Dual-recognition framework nucleic acid probe, preparation method and application

Abstract

The invention discloses a dual-recognition-framework nucleic acid probe, a preparation method and application, the dual-recognition-framework nucleic acid probe comprises a DNA tetrahedron, and a dual-recognition module and a fuel amplification module which are connected to the vertex of the DNA tetrahedron through complementary pairing of bases, ATP recognition protection and dual-target response are introduced, and the dual-recognition-framework nucleic acid probe is obtained through sequential opening of internal and external locks. The occurrence of false positive conditions can be reduced, and the specificity is effectively improved; signal amplification is realized by virtue of abnormal APE1 expression of tumor cells, so that a fluorescence signal for miRNA detection is increased, and the sensitivity and the imaging signal-to-noise ratio are effectively improved; the preparation is easy; the use is convenient; complicated equipment is not needed. The method can be applied to a target nucleic acid detection reagent or an imaging or positioning reagent of cells, tissues or organs containing target nucleic acid.

Description

Technical Field

[0001] The present invention relates to nucleic acid probes, in particular to double recognition framework nucleic acid probes, preparation methods and applications. Background Art

[0002] Nucleic acid probes are a type of short-chain DNA or RNA molecules that can specifically bind to a specific target nucleic acid sequence. They are widely used in many biomedical fields such as genetic testing, disease diagnosis, molecular imaging, and treatment. With the continuous development of molecular biology and nanotechnology, nucleic acid probes have gradually become an important tool in research and clinical applications due to their unique advantages. Compared with traditional protein probes, nucleic acid probes have stronger programmability, low-cost synthesis and modification, lower immunogenicity, and controllable thermodynamic and kinetic properties. These advantages make nucleic acid probes show great application potential in disease detection, gene expression regulation, etc.

[0003] At present, single-identification nucleic acid probes, as the core component of the detection method, have been widely used in the identification of various biomarkers, especially in the field of tumor diagnosis. Single-target probes are widely used to monitor gene expression and mutations related to cancer. However, the application of single-target probes still faces some significant limitations, which are mainly reflected in the following aspects: First, due to the heterogeneity of tumor cells in different individuals and the same tumor, a single target may not be able to fully and effectively identify all types of tumor cells, which means that the characteristic markers of certain tumor types may be expressed at a low level, and the signal intensity of a single probe may be insufficient, resulting in false-negative results; secondly, some probes may not be able to distinguish between similar markers in tumor cells and normal cells, thereby generating false-positive signals. This non-specific recognition reduces the accuracy of the detection and affects the reliability of early diagnosis; and before the nucleic acid probe enters the cell, if premature activation or non-specific binding occurs, false-positive signals may also be generated. The above problems challenge the accuracy and sensitivity of the probe during the delivery process inside and outside the cell.

[0004] In recent years, the rise of framework nucleic acid (FNA) technology has brought new breakthroughs in the design and application of nucleic acid probes. Framework nucleic acid is a structure based on DNA nanotechnology that forms a specific spatial structure by precisely controlling the self-assembly of DNA molecules. Through engineering design, framework nucleic acids can precisely regulate the interaction and signal transmission between molecules, which provides strong support for biosensing, drug delivery and disease diagnosis. The use of framework nucleic acid technology to construct a probe system is expected to effectively solve the problems of traditional probes in terms of reliability and accuracy. Summary of the invention

[0005] Purpose of the invention: The purpose of the present invention is to provide a highly specific and sensitive ATP-responsive protected dual-recognition framework nucleic acid probe; the second purpose is to provide a preparation method and application of the dual-recognition framework nucleic acid probe.

[0006] Technical solution: The dual recognition framework nucleic acid probe of the present invention comprises a DNA tetrahedron and a dual recognition module and a fuel amplification module connected at its vertices through complementary base pairing;

[0007] Wherein, the DNA tetrahedron is composed of 4 single-stranded DNAs through complementary base pairing;

[0008] The dual recognition module is composed of three or more single-stranded DNAs through complementary base pairing;

[0009] The fuel amplification module is a single-stranded DNA with a hairpin structure having a depurinic and / or depyrimidinic site.

[0010] Preferably, the four single-stranded DNAs of the DNA tetrahedron include:

[0011] S1, having the sequence shown in SEQ ID No: 1;

[0012] S2, having the sequence shown in SEQ ID No: 2;

[0013] S3, having the sequence shown in SEQ ID No: 3;

[0014] S4 has the sequence shown in SEQ ID No:4.

[0015] Preferably, the dual recognition module is composed of three complementary pairs of single-stranded DNAs, including:

[0016] L1, having the sequence shown in SEQ ID No: 5;

[0017] L2, having the sequence shown in SEQ ID No: 6;

[0018] D2, having the sequence shown in SEQ ID No: 7;

[0019] Among them, L2 is partially complementary to the bases of a single-stranded DNA in the DNA tetrahedron; L1 is partially complementary to the bases of L2 to form an ATP recognition probe; D2 is partially complementary to the bases of L2 to form a miRNA recognition probe.

[0020] Preferably, the fuel amplification module is AF, having a sequence as shown in SEQ ID No: 8, and is complementary to a portion of the bases of S1 and / or S2.

[0021] Preferably, the dual recognition framework nucleic acid probe includes two quenching groups selected from BHQ1, BHQ2, and Dabcyl; and the dual recognition module has two fluorescent groups selected from FAM, Cy5, Cy3, ROX, and TAMRA.

[0022] Preferably, there are one or more fuel amplification modules.

[0023] The method for preparing the dual recognition framework nucleic acid probe of the present invention comprises:

[0024] (1) S1, S2, S3, and S4 are mixed in equal molar ratios, incubated at 90-100°C for 2-8 min, and rapidly cooled to 2-8°C in 20-40 s to obtain DNA tetrahedrons;

[0025] (2) incubating AF at 90-100° C. for 2-8 min and cooling naturally to obtain a fuel amplification module;

[0026] (3) Mix the DNA tetrahedron obtained in step 1, the fuel amplification module obtained in step 2, L1, L2, and D2, and react at 25-37° C. for 30-120 min to obtain a dual recognition framework nucleic acid probe.

[0027] Application of the dual recognition framework nucleic acid probe described in the present invention in a target nucleic acid detection reagent.

[0028] The dual recognition framework nucleic acid probe of the present invention is used as an imaging or positioning agent for cells, tissues or organs containing target nucleic acids.

[0029] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: 1. By introducing ATP recognition protection and dual-target response, this detection technology can reduce the occurrence of false positives and effectively improve specificity by sequentially opening the inner and outer locks; 2. By utilizing the abnormal expression of APE1 in tumor cells, signal amplification is achieved, the fluorescence signal of miRNA detection is increased, and the sensitivity and imaging signal-to-noise ratio are effectively improved; 3. It is easy to prepare and use without the need for complex equipment, and can realize on-site instant detection of target tumor markers, greatly saving detection time. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Schematic diagram of the working principle of the dual recognition framework nucleic acid probe protected by ATP response;

[0031] Figure 2 The polyacrylamide gel electrophoresis diagram of the assembly process of the multi-frame nucleic acid delivery module: Lane M is a 20 bp marker, Lane 1 is S1, Lane 2 is S1+S2, Lane 3 is S1+S2+S3, Lane 4 is S1+S2+S3+S4, Lane 5 is AF, and Lane 6 is S1+S2+S3+S4+AF;

[0032] Figure 3 The test results of ATP recognition and its protective effect in the dual recognition module;

[0033] Figure 4 The test results of miRNA recognition (taking miRNA21 as an example) and ATP external lock recognition protection in the dual recognition module;

[0034] Figure 5 The comparison test results of the amplification performance of the fuel amplification module;

[0035] Figure 6 It is a fluorescence intensity change diagram and detection curve diagram of the dual recognition framework nucleic acid probe in response to ATP performance;

[0036] Figure 7 It is the fluorescence intensity change diagram and detection curve diagram of the dual recognition framework nucleic acid probe responding to miRNA performance. DETAILED DESCRIPTION

[0037] The technical solution of the present invention is further described below.

[0038] Example 1: Preparation of dual recognition framework nucleic acid probe

[0039] 1. Design a nucleic acid chain displacement recognition module for the target nucleic acid (taking miRNA21 as an example)

[0040] 2. S1, S2, S3, S4, L1, L2, D2, and AF were synthesized by Sangon Biotech. Inc. The specific sequences are shown in Table 1:

[0041] Table 1 Synthetic single-stranded nucleotide sequence list

[0042]

[0043]

[0044] As shown in Table 1, the 78th position of S3 is modified with a deoxythymidine with a BHQ2 quenching group, the 5' end of L1 is modified with a FAM fluorescent group, the 14th position of L2 is modified with a deoxythymidine with a BHQ1 quenching group, the 5' end of D2 is modified with a Cy5 fluorescent group, and the 32nd position of AF is vacant.

[0045] 3. Mix the four DNA chains (S1, S2, S3, S4) in an equimolar ratio in Tris buffer, incubate at 95°C for 5 min, and quickly cool the mixture to 4°C within 30 s using a PCR instrument to obtain a framework nucleic acid delivery module;

[0046] 4. Add the AF chain to Tris buffer, place in a 95°C water bath for 5 min, and then keep in the water bath to slowly anneal to room temperature to obtain a fuel amplification module;

[0047] 5. The modules obtained in steps 2 and 3 were incubated with L1, L2, and D2 in Tris buffer, reacted at 37°C for 90 minutes to obtain a dual recognition framework nucleic acid probe, and stored at 4°C in the dark;

[0048] The Tris buffer contains 20 mM Tris-HCl, 50 mM MgCl2, 100 mM KCl, pH = 8.0.

[0049] Example 2: Polyacrylamide gel electrophoresis to verify the assembly of framework nucleic acid probes

[0050] 1. Prepare 8% polyacrylamide gel by mixing 5 mL of 40% acrylamide / bisacrylamide solution (volume ratio 19:1), 5 mL of TBE buffer, 15 mL of deionized water, 180 μL of APS and 18 μL of TEMED;

[0051] 2. Mix 10 μL of sample and 2 μL of 6× loading buffer and load on the sample. Perform electrophoresis at 100 V for 2 h in TBE buffer (89 mM Tris, 89 mM boric acid, 2 mM EDTA, pH = 8.0).

[0052] 3. After electrophoresis, use Gel-Red staining and take pictures with a gel imager.

[0053] like Figure 2 As shown, the migration rate in lane 4 is slower than that in lanes 1-3, indicating the formation of DNA tetrahedral structure, while lane 6 indicates the successful assembly of the delivery and amplification modules.

[0054] Example 3: Dual recognition framework nucleic acid probe for ATP detection

[0055] The dual recognition framework nucleic acid probe prepared in Example 1 (final concentration of 100 nM) was incubated with ATP at 37°C for 60 min with or without the addition of miRNA21 and with or without the addition of APE1. A fluorescence spectrophotometer was used with excitation at 494 nm and data at 520 nm to plot a fluorescence intensity bar graph.

[0056] like Figure 3 As shown, when there is no ATP, the fluorescence of the FAM fluorophore is quenched by the quenching group BHQ-1, and the fluorescence intensity is low; when ATP is present, the L1 chain binds to ATP, and the fluorescence intensity of FAM at 520nm increases significantly, proving that the framework nucleic acid probe can effectively recognize ATP and respond to it fluorescently.

[0057] Example 4: Dual recognition framework nucleic acid probe for miRNA21 detection and protection performance of ATP recognition module

[0058] The dual recognition framework nucleic acid probe prepared in Example 1 (final concentration of 100 nM) was incubated with miRNA21 at 37°C for 120 min with / without ATP and with / without APE1. A fluorescence spectrophotometer was used with excitation at 635 nm, data at 660 nm was measured, and a fluorescence intensity bar graph was plotted.

[0059] like Figure 4 As shown, when there is no ATP and miRNA21, the fluorescence of the Cy5 fluorophore is quenched by the quenching group BHQ-2, and the fluorescence intensity is low; when only miRNA21 is present, due to the presence of the outer recognition module, miRNA21 cannot react with the inner recognition module, and no Cy5 fluorescence signal is released; when ATP and miRNA21 are present at the same time, the outer recognition module is opened, and the inner recognition module can work normally, the L2 chain binds to miRNA21, and the fluorescent signal chain D2 is displaced and released, and the fluorescence intensity is significantly increased, indicating that the framework nucleic acid probe can effectively recognize miRNA21 and respond to it fluorescently, and the ATP recognition module can effectively protect the inner tumor marker recognition module, effectively avoiding false positives of the probe to improve the accuracy and reliability of probe diagnosis.

[0060] Example 5: Amplification performance of the dual recognition framework nucleic acid probe fuel amplification module

[0061] The dual recognition framework nucleic acid probe prepared in Example 1 (final concentration of 100 nM) was incubated with miRNA21, ATP, and APE1 at 37°C for 120 min with or without the addition of APE1. A fluorescence spectrophotometer was used with excitation at 635 nm, data at 660 nm was measured, and a fluorescence intensity bar graph was plotted.

[0062] like Figure 5 As shown, when APE1 is not present, the fluorescence of the Cy5 fluorophore does not undergo cyclic amplification and the fluorescence intensity is low; when APE1 is present, the AP site of the AF chain is cleaved to release the amplified fuel chain, replacing the target miRNA21, allowing the system to be cyclically amplified and the fluorescence intensity is significantly increased, indicating that the dual-recognition framework nucleic acid probe can effectively recognize APE1 and amplify the detection signal of miRNA21.

[0063] Example 6: Characterization of ATP detection performance of dual recognition framework nucleic acid probe

[0064] The dual recognition framework nucleic acid probe prepared in Example 1 (final concentration of 100 nM) was incubated with ATP of different concentrations (0 μM, 1 μM, 5 μM, 10 μM, 50 μM, 100 μM, 200 μM, 500 μM, 1 mM, 5 mM) at 37° C. for 60 min. A fluorescence spectrophotometer was used with excitation at 494 nm, data at 520 nm were measured, and a fluorescence intensity bar graph was plotted.

[0065] like Figure 6 As shown, with the increase of ATP concentration, the fluorescence intensity also gradually increases, that is, the fluorescence intensity shows a good linear relationship when the ATP concentration is 10μM~200μM.

[0066] Example 7: Characterization of miRNA detection performance of dual recognition framework nucleic acid probe

[0067] The dual recognition framework nucleic acid probe prepared in Example 1 (final concentration of 100 nM) was incubated with different concentrations (0 pM, 10 pM, 50 pM, 100 pM, 500 pM, 1 nM, 5 nM, 10 nM, 50 nM) of miRNA21 at 37° C. for 120 min. A fluorescence spectrophotometer was used with excitation at 635 nm, and data from 650 to 750 nm were measured, and a fluorescence curve was plotted.

[0068] like Figure 7 As shown in the figure, as the concentration of miRNA21 increases, the fluorescence intensity also gradually increases. Among them, the logarithm of the fluorescence intensity shows a good linear relationship when the concentration of miRNA21 is 50pM~50nM, and the following equation is obtained by fitting:

[0069] F(×10 6 )=1.06341log 10 c miRNA21 +1.9034(R 2 =0.9481)

[0070] Further calculations showed that the detection limit of the probe was 65.14 pM.

Claims

1. A dual recognition framework nucleic acid probe, characterized in that: It includes a DNA tetrahedron and a double recognition module and a fuel amplification module connected at its vertices through complementary base pairing; Wherein, the DNA tetrahedron is composed of 4 single-stranded DNAs through complementary base pairing; The dual recognition module is composed of three or more single-stranded DNAs through complementary base pairing; The fuel amplification module is a single-stranded DNA with a hairpin structure having a depurinic and / or depyrimidinic site.

2. The dual recognition framework nucleic acid probe according to claim 1, characterized in that: The four single-stranded DNAs of the DNA tetrahedron include: S1, having the sequence shown in SEQ ID No: 1; S2, having the sequence shown in SEQ ID No: 2; S3, having the sequence shown in SEQ ID No: 3; S4 has the sequence shown in SEQ ID No:

4.

3. The dual recognition framework nucleic acid probe according to claim 1, characterized in that: The dual recognition module is composed of three complementary single-stranded DNA pairs, including: L1, having the sequence shown in SEQ ID No: 5; L2, having the sequence shown in SEQ ID No: 6; D2, having the sequence shown in SEQ ID No: 7; Among them, L2 is partially complementary to the bases of a single-stranded DNA in the DNA tetrahedron; L1 is partially complementary to the bases of L2 to form an ATP recognition probe; D2 is partially complementary to the bases of L2 to form a miRNA recognition probe.

4. The dual recognition framework nucleic acid probe according to claim 1, characterized in that: The fuel amplification module is AF, having a sequence as shown in SEQ ID No: 8, and is complementary to a portion of the bases of S1 and / or S2.

5. The dual recognition framework nucleic acid probe according to claim 1, characterized in that: There are one or more fuel amplification modules.

6. The dual recognition framework nucleic acid probe according to claim 1, characterized in that: The dual recognition module has two fluorescent groups selected from FAM, Cy5, Cy3, ROX and TAMRA.

7. The dual recognition framework nucleic acid probe according to claim 1, characterized in that: The dual recognition framework nucleic acid probe includes two quenching groups among BHQ1, BHQ2 and Dabcyl.

8. A method for preparing a dual recognition framework nucleic acid probe according to any one of claims 1 to 7, characterized in that: include: (1) S1, S2, S3, and S4 are mixed in equal molar ratios, incubated at 90-100°C for 2-8 min, and rapidly cooled to 2-8°C in 20-40 s to obtain DNA tetrahedrons; (2) incubating AF at 90-100° C. for 2-8 min and cooling naturally to obtain a fuel amplification module; (3) Mix the DNA tetrahedron obtained in step 1, the fuel amplification module obtained in step 2, L1, L2, and D2, and react at 25-37° C. for 30-120 min to obtain a dual recognition framework nucleic acid probe.

9. Use of the dual recognition framework nucleic acid probe according to any one of claims 1 to 7 in a target nucleic acid detection reagent.

10. Use of the dual recognition framework nucleic acid probe according to any one of claims 1 to 7 as an imaging or localization agent for cells, tissues or organs containing target nucleic acids.

Images