Two-photon Fluorescent Probes For RNA And Mitochondria-localized Probe For Active Molecules

A variety of biomolecules play a pivotal role in many cellular processes. In order to study their functions, fluorescence microscopy, monitoring and visualizing them, is a direct method. Exploiting fluorescent probes for active biomolecules has attracted a large number of reseachers.Optical imaging with fluorescence microscopy is a vital tool in the study of living systems. Whereas one-photon fluorescence microscopy (OPM) employing one photon of higher energy for the excitation is the most common method for cell imaging, two-photon fluorescence microscopy (TPM) uses two photons of lower energy as the excitation source and is becoming more popular among biologists due to the advantages it provides. They include deeper penetration depth (>500μm), lower tissue autofluorescence and selfabsorption, and reduced photodamage and photobleaching, in addition to the intrinsically localized excitation. This allows imaging deep inside the intact tissue for a long period of time without the tissue preparation artifacts such as the damaged cells that can extend>70μm into the brain slice interior. However, the progress in this field is hindered by the lack of two-photon (TP) probes for specific applications. Moreover, most of the one-photon fluorescent probes used for TPM have low TP cross sections (δ<50GM) that limit their use in TPM. Therefore, there is a pressing need to develop efficient TP probes with larger TP cross sections for in vivo imaging.Nucleic acids (NA) are large biological molecules essential for all known forms of life. They include DNA (deoxyribonucleic acid) and RNA (ribonucleic acid); each is found in abundance in all living things, where they function in encoding, transmitting and expressing genetic information. However, compared to the many fluorescent probes imaging DNA, RNA probes for cell imaging are rarely reported. The possible reasons why RNA probes are rare could be at least three-fold:first, small nucleic-acid binding molecules generally have better affinity for double-stranded DNA than for RNA; second, hydrophobic biosensors including NA probes may also non-specifically bind with proteins and membranes in cells; third, knowledge of the interaction mechanism between RNA and fluorescent probes is still not enough compared with the wealth of deeper understanding of DNA biosensors, including outside, groove and intercalative binding. Thus it is necessary to find new RNA fluorescent probes.Intracellular thiols such as cysteine (Cys), homocysteine (Hey), and glutathione (GSH) play vital roles in biology. They maintain higher-order structures of proteins and control redox homeostasis through the equilibrium between thiols (RSH) and disulfides (RSSR). In mitochondria, a primary site of oxygen consumption and the major source of reactive oxygen species (ROS), GSH plays a key role in maintaining the redox environment to avoid or repair oxidative damage leading to dysfunction and cell death. Mitochondrial GSH (mGSH) exists predominantly in the reduced form, with a GSH:GSSG ratio of>100:1. An increase in the GSSG-to-GSH ratio is considered to be indicative of oxidative stress conditions. To understand the roles of RSH in biology, it is crucial to monitor RSH at the cell, tissue and organism level.To exploit fluorescent probes detecting and imaging RNA and mitochondrial RSH, we carried out two aspects of research:1. A series of RNA probes was developed, especially two-photon excited fluorescent RNA probes. IN and PY can enter live cells within a short time and image nucleolus in live cells; INS1, IND1,1NS2and IND2have large TP action cross section in RNA solution possessing the potential on imaging RNA in nucleoli and cytoplasm in fixed HeLa cells in TPM; INSC6can be applied to RNA imaging in live cells in TPM and exhibits good counterstain compatibility with classic fluorescent nucleic dye Hoechst33342.2. A biosensor for mitochondria RSH was obtained. CAP1derived from an aldehyde group as the reaction site and triphenylphosphonium salt (TPP) as the mitochondrial targeting site. As results, CAP1targeted mitochondria successfully and responsed to a few kinds of amino acids and RSH. Its selectivity need to be improved.