Isothermal amplification probe composition, kit and application

Abstract

The invention discloses an isothermal amplification probe composition, a kit and application. The isothermal amplification probe composition is composed of a parallel line crossing DNA analogue probe PX1 and a probe PX2. The probe composition is high in universality and suitable for detection of nucleic acid molecules of different sequences, lengths and types; the response is quick during detection, and quick on-site colorimetric detection can be realized. Meanwhile, when the isothermal amplification probe composition is used, detection can be performed in a test tube without noble metal nano materials or magnetic beads or large instrument conditions, so that the detection cost can be remarkably reduced.

Description

Technical Field

[0001] This invention relates to isothermal amplification technology, and more particularly to an isothermal amplification probe composition, kit, and application. Background Technology

[0002] Hybridization chain reaction (HCR) is a nucleic acid amplification technique triggered by target nucleic acids at room temperature without enzyme involvement. Due to its simplicity and mild conditions, it has become an important tool in the biomedical field. Researchers have modified the stem-loop substrate of HCR with fluorescent molecule pairs that generate fluorescence resonance energy transfer (FRET) to monitor changes in fluorescence signals during the reaction process in real time. This enables highly sensitive detection of biomarkers such as nucleic acids, proteins, and small molecules, and has wide applications in biosensing and imaging. Furthermore, by combining DNA cross-linked hydrogels with HCR technology, researchers have further achieved the controlled release of small nucleic acid drugs, expanding their potential applications in the therapeutic field.

[0003] Besides classic linear hematoxylin and eosin (HCR), HCR technology incorporating DNA nanostructures has also attracted considerable attention in recent years. For example, immobilizing HCR substrates on DNA tetrahedra for chain amplification not only significantly improves the reaction rate but also enhances the structural stability of the product, demonstrating significant advantages in intracellular imaging and therapeutic applications. However, traditional methods based on DNA probes to recognize RNA through complementary base pairing still suffer from low recognition efficiency and long detection times; while RNA probes themselves are unstable, easily degraded by ribozymes, and prone to forming unintended secondary structures through mechanisms such as kissing loops. These factors limit the further development of detection technologies based on isothermal amplification. Summary of the Invention

[0004] Objectives of the invention: The first objective is to provide an isothermal amplification probe composition based on parallel-line crossed DNA analogs; the second objective is to provide a kit containing the isothermal amplification probe composition; and the third objective is to provide applications of the isothermal amplification probe composition and the kit.

[0005] Technical solution: The isothermal amplification probe composition of the present invention consists of parallel crossover DNA analog probe PX1 and probe PX2. The paraanemic crossover DNA analog consists of a first strand and a second strand; the first strand contains a first terminal stem-loop structure, and the second strand contains a second terminal stem-loop structure; there are one or more paraanemic cohesion structures between the first and second strands; each of the first and second strands contains one or more intramolecular double helix regions; and one end of the first strand contains a toehold structure. The probe PX1 is composed of a PX1-1 chain and a PX1-2 chain, and the probe PX2 is composed of a PX2-1 chain and a PX2-2 chain. One end of the PX1-1 chain is a toehold structure with multiple bases complementary to the target nucleic acid molecule, and one end of the PX2-1 chain is a toehold structure with multiple bases complementary to either the PX1-1 or PX1-2 chain; the stem-loop structure in the PX2-1 or PX2-2 chain is complementary to the toehold structure in the PX1-1 chain. The probe PX1 can form a parallel-line cross DNA structure with the target nucleic acid molecule, replacing the stem-loop structure and its upstream fragment in the PX1-1 or PX1-2 chain. The replaced fragment can form a parallel-line cross DNA structure with the probe PX2, replacing the stem-loop structure and its downstream fragment in the PX2-1 or PX2-2 chain. The replaced fragment can form a parallel-line cross DNA structure with the probe PX1, forming a chain substitution chain reaction. The PX1-1 chain or PX1-2 chain modifies fluorescent group A, and the PX2-1 chain or PX2-2 chain modifies fluorescent group B, which can generate fluorescence resonance energy transfer with fluorescent group A.

[0006] Preferably, the molar ratio of probe PX1 to probe PX2 is 0.5~1:0.5~1.

[0007] Preferably, the target nucleic acid molecule is a single-stranded DNA or RNA of 20-40 base pairs in length, and its A, T or U, C, G content is 10-70%; Preferably, in probe PX1 or probe PX2, each paraanemic cohesion structure consists of two sets of interchain paired structures with 4 to 6 base pairs; each duplex region structure consists of intrachain paired structures with 5 to 9 base pairs.

[0008] Preferably, the toehold structure at the 3' end of the PX1-1 chain, which is complementary to the target nucleic acid molecule, has a length of 5 to 9 bases.

[0009] Preferably, in both the PX1-1 chain and the PX2-1 chain, the toehold structure length is 5 to 9 bases.

[0010] Preferably, the stem-loop structure length in the PX1-1 chain, PX1-2 chain, PX2-1 chain, and PX2-2 chain is 5 to 9 bases.

[0011] Preferably, the PX1-1 chain, PX1-2 chain, PX2-1 chain, and PX2-2 chain contain one or more chemically modified nucleotides; more preferably, the chemically modified nucleotides include any one or more of 2'-methoxy modified nucleotides, 2'-fluoro modified nucleotides, locked nucleic acids, and peptide nucleic acids; more preferably, the number of chemically modified nucleotides does not exceed 50% of the total number of bases.

[0012] Preferably, the PX1-1 chain, PX1-2 chain, PX2-1 chain, and PX2-2 chain contain one or more uridines that substitute for thymidine.

[0013] Preferably, when the target nucleic acid molecule is DNA with the sequence shown in SEQ ID NO: 1 or RNA with the sequence shown in SEQ ID NO: 2, the isothermal amplification probe composition comprises: probe PX1 composed of a PX1-1 chain with the sequence shown in SEQ ID NO: 3 and a PX1-2 chain with the sequence shown in SEQ ID NO: 4, and probe PX2 composed of a PX2-1 chain with the sequence shown in SEQ ID NO: 5 and a PX2-2 chain with the sequence shown in SEQ ID NO: 6, wherein a Cy3 fluorescent group is inserted between positions 28 and 29 of the PX1-2 chain, and a Cy5 fluorescent group is modified at the 5' end of the PX2-1 chain; more preferably, the PX1-1 chain sequence is shown in SEQ ID NO: 7, wherein positions 51 to 56 are nucleotides modified with 2'-methoxy groups; more preferably, the PX1-1 chain sequence is shown in SEQ ID NO: 8, wherein bases 46 to 56 are nucleotides modified with 2'-methoxy groups.

[0014] Preferably, the target nucleic acid molecule is RNA with the sequence shown in SEQ ID NO: 9, and the isothermal amplification probe composition comprises: probe PX1 consisting of a PX1-1 chain with the sequence shown in SEQ ID NO: 10 and a PX1-2 chain with the sequence shown in SEQ ID NO: 11, and probe PX2 consisting of a PX2-1 chain with the sequence shown in SEQ ID NO: 12 and a PX2-2 chain with the sequence shown in SEQ ID NO: 13, wherein a Cy3 fluorescent group is inserted between positions 22 and 23 of the PX1-1 chain, and a Cy5 fluorescent group is modified at the 5' end of the PX2-1 chain; more preferably, the PX1-1 chain sequence is shown in SEQ ID NO: 14, wherein positions 39 to 44 are nucleotides modified with 2'-methoxy groups; more preferably, the PX1-1 chain sequence is shown in SEQ ID NO: 15, wherein bases at positions 34 to 44 are nucleotides modified with 2'-methoxy groups.

[0015] The kit described in this invention contains the isothermal amplification probe composition as described in any one of claims 1 to 8.

[0016] Preferably, the kit further contains a TAE-Mg buffer solution with pH = 8.0~8.6, wherein the TAE-Mg buffer solution contains 40 mM tris(hydroxymethyl)aminomethane acetate, 1 mM ethylenediaminetetraacetic acid, 12.5 mM magnesium acetate, and the remainder is water.

[0017] The application of the isothermal amplification probe composition or kit described in this invention in nucleic acid detection.

[0018] Preferably, the application steps include: mixing the isothermal amplification probe composition and the sample to be tested, mixing in a reaction buffer, and incubating at room temperature for 5 to 240 minutes; after the reaction, using a fluorescence spectrophotometer to scan the fluorescence spectrum of the reaction solution, recording the emission fluorescence intensity, and obtaining the detection result.

[0019] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: 1. The isothermal amplification probe composition is highly versatile and suitable for the detection of nucleic acid molecules of different sequences, lengths and types; 2. The isothermal amplification probe composition has a rapid response for detecting nucleic acid molecules and can realize rapid colorimetric detection on site; 3. The isothermal amplification probe composition does not require precious metal nanomaterials or magnetic beads, nor does it require large-scale instrument conditions. It can be detected in test tubes, which can significantly reduce detection costs. Attached Figure Description

[0020] Figure 1 A schematic diagram of the structure of a parallel crossover DNA analog with a closed stem-loop at the end; Figure 2This is a schematic diagram of the structure of probes PX1 (A) and PX2 (B); Figure 3 This is a schematic diagram of the chain displacement chain reaction between probes PX1 and PX2 and the target nucleic acid molecule; Figure 4 Polyacrylamide gel electrophoresis images of amplification results at different target DNA concentrations; Figure 5 The kinetic curve (a) and colorimetric results (b) of the isothermal amplification probe composition for DNA / RNA detection are shown. Figure 6 Kinetic curve (a) and colorimetric results (b) of the isothermal amplification probe composition after 2'-methoxy modification for DNA / RNA detection. Figure 7 A schematic diagram (a) and a colorimetric result (b) of the flow chart for the isothermal amplification probe composition and the 2'-methoxy modified amplification probe composition for the detection of microRNA-21. Detailed Implementation

[0021] The technical solution of the present invention will be further described below.

[0022] Example 1: Structural Design of Isothermal Amplification Probe Composition The isothermal amplification probe composition of the present invention consists of a parallel crossover DNA analog probe PX1 and a probe PX2. The parallel-line cross DNA analogue, such as Figure 1 As shown, it consists of a first chain 1 and a second chain 2; the first chain 1 contains a first terminal stem-loop structure 11, and the second chain 12 contains a second terminal stem-loop structure 21; there are one or more parallel-line cross-binding sites 3 between the first chain 1 and the second chain 2; the first chain 1 and the second chain 2 each contain one or more intramolecular double helix (duplex region) structures 4; one end of the first chain 11 contains a toehold structure 5; The structural diagram of probe PX1 is shown below. Figure 2 As shown in Figure A, the structural schematic diagram of probe PX2 is as follows: Figure 2 As shown in B, probe PX1 consists of a PX1-1 chain and a PX1-2 chain, and probe PX2 consists of a PX2-1 chain and a PX2-2 chain.

[0023] One end of the PX1-1 chain is a toehold structure that has multiple complementary base pairs with the target nucleic acid molecule, while one end of the PX2-1 chain is a toehold structure that has multiple complementary base pairs with either the PX1-1 or PX1-2 chain.

[0024] The PX1-1 chain, PX1-2 chain, PX2-1 chain, and PX2-2 chain all contain terminal stem-loop structures, and the stem-loop structure in the PX2-1 chain or PX2-2 chain is complementary to the toehold structure in the PX1-1 chain. Probe PX1 can form a parallel-line crossed DNA structure with the target nucleic acid molecule, displacing the stem-loop structure and its upstream fragment in the PX1-1 or PX1-2 strand; the displaced fragment can form a parallel-line crossed DNA structure with probe PX2, displacing the stem-loop structure and its downstream fragment in the PX2-1 or PX2-2 strand; the displaced fragment can then form a parallel-line crossed DNA structure with probe PX1, forming a chain displacement cascade reaction, as illustrated in the diagram below. Figure 3 As shown; PX1-1 or PX1-2 chains modify fluorescent group A, and PX2-1 or PX2-2 chains modify fluorescent group B, which can generate fluorescence resonance energy transfer with fluorescent group A.

[0025] Example 2: Design, preparation and validation of isothermal amplification probe compositions 1. Design and preparation of isothermal amplification probe compositions The target nucleic acid molecule sequence is shown in Table 1 below and was synthesized by Shanghai Sangon Biotech Co., Ltd.

[0026] Table 1 Target Nucleic Acid Molecular Sequences

[0027] Based on the target nucleic acid molecules, isothermal amplification probe compositions were designed to form chains, which were then synthesized by Shanghai Sangon Biotech Co., Ltd. The chain sequences are shown in Table 2 below: Table 2. Strand sequence of isothermal amplification probe compositions

[0028] In PX1-2, a Cy3 fluorescent group is inserted between positions 28 and 29, and the 5' end of PX2-1 is modified with a Cy5 fluorescent group.

[0029] PX1-1 and PX1-2 chains were mixed in TAE-Mg buffer at pH 8.0–8.6, with a final concentration of 10 μM for both chains. The mixture was heated at 95°C for 5 min and then incubated at 65°C, 45°C, 37°C, and 25°C for 20 min at each temperature gradient to obtain a 10 μM parallel crossover DNA analog probe PX1, which was stored at 4°C for later use. PX2-1 and PX2-2 chains were mixed in TAE-Mg buffer at pH 8.0–8.6, with a final concentration of 10 μM for both PX1-1 and PX1-2 chains. The mixture was heated at 95°C for 5 min and then incubated at 65°C, 45°C, 37°C, and 25°C for 20 min at each temperature gradient to obtain a 10 μM parallel crossover DNA analog probe PX2, which was stored at 4°C for later use. The TAE-Mg buffer solution contains 40 mM tris(hydroxymethyl)aminomethane acetate, 1 mM ethylenediaminetetraacetic acid, 12.5 mM magnesium acetate, and the remainder is water.

[0030] 2. Validation of the isothermal amplification probe composition 2.1 Validation at different target DNA concentrations (1) Take 4 μL of target DNA solution diluted with TAE-Mg buffer at pH = 8.0~8.6 with concentrations of 0, 2.5, 5, 7.5, 10, 20, 25, and 40 μM, respectively, mix with 8 μL each of probes PX1 and PX2 at a concentration of 10 μM prepared above, and incubate at room temperature for 30 min. (2) Take 5 μL of each reaction solution from step 1 and mix them with 1 μL of PAGE loading buffer (TAE-Mg buffer containing 50% glycerol and trace amounts of bromophenol blue and xylene cyanol). Load the mixture onto a 10% polyacrylamide gel for electrophoresis. The electrophoresis conditions are 100 V and 70 min. (3) After electrophoresis, the gel was stained in Sangon Biotech's full staining agent (product number A606359) for 5 min. After fading under light, the gel was imaged and acquired using a gel imaging system.

[0031] Gel electrophoresis results as follows Figure 4 As shown, the different DNA concentrations mentioned above can all induce chain amplification reactions of PX1 and PX2.

[0032] 2.2 Validation of the kinetics for DNA / RNA detection (1) Take 4 μL of target DNA solution or target RNA solution diluted with 20 μM TAE-Mg buffer (pH = 8.0~8.6), mix it with 8 μL of probe PX1 and PX2 (10 μM) prepared above, and react at room temperature. (2) At 5, 30, 60, 90, 120 and 240 min of reaction, the fluorescence intensity was measured at 564 nm wavelength using a fluorescence spectrometer at 490 nm wavelength, and the corresponding colorimetric changes at 5, 30, 120 and 240 min were recorded.

[0033] Based on the fluorescence intensity test kinetic curve, the results are as follows: Figure 5 As shown in A, the colorimetric changes are as follows: Figure 5 As shown in Figure B, the results indicate that this isothermal amplification probe composition successfully detected the target DNA / RNA. 1 / 2 DNA = 5.23 min, t 1 / 2 RNA = 176.04 min.

[0034] 3. Optimization of isothermal amplification probe compositions The PX1-1 chain underwent partial nucleotide chemical modification with 2'-methoxy (2'-OMe) and was synthesized by Shanghai Sangon Biotech Co., Ltd. The sequence is shown in Table 3 below: Table 3 Optimized PX1-1 chain sequence

[0035] In the 2'-methoxy modified PX1-1 chain, positions 51-56 are nucleotides modified with 2'-methoxy, and thymidine is replaced by uridine at positions 51, 53, and 54. In the extended 2'-methoxy modified PX1-1 chain, positions 46-56 are nucleic acids modified with 2'-methoxy, and thymidine is replaced by uridine at positions 48, 50, 51, 53, and 54. These are shown in underlined italics in the table.

[0036] Following the aforementioned method, probe PX1 containing 2'-methoxy groups and probe PX1 with extended 2'-methoxy groups were prepared. (1) Take 4 μL of target RNA solution diluted with 20 μM TAE-Mg buffer (pH = 8.0~8.6) and mix it with 16 μL of TAE-Mg buffer (Blank), or 8 μL each of probe PX1 and probe PX2 (DNA Toehold) prepared above at a concentration of 10 μM, or 8 μL each of 2'-methoxy modified probe PX1 and probe PX2 (2'-OMeToehold) at a concentration of 10 μM, or 8 μL each of elongated 2'-methoxy modified probe PX1 and probe PX2 (Elongated 2'-OMe Toehold) at a concentration of 10 μM, and react at room temperature; (2) At 5, 30, 60, 90, 120 and 240 min of reaction, the fluorescence intensity was measured at 564 nm wavelength using a fluorescence spectrometer at 470 nm wavelength, and the corresponding colorimetric changes at 5, 30, 120 and 240 min were recorded.

[0037] The kinetic curve was fitted based on the fluorescence intensity test results, and the results are as follows: Figure 6 As shown in A, the colorimetric changes are as follows: Figure 6As shown in Figure B, the results indicate that lengthening the 2'-methoxy modified probe PX1 effectively improved the detection rate of RNA by the isothermal amplification probe composition. 1 / 2 RNA = 4.13 min.

[0038] Example 3: Design, preparation and validation of isothermal amplification probe compositions based on microRNA-21 Using microRNA-21 (miR-21) with the sequence shown in SEQ ID NO: 9 as the target nucleic acid molecule, an isothermal amplification probe composition was designed to form a strand, which was synthesized by Shanghai Sangon Biotech Co., Ltd. The strand sequence is shown in Table 4 below: Table 4. Compositional sequences of target nucleic acid molecules and isothermal amplification probe compositions

[0039] In the PX1-1 chain, a Cy3 fluorescent group is inserted between the bases at positions 22 and 23. The 5' end of the PX2-1 chain is modified with a Cy5 fluorescent group. The nucleotides at positions 39-44 of the PX1-1 chain that are modified with 2'-methoxy groups are 2'-methoxy modified, and thymidine is replaced with uridine at position 43. The nucleotides at positions 34-44 of the PX1-1 chain that are modified with 2'-methoxy groups are lengthened, and thymidine is replaced with uridine at positions 35, 38, and 43. These are shown in underlined italics in the table.

[0040] According to the method described in Example 2, probes PX1, PX2, 2'-methoxy modified probe PX1, and extended 2'-methoxy modified probe PX1 were prepared at a concentration of 10 μM.

[0041] Take 4 μL of miR-21 solution diluted with 20 μM TAE-Mg buffer (pH = 8.0–8.6) and mix it with 16 μL of TAE-Mg buffer (Blank), or 8 μL each of the previously prepared 10 μM probe PX1 and probe PX2 (DNAToehold), or 8 μL each of the 10 μM 2'-methoxy modified probe PX1 and probe PX2 (2'-OMeToehold), or 8 μL each of the 10 μM elongated 2'-methoxy modified probe PX1 and probe PX2 (Elongated 2'-OMeToehold). React at room temperature and record the corresponding colorimetric changes at 5, 10, and 15 min.

[0042] A schematic diagram of the detection process for miR-21 using extended 2'-methoxy modified probes PX1 and PX2 is shown below. Figure 7 As shown in A, the results are as follows: Figure 7As shown in Figure B, with the extended 2'-methoxy modification scheme, miR-21 can be identified and a colorimetric signal change can be generated within 5 minutes, and a significant colorimetric signal change appears within 15 minutes. This result demonstrates the speed, convenience, and versatility of the method of this invention for detecting nucleic acids of different sequences and lengths.

Claims

1. An isothermal amplification probe composition, characterized in that, It consists of parallel-line crossed DNA analog probes PX1 and PX2; The parallel-line crossover DNA analog consists of a first strand (1) and a second strand (2); the first strand (1) contains a first terminal stem-loop structure (11), and the second strand (2) contains a second terminal stem-loop structure (21); there is one or more parallel-line crossover binding site structures (3) between the first strand (1) and the second strand (2); the first strand (1) and the second strand (2) each contain one or more intramolecular double helix structures (4); one end of the first strand (1) contains a foothold structure (5); The probe PX1 is composed of a PX1-1 chain and a PX1-2 chain, and the probe PX2 is composed of a PX2-1 chain and a PX2-2 chain. One end of the PX1-1 chain is a foothold structure with multiple bases complementary to the target nucleic acid molecule, and one end of the PX2-1 chain is a foothold structure with multiple bases complementary to the PX1-1 chain or the PX1-2 chain; the stem-loop structure in the PX2-1 chain or the PX2-2 chain is complementary to the foothold structure in the PX1-1 chain. The probe PX1 can form a parallel-line cross DNA structure with the target nucleic acid molecule, replacing the stem-loop structure and its upstream fragment in the PX1-1 or PX1-2 chain. The replaced fragment can form a parallel-line cross DNA structure with the probe PX2, replacing the stem-loop structure and its downstream fragment in the PX2-1 or PX2-2 chain. The replaced fragment can form a parallel-line cross DNA structure with the probe PX1, forming a chain substitution chain reaction. The PX1-1 chain or PX1-2 chain modifies fluorescent group A, and the PX2-1 chain or PX2-2 chain modifies fluorescent group B, which can generate fluorescence resonance energy transfer with fluorescent group A.

2. The isothermal amplification probe composition according to claim 1, characterized in that, The target nucleic acid molecule is a single-stranded DNA or RNA of 20-40 base pairs in length, with A, T or U, C, G content of 10-70%.

3. The isothermal amplification probe composition according to claim 1, characterized in that, In probe PX1 or probe PX2, each parallel-line cross-binding site structure consists of two sets of interchain pairing structures with 4 to 6 base pairs; each intramolecular double helix structure consists of intrachain pairing structures with 5 to 9 base pairs.

4. The isothermal amplification probe composition according to claim 1, characterized in that, In both the PX1-1 and PX2-1 chains, the length of the anchor structure is 5 to 9 bases.

5. The isothermal amplification probe composition according to claim 1, characterized in that, The stem-loop structure length in the PX1-1 chain, PX1-2 chain, PX2-1 chain, and PX2-2 chain is 5 to 9 bases.

6. The isothermal amplification probe composition according to claim 1, characterized in that, The PX1-1 chain, PX1-2 chain, PX2-1 chain, and PX2-2 chain contain one or more chemically modified nucleotides.

7. The isothermal amplification probe composition according to claim 6, characterized in that, The chemically modified nucleotides include any one or more of 2'-methoxy modified nucleotides, 2'-fluoro modified nucleotides, locked nucleic acids, and peptide nucleic acids.

8. The isothermal amplification probe composition according to claim 6, characterized in that, The number of chemically modified nucleotides shall not exceed 50% of the total number of nucleotides.

9. A reagent kit, characterized in that, The kit contains the isothermal amplification probe composition according to any one of claims 1 to 8.

10. The use of the isothermal amplification probe composition according to any one of claims 1 to 8 or the kit according to claim 9 in nucleic acid detection.

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