands in the solution. In some embodiments, de-armoring refers to the reduction of armored strand concentration or the absence of armored strands in the reaction. Furthermore, depending on the implementation and performance requirements of the assay, some embodiments can utilize one or more armored primers in a one-pot reaction.

[0021] Threshold Complex In some embodiments, thresholding can be achieved by the introduction of a pre-annealed oligodimer, called a "threshold complex," which is designed to consume the input strand up to a predetermined threshold level before the input strand can effectively trigger any downstream reactions. In some embodiments, the input to the threshold complex can be a target transduction strand (released from an upstream transduction protocol) that serves as a primer for a downstream amplification protocol. The thresholding mechanism is designed to suppress potential background leakage (i.e., unintended triggering of downstream reactions due to spurious exposure of binding domains from upstream reaction components) that can occur in the cascade reaction pathway of the assay due to factors such as misformed or unannealed nucleic acid complexes.

[0022] The input strand consumed by thresholding (which serves as a "primer" for the downstream amplification protocol) is rendered in the form of an inactive waste species, which in some embodiments may simply be a blunt-ended duplex. The reaction between the primer and the threshold complex also results in the release of a single-stranded nucleic acid oligo, called the "moderator strand," from the threshold complex. Because the moderator strand contains a subsequence identical to the 5' segment of the primer sequence, the moderator strand (once released from the threshold complex) may compete with the primer for binding to the downstream amplification priming site. Such competitive binding may also be designed to limit spurious amplification initiation by placing 3' modifications (e.g., short overhang toehold, 3' inverted dT, 3' ddC, 3' C3 spacer, 3' amino, 3' phosphorylation, non-canonical nucleobases, or G-quadruplexes) on the moderator strand that prevent polymerase extension. These molecular mechanisms reduce leakage and simultaneously increase specificity.

[0023] The degree of thresholding can be controlled by adjusting the initial concentration of the threshold complex in solution. In some embodiments, de-thresholding refers to a reduction in the threshold complex concentration, or the absence of a threshold complex in a reaction. Additionally, depending on the implementation and performance requirements of the assay, some embodiments may utilize a threshold complex for one or more primers or transduction strands in a one-pot reaction.

[0024] In some embodiments, the thresholding mechanism may be combined with the sheathing mechanism in a single assay and / or reaction mixture.

[0025] Deactivation / Reactivation Primers In some embodiments, the primer for the downstream amplification protocol is first deactivated by annealing to the deactivated strand to form a "deactivated primer" dimer that contains a hybridized domain of sufficient length to completely capture the primer at its 3' end. This pre-annealed dimer has an extended overhang at the 3' end of the deactivated strand. In some embodiments, the transduction strand released from the upstream detection and transduction protocol is designed to function as the "reactivated strand", which recognizes the exposed overhang of the deactivated primer and causes strand displacement to release the originally bound primer from the deactivated primer dimer. Because the reactivated strand contains a subsequence identical to the 3' segment of the primer sequence, the reactivated strand (once released from the upstream detection or transduction protocol) can compete with the primer for binding to the downstream amplification priming site. Such competitive binding can also be designed to limit spurious amplification initiation by placing 3' modifications (e.g., short overhang toeholds, 3' inverted dT, 3' ddC, 3' C3 spacers, 3' amino, or 3' phosphorylations, non-canonical nucleobases, or G-quadruplexes) on the reactivated strand that prevent polymerase extension.

[0026] In some embodiments, the deactivated primer mechanism can be used in parallel with the armoring mechanism and / or thresholding mechanism to provide another layer of protection against the generation of false positive amplicons in a molecular assay. Additionally, depending on the implementation and performance requirements of the assay, some embodiments can utilize one or more deactivated primers in a one-pot reaction.

[0027] Exemplary embodiments Exterior / De-exterior In the diagrams and accompanying description disclosed below, an asterisk (*) indicates the reverse complement of a sequence, e.g., the complementary sequence of oligo a is designated a*.

[0028] In Figures 1A-1C, an exemplary scheme of an armored primer and an amplification protocol is illustrated. Figure 1A illustrates the sequence composition of the primer and armor strand. The primer contains two subsequences (also conceptually considered functional "domains") a and b, listed in the 5' to 3' direction. The armor strand contains a domain b*, which is reverse-complementary to domain b on the primer. The armor strand also contains a simple 3' modification (indicated by the letter t in the figures), such as a short overhang toehold or inverted dT, or other 3' modifications disclosed herein, to prevent polymerase extension. The armor strand may also contain additional subsequences at its prefix or suffix, which are not shown in this figure for clarity.

[0029] A portion of the armor strand is initially unbound and free in solution at moderately high concentrations. Hybridization between the primer and the free armor strand leaves the 5' subsequence of the primer (i.e., domain a) exposed, forming an armored primer that is free to bind to the target DNA or RNA via the corresponding subsequence of the priming site (i.e., domain a*). Figure 1B illustrates that the armor strand and the target sequence compete to bind to the primer in solution. As soon as the armored primer binds to the target sequence, a process of branch migration proceeds, eventually releasing the single-stranded armor strand into solution by strand displacement. This process is thermodynamically highly specific and favorably driven by the formation of a perfect match between the primer and the target sequence. As shown in Figure 1C, the polymerase now initiates DNA synthesis from the 3' end of the primer on the fully hybridized target to initiate the downstream amplification protocol.

[0030] Figure 2 illustrates the reaction components of an exemplary scheme of the armored LAMP protocol. For simplicity, common reagents such as reaction buffer and nuclease-free water are not depicted in the drawing. Component (A) illustrates the sequence composition of the target DNA or RNA template for the LAMP reaction. The subsequences F3*, F2*, F1*, B1, B2, B3 listed in the 3' to 5' direction on the target sequence correspond to the six primer binding sites described in the conventional LAMP protocol.

[0031] Component (B) is a set of LAMP primers designed for isothermal amplification of a target. Specifically, the set includes an armored FIP primer and an armored BIP primer, together with an F3 primer, a B3 primer, a LoopF ​​primer, and a LoopB primer. The armored FIP primer is formed by a primer nucleic acid strand consisting of subsequences F1* and F2 (listed in the 5' to 3' direction) and an armor strand that hybridizes to the F2.2 subdomain of the F2 subsequence on the primer nucleic acid strand. The armor strand contains a simple 3' modification, such as a short overhang toehold to prevent polymerase extension, an inverted dT, or other 3' modifications disclosed herein.

[0032] Similarly, an armored BIP primer is formed by a primer nucleic acid strand consisting of subsequences B1* and B2 (listed in the 5' to 3' direction) and an armor strand that hybridizes to the B2.2 subdomain of the B2 subsequence on the primer nucleic acid strand. The armor strand contains a simple 3' modification such as a short overhang toehold to prevent polymerase extension, an inverted dT, or other 3' modifications disclosed herein. In some embodiments, other essential primers from the LAMP primer set (e.g., F3 primer and / or B3 primer) can be armored in a similar manner. Component C indicates that the reaction contains a strand-displacing polymerase such as Bst2.0. Component D indicates that the reaction can also contain a reverse transcriptase enzyme if the amplification target is an RNA sequence.

[0033] 3A and 3B illustrate the reaction pathway of an exemplary scheme of the armored LAMP protocol. Step (1) illustrates that the armored FIP primer hybridizes to the F2.1* subsequence of the F2* priming site on the target template. Step (2) shows the process of branch migration and strand displacement that results in complete hybridization between the F2 subsequence on the FIP primer and the F2* subsequence on the target template. At the same time, the armored strand is released from the armored FIP primer and becomes free to armor another unbound FIP primer in solution.

[0034] Steps (3-10) illustrate the sequence of primer extension and strand displacement polymerization reactions of LAMP that lead to the formation of a basic dumbbell DNA structure that serves as the starting material for the self-priming autocyclic amplification reaction of LAMP. Specifically, steps (3-5) illustrate the process including primer extension from the 3' end of the fully hybridized FIP primer on the target, followed by binding of the F3 primer on the target that primes strand displacement polymerization to release the newly synthesized strand primed by FIP. Step (6) illustrates that the armored BIP primer hybridizes to the B2.1* subsequence of the B2* priming site on the newly formed DNA structure from the previous step. Step (7) illustrates the process of branch migration and strand displacement that results in complete hybridization between the B2 subsequence of the BIP primer and the B2* subsequence of the newly formed DNA structure. At the same time, the armored strand is released from the armored BIP primer and becomes free to armor another unbound BIP primer in solution. Steps (8–10) show the process involving primer extension from the 3′ end of the fully hybridized BIP primer, followed by binding of the B3 primer to the priming site B3*, which primes strand displacement polymerization and releases the newly synthesized strand primed by BIP, resulting in the formation of a basic dumbbell DNA structure.

[0035] Steps (11-13) show the initialization of LAMP autocycle amplification. These downstream reactions involve armored FIP primer, armored BIP primer, and loop primers LoopF ​​and LoopB. Note that the effect of armoring on FIP and BIP is also present in these reactions, which helps to maintain specificity during exponential amplification. For simplicity, the hybridization, strand displacement, and strand displacement polymerization reactions involved in these processes, which are well known in the art, are not shown in detail.

[0036] In Figure 4, the reaction components of an exemplary scheme of the armored LAMP protocol are illustrated. In the exemplary implementation demonstrated herein, the principle underlying nucleic acid target transduction is similar to that described in US Patent Application No. 17 / 749,858, which is incorporated herein by reference in its entirety, except that the armoring mechanism is applied to one or more of the primers used during transduction. In brief, the transduction protocol can be used to detect the presence of any target DNA or RNA sequence in the release of one or more copies of a universal single-stranded nucleic acid oligo that serves as one of the essential primers capable of inducing a downstream universal LAMP protocol that is pre-designed, independent of the target sequence, and highly optimized in performance.

[0037] Component (A) illustrates that the target DNA or RNA sequence contains three adjacent hybridization sites recognized by the transduction protocol, including P2, P1, and P3 (listed in the 3' to 5' direction). Component (B) illustrates the armored primer scheme of loaded primer A, consisting of a primer with subsequences U* and P1* (listed in the 3' to 5' direction) hybridized to the transduction strand U on the 5' end and the armored strand on the 3' end, respectively. The armored strand also contains a simple 3' modification (indicated by the letter t in the drawing), such as a short overhang toehold or inverted dT, to prevent polymerase extension. The armored strand is initially unbound and free in solution at moderately high concentrations. Hybridization between loaded primer A and the armored strand leaves the P1.1* subsequence of the primer exposed, forming an armored primer that is free to bind to the target DNA or RNA.

[0038] Component (C) illustrates primer B consisting of nucleic acid sequence P2*. Component (D) illustrates primer C consisting of nucleic acid sequence P3*. In some embodiments, primer B and / or primer C may be armored in a manner similar to that described above. Component (E) illustrates that the reaction contains a strand displacing polymerase such as Bst2.0. Component (F) illustrates that the reaction may also contain a reverse transcriptase enzyme if the transduction target is an RNA sequence.

[0039] In Figure 5, the reaction pathway of an exemplary scheme of the armored transduction protocol is illustrated. In the exemplary implementation demonstrated herein, the underlying transduction reaction pathway is similar to that described in US Patent Application Serial No. 17 / 749,858, which is incorporated herein by reference in its entirety, except that here the armoring mechanism is applied to the loaded primer A, whereby complete hybridization to the target priming site requires initial partial binding, followed by branch migration and strand displacement to release the armored strand, and then binding such that primer extension can proceed from the 3' end of the loaded primer A.

[0040] In FIG. 5, step (1) illustrates that armored loading primer A hybridizes to a subsequence of the P1 priming site on the target via the exposed P1.1* subsequence of the armored loading primer, and can then strand-displace following hybridization of the P.1.2* subsequence to the target. Step (2) illustrates that strand displacement results in the release of the armored strand from the armored loading primer, which can then initiate polymerization from 3' of P1*. Step (3) illustrates that primer B binds to priming site P2 on the target. Step (4) shows a strand-displacement polymerization reaction initiated by primer B. Step (5) illustrates the resulting nucleic acid species generated from the strand-displacement polymerization reaction. Step (6) illustrates that primer C binds to priming site P3* on the resulting nucleic acid species. Step (7) illustrates a strand-displacement polymerization reaction initiated by primer C. Step (8) illustrates the resulting nucleoids generated from the strand displacement polymerization reaction and includes the release of the transducing strand U designed to trigger the downstream universal LAMP protocol.

[0041] In Figures 6A and 6B, the initialization of the universal LAMP protocol by the non-armored transduction strand is illustrated. The exemplary reaction pathway illustrated herein is similar to the conventional LAMP protocol, except that the transduction strand U released from the upstream transduction protocol replaces the F3 primer in this implementation. In some embodiments, the transduction strand U may function as one of the other essential primers in the conventional LAMP protocol. Step (1) illustrates the hybridization of FIP to its priming site on the universal LAMP template. Step (2) illustrates the polymerization initiated from the 3' end of FIP. Step (3) illustrates the hybridization of U to its priming site on the universal LAMP template. Step (4) illustrates the strand displacement polymerization initiated from the 3' end of U, releasing the newly synthesized strand primed with FIP. Step (5) illustrates that the BIP primer and the B3 primer hybridize to their respective priming sites on the newly synthesized strand released from the previous step. Step (6) illustrates polymerization initiated from the 3' end of BIP and strand displacement polymerization initiated from the 3' end of B3, resulting in the formation of the basic dumbbell DNA structure of LAMP. Steps (7-8) show the initialization of LAMP autocycle amplification, driven by self-priming mutagenesis using FIP, BIP, LoopF, and LoopB primers.

[0042] Threshold Complex In Figures 7A-7C, an exemplary scheme of the threshold transduction and amplification protocol is illustrated. Step (A) illustrates that the presence of a target DNA or RNA sequence is detected by a transduction protocol (e.g., any of those disclosed herein), resulting in the release of the transduction strand U. Step (B) illustrates the thresholding mechanism that converts a predetermined amount of U into an inactive DNA species. Specifically, the transduction strand U released from the upstream reaction contains the functional domains U.1 and U.2, listed in the 3' to 5' direction. Also present in the reaction mixture is a pre-annealed DNA dimer, called the "threshold complex," which is formed by a "moderator strand" that hybridizes to the U.2* domain on a nucleic acid strand consisting of the subsequences U.1* and U.2*, listed in the 5' to 3' end direction. The moderator strand contains domain U.2 and a simple 3' modification (indicated by the letter t in the figures) such as a toehold or inverted dT in the short overhang, or other 3' modifications disclosed herein, to prevent polymerase elongation.

[0043] During thresholding, the transduced strand U binds to the exposed U.1* domain on the threshold complex, releasing the moderator strand from the threshold complex via strand displacement while simultaneously consuming the single-stranded transduced U to form a blunt-ended duplex that is functionally inactive and can be considered a waste species in solution. In some embodiments, the threshold level can be adjusted simply by changing the concentration of the threshold complex.

[0044] Step (C1) illustrates that the moderator strand released from the thresholding reaction can compete with transducing strand U (which serves as an essential primer for the downstream amplification protocol) for binding to the target priming site. However, competitive binding of the moderator strand does not result in spurious amplification because of the presence of a 3' modification that prevents polymerase extension.

[0045] Step (C2) illustrates that after thresholding, remaining transduction strand U from solution can fully hybridize to the target priming site and trigger downstream amplification. Such initialization of the amplification protocol is only effective if the total amount of U released from the upstream transduction protocol is sufficient to exceed the threshold level set by the thresholding reaction.

[0046] Deactivation / Reactivation Primers 8A-8C illustrate an exemplary scheme for reactivation and amplification of inactivated primers. Step (A) illustrates the formation of an inactivated primer by annealing a primer (comprising subsequences a and b, listed in the 5' to 3' direction) to an inactivated strand (comprising subsequences e* and b*, listed in the 3' to 5' direction). The annealed inactivated primer contains a fully hybridized b / b* domain, while both subsequences a and e* remain unhybridized.

[0047] Step (B) illustrates that the presence of a target DNA or RNA sequence is detected by a transduction protocol (e.g., as disclosed herein), resulting in the release of a transduced strand that functions as a "reactivated strand" in a subsequent reaction. For example, the reactivated strand consists of subsequences e, b, and t, listed in the 5' to 3' direction, and the binding of subsequence e of the reactivated strand to the extended 3' overhang sequence e* of the inactivated primer can proceed a strand displacement reaction, leading to the release of the primer from the inactivated primer dimer. This released primer is now considered reactivated and becomes free to hybridize to its target priming site (i.e., a*b*) on the downstream amplification template, which in some embodiments can be a universal template for highly optimized amplification protocols. The reactivation reaction also generates an inactive duplex, which is functionally considered a waste species. Step (C) illustrates that the reactivated primer hybridizes fully to the target template to initiate the downstream amplification protocol.

[0048] Identification of specific nucleic acids 9A and 9B illustrate the mechanism of specific nucleic acid target discrimination by the use of armoured primers. Specifically, the thermodynamic properties of competitive hybridisation and strand displacement can be exploited to discriminate between single base mismatches between the primer and the target sequence.

[0049] Figure 9A illustrates the case where the primer and target sequences are perfectly matched. Under such conditions, fast forward strand displacement is promoted, resulting in rapid release of the armored strand from the armored primer. In contrast, Figure 9B illustrates that strand displacement by one or more nucleotide mismatches is thermodynamically unfavorable and therefore cannot efficiently proceed forward to result in perfect hybridization between the primer and the target to promote amplification. The position of the mismatched nucleotide(s) may be different in different embodiments and may be strategically selected to maximize the signal-to-noise ratio for single nucleotide discrimination.

[0050] Additional Terms and Definitions Although particular embodiments of the present disclosure have been described in detail with reference to particular configurations, parameters, components, elements, etc., the description is illustrative and should not be construed as limiting the scope of the claimed invention.

[0051] Furthermore, for any given element of a component of a described embodiment, unless stated otherwise implicitly or explicitly, it is to be understood that any of the possible alternatives listed for that element or component may be used generally, individually, or in combination with each other.

[0052] Additionally, numbers expressing amounts, components, distances, or other measurements used in the specification and claims may be optionally modified by the term "about" or its synonyms. When terms such as "about," "approximately," or "substantially" are used in conjunction with a stated amount, value, or condition, it may be interpreted to mean that the amount, value, or condition deviates by less than 20%, less than 10%, less than 5%, less than 1%, less than 0.1%, or less than 0.01% of the stated amount, value, or condition.

[0053] The embodiments disclosed herein should be understood to comprise / include the disclosed components and therefore may include additional components not specifically described. Optionally, the embodiments disclosed herein are essentially free or completely free of components not specifically described. That is, non-disclosed components may optionally be completely or essentially omitted from the disclosed embodiments. For example, components of an amplification reaction mixture that are not specifically disclosed and / or are not necessarily required to carry out the disclosed methods may be completely or essentially omitted from the disclosed embodiments.

[0054] Embodiments that are "essentially omitted" or "essentially free" of a component may include trace and / or non-functional amounts of the component. For example, an "essentially omitted" component may be present in an amount of 2.5% or less, 1% or less, 0.1% or less, or 0.01% or less by total weight or volume of the reaction mixture. This is equally applicable to other negative modifiers such as "essentially omitted," "essentially free," and similar phrases using other synonyms of "substantially" or "essentially."

[0055] Any headings and sub-headings used herein are for organizational purposes only and are not meant to be used to limit the scope of the specification and the claims. All references provided herein are incorporated herein by reference.

[0056] It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" do not exclude plural referents unless the context clearly dictates otherwise.

[0057] It should also be understood that the embodiments described herein may include properties and / or characteristics (e.g., components, components, members, elements, parts, and / or portions) described in one or more separate embodiments, and are not necessarily limited to the precise features explicitly described for that particular embodiment. Thus, various features of a given embodiment can be combined with and / or incorporated into other embodiments of the present disclosure. Thus, the disclosure of a particular feature for a particular embodiment of the present disclosure should not be construed as limiting the application or inclusion of the said feature to the particular embodiment. Rather, it will be understood that other embodiments may also include such features. 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Claims

1. 1. A composition for isothermal amplification of a target nucleic acid, said composition comprising: a first primer comprising a 5' section and a 3' section; an armor strand comprising a complementary section and a 3' modification, the complementary section being complementary to the 3' section of the first primer, such that when the armor strand hybridizes to the first primer, the 3' section of the first primer is separated while the 5' section of the first primer is free to hybridize to a first target position of a target nucleic acid; and at least a second primer that associates with a second target location of the target nucleic acid and, together with the first primer, enables amplification of the target nucleic acid; The composition, wherein during the amplification, the 3' modification of the outer strand prevents polymerase extension of the outer strand.

2. 2. The composition of claim 1, wherein the 3' modification is a 3' overhang, a 3' inverted dT, a 3' ddC, a 3' C3 spacer, a 3' amino, a 3' phosphorylation that blocks extension by a polymerase, one or more non-canonical nucleobases, or a G-quadruplex.

3. The composition of claim 1 further comprising a DNA polymerase.

4. The composition of claim 1 further comprising a reverse transcriptase.

5. 10. The composition of claim 1, further comprising additional primers which, together with the first and second primers, form a complete set of primers for carrying out an amplification reaction.

6. 2. The composition of claim 1, wherein the first and second primers, optionally with one or more additional primers, are configured to enable a transduction reaction to generate a transduction product in the presence of the target nucleic acid, and the transduction product functions to regulate a downstream amplification reaction.

7. The composition of claim 6, wherein the downstream amplification reaction is a LAMP or LAMP-like reaction, and the composition further comprises an incomplete set of primers for the LAMP or LAMP-like reaction, and the transfection products function as the necessary primers to enable the downstream LAMP or LAMP-like reaction.

8. 1. A method for amplifying a target nucleic acid, comprising: Providing a sample; Mixing said sample or a part or extract thereof with a composition according to any one of claims 1 to 7 to allow amplification of said target nucleic acid.

9. The method of claim 8, wherein the amplification of the target nucleic acid comprises a LAMP or a LAMP-like isothermal amplification reaction.

10. The method according to claim 9, wherein the LAMP reaction is a reverse transcription LAMP (RT-LAMP) reaction.

11. 9. The method of claim 8, wherein the amplification of the target nucleic acid comprises a transduction reaction, the transduction reaction functions to generate a transduction product if the target nucleic acid is present in the sample, and the transduction product functions to regulate a downstream amplification reaction.

12. The method of claim 11, wherein the downstream amplification reaction is a LAMP or a LAMP-like isothermal amplification reaction, and the transfection products function as necessary primers for the downstream amplification reaction.

13. The method of claim 12, wherein the downstream amplification reaction is a RT-LAMP reaction.

14. The method of claim 8 , wherein the target nucleic acid is associated with an infectious disease.

15. 9. The method of claim 8, wherein the sample is an environmental sample and the method is performed as part of an environmental monitoring or assessment process.

16. The method of claim 15, wherein the environmental sample is a water sample or a soil sample.

17. 1. A composition for isothermal amplification of a target nucleic acid, said composition comprising: a set of oligonucleotide primers configured to enable a transduction reaction that produces a transduction product in the presence of said target nucleic acid; a threshold complex comprising a dimer between the moderator strand and the transduction product complement; the transduction product complement comprises a 5' section and a 3' section, the moderator strand comprises (i) a section complementary to the 3' section of the transfection product complement, and (ii) a 3' modification that prevents polymerase extension of the moderator strand; when the moderator strand hybridizes to the transduction product complement, the 3' section of the transduction product complement is separated, while the 5' section of the transduction product complement is free to hybridize to the transduction product; The composition, wherein release of the moderator strand from the threshold complex regulates a downstream amplification reaction.

18. The composition of claim 17, wherein the downstream amplification reaction is a LAMP or a LAMP-like isothermal amplification reaction.

19. 19. The composition of claim 18, wherein the transduction product promotes the LAMP or LAMP-like isothermal amplification reaction and the moderator strand inhibits the amplification reaction.

20. The composition according to any one of claims 17 to 19, wherein a reaction mixture for the transduction reaction comprises the composition according to any one of claims 1 to 7.

21. 1. A method for amplifying a nucleic acid using a thresholding mechanism, the method comprising: Providing a sample; mixing said sample or a part or extract thereof with a composition according to any one of claims 17 to 19 to allow amplification of said target nucleic acid in said transduction reaction, The method, wherein the transduction reaction produces the transduction product, and the threshold complex limits activation of the downstream amplification reaction by the transduction product until sufficient transduction product has been produced.

22. 22. The method of claim 21, wherein the target nucleic acid is associated with an infectious disease.

23. 22. The method of claim 21 , wherein the sample is an environmental sample and the method is performed as part of an environmental monitoring or assessment process.

24. 24. The method of claim 23, wherein the environmental sample is a water sample or a soil sample.

25. 1. A composition for isothermal amplification of a target nucleic acid, said composition comprising: a first set of oligonucleotide primers configured to enable a transduction reaction to generate a transduction product in the form of a reactivated strand in the presence of said target nucleic acid; a second set of oligonucleotide primers configured to enable a downstream amplification reaction; and a non-activation strand that anneals to an activatable primer of the second set of primers to deactivate the activatable primer, the non-activation strand comprising (i) a complementary section hybridized to at least a portion of the activatable primer, and (ii) a 3' overhang; The reactivated strand is complementary to the non-activated strand, such that the activatable primer is released from the non-activated strand upon hybridization of the reactivated strand with the non-activated strand.

26. The composition of claim 25 , wherein the downstream amplification reaction comprises a universal template.

27. 26. The composition of claim 25, wherein the reactivated strand comprises a 3' overhang when hybridized to the non-activated strand.

28. The composition according to any one of claims 25 to 27, wherein a reaction mixture for the transduction reaction comprises the composition according to any one of claims 1 to 7 or 17 to 19.

29. 29. The composition of claim 28, wherein the downstream amplification reaction is a LAMP or a LAMP-like isothermal amplification reaction.

30. 1. A method for amplifying a nucleic acid by reactivating a deactivated primer, the method comprising: Providing a sample; mixing said sample or a part or extract thereof with a composition according to any one of claims 25 to 27 to allow amplification of said target nucleic acid in said transduction reaction, The method, wherein the transduction reaction produces the reactivated strand, which hybridizes to the non-activated strand to release the activatable primer, thereby enabling the downstream amplification reaction.

31. 31. The method of claim 30, wherein the target nucleic acid is associated with an infectious disease.

32. 31. The method of claim 30, wherein the sample is an environmental sample and the method is performed as part of an environmental monitoring or assessment process.

33. 33. The method of claim 32, wherein the environmental sample is a water sample or a soil sample.

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