Circular single-stranded DNA as a programmable vector for gene regulation in cell-free protein expression systems

• Published in: Nature communications Vol. 15; no. 1; pp. 4635 - 11
• Main Authors: Tian, Zhijin, Shao, Dandan, Tang, Linlin, Li, Zhen, Chen, Qian, Song, Yongxiu, Li, Tao, Simmel, Friedrich C., Song, Jie
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 31.05.2024; Nature Publishing Group; Nature Portfolio
• Subjects: 38/35; 38/44; 631/1647/338/552; 631/337/572; 631/61/185; 639/925/926; 82/80; 82/83; Biology; Cell-Free System; Deoxyribonucleic acid; DNA; DNA, Circular - genetics; DNA, Circular - metabolism; DNA, Single-Stranded - genetics; DNA, Single-Stranded - metabolism; DNA-Directed RNA Polymerases - genetics; DNA-Directed RNA Polymerases - metabolism; Double-stranded RNA; Expression vectors; Gene expression; Gene Expression Regulation; Gene regulation; Genetic Vectors - genetics; Genetic Vectors - metabolism; Humanities and Social Sciences; Logic circuits; multidisciplinary; Nanotechnology; Protein expression; Proteins; Ribonucleic acid; RNA; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; Science; Science (multidisciplinary); Single-stranded DNA; Synthetic biology; Synthetic Biology - methods; Yeasts
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-024-49021-6

Cell-free protein expression (CFE) systems have emerged as a critical platform for synthetic biology research. The vectors for protein expression in CFE systems mainly rely on double-stranded DNA and single-stranded RNA for transcription and translation processing. Here, we introduce a programmable vector - circular single-stranded DNA (CssDNA), which is shown to be processed by DNA and RNA polymerases for gene expression in a yeast-based CFE system. CssDNA is already widely employed in DNA nanotechnology due to its addressability and programmability. To apply above methods in the context of synthetic biology, CssDNA can not only be engineered for gene regulation via the different pathways of sense CssDNA and antisense CssDNA, but also be constructed into several gene regulatory logic gates in CFE systems. Our findings advance the understanding of how CssDNA can be utilized in gene expression and gene regulation, and thus enrich the synthetic biology toolbox. Protein expression vectors in cell-free systems mainly rely on double-stranded DNA and single-stranded RNA. Here, authors use circular single-stranded DNA as a programmable vector for gene regulation in a cell-free expression system by identifying its expression pathways.