CN113564229B

CN113564229B - Reagent and method for detecting microRNA (microribonucleic acid) based on click chemical ligation and hairpin stacking assembly

Info

Publication number: CN113564229B
Application number: CN202110857579.3A
Authority: CN
Inventors: 赵爽; 陈鸣; 唱凯; 阳莎; 汤晓琦; 余恋雨
Original assignee: Third Military Medical University TMMU
Current assignee: Third Military Medical University TMMU
Priority date: 2021-07-28
Filing date: 2021-07-28
Publication date: 2022-04-01
Anticipated expiration: 2041-07-28
Also published as: CN113564229A

Abstract

The invention discloses a reagent and a method for detecting microRNA (ribonucleic acid) based on click chemical connection and hairpin stacking assembly, wherein the reagent for detecting microRNA comprises a target recognition probe and a hairpin probe, so that the target microRNA can be accurately separated from a homologous sequence, and the detection process of the method does not need participation of various enzymes; the sensitivity is high, the lower limit of target detection is 8.63pM, the specificity is good, the recovery rate of serum standard addition is 92.45-108.92%, and a brand new thought is provided for applying click chemistry reaction and enzyme-free DNA loop to the field of biological detection in future.

Description

Reagent and method for detecting microRNA (microribonucleic acid) based on click chemical ligation and hairpin stacking assembly

Technical Field

The invention relates to a reagent and a method for detecting and quantifying nucleic acid, in particular to a reagent and a method for detecting microRNA based on click chemistry connection and hairpin stacking assembly.

Background

microRNAs are non-coding RNAs with the length of 18 to 24 bases, are secreted to the extracellular space by living cells and play an important role in the post-transcriptional regulation of genes, so that the microRNAs are closely related to a plurality of pathophysiological processes, such as the differentiation and the apoptosis of cells and the occurrence of organs. A series of researches find that the microRNA is abnormally expressed in body fluid of patients suffering from tumors, cardiovascular diseases and even psychological diseases. It is therefore considered to be a promising biomarker that can assist in disease diagnosis and treatment.

However, due to the characteristics of short sequence and low abundance of microRNA, some enzyme-dependent nucleic acid amplification methods, such as reverse transcription polymerase chain reaction (RT-PCR), isothermal exponential amplification (EXPAR), Rolling Circle Amplification (RCA), and the like, are generally required in the conventional detection strategies at present. Although these methods are effective and sensitive, they inevitably increase the cost of detection, making the detection process more complex, due to the need for the participation of one or more enzymes. More importantly, the activity of the enzyme is highly susceptible to environmental factors, which undoubtedly affect the reproducibility of the experiment. In contrast, some enzyme-free detection formats will be easier to control, simpler to operate, and more economical. Efficient detection without enzymes therefore still needs to be considered.

Due to the inherent disadvantages of the enzyme-dependent detection mode, the invention of a novel enzyme-free microRNA detection mode is hopeful to realize more economical and convenient detection. In recent years, there has been much interest in signal amplification by ligation of oligonucleotide probes by click chemistry, which has higher ligation efficiency and better versatility and compatibility than those relying on DNA ligase or RNase ligase, because it relies on chemical reactions of azido (N3) and Dibenzocyclooctynyl (DBCO). Moreover, the short sequence of microRNA is suitable for being used as a template of click chemistry reaction, and the click chemistry ligation reaction becomes a promising signal amplification strategy in microRNA detection. However, it is necessary to consider how to convert a large amount of ligation products into signal output, and the methods reported so far mostly require the use of magnetic bead separation and even the re-use of DNA polymerase, which complicates the detection step again. We found in preliminary experiments that chemical groups in the click chemistry-generated ligation products made it difficult for the ligation products to be recognized by the enzyme again like natural nucleic acids, but did not affect their participation in the strand displacement reaction as an invasive strand. Based on the inspiration, a hairpin stacking circulation reaction is designed after the click chemical reaction to realize the second signal amplification, and different from catalysis hairpin self-assembly, the circulation hairpin stacking can form Y-shaped DNA tripolymer with three arms and has continuous signal turning capacity. The two-step coupling is expected to make the microRNA detection process simpler, the detection cost lower, the detection condition easier to control without depending on enzyme, and the reproducibility is better.

Disclosure of Invention

In view of the above, an object of the present invention is to provide a reagent for detecting microRNA based on click chemistry ligation and hairpin stacking assembly; the invention also aims to provide a method for detecting microRNA based on click chemistry connection and hairpin stacking assembly.

In order to achieve the purpose, the invention provides the following technical scheme:

1. a reagent for detecting microRNA based on click chemistry ligation and hairpin stacking assembly, comprising:

a target recognition probe and a hairpin probe;

the target recognition probe comprises a microRNA recognition sequence I and a microRNA recognition sequence II;

the miRNA recognition sequence I is complementary with a 3 'end half sequence of a microRNA sequence to be detected, and the microRNA recognition sequence II is complementary with a 5' end half sequence of the microRNA sequence to be detected;

an azide group is modified at the 3' end of the microRNA recognition sequence I;

an alkynyl group is modified at the 5' end of the microRNA recognition sequence II;

the hairpin probe consists of 3 metastable DNA hairpin structures which are a hairpin probe HA, a hairpin probe HB and a hairpin probe HC respectively, the 5 ' end of the hairpin probe HA is complementary with a sequence formed by connecting the miRNA recognition sequence I and the microRNA recognition sequence II through an azide group and an alkynyl group and can be opened through a toe-mediated strand displacement reaction, the 5 ' end of the hairpin probe HB is complementary with the 3 ' end of the hairpin probe HA and can be opened through the toe-mediated strand displacement reaction, the 5 ' end of the hairpin probe HC is complementary with the 3 ' end of the hairpin probe HB and can be opened through the toe-mediated strand displacement reaction, the stem of the hairpin probe HC is modified with a fluorescent group and a quenching group, and the 3 'end of hairpin probe HC is complementary to the 5' end of hairpin probe HA, and hairpin probe HA, hairpin probe HB and hairpin probe HC form a Y-type complex through complementation.

In the invention, preferably, the fluorophore Cy3 modified on the hairpin probe HC and the quenching group is BHQ-2.

Preferably, the sequence of the microRNA to be detected is let-7a, the microRNA recognition sequence I is shown as SEQ ID NO.1, and the microRNA recognition sequence II is shown as SEQ ID NO. 2; the hairpin probe HA sequence is shown in SEQ ID NO.3, and the hairpin probe HB sequence is shown in SEQ ID NO. 4; the hairpin probe HC sequence is shown in SEQ ID NO. 6.

In the present invention, preferably, the reagent further comprises: PBS buffer solution and Tween-20.

2. A method for detecting microRNA by using the reagent of any one of claims 1 to 4, comprising the following steps:

mixing a target recognition probe with a sample to be detected, hybridizing a microRNA recognition sequence I and a microRNA recognition sequence II with the sample to be detected to form a hybrid complex, enabling an azide group and an alkynyl group to approach to each other to generate an efficient click chemical ligation reaction, heating to denature the hybrid complex to enable the sample to be detected to be dissociated, cooling to enable the sample to be detected to serve as a template to perform the click chemical ligation reaction, and repeatedly heating and cooling to obtain a product obtained by connecting the microRNA recognition sequence I and the microRNA recognition sequence II;

adding a hairpin probe into a connecting product of the microRNA recognition sequence I and the microRNA recognition sequence II, carrying out hybridization reaction, hybridizing the connecting product with the 5 ' end of the hairpin probe HA, hybridizing the 3 ' end of the hairpin probe HA with the 5 ' end of the HB, hybridizing the 3 ' end of the hairpin probe HB with the 5 ' end of the hairpin probe HC, hybridizing the 3 ' end of the hairpin probe HC with the 5 ' end of the hairpin probe HA to form a Y-shaped complex, simultaneously opening a fluorescent group and a quenching group on the stem of the hairpin probe HC to generate a fluorescent signal, and judging the microRNA content according to the intensity of the fluorescent signal.

Preferably, in the invention, the temperature is raised to 95 ℃; the temperature reduction is to reduce the temperature to 30 ℃.

In the present invention, it is preferable that the number of times of the cycle of repeating the temperature increase and decrease is 70.

In the present invention, the hybridization reaction temperature is preferably 30 ℃ and the time is preferably 1.5 hours.

In the invention, the sample to be detected is a solution containing target let-7a, and the nucleic acid sequence of the target let-7a is shown in SEQ ID No. 7.

The hairpin probe HC is modified by a fluorescent group Cy3, and the quenching group is BHQ-2.

Further preferably, the microRNA recognition sequence I is shown as SEQ ID NO. 1; the microRNA recognition sequence II is shown as SEQ ID NO. 2; the hairpin probe HA sequence is shown in SEQ ID NO. 3; the hairpin probe HB sequence is shown in SEQ ID NO. 4; the hairpin probe HC sequence is shown in SEQ ID NO. 6.

The invention has the beneficial effects that:

compared with some existing exosome-derived miRNA detection modes, the detection method mainly has the following three advantages:

firstly, thanks to the participation of click chemistry, the method does not need conventional reverse transcription and other processes for detecting short-chain RNA molecules such as miRNA, so that the whole detection process does not need the participation of various enzymes, the requirement on detection environment is relatively low, a reaction system is relatively simple and only has a few nucleic acid probes, the detection condition is easier to control, and the whole process is more convenient and easier.

Secondly, the method has satisfactory sensitivity and specificity, has higher sensitivity than some reported methods limited to nanomole, has certain detection capability on some biological samples with reduced abundance, has the capability of distinguishing single base mutation, and can play an ideal role in simulating the detection of clinical samples. The experiments show that the detection mode has excellent performance and can meet the requirements in practical application.

Finally, the method firstly tries to combine click chemistry connection and hairpin stacking assembly to realize double-cycle amplification, proves that click chemistry is a unique and effective target sequence amplification mode, is not limited to the nucleic acid types of targets and products, and even can generate DNA-RNA mixed products, thereby providing a brand new thought for applying click chemistry reaction and enzyme-free DNA loops to the field of biological detection in the future.

Drawings

In order to make the object, technical scheme and beneficial effect of the invention more clear, the invention provides the following drawings for explanation:

FIG. 1 is a flow chart of a dual cycle signal amplification detection of microRNA in combination with cyclic click chemistry ligation and hairpin stacking assembly;

FIG. 2(A) is an electrophoretic verification of the reaction process; (B) atomic force microscope scanning verification of the final product; (C) fluorescence measurement validation for the reaction;

FIG. 3(A) is the fluorescence response signals for different concentrations of target (blank, 20pM, 50pM, 100pM, 250pM, 500pM, 1nM, 1.5nM, 2nM from a to i, respectively); (B) a linear fit equation of Δ F and C (Δ F represents fluorescence signal minus background signal, C represents target concentration);

FIG. 4(A) shows fluorescence response signals of different concentrations of microRNA detected by a cycle of hairpin stack assembly alone (blank, 2nM, 5nM, 10nM, 20nM, 50nM, 80nM, 100nM from a to h, respectively); (B) linear fitting equation of Δ F and C. (. DELTA.F for fluorescence signal minus background signal, C for target concentration);

FIG. 5(A) is the fluorescent response signal intensity for target, blank and individual interfering sequences; (B) the invention detects the fluorescence response signal intensity when the target with 1 percent of content in the mixed RNA sample (let-7a keeps the concentration of 250pM unchanged).

Detailed Description

The present invention is further described with reference to the following drawings and specific examples so that those skilled in the art can better understand the present invention and can practice the present invention, but the examples are not intended to limit the present invention.

Example 1 Synthesis of Probe sequences

Probes and other nucleic acid sequences used in the present invention were synthesized with reference to table 1.

TABLE 1 all nucleic acid sequences used in the present invention

Note: in the table, the stem sequence of the DNA hairpin probe is underlined and the loop sequence is in italics. The grey font indicates the base site where let-7a is mutated.

Example 2 detection of microRNAs in combination with click chemistry ligation and hairpin Stack Assembly

FIG. 1 illustrates the implementation process of the present invention in detail, when a target exists, two nucleic acid probes (N3 Probe, DBCO Probe) each complementary to a half sequence of a miRNA molecule Let-7a and modified with Azide and DBCO groups at the end respectively can hybridize to the target to allow a click chemical group to approach to generate an efficient click chemical ligation reaction, when the temperature is raised to 95 ℃, the hybridization complex is denatured to allow Let-7a to dissociate, thereby being able to serve as a template for the next reaction after the temperature is lowered to 30 ℃. After a plurality of cycles of temperature rise and temperature reduction are carried out, a small number of targets enable a large number of connecting products to be generated, then three exquisite metastable-state DNA hairpin structures HA, HB and HC are designed for the connected products, the connecting products generated after the first-step reaction can open a first DNA hairpin HA through a toe-mediated strand displacement reaction, a sequence exposed after the first hairpin is opened can open the hairpin HB according to the same principle, the same connecting product HA-HB complex can open the HC hairpin, when the HC hairpin is opened, the connecting products are displaced to finally form a Y-type complex of the HA-HB-HC, and the displaced connecting products can drive the next Y-type complex to generate so as to achieve a second cycle amplification signal. Furthermore, when the stem of HC is modified with the fluorophore Cy3 and the quencher group BHQ-2, a strong fluorescent signal can be generated when the modified stem is opened. And finally, the intensity of the fluorescence signal is in a positive correlation with the content of the microRNA, so that the unique design of two-step signal amplification is expected to realize enzyme-free, sensitive and specific detection of miRNA.

The specific detection method comprises the following steps: mixing a target recognition Probe N3 Probe (SEQ ID NO.1) and a DBCO Probe (SEQ ID NO.2) with a sample to be detected containing a target let-7a (SEQ ID NO.7), adding PBS (phosphate buffer solution), Tween-20, N3 Probe and the DBCO Probe into the sample to be detected to hybridize to form a hybridization complex, enabling an azide group and an alkynyl group to approach to generate efficient click chemical ligation reaction, heating to 95 ℃ to denature the hybridization complex to enable the sample to be detected to dissociate, cooling to 30 ℃ to enable the sample to be detected to serve as a template to perform click chemical ligation reaction, repeatedly heating and cooling for 70 times to obtain a N3 Probe and DBCO Probe ligation product;

adding hairpin probes HA (SEQ ID NO.3), HB (SEQ ID NO.4) and HC (SEQ ID NO.6) into the N3 Probe and DBCO Probe connecting product, carrying out hybridization reaction at 30 ℃ for 1.5 hours, hybridizing the connecting product with the 5 ' end of the hairpin Probe HA, hybridizing the 3 ' end of the hairpin Probe HA with the 5 ' end of the HB, hybridizing the 3 ' end of the hairpin Probe HB with the 5 ' end of the hairpin Probe HC, hybridizing the 3 ' end of the hairpin Probe HC with the 5 ' end of the hairpin Probe HA to form a Y-type complex, simultaneously opening a fluorescent group Cy3 and a quenching group Q-2 on the stem of the hairpin Probe HC to generate a fluorescent signal, and judging the content of the microRNA according to the intensity of the fluorescent signal. To verify the feasibility of the method, we validated and characterized the entire reaction process using PAGE electrophoresis, atomic force microscopy and fluorescence detection. In FIG. 2, A is the result of PAGE electrophoresis, and as shown in the figure, in lanes 2-4, the DNA probes modified with azide and DBCO and the target miRNA to be detected are respectively: let-7a, a miRNA closely related to the development of lung cancer. Lanes 5 and 6 are the products of the click chemistry reaction, and it can be seen that in lane 5, both probes are difficult to react in the absence of the target, while in lane 6, the chemical groups on both probes react in the presence of the target to form a ligation product. Lanes 7-9 show the location of the 3 hairpins, lane 10 shows that the three hairpins are stable, while in lane 11, a very light band is seen as a result of the reaction of the solution reacted in lane 5 with the three hairpins, indicating that although there is no visible product band in lane 5, there is actually a small amount of ligation product produced, driving a small amount of hairpins to produce a Y-structured DNA product, which is the main source of background signal. While 12-14 are the results of the reaction between the reaction solution in lane 5 and HA, HA + HB + HC at 30 ℃ for 1.5 hours. It can be seen that the three hairpins can be opened by click chemistry ligation products in sequence, eventually forming the large number of Y-type DNA structures we need. After that, we photographed the resulting product with atomic force microscope (fig. 2, B), and a large number of "Y" type structures could be seen, which further proved the generation of the product. Then, we change the HC hairpin into the HC hairpin modified by the fluorophore Cy3 and the quenching group BHQ-2, and then perform the above reaction process, and it can be seen that there is a significant difference in fluorescence signal between the target and the non-target (fig. 2, 1 is target and 2 is non-target in C), and the above results fully demonstrate the feasibility of the present invention.

Example 3 sensitivity verification

The sensitivity of the detection method is verified, the experimental result is shown in figure 3, in the detection strategy, when the target concentration is in the range of 20pM to 2nM, the fluorescence signal response is in a good linear relation, and the linear fitting equation is that DeltaF is 0.3336C +13.544, R²0.9981, the lower limit of detection reached 8.63 pM. Further, in order to verify that the double-cycle signal amplification provided by us really achieves the effect of amplifying signals twice, the sensitivity of detecting microRNA by single hairpin stacking assembly is verified, and the miR-simulation (SEQ ID No.14) is used as a trigger target, so that the result is shown in FIG. 4, the linear range is 2nM-100nM, the lower detection limit is only 1.4nM, and further, the addition of the cycle click chemical ligation reaction is proved to amplify the signals by more than 100 times, thereby achieving a more sensitive actual detection effect.

Example 4 specificity verification

The specificity of the detection method is verified, and the result is shown in fig. 5, a, and it can be seen that when the interfering microRNA is used: the fluorescence signals of miR-21(SEQ ID NO.11), miR128(SEQ ID NO.12) and miR145(SEQ ID NO.13) are almost the same as the blank signal value (blank), while the fluorescence signals are significantly reduced when interference sequences miss-1(SEQ ID NO.8), miss-2(SEQ ID NO.9) and miss-3(SEQ ID NO.10) which are different from the target sequence in only 1 to 3 base sites are used, further illustrating that the used probes have very excellent performance in distinguishing single base mutations.

Further, since there may be some high expression of micrornas that are not targets to be detected in a natural serum sample, it is very important for a new method to accurately detect target sequences in an RNA-contaminated environment. Therefore, we verified that the result is shown in FIG. 5, B, and when the let-7a concentration is fixed to 250pM and mixed into the interfering RNA sequence with 99 times concentration, the detection method still has accurate quantitative effect. This point shows that it has the potential of practical clinical application.

Example 5 recovery rate measurement

Finally, we verified the feasibility of the invention for testing serum samples. Standing whole blood of healthy people for layering, and taking upper serum. To avoid the influence of complex components in serum, the serum was diluted ten-fold with hybridization buffer (PBS buffer, Tween-20) and used. A quantitative known concentration of target let-7a (1.2nM, 800pM, 200pM, 40pM) was then added to the diluted serum and the assay concentration calculated using the assay of the invention was compared to the actual concentration. As shown in Table 2, the recovery rate is 92.45-108.92%, and the relative standard deviation is 2.14-5.30%, which indicates that the invention can be applied to the detection of actual clinical specimens.

TABLE 2 comparison of the detected concentration and the actual concentration of let-7a in the clinical simulation specimen in the method

The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.

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Claims

a target recognition probe and a hairpin probe;

the hairpin probe consists of 3 metastable DNA hairpin structures which are a hairpin probe HA, a hairpin probe HB and a hairpin probe HC respectively, the 5 ' end of the hairpin probe HA is complementary with a sequence formed by connecting the miRNA recognition sequence I and the microRNA recognition sequence II through an azide group and an alkynyl group and can be opened through a toe-mediated strand displacement reaction, the 5 ' end of the hairpin probe HB is complementary with the 3 ' end of the hairpin probe HA and can be opened through the toe-mediated strand displacement reaction, the 5 ' end of the hairpin probe HC is complementary with the 3 ' end of the hairpin probe HB and can be opened through the toe-mediated strand displacement reaction, the stem of the hairpin probe HC is modified with a fluorescent group and a quenching group, the 3 'end of the hairpin probe HC is complementary with the 5' end of the hairpin probe HA, and the hairpin probe HA, the hairpin probe HB and the hairpin probe HC are complementary to form a Y-type complex;

the sequence of the microRNA to be detected is let-7a, the microRNA recognition sequence I is shown as SEQ ID NO.1, and the microRNA recognition sequence II is shown as SEQ ID NO. 2; the hairpin probe HA sequence is shown in SEQ ID NO.3, and the hairpin probe HB sequence is shown in SEQ ID NO. 4; the hairpin probe HC sequence is shown in SEQ ID NO. 6.

2. The reagent for detecting microRNA based on click chemistry ligation and hairpin stacking assembly of claim 1, wherein: the hairpin probe HC is modified by a fluorescent group Cy3, and the quenching group is BHQ-2.

3. The reagent for detecting microRNA according to claim 1, further comprising: PBS buffer solution and Tween-20.

4. Method for detecting microRNA using a reagent according to any one of claims 1 to 3 for non-diagnostic purposes, characterized in that it comprises the following steps:

5. The method for detecting microRNA according to claim 4, wherein: the temperature is increased to 95 ℃; the temperature reduction is to reduce the temperature to 30 ℃.

6. The method of claim 4, wherein: the number of the cycles of repeating the temperature increase and decrease was 70.

7. The method of claim 4, wherein: the hybridization reaction temperature was 30 ℃ and the time was 1.5 hours.

8. The method of claim 4, wherein: the sample to be detected is a solution containing target let-7a, and the nucleic acid sequence of the target let-7a is shown in SEQ ID NO. 7.