Purging Mutant mtDNA Using MitochondriallyâTargeted Gamma Peptide Nucleic Acids

项目摘要 许多疾病都是由正常和突变形式的线粒体DNA（mtDNA）的存在引起的。 这种异质性中有利于正常mtDNA的适度变化可以对患者产生显著的益处。我们 我建议使用cDNA-PNA寡聚体选择性地结合突变mtDNA并阻断其复制，导致 逐渐向正常的mtDNA转变。BMPNA是唯一能够结合到 双链DNA的任何序列，最近已被证实可有效靶向活体细胞核DNA 成年小鼠以及子宫内。我们将用穿膜肽功能化TNF-PNAs， 细胞摄取和定位。将通过标准固相方法合成α-PNA，然后表征， 生物物理（凝胶迁移率变化）和生物化学（引物延伸的抑制）实验。展示了 然后将在细胞培养中研究聚合酶活性的最高亲和力和最大有效阻断，其中 吸收，定位和表型对异质性和线粒体耗氧量的影响将是 测定

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.