[1S04e] 14. Chemical Biotechnology - Part 3

5:30 PM - 5:45 PM JST(8:30 AM - 8:45 AM UTC)

[1Chemi-18] Manipulation of gene expressions in mitochondria through G-quadruplex DNA formation depending on various intra-organellular environment

○Lutan Liu¹, Shuntaro Takahashi^1,2, Naoto Yoshinaga^3,4, Keiji Numata^3,4,5, Naoki Sugimoto¹ (1. Konan University FIBER (Japan), 2. Konan University FIRST (Japan), 3. RIKEN (Japan), 4. Keio University (Japan), 5. Kyoto University (Japan))

Keywords:

DNA secondary structure,Mitochondrial G-quadruplex,Intracellular crowding condition,Structural prediction,Mitochondrial gene expression

Manipulation of gene expressions in a spatio-temporal manner in cells is one of the key biotechnologies to regulate cell fate. One of the promising targets for controlling gene expression is non-duplex nucleic acid structure. Guanine (G)-quadruplexes form tetraplex structures from a tandem repeat of guanine sequences of DNA and RNA. This motif can reversibly form depending on the molecular crowding in cells because it alters the physicochemical properties of the solution and affects profoundly the stabilities of G4 structures. In particular, molecular crowding in the mitochondrial matrix has been suggested to be much higher than that of the nucleus and cytosol. G4s on mitochondrial DNA are accumulated much more than that on the nucleus DNA, which participate in regulatory functions of mitochondria. As mitochondria are the energy source of cellular function, manipulation of the G4 formation in mitochondria is critical for cancer therapeutics and anti-aging treatment. However, it has been limited to accessing the information about the formation of G4 in mitochondria. In this study, we developed a detection technique for G4 formations in mitochondria based on fluorescence protein expression (L. Liu et al., Commun. Chem., 8, 135 (2025)). We constructed a cassette of GFP transcribed by mitochondrial RNA polymerase and placed the G4-forming sequence between the promoter and GFP gene. The combination of the mitochondrial targeting peptide with a cationic peptide enables the plasmid DNA encoding the GFP cassette to be delivered specifically to mitochondria in cells. As GFP expression depends on G4 formation, which can act as a steric obstacle to RNA polymerase, information on G4 formation in mitochondria can be obtained from GFP expression levels. By using this technique, we observed the GFP expression in mitochondria in human cancer cells and quantitatively analyzed the GFP expression level by the confocal microscopy or RT-qPCR. Interestingly, the expression level was reduced efficiently more than that expressed in the nucleus. These results indicate that the thermodynamic stability of G4 in mitochondria is higher than that of the nucleus due to the dense molecular crowding. From the information, we could estimate the effect on the stability of DNA (L. Liu et al., JACS, 146, 32479 (2024)) and develop prediction parameters in mitochondrial conditions. Moreover, we analyzed the G4 formation in mitochondria in different molecular crowding condition changed by chloramphenicol and under aggressive malignant cancer cells, which provides that the G4 stabilities in nucleus and mitochondria show different response to crowding and cell types, possibly reflecting the effect of confinement of small quantity of molecules. This study develops a method to assess biomolecular stabilities within cellular organelles under various conditions. The study highlights how sequence-dependent G-quadruplex stabilities are controlled in separate organelles through unique properties of compartmentalized DNA existing in sub-molar particle numbers.