A gene expression control technology for cell-free systems and synthetic cells via targeted gene silencing and transfection,Biotechnology and Bioengineering

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A gene expression control technology for cell-free systems and synthetic cells via targeted gene silencing and transfection
Biotechnology and Bioengineering ( IF 3.5 ) Pub Date : 2023-05-09 , DOI: 10.1002/bit.28422
Wakana Sato ₁ , Melanie Rasmussen ₁ , Nathaniel Gaut ₁ , Mahima Devarajan ₁ , Kaitlin Stokes ₁ , Christopher Deich ₁ , Aaron E Engelhart ₁ , Katarzyna P Adamala ₁

Affiliation

Synthetic cells, expressing proteins using cell-free transcription-translation (TXTL), is a technology utilized for a variety of applications, such as investigating natural gene pathways, metabolic engineering, drug development or bioinformatics. For all these purposes, the ability to precisely control gene expression is essential. Various strategies to control gene expression in TXTL have been developed; however, further advancements on gene-specific and straightforward regulation methods are still needed. Here, we present a method of control of gene expression in TXTL using a “silencing oligo”: a short oligonucleotide, designed with a particular secondary structure, that binds to the target messenger RNA. We demonstrated that silencing oligo inhibits protein expression in TXTL in a sequence-dependent manner. We showed that silencing oligo activity is associated with RNase H activity in bacterial TXTL. To complete the gene expression control toolbox for synthetic cells, we also engineered a first transfection system. We demonstrated the transfection of various payloads, enabling the introduction of RNA and DNA of different lengths to synthetic cell liposomes. Finally, we combined the silencing oligo and the transfection technologies, demonstrating control of gene expression by transfecting silencing oligo into synthetic minimal cells.

中文翻译：

通过靶向基因沉默和转染的无细胞系统和合成细胞的基因表达控制技术

合成细胞使用无细胞转录-翻译 (TXTL) 表达蛋白质，是一种用于多种应用的技术，例如研究自然基因通路、代谢工程、药物开发或生物信息学。对于所有这些目的，精确控制基因表达的能力是必不可少的。已经开发了多种控制 TXTL 中基因表达的策略；然而，仍然需要进一步改进基因特异性和直接调控方法。在这里，我们提出了一种使用“沉默寡核苷酸”控制 TXTL 中基因表达的方法：一种短寡核苷酸，设计有特定的二级结构，可与目标信使 RNA 结合。我们证明沉默寡核苷酸以序列依赖的方式抑制 TXTL 中的蛋白质表达。我们发现沉默寡核苷酸活性与细菌 TXTL 中的 RNase H 活性相关。为了完成合成细胞的基因表达控制工具箱，我们还设计了第一个转染系统。我们演示了各种有效载荷的转染，从而能够将不同长度的 RNA 和 DNA 引入合成细胞脂质体。最后，我们结合了沉默寡核苷酸和转染技术，通过将沉默寡核苷酸转染到合成的最小细胞中来证明对基因表达的控制。

更新日期：2023-05-09

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