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CN113980959B - Y-shaped multifunctional DNA nano assembly, preparation method and application thereof - Google Patents

Y-shaped multifunctional DNA nano assembly, preparation method and application thereof Download PDF

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CN113980959B
CN113980959B CN202111255453.5A CN202111255453A CN113980959B CN 113980959 B CN113980959 B CN 113980959B CN 202111255453 A CN202111255453 A CN 202111255453A CN 113980959 B CN113980959 B CN 113980959B
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陈珊
李婧影
梁虹
张晨
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Minjiang University
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Abstract

本发明公开了一种“Y型”多功能DNA纳米组装体、制备方法及其应用，该多功能DNA纳米组装体由四条DNA杂交形成“Y型”结构，包括链ab，链cb*，链d，以及链e；且链d与链ab的部分序列互补，链e与链cb*的部分序列互补，链ab和链cb*上均修饰有细胞膜锚定基团，链d上带有邻硝基苄基光切割基团。制备方法包括步骤：S1.合成ab序列；S2.合成cb*序列；S3.合成d序列；S4.合成序e序列；S5.将四条DNA序列混合加热退火杂交。该多功能DNA纳米组装体能快速地锚定在细胞膜表面，实现对细胞膜表面的c‑Met受体功能的高效光控调控，实现对c‑Met受体抑制效果的实时监测。

The invention discloses a "Y-shaped" multifunctional DNA nano-assembly, a preparation method and an application. The multi-functional DNA nano-assembly is formed by the hybridization of four DNAs to form a "Y-shaped" structure, including chain ab, chain cb*, chain d, and chain e; and chain d is complementary to part of the sequence of chain ab, chain e is complementary to part of the sequence of chain cb*, chain ab and chain cb* are both modified with cell membrane anchoring groups, and chain d has adjacent Nitrobenzyl photocleavable group. The preparation method includes steps: S1. Synthesize ab sequence; S2. Synthesize cb* sequence; S3. Synthesize d sequence; S4. Synthesize e sequence; S5. Mix four DNA sequences, heat, anneal and hybridize. The multifunctional DNA nanoassembly can be quickly anchored on the cell membrane surface to achieve efficient light-controlled regulation of the c-Met receptor function on the cell membrane surface and real-time monitoring of the c-Met receptor inhibitory effect.

Description

一种“Y型”多功能DNA纳米组装体、制备方法及其应用A "Y-shaped" multifunctional DNA nanoassembly, preparation method and application

技术领域Technical field

本发明属于分子生物学技术领域，具体涉及一种细胞膜锚定的“Y型”多功能DNA纳米组装体、制备方法及其应用。The invention belongs to the technical field of molecular biology, and specifically relates to a cell membrane-anchored "Y-shaped" multifunctional DNA nanoassembly, a preparation method and its application.

背景技术Background technique

细胞的多种蛋白共同构筑成复杂的信号网络参与各种细胞活动，在维持细胞正常生理活动以及疾病的发生发展过程中都起到重要的作用。这个信号网络中的蛋白都不是孤立地存在，它们的功能与表达水平受到精确的调控，其中某种蛋白功能变化所引发的信号级联反应影响着其他蛋白的功能和表达，实现对细胞行为的精准调控。在细胞间质上皮转化因子(c-Met)介导的细胞信号转导过程中，c-Met受体能够感知并整合外界环境的刺激信号，其配体肝细胞生长因子(HGF)与c-Met受体结合诱导受体二聚通常被认为是c-Met信号转导过程的第一步。激活的c-Met受体将信号传递到细胞内，通过选择性地激活或抑制特定信号分子，从而促进细胞的增殖、迁移、侵袭和血管新生等多种细胞行为。例如，c-Met信号通路激活后能够提高血管内皮生长因子(VEGF)的分泌水平，从而促进血管新生过程。由于c-Met信号通路在生理功能和疾病进展中的重要作用，通过按需调节c-Met受体功能实现细胞功能调控已经引起了广泛的关注。考虑到相关信号分子之间的密切联系，特定信号分子的变化可以作为评价上游调控效果的反馈。因此，在保持蛋白的结构和状态不受影响前提下，对信号网络中的蛋白进行精准按需调控和实时监测有助于系统地阐明蛋白的生物功能和作用机制，并促进智能治疗药物的开发，对精准医疗的发展具有重要意义。Multiple proteins in cells work together to form a complex signaling network that participates in various cellular activities and plays an important role in maintaining normal physiological activities of cells and in the occurrence and development of diseases. The proteins in this signaling network do not exist in isolation. Their functions and expression levels are precisely regulated. The signaling cascade triggered by changes in the function of a certain protein affects the function and expression of other proteins, thereby controlling cell behavior. Precise control. In the process of cell signal transduction mediated by cell-mesenchymal epithelial transition factor (c-Met), the c-Met receptor can sense and integrate stimulating signals from the external environment. Its ligand hepatocyte growth factor (HGF) and c- Met receptor binding-induced receptor dimerization is generally considered to be the first step in the c-Met signaling process. Activated c-Met receptors transmit signals into cells and promote various cell behaviors such as cell proliferation, migration, invasion, and angiogenesis by selectively activating or inhibiting specific signaling molecules. For example, activation of the c-Met signaling pathway can increase the secretion level of vascular endothelial growth factor (VEGF), thereby promoting the angiogenesis process. Due to the important role of the c-Met signaling pathway in physiological functions and disease progression, the regulation of cell functions by regulating c-Met receptor function on demand has attracted widespread attention. Considering the close connection between related signaling molecules, changes in specific signaling molecules can serve as feedback to evaluate the effect of upstream regulation. Therefore, without affecting the structure and status of the protein, precise on-demand regulation and real-time monitoring of proteins in the signaling network can help to systematically elucidate the biological functions and mechanisms of action of proteins, and promote the development of intelligent therapeutic drugs. , is of great significance to the development of precision medicine.

目前，免疫印迹和酶联免疫吸附测定等传统策略是广泛应用于验证信号分子变化(表达水平或翻译后修饰等)的检测手段。然而，这些方法在分析之前需要繁琐的实验操作，且无法对活细胞进行实时监测。尽管基于基因编辑策略的荧光生物传感器能够用于细胞信号分子的监测，但向细胞导入新的基因需要复杂耗时的步骤，且仍面临因过表达重组成分而干扰细胞内源信号通路的风险，不利于实际应用。此外，由于缺乏能够实现多任务处理的策略，现有的研究方法无法同时实现蛋白功能调控和实时监控调控结果。Currently, traditional strategies such as Western blotting and enzyme-linked immunosorbent assay are widely used as detection methods to verify changes in signaling molecules (expression levels or post-translational modifications, etc.). However, these methods require tedious experimental operations before analysis and cannot provide real-time monitoring of living cells. Although fluorescent biosensors based on gene editing strategies can be used to monitor cell signaling molecules, introducing new genes into cells requires complex and time-consuming steps, and there is still a risk of interfering with the endogenous signaling pathways of cells due to overexpression of recombinant components. Not conducive to practical application. In addition, due to the lack of strategies that can achieve multi-tasking, existing research methods cannot simultaneously regulate protein function and monitor the regulation results in real time.

功能核酸具有突出的可编程性和多样性，在构建多功能纳米组装体方面有突出潜力。其中，核酸适配体作为一种能够结合特定目标分子的功能核酸，已经被设计用于精准探测活细胞中的信号分子。一些核酸适配体被报道可通过阻碍蛋白-蛋白相互作用来干扰细胞信号通路的激活过程。然而，迄今为止，基于功能核酸的纳米组装体在活细胞蛋白功能调控与监测的多功能应用仍然存在挑战。第一方面，由于核酸适配体本身的尺寸较小，与靶标蛋白结合后容易发生解离，受环境影响较大。第二方面，活细胞的细胞内吞作用也使得单个游离核酸适配体的调控和检测效率仍然不理想，尤其是针对细胞表面的受体和分泌信号蛋白。第三方面，目前所构建的探针通常只包含单一的功能模块，只能执行调控或监测的单一操作，无法满足对细胞信号网络中的多个蛋白实现多功能响应的需求，无法对调控结果进行实时监测。实现为了解决上述技术问题，现在亟需研发出一种稳定、高效的多功能DNA纳米组装体来实现蛋白功能的精准调控及信号转导关键分子的实时监测。Functional nucleic acids have outstanding programmability and diversity and have outstanding potential in constructing multifunctional nanoassemblies. Among them, nucleic acid aptamers, as functional nucleic acids that can bind specific target molecules, have been designed to accurately detect signaling molecules in living cells. Some nucleic acid aptamers have been reported to interfere with the activation process of cell signaling pathways by hindering protein-protein interactions. However, so far, there are still challenges in the multifunctional application of functional nucleic acid-based nanoassemblies in the regulation and monitoring of protein function in living cells. First, due to the small size of the nucleic acid aptamer itself, it is easy to dissociate after binding to the target protein, and is greatly affected by the environment. Secondly, the endocytosis of living cells also makes the regulation and detection efficiency of a single free nucleic acid aptamer still unsatisfactory, especially for receptors and secreted signaling proteins on the cell surface. Thirdly, the currently constructed probes usually only contain a single functional module and can only perform a single operation of regulation or monitoring. They cannot meet the needs of multi-functional responses to multiple proteins in the cell signaling network and cannot predict the results of regulation. Perform real-time monitoring. In order to solve the above technical problems, it is urgent to develop a stable and efficient multifunctional DNA nanoassembly to achieve precise regulation of protein function and real-time monitoring of key molecules in signal transduction.

发明内容：Contents of the invention:

本发明目的是提供了一种“Y型”多功能DNA纳米组装体、制备方法及其应用，要解决的技术问题包括但不限于以下任一技术问题：第一方面，如何构建具备多任务处理能力的DNA纳米组装体，第二方面，如何实现可控、高效调控c-Met受体蛋白功能以及信号转导过程；第三方面，如何实现实时监测对c-Met受体蛋白功能的调控效果。为实现以上目的，本发明采用如下技术方案：The purpose of the present invention is to provide a "Y-shaped" multifunctional DNA nanoassembly, a preparation method and its application. The technical problems to be solved include but are not limited to any of the following technical problems: First, how to construct a multi-tasking device The second aspect is how to achieve controllable and efficient regulation of c-Met receptor protein function and signal transduction process; the third aspect is how to achieve real-time monitoring of the regulatory effect on c-Met receptor protein function. . In order to achieve the above objectives, the present invention adopts the following technical solutions:

一种细胞膜锚定的“Y型”多功能DNA纳米组装体，由四条DNA杂交形成“Y型”结构，具体包括序列如SEQ ID NO.1所示的链ab，序列如SEQ ID NO.2所示的链cb*，序列如SEQ IDNO.3所示的链d，以及序列如SEQ ID NO.4所示的链e；且所述链d与所述链ab的部分序列互补，所述链e与所述链cb*的部分序列互补，所述链ab和所述链cb*上均修饰有所述细胞膜锚定基团，所述链cb*上带有Cy3荧光基团，所述链d上带有邻硝基苄基光切割基团(Photocleavable linker，PC-linker)，所述链e上带有BHQ-2荧光猝灭基团。本方案中，所述杂交形成“Y型”结构的四条DNA组成锚定模块、调控模块和监测模块三个功能模块，所述锚定模块为包含有细胞膜锚定基团修饰的双链DNA，所述调控模块包含有识别c-Met受体蛋白的核酸适配体和光切割基团修饰的阻挡探针，所述监测模块包括一个识别VEGF蛋白的核酸适配体和阻挡探针。A cell membrane-anchored "Y-shaped" multifunctional DNA nanoassembly, which is composed of four DNA hybrids to form a "Y-shaped" structure, specifically including chain ab with a sequence as shown in SEQ ID NO.1, and a sequence as shown in SEQ ID NO.2 The chain cb* shown, the chain d with the sequence shown in SEQ ID NO.3, and the chain e with the sequence shown in SEQ ID NO.4; and the chain d is complementary to the partial sequence of the chain ab, the The chain e is complementary to a partial sequence of the chain cb*, the chain ab and the chain cb* are both modified with the cell membrane anchoring group, the chain cb* has a Cy3 fluorescent group, and the The chain d has a photocleavable linker (PC-linker), and the chain e has a BHQ-2 fluorescence quenching group. In this solution, the four DNAs hybridized to form a "Y-shaped" structure form three functional modules: an anchoring module, a regulatory module, and a monitoring module. The anchoring module is a double-stranded DNA modified with a cell membrane anchoring group. The control module includes a nucleic acid aptamer that recognizes c-Met receptor protein and a blocking probe modified with a photocleavage group, and the monitoring module includes a nucleic acid aptamer that recognizes VEGF protein and a blocking probe.

在上述方案的基础上，在另一改进的方案中，所述链ab包含有靶向识别c-Met受体蛋白的核酸适配体序列，所述链cb*包含有靶向识别VEGF蛋白的核酸适配体序列。On the basis of the above scheme, in another improved scheme, the chain ab contains a nucleic acid aptamer sequence that targets the c-Met receptor protein, and the chain cb* contains a nucleic acid aptamer sequence that targets the VEGF protein. Nucleic acid aptamer sequence.

在上述方案的基础上，在另一改进的方案中，所述链ab上带有Cy5荧光基团，所述链d上带有BHQ-2荧光猝灭基团Based on the above scheme, in another improved scheme, the chain ab has a Cy5 fluorescent group, and the chain d has a BHQ-2 fluorescence quenching group.

在上述方案的基础上，在另一改进的方案中，所述细胞膜锚定基团为疏水性分子中的一种，所述疏水性分子包括胆固醇分子、生育酚分子和二酰基脂质体。Based on the above solution, in another improved solution, the cell membrane anchoring group is one of hydrophobic molecules, and the hydrophobic molecules include cholesterol molecules, tocopherol molecules and diacyl liposomes.

在上述方案的基础上，在另一改进的方案中，所述四条DNA为人工合成，或任何其他来源的如SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3和SEQ ID NO.4所示序列的核酸序列。Based on the above scheme, in another improved scheme, the four DNAs are artificially synthesized or from any other source such as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO. .Nucleic acid sequence of the sequence shown in .4.

本发明还提供一种上述的细胞膜锚定的“Y型”多功能DNA纳米组装体的制备方法，包括以下步骤：The invention also provides a method for preparing the above-mentioned cell membrane-anchored "Y-shaped" multifunctional DNA nanoassembly, which includes the following steps:

S1.合成序列如SEQ ID NO.1所示的所述ab序列；S1. The synthetic sequence is the ab sequence shown in SEQ ID NO.1;

S2.合成序列如SEQ ID NO.2所示的所述cb*序列；S2. The synthetic sequence is the cb* sequence shown in SEQ ID NO.2;

S3.合成序列如SEQ ID NO.3所示的所述d序列；S3. The synthetic sequence is the d sequence shown in SEQ ID NO.3;

S4.合成序列如SEQ ID NO.4所示的所述e序列；S4. The synthetic sequence is the e sequence shown in SEQ ID NO.4;

S5.将步骤S1至S4中的四条DNA序列按照摩尔浓度1:1:1:1进行混合，混合后于95℃加热5分钟进行退火相互杂交，缓慢冷却至室温。S5. Mix the four DNA sequences in steps S1 to S4 at a molar concentration of 1:1:1:1. After mixing, heat at 95°C for 5 minutes for annealing and hybridization, and slowly cool to room temperature.

在上述方案的基础上，在另一改进的方案中，分别将步骤S1至S4中的四条DNA序列配备成浓度为10μM的溶液后再按照比例混合。Based on the above scheme, in another improved scheme, the four DNA sequences in steps S1 to S4 are respectively prepared into solutions with a concentration of 10 μM and then mixed according to the proportion.

本发明还提供一种上述的细胞膜锚定的“Y型”多功能DNA纳米组装体在核酸适配体抑制剂药物中的应用。The present invention also provides an application of the above-mentioned cell membrane-anchored "Y-shaped" multifunctional DNA nanoassembly in nucleic acid aptamer inhibitor drugs.

本发明还提供一种上述的细胞膜锚定的“Y型”多功能DNA纳米组装体在实时监测细胞膜表面受体功能调控效果相关研究中的应用。The present invention also provides an application of the above-mentioned cell membrane-anchored "Y-shaped" multifunctional DNA nanoassembly in research related to real-time monitoring of the functional regulation effect of cell membrane surface receptors.

本发明还提供一种上述的细胞膜锚定的“Y型”多功能DNA纳米组装体在蛋白功能调控和细胞信号传导相关研究中的应用。The present invention also provides an application of the above-mentioned cell membrane-anchored "Y-shaped" multifunctional DNA nanoassembly in research related to protein function regulation and cell signaling.

本发明的技术方案至少具有以下的有益效果：本发明提出一个“Y型”多功能DNA纳米组装体来调控和实时监测活细胞的信号转导过程。本发明以间充质上皮转化(c-Met)信号通路作为模型进行说明。第一方面，多功能DNA纳米组装体能够快速地锚定在细胞膜表面，利用锚定功能提高了核酸适配体与c-Met受体的结合稳定性，进而提高了核酸适配体对c-Met受体的调控效果。进一步的，利用光控可远程操纵和无损快速的调控优势，多功能DNA纳米组装体能够实现时空分辨的c-Met受体蛋白功能调控。进一步的，多功能DNA纳米组装体能够实时监测由c-Met受体蛋白功能改变引发的关键信号分子VEGF的分泌变化，实现对c-Met受体调控效果的实时监测。因此，这种模块化设计的多功能DNA纳米组装体能同时实现高效、可控的蛋白功能调节和信号转导过程中关键分子的实时监测，从单个信号分子的独立研究转为信号转导中多个信号分子的关联研究，有助于深入研究活细胞蛋白在复杂信号网络中的生理功能和作用机制，可在肿瘤的早期诊断、治疗以及疗效评价等研究中发挥重要作用。The technical solution of the present invention has at least the following beneficial effects: The present invention proposes a "Y-shaped" multifunctional DNA nanoassembly to regulate and real-time monitor the signal transduction process of living cells. The present invention is explained using the mesenchymal-to-epithelial transition (c-Met) signaling pathway as a model. First, multifunctional DNA nanoassemblies can be quickly anchored on the cell membrane surface, using the anchoring function to improve the binding stability of nucleic acid aptamers and c-Met receptors, thereby improving the binding stability of nucleic acid aptamers to c-Met receptors. Met receptor regulatory effects. Furthermore, taking advantage of the advantages of light control, remote control and non-destructive rapid regulation, multifunctional DNA nanoassemblies can achieve spatial and temporal resolution of c-Met receptor protein function regulation. Furthermore, the multifunctional DNA nanoassembly can real-time monitor the secretion changes of the key signaling molecule VEGF caused by the functional changes of the c-Met receptor protein, achieving real-time monitoring of the regulatory effect of the c-Met receptor. Therefore, this modularly designed multifunctional DNA nanoassembly can simultaneously achieve efficient and controllable protein function regulation and real-time monitoring of key molecules in the signal transduction process, transforming from independent research on a single signaling molecule to multiple signals in signal transduction. Association studies on signaling molecules can help to further study the physiological functions and mechanisms of living cell proteins in complex signaling networks, and can play an important role in early diagnosis, treatment and efficacy evaluation of tumors.

附图说明Description of the drawings

图1为本发明实施例中的“Y型”多功能DNA纳米组装体的结构示意图；Figure 1 is a schematic structural diagram of a "Y-shaped" multifunctional DNA nanoassembly in an embodiment of the present invention;

图2为本发明实施例中的“Y型”多功能DNA纳米组装体用于蛋白功能调控及信号转导关键分子监测的原理示意图；Figure 2 is a schematic diagram of the principle of using the "Y-shaped" multifunctional DNA nanoassembly in an embodiment of the present invention to regulate protein functions and monitor key molecules in signal transduction;

图3为本发明实验例1中(a)12％非变性聚丙烯酰胺凝胶电泳和(b)共聚焦成像分析DNA纳米组装体2CH-ab:b*:d的光响应性能，标尺：20μm；Figure 3 shows (a) 12% non-denaturing polyacrylamide gel electrophoresis and (b) confocal imaging analysis of the light response performance of DNA nanoassembly 2CH-ab:b*:d in Experimental Example 1 of the present invention, scale bar: 20 μm ;

图4为本发明实验例2中免疫印迹分析2CH-b:b*对c-Met受体功能的影响。Figure 4 is an immunoblot analysis of the effect of 2CH-b:b* on c-Met receptor function in Experimental Example 2 of the present invention.

图5为本发明实验例3中(a)免疫印迹分析DNA纳米组装体2CH-ab:b*:d光控调控c-Met受体功能的效果，(b)免疫印迹分析DNA纳米组装体2CH-ab:b*:d或ab:b*:d与DU145细胞孵育24小时后光控调控c-Met受体功能的效果；Figure 5 shows (a) Western blot analysis of the effect of light-controlled c-Met receptor function of DNA nanoassembly 2CH-ab:b*:d in Experimental Example 3 of the present invention, (b) Western blot analysis of DNA nanoassembly 2CH -The effect of light control on c-Met receptor function after incubation of ab:b*:d or ab:b*:d with DU145 cells for 24 hours;

图6为本发明实验例3中(a)不同浓度的VEGF与200nM 2CH-cb*:b:e探针反应30分钟的荧光光谱图，(b)5μg/mL的不同蛋白200nM与2CH-cb*:b:e探针反应30分钟的荧光光谱图；Figure 6 shows the fluorescence spectra of (a) different concentrations of VEGF and 200nM 2CH-cb*:b:e probe reacted for 30 minutes in Experimental Example 3 of the present invention, (b) 200nM of different proteins at 5 μg/mL and 2CH-cb *:b:e Fluorescence spectrum of probe reacted for 30 minutes;

图7为本发明实验例4中(a)酶联免疫吸附剂测定DU145细胞由c-Met受体激活介导的促进VEGF分泌情况，(b)酶联免疫吸附剂测定和(c)共聚焦成像分析2CH-ab:b*对DU145细胞由c-Met受体激活介导的VEGF分泌影响，标尺：20μm；Figure 7 shows the promotion of VEGF secretion in DU145 cells mediated by c-Met receptor activation in Experimental Example 4 of the present invention (a) enzyme-linked immunosorbent assay, (b) enzyme-linked immunosorbent assay and (c) confocal Imaging analysis of the effect of 2CH-ab:b* on VEGF secretion mediated by c-Met receptor activation in DU145 cells, scale bar: 20 μm;

图8本发明实验例5中“Y型”多功能DNA纳米组装体2CH-ab:b*:d:e的凝胶电泳图。Figure 8 is a gel electrophoresis pattern of the "Y-shaped" multifunctional DNA nanoassembly 2CH-ab:b*:d:e in Experimental Example 5 of the present invention.

图9为本发明实验例6中“Y型”多功能DNA纳米组装体2CH-ab:b*:d:e实现DU145细胞上光控c-Met受体功能并实时监测VEGF分泌的共聚焦图像，标尺：20μm。Figure 9 is a confocal image of the "Y-shaped" multifunctional DNA nanoassembly 2CH-ab:b*:d:e in Experimental Example 6 of the present invention to realize the light-controlled c-Met receptor function on DU145 cells and monitor VEGF secretion in real time. ,Scale bar: 20μm.

具体实施方式Detailed ways

以下提供本发明的优选实施例，以助于进一步理解本发明。本领域技术人员应了解到，本发明实施例的说明仅是示例性的，并不是为了限制本发明的方案。Preferred embodiments of the present invention are provided below to help further understand the present invention. Those skilled in the art should understand that the description of the embodiments of the present invention is only exemplary and is not intended to limit the solution of the present invention.

参见附图1的示意，在本实施例中，我们利用功能核酸独特的分子识别机制和核酸结构精确的自组装特性来构建多功能模块的“Y型”DNA纳米组装体。本发明实施例中的“Y型”多功能DNA纳米组装体，由四条DNA杂交形成“Y型”结构，具体包括序列如SEQ ID NO.1所示的链ab，序列如SEQ ID NO.2所示的链cb*，序列如SEQ ID NO.3所示的链d，以及序列如SEQ ID NO.4所示的链e；且链d与链ab的部分序列互补，链e与链cb*的部分序列互补，链ab和链cb*上均修饰有细胞膜锚定基团，链cb*上带有Cy3荧光基团，链d上带有邻硝基苄基光切割基团(Photocleavable linker，PC-linker)，链e上带有BHQ-2荧光猝灭基团。其中链ab包含有靶向识别c-Met受体蛋白的核酸适配体序列，链cb*包含有靶向识别VEGF蛋白的核酸适配体序列；链ab上带有Cy5荧光基团，链d上带有BHQ-2荧光猝灭基团。四条DNA链上如何修饰上述各基团的技术，在本领域属于常规操作，不属于本申请的改进点，在说明书中不进行赘述。Referring to the diagram in Figure 1, in this example, we utilize the unique molecular recognition mechanism of functional nucleic acids and the precise self-assembly properties of nucleic acid structures to construct "Y-shaped" DNA nanoassemblies of multifunctional modules. The "Y-shaped" multifunctional DNA nanoassembly in the embodiment of the present invention is formed by the hybridization of four DNAs to form a "Y-shaped" structure, specifically including chain ab with a sequence as shown in SEQ ID NO.1, and a sequence as shown in SEQ ID NO.2 The chain cb* shown, the chain d whose sequence is shown in SEQ ID NO.3, and the chain e whose sequence is shown in SEQ ID NO.4; and chain d is complementary to the partial sequence of chain ab, and chain e is complementary to chain cb The partial sequences of * are complementary. Both chains ab and cb* are modified with cell membrane anchoring groups. Chain cb* has a Cy3 fluorescent group, and chain d has an o-nitrobenzyl photocleavable group (Photocleavable linker). , PC-linker), with a BHQ-2 fluorescence quenching group on chain e. Chain ab contains a nucleic acid aptamer sequence that targets c-Met receptor protein, chain cb* contains a nucleic acid aptamer sequence that targets VEGF protein; chain ab has a Cy5 fluorescent group, and chain d It contains BHQ-2 fluorescence quenching group. The technology of how to modify the above-mentioned groups on the four DNA strands is a routine operation in this field and does not belong to the improvement point of this application, and will not be described in detail in the specification.

在本实施例中细胞膜锚定基团为胆固醇分子；在另一改进的实施例中，细胞膜锚定基团为其它疏水性分子中的一种，比如生育酚分子或二酰基脂质体。In this embodiment, the cell membrane anchoring group is a cholesterol molecule; in another improved embodiment, the cell membrane anchoring group is one of other hydrophobic molecules, such as tocopherol molecules or diacyl liposomes.

本实施例中的“Y型”多功能DNA纳米组装体实现蛋白功能调控及信号转导关键分子监测的原理可参见附图2的示意。以HGF/c-Met信号转导相关的c-Met和VEGF作为实验对象来验证策略的可行性。当c-Met被HGF激活后引发的信号级联反应表现出促进VEGF分泌的作用，进而在血管新生过程中发挥重要作用。本实施例中的“Y型”多功能DNA纳米组装体2CH-ab:cb*:d:e共分为3个功能模块，由不同功能的DNA序列组装构成(参见附图1)。其中区域2CH-b:b*发挥锚定功能，将该多功能DNA纳米组装体锚定在细胞膜表面。区域a和d组成发挥光响应的调控受体蛋白活性功能。区域a为识别c-Met受体的核酸适配体，能够特异性结合并抑制c-Met受体的功能活性。探针d被设计为含有一个邻硝基苄基光切割基团(PC-linker)和一个BHQ-2猝灭基团的序列，它能与区域a的部分序列完全杂交，使区域a末端修饰的Cy5荧光被猝灭，在没有光照驱动时阻止区域a发挥调控功能。当多功能DNA纳米组装体被光照触发，探针d的断裂使区域a恢复为可结合c-Met受体的三维空间构型，进而抑制蛋白功能，Cy5荧光恢复。区域c和e用于实现实时监测功能。区域c为VEGF的核酸适配体序列，修饰BHQ-2猝灭基团的探针e与区域c的部分序列完全互补，猝灭区域c修饰的Cy3荧光。当有VEGF存在时，由于VEGF与核酸适配体的结合使区域c的构型发生改变，从而与探针e解离，恢复区域c末端的Cy3荧光信号。因此，多功能DNA纳米组装体能实现复杂功能操作，实现高效、可控的活细胞c-Met受体功能调控，并能同时监测由c-Met受体功能变化引起的关键信号分子VEGF分泌的变化。The principle of using the "Y-shaped" multifunctional DNA nanoassembly in this embodiment to regulate protein functions and monitor key molecules in signal transduction can be seen in the schematic diagram in Figure 2. c-Met and VEGF, which are related to HGF/c-Met signal transduction, were used as experimental subjects to verify the feasibility of the strategy. The signaling cascade reaction triggered when c-Met is activated by HGF shows the effect of promoting VEGF secretion, thereby playing an important role in the angiogenesis process. The "Y-shaped" multifunctional DNA nanoassembly 2CH-ab:cb*:d:e in this example is divided into three functional modules, which are assembled from DNA sequences with different functions (see Figure 1). Region 2CH-b:b* plays an anchoring function to anchor the multifunctional DNA nanoassembly on the cell membrane surface. Regions a and d are composed of regions that function to regulate the activity of the receptor protein in response to light. Region a is a nucleic acid aptamer that recognizes c-Met receptor and can specifically bind and inhibit the functional activity of c-Met receptor. Probe d is designed to contain a sequence of an ortho-nitrobenzyl photocleavage group (PC-linker) and a BHQ-2 quenching group, which can completely hybridize with the partial sequence of region a and modify the end of region a. The Cy5 fluorescence is quenched, preventing region a from exerting its regulatory function when there is no light driving. When the multifunctional DNA nanoassembly is triggered by light, the breakage of probe d restores region a to a three-dimensional spatial configuration that can bind to the c-Met receptor, thereby inhibiting the protein function and restoring Cy5 fluorescence. Areas c and e are used to implement real-time monitoring functions. Region c is the nucleic acid aptamer sequence of VEGF. The probe e modified with the BHQ-2 quenching group is completely complementary to the partial sequence of region c and quenches the fluorescence of Cy3 modified in region c. When VEGF is present, the binding of VEGF to the nucleic acid aptamer changes the configuration of region c, thereby dissociating from probe e and restoring the Cy3 fluorescence signal at the end of region c. Therefore, multifunctional DNA nanoassemblies can realize complex functional operations, achieve efficient and controllable functional regulation of c-Met receptors in living cells, and can simultaneously monitor changes in the secretion of the key signaling molecule VEGF caused by changes in c-Met receptor functions. .

在本实施例中，链ab包含有识别c-Met受体蛋白的核酸序列(属于调控模块的一部分)和带有细胞膜锚定基团修饰的锚定序列(属于锚定模块的一部分)，链cb*包含有识别VEGF蛋白的核酸序列(属于监测模块的一部分)和带有细胞膜锚定基团修饰的锚定序列(属于锚定模块的一部分)，所述链d为修饰有邻硝基苄基光切割基团的光控序列(属于调控模块的一部分)，所述链e为阻挡探针序列(属于监测模块的一部分)。In this embodiment, chain ab includes a nucleic acid sequence that recognizes c-Met receptor protein (part of the regulatory module) and an anchor sequence modified with a cell membrane anchoring group (part of the anchoring module). cb* contains a nucleic acid sequence that recognizes VEGF protein (part of the monitoring module) and an anchor sequence modified with a cell membrane anchoring group (part of the anchoring module). The chain d is modified with o-nitrobenzyl The light control sequence of the base photocleavage group (belongs to a part of the regulation module), and the chain e is a blocking probe sequence (belongs to a part of the monitoring module).

本实施例中的四条DNA为人工合成，在其它实施例中也可以是任何其他来源的如SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3和SEQ ID NO.4所示序列的核酸序列。The four DNAs in this embodiment are artificially synthesized. In other embodiments, they can also be from any other source, such as the sequences shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4. nucleic acid sequence.

本实施例中的细胞膜锚定的“Y型”多功能DNA纳米组装体的制备方法，包括以下步骤：The preparation method of cell membrane-anchored "Y-shaped" multifunctional DNA nanoassemblies in this example includes the following steps:

S1.合成序列如SEQ ID NO.1所示的所述ab序列；S1. The synthetic sequence is the ab sequence shown in SEQ ID NO.1;

S2.合成序列如SEQ ID NO.2所示的所述cb*序列；S2. The synthetic sequence is the cb* sequence shown in SEQ ID NO.2;

S3.合成序列如SEQ ID NO.3所示的所述d序列；S3. The synthetic sequence is the d sequence shown in SEQ ID NO.3;

S4.合成序列如SEQ ID NO.4所示的所述e序列；S4. The synthetic sequence is the e sequence shown in SEQ ID NO.4;

S5.将步骤S1至S4中的四条DNA序列按照摩尔浓度1:1:1:1进行混合，混合后于95℃加热5分钟进行退火相互杂交，缓慢冷却至室温。S5. Mix the four DNA sequences in steps S1 to S4 at a molar concentration of 1:1:1:1. After mixing, heat at 95°C for 5 minutes for annealing and hybridization, and slowly cool to room temperature.

在上述实施例的基础上，在另一改进的实施例中，分别将步骤S1至S4中的四条DNA序列配备成浓度为10μM的溶液后再按照比例混合。Based on the above embodiment, in another improved embodiment, the four DNA sequences in steps S1 to S4 are respectively prepared into solutions with a concentration of 10 μM and then mixed according to the proportion.

本发明还提供一种上述的细胞膜锚定的“Y型”多功能DNA纳米组装体在核酸适配体抑制剂药物中的应用。The present invention also provides an application of the above-mentioned cell membrane-anchored "Y-shaped" multifunctional DNA nanoassembly in nucleic acid aptamer inhibitor drugs.

本发明还提供一种上述的细胞膜锚定的“Y型”多功能DNA纳米组装体在实时监测细胞膜表面受体功能调控效果相关研究中的应用。The present invention also provides an application of the above-mentioned cell membrane-anchored "Y-shaped" multifunctional DNA nanoassembly in research related to real-time monitoring of the functional regulation effect of cell membrane surface receptors.

本发明还提供一种上述的细胞膜锚定的“Y型”多功能DNA纳米组装体在蛋白功能调控和细胞信号传导相关研究中的应用。The present invention also provides an application of the above-mentioned cell membrane-anchored "Y-shaped" multifunctional DNA nanoassembly in research related to protein function regulation and cell signaling.

实验例1至实验例6所用到的主要仪器为：The main instruments used in Experimental Examples 1 to 6 are:

SH-1000UV-Vis分光光度计(Corona Electric公司，日本)；A1共聚焦激光扫描显微镜(尼康公司，日本)；CHB 202恒温金属浴(杭州博日科技公司，中国)；Cary Eclipse荧光光谱仪(Agilent Technologies公司，美国)；Milli-Q Integarl纯水/超纯水一体化系统(Millipore公司，美国)；小垂直电泳槽(Bio-Rad公司，美国)；ChemiDocTM Touch凝胶成像系统(Bio-Rad公司，美国)；层析实验冷柜(北京德天佑科技公司，中国)。SH-1000UV-Vis spectrophotometer (Corona Electric Co., Japan); A1 confocal laser scanning microscope (Nikon Co., Japan); CHB 202 constant temperature metal bath (Hangzhou Bioer Technology Co., Ltd., China); Cary Eclipse fluorescence spectrometer (Agilent Technologies, USA); Milli-Q Integarl pure water/ultrapure water integrated system (Millipore, USA); Small vertical electrophoresis tank (Bio-Rad, USA); ChemiDocTM Touch gel imaging system (Bio-Rad, USA) , United States); chromatography experimental freezer (Beijing Detianyou Technology Co., Ltd., China).

实验例1至实验例6所用到的主要试剂为：The main reagents used in Experimental Examples 1 to 6 are:

实验例1至实验例6中所用的DNA序列(表1)均由中国上海生工生物工程技术有限公司合成并进行HPLC纯化。VEGF的ELISA检测试剂盒购于美国Abcam公司。重组人HGF购自美国PeproTech公司。重组人VEGF和重组人PDGF-BB购自中国近岸蛋白质科技有限公司。本实验所使用的抗体购自美国Cell Signaling Technology公司。MEM培养基、RPMI1640培养基、BSA、PBS和FBS等购自美国Gibco公司。The DNA sequences (Table 1) used in Experimental Examples 1 to 6 were synthesized by Shanghai Sangon Bioengineering Technology Co., Ltd. in China and subjected to HPLC purification. The ELISA detection kit for VEGF was purchased from Abcam Company in the United States. Recombinant human HGF was purchased from PeproTech Company of the United States. Recombinant human VEGF and recombinant human PDGF-BB were purchased from China Nearshore Protein Technology Co., Ltd. The antibodies used in this experiment were purchased from Cell Signaling Technology Company in the United States. MEM culture medium, RPMI1640 culture medium, BSA, PBS and FBS were purchased from Gibco Company of the United States.

表1本发明中所用到的寡核苷酸序列Table 1 Oligonucleotide sequences used in the present invention

上表1中，链CH-ab包含有c-Met的核酸适配体序列；链CH-cb*包含有VEGF的核酸适配体序列；“-CH-”代表胆固醇分子以及“//”代表光切割基团PC-linker。In Table 1 above, chain CH-ab contains the nucleic acid aptamer sequence of c-Met; chain CH-cb* contains the nucleic acid aptamer sequence of VEGF; "-CH-" represents cholesterol molecules and "//" represents Photo-cleavable group PC-linker.

实验例1Experimental example 1

本实验例1验证了由“锚定模块”和“调控模块”构成的DNA纳米组装体2CH-ab:b*:d在溶液中以及活细胞表面的光控响应性能。从附图3a的非变性聚丙烯酰胺凝胶电泳分析可以看到，当d与2CH-ab:b*组装成功后，出现了一条分子量更高的条带2CH-ab:b*:d(泳道2)。当用紫外手电筒光照5分钟后，由于PC-linker的断裂使区域a恢复本身的核酸适配体构型，同时出现断裂的d条带(泳道3)。我们在HeLa细胞表面考察了这一光响应过程，将Cy3荧光基团修饰的2CH-ab:b*与BHQ-2猝灭基团修饰的d组装形成2CH-ab:b*:d，将2CH-ab:b*:d与HeLa细胞进行孵育。如附图3b的共聚焦图像所示，只有在光照触发的条件下，HeLa细胞膜上出现一圈明显的Cy3荧光，而未光照组无明显荧光信号，指示了2CH-ab:b*:d的光响应性能。This experimental example 1 verified the light-controlled response performance of the DNA nanoassembly 2CH-ab:b*:d composed of the "anchoring module" and the "regulatory module" in solution and on the surface of living cells. It can be seen from the non-denaturing polyacrylamide gel electrophoresis analysis in Figure 3a that when d and 2CH-ab:b* are successfully assembled, a higher molecular weight band 2CH-ab:b*:d (lane 2). When illuminated with a UV flashlight for 5 minutes, the fragmentation of the PC-linker caused region a to restore its own aptamer configuration, and a fragmented band d appeared (lane 3). We investigated this photoresponse process on the surface of HeLa cells. 2CH-ab:b* modified with Cy3 fluorescent group and d modified with BHQ-2 quenching group were assembled to form 2CH-ab:b*:d. 2CH -ab:b*:d was incubated with HeLa cells. As shown in the confocal image in Figure 3b, only under light-triggered conditions, an obvious circle of Cy3 fluorescence appeared on the HeLa cell membrane, while there was no obvious fluorescence signal in the non-illuminated group, indicating the 2CH-ab:b*:d Photoresponsiveness.

具体操作过程如下：(a)将200nM Cy3-CH-ab、BHQ-2-d、CH-b*按照摩尔浓度比1:1:1进行自组装，构筑2CH-ab:b*:d用于凝胶电泳实验。实验分为光照组和未光照组，其中光照组的溶液在365nm的紫外灯(5mW/cm2)照射5分钟后，反应10分钟。将2CH-ab:b*作为阳性对照组。(b)将组装好的200nM2CH-ab:b*:d与HeLa细胞孵育15分钟，用PBS洗去未锚定的探针。实验分为光控组和未光控组，其中光控组的条件是光照细胞5分钟后，再孵育10分钟后进行共聚焦成像。The specific operation process is as follows: (a) 200nM Cy3-CH-ab, BHQ-2-d, CH-b* are self-assembled according to the molar concentration ratio of 1:1:1 to construct 2CH-ab:b*:d for Gel electrophoresis experiment. The experiment was divided into an illuminated group and a non-illuminated group. The solution in the illuminated group was irradiated with a 365nm ultraviolet lamp (5mW/cm 2 ) for 5 minutes and then reacted for 10 minutes. 2CH-ab:b* was used as a positive control group. (b) Incubate the assembled 200nM2CH-ab:b*:d with HeLa cells for 15 minutes, and wash away the unanchored probe with PBS. The experiment was divided into a light-controlled group and a non-light-controlled group. The conditions of the light-controlled group were to illuminate the cells for 5 minutes and then incubate them for 10 minutes before performing confocal imaging.

实验例2Experimental example 2

为了进一步考察DNA纳米组装体2CH-ab:b*:d的光响应调控c-Met蛋白功能的能力，本实验例2中利用经典的免疫印迹实验对不同实验条件下细胞p-Met蛋白的表达水平进行考察，p-Met的表达水平是验证c-Met是否被激活的最直接手段。如附图4所示，将单独的“锚定模块”2CH-b:b*与DU145细胞进行孵育，并不会影响由20ng/mL HGF与c-Met受体结合引起的蛋白磷酸化过程。如附图5a所示，将2CH-ab:b*:d与DU145细胞孵育后，在未光照时表现出与仅加入HGF的阳性对照组中类似的p-Met表达水平。同时，仅对细胞进行光照处理也不会影响HGF与c-Met蛋白的结合。当光照触发“调控模块”的调控性能后，几乎没有检测到p-Met的蛋白条带，说明该DNA纳米组装体能够高效地实现光控调节c-Met受体功能。如附图5b所示，本实验例2还验证了2CH-ab:b*:d在长时间孵育后的光响应调控性能。将2CH-ab:b*:d先与DU145细胞孵育24小时后，再进行光照调控。光照触发2CH-ab:b*:d时依然能表现出明显的抑制p-Met蛋白表达的效果，而没有光照时对由HGF刺激诱导的p-Met蛋白表达没有明显影响。但是，没有锚定功能的ab:b*:d在与细胞孵育24小时之后，无论是否光照都无法抑制由HGF介导的c-Met受体磷酸化。实验结果表明，2CH-ab:b*:d能够在长时间孵育后依然能响应光照而触发调控功能，实现对c-Met受体的光响应功能调控。In order to further investigate the ability of the light response of the DNA nanoassembly 2CH-ab:b*:d to regulate the function of c-Met protein, in this experimental example 2, classic immunoblotting experiments were used to measure the expression of cellular p-Met protein under different experimental conditions. The expression level of p-Met is the most direct way to verify whether c-Met is activated. As shown in Figure 4, incubating the "anchoring module" 2CH-b:b* alone with DU145 cells does not affect the protein phosphorylation process caused by the binding of 20ng/mL HGF to the c-Met receptor. As shown in Figure 5a, after incubation of 2CH-ab:b*:d with DU145 cells, the p-Met expression level in the absence of light was similar to that in the positive control group with only HGF added. At the same time, simply treating the cells with light will not affect the binding of HGF to c-Met protein. When light triggered the regulatory performance of the "regulatory module", almost no p-Met protein band was detected, indicating that the DNA nanoassembly can efficiently realize light-controlled regulation of the c-Met receptor function. As shown in Figure 5b, this experimental example 2 also verified the light response regulation performance of 2CH-ab:b*:d after long-term incubation. 2CH-ab:b*:d was first incubated with DU145 cells for 24 hours, and then controlled by light. When light triggers 2CH-ab:b*:d, it still shows a significant inhibitory effect on p-Met protein expression, while in the absence of light, it has no obvious effect on p-Met protein expression induced by HGF stimulation. However, ab:b*:d without anchoring function was unable to inhibit HGF-mediated c-Met receptor phosphorylation after incubation with cells for 24 hours, regardless of whether it was illuminated or not. Experimental results show that 2CH-ab:b*:d can still trigger the regulatory function in response to light after long-term incubation, achieving the regulation of the light response function of the c-Met receptor.

具体操作过程如下：将DU145细胞(每孔3×105)接种于6孔板中培养24小时后，将培养液更换为含有0.5％BSA的MEM培养基饥饿24小时，进行实验。The specific operation process is as follows: DU145 cells (3×10 5 per well) were inoculated into a 6-well plate and cultured for 24 hours, then the culture medium was replaced with MEM medium containing 0.5% BSA and starved for 24 hours before conducting the experiment.

(1)将不同浓度的2CH-b:b*与DU145细胞孵育15分钟，然后加入20ng/mL HGF与细胞孵育30分钟。用PBS洗涤细胞3次后，裂解细胞提取蛋白进行免疫印迹实验。(1) Incubate different concentrations of 2CH-b:b* with DU145 cells for 15 minutes, then add 20ng/mL HGF and incubate with the cells for 30 minutes. After washing the cells three times with PBS, the cells were lysed and proteins were extracted for Western blotting experiments.

(2)光控调控c-Met蛋白活性实验分为光照组和未光照组进行实验。将DU145细胞与30nM 2CH-ab:b*:d孵育15分钟，其中光照组的实验条件为光照细胞5分钟后再孵育10分钟。然后加入20ng/mL HGF与细胞孵育30分钟。用PBS洗涤细胞3次后，裂解细胞提取蛋白进行免疫印迹实验。(2) The experiment of light-controlled regulation of c-Met protein activity was divided into an illuminated group and a non-illuminated group for experiments. DU145 cells were incubated with 30 nM 2CH-ab:b*:d for 15 minutes. The experimental conditions of the illumination group were to illuminate the cells for 5 minutes and then incubate for 10 minutes. Then add 20ng/mL HGF and incubate with cells for 30 minutes. After washing the cells three times with PBS, the cells were lysed and proteins were extracted for Western blotting experiments.

(3)为了考察该组装体的锚定作用使“调控模块”能够在长时间孵育后仍响应光控调控c-Met蛋白活性的性能，将250nM 2CH-ab:b*:d或ab:b*:d加入DU145细胞后，将孔板置于在细胞培养箱内孵育24小时后再进行光照处理。然后加入20ng/mL HGF与细胞孵育30分钟后提取蛋白。(3) In order to examine the anchoring effect of the assembly so that the "regulatory module" can still respond to light-controlled regulation of c-Met protein activity after long-term incubation, 250nM 2CH-ab:b*:d or ab:b *:d After adding DU145 cells, place the well plate in a cell culture incubator and incubate it for 24 hours before lighting. Then add 20ng/mL HGF and incubate with the cells for 30 minutes before extracting the protein.

实验例3Experimental example 3

本实验例3考察了由“锚定模块”和“监测模块”构成的DNA纳米组装体2CH-cb*:b:e在溶液中检测VEGF的性能。如附图6a所示，2CH-cb*:b:e的荧光信号随着VEGF浓度的增强而增强，指示该DNA纳米组装体检测VEGF的可行性。此外，当存在其他蛋白，包括BSA，HGF和PDGF-BB，不能观察到明显的荧光信号响应，证明了2CH-cb*:b:e的选择性(附图6b)。This experimental example 3 examined the performance of the DNA nanoassembly 2CH-cb*:b:e composed of the "anchoring module" and the "monitoring module" in detecting VEGF in solution. As shown in Figure 6a, the fluorescence signal of 2CH-cb*:b:e increases with the increase of VEGF concentration, indicating the feasibility of this DNA nanoassembly to detect VEGF. In addition, when other proteins, including BSA, HGF and PDGF-BB, were present, no obvious fluorescence signal response was observed, demonstrating the selectivity of 2CH-cb*:b:e (Figure 6b).

具体操作过程如下：将200nM Cy3-CH-cb*、BHQ2-e、CH-b按照摩尔浓度比1:1:1进行自组装，构筑2CH-cb*:b:e用于荧光检测实验。加入不同浓度的重组人VEGF(0-6000ng/mL)反应30分钟。用荧光光谱仪记录各组的光谱数据。为了验证2CH-cb*:b:e的选择性，将5000ng/mL VEGF，BSA，HGF和PDGF-BB分别与200nM 2CH-cb*:b:e反应30分钟。用荧光光谱仪记录各组的光谱数据。The specific operation process is as follows: 200nM Cy3-CH-cb*, BHQ2-e, and CH-b are self-assembled according to the molar concentration ratio of 1:1:1 to construct 2CH-cb*:b:e for fluorescence detection experiments. Add different concentrations of recombinant human VEGF (0-6000ng/mL) and react for 30 minutes. Use a fluorescence spectrometer to record the spectral data of each group. To verify the selectivity of 2CH-cb*:b:e, 5000ng/mL VEGF, BSA, HGF and PDGF-BB were reacted with 200nM 2CH-cb*:b:e for 30 minutes respectively. Use a fluorescence spectrometer to record the spectral data of each group.

实验例4Experimental example 4

本实验例4考察了在活细胞上考察细胞膜锚定的“监测模块”2CH-cb*:b:e的VEGF响应性能。通过酶联免疫吸附测定考察了DU145细胞由c-Met受体蛋白激活介导的促进VEGF分泌情况。如附图7a所示，与空白对照组相比，HGF的刺激促进DU145细胞分泌VEGF。如附图7b所示，当用2CH-ab:b*抑制c-Met受体活性后再加入HGF刺激则表现出VEGF分泌减少的效果。附图7c的共聚焦图像也验证了这一现象。This experimental example 4 examines the VEGF response performance of the cell membrane-anchored "monitoring module" 2CH-cb*:b:e on living cells. Enzyme-linked immunosorbent assay was used to examine the promotion of VEGF secretion in DU145 cells mediated by c-Met receptor protein activation. As shown in Figure 7a, compared with the blank control group, stimulation by HGF promoted the secretion of VEGF in DU145 cells. As shown in Figure 7b, when 2CH-ab:b* is used to inhibit c-Met receptor activity and then HGF is added to stimulate, the effect of reducing VEGF secretion is shown. The confocal image in Figure 7c also verified this phenomenon.

具体操作过程为：将DU145细胞(每孔3×104)接种于48孔板中。为了验证HGF/c-Met通路促进VEGF分泌的能力，实验分为2组，第一组为空白对照组；第二组为由20ng/mLHGF刺激的实验组；将孔板置于37℃细胞培养箱内孵育2，4，8或12小时。取各组细胞的培养液上清，利用ELISA检测试剂盒测定VEGF的含量。为了验证2CH-ab:b*抑制VEGF分泌的能力，实验分为3组：第一组为空白对照组；第二组为仅由20ng/mL HGF刺激的阳性对照组；第三组细胞先与200nM 2CH-ab:b*孵育15分钟后再加入20ng/mL HGF刺激。将孔板置于37℃细胞培养箱内孵育4或8小时。取各组细胞的培养液上清，利用ELISA检测试剂盒测定VEGF的含量。为了验证2CH-cb*:b:e的检测VEGF性能，将DU145细胞与200nM 2CH-ab:b*反应15分钟后，加入20ng/mL HGF反应4个小时。以只加入20ng/mL HGF的DU145细胞作为阳性对照组。加入Cy3-2CH-cb*:b:e与DU145细胞孵育30分钟，进行共聚焦成像。The specific operation process is as follows: DU145 cells (3×10 4 per well) are seeded in a 48-well plate. In order to verify the ability of the HGF/c-Met pathway to promote VEGF secretion, the experiment was divided into two groups. The first group was the blank control group; the second group was the experimental group stimulated by 20ng/mL HGF; the well plates were placed in 37°C cell culture Incubate in the chamber for 2, 4, 8 or 12 hours. The culture medium supernatant of each group of cells was taken, and the VEGF content was measured using an ELISA detection kit. In order to verify the ability of 2CH-ab:b* to inhibit VEGF secretion, the experiment was divided into 3 groups: the first group was the blank control group; the second group was the positive control group stimulated only by 20ng/mL HGF; the third group of cells were first treated with After incubation with 200nM 2CH-ab:b* for 15 minutes, 20ng/mL HGF was added for stimulation. Place the well plate in a 37°C cell culture incubator and incubate for 4 or 8 hours. The culture medium supernatant of each group of cells was taken, and the VEGF content was measured using an ELISA detection kit. In order to verify the VEGF detection performance of 2CH-cb*:b:e, DU145 cells were reacted with 200nM 2CH-ab:b* for 15 minutes, and then 20ng/mL HGF was added for 4 hours. DU145 cells with only 20ng/mL HGF added were used as the positive control group. Add Cy3-2CH-cb*:b:e and incubate with DU145 cells for 30 minutes for confocal imaging.

实验例5Experimental example 5

本实验例5考察了“Y型”多功能DNA纳米组装体2CH-ab:cb*:d:e在缓冲溶液中的组装情况。如附图8所示，2CH-ab:cb*:d:e能够实现成功组装。This experimental example 5 examined the assembly of the "Y-shaped" multifunctional DNA nanoassembly 2CH-ab:cb*:d:e in a buffer solution. As shown in Figure 8, 2CH-ab:cb*:d:e can be successfully assembled.

具体操作步骤如下：将CH-ab，CH-cb*，d和e的DNA干粉用超纯水溶解后分别制备成100μM母液，用PBS将DNA母液稀释到10μM。将DNA溶液置于恒温金属浴，95℃加热5分钟进行退火，缓慢冷却至室温。将CH-ab和d、CH-cb*和e按照摩尔浓度比1:1进行自组装，分别构筑CH-ab:d和CH-cb*:e；将CH-ab:d和CH-cb*:e按照摩尔浓度比1:1进行自组装，构筑2CH-ab:cb*:d:e，终浓度均为1μM，用于凝胶电泳实验。The specific steps are as follows: Dissolve the dry DNA powders of CH-ab, CH-cb*, d and e in ultrapure water and prepare 100 μM mother solution respectively, and dilute the DNA mother solution to 10 μM with PBS. Place the DNA solution in a constant-temperature metal bath, heat at 95°C for 5 minutes for annealing, and slowly cool to room temperature. Self-assemble CH-ab and d, CH-cb* and e according to the molar concentration ratio of 1:1 to construct CH-ab:d and CH-cb*:e respectively; combine CH-ab:d and CH-cb* :e was self-assembled according to the molar concentration ratio of 1:1 to construct 2CH-ab:cb*:d:e. The final concentration was 1 μM for gel electrophoresis experiments.

实验例6Experimental example 6

本实验例6将“Y型”多功能DNA纳米组装体2CH-ab:cb*:d:e用于光控响应的c-Met蛋白功能调控以及由相关信号转导引发的VEGF分泌改变的监测。多功能DNA纳米组装体的区域a和区域c分别标记了Cy5和Cy3荧光，在初始阶段，Cy5和Cy3荧光分别被探针d和e上的BHQ-2猝灭。如附图9所示，将2CH-ab:cb*:d:e与DU145细胞进行孵育，由于光照触发“调控模块”，细胞膜上出现Cy5荧光信号。“调控模块”抑制了c-Met蛋白的功能，进而抑制了VEGF的分泌，细胞孵育2，3，4小时后没有观察到明显的Cy3荧光信号。而在没有光照的对照组实验中，没有明显的Cy5荧光信号。当细胞孵育2，3，4小时后，由于VEGF的分泌而产生了逐渐增强的Cy3荧光信号。以上结果证明，光照触发多功能模块的多功能DNA纳米组装体2CH-ab:cb*:d:e中区域a与c-Met蛋白的结合，可以有效抑制HGF与c-Met蛋白的结合，实现光控调控c-Met蛋白功能，同时，多功能DNA纳米组装体能实现对由HGF/c-Met通路信号转导变化引发的VEGF分泌改变的监测分析。In this experimental example 6, the "Y-shaped" multifunctional DNA nanoassembly 2CH-ab:cb*:d:e was used to regulate the function of c-Met protein in light-controlled response and to monitor the changes in VEGF secretion caused by related signal transduction. . Region a and region c of the multifunctional DNA nanoassembly are labeled with Cy5 and Cy3 fluorescence respectively. In the initial stage, the Cy5 and Cy3 fluorescence are quenched by BHQ-2 on probes d and e, respectively. As shown in Figure 9, 2CH-ab:cb*:d:e was incubated with DU145 cells. As the "regulatory module" was triggered by light, a Cy5 fluorescence signal appeared on the cell membrane. The "regulatory module" inhibited the function of c-Met protein, thereby inhibiting the secretion of VEGF. No obvious Cy3 fluorescence signal was observed after the cells were incubated for 2, 3, and 4 hours. In the control experiment without light, there was no obvious Cy5 fluorescence signal. When the cells were incubated for 2, 3, and 4 hours, a gradually increasing Cy3 fluorescence signal was produced due to the secretion of VEGF. The above results prove that the binding of region a in the multifunctional DNA nanoassembly 2CH-ab:cb*:d:e of the light-triggered multifunctional module to c-Met protein can effectively inhibit the binding of HGF to c-Met protein, achieving Light control regulates the function of c-Met protein. At the same time, the multifunctional DNA nanoassembly can monitor and analyze changes in VEGF secretion caused by changes in signal transduction of the HGF/c-Met pathway.

具体操作步骤如下：将200nM多功能DNA纳米组装体2CH-ab:cb*:d:e与DU145细胞孵育15分钟，将其锚定于DU145细胞膜表面。其中，光照组的细胞反应条件为先光照细胞5分钟后再孵育10分钟。加入20ng/mL HGF分别孵育2，3和4个小时后进行共聚焦成像。The specific steps are as follows: Incubate 200nM multifunctional DNA nanoassembly 2CH-ab:cb*:d:e with DU145 cells for 15 minutes, and anchor it on the surface of DU145 cell membrane. Among them, the cell reaction conditions in the illumination group were to illuminate the cells for 5 minutes and then incubate them for 10 minutes. Confocal imaging was performed after adding 20ng/mL HGF and incubating for 2, 3 and 4 hours respectively.

最后应当说明的是，以上实施例仅用于说明本申请的技术方案而非对其保护范围的限制，尽管参照上述实施例对本申请进行了详细的说明，所述领域的普通技术人员应当理解：本领域技术人员阅读本申请后依然可对申请的具体实施方式进行种种变更、修改或等同替换，但以上变更、修改或等同替换，均在本申请的待授权或待批准之权利要求保护范围之内。Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application and do not limit the scope of protection. Although the present application has been described in detail with reference to the above embodiments, those of ordinary skill in the field should understand that: Those skilled in the art can still make various changes, modifications or equivalent substitutions to the specific implementation modes of the application after reading this application, but the above changes, modifications or equivalent substitutions are all within the protection scope of the claims to be authorized or approved in this application. Inside.

序列表sequence list

<110> 闽江学院<110> Minjiang College

<120> 一种“Y型”多功能DNA纳米组装体、制备方法及其应用<120> A "Y-shaped" multifunctional DNA nanoassembly, preparation method and application

<160> 4<160> 4

<170> SIPOSequenceListing 1.0<170> SIPOSequenceListing 1.0

<210> 1<210> 1

<211> 69<211> 69

<212> DNA<212> DNA

<213> 人工序列<213> Artificial sequence

<400> 1<400> 1

ggaacgttat gactacaata tcaggctgga tggtagctcg gtcggggtgg gtgggttggc 60ggaacgttat gactacaata tcaggctgga tggtagctcg gtcggggtgg gtgggttggc 60

aagtctgat 69aagtctgat 69

<210> 2<210> 2

<211> 43<211> 43

<212> DNA<212> DNA

<213> 人工序列<213> Artificial sequence

<400> 2<400> 2

tgtgggggtg gacgggccgg gtagattgta gtcataacgt tcc 43tgtgggggtg gacgggccgg gtagattgta gtcataacgt tcc 43

<210> 3<210> 3

<211> 18<211> 18

<212> DNA<212> DNA

<213> 人工序列<213> Artificial sequence

<400> 3<400> 3

atcagacttgccaaccca 18atcagacttgccaaccca 18

<210> 4<210> 4

<211> 12<211> 12

<212> DNA<212> DNA

<213> 人工序列<213> Artificial sequence

<400> 4<400> 4

tctacccggccc 12tctacccggccc 12

Claims (6)

1. A cell membrane anchored 'Y-shaped' multifunctional DNA nano-assembly is characterized in that four DNA are hybridized to form a 'Y-shaped' structure, and the 'Y-shaped' structure specifically comprises a chain ab with a sequence shown as SEQ ID NO.1, a chain cb with a sequence shown as SEQ ID NO.2, a chain d with a sequence shown as SEQ ID NO.3 and a chain e with a sequence shown as SEQ ID NO. 4; and said strand d is complementary to a partial sequence of said strand ab, said strand e is complementary to a partial sequence of said strand cb, both said strand ab and said strand cb are modified with a cell membrane anchoring group, said strand cb carries a Cy3 fluorescent group, said strand d carries an orthonitrobenzyl photocleavage group, and said strand e carries a BHQ-2 fluorescence quenching group;
the strand ab comprises a nucleic acid aptamer sequence which targets and recognizes a c-Met receptor protein, and the strand cb comprises a nucleic acid aptamer sequence which targets and recognizes a VEGF protein;
the chain ab is provided with a Cy5 fluorescent group, and the chain d is provided with a BHQ-2 fluorescence quenching group.
2. The cell membrane anchored "Y-type" multifunctional DNA nanoessembly of claim 1, wherein the cell membrane anchoring group is one of hydrophobic molecules including cholesterol molecules, tocopherol molecules, and diacyl liposomes.
3. The membrane anchored "Y-type" multifunctional DNA nanoassembly of any of claims 1-2, wherein the four DNA strands are artificially synthesized or any other source of nucleic acid sequences as set forth in SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 and SEQ ID No. 4.
4. A method for preparing a cell membrane anchored "Y-type" multifunctional DNA nano-assembly according to any one of claims 1-2, comprising the steps of:
s1, synthesizing the chain ab with a sequence shown as SEQ ID NO. 1;
s2, synthesizing the chain cb with the sequence shown as SEQ ID NO. 2;
s3, synthesizing the chain d with the sequence shown as SEQ ID NO. 3;
s4, synthesizing the chain e with the sequence shown as SEQ ID NO. 4;
s5, mixing the four DNA sequences in the steps S1 to S4 according to the molar concentration of 1:1:1:1, heating at 95 ℃ for 5 minutes for annealing and hybridizing, and slowly cooling to room temperature.
5. The method for preparing a membrane-anchored "Y-type" multifunctional DNA nano-assembly according to claim 4, wherein the four DNA sequences in the steps S1 to S4 are prepared into a solution with a concentration of 10. Mu.M, respectively, and then mixed according to a ratio.
6. Use of a cell membrane anchored "Y-type" multifunctional DNA nano-assembly according to any of claims 1-2 for the preparation of a nucleic acid aptamer comprising a target recognition of c-Met receptor protein.
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