合成生物学与荧光成像技术

doi:10.12211/2096-8280.2021-060

合成生物学 ›› 2022, Vol. 3 ›› Issue (2): 369-384.DOI: 10.12211/2096-8280.2021-060

合成生物学与荧光成像技术

武伟红¹, 李炜², 张先恩³, 崔宗强²

^1.中国科学技术大学生命科学与医学部，安徽合肥 230026
^2.中国科学院武汉病毒研究所，病毒学国家重点实验室，湖北武汉 430071
^3.中国科学院生物物理研究所，生物大分子国家重点实验室，北京 100101

收稿日期:2021-05-13 修回日期:2021-09-06 出版日期:2022-04-30 发布日期:2022-05-11
通讯作者: 崔宗强
作者简介:武伟红（1998—），女，硕士研究生。研究方向为病毒与合成生物学。 E-mail：wuweihong@mail.ustc.edu.cn
崔宗强（1977—），男，研究员，博士生导师。研究方向为病毒学研究新技术如单病毒示踪、纳米疫苗等。创建了多种荧光纳米标记及单病毒示踪新技术，实时示踪了单个艾滋病毒、单个流感病毒脱壳过程，并实现了单拷贝艾滋病毒基因原位成像、多蛋白复合体超高分辨成像等。 E-mail：czq@wh.iov.cn
基金资助:
国家自然科学基金(31925025)

Synthetic biology for fluorescent bioimaging

Weihong WU¹, Wei LI², Xian’en ZHANG³, Zongqiang CUI²

^1.Division of Life Sciences and Medicine，University of Science and Technology of China，Hefei 230026，Anhui，China
^2.State Key Laboratory of Virology，Wuhan Institute of Virology，Chinese Academy of Sciences，Wuhan 430071，Hubei，China
^3.National Key Laboratory of Biomacromolecules，Institute of Biophysics，Chinese Academy of Sciences，Beijing 100101，China

Received:2021-05-13 Revised:2021-09-06 Online:2022-04-30 Published:2022-05-11
Contact: Zongqiang CUI

摘要/Abstract

摘要：

合成生物学的迅速发展为分子荧光标记与生物成像技术提供了新的机遇。基于合成生物学原理，可以建立材料生物合成新方法，开发性能优异的荧光纳米材料和探针，发展新的荧光成像技术。合成生物学应用于生物荧光成像，多涉及荧光材料与探针的设计合成、对生物靶标分子进行定点改造和修饰、荧光探针和靶标分子的可控时空耦合等以实现生物分子的精准特异性标记。这些荧光纳米材料和生物分子标记技术可应用于细胞内分子的荧光标记、成像和动态示踪，可视化解析相关的关键分子事件，从而深入揭示细胞内分子运动机制和病原致病机理等。本文主要综述了近年来合成生物学技术在生物荧光成像方面的应用，包括利用合成生物学技术合成量子点等荧光纳米材料与探针、对蛋白质和核酸分子的精准标记及其用于病毒荧光成像和示踪。最后，也对该领域面临的问题如荧光杂合生物材料可控合成、分子原位多重标记等进行了探讨和展望。合成生物学与荧光成像技术的交叉融合，将推动荧光成像技术发展和进步，并拓展合成生物学的研究领域。

关键词: 合成生物学, 生物探针, 分子标记, 荧光成像, 病毒示踪

Abstract:

The rapid development of synthetic biology provides new opportunities for fluorescent bioimaging. Fluorescence imaging plays an important role in the visualization of biomolecules inside cells. Traditional visualization research methods have many disadvantages, such as the influence of excessive molecular weight of traditional fluorescent protein on the research object, and the low resolution of traditional fluorescence imaging observation system. The application of synthetic biology can develop new fluorescent nanomaterials, which have various advantages such as high stability, low toxicity and so on, and can be more widely used in cell internal visualization. Based on the principles of synthetic biology, we can establish new methods of material biosynthesis, develop fluorescent nanomaterials and probes with excellent performance, and develop new fluorescence imaging technologies. In the field of fluorescent biological imaging, synthetic biology mainly involves the design and synthesis of fluorescent materials, site-specific modification and labeling of biological target molecules, and controllable coupling of fluorescent probes and other macromolecules in different spatial relations. These novel fluorescent materials and molecular labeling techniques could be applied to molecular imaging and single particle tracking to explore intracellular molecular dynamic mechanisms. And new fluorescent nanomaterials developed in synthetic biology also can be used to tag different parts of viruses to trace the mechanism of their invasion so as to reveal pathogen infection and pathogenesis. This review summarizes advances of the synthetic biotechnology and the application in fluorescent imaging, including the synthesis of fluorescent nanomaterials and probes such as quantum dots, accurate labeling of proteins and nucleic acids, and the application in virus fluorescence imaging and tracing. We also discuss some existing problems and prospects in the field, such as controllable synthesis of fluorescent heterozygous biomaterials and multiple molecular labeling in situ. Synthetic biology has great development potential in the next decade. The multidisciplinary fusion of synthetic biology and fluorescence imaging technology will promote the development and progress of fluorescence imaging technology and expand the research field of biosynthesis.

Key words: synthetic biology, biological probes, molecular labeling, fluorescent imaging, virus tracing

中图分类号:

Q 81

武伟红, 李炜, 张先恩, 崔宗强. 合成生物学与荧光成像技术[J]. 合成生物学, 2022, 3(2): 369-384.

Weihong WU, Wei LI, Xian’en ZHANG, Zongqiang CUI. Synthetic biology for fluorescent bioimaging[J]. Synthetic Biology Journal, 2022, 3(2): 369-384.

图/表 12

图1 CdSe在酵母细胞中的可控合成［45］

Fig. 1 Controllable CdSe synthesis in yeast cells[45]

图2 在蚯蚓细胞中CdTe的生物合成［50］

Fig. 2 CdTe biosynthesis in earthworm cells[50]

图3 大肠杆菌细胞中合成CdSeZn、PrGd纳米晶体［66］

Fig. 3 CdSeZn and PrGd nanocrystals synthesized in E.coil cells [66]

表1 不同荧光材料性能参数［74-76］

Tab. 1 Properties of different fluorescent materials [74-76]

荧光材料	大小	荧光强度	荧光量子效率	荧光寿命
量子点	2～20 nm	+++++	0.1～0.85	20 min
荧光蛋白	4 nm	+	0.79	100 ms
荧光RNA	40 bp	+++	0.30～0.70	> 15 min

图4 Venus-TFFC系统与mIrisFP-TFFC系统成像［83-84］

Fig. 4 Schematic diagram for imaging of Venus-TFFC(a) and mIrisFP-TFFC systems(b)[83-84]

图5 核酸适配子Corn荧光基团成像单细胞中聚合酶活性示意图［87］（转入报告质粒的HEK293T细胞加入荧光团DFHO可产生黄色荧光，加入RNA聚合酶抑制剂放线菌素D后荧光消失）

Fig. 5 Schematic diagram for imaging polymerase activity in single cells through aptamer corn fluorophore[87](HEK293T cells transformed with the reporter plasmid could produce yellow fluorescence by adding fluorophore DFHO, but the fluorescence disappears after adding the RNA polymerase inhibitor actinomycin D.)

图6 细菌核糖开关原理及结构［89］

Fig. 6 Diagram for the working principle and structure diagram of bacterial riboswitch[89]

图7 荧光RNA复合体在HeLa细胞成像［76］（合成染料HBC的一系列类似物与Pepper结合可发出不同颜色荧光，HBC类似物与Pepper结合物在HeLa细胞中进行成像，用空载细胞做对比，在共聚焦显微镜和双光子显微镜下进行成像观察）

Fig. 7 Schematic diagram for imaging of the fluorescent RNA complex in HeLa cells[76]（A series of analogues of synthetic dye HBC combined with Pepper can emit fluorescence with different colors. HBC analogues were combined with Pepper in HeLa cells and are compared with mock cells. Imaging observation is carried out with confocal microscope and two-photon microscope.）

图8 HIV-1通过内吞作用进入细胞［77］

Fig. 8 Schematic diagram for HIV-1 entering cell through endocytosis[77]

图9 流感病毒脱壳过程实时动态示踪［75］［QD625在电镜下又黑又圆，而QD705为三角形。用QD625（红色）和QD705（绿色）分别标记vRNP，结果显示在脱壳过程中QD625和QD705分别释放。IAV释放的vRNPs经过三阶段的主动运输到达细胞核，在细胞核经历两种扩散模式］

Fig. 9 Real-time dynamic tracking of the influenza virus unpacking process[75][Under electron microscope, QD625 is black and round, while QD705 is triangular. vRNP is labeled with QD625 (red) and QD705 (green) respectively, and the results showed that QD625 and QD705 are released during the hulling process, respectively. IAV-released vRNPs released from IAV reach the nucleus through a three-stage active transport, where they undergo two diffusion modes.]

图10 量子点标记的TALEs用于基因组成像［95］

Fig. 10 TALEs labeled with quantum dots are used for gene composition imaging[95]

图11 SV40-QD VLPs用于靶向小鼠动脉粥样硬化斑块成像［100］（SV40 VLPs 含有动脉粥样硬化靶向单元，凝血酶抑制剂并可以封装QD800。FH-QDs可靶向集中在小鼠体内动脉粥样硬化形成区）

Fig. 11 SV40-QD VLPs for imaging atherosclerotic plaques in targeted mice[100](SV40 VLPs contain atherosclerotic targeting units, thrombin inhibitors, which can encapsulate as QD800. FH-QDS can target atherosclerotic areas in mice.)

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合成生物学与荧光成像技术

Synthetic biology for fluorescent bioimaging

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