阅读说明：本技术 一种熵驱动的dna纳米回路及其应用 (Entropy-driven DNA nano loop and application thereof ) 是由 赵永席 陈锋 白敏� 于 2020-03-30 设计创作，主要内容包括：本发明公开了一种熵驱动的DNA纳米回路及其应用,属于活细胞成像技术领域,利用熵驱动的DNA纳米回路,通过链置换反应和目标物循环反应实现对信号放大,最终实现活细胞内RNA和酶催化联合成像分析。(The invention discloses an entropy-driven DNA nano loop and application thereof, belonging to the technical field of living cell imaging.)

1. An entropy-driven DNA nanocircuit, comprising one of three circuits:

a monovalent DNA nanocircuit comprising a fluorescent output signal, and telomerase and miRNA21 as input molecules;

a bivalent DNA nanocircuit comprising one or two fluorescent output signals, and telomerase and miRNA21 as input molecules;

the trivalent DNA nano-loop comprises three fluorescence output signals, and telomerase, miRNA21 and miRNA31 which are used as input molecules.

2. An entropy driven DNA nanocircuit according to claim 1, characterized in that said monovalent DNA nanocircuit comprises an OR logic gate AND an AND logic gate:

for an OR logic gate, telomerase and miRNA21 are used as input molecules, and when one OR two target substances exist, the monovalent DNA nano loop outputs a fluorescence signal which is the fluorescence of a fluorescence molecule FAM;

for the AND logic gate, telomerase AND miRNA21 are used as input molecules, AND only when two targets exist simultaneously, the monovalent DNA nano-loop outputs a fluorescence signal, wherein the fluorescence signal is the fluorescence of a fluorescent molecule TAMRA.

3. An entropy driven DNA nanocircuit according to claim 1, characterized in that said bivalent DNA nanocircuit comprises an OR logic gate AND an AND logic gate:

for an OR logic gate, telomerase and miRNA21 are used as input molecules, the telomerase triggers the release of a fluorescent molecule FAM signal, and miRNA21 triggers the release of a fluorescent molecule TAMRA signal;

for the AND logic gate, with telomerase AND miRNA21 as input molecules, the bivalent DNA nano-loop outputs a fluorescence signal, which is the fluorescence of the fluorescent molecule TAMRA.

4. An entropy-driven DNA nanocircuit according to claim 1, wherein the trivalent DNA nanocircuit is capable of outputting three fluorescent signals, telomerase, miRNA21 and miRNA31 are used as input molecules, telomerase triggers the release of fluorescent molecule FAM signal, miRNA21 triggers the release of fluorescent molecule TAMRA signal, and miRNA31 triggers the release of fluorescent molecule Cy5 signal.

5. The application of the entropy-driven DNA nano-loop in living cell RNA and enzyme-linked catalysis combined analysis is characterized in that the entropy-driven DNA nano-loop is used for realizing signal amplification through strand displacement reaction and target object circulation reaction, and finally realizing living cell RNA and enzyme catalysis combined imaging analysis.

6. Use of an entropy driven DNA nanocircuit as claimed in any one of claims 1 to 4 in combination with a microfluidic system for identifying cell types in a mixed cultured cell sample.

7. Use of an entropy-driven DNA nanocircuit as claimed in any one of claims 1 to 4 in combination with a microfluidic system for analysing the performance of an anti-cancer drug.

Technical Field

The invention belongs to the technical field of living cell imaging, and particularly relates to an entropy-driven DNA nano loop and application thereof.

Background

Gene expression and enzyme catalysis are dynamically variable in different cell states and cell types, and changes in genes are closely related to human diseases. For example, there is a difference in expression of non-coding mirnas and telomerase activity in both cancerous and normal tissues. Thus, monitoring such dynamic changes can determine the state or type of cells for biological research and clinical applications. However, conventional multiplex RNA detection methods including reverse transcription PCR and sequencing are not suitable for enzyme activity analysis, and they cannot observe dynamic changes of RNA and enzyme catalysis in living cells, and spatiotemporal information is also overlooked. In addition, there are few reports on how to convert these multigene expression and enzyme-catalyzed characteristics into cell type classification and cancer assessment. Therefore, there is an urgent need to develop efficient methods for detecting multiple RNAs and enzyme catalysis in cells.

Emerging DNA nanotechnology plays an important role in cellular imaging analysis because of its programming and assembly. There are currently relevant studies to observe molecular imaging in living cells by constructing DNA nanostructures. For example, DNA tetrahedral nano-hybridators have been established by FRET techniques to accurately image tumor-associated mrnas in living cells; gold nanoparticles based on molecular beacon probes and biofunctionalization were also designed for intracellular telomerase analysis; in addition, various DNA nanodevices and signal amplification circuits also significantly improve detection sensitivity in living cells, they avoid multi-step diffusion and assembly procedures, and achieve rapid and uniform signal amplification in a molecularly crowded cellular environment.

However, due to the complexity of the design of these DNA nanostructures, it is difficult to use them for the analysis of differential gene expression and changes in enzyme activity in living cells.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention aims to provide an entropy-driven DNA nano-loop and an application thereof, wherein the multivalent DNA nano-loop has simple structural design and can realize the intracellular RNA and enzyme catalysis combined imaging analysis.

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

the invention discloses an entropy-driven DNA nano loop, which comprises one of the following three loops:

a monovalent DNA nanocircuit comprising a fluorescent output signal, and telomerase and miRNA21 as input molecules;

a bivalent DNA nanocircuit comprising one or two fluorescent output signals, and telomerase and miRNA21 as input molecules;

the trivalent DNA nano-loop comprises three fluorescence output signals, and telomerase, miRNA21 and miRNA31 which are used as input molecules.

Preferably, the monovalent DNA nanoloop comprises an OR logic gate AND an AND logic gate:

for an OR logic gate, telomerase and miRNA21 are used as input molecules, and when one OR two target substances exist, the monovalent DNA nano loop outputs a fluorescence signal which is the fluorescence of a fluorescence molecule FAM;

for the AND logic gate, telomerase AND miRNA21 are used as input molecules, AND only when two targets exist simultaneously, the monovalent DNA nano-loop outputs a fluorescence signal, wherein the fluorescence signal is the fluorescence of a fluorescent molecule TAMRA.

Preferably, the bivalent DNA nanocircuit comprises an OR logic gate AND an AND logic gate:

for an OR logic gate, telomerase and miRNA21 are used as input molecules, the telomerase triggers the release of a fluorescent molecule FAM signal, and miRNA21 triggers the release of a fluorescent molecule TAMRA signal;

for the AND logic gate, with telomerase AND miRNA21 as input molecules, the bivalent DNA nano-loop outputs a fluorescence signal, which is the fluorescence of the fluorescent molecule TAMRA.

Preferably, the trivalent DNA nano-loop can output three fluorescent signals, and with telomerase, miRNA21, and miRNA31 as input molecules, the telomerase triggers the release of a fluorescent molecule FAM signal, miRNA21 triggers the release of a fluorescent molecule TAMRA signal, and miRNA31 triggers the release of a fluorescent molecule Cy5 signal.

The invention also discloses application of the entropy-driven DNA nano loop in living cell RNA and enzyme catalysis combined analysis, wherein the entropy-driven DNA nano loop is utilized to amplify signals through strand displacement reaction and target object circulating reaction, and finally RNA and enzyme catalysis combined imaging analysis in living cells is realized.

Specifically, the following operations are included:

1) preparing multivalent DNA nano-Substrates (SPs);

2) carrying out lipofection on multivalent DNA nano Substrates (SPs), amplification probes (AMPs) and telomerase recognition primers (TRprimer) to enter living cells;

3) after 4 hours of transfection, cells were washed three times with 1X PBS;

4) observation was performed by a laser confocal fluorescence microscope.

The triggering process of the entropy-driven DNA nano-loop is as follows:

at 10. mu. LTEMg reaction buffer (10mM Tris-HCl, 2mM EDTA, 1mM Mg)²⁺pH 8.0), adding 0.4mM polyvalent DNA nano-substrate and 0.5mM amplification probe, and different concentrations of target probe, incubating at 37 deg.C for 60min, and monitoring the signal amplification process in real time by L ightCycler 96 instrument.

The entropy-driven multivalent DNA nanocircuit system mixed solution was introduced into living cells by seeding cells in an octawell plate at a density of 50000 cells per well, overnight growth in an incubator at 37 deg.C, transfection of 100nM multivalent DNA nanomaterials (SPs), 125nM amplification probes (AMPs) and 100nM telomerase recognition primers (TR primer) into living cells by L ipofectamine 3000, incubation at 37 deg.C for 4h, washing of the cells with 1 XPBS three times, and observation by confocal laser fluorescence microscopy.

The invention also discloses application of the entropy-driven DNA nano-circuit in combination with a microfluidic system to identification of cell types in mixed-culture cell samples.

The invention also discloses application of the entropy-driven DNA nano-circuit combined with a microfluidic system in analyzing the performance of anticancer drugs

Compared with the prior art, the invention has the following beneficial effects:

1) the entropy-driven multivalent DNA loop disclosed by the invention is simple in design and strong in operability, and the designed multivalent loop can realize simultaneous detection of multiple targets in a short time through simple strand displacement; the trivalent and bivalent loops are added in valence state based on the nucleic acid unit of the monovalent loop, and the N-valent loop can be easily designed by the invention under the condition of enough detection signals.

2) The invention realizes the obvious amplification of signals through simple strand displacement reaction and target object circulation reaction, and finally realizes the RNA and enzyme catalysis combined imaging analysis in living cells.

3) When the entropy-driven multivalent DNA nano-loop is implemented on a micro-microfluidic system, 100% of three mixed-culture mammary gland cells (MCF-10A, MCF-7 and MDA-MB-231) can be distinguished through the entropy-driven multivalent DNA loop.

4) When the entropy-driven multivalent DNA nano-loop is implemented on a micro microfluidic system, the performance analysis of the breast cancer drug can be realized through the entropy-driven multivalent DNA loop. Tamoxifen (TAM) is an Estrogen Receptor (ER) antagonist, and when co-cultured with triple negative MDA-MB-231, it was found by the method of the invention that ER-positive MCF-7 can resist TAM. This suggests that in a complex tumor microenvironment, triple negative and metastatic cells may interfere with the inhibition of ER positive cell growth by TAMs.

5) The entropy-driven multivalent DNA nanocircuit is used for carrying out combined analysis on RNA and enzyme catalysis in cells, so that not only can the cell type be accurately identified, but also a basis can be provided for other measurement methods for disease diagnosis and treatment.

Drawings

FIG. 1 is a schematic diagram of the preparation of a DNA nanocircuit;

FIG. 2 is a multivalent DNA nanocircuit observed under a cryoelectron microscope;

FIG. 3 is a gel electrophoresis analysis of different valency DNA nanostructure assembly;

FIG. 4 is a schematic representation of the multivalent DNA nanocircuit system for identifying metastatic potential of breast cancer;

FIG. 5 is a representative fluorescence microscope image of three different breast cell lines under different channels;

FIG. 6 is a fluorescence microscope image of a representative microchamber in a multi-microchamber chip.

Detailed Description

In order to make the technical solutions of the present invention better understood, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

It should be noted that the terms "first," "second," and the like in the description and claims of the present invention and in the drawings described above are used for distinguishing between similar elements and not necessarily for describing a particular sequential or chronological order. It is to be understood that the data so used is interchangeable under appropriate circumstances such that the embodiments of the invention described herein are capable of operation in sequences other than those illustrated or described herein. Furthermore, the terms "comprises," "comprising," and "having," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed, but may include other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.

The invention is described in further detail below with reference to the accompanying drawings: