阅读说明：本技术 基于量子点胶束球形核酸的DNA机器用于循环miRNA检测的方法 (Method for circulating miRNA detection by using DNA machine based on quantum dot micelle spherical nucleic acid ) 是由 孙清江 于 2020-05-07 设计创作，主要内容包括：本发明公开了一种基于量子点胶束球形核酸的DNA机器用于循环miRNA检测的方法。具体实施方法由三部分组成：(1)制备负载DNA酶序列和猝灭剂修饰的底物序列的多色量子点胶束球形核酸(QM-SNA)；(2)在靶标触发且金属离子辅助条件下,DNA酶可实现沿QM表面DNA轨道(底物序列)的自动化行走；(3)采用荧光分光光度计测量行走结束后多色QM的荧光信号,实现对miRNA的检测分析。本发明中基于多色QM-SNA的DNA机器用于循环miRNA的检测方法,采用DNA酶步行器介导QM荧光信号放大,实现miRNA高灵敏检测；采用多色QM-SNA实现miRNA的多元检测,为循环miRNA精确检测提供了新方法。(The invention discloses a method for detecting circulating miRNA (micro ribonucleic acid) by using a DNA machine based on quantum dot micelle spherical nucleic acid. The specific implementation method comprises three parts: (1) preparing multicolor quantum dot micelle spherical nucleic acid (QM-SNA) loaded with a DNase sequence and a quencher-modified substrate sequence; (2) under the conditions of target triggering and metal ion assistance, the DNase can realize automatic walking along a QM surface DNA track (substrate sequence); (3) and (3) measuring the fluorescent signal of the multicolor QM after walking by using a fluorescence spectrophotometer to realize the detection and analysis of miRNA. The invention relates to a method for detecting circulating miRNA by using a DNA machine based on multicolor QM-SNA, which adopts DNA enzyme walker to mediate QM fluorescence signal amplification to realize high-sensitivity miRNA detection; the multicolor QM-SNA is adopted to realize the multiple detection of miRNA, and a new method is provided for the accurate detection of circulating miRNA.)

1. A method for circulating miRNA detection by using a DNA machine based on quantum dot micelle spherical nucleic acid is characterized by comprising the following steps:

(1) preparing multicolor quantum dot micelle spherical nucleic acid loaded with a DNA enzyme sequence and a quencher modified substrate sequence;

(2) under the conditions of target triggering and metal ion assistance, the DNA enzyme automatically walks along the substrate sequence on the surface of the quantum dispensing bundle;

(3) and (3) measuring the fluorescence signal of the quantum dot micelle after the walking in the step (2) by using a fluorescence spectrophotometer to realize the detection and analysis of miRNA.

2. The method for circulating miRNA detection by quantum dot micelle spherical nucleic acid based DNA machinery according to claim 1, wherein: the kit comprises quantum dot micelles with three colors, three targets, three DNases, corresponding substrates thereof and metal ions; the three-color quantum dot micelles are respectively green, yellow and red; the three target molecules are three circulating miRNAs in serum; the three DNAzymes comprise DNAzyme1, DNAzyme 2 and DNAzyme 3, and amino groups are modified at the 5' ends of the three DNAzymes; the three substrates comprise Substrate 1, Substrate2 and Substrate 3, wherein the 5 'end of the three substrates is modified with a quencher, and the 3' end of the three substrates is modified with an amino; the metal ions are zinc ions or magnesium ions.

3. The method for circulating miRNA detection by quantum dot micelle spherical nucleic acid based DNA machinery according to claim 2, wherein: the three circulating miRNAs are non-small cell lung cancer markers miRNA-196a, miRNA-25 and miRNA-21.

4. The method for circulating miRNA detection by quantum dot micelle spherical nucleic acid based DNA machinery according to claim 2, wherein: the multicolor quantum dot micelle spherical nucleic acid loaded with the DNase sequence and the quencher modified substrate sequence in the step (1) is formed by respectively connecting three DNases and substrates thereof to the surfaces of quantum dot micelle beams with three colors by a chemical crosslinking method.

5. The method for circulating miRNA detection by quantum dot micelle spherical nucleic acid based DNA machinery according to claim 1, wherein: triggering the target miRNA in the step (2) and automatically walking the DNase along the DNA track under the metal ion auxiliary condition; hybridizing the target with DNA enzyme on the surface of the quantum dot dispensing bundle to release the upper arm and the lower arm of the DNA enzyme to identify adjacent substrates, activating rA sites on the DNA enzyme activity specificity shearing substrates by combining metal ions, and releasing the DNA enzyme and a quencher after shearing; the released DNase automatically recognizes and cleaves the next substrate sequence, and multi-step recognition-cleavage-release walking along the DNA track is realized.

6. The method for circulating miRNA detection by quantum dot micelle spherical nucleic acid based DNA machinery according to claim 1, wherein: detecting the fluorescent signal of the three-color quantum dot micelle after the DNase in the step (2) is finished walking by using a fluorescence spectrophotometer in the step (3); three-color quantum dispensing bundle fluorescence emission wavelength marks three target miRNAs, and quantum dispensing bundle fluorescence intensity marks the number of the target miRNAs.

Technical Field

The invention relates to a method for detecting circulating miRNA (micro ribonucleic acid) by using a DNA machine based on quantum dot micelle spherical nucleic acid, belonging to the technical field of biomedicine.

Background

microRNA (miRNA) is endogenous non-coding RNA with the length of about 22 bases, can regulate and control the expression level of a transcribed gene by inhibiting the expression of targeted mRNA, can regulate and control important physiological processes such as cell generation, cell differentiation, immune response and the like, and can also be used as abnormal expression of protooncogenes or cancer suppressor genes in different cancer cells to regulate and control corresponding pathological processes. Multiple studies show that the abnormal expression of circulating miRNA stably existing in blood and body fluid is closely related to the occurrence and development of various cancers, and the circulating miRNA is an important tumor molecular marker at present. The circulating miRNA has the characteristics of short sequence, high similarity and low abundance, one miRNA can act on multiple mRNAs, and multiple miRNAs can act on one mRNA molecule at the same time. Therefore, how to develop a circulating miRNA detection method with high sensitivity, high specificity and multiplex detection capability becomes a great challenge at this stage.

Traditional miRNA detection methods include PCR methods, microarray methods, and sequencing methods. The PCR method has higher sensitivity and accuracy in miRNA detection, but the detection process comprises primer design, temperature regulation and the like which are relatively complex. Microarrays and sequencing methods have the advantage of high throughput detection, but are generally incompatible with amplification processes, and thus have yet to be improved in terms of sensitivity and specificity, and are relatively costly. The fluorescence sensor has the advantages of simple preparation, high detection sensitivity and the like, and is a research hotspot of miRNA detection at present. As a novel fluorophore, the quantum dot has excellent optical properties such as good photobleaching resistance, high brightness, single-wavelength excitation and multicolor emission, and can be used for conveniently preparing a fluorescence sensor through surface functionalization. Most of the existing quantum dot fluorescence sensors are endowed with good water solubility by a ligand exchange method, but the preparation time is long, and the fluorescence quantum yield is greatly reduced. More importantly, the water-soluble quantum dots prepared by the ligand exchange method have small particle size and are inconvenient for further integration of functional nucleic acid. Compared with the prior art, the quantum dot micelle (QM) method, namely the method for preparing the micelle by directly coating phospholipid on the surface of the oil-soluble quantum dot through hydrophobic effect, has the advantage of simple preparation, and keeps the high fluorescence quantum yield of the inner core oil-soluble quantum dot. Meanwhile, the quantum dot micelle effectively increases the surface area, and is convenient for further functionalization such as integration of functional nucleic acid and the like, so that the application of the quantum dot micelle in the field of biosensing is more convenient.

The low abundance of miRNA requires the detection of miRNA to be assisted by signal amplification technology. Various isothermal amplification techniques such as chain hybridization reaction (HCR), Rolling Circle Amplification (RCA), chain displacement reaction (SDA), catalytic hairpin self-assembly (CHA), and DNA nanomachines (e.g., dnase walker), etc. have been developed to achieve efficient signal amplification. The DNase walker utilizes the catalytic activity of metal ion DNase to shear a substrate and realize automatic walking along a specific orbit without the participation of protease. Compared with protease, the metal ion DNA enzyme has the obvious advantages of low synthesis cost and stable structure. The metal ion DNA enzyme walking has the advantages of mild condition, automation, high efficiency and the like, and provides a new method for detecting miRNA.

Disclosure of Invention

The technical problem is as follows: aiming at the problems, the invention provides a method for detecting circulating miRNA by using a DNA machine based on quantum dot micelle spherical nucleic acid (QM-SNA), which can realize high sensitivity and multi-element detection of circulating miRNA.

The technical scheme is as follows: in order to achieve the purpose, the invention adopts the following technical scheme, and the specific steps comprise:

(1) preparing multicolor quantum dot micelle spherical nucleic acid (QM-SNA) loaded with substrates modified by DNase and quencher;

(2) the target miRNA triggers the automatic walking of the metal ion DNase along the QM (quantum dot micelle) surface DNA track (substrate sequence);

(3) and (3) measuring the fluorescence signal of QM after the walking in the step (2) by using a fluorescence spectrophotometer, and performing multivariate detection analysis on the circulating miRNA.

The QM-SNA loaded with DNase and substrates thereof in the step (1) comprises three quantum dot micelles (QMs), three targets, three DNase sequences and substrate sequences thereof and metal ions. The three QMs are respectively prepared from green, yellow and red quantum dots; the three target molecules are three circulating miRNAs in serum, such as non-small cell lung cancer markers miRNA-196a, miRNA-25 and miRNA-21; the three DNAzymes and corresponding substrates are stem-loop structures, the sequences of the three DNAzymes are DNAzyme1/Substrate 1, DNAzyme 2/Substrate 2 and DNAzyme 3/Substrate 3, the 5 ' end of the DNAzyme is modified with amino, and the 5 ' end and the 3 ' end of the Substrate are modified with quencher (BHQ) and amino respectively. Wherein DNAzyme1/Substrate 1 is used for detecting miRNA1 and is coupled to a green QM surface; DNAzyme 2/Substrate 2 for detection of miRNA 2, coupled to a yellow QM surface; DNAzyme 3/Substrate 3 was used to detect miRNA3, coupled to a red QM surface. The fluorescence of all three QMs is in a quenched state due to coupling of quencher-modified substrates. The metal ions used are zinc ions or magnesium ions.

Triggering the target miRNA in the step (2) and enabling the DNase to walk along the QM surface DNA track under the metal ion auxiliary condition. In the absence of target, the upper and lower arms of the DNase are closed. Adding miRNA, hybridizing a target and DNase to open a closed area of the DNase, releasing upper and lower arms, hybridizing the upper and lower arms and an adjacent substrate, combining metal ions, activating a catalytic center of the DNase, shearing an rA site on the substrate, and releasing a substrate fragment modified by a 5' end quencher (BHQ). Because the lower arm of the DNase has weak binding force with the residual substrate fragments, the upper arm and the lower arm of the DNase are released again and walk to the next substrate to be hybridized and cut with the substrate. After the DNA enzyme executes a walking process of 'recognition-shearing-releasing' for a plurality of times, all substrate sequences on the surface of QM can be sheared finally. The QM fluorescence signal was maximally recovered due to the large amount of BHQ leaving the QM surface.

And (3) measuring the fluorescent signal of the multicolor QM after the DNase in the step (2) is finished walking by using a fluorescence spectrophotometer to realize the multi-element detection and analysis of miRNA. Three-color QM fluorescence emission wavelengths mark three target miRNAs, and the QM fluorescence intensity marks the number of the target miRNAs. A linear relation exists between the QM fluorescence intensity and the target abundance, and a standard curve is drawn. Target quantification can be achieved by measuring the fluorescence spectrum of the polychromatic QM in the actual sample.

Has the advantages that: compared with the prior art, the multicolor quantum dot micelle spherical nucleic acid (QM-SNA) fluorescence sensor loaded with the DNase walker is prepared, and the high sensitivity and the multivariate detection of circulating miRNA are realized by triggering the automatic walking-mediated QM fluorescence enhancement of DNase through the target.

Detection (walking) time: 40 minutes; detection limit: 10 femtomoles (fM); at least three target miRNAs can be detected simultaneously.

Drawings

FIG. 1 is a schematic diagram of the QM-SNA-based DNase walking process;

FIG. 2 is a characterization of the properties of QM and QM-SNA;

FIG. 3 is the time kinetics of QM surface DNase walking;

FIG. 4 is a graph of the sensitivity and standard curve for DNase walker detection of miRNAs;

FIG. 5 shows the specificity of DNase walker for the detection of miRNAs;

FIG. 6 is a multiplex capacity assessment of DNase walker for the detection of miRNAs;

FIG. 7 is a multiplex assay of circulating miRNAs in serum with a DNase walker.

Detailed Description

The invention is further illustrated by the following examples in conjunction with the accompanying drawings