MicroRNA detection method based on target-induced catalytic hairpin self-assembly and DNA motor amplification
Through target-induced catalytic card issuance self-assembly and DNA motor amplification methods, the existing microRNA detection methods have solved the problems of low sensitivity and long reaction time, and achieved efficient and rapid microRNA-92a detection.
Patent Information
- Application Number
- CN202311492491.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-11-10
- Publication Date
- 2025-05-13
AI Technical Summary
The existing microRNA detection methods have low sensitivity, expensive reagents, and long process time. Especially in two-stage amplification methods, the reaction time is usually more than 4 hours, limiting their application in actual testing.
The target-induced catalytic self-assembly and DNA motor amplification method was used to open the hairpin structure by hybridizing microRNA-92a and hairpin H4, and initiate catalytic self-assembly to form a Y-shaped DNA structure, improving the amplification efficiency and shortening the reaction time.
The detection limit is as low as 0.04fM, and the reaction time is shortened to 130 minutes, which significantly improves the detection efficiency and sensitivity, and is suitable for actual sample detection.
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Figure CN119979670A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to a microRNA detection method based on target-induced catalytic hairpin self-assembly and DNA motor amplification. Background Art
[0002] MicroRNAs (microRNAs) are a group of short endogenous noncoding RNAs, typically 19–23 nucleotides in length [1]. They play important roles in various biological processes such as cell proliferation, differentiation, carcinogenesis, aging, and cell death through posttranscriptional regulation of gene expression. Several standard methods are used for various microRNA analyses, including northern blot, microarray, and real-time quantitative polymerase chain reaction (qRT-PCR). However, their further practical applications are severely hampered by shortcomings such as low sensitivity, expensive reagents, and time-consuming processes. In order to further improve the performance of analytical techniques, several new biosensor-based microRNA detection methods have been developed, including colorimetry, fluorescence, electrochemistry, and surface plasmon resonance.
[0003] At the same time, some new isothermal amplification techniques have been combined to further improve the sensitivity, such as hybridization chain reaction (HCR), rolling circle amplification (RCA), and DNAse-based loop cleavage. Most of the methods with two-stage amplification methods spend a long reaction time in the two-stage amplification, usually more than 4 hours. Undoubtedly, the long incubation time greatly limits its application in practical testing. Therefore, how to shorten the reaction time becomes an urgent problem to be solved. Therefore, the detection of microRNA remains a challenge because of its small molecular weight, low abundance, and sequence similarity to its family members.
[0004] In order to solve the problems in the prior art, the present invention proposes a microRNA detection method based on target-induced catalytic hairpin self-assembly and DNA motor amplification. Summary of the invention
[0005] The present invention provides a method for detecting microRNA-92a (sequence: 5'-UAUUGCACUUGUCCCGGCCUGU-3') by combining target-induced catalytic hairpin self-assembly with DNA motor amplification method. MicroRNA-92a hybridizes with hairpin H4 to open its hairpin structure. The open hairpin structure can initiate catalytic hairpin self-assembly between H1, H2 and H3 to form a Y-shaped DNA structure. The two product catalytic tails of the Y-shaped DNA structure can also open the hairpin H4 structure, causing a high concentration of catalyst, and then triggering self-powered catalytic hairpin self-assembly. Therefore, the amplification efficiency of catalytic hairpin self-assembly can be greatly improved, and the reaction time can also be significantly shortened. The enzyme chain (E-DNA) tail of the Y-shaped DNA structure (sequence: 5'-CTCTTC-AGCGAT-TAAC-CAG-GTTA-CACCCATGT-TAGTGA-3') can hybridize with the substrate chain DNA (S-DNA) on gold nanoparticles (AuNPs) to form a DNA enzyme structure. Then, the autonomous cutting of the DNA motor leads to a significant recovery of fluorescence. Due to the two-level amplification method of catalytic hairpin self-assembly and DNA motor, the limit of detection (LOD) was as low as 0.04fM. Importantly, due to the self-catalytic hairpin self-assembly method, only a small amount of sample was required. In addition, due to the high amplification efficiency of the self-powered catalytic hairpin self-assembly, the reaction time of the entire method was greatly shortened to 130 minutes. In addition, this method has good application prospects in actual sample detection.
[0006] Specifically, the present invention provides a method for detecting microRNA-92a by combining target-induced catalytic hairpin self-assembly with DNA motor amplification method, which comprises the following steps:
[0007] 1) preparing a DNA motor: synthesizing gold nanoparticles, incubating FAM fluorescently labeled thiol-modified S-DNA and gold nanoparticles to fix the S-DNA on the surface of the gold nanoparticles to form a DNA motor, wherein the sequence of the S-DNA is 5'-HS-AAAAAAAAATCACTATrAGGAAGAG-FAM-3';
[0008] 2) Detection of microRNA-92a: The test solution was mixed and incubated with hairpin H1, hairpin H2, hairpin H3 and hairpin H4 in PBS buffer, wherein the sequence of hairpin H1 is 5'-AGCATAT-ATTGGA-CACTGA-GATAGA-TCATAG-CTTTCC-TCCAAT-CAATAC-GGAAAG-CTATGA-TCTATC-GAGCAA-3'; the sequence of hairpin H2 is 5'-AGCATAT-GATAGA-TCATAG-CTTTCC-GTAT The hairpin H3 sequence is 5'-CTCTTC-AGCGAT-TAAC-CAG-CTTTCC-GTATTG-ATTGGA-CACTGA-GATAGA-GGAAAG-CTATGA-TCTATC-TCAGTG-TCCAAT-GAGCAA-3', and the hairpin H4 sequence is 5'-ACAG GCCG GGAC AAGT GCAA TAGTT-GGAAAG-CTATGA-TCTATC-TCAGTG-TTCTATTGC-TCATATGCT-3'. Then, the DNA motor prepared in step 1) is added, and after further incubation, the fluorescence spectrum from 510 nm to 600 nm is monitored at an excitation wavelength of 492 nm, and the concentration of microRNA-92a is quantified by the linear relationship between the fluorescence intensity at 518 nm and the concentration of microRNA-92a.
[0009] Preferably, step 1) is specifically as follows: 1 μM FAM fluorescently labeled thiol-modified S-DNA and 10 nM gold nanoparticles are incubated overnight to fix the S-DNA on the surface of the gold nanoparticles, then 50 μM 6-mercaptohexanol is added to remove excess thiol-modified S-DNA, then 0.05% Tween 20 is used to inhibit nonspecific adsorption, and finally, the obtained gold nanoparticles are centrifuged at 10,000 rpm for 15 minutes, then washed with PBS buffer, and the gold nanoparticles are redispersed in PBS buffer solution.
[0010] Preferably, step 2) is specifically as follows: 10 μL of the microRNA-92a test solution is mixed with 200 μL of 50 nM hairpin H1, 50 nM hairpin H2, 50 nM hairpin H3 and 10 nM hairpin H4 in 10 mM PBS buffer (pH 7.5, 0.05 mM NaCl), and then the solution is incubated at 30°C for 90 minutes, and then mixed with 300 μL of 20 nM DNA step 2) The DNA motor prepared is in 10 mM PBS buffer (pH 7.5, 0.05 mM NaCl, 10 mM MgCl2), and incubated at room temperature for another 40 minutes, and then the fluorescence spectrum from 510 nm to 600 nm is monitored in a quartz test tube at an excitation wavelength of 492 nm. The concentration of microRNA-92a is quantified by the linear relationship between the fluorescence intensity at 518 nm and the concentration of microRNA-92a. BRIEF DESCRIPTION OF THE DRAWINGS
[0011] Figure 1 (A) is a schematic diagram of the catalytic hairpin self-assembly, and (B) is a schematic diagram of the detection process of the present invention.
[0012] Figure 2 The fluorescence intensity comparison chart of different samples. (1) H1, H2, H3, hairpin, DNA motor; (2) microRNA-92a, H2, H3, DNA motor; (3) microRNA-92a, H1', H2', H3', hairpin, DNA motor; (4) microRNA-92a, H1*, H2*, H3*, hairpin, DNA motor; (5) microRNA-92a, H1", H2", H3", hairpin, DNA motor. (6) microRNA-92a, H1, H2, H3, hairpin, DNA motor.
[0013] Figure 3 The fluorescence intensity of different microRNAs. Specific embodiments
[0014] The present invention will be further described in detail below in conjunction with the embodiments.
[0015] The detection method and steps of the present invention are as follows:
[0016] 1) Incubate 1 μM FAM fluorescently labeled thiol-modified S-DNA and 10 nM gold nanoparticles overnight to fix the S-DNA on the surface of the gold nanoparticles. Afterwards, add 50 μM 6-mercaptohexanol to remove excess thiol-modified S-DNA. Then, 0.05% Tween 20 inhibits nonspecific adsorption. Finally, the resulting gold nanoparticles are centrifuged at 10,000 rpm for 15 minutes, and then washed with PBS buffer, and the gold nanoparticles are redispersed in PBS buffer solution. Among them, the S-DNA sequence is 5'-HS-AAAAAAAAATCACTATrAGGAAGAG-FAM-3';
[0017] 2) 10 μL of the microRNA-92a test solution was mixed with 200 μL of 50 nM hairpin H1, 50 nM hairpin H2, 50 nM hairpin H3 and 10 nM hairpin H4 in 10 mM PBS buffer (pH 7.5, 0.05 mM NaCl). The solution was then incubated at 30 ° C for 90 minutes. Afterwards, it was mixed with 300 μL of 20 nM DNA step 2) The DNA motor prepared was mixed in 10 mM PBS buffer (pH 7.5, 0.05 mM NaCl, 10 mM MgCl2) and incubated at room temperature for another 40 minutes. Subsequently, the fluorescence spectrum from 510 nm to 600 nm was monitored in a quartz test tube with an excitation wavelength of 492 nm. The concentration of microRNA-92a was quantified by the linear relationship between the fluorescence intensity at 518 nm and the concentration of microRNA-92a.
[0018] The above method is the best embodiment of the present invention.
[0019] The principle of microRNA detection in this method is shown in Scheme 1. MicroRNA-92a can hybridize with the stem of hairpin H4 and open its structure. The exposed sequence of hairpin H4 can serve as a foothold for hybridization and opening probe H1. The newly released sticky sequence of H1 can serve as a new foothold for unfolding probe H2. H3 is then unfolded in the same way. The tail released by H3 is complementary to a part of H1. The H1-H3 double strand is more stable than the H1-hairpin 4 double strand, so the hairpin sequence can be displaced from the double strand by H2 to form a Y-shaped DNA structure. The released hairpin sequence can then start a new catalytic hairpin self-assembly cycle between H1, H2, and H3. The two separated tails of each hairpin are tightly bound to form the entire long product catalytic tail. The two product catalytic tails also automatically cycle to catalyze and power the catalytic hairpin self-assembly. With the increase of catalyst concentration, the amplification efficiency of catalytic hairpin self-assembly is greatly improved and the reaction time is shortened. At the same time, the E-DNA tail of the Y-shaped DNA structure can hybridize with the S-DNA on AuNPs to form a DNA enzyme structure. Then, the S-DNA is cleaved by the autonomous cleavage of the DNAzyme structure and leaves the AuNPs surface, leading to the recovery of the fluorescence signal that was previously quenched by the AuNPs. Finally, the concentration of the microRNA can be quantified by the recovery of the fluorescence intensity.
[0020] Feasibility characterization of the method
[0021] Some design samples were used for feasibility studies. Figure 2Comparison of fluorescence intensities of different samples. (1) Hairpin H1, hairpin H2, hairpin H3, hairpin H4, DNA motor; (2) microRNA-92a, hairpin H2, hairpin H3, DNA motor; (3) microRNA-92a, hairpin H1' (sequence: 5'-ATTGGA-CACTGA-GATAGA-TCATAG-CTTTCC-TCCAAT-CAATAC-GGAAAG-CTATGA-TCTATC-3'), hairpin H2' (sequence: 5'-GATAGA-TCATAG-CTTTCC-GTATTG-ATTGGA-TCTATC-TCAGTG-TCCAAT-CAATAC-GGAAAG-3'), hairpin H 3' (sequence: 5'-CTTTCC-GTATTG-ATTGGA-CACTGA-GATAGA-GGAAAG-CTATGA-TCTATC-TCAGTG-TCCAAT-3'), hairpin, DNA motor; (4) microRNA-92a, hairpin H1* (5'-ATTGGA-CACTGA-GATAGA-TCATAG-CTTTCC-TCCAAT-CAATAC-GGAAAG-CTATGA-TCTATC-GAGCAA-3'), hairpin H2* (sequence: 5'-AGCATAT-GATAGA-TCATAG-CTTTCC-GTATTG-ATTG GA-TCTATC-TCAGTG-TCCAAT-CAATAC-GGAAAG-GTTA-CACCCATGT-TAGTGA-3'), hairpin H3* (sequence: 5'-CTCTTC-AGCGAT-TAAC-CAG-CTTTCC-GTATTG-ATTGGA-CACTGA-GATAGA-GGAAAG-CTATGA-TCTATC-TCAGTG-TCCAAT-3'), hairpin, DNA motor; (5) microRNA-92a, hairpin H1" (sequence: 5'-AGCATAT-ATTGGA-CACTGA-GATAGA-TCATAG-CTT TCC-TCCAAT-CAATAC-GGAAAG-CTATGA-TCTATC-GAGCAA-3'), hairpin H2" (sequence: 5'-AGCATAT-GATAGA-TCATAG-CTTTCC-GTATTG-ATTGGA-TCTATC-TCAGTG-TCCAAT-CAATAC-GGAAAG-3'), hairpin H3 (sequence: 5'-CTTTCC-GTATTG-ATTGGA-CACTGA-GATAGA-GGAAAG-CTATGA-TCTATC-TCAGTG-TCCAAT-GAGCAA-3'), hairpin H4, DNA motor.(6) microRNA-92a, hairpin H1, hairpin H2, hairpin H3, hairpin H4, DNA motor.
[0022] like Figure 2 As shown, due to the absence of microRNA, the fluorescence signal of the blank sample (sample 1) is negligible. Due to the absence of catalyst, the sample without hairpin also shows a background signal (sample 2). Sample 3 without two product catalytic tails (no self-powered catalytic hairpin self-assembly) shows a weak fluorescence signal, which is attributed to the inhibition of self-powered catalytic hairpin self-assembly. Sample 4 with only one product catalytic tail shows a relatively strong fluorescence signal, which is attributed to the low amplification efficiency of self-powered catalytic hairpin self-assembly with only one product catalytic tail. Sample 5, in which the catalytic self-assembly product does not have an enzyme chain E-DNA tail, shows a negligible signal. This can be explained as the inhibition of DNA enzyme cutting. Sample 6 (i.e., the present invention) with standard conditions shows the strongest fluorescence intensity, indicating that the self-powered catalytic hairpin self-assembly and the cyclic cutting of the DNA motor are triggered as expected.
[0023] The method is selective for microRNA-92a
[0024] The selectivity of this method for microRNA-92a was further evaluated using several other microRNA sequences. Figure 3 As shown, the fluorescence intensity of the random sequence is as weak as that of the blank sample. MicroRNA-16 and microRNA-146a also show weak signal intensity. Only microRNA-92a and the mixture sample containing the target microRNA show strong fluorescence intensity, indicating that microRNA-92a can effectively initiate the self-powered catalytic hairpin self-assembly and DNA motor to amplify the fluorescence signal. These results show the great selectivity of this method for the detection of microRNA-92a.
[0025] In summary, a target-induced self-powered catalytic hairpin self-assembly combined with DNA motor amplification was developed for microRNA-92a detection. This method showed high sensitivity and reliability for microRNA-92a detection. In addition, the LOD of this method was greatly improved by the target-induced self-powered catalytic hairpin self-assembly and the two-level amplification of the DNA motor. In addition, the incubation time of the entire method has been significantly shortened to 130 minutes by the target-induced self-powered catalytic hairpin self-assembly. The actual detection of microRNA-92a in human serum also showed satisfactory results. However, this method is limited by the stability of AuNPs. It is well known that salt can induce the aggregation of AuNPs. Therefore, this method is not suitable for samples with high salt concentrations. In addition, when the target is expanded to other microRNAs, the sequences of the hairpin, H1, H2, and H3 need to be redesigned, and the detection conditions need to be optimized again.
Claims
1. A method for detecting microRNA-92a combining target-induced catalytic hairpin self-assembly with DNA motor amplification method, comprising the following steps: 1) Preparation of DNA motor: synthesizing gold nanoparticles, incubating FAM fluorescently labeled thiol-modified S-DNA and gold nanoparticles to fix S-DNA on the surface of gold nanoparticles to form a DNA motor, wherein: The S-DNA sequence was 5′-HS-AAAAAAAAATCACTATrAGGAAGAG-FAM-3′; 2) Detection of microRNA-92a: The test solution was mixed and incubated with hairpin H1, hairpin H2, hairpin H3 and hairpin H4 in PBS buffer, wherein the sequence of hairpin H1 was 5'-AGCATAT-ATTGGA-CACTGA-GATAGA-TCATAG-CTTTCC-TCCAAT-CAATAC-GGAAAG-CTATGA-TCTATC-GAGCAA-3'; the sequence of hairpin H2 was 5'-AGCATAT-GATAGA-TCATAG-CTTTCC-GTATTG-ATTGG A-TCTATC-TCAGTG-TCCAAT-CAATAC-GGAAAG-GTTA-CACCCATGT-TAGTGA-3', the hairpin H3 sequence is 5'-CTCTTC-AGCGAT-TAAC-CAG-CTTTCC-GTATTG-ATTGGA-CACTGA-GATAGA-GGAAAG-CTATGA-TCTATC-TCAGTG-TCCAAT-GAGCAA-3', and the hairpin H4 sequence is 5'-ACAGGCCGGGACAAGTGCAA TAGTT-GGAAAG-CTATGA-TCTATC-TCAGTG-TTCTATTGC-TCATATGCT-3'. Then, the DNA motor prepared in step 1) was added, and after further incubation, the fluorescence spectrum from 510 nm to 600 nm was monitored at an excitation wavelength of 492 nm, and the concentration of microRNA-92a was quantified by the linear relationship between the fluorescence intensity at 518 nm and the concentration of microRNA-92a.
2. The method according to claim 1, characterized in that Step 1) is specifically as follows: 1 μM FAM fluorescently labeled thiol-modified S-DNA and 10 nM gold nanoparticles are incubated overnight to fix the S-DNA on the surface of the gold nanoparticles, then 50 μM 6-mercaptohexanol is added to remove excess thiol-modified S-DNA, then 0.05% Tween 20 is used to inhibit nonspecific adsorption, and finally, the obtained gold nanoparticles are centrifuged at 10,000 rpm for 15 minutes, then washed with PBS buffer, and the gold nanoparticles are redispersed in PBS buffer solution.
3. The method according to claim 2, characterized in that Step 2) is specifically as follows: 10 μL of the microRNA-92a test solution is mixed with 200 μL of 50 nM hairpin H1, 50 nM hairpin H2, 50 nM hairpin H3 and 10 nM hairpin H4 in 10 mM PBS buffer (pH 7.5, 0.05 mM NaCl), and then the solution is incubated at 30°C for 90 minutes, after which it is mixed with 300 μL of 20 nM DNA motor prepared in step 2) in 10 mM PBS buffer (pH 7.5, 0.05 mM NaCl, 10 mM MgCl2), and incubated at room temperature for another 40 minutes, and then the fluorescence spectrum from 510 nm to 600 nm is monitored in a quartz test tube at an excitation wavelength of 492 nm. The concentration of microRNA-92a is quantified by the linear relationship between the fluorescence intensity at 518 nm and the concentration of microRNA-92a.