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中国生物工程杂志

China Biotechnology
China Biotechnology
China Biotechnology  2022, Vol. 42 Issue (9): 67-82    DOI: 10.13523/j.cb.2203060
    
Construction of Riboregulator in the Application of Point-of-care Testing
LIANG Shu-rui,LI Jiao-jiao,QI Hao**()
Synthetic Biology Research Platform, Tianjin Collaborative Innovation Center of Chemical Science and Engineering,Key Laboratory of Systems Bioengineering of Ministry of Education, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China
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Abstract  

Ongoing efforts in synthetic biology are focused on the design of reusable and modular fragments that demonstrate well-characterized behaviors and functionalities in biological systems. In the cell-free protein expression system, riboregulators are developed as an important sensing element in point-of-care testing, which can change their structure through the induction of target molecules, thereby regulating the expression of downstream genes. Different types of riboregulators and their action mechanisms are systematically introduced, including traditional riboregulators, toehold switch, toehold repressor, three-way junction repressor, small transcription activating RNA, and riboswitch. The main processes of constructing riboregulators are described in detail: designing with computers, testing gene expression, and analyzing the functional structure. Finally, the applications of in vitro point-of-care testing (POCT) based on riboregulators are summarized, mainly including pathogen nucleic acid detection based on toehold switch and small molecule detection mediated by riboswitch. The characteristics, challenges, and development tendencies of POCT in the cell-free system are discussed and summarized. In conclusion, the construction and application of riboregulators have opened up a new direction in the biosensing field. This article is expected to provide some helpful insight into the development of new riboregulators and POCT tools.

Key words： Riboregulator      Toehold switch      Riboswitch      Cell-free protein synthesis      Point-of-care testing (POCT)     
Received: 26 March 2022      Published: 10 October 2022
ZTFLH:  Q78  
Corresponding Authors: Hao QI     E-mail: haoq@tju.edu.cn
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Shu-rui LIANG
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Cite this article:

LIANG Shu-rui,LI Jiao-jiao,QI Hao. Construction of Riboregulator in the Application of Point-of-care Testing. China Biotechnology, 2022, 42(9): 67-82.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2203060     OR     https://manu60.magtech.com.cn/biotech/Y2022/V42/I9/67

Fig.1 Riboregulator located in 5'-UTR
Fig.2 Action mechanism of traditional riboregulator
Fig.3 Riboregulators based on the different action mechanisms (a) Toehold switch (b) Toehold repressor (c) 3WJ repressor (d) STAR
Fig.4 Regulation mechanism of riboswitch (a) Activation (b) Repression (c) Ribozyme cleavage
分类 名称 调节类型 动态范围
(激活/抑制倍数)		 设计来源 参考文献
一代核糖核酸调节子 RR10、RR12 翻译激活 8~19 从头设计 [9]
RAJ11、RAJ12 翻译激活 11.2~26.7 细菌sRNA、人工结构 [32]
RR42、RR12y 翻译激活 70~200 RR10、RR12 [33]
二代核糖核酸调节子 Toehold switch 翻译激活 >400 从头设计 [10]
功能拓展的调节子 STARs 转录激活 94~9 000 转录衰减 [19,21]
3WJ repressor 翻译抑制 43 从头设计 [16]
Toehold repressor 翻译抑制 122 从头设计 [16]
Table 1 Characteristics of riboregulators
Fig.5 Design, test and analysis cycle of engineered riboregulators
Fig.6 Cell-free sensors based on paper
Fig.7 POCT based on toehold switch
Fig.8 SNIPR design principles
Fig.9 Point-of-care testing based on riboswitch
无细胞系统 开关类型 检测对象 报告蛋白 检测限 检测时间 参考文献
PURE Toehold switch Zika virus LacZ 2.8 fmol/L 3 h [60]
PURE Toehold switch Dengue virus LacZ 5.23 nmol/L 3 h [61]
PURE Toehold switch Norovirus LacZα/LacZω 270 zmol/L 3~6 h [62]
E. coli Toehold switch RSV LacZ 91 amol/L 2.5 h [63]
N. Tabassum leaf STAR Plant pathogen C23DO 4.4 pmol/L 2.5 h [64]
E. coli Toehold switch Shiga toxin gene LacZ 5 nmol/L 1 h [65]
PURE Riboswitch Theophylline LacZ 1 mmol/L 35 min [73]
PURE Toehold switch SARS-CoV-2 GFP 1 800 copies 2 h [75]
E. coli Toehold switch SARS-CoV-2 NanoLuc 10 nmol/L 0.5 h [76]
PURE Toehold switch Gut bacteria GFP 30 amol/L 3~5 h [78]
PURE SNIPR SNV LacZ 10 fmol/L 1 h [79]
E. coli Riboswitch Fluoride C23DO 2 ppm 1 h [81]
Table 2 Cell-free biosensors with riboregulators
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