CN106868155B - Method for visually detecting miRNA (micro ribonucleic acid) by using exonuclease reaction generated primer combined with dendritic rolling circle amplification - Google Patents
Method for visually detecting miRNA (micro ribonucleic acid) by using exonuclease reaction generated primer combined with dendritic rolling circle amplification Download PDFInfo
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Abstract
The invention discloses a method for visually detecting miRNA (micro ribonucleic acid) by using exonuclease reaction generated primers and dendritic rolling circle amplification. The method comprises three parts, namely, generation of a first part primer, degradation of a single-chain part by exonuclease I after a hairpin probe I is opened by a target miRNA, degradation of DNA by ribonuclease H: RNA in the RNA hybrid strand, leaving a short strand of DNA; the second part is tree rolling circle amplification, the single-chain DNA generated in the last step is connected with a long single-chain DNA probe to form a circle as a template, then the single-chain DNA is used as a primer to carry out primary rolling circle amplification, and a hairpin stem-loop structure II introduced into a rolling circle amplification system is opened by a primary rolling circle amplification product, so that the multi-stage tree rolling circle amplification is realized; the third part is based on the detection of the G-quadruplexes contained in the products of the rolling circle amplification. The method is a sensitive, economical, convenient and in-situ visual miRNA detection method.
Description
Technical Field
The invention belongs to the field of molecular biology and nucleic acid chemistry, and relates to a method for visually detecting miRNA (micro ribonucleic acid) by using exonuclease reaction to generate a primer combined with dendritic rolling circle amplification.
Background
MicroRNA (miRNA) is an endogenous, non-coding, small RNA of about 20-24 nucleotides in length that has a number of important regulatory roles within the cell. The 5 'end of the mature miRNA is a phosphate group, and the 3' end of the mature miRNA is a hydroxyl group. The miRNA gene is usually transcribed by nuclear RNA polymerase II, the initial product is a large pri-miRNA with a cap structure and a polyadenylic tail, then processed in the nucleus by Drosha enzyme into hairpin-like pre-RNA of 60-70 bases, transported into the cytoplasm by the transporter Exprotin-5 complex, and recognized and cleaved by Dicer enzyme to form a mature miRNA. It is speculated that mirnas regulate one third of the genes in humans. In recent years, scientists have come to recognize that mirnas have a broad role in the regulation of eukaryotic gene expression.
The location of mirnas is highly conserved among different species. The miRNA is not only conserved in gene position, but also shows high homology in sequence, and the high conservation of the miRNA is closely related to the importance of the function of the miRNA. Human miRNA genes are often found in cancer-associated genomic regions and fragile sites, and it is presumed that miRNA is closely related to tumor formation and may serve as a proto-oncogene or a cancer suppressor gene. The miRNA detection has important significance in disease diagnosis and research, and the research and sequence analysis on the expression level of the miRNA related to the tumor are beneficial to the elucidation of the generation and development mechanism of the tumor, and provide a new direction and strategy for the diagnosis, treatment and prognosis of the tumor. Due to the characteristics of short sequence, high homology, low expression content and the like, the detection of the polypeptide has many challenges. At present, some methods for miRNA detection, such as the traditional northern blot method, require large sample size, long detection period and poor discrimination capability for homologous sequences to limit further application. Some emerging detection methods based on nanoparticles and electrochemistry solve the problem of detection limit well, but the preparation of nanoparticles and electrochemistry substrates needs skilled professionals, the method is not easy to popularize and apply, the RT-PCR method successfully solves the problems, the sensitivity is high, the detection range is up to 7 orders of magnitude, the miRNA with low concentration or high concentration can be detected, and homologous sequences can be distinguished well. However, the complicated primer design, expensive real-time fluorescence quantitative PCR instrument and the like limit the popularization and application of RT-PCR to a certain extent.
Rolling circle amplification, an isothermal signal amplification technique, with a linear amplification multiple of 105Exponential amplification fold greater than 109The efficient amplification enables the technology to be widely applied to the fields of trace biomolecule detection, such as DNA, RNA, protein and metal ion detection, and the like, and to be successfully applied to the detection of biomacromolecules in biological environments. Scientists have carried out a series of improvements and optimizations on the basis of this technique, and the sensitivity of this technique is further improved to supermolecule rolling circle amplification, increase enzyme cutting site and initiate second grade rolling circle amplification, etc.. The rolling circle amplification technology comprises cyclization and then signal amplification, and polymerase in the rolling circle amplification has good selectivity on base mismatch, so the rolling circle amplification technology can be well applied to miRNA detection.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a method for visually detecting miRNA by using exonuclease reaction to generate a primer combined with dendritic rolling circle amplification.
The purpose of the invention is realized by the following technical scheme:
a method for visually detecting miRNA (micro ribonucleic acid) by utilizing exonuclease reaction generated primer and dendritic rolling circle amplification comprises three parts, wherein the first part is generated, a hairpin probe I is opened by a target miRNA and then degraded by exonuclease I to form a protruding single-stranded part, and then DNA is degraded by RNase H: RNA in the RNA hybrid strand, leaving a short strand of DNA; the second part is tree rolling circle amplification, the single-chain DNA generated in the last step is connected with a phosphorylated long single-chain DNA probe to form a circle as a template, then the single-chain DNA is used as a primer to carry out primary rolling circle amplification, and a hairpin stem-loop structure II introduced into a rolling circle amplification system is opened by a primary rolling circle amplification product, so that the multi-stage dendritic rolling circle amplification is initiated; the third part is the qualitative or quantitative detection based on the G-quadruplex contained in the rolling circle amplification product.
Preferably, the method for visually detecting the miRNA by using the exonuclease reaction generated primer combined with dendritic rolling circle amplification comprises the following steps:
(1) adding a hairpin probe I, an exonuclease I buffer solution, a sample to be detected containing a target miRNA and water into the system, adding an exonuclease I into the system for reaction after pre-incubation, and inactivating the exonuclease I after the reaction is finished.
(2) T4 DNA ligase buffer, a phosphorylated single-stranded DNA probe, a ribonuclease H, T4 DNA ligase and water were added to the reaction system of the step (1) to carry out a reaction.
(3) And (3) adding the hairpin probe II, phi29 DNA polymerase, dNTPs and water into the system reacted in the step (2) for reaction, and inactivating after the reaction is finished.
(4) Adding G-quadruplex buffer solution into the reaction system in the step (3), heating for denaturation, adding hemin for incubation, and finally adding ABTS2-And H2O2And detecting the ultraviolet absorption value of 414nm and observing the color change of the system. In the system with the target miRNA, the color is changed from colorlessGreen to the naked eye and uv absorbance increases with increasing miRNA concentration.
The design principles of the hairpin probe I, the phosphorylated long single-strand DNA probe and the hairpin probe II are as follows:
the target miRNA is divided into two parts with the sequence length being close to or equal to N1 and N2, namely the target miRNA sequences N1-N2. Taking miRNA21 (sequence 5'-UAGCUUAUCAG ACUGAUGUUGA-3') as an example, UAGCUUAUCAG is a N1 portion, ACUGAUGUUGA is a N2 portion, and U is T in the DNA sequence.
The hairpin probe I comprises A, B, C, D four parts, and has the structure of A-B-C-D, A, D is a stem terminal region, and B, C is a loop region. The stem end region A, D is complementary and is 8-10 base pairs in length to ensure stable hairpin structure. The B sequence in the Loop is complementary and matched with a target miRNA, and the composition of the B sequence is N2 '-N1', which is complementary and matched with N2 and N1 respectively. C is a small single-chain sequence with the length of 4-7 bases to ensure the stability of the hairpin structure.
The phosphorylated long single-stranded DNA probe contains E, F, G, H four parts and has the structure E-F-G-H. E is a 5' terminal phosphorylation sequence which is identical to the sequence of N2; h is the 3' terminal sequence, identical to N1. F is 20-25 bases in length. G is the complementary sequence of the G-quadruplex and is used for amplifying the G-quadruplex, and the length of the G-quadruplex is 17 bases (CCCAACCCGCCCTACCC).
Hairpin probe II comprises I, J, K, L four parts, its structure is I-J-K-L, I and L can be complementary paired in stem terminal region, J, K is Loop region. Wherein J is the same as the F sequence of the phosphorylated long single-stranded DNA probe, and K-L is the same as the B sequence of the hairpin probe I.
The detection method mainly comprises three parts: the first part is a primer for generating rolling circle amplification by using an exonuclease reaction; the second part of dendritic rolling circle amplification, linear rolling circle amplification develops along the direction of the branched chain by introducing a hairpin probe II, and further amplification of signals is achieved; the third part is the detection of rolling circle amplification products. The detection principle of the present invention is shown in fig. 1. The target miRNA can be complementarily paired with a sequence in the loop region of the hairpin probe I, so that the hairpin structure is opened, and the DNA-RNA hybrid contains a section of single-stranded DNA bulgeThe structure is that exonuclease I can recognize the area and degrade single-stranded DNA from 3'-5' direction, RNA in RNA heterozygote is degraded by ribonuclease H, a short single-stranded DNA is left, then the short single-stranded DNA is used as a template, under the action of T4 DNA ligase, phosphorylated long single-stranded DNA is connected end to form a ring, then the ring-formed DNA is used as a template, the short single-stranded DNA generated in the last step is used as a primer, and primary rolling circle amplification is initiated under the catalysis of phi29 DNA polymerase. In a rolling circle amplification system, a hairpin structure II which can be complementarily paired with a repeat sequence of a primary rolling circle amplification product is introduced, and after a loop region and the complementary pairing of the primary rolling circle amplification product are opened, a stem region and a rolling circle template can be complementarily paired, so that rolling circle amplification is initiated to continue in the direction of a branched chain, signals are further amplified, and the detection limit is reduced. The designed DNA loop template contains the complementary sequence of the G-quadruplex, so that the obtained rolling circle amplification product contains a large number of repeated G-quadruplex sequences, and the G-quadruplex can be assembled with the hemin to form peroxidase to catalyze ABTS2-(2, 2-Aza-bis (3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt) oxidation to ABTS.-And the ultraviolet absorption at 414nm is enhanced, the solution is changed from colorless to green, and the content of miRNA can be quantitatively detected. Aiming at different target miRNAs, only corresponding hairpin probes I, phosphorylated long single-strand DNA probes and hairpin probes II need to be designed.
The invention provides a sensitive, economical, convenient and in-situ visual miRNA detection method based on primers generated by exonuclease reaction, signals amplified by dendritic rolling circle amplification and the activity of G-quadruplex peroxidase. The invention has the advantages and beneficial effects that:
(1) the DNA used in the invention is common fluorescence-labeled DNA, so that the interference of background signals is avoided, and under the condition that the detection condition is not allowed, the content of the target miRNA can be judged through the change of color, so that the in-situ detection is realized.
(2) Different target miRNAs can be detected by designing different probe sequences. In addition, the method firstly generates a DNA primer through enzyme digestion reaction and then carries out dendritic rolling circle amplification, thereby improving the selectivity of the method. DNA is more stable than RNA and is more suitable for use as a primer to prime rolling circle amplification for a longer period of time.
Drawings
FIG. 1 is a schematic diagram of a method for detecting miRNA by using exonuclease reaction to generate primers and combining dendritic rolling circle amplification.
Fig. 2 is a graph of the uv kinetics and visualization results of miRNA21 detection in example 1.
FIG. 3 is a graph showing the results of detection of different miRNAs by the probe in example 1.
Fig. 4 is a graph of the results of detecting miRNA155 in example 2.
FIG. 5 is a graph of the results of example 3 for the detection of miRNA21 in hela cells.
Detailed Description
The following examples are intended to further illustrate the invention but should not be construed as limiting it. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.
Example 1
This example uses miRNA21 (sequence 5'-UAGCUUAUCAG ACUGAUGUUGA-3') as the detection target.
1. Design synthesis of hairpin probe I, phosphorylated long single-stranded DNA and hairpin probe II
(1) The hairpin probe I sequence is:
5'-CCCAACCCATCAACATCAGTCTGATAAGCTATTACTAGTGGGTTGGG-3'。
wherein the underlined sequences can be complementarily paired to form a "stem" and the remainder form a loop, and the double underlined sequences can be complementarily paired with miRNA21 to allow hairpin probe I to be opened.
After complementary pairing of the hairpin probe I and miRNA21, degrading the hairpin probe I and the miRNA with exonuclease I and ribonuclease H in sequence to obtain short single-stranded DNA with the sequence of CCCAACCCA TCAACATCAGT CTGATAAGCTA.
(2) The sequence of the phosphorylated long single-stranded DNA probe is as follows:
5'-PO4-ACTGATGTTGACAGGAATTAGTAAACAATGAAGACCCAACCCGCCCTACCCTAGCTTATC AG-3'。
wherein the head and tail parts (sequences marked with wavy lines) can be degraded with hairpin probe I to form short single-stranded DNA (CCCAACCCA)TCAACATCAGT CTGATAAGCTA) Partial (sequence marked with wavy lines) complementary pairings; the dotted underlined sequence (CCCAACCCGCCCTACCC) is the complement of the G-quadruplex, which serves as a template for G-quadruplex synthesis; the remaining underlined part (CAGGAATTAGTAAACAATGAAGA) of the sequence obtained after rolling circle amplification was used to open the dendritic rolling circle amplification primer hairpin probe II.
The phosphorylated long single-stranded DNA is connected end to form a DNA loop under the action of T4 DNA ligase by taking the short single-stranded DNA as a template. The formed DNA loop is used as a template, the short single-stranded DNA is used as a primer, and the primary rolling circle amplification is initiated under the catalysis of phi29 DNA polymerase.
(3) The hairpin probe II sequence is:
5'-TAGCTTATCAGACAGGAATTAGTAAACAATGAAGATCAACATCAGTCTGATAAGCTA-3'. Wherein the underlined sequences can be complementarily paired to form a "stem" with the remainder forming a loop. The underlined sequence (CAGGAATTAGTAAACAATGAAGA) in the loop is complementary to the product obtained by the previous-stage rolling circle amplification, so that the hairpin probe II is opened, and after the hairpin probe II is opened, the sequence (TCAACATCAGTCTGATAAGCTA) can be complementary to the rolling circle sequence (TAGCTTATCAGACTGATGTTGA) to initiate dendritic rolling circle amplification.
2. Detection of miRNA21
(1) Exo-enzyme reaction
10nmol/L of hairpin probe I, a series of miRNA21 (0 fmol/L, 1fmol/L, 10fmol/L, 100fmol/L, 1pmol/L, 10pmol/L, 100pmol/L, 1 nmol/L), 1. mu.L of 10 Xexonuclease buffer, 0.5. mu.L of RNase inhibitor, supplemented with enzyme-free water to a volume of 9. mu.L, pre-incubation at 37 ℃ for 30 minutes was added to a 10. mu.L exonuclease reaction system. 10U of exonuclease I (1. mu.L) was added, incubated at 37 ℃ for 1 hour, and inactivated at 80 ℃ for 20 minutes, and then slowly cooled to 30 ℃.
(2) Ligation reaction
Directly adding 2 mu L of 10 XT 4 DNA ligase buffer solution, 1U ribonuclease H, 5U T4 DNA ligase and 30nmol/L phosphorylated long single-stranded DNA probe into the one-step exonuclease reaction system, adding enzyme-free water to supplement the volume to 20 mu L, and incubating for 2 hours at 30 ℃.
(3) Dendritic rolling circle amplification
Directly adding 3 μ L of 10 XPhi 29 DNA polymerase buffer solution, 200 μmol/L dNTPs, 200nmol/L hairpin probe II, 1U Phi29 DNA polymerase and ddH into the one-step ligation reaction system2The volume of O-filled up to 30. mu.L. After incubation at 30 ℃ for 6 hours, inactivation was carried out at 70 ℃ for 10 minutes.
(4) Result detection
Directly adding 1 XG-4 buffer (25 mmol/L HEPES, NH) into the product obtained by dendritic rolling circle amplification4OH (pH 8.0), 20mmol/L KCl, 200mmol/L NaCl) to a total volume of 200. mu.L, heating at 95 deg.C for 5 min, standing at room temperature for 0.5 hr, adding hemin (125. mu. mol/L), incubating at room temperature for 0.5 hr, adding ABTS2-（2mmol/L）、H2O2(2 mmol/L), and the 414nm ultraviolet absorption kinetics is detected. The results are shown in fig. 2, and as the concentration of miRNA increases, the uv absorption gradually increases; and in the presence of 1nmol/L of miRNA21, the system color turns green obviously.
3. Specificity detection
Other mirnas used for specific detection were as follows:
miRNA 141：5'-UAACACUGUCUGGUAAAGAUGG-3'，
miRNA155：5'-UUAAUGCUAAUCGUGAUAGGGGU-3'，
miRNA199a：5'-ACAGUAGUCUGCACAUUGGUUA-3'，
miRNA429：5'-UAAUACUGUCUGGUAAAACCGU-3'。
the used hairpin probe I, hairpin probe II, phosphorylation long single-stranded DNA probe, method and the like are the same as the detection of miRNA 21. The results are shown in fig. 3, and only the uv kinetics corresponding to miRNA21 is significantly increased, while the uv kinetics corresponding to other negative control mirnas are substantially unchanged and have no enhancement. Detecting miRNA155
Example 2
In this example, miRNA155 is used as a detection target, the detection method is the same as in example 1, and the probe sequences used are as follows:
card issuing probe I: 5'-CCCAACCCA ACCCCTATCACGATTAGCATTAA TTACTAG TGGGTTGGG-3', respectively;
phosphorylated long single-stranded DNA probes: 5' -PO4-GTGATAGGGGT CAGGAATTAGTAAACAATGAAGACCCAACCCGCCCTACCC TTAATGCTAATC-3'；
Hairpin probe II: TTAATGCTAATC CAGGAATTAGTAAACAATGAAGA ACCCCTATCACGATTAGCATTAA are provided.
The results are shown in fig. 4, with increasing miRNA155 concentration, increasing uv absorption. The method is applicable to detecting other miRNA by changing the probe sequence.
Example 3
Total RNA extracted from human cervical cancer cell line hela cells was detected according to the probe and method in example 1. The total RNA amounts used were 0.5. mu.g and 5. mu.g, respectively. The results are shown in FIG. 5, where the UV absorbance increased with the addition of total RNA.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
SEQUENCE LISTING
<110> Wuhan university
<120> method for visually detecting miRNA by using exonuclease reaction generated primer combined dendritic rolling circle amplification
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Claims (4)
1. A method for visually detecting miRNA by using exonuclease reaction generated primers combined with dendritic rolling circle amplification is characterized in that: the method comprises three parts, namely, generation of a first part primer, degradation of a protruding single-stranded part by exonuclease I after a hairpin probe I is opened by a target miRNA, and degradation of DNA by ribonuclease H: RNA in the RNA hybrid strand, leaving a short strand of DNA; the second part is dendritic rolling circle amplification, short chain DNA generated in the last step is connected with a phosphorylated long single-chain DNA probe to form a ring to be used as a template, then the single-chain DNA is used as a primer to carry out primary rolling circle amplification, and a hairpin probe II introduced into a rolling circle amplification system is opened by a primary rolling circle amplification product, so that the multi-stage dendritic rolling circle amplification is initiated; the third part is to carry out qualitative or quantitative detection according to the G-quadruplex contained in the rolling circle amplification product; the exonuclease I has the activity of degrading single-stranded DNA from 3'-5' direction;
the design principles of the hairpin probe I, the phosphorylated long single-strand DNA probe and the hairpin probe II are as follows:
dividing the target miRNA into two parts with the sequence lengths being close to or equal to each other, N1 and N2, namely the sequence of the target miRNA is 5 '-N1-N2-3';
the hairpin probe I comprises A, B, C, D four parts, and has a structure of 5'-A-B-C-D-3', A, D, B, C, wherein the stem end regions can be complementarily paired, and the loop region is B, C; the B sequence in the Loop consists of N2 '-N1', which is complementary and matched with N2 and N1 respectively; c is a short single-stranded sequence;
the phosphorylated long single-stranded DNA probe comprises E, F, G, H four parts and has the structure 5' -PO4-E-F-G-H-3'; e is a 5' terminal phosphorylation sequence which is identical to the sequence of N2; h is a 3' terminal sequence, identical to N1; f is 20-25 bases in length; g is the complementary sequence of the G-quadruplex; the C-D moiety in hairpin probe I is not complementary paired with the G or G-F moiety in the phosphorylated long single-stranded DNA probe;
the hairpin probe II comprises I, J, K, L four parts, the structure is 5'-I-J-K-L-3', I and L are stem terminal regions which can be complementarily matched, J, K is Loop region; wherein J is the same as the F sequence of the phosphorylated long single-strand DNA probe, and K-L is the same as the B sequence of the hairpin probe I;
the method for visually detecting miRNA by using exonuclease reaction generated primers and dendritic rolling circle amplification is used for non-disease diagnosis.
2. The method of claim 1, wherein: the method comprises the following steps:
(1) adding a hairpin probe I, an exonuclease I buffer solution, a sample to be detected containing a target miRNA and water into the system, pre-incubating, adding an exonuclease I for reaction, and inactivating the exonuclease I after the reaction is finished;
(2) adding a T4 DNA ligase buffer solution, a phosphorylated single-stranded DNA probe, a ribonuclease H, T4 DNA ligase and water into a system reacted in the step (1) to perform reaction;
(3) adding hairpin probe II, phi29 DNA polymerase, dNTPs and water into the system reacted in the step (2) for reaction, and inactivating after the reaction is finished;
(4) adding G-quadruplex buffer solution into the reaction system in the step (3), heating for denaturation, adding hemin for incubation, and finally adding ABTS2-And H2O2Detecting the ultraviolet absorption value of 414nm and the color change of an observation system; in the system where the target miRNA is present, the color changes from colorless to macroscopic green, and the uv absorbance increases with increasing miRNA concentration.
3. The method of claim 1, wherein: the stem end region of hairpin probe I is 8-10 base pairs in length.
4. The method of claim 1, wherein: the C portion of hairpin probe I is 4-7 bases in length.
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