Purging Mutant mtDNA Using Mitochondrially‐Targeted Gamma Peptide Nucleic Acids

项目总结 许多疾病都是由于线粒体DNA(MtDNA)的正常和突变形式的存在而引起的。 这种异质性的适度转变有利于正常的mtDNA，可能会给患者带来显著的好处。我们 建议使用pna寡聚体选择性地结合突变的mtdna并阻止其复制，从而导致 线粒体DNA逐渐向正常方向转变。核酸是唯一一种能够结合到 任何双链DNA序列，最近已被证实可有效靶向活体中的核DNA 成年小鼠和子宫内的小鼠。我们将用线粒体穿透肽对PNA进行功能化处理，以促进 细胞摄取和定位。核糖核酸将用标准固相合成方法合成，然后在 生物物理(凝胶迁移率变化)和生化(抑制引物延伸)实验。展出的PNA 然后将在细胞培养中研究对聚合酶活性的最高亲和力和最大有效阻断， 摄取、定位和表型对异质性和线粒体氧耗的影响将是 下定决心。

PROJECT SUMMARY Numerous diseases arise from the presence of both normal and mutant forms of mitochondrial DNA (mtDNA). Modest shifts in this heteroplasmy in favor of the normal mtDNA can have significant patient benefits. We propose to use PNA oligomers to bind selectively to mutant mtDNA and block its replication, resulting in a progressive shift in favor of normal mtDNA. PNA is the only synthetic oligonucleotide capable of binding to any sequence of double-stranded DNA and has recently been validated to effectively target nuclear DNA in live adult mice as well as in utero. We will functionalize PNAs with mitochondrial-penetrating peptides to promote cell uptake and localization. PNA will be synthesized by standard solid phase methods, then characterized in biophysical (gel mobility shift) and biochemical (inhibition of primer extension) experiments. PNA that exhibit highest affinity and greatest potent blockage of polymerase activity will then be studied in cell culture, where uptake, localization and phenotypic effects on heteroplasmy and mitochondrial oxygen consumption will be determined.