Hidaka T, Pandian GN, Taniguchi J, Nobeyama T, Hashiya K, Bando T, Sugiyama H. Creation of a Synthetic Ligand for Mitochondrial DNA Sequence Recognition and Promoter-Specific Transcription Suppression. J Am Chem Soc. 2017 Jun 28;139(25):8444-8447.
Synthetic ligands capable of recognizing the specific DNA sequences inside human mitochondria and modulating gene transcription are in increasing demand because of the surge in evidence linking mitochondrial genome and diseases. In the work described herein, we created a new type of mitochondria-specific synthetic ligand, termed MITO-PIPs, by conjugating a mitochondria-penetrating peptide with pyrrole-imidazole polyamides (PIPs). The designed MITO-PIPs showed specific localization inside mitochondria in HeLa cells and recognized the target DNA in a sequence-specific manner. Furthermore, MITO-PIPs that inhibit the binding of mitochondrial transcription factor A to the light-strand promoter (LSP) also triggered targeted transcriptional suppression. The tunability of PIPs' properties suggests the potential of the MITO-PIPs as potent modulators of not only mitochondrial gene transcription but also its DNA mutations.
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Although most mutations in mtDNA have a minimal effect on cellular function, recent evidence has revealed that some mutations can cause mitochondrial diseases. Furthermore, the mtDNA haplotype is related to the risk of other diseases such as diabetes. Whereas patients with Leber’s hereditary optic neuropathy have a T-to-C mutation in the ND6 gene, patients with Leigh syndrome have a G-to-A mutation. The mutated mtDNA is a prime target for disease therapy because a higher percentage of mutated mtDNA exceeding a threshold can cause cellular defects and mitochondrial diseases. MITO-PIPs could also read the sequence of a mutated region and distinguish mutated mtDNA from normal mtDNA. Therefore, MITO-PIPs can be developed further to generate a new functional compound that can exert bioactivity on either a normal or a mutated mtDNA in a defined manner. Our proof-of-concept study provides a fresh platform that opens new avenues for DNA-based functional ligands that are capable of altering the mitochondrial genome in a sequence-specific manner.
A new molecule that reads mitochondrial DNA could pave the way to treat some genetic nerve and muscle diseases
For the first time, a synthetic compound has been made that can bind to DNA in the cells’ energy powerhouses, suppressing a gene associated with nerve and muscle disease.
Pyrrole-imidazole polyamides (PIPs) are compounds that can read specific DNA sequences inside living cells and silence disease-causing genes. They prevent proteins, called transcription factors, from binding to specific parts of the DNA strand, thus suppressing the transcription of DNA into RNA.
Most DNA is found in the nucleus. But mitochondria, the cell’s powerhouses, also host a small amount of DNA. PIPs are capable of crossing the nuclear membrane to bind to nuclear DNA, but are incapable of crossing the mitochondrial membrane.
A team, led by Ganesh Pandian Namasivayam, from Kyoto University’s Institute for Integrated Cell-Material Science (iCeMS) succeeded to re-direct PIP to cross the mitochondrial membrane so that it can access its DNA and alter gene transcription.
They achieved this complex feat by complementing PIP with a ‘mitochondria-penetrating peptide’ (MPP), which is capable of overcoming the mitochondria’s energy barrier. The MPP-conjugated PIP called MITO-PIP was designed to block a specific binding site for mitochondrial transcription factor A (TFAM). TFAM is essential in governing mitochondrial metabolism and energy synthesis, playing a role in the transcription of a gene called ND6, says Takuya Hidaka, the first author of the study.
The team found that a TFAM-inhibiting MITO-PIP selectively read a mitochondrial DNA sequence and caused a 60% to 90% reduction in the expression of ND6, depending upon the concentration used. The team then labeled the MITO-PIPs with a molecule that fluoresces when exposed to light and, using special microscopes, confirmed that they localized inside the mitochondria without being present in the nuclei of treated cells.
ND6 is associated with several mitochondrial disorders, including Leber’s hereditary optic neuropathy, which causes loss of central vision, mitochondrial myopathy, muscle weakness, seizures and learning difficulties. Hence, chemical control over such disease-associated genes has clinical potential in mitochondrial gene therapy. “We plan to develop an advanced version of MITO-PIPs that can identify and localize only inside diseased mitochondria,” says Ganesh.
“Our proof-of-concept study provides a fresh platform that opens new avenues for DNA-based functional ligands that are capable of altering the mitochondrial genome in a sequence-specific manner,” concludes the principal investigator Hiroshi Sugiyama. The study was published in the Journal of the American Chemical Society.