A DNAzyme-based detection and treatment system for multiple disease targets and its applications

Abstract

The present invention discloses a detection and treatment system for multiple disease targets based on DNAzyme. This system includes DNAzyme (S1), detection target (S2), and cleavage substrate (S3); the DNAzyme (S1) includes Loop 1 and Loop 2. Loop 1 is the conserved catalytic domain of the 10-23 DNA enzyme, and Loop 2 is a nucleic acid sequence that is completely complementary to the detection target (S2); initially, the DNAzyme (S1) is in a state where its cleavage activity is inhibited. When the detection target (S2) is added to the system, the cleavage activity of the DNAzyme (S1) is activated, and the cleavage substrate (S3) releases fluorescence to achieve signal amplification. The fluorescence signal is detected in real time to achieve the detection purpose. The detection and treatment system for multiple disease targets based on DNAzyme in this application has advantages such as a simple system, strong universality, high sensitivity, and room temperature detection. Only one-step amplification by DNA enzyme can achieve the microRNA fM-level detection limit (LOD).

Description

Technical Field

[0001] The present invention relates to the field of clinical testing, and in particular to a detection and treatment system for multiple disease targets based on DNAzyme and its applications. Background Art

[0002] Deoxyribozyme (DNA enzyme) is an oligonucleotide sequence with DNA or RNA cleavage activity screened in vitro. In 1997, 8-17 and 10-23 DNA enzymes with high modularity, specificity, and efficiency were screened out. The cleavage schematic diagram of the 10-23 DNA enzyme is as Figure 1 shown, including a catalytic domain composed of 15 highly conserved deoxynucleotides. There are two substrate recognition domains on both sides of it, which are used to recognize RNA substrates or DNA-RNA complex substrates through base complementary pairing and provide binding energy to maintain the substrates within the active site of the 10-23 DNA enzyme. Divalent metal cations serve as cofactors, and the substrates cleave specific phosphodiester bonds between unpaired purines (R) and paired pyrimidines (Y), generating 5'-end and 3'-end cleavage products. The 10-23 DNA enzyme is one of the DNA enzymes with the highest cleavage activity and has been used as a detection signal amplification system for the detection of multiple disease targets, providing a new technical route for clinical test diagnosis.

[0003] Problems such as difficult design and poor universality of detection targets exist in DNA sticky-end strand displacement reactions; problems such as low cleavage activity and impaired signal amplification effect exist in split DNA enzymes. At the same time, it is necessary to combine pre-amplification systems such as hybridization chain reaction (HCR), catalytic hairpin amplification (CHA), and reverse transcription recombinase polymerase amplification (RT-RPA), resulting in complex design and poor universality.

[0004] Therefore, it is necessary to design a detection and treatment system for multiple disease targets based on DNAzyme. Summary of the Invention

[0005] In order to overcome the defects in the prior art, a detection and treatment system for multiple disease targets based on DNAzyme and its applications are provided.

[0006] The present invention is achieved through the following solutions:

[0007] A detection and treatment system for multiple disease targets based on DNAzyme, the system includes self-blocking DNAzyme (S1), detection target (S2), cleavage substrate (S3); the DNAzyme (S1) includes loop 1 and loop 2, the loop 1 is the conserved catalytic domain of the 10-23 DNA enzyme, and the loop 2 is a nucleic acid sequence that is completely complementary and paired with the detection target (S2);

[0008] The DNAzyme (S1) is initially in a state where its cleavage activity is inhibited. When the detection target (S2) is added to the system, the cleavage activity of the DNAzyme (S1) is activated, and the substrate (S3) is cleaved to release fluorescence, enabling detection and treatment purposes by detecting the fluorescence signal.

[0009] The DNAzyme (S1) folds to form a self-blocking structure, rendering its cleavage activity inhibited.

[0010] In response to the detection target (S2), changes including but not limited to fluorescence signals are induced.

[0011] On the right side of loop 1 of the DNAzyme (S1) is binding domain 1, and on the left side of loop 1 of the DNAzyme (S1) is binding domain 2. Both binding domain 1 and binding domain 2 are composed of oligonucleotides.

[0012] Binding domain 1 and binding domain 2 are composed of x oligonucleotides and y oligonucleotides respectively, where the range of x and y is 3 - 8.

[0013] On the right side of loop 2 of the DNAzyme (S1) is binding domain 3, and on the left side of loop 2 of the DNAzyme (S1) is binding domain 4;

[0014] Binding domain 3 and binding domain 4 are complementary base-paired with binding domain 1 and binding domain 2 to inhibit the cleavage activity of the DNAzyme (S1).

[0015] When the detection target (S2) is added to the system, loop 2 of the DNAzyme (S1) forms a 23bp double-stranded DNA with the detection target (S2), and the base complementary pairing between binding domain 1 and binding domain 3 is disrupted.

[0016] The detection target (S2) is a single-stranded DNA or RNA, and the length of the detection target (S2) is 13 - 60 nt.

[0017] An application of a DNAzyme-based detection and treatment system for multiple disease targets, which is applied to the detection and treatment of multiple diseases.

[0018] For the detection and treatment of multiple diseases, it can be applied to the detection of microRNA in, including but not limited to, patients and healthy people; for the detection of viruses, it can be applied to the detection of, including but not limited to, patient saliva samples, serum samples, and fecal samples.

[0019] The beneficial effects of the present invention are:

[0020] The multiple disease target detection and treatment system based on DNAzyme in this application has the advantages of a simple system, strong generality, high sensitivity, and room temperature detection. Only one-step amplification by DNAzyme can achieve an fM-level detection limit (LOD) for microRNA. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is a schematic diagram of the cleavage of 10-23 DNAzyme;

[0022] Figure 2 It is a schematic structural diagram of the multiple disease target detection and treatment system based on DNAzyme in this application;

[0023] Figure 3 It is a diagram of the verification results of the system detection limit and system generality. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0024] The preferred embodiments of the present invention will be further described below in conjunction with the drawings:

[0025] As Figure 2 shown, a multiple disease target detection and treatment system based on DNAzyme, the system includes a self-blocking DNAzyme (S1), a detection target (S2), and a cleavage substrate (S3); the DNAzyme (S1) includes Loop 1 and Loop 2, the Loop 1 is the conserved catalytic domain of 10-23 DNAzyme, and the Loop 2 is a nucleic acid sequence that is completely complementary and paired with the detection target (S2);

[0026] The DNAzyme (S1) is initially in a state where its cleavage activity is inhibited. When the detection target (S2) is added to the system, the cleavage activity of the DNAzyme (S1) is activated, and the cleavage substrate (S3) releases fluorescence to achieve the purpose of detection and treatment by detecting the fluorescence signal..

[0027] The DNAzyme (S1) folds to form a self-blocking structure, making it in a state where its cleavage activity is inhibited.

[0028] Responds to the detection target (S2), triggering changes including but not limited to fluorescence signals.

[0029] On the right side of Loop 1 of the DNAzyme (S1) is Binding Domain 1, and on the left side of Loop 1 of the DNAzyme (S1) is Binding Domain 2. Both Binding Domain 1 and Binding Domain 2 are composed of oligonucleotides.

[0030] Binding Domain 1 and Binding Domain 2 are respectively composed of x oligonucleotides and y oligonucleotides, and the ranges of x and y are 3-8.

[0031] On the right side of loop 2 of DNAzyme (S1) is binding domain 3, and on the left side of loop 2 of DNAzyme (S1) is binding domain 4;

[0032] The binding domain 3 and binding domain 4 are complementary paired with binding domain 1 and binding domain 2 to inhibit the cleavage activity of DNAzyme (S1).

[0033] When the detection target (S2) is added to the system, loop 2 of DNAzyme (S1) forms a 23bp double-stranded DNA with the detection target (S2). Due to the rigid structure of the double-stranded DNA, the base complementary pairing between binding domain 1 and binding domain 3 is disrupted, thereby activating the DNA enzyme cleavage activity and cleaving the substrate (S3) to release fluorescence for signal amplification.

[0034] The detection target (S2) is a single-stranded DNA or RNA, and the length of the detection target (S2) is 113 - 60 nt.

[0035] A detection and treatment system for multiple disease targets based on DNAzyme, which is applied to the detection and treatment of multiple diseases.

[0036] For the detection and treatment of multiple diseases, it can be applied to the detection of microRNA in, including but not limited to, patients and healthy people; for the detection of viruses, it can be applied to the detection of, including but not limited to, patient saliva samples, serum samples, and fecal samples.

[0037] After a series of optimization experiments, as Figure 3 (a) shows, the detection of fM-level miR155 can be achieved after incubating at room temperature for 1 hour after adding the substrate. To further prove the universality of this system, we simultaneously shortened or extended the lengths of loop 2 and the detection target (S2), as Figure 3 (b) shows, single-stranded DNA or RNA with a length of the detection target (S2) between 13 - 60 nt can be detected in this system.

[0038] Therefore, according to different detection target sequences, only the part of loop 2 in S1 needs to be changed, and all other sequences remain unchanged. With only two nucleic acid strands (S1, S3), the purpose of detecting multiple disease targets can be achieved, having the advantages of a simple detection system and strong universality. Compared with the systems combined with HCR, CHA,

[0039] and DNA enzyme, for different detection target sequences, multiple hairpin structures need to be redesigned to make the system work, and the detection system is more complex and has weaker universality. Although there is only one step of signal amplification, fM-level detection can also be achieved in 1 hour, realizing high-sensitivity detection in a short time, and the detection limit is close to that of the systems combined with multiple signal amplifications.

[0040] ​

[0041] Although the technical solutions of the present invention have been described and enumerated in detail, it should be understood that for those skilled in the art, making modifications to the above embodiments or adopting equivalent alternative solutions will be obvious to those skilled in the art. These modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection required by the present invention.

Claims

1. A DNAzyme-based disease target detection system, characterized in that, The system includes a self-blocking DNAzyme S1 and a cleavage substrate S3; the DNAzyme S1 includes loop 1 and loop 2, where loop 1 is the conserved catalytic domain of the 10-23 DNAzyme, and loop 2 is a nucleic acid sequence that is completely complementary to the detection target S2; the DNAzyme S1 and the cleavage substrate S3 are two nucleic acid strands, the detection target S2 is a single-stranded DNA or RNA, and the length of the detection target S2 is 13-31 nt; the sequence of the cleavage substrate S3 includes the 10-23 DNAzyme recognition site RY. On the right side of loop 1 of the DNAzyme S1 is binding domain 1, and on the left side of loop 1 of the DNAzyme S1 is binding domain 2. Both binding domain 1 and binding domain 2 are composed of oligonucleotides. Binding domain 1 and binding domain 2 are composed of x oligonucleotides and y oligonucleotides respectively, and the range of x and y is 3-8; on the right side of loop 2 of the DNAzyme S1 is binding domain 3, and on the left side of loop 2 of the DNAzyme S1 is binding domain 4; binding domain 3 and binding domain 4 are complementary to binding domain 1 and binding domain 2 to inhibit the cleavage activity of DNAzyme S1. The DNAzyme S1 is initially in a state where its cleavage activity is inhibited. When the detection target S2 is added to the system, loop 2 of the DNAzyme S1 forms a completely complementary double-stranded DNA with the detection target S2, activating the cleavage activity of the DNAzyme S1. The cleavage substrate S3 releases fluorescence, and the detection purpose is achieved by detecting the fluorescence signal.

2. The disease target detection system based on DNAzyme according to claim 1, characterized in that: When the detection target S2 is added to the system, loop 2 of the DNAzyme S1 forms a 23-bp double-stranded DNA with the detection target S2, and the base complementary pairing between binding domain 1 and binding domain 3 is disrupted.

3. Use of a DNAzyme-based disease target detection system according to any one of claims 1-2, characterized in that: This system is applied to the preparation of reagents for detecting diseases.

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