Nuclease

Abstract

The present application provides a ribozyme comprising: a) one or more catalytic domains capable of transitioning between an active state and an inactive state; b) one or more than one releasable RNA fragment wherein each of the releasable RNA fragments is flanked by two ribozyme cleavage sites wherein the cleavage at each cleavage site is catalyzed by at least one of the one or more catalytic domains in the active state; c) one or more trigger binding domains, each of which is used to bind a trigger nucleic acid molecule; wherein each of the one or more catalytic domains is linked to one of the one or more trigger binding domains; wherein the catalytic domain is in an inactive state when a trigger binding domain linked to the catalytic domain is not bound by a trigger nucleic acid molecule, and wherein the catalytic domain is in an active state when a trigger binding domain linked to the catalytic domain is bound by a trigger nucleic acid molecule; and wherein the one or more releasable RNA fragments are released from the ribozyme when both cleavage sites flanking the releasable RNA fragment are cleaved under catalysis by the one or more catalytic domains. Also disclosed are methods of detecting the presence of a trigger nucleic acid molecule in a sample, methods of detecting the presence of a target sequence or a target mutation on a target nucleic acid in a sample, and kits comprising the ribozyme thereof.

Description

Technical Field

[0001] The present disclosure broadly relates to ribozymes engineered to contain one or more than one target binding domain / trigger binding domain. Background Art

[0002] The levels and expression profiles of coding and noncoding RNAs in cells and individuals provide a wealth of information about biological and disease states.

[0003] Therefore, methods for RNA detection and quantification have been the mainstay of molecular biology and continue to evolve as the sophistication of the technology increases. Traditional RT-qPCR and in situ hybridization methods are still routinely used, supplemented by state-of-the-art single-cell RNA sequencing and spatial transcriptomics methods to answer increasingly complex biological questions. It is now possible to accurately measure the RNA content of most cells, allowing the probing of the molecular and cellular functions of the transcriptome in normal and disease states. In addition, cellular and systemic RNA biomarkers are also important for disease diagnosis and clinical decision guidance.

[0004] Despite the many important applications of in situ RNA detection, there is currently no universal method that can directly sense specific RNA signals and convert them into a second functional noncoding RNA readout. Such an approach could be genetically encoded and act as a compact genetic switch that converts the RNA environment into a functional output. RNA-sensing genetic switches have been developed for several gene regulatory systems, most notably CRISPR. For example, guide RNAs have been modified to respond to antisense blocking sequences at the guide spacer or other regions, enabling activation or inactivation in response to RNA triggers or RNA ligands through toehold-mediated strand displacement, and CRISPR machinery has been modified to be conditionally activated when microRNAs are functional, such as microRNA-guided Ago2 cleavage and sgRNA release, and miRNA-regulated Cas9 mRNA. Many of these systems have sequence limitations because their designs involve strand displacement of critical regions of the gRNA or require multiple RNA or protein components. When the guide RNA itself is modified in this way, the switching mechanism needs to be carefully designed so that the function and specificity of the sgRNA are not affected. They are also CRISPR-specific and cannot be generalized to direct RNA signaling to other functional noncoding RNA pathways, such as shRNAs, antisense-switching or splice-switching oligonucleotides, and RNA aptamers.

[0005] Therefore, there is a need for a molecular system that can transduce the binding of an RNA signal or other nucleic acid signal into the release of a functional RNA. The present disclosure provides a solution in the form of a ribozyme that is engineered to release a functional RNA upon binding a sequence-complementary trigger factor. Summary of the invention

[0006] In one aspect, a ribozyme is provided, comprising:

[0007] a) one or more catalytic domains capable of switching between an active state and an inactive state;

[0008] b) one or more releasable RNA segments, wherein each of the releasable RNA segments is flanked by two ribozyme cleavage sites, wherein cleavage at each cleavage site is catalyzed by at least one of the one or more catalytic domains in an active state;

[0009] c) one or more trigger factor binding domains, each of which is used to bind to a trigger nucleic acid molecule; each of the one or more catalytic domains is connected to one of the one or more trigger factor binding domains;

[0010] wherein when the trigger factor binding domain connected to the catalytic domain is not bound by the trigger nucleic acid molecule, the catalytic domain is in an inactive state, and wherein when the trigger factor binding domain connected to the catalytic domain is bound by the trigger nucleic acid molecule, the catalytic domain is in an active state; and

[0011] wherein one or more than one releasable RNA fragment is released from the ribozyme when both cleavage sites flanking the releasable RNA fragment are cleaved,

[0012] The ribozyme comprises an RNA strand having the following motif

[0013] Motif [A] and motif [a], wherein motif [A] and motif [a] constitute a trigger factor binding domain for binding to a trigger nucleic acid molecule;

[0014] Motif [B] and motif [b], wherein motif [B] and motif [b] constitute a linker that acts as a communication module to stabilize the catalytic domain when the trigger nucleic acid is bound, wherein motif [B] and motif [b] are independently at least 1 nucleotide in length;

[0015] Motif [C] and motif [c], wherein motif [C] and motif [c] constitute a catalytic domain;

[0016] motif [D], wherein motif [D] comprises a first cleavage site capable of being cleaved when catalyzed by the catalytic domain;

[0017] motif [D'], wherein motif [D'] comprises a second cleavage site capable of being cleaved when catalyzed by the catalytic domain;

[0018] motif [E], wherein motif [E] comprises a releasable RNA segment;

[0019] Motif [e], wherein motif [e] comprises a sequence that is partially complementary or fully complementary to the sequence of motif [E];

[0020] And wherein the motifs are connected by one or more than one optional linker regions.

[0021] In some embodiments, the linker between motif [C] and motif [D] is selected from a dual channel linker, a triple channel linker, a quad channel linker, a stem, a single nucleotide bulge, a dinucleotide bulge, a trinucleotide bulge, a multinucleotide bulge, and combinations thereof.

[0022] In some embodiments, the linker between motif [C] and motif [D] comprises a three-way linker and a stem.

[0023] In some embodiments, the stem sequence connecting the adaptor to the motif [D] is 4 to 12 nucleotides in length.

[0024] In some embodiments, the stem sequence connecting motif [C] and motif [c] to the linker is 4 to 12 nucleotides in length.

[0025] In some embodiments, the trigger nucleic acid molecule comprises a region complementary to a trigger factor binding domain, wherein the region is more than 10 nucleotides in length, optionally one or more than one trigger factor binding domain is used to bind to the same trigger nucleic acid molecule.

[0026] In some embodiments, the releasable RNA fragment is 6 to 150 nucleotides in length.

[0027] In some embodiments, the releasable RNA fragment comprises a sequence identical to at least one of the one or more trigger RNA molecules.

[0028] In some embodiments, the releasable RNA fragment is a functional RNA selected from a small guide RNA (sgRNA), a guide RNA (gRNA), a short hairpin RNA (shRNA), and an RNA aptamer.

[0029] In some embodiments, the length of motif [B] and motif [b] is independently 1 or more than 1 nucleotide, optionally 3 or more than 3 nucleotides.

[0030] In some embodiments, motif [B] and motif [b] have a motif selected from SEQ ID NO: 1 (5'-ACG / CGU-3'), SEQ ID NO: 2 (5'-ACG / CGA-3'), SEQ ID NO: 449 (5'-ACG / UGA-3'), SEQ ID NO: 450 (5'-AUG / CGA-3'), SEQ ID NO: 451 (5'-AUG / UGA-3'), SEQ ID NO: 452 (5'-CG / CG-3'), SEQ ID NO: 453 (5'-UUG / UGG-3'), SEQ ID NO: 454 (5'-UAU / AUA-3'), SEQ ID NO: 455 (5'-ACU / AGA-3'), SEQ ID NO: 456 (5'-AUG / CAA-3'), SEQ ID NO: 457 (5'-CU / AG-3'), and SEQ ID NO: 458 (5'- The sequence of NO:458 (5'-UG / CA-3').

[0031] In some embodiments, if motif [e] and motif [E] are partially complementary to each other, the complementarity between motif [e] and motif [E] is characterized by alternating regions of complementarity and non-complementarity.

[0032] In some embodiments, motif [D] comprises nucleotide N +3 Mutation of the paired nucleotide N7.

[0033] In some embodiments, the optional linker regions independently or collectively form one or more secondary structures, optionally one or more secondary structures are selected from: a single nucleotide bulge, a dinucleotide bulge, a trinucleotide bulge, a polynucleotide bulge, a stem, a stem-loop, a t-RNA type structure, a clover-type structure, a tetraloop, a pseudoknot, a symmetric internal loop, an asymmetric internal loop, a three-stem linker (3-way linker), a four-stem linker (4-way linker), a two-stem linker (2-way linker) or a coaxial stacking or a combination thereof.

[0034] In some embodiments, the ribozyme complex comprises any one or more than one sequence of SEQ ID NO:3 to SEQ ID NO:448.

[0035] In some embodiments, the ribozyme further comprises one or more modifications.

[0036] In some embodiments, the trigger nucleic acid comprises one or more modified nucleotides.

[0037] In other aspects, a method of detecting the presence of a target nucleic acid molecule / trigger nucleic acid molecule in a sample is provided, wherein the method comprises:

[0038] incubating the sample with a ribozyme as disclosed herein at a temperature T1 that allows binding of the target nucleic acid molecule / trigger nucleic acid molecule to one or more than one target binding domain / trigger binding domain contained in the ribozyme;

[0039] incubating the sample at a temperature T2 that allows the release of nucleic acid molecules and RNA fragments from the ribozyme;

[0040] The release of the releasable RNA fragment from the ribozyme is detected.

[0041] In another aspect, a method for detecting the presence of a target sequence or a target mutation on a target nucleic acid in a sample is provided, wherein the method comprises:

[0042] incubating the sample with a ribozyme as disclosed herein, thereby allowing the target nucleic acid molecule to bind to one or more than one target binding domain / trigger binding domain contained in the ribozyme;

[0043] incubating the sample to allow the nucleic acid molecules and the releasable RNA fragments to be released from the ribozyme;

[0044] detecting release of the releasable RNA fragment from the ribozyme;

[0045] The releasable RNA fragment is a sgRNA or a shRNA; wherein detection of a target sequence or a target mutation in a sample results in signal generation.

[0046] In some embodiments, the trigger nucleic acid molecule is a viral genome or a fragment thereof.

[0047] In yet another aspect, a kit comprising a ribozyme as disclosed herein is provided. DETAILED DESCRIPTION

[0048] Detection of specific RNA or other nucleic acid sequences is integral to many research, disease diagnostic and therapeutic applications. Such applications would be further enabled if the detected nucleic acid signal could be directly functionally transformed by a second signal. This need is addressed by the present invention, which is a modular ribozyme whose self-cleavage is activated by binding to a specific complementary nucleic acid trigger sequence, resulting in the release of a second embedded RNA product without altering the original trigger. The reaction is encoded entirely within the single-stranded RNA without requiring any protein or DNA cofactors.

[0049] The inventors of the present disclosure have demonstrated that the ribozyme disclosed herein is specific and sensitive. The inventors have demonstrated that the ribozyme can be modularly designed for cell-free applications and intracellular applications. Therefore, it is a versatile platform for which many potential applications can be envisioned.

[0050] The present disclosure describes a modular RNA signal transduction platform based on a modified self-cleaving ribozyme, wherein the ribozyme has a trigger factor binding site and two cleavage sites, between which a releasable RNA cleavage product is embedded. The self-cleavage activity of the ribozyme depends on the detection and binding of the complementarity of the specific trigger nucleic acid. When the trigger factor binds, the ribozyme self-cleavage is activated, thereby releasing the embedded RNA cleavage product. Therefore, the ribozyme acts as a detector and converter of a direct RNA signal at the same time.

[0051] Thus, in one aspect of the present disclosure, there is provided a ribozyme comprising:

[0052] a) one or more catalytic domains capable of switching between an active state and an inactive state;

[0053] b) one or more releasable RNA segments, wherein each of the releasable RNA segments is flanked by two ribozyme cleavage sites, wherein cleavage at each cleavage site is catalyzed by at least one of the one or more catalytic domains in an active state;

[0054] c) one or more trigger factor binding domains, each of which is used to bind to a trigger nucleic acid molecule; each of the one or more catalytic domains is connected to one of the one or more trigger factor binding domains;

[0055] wherein when the trigger factor binding domain connected to the catalytic domain is not bound by the trigger nucleic acid molecule, the catalytic domain is in an inactive state, and wherein when the trigger factor binding domain connected to the catalytic domain is bound by the trigger nucleic acid molecule, the catalytic domain is in an active state; and wherein when both cleavage sites flanking the releasable RNA fragment are cleaved, one or more releasable RNA fragments are released from the ribozyme,

[0056] The ribozyme comprises an RNA strand having the following motif

[0057] Motif [A] and motif [a], wherein motif [A] and motif [a] constitute a trigger factor binding domain for binding to a trigger nucleic acid molecule;

[0058] Motif [B] and motif [b], wherein motif [B] and motif [b] constitute a linker that acts as a communication module to stabilize the catalytic domain when the trigger nucleic acid is bound, wherein motif [B] and motif [b] are independently at least 1 nucleotide in length;

[0059] Motif [C] and motif [c], wherein motif [C] and motif [c] constitute a catalytic domain;

[0060] motif [D], wherein motif [D] comprises a first cleavage site capable of being cleaved when catalyzed by the catalytic domain;

[0061] motif [D'], wherein motif [D'] comprises a second cleavage site capable of being cleaved when catalyzed by the catalytic domain;

[0062] motif [E], wherein motif [E] comprises a releasable RNA segment;

[0063] Motif [e], wherein motif [e] comprises a sequence that is partially complementary or fully complementary to the sequence of motif [E];

[0064] And wherein the motifs are connected by one or more than one optional linker regions.

[0065] In some embodiments, one or more motifs [A], motif [B], motif [C], motif [D], motif [E], motif [D'], motif [e], motif [c], motif [b], are in the 5' to 3' direction or the 3' to 5' direction to motif [a]. In some embodiments, one or more motifs [A], motif [B], motif [D], motif [E], motif [D'], motif [e], motif [b], are in the 5' to 3' direction or the 3' to 5' direction to motif [a].

[0066] In some embodiments, a ribozyme is provided, comprising:

[0067] a) one or more catalytic domains capable of switching between an active state and an inactive state;

[0068] b) one or more releasable RNA segments, wherein each of the releasable RNA segments is flanked by two ribozyme cleavage sites, wherein cleavage at each cleavage site is catalyzed by at least one of the one or more catalytic domains in an active state;

[0069] c) one or more trigger factor binding domains, each of which is used to bind to a trigger nucleic acid molecule; each of the one or more catalytic domains is connected to one of the one or more trigger factor binding domains;

[0070] wherein when the trigger factor binding domain connected to the catalytic domain is not bound by the trigger nucleic acid molecule, the catalytic domain is in an inactive state, and wherein when the trigger factor binding domain connected to the catalytic domain is bound by the trigger nucleic acid molecule, the catalytic domain is in an active state; and wherein when both cleavage sites flanking the releasable RNA fragment are cleaved, one or more releasable RNA fragments are released from the ribozyme,

[0071] The ribozyme contains an RNA chain with the following structure:

[0072]

[0073] wherein [A] to [a] is in the 5' to 3' direction or the 3' to 5' direction, and wherein:

[0074] Motif [A] and motif [a] constitute a trigger factor binding domain for binding to a trigger nucleic acid molecule;

[0075] Motif [B] and motif [b] constitute a linker that acts as a communication module to stabilize the catalytic domain when the trigger nucleic acid is bound, wherein the length of motif [B] and motif [b] are independently at least 1 nucleotide;

[0076] Motif [C] and motif [c] constitute the catalytic domain;

[0077] Motif [D] comprises a first cleavage site capable of being cleaved when catalyzed by the catalytic domain;

[0078] The motif [D'] comprises a second cleavage site capable of being cleaved when catalyzed by the catalytic domain;

[0079] Motif [E] contains a releasable RNA segment;

[0080] Motif [e] comprises a sequence that is partially complementary or fully complementary to the sequence of motif [E];

[0081] The horizontal line across each connecting motif represents an optional linker region.

[0082] Wherein the catalytic domain refers to one or more domains comprising nucleotides required for or involved in ribozyme catalysis, or a motif comprising nucleotides required for or involved in catalysis. In some embodiments, there is no single catalytic domain that switches between an active state and an inactive state. In some embodiments, the ribozyme needs to bind a trigger factor to fold into a conformation that allows cleavage by nucleotides involved in catalysis. In some embodiments, the cleavage at the cleavage site can be catalyzed by one or more catalytic nucleotides located in the catalytic domain. As a result, when the trigger factor binding domain connected to the catalytic domain is not bound by a trigger nucleic acid molecule, the ribozyme is in an inactive state, and wherein when the trigger factor binding domain connected to the catalytic domain is bound by a trigger nucleic acid molecule, the ribozyme is in an active state.

[0083] In some embodiments, a ribozyme may comprise:

[0084] a) one or more catalytic domains capable of switching between an active state and an inactive state,

[0085] b) one or more releasable RNA segments, wherein each of the releasable RNA segments is flanked by two ribozyme cleavage sites, wherein cleavage at each cleavage site is catalyzed by at least one of the one or more catalytic domains in an active state;

[0086] c) one or more trigger factor binding domains, each of which is used to bind to a trigger nucleic acid molecule; each of the one or more catalytic domains is connected to one of the one or more trigger factor binding domains;

[0087] wherein when the trigger factor binding domain connected to the catalytic domain is not bound by the trigger nucleic acid molecule, the catalytic domain is in an inactive state, and wherein when the trigger factor binding domain connected to the catalytic domain is bound by the trigger nucleic acid molecule, the catalytic domain is in an active state; and

[0088] When two cleavage sites flanking the releasable RNA fragment are cleaved under the catalysis of one or more catalytic domains, one or more releasable RNA fragments are released from the ribozyme.

[0089] The ribozyme comprises an RNA chain having the following structure:

[0090]

[0091] wherein [A] to [a] is in the 5' to 3' direction or the 3' to 5' direction, and wherein:

[0092] Motif [A] and motif [a] constitute a trigger factor binding domain for binding to a trigger nucleic acid molecule;

[0093] Motif [B] and motif [b] constitute a linker that acts as a communication module to stabilize the catalytic domain when the trigger nucleic acid is bound, wherein the length of motif [B] and motif [b] are independently at least 1 nucleotide;

[0094] Motif [C] and motif [c] constitute the catalytic domain;

[0095] Motif [D] comprises a first cleavage site capable of being cleaved when catalyzed by the catalytic domain;

[0096] The motif [D'] comprises a second cleavage site capable of being cleaved when catalyzed by the catalytic domain;

[0097] Motif [E] contains a releasable RNA segment;

[0098] Motif [e] comprises a sequence that is partially complementary or fully complementary to the sequence of motif [E];

[0099] Each horizontal line connecting the motifs represents an optional linker region.

[0100] As used herein, the term "ribozyme" refers to an RNA molecule that can catalyze a specific biochemical reaction. Common examples of this type of reaction include the cutting or connection of RNA and DNA, and the formation of peptide bonds. As used herein, the term "ribozyme" includes natural ribozymes and artificial ribozymes. Artificial ribozymes include synthetic ribozymes and modified or engineered natural ribozymes. The term "ribozyme" also encompasses fusion ribozymes and ribozyme complexes derived from natural ribozymes or artificial ribozymes.

[0101] In some embodiments, the term "ribozyme" may include ribozymes that contain one or more than one modification to its phosphate backbone, sugar or nucleobase or have conjugations. That is, ribozymes as disclosed herein may contain changes to its phosphate backbone (e.g., replacing phosphate bonds with thiophosphate bonds). They may contain nucleotides with modified sugar moieties or sugar moiety analogs. Sugar moiety modifications include, but are not limited to, 2'-O-aminoethoxy, 2'-O-aminoethyl (2'-OAE), 2'-O-methoxy, 2-guanidineethyl (2'-OGE), 2'-O, 4'-C-methylene (LNA), 2'-O-(N-(methyl)acetamido) (2'-OMA), 2'-O-methyl, 2'-fluoro, 2'-O-(methoxyethyl) (2'-OME), etc. They can also contain base modifications, such as 5-methylcytosine or pseudouridine. Such modifications are introduced to improve the stability of ribozymes and reduce their immunogenicity. Methods for introducing such modifications into RNA (eg, ribozymes) are well known in the art.

[0102] As used herein, the terms "nucleic acid" or "polynucleotide" used interchangeably herein refer to a polymeric form of nucleotides of any length, which may be ribonucleotides or deoxyribonucleotides. Thus, the term includes, but is not limited to, single-stranded, double-stranded or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine bases and pyrimidine bases or other natural, chemically modified or biochemically modified, non-natural or derived nucleotide bases.

[0103] As used herein, the term "catalytic domain" refers to one or more than one domain in a ribozyme, which comprises nucleotides required for catalyzing the above-mentioned biochemical reactions or participating in the above-mentioned biochemical reactions, or comprises motifs required for catalysis or participating in catalysis together. The domain may not be a continuous fragment or structure of a ribozyme, but may comprise nucleotides located in different parts of the sequence or structure of the ribozyme. In some embodiments, the ribozyme needs to bind to a trigger factor to fold into a conformation that allows the nucleotides involved in catalysis to be cut. In some embodiments, the cutting at the cleavage site can be catalyzed by one or more than one catalytic nucleotides, which are generally referred to as catalytic domains. As a result, when the trigger factor binding domain connected to the catalytic domain is not bound by the trigger nucleic acid molecule, the ribozyme is in an inactive state, and wherein when the trigger factor binding domain connected to the catalytic domain is bound by the trigger nucleic acid molecule, the ribozyme is in an active state. In one embodiment, in a ribozyme capable of cutting RNA, one or more than one catalytic domains participate in the cutting of the RNA backbone at the catalytic ribozyme cleavage site.

[0104] The term "ribozyme cleavage site" refers to a sequence that is recognized and cleaved by one or more ribozyme catalytic domains. Unless otherwise indicated, the term "cleavage site" as used herein refers to a ribozyme cleavage site. When the ribozyme is able to catalyze a biochemical reaction, the ribozyme is in an "active state"; and when the ribozyme is unable to catalyze a biochemical reaction, the ribozyme is in an "inactive state". In other embodiments, when the ribozyme is able to cleave the ribozyme cleavage site, the ribozyme is in an "active state"; and when the ribozyme is unable to cleave the ribozyme cleavage site, the ribozyme is in an "inactive state".

[0105] The term "target binding domain" refers to a domain that can bind to a target nucleic acid molecule. In some embodiments, the term "target binding domain" can be used interchangeably with the term "trigger binding domain". In some embodiments, the binding between the target nucleic acid molecule / trigger nucleic acid molecule and the target binding domain / trigger binding domain occurs through the renaturation of complementary sequences between the two. Therefore, the sequence of one or more target binding domains / trigger binding domains can be designed or modified to enable them to bind to different target nucleic acid molecules with specific sequences. In some embodiments, the target nucleic acid molecule can be a target RNA molecule and / or a target DNA molecule.

[0106] As used herein, the term "complementarity" describes the relationship between two nucleotides or two polynucleotides. When referring to nucleic acid complementarity, nucleotide A is complementary to nucleotide U, and vice versa, and nucleotide C is complementary to nucleotide G, and vice versa. Complementary nucleotides include nucleotides that perform Watson-Crick base pairing, and nucleotides that perform base pairing in an alternative mode, such as G:U wobble base pairs. It should be understood that unless explicitly stated (for example, by specifying a percentage or the term "completely" or the term "partially"), the term "complementarity" or "complementary" includes varying degrees of complementarity when associated with nucleotides. As used herein, the term "complementarity" refers to the degree and pattern of complementarity between a nucleic acid chain or nucleic acid fragment and another nucleic acid chain or nucleic acid fragment. When a percentage is specified for "complementarity" or "complementarity" between two polynucleotides (or its fragments), the percentage refers to the percentage of nucleotides complementary to other polynucleotides (or its fragments) in a polynucleotide (or its fragment). Therefore, it should be understood that fully complementary and partially complementary are covered when referring to two polynucleotide chains as "complementary".

[0107] The term "target nucleic acid molecule" and the term "trigger nucleic acid molecule" can be used interchangeably in the present disclosure. As used herein, both terms refer to target nucleic acid molecules that are sensed and bound by a target binding domain or a trigger factor binding domain. In some embodiments, when bound to a trigger factor binding domain, the trigger nucleic acid converts the ribozyme from an inactive state to an active state. Without wishing to be bound by theory, the present disclosure also includes that the binding of a trigger factor can stabilize the ribozyme so that the ribozyme can fold into a conformation required for cutting the cleavage site. Therefore, in some embodiments, the binding of a trigger factor can be used to stabilize the ribozyme to allow cutting by nucleotides involved in catalysis. In some embodiments, the binding of a trigger factor can include two situations: the binding of a trigger factor converts the catalytic domain from an inactive state to an active state, and the binding of a trigger factor stabilizes the ribozyme to allow the structural conformation required for cutting.

[0108] The term "connected" refers to the relationship between two domains, and can refer to a physical connection, a functional connection, or both. In one embodiment of the invention, the catalytic domain is connected to the target binding domain when the ability of the catalytic nucleotide to perform catalysis is determined by the state of the target binding domain, in particular, by whether the target binding domain binds to its corresponding target RNA molecule.

[0109] The term "flanked" refers to a polynucleotide sequence adjacent to another sequence, or a polynucleotide sequence located between an upstream polynucleotide sequence and / or a downstream polynucleotide sequence, i.e., a polynucleotide sequence located 5' and / or 3' relative to the sequence. For example, "a releasable RNA fragment flanked by two cleavage sites" means that one cleavage site is located at the 5' of the releasable RNA fragment, and the other cleavage site is located at the 3' of the releasable RNA fragment; however, there may be a spacer sequence between them.

[0110] Since the cleavage site is contained in the ribozyme itself, the ribozyme of the present invention is considered to be a self-cleaving ribozyme, and "releasable RNA fragments" can be considered to be the cleavage products of self-cleavage activity. Since the release of releasable RNA fragments from the ribozyme is the result of the ribozyme binding to one or more than one target nucleic acid molecules, the ribozyme can be used to detect the presence of the target nucleic acid molecule. Since the target nucleic acid molecule can be released from the ribozyme to activate more ribozymes and trigger the release of more "releasable RNA fragments", the presence of the target nucleic acid molecule can be amplified by releasing the "releasable RNA fragments" with a higher copy number. "Releasable RNA fragments" are variable and can be designed to include multiple sequences. In some embodiments where "releasable RNA fragments" include the same sequence as the target nucleic acid molecule, the target nucleic acid molecule (or its sequence) is amplified using the ribozyme of the present invention. In certain embodiments, the releasable RNA fragments can include any one or more than one sequence SEQ ID NO:307 to SEQ ID NO:409.

[0111] In some embodiments, binding of the target nucleic acid molecule to the target binding domain enables catalysis by the catalytic nucleotide, which results in cleavage of the cleavage site and subsequent release of a releasable nucleic acid fragment.

[0112] In view of the definition of "complementary" provided above in this specification, it should be understood by those skilled in the art that the expression "optionally complementary" as used herein and in this specification not only covers complete complementarity (100% complementary) and partial complementarity (0% to 100% complementary), but also covers non-complementarity (0% complementary). In some embodiments, the present disclosure includes about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% complementary.

[0113] The term "direction" as used herein refers to the end-to-end chemical orientation of a single strand of an RNA molecule. In a single strand of RNA, the chemical convention for naming the carbon atoms in the nucleotide sugar ring means that there will be a 5' end, which contains a phosphate (or modified phosphate) group attached to the 5' carbon of the ribose ring, and a 3' end, which in native RNA contains an -OH at the 2' position, but can also contain a variety of modifications, including but not limited to 2'-fluoro, 2'-O-methyl, 2'-methoxyethyl, etc. For example, when [A] to [A'] of chain S1 is in the 5' to 3' direction, [a] to [a'] of chain S2 will be in the 3' to 5' direction.

[0114] As used herein, the term "motif" refers to a region on an RNA strand that has a specific structure or is involved in a specific function. As used herein, the term "domain" refers to a region of a ribozyme that has a specific structure or is involved in a specific function. As used herein, the term "domain" is used when referring to a functional entity formed by more than one RNA strand or more than one motif of an RNA strand. For example, a target binding domain comprises both motif [A] and motif [a], and the target binding domain is considered to "bind" to a target RNA molecule only when both motifs bind to the target RNA molecule.

[0115] In some embodiments, the ribozyme as disclosed herein further comprises one or more inhibition domains; each of the one or more catalytic domains is functionally connected to one of the one or more inhibition domains, wherein the catalytic domain is in an inactive state due to inhibition from the inhibition domain, and the inhibition domain is connected to one of the one or more target binding domains; wherein when one of the one or more target binding domains binds to a target nucleic acid molecule, the inhibition domain connected to the target binding domain stops inhibiting the catalytic domain connected to the inhibition domain, which causes the catalytic domain to be converted to an active state. In this embodiment, the connection between the target binding domain and the catalytic domain is completed by the inhibition domain, which is connected to both the target binding domain and the catalytic domain.

[0116] As is known in the art, secondary structures are typically formed within ribozymes. In embodiments of the present disclosure, one or more secondary structures are independently formed by any motif or optional joint region, or are jointly formed by a motif, a joint region, or a combination thereof. In certain embodiments, the optional joint region independently or jointly forms one or more secondary structures.

[0117] As used herein, the term "secondary structure" refers to a structure formed by the interaction between the nucleotides in one or more than one polynucleotide. The embodiment of the secondary structure includes but is not limited to a single nucleotide protrusion, a trinucleotide protrusion, a stem, a stem loop, a t-RNA type structure, a clover type structure, a tetracycle, a pseudoknot, a symmetrical inner ring, an asymmetric inner ring, a three-stem joint (3-channel joint), a four-stem joint (4-channel joint), a two-stem joint (2-channel joint) or a coaxial stacking or a combination thereof. The specific embodiment of the secondary structure includes a stem, a stem loop, a t-RNA type structure, a clover type structure, a tetracycle, a pseudoknot or a combination thereof. As used herein, the term "stem loop", also referred to as "hairpin loop", refers to two regions of the same chain, which are generally two regions complementary in a nucleotide sequence when read in opposite directions, when base pairing to form a double helix ending with an unpaired loop, the formed secondary nucleic acid structure.

[0118] Thus, in some embodiments, the optional linker regions in the ribozymes disclosed herein can independently or collectively form one or more secondary structures. In some embodiments, one or more secondary structures can include, but are not limited to, single nucleotide bulges, dinucleotide bulges, trinucleotide bulges, polynucleotide bulges, stems, stem loops, t-RNA type structures, clover-type structures, tetraloops, pseudoknots, symmetric inner loops, asymmetric inner loops, three stem linkers (3-way linkers), four stem linkers (4-way linkers), two stem linkers (2-way linkers), or coaxial stacking or a combination thereof.

[0119] As illustrated in the experimental section and the accompanying drawings, the ribozyme complex as disclosed herein may comprise a linker having a motif such as, but not limited to, a dual channel linker, a triple channel linker, a quad channel linker, a stem, a single nucleotide bulge, a dinucleotide bulge, a trinucleotide bulge, a polynucleotide bulge, and combinations thereof. In some embodiments, the linker between motif [C] and motif [D] (i.e., helix 2 and helix 3) may be, but not limited to, a dual channel linker, a triple channel linker, a quad channel linker, a stem, a single nucleotide bulge, a dinucleotide bulge, a trinucleotide bulge, a polynucleotide bulge, and combinations thereof. In some embodiments, the linker between motif [C] and motif [D] may comprise a triple channel linker and a stem (or may be referred to as a quad channel linker).

[0120] In some embodiments, the stem sequence connecting the adaptor to the motif [D] is 4 to 12 nucleotides in length.

[0121] In some embodiments, the length of the stem sequence connecting the adapter to the motif [D] is 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more nucleotides. In some embodiments, the length of the stem sequence connecting the adapter to the motif [D] is about 4, 5, 6, 7, 8, 9, 10, 11 or 12 nucleotides. In some embodiments, the length of the stem sequence connected to the adapter to the motif [D] is about 5, 6, 7, 8, 10, 11 or 12 nucleotides.

[0122] In some embodiments, the linker is selected from helix-helix-helix (HHH), helix [minus 1 nucleotide] - strand [7 nucleotides long] - helix (H1S7H), helix-helix-strand [4 nucleotides long] - helix (HHS4H), and helix-helix-strand [2 nucleotides long] - helix (HHHS2H), wherein the strand represents an unpaired nucleotide.

[0123] In some embodiments, the length of the stem sequence connecting the motif [C] and the motif [c] to the linker is 4 to 15 nucleotides. In some embodiments, the length of the stem sequence connecting the linker to the motif [C] and the motif [c] is 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 13 or more, 14 or more, 15 or more, 15 nucleotides. In some embodiments, the length of the stem sequence connecting the linker to the motif [C] and the motif [c] is about 4, 5, 6, 7, 8, 9, 10, 11 or 12 nucleotides. In some embodiments, the stem sequence connecting motif [C] and motif [c] to the linker is about 4, 5, 6, 7, 8, 10, 11, 12, 13, 14, or 15 nucleotides in length.

[0124] In some embodiments, the linker is selected from helix-helix-helix (HHH), helix [minus 1 nucleotide] - strand [7 nucleotides long] - helix (H1S7H), helix-helix-strand [4 nucleotides long] - helix (HHS4H), and helix-helix-strand [2 nucleotides long] - helix (HHHS2H), wherein the strand represents an unpaired nucleotide.

[0125] In some embodiments, the trigger nucleic acid molecule comprises a region that is complementary to a trigger factor binding domain, wherein the region is more than 10, more than 20, more than 30, more than 40, more than 50, more than 60, more than 70, more than 80, more than 90, more than 100, more than 110, more than 120, more than 130, more than 140, more than 150, more than 160, more than 170, more than 180, more than 190, or 200 nucleotides in length. In some embodiments, the length of the region is no more than 200, no more than 190, no more than 180, no more than 170, no more than 160, no more than 150, no more than 140, no more than 130, no more than 120, no more than 110, no more than 100, no more than 90, no more than 80, no more than 70, no more than 60, no more than 50, no more than 40, no more than 30, no more than 20, or no more than 10 nucleotides. In some embodiments, the trigger nucleic acid molecule can be any size from no more than 5 nucleotides in length to up to thousands or tens of thousands of nucleotides or more in length. Without wishing to be bound by theory, it will be understood by those skilled in the art that the ribozyme as described herein can bind to a trigger nucleic acid of any size as long as the ribozyme can bind to the binding site of the trigger nucleic acid.

[0126] In some embodiments, the nucleotides of motif [A] and motif [a] can bind to any site of the target nucleic acid molecule / trigger nucleic acid molecule to which the motif can bind. For example, if the nucleotides of motif [A] and motif [a] are 10 nucleotides each, and the trigger nucleic acid is 2000 nucleotides long, then [A] and [a] can bind to any two 10-nucleotide regions to which the motif can bind. In some embodiments, motif [A] and motif [a] can bind to any two regions of the target nucleic acid / trigger nucleic acid that are not sequentially adjacent to each other.

[0127] In some embodiments, the nucleotides of motif [A] and motif [a] can be complementary to each other at opposite ends of the target nucleic acid molecule. For example, motif [A] can be complementary to the 5' end of the target nucleic acid molecule, while motif [a] can be complementary to the 3' end of the target nucleic acid molecule, or vice versa.

[0128] The lengths of motif [A] and motif [a] are independent of each other and can be the same or different. For example, the length of each of motif [A] and motif [a] can be 3 to 5, or 5 to 10, or 10 to 15, or 15 to 20, or 20 to 30, or 30 to 40, or 40 to 50, or 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 nucleotides. In some other embodiments, each of motif [A] or motif [a] has a length of 1 to 5, or 5 to 10, or 10 to 15, or 15 to 20, or 20 to 30, or 30 to 40, or 40 to 50, 50 to 60, 60 to 70, 70 to 80, 80 to 90, 90 to 100, 100 to 110, 110 to 120, 130 to 140, 140 to 150, 150 to 160 nucleotides. In some embodiments, motif [a] has a length of 11 nucleotides. In some embodiments, the above description of motif [A] and motif [a] also applies to motif [A'] and motif [a'].

[0129] Complementary binding is partial complementarity or full complementarity. For example, motif [A] is complementary to a region of a target nucleic acid molecule / trigger nucleic acid molecule by about 70% to about 80%, or about 80% to about 90%, or about 90% to about 100%, or about 75% to about 85%, or about 85% to about 95%, or about 95% to about 100%, or about 88% to about 98%, or about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100%; and the motif [A] is complementary to a region of a target nucleic acid molecule / trigger nucleic acid molecule by about 70% to about 80%, or about 80% to about 90%, or about 90% to about 100%, or about 95% to about 95%, or about 95% to about 100%, or about 88% to about 98%, or about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100%; [a] is about 70% to about 80%, or about 80% to about 90%, or about 90% to about 100%, or about 75% to about 85%, or about 85% to about 95%, or about 95% to about 100%, or about 88% to about 98%, or about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% complementary to a region of another target RNA molecule / trigger RNA molecule, or vice versa.

[0130] In some embodiments, one or more than one trigger factor binding domain is used to bind to the same target nucleic acid molecule / trigger nucleic acid molecule. Examples of target nucleic acid molecules or trigger nucleic acid molecules may include, but are not limited to, viral nucleic acids, bacterial nucleic acids, modified nucleic acids (such as mutations in nucleic acids), messenger nucleic acids, coding nucleic acids, genomic nucleic acids, etc.

[0131] Examples of target DNA molecules or trigger DNA molecules may include, but are not limited to, viral DNA, cDNA, circulating DNA, cell-free DNA (cfDNA), fetal DNA, modified DNA (e.g., mutations), single-stranded DNA, double-stranded DNA, mitochondrial DNA, etc.

[0132] In some embodiments, the target nucleic acid / trigger nucleic acid may include, but is not limited to, viral RNA, microRNA (miRNA), short interfering RNA (siRNA), small RNA (sRNA), messenger RNA (mRNA), non-coding RNA (ncRNA), short non-coding RNA, transfer RNA (tRNA), ribosomal RNA (rRNA), transfer-messenger RNA (tmRNA), clustered regularly interspaced short palindromic repeat RNA (CRISPR RNA), antisense RNA, pre-mRNA, circular RNA or pre-miRNA, or fragments thereof. In some embodiments, the trigger RNA molecule is a microRNA, or a precursor thereof, or a fragment thereof. In some embodiments, the trigger RNA can be a Let-7 microRNA precursor. In some embodiments, the trigger nucleic acid can include any one or more than one sequence SEQ ID NO: 221 to SEQ ID NO: 306.

[0133] In some embodiments, the target nucleic acid molecule / trigger nucleic acid molecule may include one or more than one modified nucleotides. In some embodiments, the target nucleic acid molecule / trigger nucleic acid molecule may include nucleotides with modified phosphate backbones, modified sugar moieties or sugar moiety analogs or modified bases. Sugar moiety modifications include but are not limited to 2'-O-aminoethoxy, 2'-O-aminoethyl (2'-OAE), 2'-O-methoxy, 2-guanidineethyl (2'-OGE), 2'-O, 4'-C-methylene (LNA), 2'-O-(N-(methyl)acetamido) (2'-OMA), 2'-O-methyl, 2'-fluoro, 2'-O-(methoxyethyl) (2'-OME) etc. The modified phosphate bond includes thiophosphate bond and thiophosphorylsulfide bond. The modified base can include hundreds of naturally occurring and synthetic modification examples, such as 5-methylcytosine, methyladenosine and pseudouridine, just to name a few of the most common examples.

[0134] As used herein, the term "microRNA" (abbreviated as miRNA) refers to a non-coding small RNA molecule. It generally plays a role in RNA silencing and post-transcriptional regulation of gene expression. Although most miRNAs are located in cells, some miRNAs have also been found in the extracellular environment, including various biological fluids and cell culture media, and are generally referred to as circulating miRNAs or extracellular miRNAs.

[0135] In some embodiments, the length of the releasable RNA fragment is more than 5, more than 10, more than 15, more than 20, more than 30, more than 40, more than 50, more than 60, more than 70, more than 80, more than 90, more than 100, more than 110, more than 120, more than 130, more than 140, or 150 nucleotides. In some embodiments, the length of the releasable RNA fragment is no more than 150, no more than 140, no more than 130, no more than 120, no more than 110, no more than 100, no more than 90, no more than 80, no more than 70, no more than 60, no more than 50, no more than 40, no more than 30, no more than 20, or no more than 10 nucleotides. In some embodiments, the length of the releasable RNA fragment can be about 1 to about 200, 1 to 20, 21 to 30, 31 to 40, 41 to 50, 51 to 60, 61 to 70, 71 to 80, 81 to 90, 91 to 100, 101 to 110, 111 to 120, 121 to 130, 131 to 140, or 141 to 150 nucleotides. In some embodiments, the length of the releasable RNA fragment is 6 to 150 nucleotides.

[0136] Some ribozymes as disclosed herein are capable of functioning in the presence of complete or partial complementarity between the releasable cleavage product and its complementary strand.In some embodiments, the releasable RNA fragment comprises a sequence identical to at least one of one or more than one target nucleic acid molecule / trigger nucleic acid molecule.

[0137] In some embodiments, each of motif [E] and motif [e] is 6 to 160 nucleotides in length. In some embodiments, each of motif [E] and motif [e] is 5 to 10, or 10 to 15, or 15 to 20, or 20 to 30, or 30 to 40, or 40 to 50, 50 to 60, 60 to 70, 70 to 80, 80 to 90, 90 to 100, 100 to 110, 110 to 120, 130 to 140, 150 to 200, 200 to 300, 200 to 400, 200 to 500, 200 to 600, 200 to 700, 200 to 800, 200 to 900, 200 to 1000, 200 to 1100, 200 to 1200, 200 to 1300, 200 to 1400, 200 to 1500, 200 to 1600, 200 to 1 10 to 120, 120 to 130, 130 to 140, 140 to 150, or 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 nucleotides.

[0138] In some embodiments, if motif [e] and motif [E] are partially complementary to each other, the complementarity between motif [e] and motif [E] is characterized by alternating regions of complementarity and non-complementarity.

[0139] In some embodiments, motif [E] and motif [e] are less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, or less than 30%, or about 10%, or about 20%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90% complementary to each other. In some embodiments, the total complementarity between motif [E] and motif [e] is at least 20%.

[0140] In some embodiments, the complementarity between motif [E] and motif [e] is characterized by alternating regions of complementarity and non-complementarity. The inventors contemplate that the complementarity of nucleotides may include an unlimited complementarity pattern. A non-exhaustive list of possible complementarity patterns includes, but is not limited to, 5'–[1 nucleotide complementary–1 nucleotide non-complementary]n–3'; 5'–[1 nucleotide complementary–2 nucleotides non-complementary]n–3'; 5'–[1 nucleotide complementary–3 nucleotides non-complementary]n–3'; 5'–[1 nucleotide complementary–4 nucleotides non-complementary]n–3'; 5'–[2 nucleotide complementary–1 nucleotide non-complementary]n–3'; 5'–[2 nucleotide complementary–2 nucleotides non-complementary]n–3'; 5'–[2 nucleotide complementary–3 nucleotides non-complementary]n–3'; 5'–[2 nucleotide complementary–4 nucleotides non-complementary]n–3'; 5'–[3 nucleotide complementary–1 nucleotide non-complementary]n–3'; 5'–[3 nucleotide complementary–2 nucleotides non-complementary]n–3'; 5'– [3 nucleotides complementary–3 nucleotides non-complementary]n–3’; 5’–[3 nucleotides complementary–4 nucleotides non-complementary]n–3’; 5’–[4 nucleotides complementary–1 nucleotide non-complementary]n–3’; 5’–[4 nucleotides complementary–2 nucleotides non-complementary]n–3’; 5’–[4 nucleotides complementary–3 nucleotides non-complementary]n–3’; 5’–[4 nucleotides complementary–4 nucleotides non-complementary]n–3’; 5’–[2 nucleotides complementary–2 nucleotides non-complementary-1 nucleotide complementary–1 nucleotide non-complementary]n–3’; 5’–[3 nucleotides complementary–3 nucleotides non-complementary-1 nucleotide complementary–1 nucleotide non-complementary]n–3’; 5’–[4 nucleotides complementary–4 nucleotides non-complementary-1 nucleotide complementary–1 nucleotide non-complementary]n–3’; etc. In some embodiments, n can be an integer from 1 to 200.

[0141] Examples of 5'–[3 nucleotides complementary–2 nucleotides non-complementary]n–3' are SEQ ID NO:66, SEQ ID NO:106, etc.; an example of 5'–[3 nucleotides complementary–3 nucleotides non-complementary]n–3' is SEQ ID NO:99; an example of 5'–[4 nucleotides complementary–1 nucleotide non-complementary]n–3' is SEQ ID NO:108; an example of complete complementarity is SEQ ID NO:130.

[0142] In some embodiments, the alternating complementary and non-complementary regions may not have a pattern (or may be random in nature). When designing a target ribozyme, the combination can be determined as needed. In some embodiments, the complementary pattern can be determined by the target nucleic acid / trigger nucleic acid of interest. In some embodiments, the complementary pattern can be determined by the cleavage product.

[0143] In some embodiments, each complementary region is no longer than 3 consecutive nucleotides, and each non-complementary region is at least 3 consecutive nucleotides in length. In some embodiments, each complementary region between motif [E] and motif [e] is no longer than 3 consecutive nucleotides, wherein each non-complementary region between motif [E] and motif [e] is at least 2 consecutive nucleotides in length.

[0144] As used herein, "complementary region" refers to the region in which the first RNA chain and the second RNA chain of the ribozyme are completely complementary to each other; "non-complementary region" refers to the region in which the first RNA chain and the second RNA chain of the ribozyme are not complementary to each other.

[0145] In some embodiments, the complementarity follows the pattern of 5'-3 nucleotides complementary-3 nucleotides non-complementary [2 nucleotides complementary-3 nucleotides non-complementary-2 nucleotides complementary]n-3 nucleotides non-complementary-3 nucleotides complementary-3'; where n is the number of pattern repetitions required to cover the length of motif [E] and motif [e]. In some embodiments, n can be a number from 1 to 50, or 1 to 10, 10 to 20, 20 to 30, 30 to 40, 40 to 50, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200.

[0146] In some embodiments, the releasable RNA fragment can be a functional RNA, such as but not limited to small guide RNA (sgRNA), guide RNA (gRNA), short hairpin RNA (shRNA), RNA aptamer, etc.

[0147] Without wishing to be bound by theory, it is observed that various forms of motif [B] and motif [b] (i.e., helix 4 communication modules) as described herein advantageously impart the properties of very low background cleavage, low leakage, and high cleavage signal, which is ideal for a variety of applications.

[0148] Without wishing to be bound by theory, it is observed that modified RNA (e.g., locked nucleic acid modified as disclosed herein) can bind ribozymes more strongly than unmodified RNA. In some embodiments, modified RNA can bind modified ribozymes (e.g., ribozymes modified with locked nucleic acid nucleotides as disclosed herein) more strongly. Therefore, in some embodiments, the length of motif [B] and motif [b] can be as short as 1 nucleotide. In some embodiments, the length of motif [B] and motif [b] is independently 1 or more than 1 nucleotide, optionally 2 or more than 2 nucleotides, optionally 3 or more than 3 nucleotides. In some embodiments, the length of motif [B] and motif [b] is independently 1 or more than 1, 2 or more than 2, 3 or more than 3, or 4 or no more than 4 nucleotides. In some embodiments, the length of motif [B] and motif [b] is independently 1, 2, 3 or 4 nucleotides.

[0149] In some embodiments, motif [B] and motif [b] have a motif selected from SEQ ID NO: 1 (5'-ACG / CGU-3'), SEQ ID NO: 2 (5'-ACG / CGA-3'), SEQ ID NO: 449 (5'-ACG / UGA-3'), SEQ ID NO: 450 (5'-AUG / CGA-3'), SEQ ID NO: 451 (5'-AUG / UGA-3'), SEQ ID NO: 452 (5'-CG / CG-3'), SEQ ID NO: 453 (5'-UUG / UGG-3'), SEQ ID NO: 454 (5'-UAU / AUA-3'), SEQ ID NO: 455 (5'-ACU / AGA-3'), SEQ ID NO: 456 (5'-AUG / CAA-3'), SEQ ID NO: 457 (5'-CU / AG-3'), and SEQ ID NO: 458 (5'- The sequence of NO:458 (5'-UG / CA-3').

[0150] In some embodiments, each of the motifs [C], [c], [D], and [d] is independently 1 to 100 nucleotides in length. In some embodiments, each of [C], [c], [D], and [d] is 1 to 5, or 5 to 10, or 10 to 15, or 15 to 20, or 20 to 30, or 30 to 40, or 40 to 50, 50 to 60, 60 to 70, 70 to 80, or 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides in length. In some specific embodiments, motif [C] has the same length as motif [c].

[0151] As shown in the experimental section, the inventors of the present disclosure have found that mutations at specific sites of the motif advantageously promote cleavage by the ribozyme. +3 A mutation at the paired nucleotide N7, wherein the mutation increases cleavage when the sequence at the cleavage site deviates from the canonical sequence. In some embodiments, motif [D] comprises a mutation at nucleotide N7 paired with nucleotide N+3.

[0152] Without wishing to be bound by theory, it is believed that one or more than one or all of the above features allow the formation of a trigger-activated ribozyme (or binuclear enzyme). That is, in some embodiments, the disclosure provides a (binuclear) ribozyme comprising one or more than one feature, such as a circularly arranged (binuclear) ribozyme, reduced complementarity of the cleavage product, a 4-way adapter, a shortened helix 4, and a helix 4 of the trigger region that is altered to include a branched nucleic acid binding. In some embodiments, the disclosure includes a (binuclear) ribozyme comprising two or more than two features, or three or more than three features, or all four features as disclosed herein. As disclosed herein, each element of the ribozyme disclosed herein can be combined with other elements in different combinations.

[0153] Thus, as disclosed herein are embodiments of ribozymes having one or more than one motif as disclosed herein:

[0154] 1) A sequence motif in the helix 4 communication module of the ribozyme that results in very low background cleavage of the ribozyme in the absence of target RNA / trigger RNA and high cleavage rate in the presence of target nucleic acid / trigger nucleic acid, which is applicable to a range of ribozymes.

[0155] 2) The helix 2 domain was altered and lengthened, which reduced background cleavage at the cleavage site proximal to the catalytic domain.

[0156] 3) Ribozymes with increased and complete complementarity between the releasable cleavage product and its complementary strand, which reduces the release of background cleavage products.

[0157] 4) With nucleotide N +3 Mutation of the paired nucleotide N7, which promotes cleavage when the sequence at or near the cleavage site deviates from the canonical cleavage site sequence.

[0158] 5) The helix 3 domain was altered and lengthened, which reduces background cleavage at the cleavage site proximal to the catalytic domain.

[0159] In some embodiments, a ribozyme as disclosed herein may comprise two or more than two, three or more than three, or all four motifs as disclosed herein.

[0160] In some embodiments, the ribozyme may comprise a sequence as disclosed herein. In some embodiments, the ribozyme may comprise a sequence as disclosed in Table 1. In some embodiments, the ribozyme can comprise SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO:72, SEQ ID NO:73, SEQ IDNO:74、SEQID NO:75、SEQ ID NO:76、SEQ ID NO:77、SEQ ID NO:78、SEQ ID NO:79、SEQ ID NO:80、SEQIDNO:81、SEQ ID NO:82、SEQ ID NO:83、SEQ ID NO:84、SEQ ID NO:85、SEQ ID NO:86、SEQID NO:87、SEQ ID NO:88、SEQ ID NO:89、SEQ ID NO:90、SEQ ID NO:91、SEQ ID NO:92、SEQID NO:93、SEQ ID NO:94、SEQ ID NO:95、SEQ ID NO:96、SEQ ID NO:97、SEQ ID NO:98、SEQID NO:99、SEQ ID NO:100、SEQ ID NO:101、SEQ ID NO:102、SEQ ID NO:103、SEQ ID NO:104、SEQ ID NO:105、SEQ ID NO:106、SEQ ID NO:107、SEQ ID NO:108、SEQ ID NO:109、SEQID NO:110、SEQ ID NO:111、SEQ ID NO:112、SEQ ID NO:113、SEQ ID NO:114、SEQ ID NO:115、SEQ ID NO:116、SEQ ID NO:117、SEQ ID NO:118、SEQ ID NO:119、SEQ ID NO:120、SEQID NO:121、SEQ ID NO:122、SEQ ID NO:123、SEQ ID NO:124、SEQ ID NO:125、SEQ ID NO:126、SEQ ID NO:127、SEQ ID NO:128、SEQ ID NO:129、SEQ ID NO:130、SEQ ID NO:131、SEQID NO:132、SEQ ID NO:133、SEQ ID NO:134、SEQ ID NO:135、SEQ ID NO:136、SEQ ID NO:137、SEQ ID NO:138、SEQ ID NO:139、SEQ ID NO:140、SEQ ID NO:141、SEQ ID NO:142、SEQID NO:143、SEQ ID NO:144、SEQ ID NO:145、SEQ ID NO:146、SEQ ID NO:147、SEQ IDNO:148、SEQ ID NO:149、SEQ ID NO:150、SEQ ID NO:151、SEQ ID NO:152、SEQ ID NO:153、SEQID NO:154、SEQ ID NO:155、SEQ ID NO:156、SEQ ID NO:157、SEQ ID NO:158、SEQ ID NO:159、SEQ ID NO:160、SEQ ID NO:161、SEQ ID NO:162、SEQ ID NO:163、SEQ ID NO:164、SEQID NO:165、SEQ ID NO:166、SEQ ID NO:167、SEQ ID NO:168、SEQ ID NO:169、SEQ ID NO:170、SEQ ID NO:171、SEQ ID NO:172、SEQ ID NO:173、SEQ ID NO:174、SEQ ID NO:175、SEQID NO:176、SEQ ID NO:177、SEQ ID NO:178、SEQ ID NO:179、SEQ ID NO:180、SEQ ID NO:181、SEQ ID NO:182、SEQ ID NO:183、SEQ ID NO:184、SEQ ID NO:185、SEQ ID NO:186、SEQID NO:187、SEQ ID NO:188、SEQ ID NO:189、SEQ ID NO:190、SEQ ID NO:191、SEQ ID NO:192、SEQ ID NO:193、SEQ ID NO:194、SEQ ID NO:195、SEQ ID NO:196、SEQ ID NO:197、SEQID NO:198、SEQ ID NO:199、SEQ ID NO:200、SEQ ID NO:201、SEQ ID NO:202、SEQ ID NO:203、SEQ ID NO:204、SEQ ID NO:205、SEQ ID NO:206、SEQ ID NO:207、SEQ ID NO:208、SEQID NO:209、SEQ ID NO:210、SEQ ID NO:211、SEQ IDNO:212、SEQ ID NO:213、SEQ ID NO:214、SEQ ID NO:215、SEQ ID NO:216、SEQ ID NO:217、SEQ ID NO:218、SEQ ID NO:219、SEQIDNO:220、SEQ ID NO:221、SEQ ID NO:222、SEQ ID NO:223、SEQ ID NO:224、SEQ ID NO:225、SEQ ID NO:226、SEQ ID NO:227、SEQ ID NO:228、SEQ ID NO:229、SEQ ID NO:230、SEQID NO:231、SEQ ID NO:232、SEQ ID NO:233、SEQ ID NO:234、SEQ ID NO:235、SEQ ID NO:236、SEQ ID NO:237、SEQ ID NO:238、SEQ ID NO:239、SEQ ID NO:240、SEQ ID NO:241、SEQID NO:242、SEQ ID NO:243、SEQ ID NO:244、SEQ ID NO:245、SEQ ID NO:246、SEQ ID NO:247、SEQ ID NO:248、SEQ ID NO:249、SEQ ID NO:250、SEQ ID NO:251、SEQ ID NO:252、SEQID NO:253、SEQ ID NO:254、SEQ ID NO:255、SEQ ID NO:256、SEQ ID NO:257、SEQ ID NO:258、SEQ ID NO:259、SEQ ID NO:260、SEQ ID NO:261、SEQ ID NO:262、SEQ ID NO:263、SEQID NO:264、SEQ ID NO:265、SEQ ID NO:266、SEQ ID NO:267、SEQ ID NO:268、SEQ ID NO:269、SEQ ID NO:270、SEQ ID NO:271、SEQ ID NO:272、SEQ ID NO:273、SEQ ID NO:274、SEQID NO:275、SEQ ID NO:276、SEQ ID NO:277、SEQ ID NO:278、SEQ ID NO:279、SEQ ID NO:280、SEQ ID NO:281、SEQ ID NO:282、SEQ ID NO:283、SEQ ID NO:284、SEQ ID NO:285、SEQID NO:286、SEQ ID NO:287、SEQ ID NO:288、SEQ ID NO:289、SEQ ID NO:290、SEQ ID NO:291、SEQID NO:292、SEQ ID NO:293、SEQ ID NO:294、SEQ ID NO:295、SEQ ID NO:296、SEQID NO:297、SEQ ID NO:298、SEQ ID NO:299、SEQ ID NO:300、SEQ ID NO:301、SEQ ID NO:302、SEQ ID NO:303、SEQ ID NO:304、SEQ ID NO:305、SEQ ID NO:306、SEQ ID NO:307、SEQID NO:308、SEQ ID NO:309、SEQ ID NO:310、SEQ ID NO:311、SEQ ID NO:312、SEQ ID NO:313、SEQ ID NO:314、SEQ ID NO:315、SEQ ID NO:316、SEQ ID NO:317、SEQ ID NO:318、SEQID NO:319、SEQ ID NO:320、SEQ ID NO:321、SEQ ID NO:322、SEQ ID NO:323、SEQ ID NO:324、SEQ ID NO:325、SEQ ID NO:326、SEQ ID NO:327、SEQ ID NO:328、SEQ ID NO:329、SEQID NO:330、SEQ ID NO:331、SEQ ID NO:332、SEQ ID NO:333、SEQ ID NO:334、SEQ ID NO:335、SEQ ID NO:336、SEQ ID NO:337、SEQ ID NO:338、SEQ ID NO:339、SEQ ID NO:340、SEQID NO:341、SEQ ID NO:342、SEQ ID NO:343、SEQ ID NO:344、SEQ ID NO:345、SEQ ID NO:346、SEQ ID NO:347、SEQ ID NO:348、SEQ ID NO:349、SEQ ID NO:350、SEQ ID NO:351、SEQID NO:352、SEQ ID NO:353、SEQ ID NO:354、SEQ ID NO:355、SEQ ID NO:356、SEQ ID NO:357、SEQ ID NO:358、SEQ ID NO:359、SEQ ID NO:360、SEQ ID NO:361、SEQ ID NO:362、SEQID NO:363、SEQID NO:364、SEQ ID NO:365、SEQ ID NO:366、SEQ ID NO:367、SEQ ID NO:368、SEQ ID NO:369、SEQ ID NO:370、SEQ ID NO:371、SEQ ID NO:372、SEQ ID NO:373、SEQID NO:374、SEQ ID NO:375、SEQ ID NO:376、SEQ ID NO:377、SEQ ID NO:378、SEQ ID NO:379、SEQ ID NO:380、SEQ ID NO:381、SEQ ID NO:382、SEQ ID NO:383、SEQ ID NO:384、SEQID NO:385、SEQ ID NO:386、SEQ ID NO:387、SEQ ID NO:388、SEQ ID NO:389、SEQ ID NO:390、SEQ ID NO:391、SEQ ID NO:392、SEQ ID NO:393、SEQ ID NO:394、SEQ ID NO:395、SEQID NO:396、SEQ ID NO:397、SEQ ID NO:398、SEQ ID NO:399、SEQ ID NO:400、SEQ ID NO:401、SEQ ID NO:402、SEQ ID NO:403、SEQ ID NO:404、SEQ ID NO:405、SEQ ID NO:406、SEQID NO:407、SEQ ID NO:408、SEQ ID NO:409、SEQ ID NO:410、SEQ ID NO:411、SEQ ID NO:412、SEQ ID NO:413、SEQ ID NO:414、SEQ ID NO:415、SEQ ID NO:416、SEQ ID NO:417、SEQID NO:418、SEQ ID NO:419、SEQ ID NO:420、SEQ ID NO:421、SEQ ID NO:422、SEQ ID NO:423、SEQ ID NO:424、SEQ ID NO:425、SEQ ID NO:426、SEQ ID NO:427、SEQ ID NO:428、SEQID NO:429、SEQ ID NO:430、SEQ ID NO:431、SEQ ID NO:432、SEQ ID NO:433、SEQ ID NO:434、SEQ IDNO:435, SEQ ID NO:436, SEQ ID NO:437, SEQ ID NO:438, SEQ ID NO:439, SEQ ID NO:440, SEQ ID NO:441, SEQ ID NO:442, SEQ ID NO:443, SEQ ID NO:444, SEQ ID NO:445, SEQ ID NO:446, SEQ ID NO:447, and SEQ ID NO:448. In some embodiments, the ribozyme complex comprises any one or more of SEQ ID NO:3 to SEQ ID NO:448.

[0161] As will be appreciated by those skilled in the art, ribozymes as disclosed herein can be used to detect and / or amplify trigger / target nucleic acid molecules in a sample. Thus, in a second aspect of the present disclosure, a method for detecting the presence of a trigger nucleic acid molecule in a sample is provided, wherein the method comprises:

[0162] incubating the sample with a ribozyme as disclosed herein at a temperature T1 that allows binding of the trigger nucleic acid molecule to one or more trigger factor binding domains contained in said ribozyme;

[0163] incubating the sample at a temperature T2 that allows the release of nucleic acid molecules and RNA fragments from the ribozyme;

[0164] The release of the releasable RNA fragment from the ribozyme is detected.

[0165] An exemplary method of using a ribozyme disclosed herein includes a method of detecting the presence of a trigger nucleic acid molecule in a sample, wherein the method comprises:

[0166] a) incubating the sample with a ribozyme as disclosed herein at a temperature T1 that allows the binding of the trigger RNA molecule to one or more trigger factor binding domains contained in the ribozyme;

[0167] b) incubating the sample at a temperature T2 that allows the nucleic acid molecules and releasable RNA fragments to be released from the ribozyme;

[0168] c) Detecting the release of the releasable RNA fragment from the ribozyme.

[0169] In some embodiments, step c) is performed by detecting the presence of releasable RNA fragments in the sample. Any RNA detection method or RNA detection system known in the art can be used. Exemplary and non-exhaustive examples of RNA detection methods include: reverse transcription polymerase chain reaction (RT-PCR), quantitative RT-PCR (RT-qPCR), probe-based RNA detection (such as Northern blot, microarray and molecular beacon). Exemplary and non-exhaustive examples of RNA detection systems include: NanoString Technologies' miRNA expression analysis and Exiqon's Smart Flares, RNA-activated fluorescent sensors such as Pandan fluorescent sensors (PCT patent PCT / SG 2017 / 050086; Aw et al., Nucleic Acids Research 2016), and CRISPR-Cas based nucleic acid detection systems such as DETECTR (Chen, JS et al. CRISPR-Cas12a target-binding unleashes indiscriminate single-stranded DNase activity. Science 360, 436-439, doi: 10.1126 / science.aar6245 (2018)) and SHERLOCK (Gootenberg, JS et al. Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6. Science 360, 439-444, doi: 10.1126 / science.aaq0179 (2018)).

[0170] In some embodiments, before performing step c), steps a) to b) are repeated once or more than once, wherein the RNA molecules released from step b) are used to bind to another copy of the ribozyme. In the present embodiment, when step a) is performed for the first time, the sample can be incubated with an excess of ribozymes so that when step a) is performed for the second time or the number of times after the second time, no additional ribozymes may be added to the sample. Since the target nucleic acid molecules released from the ribozymes in step b) can further bind to new ribozymes, the number of copies of the released cleavage products ("releasable RNA fragments") can be many times higher than the number of copies of the target molecules / trigger molecules in the sample. Therefore, the presence or amount of the released "releasable RNA fragments" can be used as an amplified signal for the presence or amount of the target nucleic acid molecules / trigger nucleic acid molecules in the sample. Therefore, when used in combination, this method can improve the sensitivity of existing nucleic acid detection technologies.

[0171] The releasable RNA fragment can contain the same sequence as the target nucleic acid / trigger nucleic acid, in which case the released "releasable RNA fragment" can further bind to a new ribozyme as the target nucleic acid molecule itself. By repeating steps a) to b) as above, a significant amplification of the target nucleic acid molecule / trigger nucleic acid molecule (or the sequence of the target nucleic acid molecule / trigger nucleic acid molecule) can be achieved. Therefore, the method of amplifying the target nucleic acid molecule / trigger nucleic acid molecule is also considered to be part of the present disclosure. This method may include the following steps: a) incubating the target nucleic acid molecule / trigger nucleic acid molecule with the ribozyme described in the present disclosure at a temperature T1 that allows the target nucleic acid molecule / trigger nucleic acid molecule to bind to one or more target binding domains contained in the ribozyme, and wherein the releasable RNA fragment contains the same sequence as the target nucleic acid molecule / trigger nucleic acid molecule; b) incubating the ribozyme bound to the target nucleic acid molecule / trigger nucleic acid molecule at a temperature T2 that allows the target nucleic acid molecule / trigger nucleic acid molecule and the RNA fragment to be released from the ribozyme. In some embodiments, steps a) to b) are repeated once or more than once, wherein the target nucleic acid molecule / trigger nucleic acid molecule and the releasable RNA fragment released from step b) are used to bind another copy of the ribozyme.

[0172] In some embodiments, the releasable RNA fragment comprises a sequence identical to at least one of one or more than one trigger nucleic acid molecules. In some embodiments, the ribozyme comprises two trigger factor binding domains for binding to a specific trigger nucleic acid molecule, wherein the releasable RNA fragment comprises a sequence identical to the trigger nucleic acid molecule. In some embodiments, the binding of the trigger nucleic acid molecule results in the release of an RNA molecule comprising a sequence identical to the trigger nucleic acid molecule.

[0173] In some embodiments, the binding of the trigger nucleic acid molecule to one or more trigger factor binding domains of the ribozyme occurs at a temperature T1. In some embodiments, T1 is a temperature of no more than 50°C. In some embodiments, T1 is a temperature of 0°C to 50°C, a temperature of 15°C to 45°C, a temperature of 25°C to 45°C, a temperature of 30°C to 40°C, a temperature of 35°C to 38°C, a temperature of 36.5°C to 37.5°C. In a specific embodiment, T1 is a temperature of about 37°C.

[0174] In some embodiments, temperature T1 and temperature T2 are the same. In another embodiment, temperature cycling does not occur and / or is not required. Instead, the method requires incubating the sample at a specific temperature. In some embodiments, the ribozyme is incubated at 37° C. for 4 hours. In one embodiment, the method is a cleavage assay.

[0175] In some embodiments, the trigger nucleic acid molecule bound to the trigger factor binding domain of the ribozyme is released from the target binding domain at temperature T2. In some embodiments, the two cleavage sites flanking the releasable RNA fragment are cut, and the releasable RNA fragment is released at a preferred temperature T2. In some embodiments, T2 is a temperature of 20°C to 100°C, a temperature of 25°C to 80°C, a temperature of 30°C to 80°C, a temperature of 35°C to 80°C, a temperature of 40°C to 80°C, a temperature of 45°C to 80°C, a temperature of 50°C to 80°C, a temperature of 55°C to 75°C, or a temperature of 57°C to 63°C. In a specific embodiment, T2 is a temperature of about 60°C.

[0176] It should be understood that T1 and T2 as described above refer to temperatures that allow binding (binding of target RNA molecules) and release (release of target nucleic acid molecules and releasable RNA fragments), respectively. T1 should not be understood as a temperature at which no trigger nucleic acid molecules or releasable RNA fragments can be released; and T2 should not be understood as a temperature at which no target nucleic acid molecules can bind to the trigger factor binding domain. It is generally understood in the art that the binding (also referred to as "renaturation") and release (also referred to as "melting") of complementary RNA chains can occur simultaneously at a wide range of temperatures, although the kinetics are different, so T1 and T2 can be the same or different. In some embodiments, the trigger nucleic acid molecule can bind to the trigger factor binding domain of the ribozyme, trigger the cutting and release of the releasable RNA fragment, and release from the ribozyme, all of which are at a temperature of 35°C to 38°C. However, when the ribozyme is used to detect and amplify the trigger molecule, the implementation of renaturation (at T1) and melting (at T2), where T2 is higher than T1, drives the reaction forward and can result in an increase in the number of released releasable RNA products.

[0177] In another aspect, a method for detecting the presence of a target sequence or a target mutation on a target nucleic acid in a sample is provided, wherein the method comprises:

[0178] incubating the sample with a ribozyme as disclosed herein, thereby allowing the trigger nucleic acid molecule to bind to one or more trigger factor binding domains contained in the ribozyme;

[0179] incubating the sample, which allows the nucleic acid molecules and releasable RNA fragments to be released from the ribozyme;

[0180] detecting the release of the releasable RNA fragment from the ribozyme;

[0181] The releasable RNA fragment is a sgRNA or a shRNA; wherein detection of a target sequence or a target mutation in a sample results in signal generation.

[0182] In some embodiments, the trigger nucleic acid molecule is a nucleic acid molecule obtained from an animal, virus, bacteria, yeast or plant. Therefore, in some embodiments, the target nucleic acid molecule or trigger nucleic acid molecule may include but is not limited to viral nucleic acid, bacterial nucleic acid, modified nucleic acid (such as mutation in nucleic acid), messenger nucleic acid, coding nucleic acid, genomic nucleic acid, etc.

[0183] Examples of target DNA molecules or trigger DNA molecules may include, but are not limited to, viral DNA, cDNA, circulating DNA, cell-free DNA (cfDNA), fetal DNA, modified DNA (e.g., mutations), single-stranded DNA, double-stranded DNA, mitochondrial DNA, etc.

[0184] In some embodiments, the target nucleic acid / trigger nucleic acid may include but is not limited to viral RNA, microRNA (miRNA), short interfering RNA (siRNA), small RNA (sRNA), messenger RNA (mRNA), non-coding RNA (ncRNA), short non-coding RNA, transfer RNA (tRNA), ribosomal RNA (rRNA), transfer-messenger RNA (tmRNA), clustered regularly spaced short palindromic repeat RNA (CRISPR RNA), antisense RNA, pre-mRNA, circular RNA or pre-miRNA, or fragments thereof. In some embodiments, the trigger RNA molecule is a microRNA, or a precursor thereof, or a fragment thereof. The trigger nucleic acid molecule is an RNA or DNA molecule, or a modified RNA or DNA molecule.

[0185] In some embodiments, the trigger nucleic acid molecule is a viral genome or fragment thereof. In some embodiments, the virus may include, but is not limited to, Retroviridae, Lentiviridae, Coronaviridae, Picornaviridae, Caliciviridae, Flaviviridae, Togaviridae, Bornaviridae, Filoviridae, Paramyxoviridae, Pneumoviridae, Rhabdoviridae, Arenaviridae, Bunyaviridae, Orthomyxoviridae, Delta virus.

[0186] In some embodiments, the virus may include, but is not limited to, lymphocytic choriomeningitis virus, coronavirus, human immunodeficiency virus (HIV), severe acute respiratory syndrome virus (SARS), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), poliovirus, rhinovirus, hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, Norwalk virus, yellow fever virus, West Nile virus, dengue virus, Zika virus, rubella virus, Ross River virus, rhinovirus C, Sindbis virus, chikungunya virus, coxsackie virus, Borna disease virus, Ebola virus, Marburg virus, measles virus, mumps virus, Nipah virus, Hendra virus, Newcastle disease virus, human respiratory syncytial virus, rabies virus, Lassa virus, Hanta virus, Crimean-Congo hemorrhagic fever virus, influenza virus (e.g., influenza A virus), smallpox virus (variola virus)), Japanese encephalitis virus, Seoul virus, adenovirus, herpes simplex virus.

[0187] The inventors illustrate in the experimental part the potential application of the ribozyme for cell-free RNA signal amplification in fish cells and human cells and as a gene reporter and signal transducer in vitro and in vivo.

[0188] Thus, in some embodiments, methods or ribozymes as disclosed herein can be used in any of mammalian cells, mammalian cell lines, human cells, human cell lines, zebrafish cells, and zebrafish embryos. In some embodiments, methods or ribozymes as disclosed herein can be used in vitro or in vivo.

[0189] In some embodiments, ribozymes as disclosed herein can be used in any of mammalian cells, mammalian cell lines, human cells, human cell lines, zebrafish cells, and zebrafish embryos.

[0190] In another other aspect, polynucleotides encoding ribozymes as disclosed herein are provided. In some embodiments wherein the ribozyme comprises more than one RNA chain, the RNA chains can be encoded together on one polynucleotide or can be encoded separately on several polynucleotides.

[0191] As disclosed herein, the isolated nucleotide sequence can be used to transduce the binding signal of the target trigger nucleic acid sequence into the release of the second functional nucleic acid sequence. As disclosed herein, the isolated nucleic acid sequence contains a trigger factor binding domain comprising two trigger factor binding arms, and the trigger factor binding arm is sequence complementary to the target nucleic acid trigger factor, and can form a bimolecular interaction with the target trigger nucleic acid sequence. As disclosed herein, the isolated nucleic acid sequence also forms a ternary complex, which allows its own self-cleavage when combined with the target nucleic acid sequence, releasing RNA cleavage product fragments, which can take the form of functional RNA with a tertiary structure, such as CRISPR guide RNA (gRNA or sgRNA), short hairpin RNA (shRNA), RNA aptamer, etc. In some embodiments, as disclosed herein, ribozymes release CRISPR gRNA, shRNA or RNA aptamers in the presence of trigger nucleic acids. Therefore, as disclosed herein, ribozymes have the ability to play a gene regulation role in vitro and in vivo. For example, in fish and mammalian cells, the experimental part has demonstrated the functional gene regulation of ribozymes in vitro and in vivo.

[0192] The isolated nucleotide sequence also comprises one or both of the following distinct features: A) a catalytic domain that is stabilized by binding to a trigger nucleic acid, and B) a communication module between the catalytic domain and the trigger arm. Although it is envisioned that a range of sequence motifs will work, and indeed the optimal module for any particular ribozyme may need to be optimized empirically, 5 to 10 sequence motifs have been identified in helix 4 that allow for optimized low background / high cleavage activity in the absence and presence of trigger nucleic acids, respectively. It has also been found that extending helix 2 or helix 3 reduces background cleavage of the catalytic domain proximal cleavage site in the absence of a trigger factor.

[0193] In yet another aspect, a kit comprising a ribozyme as disclosed herein is provided.

[0194] In some embodiments, the kit further comprises a nucleic acid detection system. In some embodiments, the nucleic acid detection system comprises an RNA-activated fluorescent sensor. In some embodiments, the sensor is a Pandan fluorescent sensor or detection system (PCT patent PCT / SG2017 / 050086; Aw et al., Nucleic Acids Research 2016). In some embodiments, the nucleic acid detection system is a CRISPR-Cas-based nucleic acid detection system. Methods and systems for nucleic acid detection based on CRISPR Cas are known in the art and are disclosed in, for example, Chen, JS et al., CRISPR-Cas12a target-binding unleashes indiscriminate single-stranded DNase activity. Science 360, 436-439, doi: 10.1126 / science.aar6245 (2018) and Gootenberg, JS et al., Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6. Science 360, 439-444, doi: 10.1126 / science.aaq0179 (2018).

[0195] In some embodiments, ribozymes as disclosed herein may also function as nucleic acid signal transducers and amplifiers.

[0196] In some embodiments, the ribozyme is modified from a naturally occurring ribozyme. In some embodiments, the ribozyme is modified from an artificial ribozyme, a fusion ribozyme, a fragment thereof, and a derivative thereof. In some embodiments, the ribozyme has the characteristics of a hairpin ribozyme or a hammerhead ribozyme or a fragment thereof and a fusion thereof. In some embodiments, the ribozyme is a twin ribozyme or a double-chain ribozyme. In some embodiments, the ribozyme comprises a twin hairpin ribozyme structure. The design of ribozymes as described herein is described in an earlier application PCT / SG2020 / 050226, the contents of which are incorporated herein and briefly described below.

[0197] As is known in the art, a ribozyme can comprise one or more than one RNA strand. In a specific embodiment, the ribozyme comprises a first RNA strand and a second RNA strand. When the ribozyme comprises two RNA strands, the two RNA strands have sufficient complementarity so that they bind to each other. Typically, the two RNA strands are not completely complementary over their entire length. Each RNA strand can independently form a secondary structure, as is known in the art. In one embodiment, the ribozyme comprises the following structure:

[0198]

[0199] Wherein: S1 is the first RNA strand, S2 is the second RNA strand, wherein [A] to [A'] and [a] to [a'] represent opposite directions; motif [A] and motif [a] constitute a first trigger factor binding domain for binding to a first trigger nucleic acid molecule, motif [C] and motif [c] constitute a first catalytic domain, motif [D] comprises a first cleavage site that can be cleaved, motif [A'] and motif [a'] constitute a second trigger factor binding domain for binding to a second trigger nucleic acid molecule, motif [C'] and motif [c'] The invention relates to a method for preparing an RNA-binding domain comprising: constituting a second catalytic domain, wherein the motif [D'] comprises a second cleavage site capable of being cleaved by the second catalytic domain, the motif [E] comprises a releasable RNA fragment, the motif [e] comprises a sequence that is optionally complementary to the sequence of the motif [E], and each horizontal line connecting the motifs represents an optional linker region; and wherein when the first target binding domain binds to the first target RNA molecule, the first catalytic domain is in an active state; and wherein when the second target binding domain binds to the second target nucleic acid molecule, the second catalytic domain is in an active state.

[0200] In another embodiment, the ribozyme comprises the following structure:

[0201]

[0202] wherein: S1 is the first RNA strand, S2 is the second RNA strand, wherein [A] to [A'] and [a] to [a'] represent opposite directions; wherein motif [A] and motif [a] constitute a first trigger factor binding domain for binding to a first trigger nucleic acid molecule, motif [B] and motif [b] constitute a linker that acts as a communication module to stabilize the catalytic domain when the trigger nucleic acid is bound, wherein the length of motif [B] and motif [b] are independently at least 1 nucleotide;

[0203] Alternatively, wherein motif [B] and motif [b] constitute a first inhibitory domain, motif [C] and motif [c] constitute a first catalytic domain, motif [D] comprises a first cleavage site that can be cleaved by the first catalytic domain, motif [A'] and motif [a'] constitute a second trigger binding domain for binding to a second trigger nucleic acid molecule; motif [C'] and motif [c'] constitute a second catalytic domain, motif [D'] comprises a second cleavage site that can be cleaved by the second catalytic domain, motif [E] comprises a releasable RNA fragment, motif [e] comprises a sequence that is optionally complementary to the sequence of motif [E], and each horizontal line connecting the motifs represents an optional linker region, wherein the first inhibitory domain is characterized by the following i) or ii):

[0204] i) when the first target binding domain is not bound to the first target nucleic acid molecule, motif [b] renatures with motif [C], but when the first target binding domain is bound to the first target nucleic acid molecule, motif [b] renatures with motif [B],

[0205] ii) when the first target binding domain is not bound to the first target nucleic acid molecule, motif [B] renatures with motif [c], but when the first target binding domain is bound to the first target nucleic acid molecule, motif [B] renatures with motif [b],

[0206] When motif [b] renatures with motif [B], the first catalytic domain is in an active state.

[0207] In other embodiments, the first inhibitory domain is further characterized by the following i) or ii): i) motif [b] is at least 50% complementary to motif [C] and at least 20% complementary to motif [B], and ii) motif [B] is at least 50% complementary to motif [c] and at least 20% complementary to motif [b].

[0208] In other embodiments, the first inhibitory domain is further characterized by i) or ii) below:

[0209] i) motif [b] is at least 60%, at least 70%, at least 80% or at least 90% complementary to motif [C] and at least 20% complementary to motif [B], or

[0210] ii) Motif [B] is at least 60%, at least 70%, at least 80% or at least 90% complementary to motif [c] and at least 20% complementary to motif [b].

[0211] In another embodiment, the ribozyme comprises the following structure:

[0212]

[0213] in:

[0214] S1 is a first RNA strand, and S2 is a second RNA strand, wherein [A] to [A'] and [a] to [a'] represent opposite directions; wherein motif [A] and motif [a] constitute a first trigger factor binding domain for binding to a first trigger nucleic acid molecule, and motif [B] and motif [b] constitute a linker that acts as a communication module to stabilize the catalytic domain when the trigger nucleic acid is bound, wherein the length of motif [B] and motif [b] are independently at least 1 nucleotide;

[0215] Alternatively, wherein motif [B] and motif [b] constitute a first inhibitory domain, motif [C] and motif [c] constitute a first catalytic domain, motif [D] comprises a first cleavage site that can be cleaved by the first catalytic domain, motif [A'] and motif [a'] constitute a second trigger factor binding domain for binding to a second target RNA molecule; motif [B'] and motif [b'] constitute a second linker that acts as a communication module to stabilize the catalytic domain when the trigger nucleic acid is bound, wherein the length of motif [B'] and motif [b'] are independently at least 1 nucleotide;

[0216] Alternatively, wherein motif [B'] and motif [b'] constitute a second inhibitory domain, motif [C'] and motif [c'] constitute a second catalytic domain, motif [D'] comprises a second cleavage site that can be cleaved by the second catalytic domain, motif [E] comprises a releasable RNA fragment, motif [e] comprises a sequence that is optionally complementary to the sequence of motif [E], and each horizontal line connecting the motifs represents an optional linker region; wherein the first inhibitory domain is characterized by the following i) or ii):

[0217] i) when the first target binding domain is not bound to the first target nucleic acid molecule, motif [b] renatures with motif [C], but when the first target binding domain is bound to the first target nucleic acid molecule, motif [b] renatures with motif [B],

[0218] ii) when the first target binding domain is not bound to the first target nucleic acid molecule, motif [B] renatures with motif [c], but when the first target binding domain is bound to the first target nucleic acid molecule, motif [B] renatures with motif [b],

[0219] wherein the second inhibitory domain is characterized by the following i) or ii):

[0220] i) when the second target binding domain is not bound to the second target nucleic acid molecule, motif [b'] renatures with motif [C'], but when the second target binding domain is bound to the second target nucleic acid molecule, motif [b'] renatures with motif [B'],

[0221] ii) when the second target binding domain is not bound to the second target RNA molecule, the motif [B'] renatures with the motif [c'], but when the second target binding domain is bound to the second target RNA molecule, the motif [B'] renatures with the motif [b'],

[0222] When motif [b] renatures with motif [B], the first catalytic domain is in an active state, and when motif [b'] renatures with motif [B'], the second catalytic domain is in an active state.

[0223] One or more secondary structures can be formed independently by any motif or optional linker region, or formed jointly by motif, linker region or a combination thereof. In one embodiment, the optional linker region independently or jointly forms one or more secondary structures. In some embodiments of the ribozyme, wherein the ribozyme comprises two target binding domains and two catalytic domains, the ribozyme comprises one of the following structures:

[0224]

[0225] The linker region R1 and the linker region R2 independently or jointly form one or more secondary structures, and the linker region R1 ′ and the linker region R2 ′ independently or jointly form one or more secondary structures.

[0226] Table 1. Sequence list

[0227]

[0228]

[0229]

[0230]

[0231]

[0232]

[0233]

[0234]

[0235]

[0236]

[0237]

[0238]

[0239]

[0240]

[0241]

[0242]

[0243]

[0244]

[0245]

[0246]

[0247]

[0248]

[0249]

[0250]

[0251]

[0252]

[0253]

[0254]

[0255]

[0256]

[0257]

[0258]

[0259]

[0260]

[0261]

[0262]

[0263]

[0264]

[0265]

[0266]

[0267]

[0268]

[0269]

[0270]

[0271]

[0272]

[0273]

[0274]

[0275]

[0276]

[0277]

[0278]

[0279]

[0280]

[0281]

[0282]

[0283]

[0284]

[0285]

[0286]

[0287]

[0288]

[0289]

[0290]

[0291]

[0292]

[0293]

[0294]

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[0325] Note that GGGA is introduced by T7 polymerase at the 5' end of each ribozyme sequence; in some of the examples above, this is underlined or indicated in lower case.

[0326] Unless otherwise stated, the terms "coupled" or "connected" as used in this specification are intended to encompass either direct connection or connection through one or more intermediate means.

[0327] When referring to two elements, the term "associated" as used herein refers to a broad relationship between the two elements. The relationship includes, but is not limited to, a physical, chemical, or biological relationship. For example, when element A is associated with element B, element A and element B may be directly or indirectly connected to each other, or element A may contain element B, or vice versa.

[0328] When referring to two elements, the term “adjacent” used herein means that one element is very close to the other element, and may be, but is not limited to, elements contacting each other, or may further include elements separated by one or more other elements interposed therebetween.

[0329] The term "and / or", for example, "X and / or Y" should be understood as "X and Y" or "X or Y" and should be considered as providing clear support for both meanings or just one of them.

[0330] In addition, in the description herein, whenever the word "substantially" is used, it is understood to include, but not limited to, "completely" and the like. In addition, whenever terms such as "including", "comprising", etc. are used, it is intended to express non-restrictive descriptive language, because they broadly cover the elements / components listed after these terms, except for other components that are not explicitly listed. For example, when "including" is used, a reference to "a" feature is also intended to reference "at least one" of the feature. In the appropriate context, terms such as "consisting of", "consisting of", etc. can be considered as subsets of terms such as "including", "comprising", etc. In the embodiments disclosed herein that use terms such as "including", "comprising", etc., it should be understood that these embodiments provide guidance for the corresponding embodiments that use terms such as "consisting of", "consisting of", etc. In addition, whenever terms such as "about", "approximately", "approximately", etc. are used, it is generally intended to mean a reasonable variation, such as a variation of + / -5% of the disclosed value, or a variation of + / -4% of the disclosed value, or a variation of + / -3% of the disclosed value, a variation of + / -2% of the disclosed value, or a variation of + / -1% of the disclosed value.

[0331] In addition, in the description herein, certain values ​​may be disclosed as being within a certain range. The values ​​showing the range endpoints are intended to illustrate preferred ranges. Whenever describing a range, it is intended to express that the range covers and guides all possible subranges and single numerical values ​​within the range. That is, the range endpoints should not be interpreted as inflexible restrictions. For example, the description of the range of 1% to 5% is intended to specifically disclose subranges such as 1% to 2%, 1% to 3%, 1% to 4%, 2% to 3%, and independently specifically discloses the values ​​within the range, such as 1%, 2%, 3%, 4% and 5%. It should be understood that the single numerical values ​​within the range also include integers, fractions and decimals. In addition, whenever describing a range, it is also intended to express that the range covers and guides the value of up to 2 additional decimal places or significant figures (if applicable) from the numerical endpoints shown. For example, the description of a range of 1% to 5% is intended to specifically disclose the ranges of 1.00% to 5.00% and 1.0% to 5.0% and all intermediate values ​​thereof (e.g., 1.01%, 1.02% ... 4.98%, 4.99%, 5.00% and 1.1%, 1.2% ... 4.8%, 4.9%, 5.0%, etc.) The above specific disclosure is intended to apply to any depth / width of the range.

[0332] In addition, when describing some embodiments, the present disclosure may have disclosed the method and / or process as a specific order of steps. However, unless otherwise required, it should be understood that the method or process should not be limited to the specific order of steps disclosed. Other order of steps are also possible. The specific order of steps disclosed herein should not be interpreted as an undue limitation. Unless otherwise required, the method and / or process disclosed herein should not be limited to the steps performed in the written order. The order of the steps can be changed and still remain within the scope of the present disclosure.

[0333] Furthermore, it should be understood that although the present disclosure provides embodiments having one or more features / characteristics discussed herein, one or more of these features / characteristics may be disclaimed in other alternative embodiments, and the present disclosure provides support for such disclaimers and these associated alternative embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0334] The exemplary embodiments of the present disclosure will be discussed below, and in conjunction with the accompanying drawings where applicable, so that those of ordinary skill in the art will better understand and be apparent. It should be understood that other modifications related to ribozymes can be carried out without departing from the scope of the present invention. The exemplary embodiments are not necessarily mutually exclusive, because some exemplary embodiments can be combined with one or more than one embodiment to form new exemplary embodiments. The exemplary embodiments should not be construed as limiting the scope of the present disclosure.

[0335] Figure 1. Development of an RNA trigger-activated dual self-cleaving ribozyme with two trigger-binding catalytic domains.

[0336] Figure 1A Schematic diagram of a representative hairpin ribozyme consisting of two loops, loop A and loop B, each flanked by two helices. The arrow marks the cleavage site between the N-1 nucleotide and the guanine (G+1) nucleotide in loop A. The key catalytic nucleotides A38 and C25 in the catalytic loop B are marked. 5'-*G**-3' spans the cleavage site, and the most highly tolerated sequence with a cleavage activity of at least 20% of the wild-type ribozyme is shown in the box.

[0337] Figure 1Bi .Design strategies for RNA trigger-activated self-cleaving diribozymes that release embedded RNA products upon trigger-induced cleavage.

[0338] Figure 1Bii .Dual tandem ribozyme.

[0339] Figure 1Biii .Circularly permuted binuclear enzymes.

[0340] Figure 1Biv.Internal form of self-cleaving sensor ribozymes, in which the RNA trigger factor is built into the ribozyme structure.

[0341] Figure 1Bv .External form of self-cleaving sensor ribozyme, in which the trigger RNA is separated from the ribozyme structure.

[0342] Figure 1C When the length of helix 4 is at least 2 bp to 3 bp, the internal form of the sensor ribozyme retains self-cleavage activity. Figure 1C , Figure 1D and Fig.9A The helix 4 configurations tested in are shown in the upper panel. The asterisk marks the band corresponding to the predicted cleavage product of the ribozyme with a 4 bp helix 4. The right panel shows a blot of the same gel detecting the 29 nt cleavage product.

[0343] Figure 1D .Separation of the trigger from the ribozyme yields a dual ribozyme activated by the RNA trigger. The asterisk marks the band corresponding to the predicted cleavage product of the ribozyme with 8 bp helix 4. The lower panel shows a blot of the same gel detecting the 29 nt cleavage product. The label indicates a nonspecific product generated in the initial in vitro transcription that is probe negative.

[0344] Figure 2A Ribozymes with a single catalytic domain exhibit RNA-triggered dual self-cleavage. T-ban5p_Cl-29nt-clvRNA RNA-triggered dual ribozymes with A38 mutations in one or both catalytic domains showed that at least one catalytic domain is necessary and sufficient for dual self-cleavage.

[0345] Figure 2B .Sequence and structure of T-ban5p_Cl-29nt-clvRNA ribozyme with exemplary double cleavage sites with a single "right-side" wild-type catalytic domain or a single "left-side" reverse-linked catalytic domain.

[0346] Figure 2C . A monoribozyme with a double cleavage site and a single "right-side" wild-type catalytic domain or a single "left-side" reverse-linked catalytic domain (structure similar to Figure 2B ), wherein the catalytic domain has a sensing region triggered by dme-ban-5p, hsa-mir-451 or SARS-CoV-2E gene fragment, and is capable of cleaving at two cleavage sites to release the embedded 29ntRNA cleavage product.

[0347] Figure 2D.Optimization of the helix 4 communication module in the T-SARS-CoV-2-E gene_Cl-29nt-clvRNA ribozyme and identification of a 3nt motif that improves the signal-to-noise ratio of the ribozyme (other motifs were also identified).

[0348] Figure 2E The cleavage products released from the T-SARS-CoV-2-E gene_Cl-29nt-clvRNA ribozyme increased with increasing concentrations of the E gene trigger RNA. 600 nM ribozyme was used. Asterisks mark the bands corresponding to the predicted cleavage products.

[0349] Figure 2F .Sequence variants of the E gene test RNA were tested against the T-SARS-CoV-2-E gene_Cl-29nt-clvRNA ribozyme, showing that the ribozyme was able to distinguish closely related trigger RNAs with differences of 1nt to 3nt, while unrelated sequences did not trigger the ribozyme.

[0350] Figure 2G .The ribozyme is able to detect its trigger factor in a complex mixture of RNA where the nonspecific RNA outnumbers the trigger RNA by at least 1000 times.

[0351] Figure 3A Functional RNA can be embedded in ribozymes as cleavage products. Schematic diagram of ribozymes containing embedded small guide RNA (sgRNA).

[0352] Figure 3B . Schematic representation of a ribozyme containing an embedded short hairpin RNA (shRNA).

[0353] Figure 3C . Schematic representation of a ribozyme containing an embedded RNA aptamer Broccoli.

[0354] Figure 3D Cleavage assay of the ribozyme T-let-7f_Cl-sgRNAGFP. Asterisks mark the bands corresponding to the predicted cleavage products. The right panel shows a blot of the same gel detecting sgRNA cleavage products.

[0355] Figure 3Ei . Ribozyme T-let-7f_Cl-shRNAGFP cleavage assay and Figure 3Eii . Cleavage assay of ribozyme T-let-7f_Cl-shRNAGFP6. The asterisk marks the band corresponding to the predicted cleavage product. The right panel shows a blot of the same gel detecting shRNA cleavage products.

[0356] Figure 3F. Cleavage assay of ribozyme T-let-7f_Cl-Broccoli aptamer. The upper panel shows a blot of the same gel detecting aptamer cleavage products.

[0357] Figure 3G Members of the human let-7 microRNA family (top) and cleavage assay of the ribozyme T-let-7f_Cl-sgRNAGFP when triggered by each member of the let-7 family (bottom). The bottom panel shows a blot of the same gel detecting sgRNA cleavage products. Asterisks mark the bands corresponding to the predicted cleavage products.

[0358] Figure 3H . Extending helix 2 on T-let-7f_Cl-sgRNAGFP from 4 bp to 8 bp or 12 bp reduces trigger-independent cleavage at the proximal cleavage site (dashed box). Asterisks mark the bands corresponding to the predicted cleavage products of the ribozyme with 8 nt helix 2. The right panel shows a blot of the same gel detecting sgRNA cleavage products.

[0359] Figure 4A Editing rates in uninjected, positive control gRNA+Cas9 injected, ribozyme (triggered by let-7f to release gRNA targeting GFP)+Cas9 injected, and ribozyme+Cas9+let-7f morpholino injected zebrafish embryos. Mann-Whitney nonparametric test.

[0360] Figure 4B . Ribozyme (50% 2'-fluorinated C and U modified unilateral_Tlet7f_CshRNA-GFP6) detects the modified let-7f trigger factor, which increases the downregulation of GFP expression.

[0361] Figure 4C .The ribozyme (50% 2'-Fluorinated C modified single-sided_TE gene 20_CsgRNA-Stoplight_modC) detects the modified E gene trigger factor, which increases editing of the GFP locus.

[0362] Figure 5A . The original ribozyme of A7 which is located in the lower strand of the proximal cleavage loop is not modified.

[0363] Figure 5B .A7 to C7 mutation to enable pairing with the opposite strand (bottom C of the lower strand at the proximal cleavage loop).

[0364] Figure 5C : Mutation from A7 to U7 to enable pairing with the opposite strand (bottom U on the lower strand at the proximal cleavage loop).

[0365] Figure 5D : The N7 mutation paired with N+3 improves the cleavage activity of the ribozyme with the non-canonical sequence in the cleavage site (cleavage products are within the outline box).

[0366] Fig. 6A .Has a single-sided_TCel-mir-238_CsgRNA-GFP_ML_modC1_8H2-A7 ribozyme that is completely complementary (non-alternatingly complementary) to most of the cleavage products.

[0367] Figure 6B : Cleavage assay of unilateral_TCel-mir-238_CsgRNA-GFP_ML_modC1_8H2-A7 ribozyme.

[0368] Figure 6Ci to Figure 6Civ .Has a single-sided_TCel-mir-238_CsgRNA-GFP_ML_modC1(i) / or C2(ii) / or C3(iii) / or C4(iv)_8H2-A7 ribozyme that is completely complementary (non-alternatingly complementary) to most of the cleavage products.

[0369] Figure 6Di to Figure 6Div .Has a single-sided_let7f_CsgRNA-GFP_ML_modC1(i) / or C2(ii) / or C3(iii) / or C4(iv)_8H2-A7 ribozyme that is completely complementary (non-alternatingly complementary) to most of the cleavage products.

[0370] Fig. 6E : Cleavage assay of unilateral_Tlet-7f or Cel-mir-238_CsgRNA-GFP_ML_modC1_8H2-A7 ribozyme.

[0371] Figure 7 : shRNA ribozymes function in human cells. Structure of the unilateral_Tlet7f_shRNA-GFP6 ribozyme.

[0372] Fig. 8A .Ribozymes can cleave RNA aptamers. The structure and sequence of ribozymes that cleave red Broccoli fluorescent RNA aptamers.

[0373] Figure 8B .Ribozymes can cleave RNA aptamers. Trigger factors induce the cleavage and release of RNA aptamers with red Broccoli fluorescence.

[0374] Fig.9A . Ribozymes with circular arrangements of 8 nt helix 4 retain self-cleavage activity, whereas shortening of helix 4 progressively reduces self-cleavage. Asterisks mark the band corresponding to the predicted cleavage product of the ribozyme with 8 bp H2.

[0375] Fig. 9B Schematic representation of the optimal configurations of the hairpin ribozyme linker (i-iv), which were tested (v). Asterisks mark bands corresponding to predicted cleavage products of the 4WJ paired ribozyme. # labels mark unpredicted cleavage products, which occur primarily in ribozymes with strong non-complementarity between cleavage product and ribozyme (unpaired).

[0376] Fig. 9C Pairing configurations of helix 1 tested in the 4WJ(HHHS2H) 8nt helix 4 ribozyme. Top: Cleavage product sequences in the 3' to 5' direction. Bottom: Pairing configurations with cleavage products tested on five ribozymes; sequences are in the 5' to 3' direction, and the vertical line represents complementary pairing with the cleavage product above.

[0377] Fig.9D . A graph showing the amount of cleavage product released by the internal form of the ribozyme (normalized with the S2 strand) when changing the length of helix 4 from 4 bp to 1 bp (see Figure 1D ).

[0378] Fig.9E . Graph showing the fold change in cleavage products released by the external form of the ribozyme when changing the length of helix 4 from 8 bp to 1 bp (trigger lane / water lane) (see FIG. 1E ).

[0379] Fig.9F .T-let-7f_Cl-29nt-clvRNA binuclear enzyme exhibits let-7f-induced cleavage release of the embedded 29nt cleavage product.

[0380] Fig. 10A .Ribozymes with a single catalytic domain and triggered by RNA fragments of dme-mir-184, dme-mir-252, dme-mir-263a, has-let-7f, or SARS-CoV-2 Orf1ab genes are capable of self-cleavage at dual sites.

[0381] Fig. 10B Mutation of one or both catalytic domains in the binuclear enzymes triggered by ban-5p, mir-451a, or the E gene indicated that one catalytic domain was sufficient for double cleavage.

[0382] Fig. 10C .Optimization of the helix 4 communication module in T-mir-451a_Cl-29nt-clvRNA ribozyme and T-SARS-CoV-2-S gene_Cl-29nt-clvRNA ribozyme, and identification of 3-nt v1 motif and 3-nt v2 motif to improve the signal-to-noise ratio of the ribozyme.

[0383] Fig. 10D . Mutant variants of the E gene test RNA were tested against the T-SARS-CoV-2-E gene_Cl-29nt-clvRNA ribozyme, demonstrating that the ribozyme is able to distinguish closely related trigger RNAs with 1nt to 3nt differences. The figure below shows the quantification of the ratio of the intensity of the variant bands compared to the WT band, with each band intensity normalized to its 40nt spike-in control.

[0384] Fig.11A A series of GFP small guide RNAs were tested for editing efficiency. ^ represents the sgRNA encoded within the ribozyme selected for zebrafish studies.

[0385] Fig. 11B sgRNAs starting with GUC can be cleaved from ribozymes in a trigger-dependent manner (previous Fig.11A 227R in.

[0386] Fig. 11C i. Structures of ribozymes in which A7 has been changed to C7 or U7. Fig. 11C ii. Mutation of A7 to C7 or U7 restored cleavage of the ribozyme containing the GFP-149R sgRNA starting from GGGC. Fig. 11C ii) Ribozyme cleavage assay in Ci. The right panel shows a blot of the same gel detecting sgRNA cleavage products.

[0387] Fig.11D . Extending Helix 2 on T-let-7f_Cl-shRNAGFP from 4 bp to 8 bp or 12 bp reduces trigger-independent cleavage at the proximal cleavage site (dashed box). The asterisk marks the band corresponding to the predicted cleavage product of the ribozyme with 8 nt helix 2. The right panel shows a blot of the same gel detecting shRNA cleavage products.

[0388] Fig. 12A . Various helix 4 motifs were tested.

[0389] Fig. 12B Cleavage assays showed that (CM2: 5'-AUG / CGA -3'), (CM7: 5'-ACU / AGA-3') and (CM8: 5'-AUG / CAA -3') were other well-performed helix 4 motifs.

[0390] Fig.13A . Sequence of the shRNA-embedded ribozyme with the original right-hand helix 2 and the sequences of the other helix 2 alterations tested.

[0391] Fig. 13B .like Fig.13ACleavage assay of the ribozyme shown with altered helix 2. Thus, Fig. 13B It is shown that extension of helix 2 beyond 5 nt reduces background cleavage (asterisk below).

[0392] Fig.14A .Sequence of ribozyme embedded with shRNA with original pairing.

[0393] Fig. 14B Above: Tested from Fig.14A Lower panel: Flow cytometry results of shRNA knockdown of GFP fluorescence by ribozymes with different degrees of pairing between embedded shRNA and ribozyme. Thus, Figure 19B shows that the increase in complementarity between shRNA cleavage products and ribozymes increases the cleavage dependency on the embedded shRNA function.

[0394] Fig.15A . The initial design of the sgRNA-embedded ribozyme with partial complementarity between the sgRNA spacer and the ribozyme backbone, and the changes to the spacer complementarity that were tested (Modification A, Modification B, and Modification C in the right box).

[0395] Fig. 15B . Cleavage assay of ribozymes embedded with sgRNAs of modification A, modification B, and modification C from (A).

[0396] Fig. 15C .Design of sgRNA-embedded ribozymes in which a portion of the first stem-loop of the sgRNA is “flattened” to pair with the ribozyme backbone, thereby increasing the complementarity between the sgRNA and the ribozyme backbone (also known as modC and modC1 versions).

[0397] Fig.15D . Cleavage assay of ribozymes embedded in modC1 sgRNA from (C).

[0398] Fig.16A .Structure of a ribozyme with an extended right-hand helix 3 (RH3). Fig. 16B . Results of cleavage assays of ribozymes with an extended right-hand helix 3 (RH3).

[0399] Fig.17A : Detection of modified RNA triggers in cells.

[0400] Figure 17Bi : Ribozymes can be triggered by 2'MOE-modified synthetic RNA (E gene). Two shRNA ribozymes with ML and CM2 communication modules triggered by E gene can be triggered by modified E gene trigger factors to cleave embedded RNA cleavage products (circled in the gel with boxes).

[0401] 2'MOE let-7f sequence ( / 52MOErT / / i2MOErG / / i2MOErA / rGrGrU rArGrU rArGrArUrUrG rUrArU rA / i2MOErG / / i2MOErT / / 32MOErT / ) (SEQ ID NO:506)

[0402] 2'MOE E gene sequence ( / 52MOErT / / i2MOErT / / i2MOErC / rGrGrArArGrA rGrArC rArGrGrUrA / i2MOErC / / i2MOErG / / 32MOErT / ) (SEQ ID NO: 507)

[0403] Figure 17Bii : Ribozymes can be triggered by 2'MOE-modified synthetic RNA (let7f). Two shRNA ribozymes with ML and CM2 communication modules triggered by let-7f can be triggered by modified let-7f trigger factors to cleave embedded RNA cleavage products (circled in the gel with boxes).

[0404] Fig. 17C .Ribozymes can be triggered by DNA. The three ribozymes triggered by the S gene fragment, the E gene fragment and mir-451a, respectively, can detect RNA trigger factors and DNA trigger factors to cut out the embedded RNA cleavage products (circled in the gel with a box). The E gene ribozyme potentially shows a preference for RNA trigger factors over DNA trigger factors.

[0405] Fig.17D . Ribozymes partially modified with 2'-fluoro nucleotides (using Durascribe) are able to cleave. Fully modified ribozymes (100% 2'F C&U) may not cleave.

[0406] Experimental data

[0407] method

[0408] PCR amplification of template and in vitro transcription of RNA

[0409] DNA templates and primers were ordered (Integrated DNA Technologies, USA) and PCR amplification was performed using Phusion high-fidelity PCR premix (#F531L, Thermo Fisher Scientific, USA) according to the manufacturer's instructions. PCR products were purified using the QIAquick PCR purification kit (#28106, Qiagen, Germany) according to the manufacturer's instructions and used as templates for in vitro transcription using the AmpliScribe T7 rapid transcription kit (#LGLC-ASF3507, Lucigen, USA). For some templates, DNA gene fragments (Twist Bioscience, USA) were used directly for in vitro transcription. For experiments using modified ribozymes, Durascribe (#DS010925, Epicentre, USA) was used according to the manufacturer's instructions. After the reaction, 280 μL of RNase-free water (#SH30538.02, Hyclone, USA) was added to 20 μL of the in vitro transcription reaction system. RNA purification was performed by adding 300 μL of acidic phenol:chloroform, pH 4.5 (with IAA, 125:24:1) (#AM9722, Invitrogen, USA), mixing, and centrifuging at room temperature, 14000 rpm for 3 minutes. The aqueous phase was transferred to a new tube, and 300 μL of chloroform (#07278-00, Kanto Chemical, Japan) was added. The mixture was centrifuged at room temperature, 14000 rpm for 3 minutes. The aqueous phase was transferred to a new tube, and the RNA was precipitated with 1 / 10 volume (25 μL) of 3M sodium acetate (pH 5.2) and 2.5 volumes (625 μL) of anhydrous ethanol. After precipitation at -20°C overnight, the sample was centrifuged at 4°C, 13000 rpm for 20 minutes. The supernatant was discarded, the RNA precipitate was washed with cold 75% ethanol, and centrifuged at 4°C, 13000 rpm for 20 minutes. After centrifugation, the supernatant was completely removed and the pellet was air-dried. The RNA was resuspended in 50 μL RNase-free water (#SH30538.02, Hyclone, USA) and its concentration was measured using a NanoDrop spectrophotometer (Thermo Fisher Scientific, USA).

[0410] Cell-free cleavage assay

[0411] 200nM ribozyme RNA (in vitro transcribed) and 50nM trigger RNA (Integrated DNA Technologies, USA) in 1× cleavage buffer (10mM Tris, 7mM magnesium chloride, 5mM spermine, 2mM sodium chloride, pH 6.4) were used for the assay. 200nM of an inert 40nt RNA sequence (5'-GGGACAUGGAAGUCACACCUUCGGGAACGUGGUUGACCUA-3') (SEQ ID NO: 461) was added to the reaction system as a loading control. The reaction system was incubated at 37°C for 4 hours.

[0412] Detection and visualization of cleavage products

[0413] RNA was heat denatured at 70°C for 10 minutes, loaded with 2× RNA loading dye (#B0363S, New England Biolabs, USA), and separated in a 10% denaturing polyacrylamide gel (#EC-833, National Diagnostics, USA) in 1×TBE (Tris / boric acid / EDTA) buffer (#1610770, Bio-Rad, USA) at 200 V for 1 hour or until the dye front migrated to the bottom of the gel. A low-range ssRNA ladder (#N0364S, New England Biolabs, USA) was loaded as a size reference, and 25 ng of a 29 nt oligonucleotide (5'-GUCCUUAGUCGAAAGUUUUACUAGAGUCA-3') (SEQ ID NO: 462) (Integrated DNA Technologies, USA) or an in vitro transcribed RNA sequence corresponding to the expected cleavage product size was added to the ladder as an additional size reference. Where appropriate, 25 ng of trigger factor and spike sequence were also added to the ladder as size reference. The gel was stained with SYBR Gold at a dilution of 1:10000 (#S11494, Invitrogen, USA) and visualized using the ChemiDoc imaging system (Bio-Rad, USA). Images were analyzed using Image Lab software (Bio-Rad, USA).

[0414] In a cold room, RNA was transferred to a Hybond-N+ membrane (#RPN303B, GE Healthcare, USA) using a Transblot SD semi-dry transfer tank (Bio-Rad, USA) in 1×TBE (#1610770, Bio-Rad, USA) at 10 V and 300 mA for 1 hour. The membrane was cross-linked using a UV cross-linker (Analytik Jena, USA) and pre-hybridized for 5 minutes at 45°C with rotation in PerfectHybPlus hybridization buffer (#H7033-1L, Merck, USA). A 5'Alexa Fluor647 or Cy5-labeled DNA probe (Integrated DNA Technologies, USA) for the cleavage product was added to the pre-hybridization solution and incubated for 3 hours in a hybridization oven at 45°C with rotation. After hybridization, the membrane was washed with wash buffers of increasing stringency stepwise (low stringency wash buffer: 2×SSC, 0.1% SDS; high stringency wash buffer: 0.5×SSC, 0.1% SDS; ultra-high stringency wash buffer: 0.1×SSC, 0.1% SDS). All washing steps were performed in a hybridization oven at 45°C with rotation (except for low stringency washes, which were performed with rotation at room temperature). The membrane was visualized using the ChemiDoc MP imaging system (Bio-Rad, USA) and analyzed using Image Lab software (Bio-Rad, USA).

[0415] Table 2.

[0416]

[0417] Maintenance of cell cultures

[0418] HEK293 cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (Gibco #10270106), 1% penicillin / streptomycin (Gibco #15140122). HEK293-GFP cells (#SC001, Amsbio, USA) were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (Gibco #10270106), 1% penicillin / streptomycin (Gibco #15140122), 2 mM L-glutamine 0.1 mM MEM non-essential amino acids (Gibco #11140050) and 10 μg / ml blasticidin (Gibco #11110). All cultures were maintained at 37°C and 5% CO2.

[0419] Electroporation

[0420] According to the manufacturer's instructions, RNA was electroporated into HEK293-GFP cells using the Neon transfection system (#MPK1096, Invitrogen, USA), with parameters set to 1500V, 30ms pulse width, 1 pulse. Using the Alt-R CRISPR-Cas9 system (Integrated DNA Technologies, USA), the ribonucleoprotein complex was delivered by electroporation using the Neon kit. After electroporation, cells were seeded in 96-well plates and incubated for 48 hours until analysis.

[0421] miRNA inhibitors

[0422] miRNA inhibitors and control inhibitors were purchased from Integrated DNA Technologies, USA.

[0423]

[0424] Flow cytometry

[0425] The cells were washed with 1×PBS (#10010023, Gibco, USA) and removed from the cell culture plate with trypsin-EDTA (0.05%) and phenol red (#25300054, Gibco, USA). Complete medium was added to neutralize trypsin, and the entire volume of each well was transferred to a U-shaped bottom 96-well plate. The plate was centrifuged at 300g for 4 minutes and the supernatant was discarded. The cells were stained at room temperature for 15 minutes using Live / Dead fixable near-infrared dye (#L34975, Invitrogen, USA) diluted 1:1000 in 1×PBS (#10010023, Gibco, USA). After staining, the plate was centrifuged at 300g for 4 minutes and the supernatant was discarded. The cells were resuspended in 1×PBS (#10010023, Gibco, USA) and analyzed on a BD FACSymphony high-throughput sampler (HTS). Data analysis was performed using FlowJo (BD Biosciences, USA).

[0426] Plasmid cloning

[0427] pSpCas9(BB)-2A-Puro V2.0 vector (#62988, Addgene, USA) was used as a basic vector for cloning gRNA sequences or ribozyme sequences. The vector was digested with BbsI (#R0539S, New England Biolabs, USA) and connected to the desired insert using T4 DNA ligase (#10716359001, Roche, USA). The gRNA insert was prepared by oligonucleotide renaturation of the sense and antisense strands of the gRNA sequence, with BbsI single-stranded overhangs. In order to clone the ribozyme into the same vector, a gblock (Integrated DNA technologies, USA) was inserted between the AflIII site and the XbaI site, which removed the gRNA scaffold sequence and included a new BamHI restriction site. The modified plasmid was then digested with BbsI and connected to the desired ribozyme insert. Because the ribozyme sequence contains an internal BbsI restriction site, the ribozyme insert was prepared by PCR amplification using Phusion High Fidelity PCR Master Mix (#F531L, Thermo Fisher Scientific, USA), including the BbsI single-stranded overhang by creating a PaqCI site, and then digested with PaqCI (#R0745S, New England Biolands, USA).

[0428] Fish microinjection

[0429] A ribonucleoprotein mixture (RNP) (Integrated DNA Technologies, USA) containing 150 ng of in vitro transcribed ribozyme and 0.5 μg of Cas9 protein was incubated at 37°C for 10 minutes and then cooled to room temperature. Each embryo was injected with 0.75 nL of the RNP mixture.

[0430] 24 hours after microinjection, genomic DNA was extracted from individual embryos. Each embryo was first rinsed with 1×PBS (#10010023, Gibco, USA), then 20 μL of alkaline lysis solution (25 mM sodium hydroxide, 0.2 mM EDTA) was added and incubated at 95°C for 30 minutes. The test tubes were vortexed to check for complete dissolution of the embryos, and then incubated at 95°C for another 10 minutes. 20 μL of neutralization buffer (40 mM Tris-HCl, pH 8.0) was added to each tube, and 2 μL of extracted genomic DNA was used as a template to perform PCR amplification of GFP using the following primers: SAW907_GFPF2 and SAW908_GFPR2.

[0431]

[0432] Genome editing and detection

[0433] sgRNA design was performed using http: / / crispor.tefor.net / . Genomic DNA was extracted from cells using QuickExtract DNA Extraction Solution (#QE09050, Lucigen, USA) and used as a template for PCR amplification of GFP. The desired bands were gel extracted using the ZR-96 Zymoclean Gel DNA Recovery Kit (#D4022, Zymo Research, USA). DNA was eluted in RNase-free water (#SH30538.02, Hyclone, USA) and DNA concentration was measured using a NanoDrop spectrophotometer (Thermo Fisher Scientific, USA).

[0434] 50ng DNA was used as the template for the second PCR using fluorescently labeled primers. Capillary electrophoresis was performed on an ABI3730xl DNA analyzer (Applied Biosystems, the U.S.). By comparing with size standards (#4322682, Applied Biosystems, the U.S.), the size of the fluorescently labeled DNA fragments was determined. GeneMapper (Applied Biosystems, the U.S.) was used for data analysis.

[0435]

[0436] result

[0437] RNA trigger-activated self-cleaving binuclease

[0438] Pandan is a miRNA sensor that is fluorescently activated upon binding to a specific target miRNA. Pandan was previously developed by altering the structure of the fluorescent RNA Spinach2, so that hybridization of the target miRNA with its cognate RNA sensor backbone is required to stabilize the binding of the fluorophore DFHBI (3,5-difluoro-4-hydroxybenzylidene imidazolinone). Pandan was designed by removing part of the structure of Spinach2 and replacing it with a sequence complementary to the target miRNA, so that stabilization of the G-quadruplex structure of the fluorophore DFHBI with Spinach2 can only occur upon binding to the target RNA. The fluorescence of the Pandan sensor is significantly enhanced by 50 times in the presence of 1uM of the target miRNA, but unlike other RNA detection methods, the Pandan sensor system does not include an amplification step that would enable more sensitive applications.

[0439] It is speculated that the principles used to design Pandan can be used to modify ribozymes that can amplify and convert RNA signals: if the structural elements required to stabilize RNA folding into a catalytic conformation are only reconstructed when base pairing occurs between the ribozyme sensor and its trigger RNA, then molecules with ribozyme activity can be assembled from two or more different RNA molecules. This can be achieved if a portion of the ribozyme structure is removed and replaced by a sequence complementary to the target trigger RNA. In addition, the inventors of the present disclosure hypothesize that the connection of two such RNA-activated self-cleaving ribozymes can achieve amplification and conversion of RNA signals by releasing a second RNA molecule from the dual ribozyme.

[0440] After considering the structural features of various ribozymes, the inventors chose the self-cleaving hairpin ribozyme (HpRz) as a proof of concept because mutagenesis and crystallographic studies showed that it has the required structural elements. HpRz consists of two independently folded domains (domain A and domain B; Figure 1A ), each domain consists of a loop region containing the key catalytic nucleotide, flanked by two helices. The phosphodiester bond cleavage reaction occurs in the N -1 and guanine (G +1 ) between nucleotides ( Figure 1A The black arrow in the figure). Many groups have systematically studied the sequence requirements for the cleavage site; the most highly tolerated sequences with at least 20% of wild-type ribozyme activity are Figure 1A shown.

[0441] Figure 1Bi Our initial design approach is shown. Two HpRz, each with a modified helix 4 containing complementary sequences to the trigger miRNA, will be arranged in tandem, one in the wild-type configuration ("right side") and the other in the reverse ligated configuration ("left side"). In the absence of trigger RNA, catalytic loop B is conformationally unstable and ribozyme self-cleavage does not occur. Binding of the RNA trigger stabilizes loop B, activating cleavage to release the RNA cleavage product ( Figure 1Bi This strategy requires three modifications to the original wild-type binuclear enzyme ( Figure 1Bii First, the inventors will perform a circular arrangement so that helix 4 is no longer a closed stem loop; instead, the positions of the 5' and 3' ends will be changed to be located within each helix 4 ( Figure 1Biii ; different linker configurations are possible, and a 3-way linker is shown). This binuclear enzyme will consist of two strands, the first containing the cleavage site and cleavage products (strand 1; S1) and the second being the non-cleavage strand (strand 2; S2). Next, the inventors will introduce a second sequence-variable stem-loop branching from helix 4 (internal form; Figure 1Biv); this mimics the structure formed by the binding of the trigger RNA. Finally, the inventors will assemble a functional self-cleaving ribozyme from three RNAs (S1, S2 and trigger RNA) that are sequence-complementary in the helix 4 of the new branch (OUT form; Figure 1Bv The inventors systematically tested the feasibility of this strategy and optimized each feature of the ribozyme.

[0442] The inventors first selected an example of a trigger miRNA and an example of a cleavage product to start the development. Since the Pandan sensor has a wide range of performance efficacy, with fluorescence multiples varying from 4 to 118 times, the inventors decided to first select Drosophila microRNA bantam-5p (ban-5p) as the trigger RNA, which is the trigger RNA of the best-performing Pandan sensor. It is speculated that trigger RNAs that bind well to their homologous Pandan sensors can also effectively bind to other similar branched sensor structures. The inventors selected a 29nt RNA fragment (29nt-clvRNA) as an example of a cleavage product, which is long enough to be distinguished from the 23nt ban-5p trigger miRNA on the gel.

[0443] The first step is to perform circular permutations of the ribozymes ( Figure 1Biii The inventors observed that the circularly arranged ribozymes retained self-cleavage activity to release the embedded 29nt cleavage product ( Fig.9A In order for the trigger-activated cleavage strategy to work, the inventors reasoned that they should be able to destabilize helix 4 by shortening its length; indeed, although the ribozyme with an 8-bp helix 4 showed clear release of cleavage products, shortening helix 4 to less than 3 to 4 nucleotides impeded release of cleavage products ( Fig.9A These initial experiments were performed with the ribozyme in a 3-way adaptor configuration ( Figure 1Biii ), and the inventors observed that even when the length of helix 4 was 8 nt, the rate of formation of cleavage products was low and a large portion of the S1 strand remained intact ( Fig.9A ). Therefore, the inventors took two approaches to promote cleavage. First, since the religation rate increases when the cleavage products are highly complementary within helix 1, the inventors reduced the base pairing between the cleavage products and S2 to facilitate the release of the cleavage products. In addition, studies have shown that HpRz with a 3-channel linker (HHH) configuration (Figure 1bii, Fig.9A , Fig. 9B i) Poor cleavage with a modified 2-channel configuration (H -1 S7H)( Fig. 9Bii), a modified 3-channel configuration (HHS4H) ( FIG. 9bii ) or a modified 4-channel configuration (HHHS2H) ( Fig. 9B Therefore, the inventors tested the cleavage activity of ribozymes with these modified linkers, wherein the cleavage products were paired or unpaired with the ribozyme ( Fig. 9C ). These modifications greatly promoted the release of cleavage products, and the unpaired 3-channel linker (HHS4H) and 4-channel linker (HHHS2H) showed the highest cleavage rates ( Fig. 9C ). However, the completely unpaired ribozyme showed unexpected cleavage bands ( Fig. 9C , # tag), the inventors hypothesized that the non-complementarity of the cleavage products disrupted the ribozyme folding and caused off-target cleavage. Therefore, the inventors partially restored the complementarity between the cleavage products and the ribozyme ( Fig. 9C The configuration of 3 / 4 unpaired nucleotides and 2 paired nucleotides repeated along the cleavage product-ribozyme region is very effective for the release of the cleavage product ( Fig. 9C , configuration #5).

[0444] Next, to determine whether the ribozyme could accommodate an RNA-bound trigger region, the inventors introduced a branched stem into helix 4 to mimic this structure (an internal trigger format, in which the trigger RNA is introduced as part of the ribozyme, Figure 1Biv ). An optimized 4-channel adapter ribozyme with an alternating base pairing configuration was used ( Fig. 9C , configuration #5). Since the stem of helix 4 is longer than 4 bp, it will release the cleavage product in the absence of trigger factors ( Fig.9A ), so the inventors tested stems of helix 4 ranging from 1 bp to 4 bp. When the length of helix 4 was at least 3 bp ( Figure 1C ), the internally triggered ribozyme can self-cleave to release a large number of 29nt cleavage products, and the increase in the length of helix 4 is associated with the release of more cleavage products ( Fig.9D ). Thus, trigger-activated binozymes can be achieved by cyclically permuting the binozymes, reducing the complementarity of the cleavage products, introducing a 4-way linker, shortening helix 4 and altering it to include a branched RNA-binding trigger region.

[0445] RNA-triggered cleavage in cell-free assays

[0446] The sequences of the two helices branching off from helix 4 can vary. This allowed the inventors to encode into the ribozyme backbone a sequence complementary to the target RNA. Therefore, the possibility of reconstructing ribozyme activity from three isolated RNAs was investigated by: a binuclear ribozyme with a ban-5p trigger factor binding domain (external form, Figure 1Bv), and a short RNA complementary to the trigger domain of the ribozyme (ban-5p) ( Figure 1D This ribozyme was named trigger RNA-ban-5p_cleavage product-29nt-clvRNA (T-ban-5p_Cl-29nt-clvRNA). In the external form, when helix 4 is shorter than 2 bp, the ribozyme releases very little cleavage product even in the presence of the trigger factor (most of the S1 strand remains uncleaved; Figure 1D ). Helix 4 with a length of 2 to 3 bp exhibited trigger-activated self-cleavage, releasing more cleavage products in the presence of a trigger than in its absence. Increasing the length of helix 4 to 8 bp resulted in high background cleavage in the absence of a trigger and no obvious trigger-activated cleavage ( Figure 1D , Fig.9E A second ribozyme was tested that was triggered by a different miRNA, let-7f, to release the same 29nt cleavage product (T-let-7f_Cl-29nt-clvRNA). This ribozyme also released the cleavage product in a trigger-dependent manner ( Fig.9F ).

[0447] Therefore, the present inventors developed a ribozyme with two cleavage sites and two trigger factor binding regions, which is activated by the target trigger RNA to release the embedded RNA cleavage product.

[0448] A single catalytic domain is sufficient for RNA-triggered dual self-cleavage

[0449] The observed cleavage patterns suggest that the cleavage rates between the two sites are different ( Figure 1C , Figure 1D ). Therefore, each catalytic domain was mutated independently to determine whether one was more active. 38 is a key base involved in stabilizing the transition state during catalysis, and mutation of this nucleotide abolishes cleavage. 38 →U 38 mutations, either in the "right" wild-type catalytic domain or in the "left" reverse-linked catalytic domain in the T-ban-5p_Cl-29nt-clvRNA binuclear enzyme ( Figure 1Bv , Figure 1D , with an 8nt H4), did not abolish cleavage at either cleavage site or significantly reduce release of cleavage products, whereas simultaneous mutation of both catalytic domains completely abolished cleavage ( Figure 2A ). This indicates that any single catalytic domain can cleave at two cleavage sites.

[0450] To test this, dual-cleavage site ribozymes were designed with either a single "right-side" wild-type catalytic domain or a single "left-side" inverse-linked catalytic domain. Figure 2B ). In this case, these ribozymes can be encoded by single-stranded RNA. Surprisingly, both monoribozymes are able to cleave at two sites to release cleavage products ( Figure 2C Addition of trigger RNA increases release of cleavage products from the ban-5p single "right-side" ribozyme ( Figure 2C ). The inventors chose to proceed with a single "right-side" wild-type catalytic domain, speculating that it could be rationally optimized based on the extensive functional studies of this classic structure. The "right-side" mononuclear enzymes triggered by microRNAs dme-mir-184, dme-mir-252, dme-mir-263a, hsa-let-7f, and fragments of the E gene and Orf1ab transcripts of the SARS-CoV-2 genome can self-cleave at two sites to release the cleavage products ( Figure 2C , Fig. 10A ), confirming that one catalytic domain is sufficient for double cleavage. 38 →U 38 The mutation abolished the cleavage ( Figure 2C Several ribozymes showed additional cleavage product release when trigger RNA was added; however, most ribozymes showed cleavage product release even in the absence of trigger factors ( Figure 2C , 10A).

[0451] To optimize the signal-to-noise ratio in the presence and absence of trigger factors, helix 4 of the trigger-responsive communication module was varied to identify the optimal stem length and sequence. Ribozymes with helix 4 longer than 5 nt cleaved in a trigger-independent manner, while ribozymes with helix 4 longer than 2 nt were resistant to cleavage even in the presence of trigger factors ( Figure 2D ). Two 3nt motifs 5'-ACG / CGU-3' (3-nt v1) and 5'-ACG / CGA-3' (3-nt v2) greatly improved the signal-to-noise ratio of several ribozymes ( Figure 2D , Fig. 10B The cleavage products increase with the increase of trigger RNA concentration ( Figure 2E The inventors named this trigger-activated single-chain dual-ribozyme platform that can self-cleave and release the embedded RNA product upon trigger RNA binding as "Unlocked By Activating RNA (UNBAR)".

[0452] The inventors next investigated the ability of the ribozyme to discriminate between unrelated and closely related sequences by introducing sequence changes into the test RNA ( Figure 2F ). Unrelated RNA (S gene fragment) failed to trigger cleavage. The effect of single nucleotide mismatches depended on the position of the mismatch, with position 4 reducing the formation of cleavage products by more than 80%. Double and triple mutants showed a strong reduction in cleavage activity ( Figure 2F , Fig. 10D ). These results suggest that these ribozymes can be optimized to discriminate between similar target sequences.

[0453] For these ribozymes to be useful in biological samples, they must recognize their trigger in a complex mixture. Total RNA from HEK293T cells was prepared and the ability of the ribozymes to detect trigger RNA spiked into the RNA mixture over a range of concentrations was determined. The ribozymes could detect their trigger in the presence of at least a 1000-fold excess of competing RNA ( Figure 2G ).

[0454] Functional RNA as cleavage products: CRISPR sgRNA and shRNA

[0455] To explore whether these ribozymes can be used for RNA environment-specific gene regulation in vivo, the inventors engineered ribozymes to encode CRISPR small guide RNA (sgRNA) targeting GFP. GFP ; Figure 3A ), short hairpin RNA (shRNA) targeting GFP GFP ; Figure 3B ), or RNA aptamers (Broccoli, Figure 3C ) is encoded as a cleavage product. The inventors first performed a cell-based screen to identify effective sgRNAs; the inventors found that three sgRNAs ( Figure 1A ) is inefficient in inducing editing ( Fig.11A ), although they can be encoded within ribozymes and released in a trigger-dependent manner ( Fig. 11B ). Therefore, the inventors selected sgRNA GFP-149R, which is effective ( Fig.11A) and has been characterized previously. The inventors first retained a conserved hairpin ribozyme sequence at the cleavage site to test cleavage of these more complex products; therefore, there are 1 to 4 nucleotide "residues" at the 5' and 3' ends of the sgRNA and shRNA that will be included in the cleavage product. The additional nucleotides at the 3' end of the gRNA chain should not alter Cas9 targeting and DNA cleavage. Although unpaired nucleotides at the 5' end of the 20nt protospacer sequence of the sgRNA can inhibit DNA cleavage, these effects are context-dependent, and the sgRNA we selected can tolerate unpaired nucleotides at its 5' end. The inventors later discovered that although this sgRNA starts with GGG, by mutating A7 in the WT cleavage loop to U7 or C7 to align with G +3 pairing, and also enables efficient and precise cleavage without "residual" nucleotides at the 5' end ( Fig. 11C ).

[0456] The inventors designed shRNA ribozymes to be similar to primary microRNAs, with a single-stranded RNA (ssRNA) primary segment and a double-stranded (dsRNA) segment predicted to be a Drosha cleavage substrate, which binds to GFP. shRNA This is achieved by designing and screening transcripts that are optimized to have features important for microRNA processing. 49 , which also included 5' and 3' ribozyme scar sequences that enabled subsequent insertion into the ribozyme. The inventors identified potential cleavage products that strongly reduced GFP expression. They then designed a ribozyme (T-let-7f_Cl-shRNA) containing the most effective cleavage product shGFP6 GFP6 )( Figure 7 Before cleavage, the ssRNA base fragments base-pair with the ribozyme ( Figure 3B ), since the single-stranded base strand is essential for Drosha processing, the uncut shRNA embedded in the ribozyme should not be processed for RNAi. After the trigger-induced ribozyme self-cleavage, the cleavage product is released, becomes a potential substrate for Drosha, and enters the miRNA maturation pathway. Since Drosha cleavage does not require a specific conserved sequence, but requires a "count" starting from the junction of ssRNA and dsRNA, the inventors hypothesized that the incorporation of 5' and 3' scar nucleotides would not affect the processing of shRNA cleavage products. Finally, the inventors also designed and tested a ribozyme that cleaves the fluorescent aptamer Broccoli ( Figure 3C ).

[0457] The inventors chose let-7f as the trigger miRNA because the let-7 family is highly conserved and let-7f is one of its most abundant members (www.mirbase.org), which enabled the inventors to test these ribozymes in zebrafish embryos and human cell lines. GFP 、T-let-7f_Cl-shRNA GFP Trigger-activated cleavage was tested in T-let-7f_Cl-Broccoli (tested for cleavage by two different shRNAs for GFP). For all three ribozymes, significantly more cleavage products were released in the presence of the trigger than in the absence of the trigger ( Figures 3D to 3F ). Therefore, these ribozymes can be triggered to release longer functional RNAs with secondary structures, including sgRNAs, shRNAs, and aptamers.

[0458] The let-7 miRNA family consists of 12 closely related members ( Figure 3G We studied T-let-7f_Cl-sgRNA GFP Can they be distinguished? T-let-7f_Cl-sgRNA GFP Most let-7 family members that differ from let-7f by at least 3 nt (i.e., let-7b, let-7i, and mir-98) can be distinguished. Let-7d, despite being 3 nt different, cannot be distinguished, probably because the nucleotide at position 1 may have a weaker effect ( Figure 3G ).

[0459] Although the release of ribozyme cleavage products depends on the presence of trigger RNA, in the absence of trigger factors, a considerable portion of ribozymes cleave at sites proximal to the catalytic domain ( Figures 2D to 2F ), which may lead to active leakage of sgRNA or shRNA cleavage products in vivo. However, the distal cleavage site was not cleaved in the absence of a trigger, and the inventors hypothesized that this was due to its increased distance from the catalytic loop b. To test this, the inventors used T-let-7f_Cl-sgRNA GFP Helix 2 is extended from 4 bp to 8 bp or 12 bp ( Figure 3H This greatly reduces trigger-independent cleavage at the proximal site ( Figure 3H To investigate whether this is generalizable, the inventors similarly altered T-let-7f_Cl-shRNA GFP This also reduces trigger-independent cleavage at the proximal site ( Fig.11D). Therefore, the length of helix 2 can be adjusted to optimize the regulation of cleavage by the trigger RNA.

[0460] RNA signaling in vivo

[0461] The inventors then investigated whether these ribozymes can perform cellular RNA environment-dependent gene regulation in vivo. CRISPR-Cas9 editing can be performed in zebrafish embryos by microinjection of RNA-Cas9 complexes. T-let-7f_Cl-sgRNA GFP The ribozyme can detect let-7a, let-7c, let-7d, let-7e, let-7f, and let-7g (Figure 3E); let-7a, let-7c, and let-7f account for 72% of these miRNAs in early fish embryos (www.mirbase.org). Single cell injection of Cas9 protein and positive control sgRNA GFP This resulted in a high genomic deletion rate of approximately 88% per embryo ( Figure 4A ). T-let-7f_Cl-sgRNA GFP Co-injection with Cas9 also resulted in robust editing, although less than with gRNA alone, at ∼29% per embryo (p<0.001) ( Figure 4A ). This provides evidence that ribozymes encoding sgRNAs are stable in vivo and are able to cause gene editing when delivered in RNA form. To determine whether editing is let-7 dependent, the inventors co-expressed the ribozyme with a morpholino antisense inhibitor against let-7f. Co-injection of the inhibitor with the ribozyme reduced ribozyme-induced editing at the GFP locus from about 29% to about 12% (p<0.05), providing support that gRNA function is let-7f dependent ( Figure 4A ).

[0462] The inventors next investigated whether these ribozymes could function in human cells. Durascribe-modified (50% C and U modified) T-let-7f_Cl-shRNA was expressed in HEK293-GFP cells GFP6 ribozyme, and co-expression of 2'MOE-modified let-7f resulted in an approximately 9% increase in GFP knockdown, whereas expression of 2'MOE-modified E gene had no effect ( Figure 4B ).

[0463] Similarly, expressing the Durascribe-modified (50% C modified) TE gene 20_Cl-Stoplight_modC ribozyme in Stoplight cells (de Jong et al., Nature Communications 2020) and simultaneously expressing the 2'MOE-modified E gene increased gene editing, whereas expression of the 2'MOE-modified let-7f had no effect ( Figure 4C ). Therefore, in mammalian cells, the regulation of ribozymes is trigger-dependent.

[0464] Sequence containing A7: (Note: See Figure 11, as described above)

[0465]

[0466] Sequence containing C7:

[0467]

[0468] For the sake of clarity, specific nucleotide sequences are identified in the art by numerical positions based on the original hairpin ribozymes. Therefore, in one embodiment, A7 and C7 as mentioned herein are named based on the annotations of the original hairpin ribozymes, i.e., they (the positions mentioned) do not depend on their specific positions in the specific ribozymes, but rather the positions refer to the nucleotides in the protuberances relative to the cleavage sites in the classical hairpin ribozymes. Fig. 1 shows a schematic diagram of a classical hairpin ribozyme, and an exemplary N7 position is indicated. An influential modification disclosed herein on the cutting efficacy is a change in the identity of the N7 nucleotides, making it identical to the nucleotide N7 in the cleavage product. +3 This is useful because it allows for differences in the sequence of the cleavage products. So, for example, if nucleotide N +3 is a G in the cleavage product, then N7 can be changed to C (to pair with this G). However, for example, if N +3 becomes A, then N7 can be changed to U (pairing with A) to improve the cleavage activity.

[0469] Disclosed herein are features of ribozymes whereby these features are demonstrated to improve the signal-to-noise ratio of the ribozyme, i.e., reduce its background cleavage in the absence of target RNA / trigger RNA, and increase its cleavage rate in the presence of target RNA / trigger RNA. Preferably, any such modification should be applicable to a broad range of ribozymes. To this end, the inventors tested several motifs for the communication module of the ribozyme, i.e., the region between the catalytic domain and the trigger arm ( Figure 1A The inventors found that the 2nt communication module is too short ( Fig. 12B), while 3nt motifs were generally preferred over other lengths tested. Specifically, several 3nt communication module dsRNA motifs, 5'-ACG / CGU-3' (SEQ ID NO: 1) and 5'-ACG / CGA-3' (SEQ ID NO: 2) were highly efficient ( Figure 2D , Fig. 10C ),besides Fig. 12A and Fig. 12B Several other variants are shown, particularly (CM2: 5'-AUG / CGA-3') (SEQ ID NO: 450), (CM7: 5'-ACU / AGA -3') (SEQ ID NO: 455) and (CM8: 5'-AUG / CAA-3') (SEQ ID NO: 456), 5'-UAU / AUA-3' (SEQ ID NO: 454), and 5'-CU / AG-3' (SEQ ID NO: 457) ( Fig. 12A , Fig. 12B ). Therefore, changing the communication module to these variants can significantly improve the signal-to-noise ratio of the ribozyme.

[0470] Next, using these optimized communication modules, the inventors developed ribozymes triggered by short RNA to release shRNA or sgRNA targeting green fluorescent protein (GFP), or fluorescent aptamers (Figures A to C). When triggered by RNA let-7f, all of these ribozymes released the embedded shRNA, sgRNA, or aptamer with a high signal-to-noise ratio ( Figures 3D to 3F ).

[0471] However, the inventors noticed that there was a lot of background cutting at the proximal cleavage site of the catalytic domain, while there was little or no background cutting at the distal cleavage site. The inventors wondered whether the lower background cutting at the distal site was due to its greater distance from the catalytic domain. Therefore, the inventors studied whether extending the length of helix 2 could reduce the level of background cutting at the proximal site. Therefore, the inventors extended helix 2 from its normal 4bp configuration to 8bp or 12bp. This significantly reduced background cutting at the proximal site in the absence of a trigger factor ( Figure 3H ). Extending the length of helix 2 to 5 to 10 bp also had similar benefits ( Fig.13A ). Therefore, the extension of helix 2 can reduce background cleavage in the absence of triggering factors.

[0472] The inventors also investigated whether the extension of helix 3 would affect cleavage. They found that changes in helix 3 also significantly reduced background cleavage in the absence of triggering factors (Figure 16).

[0473] Optimization and other experimental data

[0474] 1) A novel sequence motif in helix 4 of the ribozyme that generates an Rz with a very high signal-to-noise ratio in the presence of trigger RNA, generalizable to many Rzs

[0475] Fig. 12A Ten other motifs in helix 4 of ribozymes with very high signal-to-noise ratios in the presence of trigger RNA are exemplified. These other motifs include, but are not limited to, (5'-ACG / UGA-3') (SEQ ID NO: 449), (5'-AUG / CGA-3') (SEQ ID NO: 450), (5'-AUG / UGA-3') (SEQ ID NO: 451), (5'-CG / CG-3') (SEQ ID NO: 452), (5'-UUG / UGG-3') (SEQ ID NO: 453), (5'-UAU / AUA-3') (SEQ ID NO 454), (5'-ACU / AGA-3') (SEQ ID NO: 455), (5'-AUG / CAA -3') (SEQ ID NO: 456), (5'-CU / AG-3') (SEQ ID NO: 457), and (5'-UG / CA -3') (SEQ ID NO: 458).

[0476] Cleavage assays showed that at least three of these motifs were as effective or better than the motifs of SEQ ID1 and SEQ ID2 ( Fig. 12B )(CM2: 5'-AUG / CGA-3')(SEQ ID NO: 450), (CM7: 5'-ACU / AGA-3')(SEQ ID NO: 455) and (CM8: 5'-AUG / CAA-3')(SEQ ID NO: 456). In addition, the effects were weaker with 5'-UAU / AUA -3'(SEQ ID NO: 454) and 5'-CU / AG-3'(SEQ ID NO: 457), indicating that the 2nt helix 4 can also operate in some ribozyme environments.

[0477] 2) A new extended helix 2 (we previously tested 8nt and 12nt) that reduces background cleavage at the catalytic domain proximal cleavage site in the absence of the trigger factor

[0478] Figure 13 shows that other modified helix 2 lengths were tested, from 5 nt to 10 nt, some of which also showed reduced background cleavage, for example 6 nt to 10 nt.

[0479] 3) Ribozymes with full complementarity show increased cleavage dependence of some functional ribozymes

[0480] The inventors of the present disclosure prepared shRNA-releasing ribozymes with increased complementarity between the cleavage product and the ribozyme, including complete complementarity ( Fig.14A , Fig. 14B , D2 design). These greatly improve the weak cutting dependence of the original design ( Fig. 14B ). sgRNA-releasing ribozymes with increased complementarity between the cleavage product and the ribozyme were also prepared and found to be functional ( FIG. 15 ).

[0481] 4) Rz embedded in CRISPR sgRNA, shRNA, and aptamers, releasing them in the presence of trigger RNA

[0482] The inventors provide examples of ribozymes embedded in shRNA and sgRNA. For shRNA, see Figures 3, 12, 13 and 14. New sgRNA examples are shown in Figures 3 and 15.

[0483] 5) Use sgRNA to release ribozymes and shRNA to release ribozymes in vivo and in vitro in zebrafish and mammalian cells, respectively.

[0484] Other examples of shRNA-released ribozymes and sgRNA-released ribozymes are listed in Table 1 above. FIG. 4A to FIG. 4C The functional efficacy of shRNA ribozymes and sgRNA ribozymes in cells and in vivo is shown.

[0485] application

[0486] Embodiments of the methods disclosed herein provide sensitive, low to no background, specific, and functional ribozymes.

[0487] Advantageously, the ribozymes disclosed herein include one or more unexpected improvements, including: 1) a novel sequence motif in the helix 4 communication module of the ribozyme, which results in a ribozyme with very low background cleavage in the absence of target RNA / trigger RNA and a high cleavage rate in the presence of target RNA / trigger RNA, which is applicable to a range of ribozymes, 2) a novel extended helix 2 domain, which reduces background cleavage at the catalytic domain proximal cleavage site, 3) a ribozyme with complete complementarity between the releasable cleavage product and its complementary strand, which reduces the release of background cleavage products, 4) a ribozyme with nucleotide N +3 Mutation of the paired nucleotide N7, which promotes cleavage when the sequence at the cleavage site deviates from the canonical cleavage site sequence, and 5) a new extended helix 3 domain, which reduces background cleavage at the catalytic domain proximal cleavage site.

[0488] Even more advantageously, ribozymes as disclosed herein have very low background cleavage and leakage, making them useful for a variety of cell-free, in vitro, and in vivo applications.

[0489] The ribozymes disclosed herein can be advantageously designed to contain functional RNA, such as but not limited to small guide RNA (sgRNA), short hairpin RNA (shRNA) or RNA aptamer, etc. Therefore, the ribozymes disclosed herein are capable of releasing functional RNA in the presence of trigger RNA.

[0490] Ribozymes as disclosed herein are also advantageously capable of releasing functional RNAs with secondary structures, such as, but not limited to, small guide RNAs (sgRNAs), short hairpin RNAs (shRNAs), RNA aptamers, and the like.

[0491] Ribozymes as disclosed herein can be used in vitro and in vivo (such as, for example, in zebrafish and mammalian cells) to trigger gene knockdown (as in the case of shRNA ribozymes) or gene editing (in the case of sgRNA ribozymes) in the presence of a trigger RNA. Thus, ribozymes as disclosed herein can also be used for gene regulation, the output of which depends on the type of functional RNA that encodes a releasable cleavage product.

[0492] Disclosed herein are features of ribozymes whereby these features are demonstrated to improve the signal-to-noise ratio of the ribozyme, i.e., reduce its background cleavage in the absence of target RNA / trigger RNA, and increase its cleavage rate in the presence of target RNA / trigger RNA. Preferably, any such modification should be applicable to a wide range of ribozymes.

[0493] Those skilled in the art will appreciate that other changes and / or modifications may be made to the embodiments disclosed herein without departing from the spirit or scope of the present disclosure as broadly described. For example, in the description herein, features of different exemplary embodiments may be mixed, combined, interchanged, merged, adopted, modified, included, etc., or similar operations may be performed between different exemplary embodiments. Therefore, the present embodiments are considered to be illustrative and not restrictive in all respects.

Claims

1. A ribozyme comprising: a) one or more catalytic domains capable of switching between an active state and an inactive state; b) one or more releasable RNA segments, wherein each of the releasable RNA segments is flanked by two ribozyme cleavage sites, wherein cleavage at each cleavage site is catalyzed by at least one of the one or more catalytic domains in an active state; c) one or more trigger factor binding domains, each of which is used to bind to a trigger nucleic acid molecule; each of the one or more catalytic domains is connected to one of the one or more trigger factor binding domains; wherein when the trigger factor binding domain connected to the catalytic domain is not bound by the trigger nucleic acid molecule, the catalytic domain is in an inactive state, and wherein when the trigger factor binding domain connected to the catalytic domain is bound by the trigger nucleic acid molecule, the catalytic domain is in an active state; and wherein one or more than one releasable RNA fragment is released from the ribozyme when both cleavage sites flanking the releasable RNA fragment are cleaved, The ribozyme comprises an RNA strand having the following motif Motif [A] and motif [a], wherein motif [A] and motif [a] constitute a trigger factor binding domain for binding to a trigger nucleic acid molecule; Motif [B] and motif [b], wherein motif [B] and motif [b] constitute a linker that acts as a communication module to stabilize the catalytic domain when the trigger nucleic acid is bound, wherein motif [B] and motif [b] are independently at least 1 nucleotide in length; Motif [C] and motif [c], wherein motif [C] and motif [c] constitute a catalytic domain; motif [D], wherein motif [D] comprises a first cleavage site capable of being cleaved when catalyzed by the catalytic domain; motif [D'], wherein motif [D'] comprises a second cleavage site capable of being cleaved when catalyzed by the catalytic domain; motif [E], wherein motif [E] comprises a releasable RNA segment; Motif [e], wherein motif [e] comprises a sequence that is partially complementary or fully complementary to the sequence of motif [E]; And wherein the motifs are connected by one or more than one optional linker regions.

2. The ribozyme complex of claim 1, wherein the linker between motif [C] and motif [D] is selected from a dual channel linker, a triple channel linker, a quadruple channel linker, a stem, a single nucleotide bulge, a dinucleotide bulge, a trinucleotide bulge, a multinucleotide bulge, and combinations thereof.

3. The ribozyme complex according to claim 1 or 2, wherein the linker between motif [C] and motif [D] comprises a three-way linker and a stem.

4. The ribozyme complex according to claim 3, wherein the stem sequence connecting the linker to the motif [D] is 4 to 12 nucleotides in length.

5. The ribozyme complex according to claim 3, wherein the stem sequence connecting motif [C] to motif [c] to the linker is 4 to 12 nucleotides in length.

6. A ribozyme according to any of the preceding claims, wherein the trigger nucleic acid molecule comprises a region complementary to a trigger factor binding domain, wherein the length of the region exceeds 10 nucleotides, and optionally one or more than one trigger factor binding domains are used to bind to the same trigger nucleic acid molecule.

7. The ribozyme of any one of the preceding claims, wherein the releasable RNA fragment is 6 to 150 nucleotides in length.

8. The ribozyme of any of the preceding claims, wherein the releasable RNA fragment comprises a sequence identical to at least one of the one or more trigger RNA molecules.

9. The ribozyme according to any one of the preceding claims, wherein the releasable RNA fragment is a functional RNA selected from a small guide RNA (sgRNA), a guide RNA (gRNA), a short hairpin RNA (shRNA) and an RNA aptamer.

10. The ribozyme according to any one of the preceding claims, wherein the length of motif [B] and motif [b] are independently 1 or more than 1 nucleotide, optionally 3 or more than 3 nucleotides.

11. The ribozyme according to any of the preceding claims, wherein motif [B] and motif [b] have a motif selected from the group consisting of SEQ ID NO: 1 (5'-ACG / CGU-3'), SEQ ID NO: 2 (5'-ACG / CGA-3'), SEQ ID NO: 449 (5'-ACG / UGA-3'), SEQ ID NO: 450 (5'-AUG / CGA-3'), SEQ ID NO: 451 (5'-AUG / UGA-3'), SEQ ID NO: 452 (5'-CG / CG-3'), SEQ ID NO: 453 (5'-UUG / UGG-3'), SEQ ID NO: 454 (5'-UAU / AUA-3'), SEQ ID NO: 455 (5'-ACU / AGA-3'), SEQ ID NO: 456 (5'-AUG / CAA-3'), SEQ ID NO: 457 (5'-CU / AG-3'), and SEQ ID NO: 458 (5'- The sequence of NO:458 (5'-UG / CA-3').

12. The ribozyme according to any one of the preceding claims, wherein if motif [e] and motif [E] are partially complementary to each other, the complementarity between motif [e] and motif [E] is characterized by alternating complementary and non-complementary regions.

13. A ribozyme according to any one of the preceding claims, wherein the motif [D] comprises +3 Paired nucleotide N 7 mutation.

14. The ribozyme of any of the preceding claims, wherein the optional linker regions independently or collectively form one or more secondary structures, optionally one or more secondary structures selected from: a single nucleotide bulge, a dinucleotide bulge, a trinucleotide bulge, a polynucleotide bulge, a stem, a stem-loop, a t-RNA type structure, a cloverleaf type structure, a tetraloop, a pseudoknot, a symmetric internal loop, an asymmetric internal loop, a three-stem linker (3-way linker), a four-stem linker (4-way linker), a two-stem linker (2-way linker) or a coaxial stacking or a combination thereof.

15. The ribozyme according to any one of the preceding claims, wherein the ribozyme complex comprises any one or more than one sequence of SEQ ID NO: 3 to SEQ ID NO:

448.

16. The ribozyme of any one of the preceding claims, wherein the ribozyme further comprises one or more than one modification.

17. The ribozyme of any one of the preceding claims, wherein the trigger nucleic acid comprises one or more than one modified nucleotide.

18. A method for detecting the presence of a target nucleic acid molecule / trigger nucleic acid molecule in a sample, wherein the method include: incubating the sample with the ribozyme according to any one of claims 1 to 17 at a temperature T1 that allows binding of the target nucleic acid molecule / trigger nucleic acid molecule to one or more target binding domains / trigger binding domains contained in the ribozyme; incubating the sample at a temperature T2 that allows the release of nucleic acid molecules and RNA fragments from the ribozyme; The release of the releasable RNA fragment from the ribozyme is detected.

19. A method for detecting the presence of a target sequence or a target mutation on a target nucleic acid in a sample, wherein the method include: incubating a sample with a ribozyme according to any one of claims 1 to 17, thereby allowing the target nucleic acid molecule to bind to one or more target binding domains / trigger binding domains contained in the ribozyme; incubating the sample to allow the nucleic acid molecules and the releasable RNA fragments to be released from the ribozyme; detecting release of the releasable RNA fragment from the ribozyme; The releasable RNA fragment is a sgRNA or a shRNA; wherein detection of a target sequence or a target mutation in a sample results in signal generation.

20. The ribozyme of any one of claims 1 to 17 or the method of any one of claims 18 to 19, wherein the trigger nucleic acid molecule is a viral genome or a fragment thereof.

21. A kit comprising the ribozyme according to any one of claims 1 to 17 or 20.

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