Design of an Artificial Peptide Inspired by Transmembrane Mitochondrial Protein for Escorting Exogenous DNA into the Mitochondria to Restore their Functions by Simultaneous Multiple Gene Expression

新規ミトコンドリア膜貫通ペプチドによる遺伝子送達 --ミトコンドリア内部で効率的な多重遺伝子発現を達成--. 京都大学プレスリリース. 2023-11-02.Mitochondria are vital organelles regulating essential cellular functions. Human mitochondrial DNA (mtDNA) consists of 37 genes, 13 of which encode mitochondrial proteins, and the remaining 24 genes encode two ribosomal RNAs and 22 transfer RNAs needed for the translation of the mtDNA-encoded 13 proteins. However, mtDNA often impairs the expression and function of these genes due to various mutations, ultimately causing mitochondrial dysfunction. To recover from this desperate condition, developing the technology to supply all mitochondrial proteins encoded by mtDNA at once is an urgent task, but there is no established strategy for this purpose. In this study, a simple yet effective mitochondrial gene delivery system is proposed comprising an artificial peptide inspired by a transmembrane mitochondrial membrane protein. The designed mitochondria-targeting peptides presented on the carrier surface effectively guide the encapsulated plasmid to the mitochondria, facilitating mitochondrial uptake and gene expression. The developed system successfully delivers exogenous mtDNA to mtDNA-depleted cells and leads to simultaneous multigene expression, ultimately restoring mitochondrial functions, including the mitochondrial respiration rate. The established multiple gene expression system in each mitochondrion is a game-changing technology that can accelerate the development of mitochondrial engineering technologies as well as clinical applications for mitochondrial diseases

In vitro amplification of whole large plasmids via transposon-mediated oriC insertion

DNA amplification is a fundamental technique in molecular biology. The replication cycle reaction is a new method for amplification of large circular DNA having oriC sequences, which is a replication initiation site of the Escherichia coli chromosome. We here developed a replication cycle reaction-based method useful for amplification of various circular DNAs lacking oriC, even in the absence of any sequence information, via transposon-mediated oriC insertion to the circular DNA template. A 15-kb non-oriC plasmid was amplified from a very small amount of starting DNA (50 fg, 1 fM). The method was also applicable to GC-rich plasmid (69%) or large F-plasmid (230 kb). This method thus provides a powerful tool to amplify various environmental circular DNAs

Design of an Artificial Peptide Inspired by Transmembrane Mitochondrial Protein for Escorting Exogenous DNA into the Mitochondria to Restore their Functions by Simultaneous Multiple Gene Expression

Mitochondria are vital organelles regulating essential cellular functions. Human mitochondrial DNA (mtDNA) consists of 37 genes, 13 of which encode mitochondrial proteins, and the remaining 24 genes encode two ribosomal RNAs and 22 transfer RNAs needed for the translation of the mtDNA-encoded 13 proteins. However, mtDNA often impairs the expression and function of these genes due to various mutations, ultimately causing mitochondrial dysfunction. To recover from this desperate condition, developing the technology to supply all mitochondrial proteins encoded by mtDNA at once is an urgent task, but there is no established strategy for this purpose. In this study, a simple yet effective mitochondrial gene delivery system is proposed comprising an artificial peptide inspired by a transmembrane mitochondrial membrane protein. The designed mitochondria-targeting peptides presented on the carrier surface effectively guide the encapsulated plasmid to the mitochondria, facilitating mitochondrial uptake and gene expression. The developed system successfully delivers exogenous mtDNA to mtDNA-depleted cells and leads to simultaneous multigene expression, ultimately restoring mitochondrial functions, including the mitochondrial respiration rate. The established multiple gene expression system in each mitochondrion is a game-changing technology that can accelerate the development of mitochondrial engineering technologies as well as clinical applications for mitochondrial diseases.新規ミトコンドリア膜貫通ペプチドによる遺伝子送達 --ミトコンドリア内部で効率的な多重遺伝子発現を達成--. 京都大学プレスリリース. 2023-11-02