The methodologies of controlling protein functions by intervening central dogma processes are evolving as a new cancer therapy modality. The typical examples are DNA editing, RNAi knock down, and protein-targeted missile therapy. However, there still remain the issues of safety ethics, selective delivery to the target cells or tissues, and the high cost. To resolve these issues at once, it is required to be able to remote-control the ON/OFF of the intervention of central dogma processes in a spatiotemporal and reversible manner.
Among the media potentially capable of the remote control, i.e., ultrasound, magnetism, electromagnetic waves including light, and radiation, light is the best candidate because it has sufficient energy to rearrange the covalent bonds in the molecules with photo reactive groups and yet is intact toward the biomolecules without such photo responsive parts. The light signals can be exploited by photo catalysts, which enable an enzyme-like selective conversion of biomolecules and thus permitting the spatiotemporal, reversible photo remote-control of central dogma molecules.
The photo catalyst we focused on in this project is the biphenyl (BP) photosensitizer (PS) developed on our own (Fig. 1). BP can convert the oxygen molecule (3O2) in air by photo irradiation into the reactive singlet oxygen (1O2), which promotes the oxidation of biomolecules as the general PSs do. Compared with porphyrins, the most typical PS group, the size of the BP molecule is less than half and thus it can sneak into a DNA major groove (Fig. 2) and the hole of channel proteins as suggested from molecular models. BP shows as large photosensitizing efficiency as that of porphyrins in non-water environment, enabling the oxidations of guanine base (G) into oxo-guanine (OG) and amino acids like tryptophan leading to decomposition. Interestingly, while G forms a stable base-pair with cytosine (C), for OG it is adenosine (A) that wins the best base-pair partner. Therefore, if a certain G in DNA can be selectively oxidized into OG, a C-to-A point mutation in RNA is possible through the transcription. Also, the existence of OG in RNA is known to cause the stall of protein syntheses in the translation. Furthermore, a photo catalytic conjugate consisting of a PS and a ligand molecule for a target protein, has been confirmed to oxidatively inactivate the target protein by 1O2 produced from the PS in live cells.
The purpose of our project is to establish a new modality of safe, concise cancer therapy by controlling the disease-related functions of a target protein spatiotemporally and reversibly with the help of BP-based photo catalytic reactions toward DNA, RNA, and proteins by the oligonucleotides and ligands covalently attached with BP. The protein modification (Fig. 3) will be performed by the collaborative researcher Professor Hiroyuki Nakamura (Institute of Innovative Research, Tokyo Institute of Technology). The other members of this project are Assistant Professor Takashi Kanamori, Technical assistants Selvakumaran Paulsi and Sachiko Nishida (Fig. 4; All at Tokyo Institute of Technology).
Figure 1: The perspective of our project. Photo editing or damage of central dogma biomolecules: RNA point mutation by photo editing of DNA, translation inhibition by photo editing of RNA, and photo oxidative inactivation of protein. The construction of the technologies for photo gene manipulation and photo protein inactivation and light source engineering, which is required to apply the above methodology for human and eventually out-licensing.
Figure 2: Triplex and duplex of dsDNA and RNA, respectively, combined with a BP-oligonucleotide.
Figure 3: Mechanism of oxidative inactivation of intracellular proteins by BP-protein ligand conjugates.Photo-switching inactivates intracellular target proteins post-translationally using 1O2 produced by photosensitizers. Spatiotemporal dynamics of disease-causing proteins can be observed by this technology.
Figure 4: Group photo of our project members.