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

Autor: Sato W; Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, Minnesota, USA., Rasmussen M; Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, Minnesota, USA., Gaut N; Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, Minnesota, USA., Devarajan M; Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, Minnesota, USA., Stokes K; Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, Minnesota, USA., Deich C; Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, Minnesota, USA., Engelhart AE; Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, Minnesota, USA., Adamala KP; Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, Minnesota, USA.
Jazyk: angličtina
Zdroj: Biotechnology and bioengineering [Biotechnol Bioeng] 2023 Jul; Vol. 120 (7), pp. 1986-1997. Date of Electronic Publication: 2023 May 09.
DOI: 10.1002/bit.28422
Abstrakt: 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.
(© 2023 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals LLC.)
Databáze: MEDLINE
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