Ddiels Alder Reaction Rates And 'Bioorthogonal Engineering' Based Membrane Potential Probes In The Far Red Zone

On April 15, 2021, Peng Zou's group and Peng Chen's group in the School of Chemical and Molecular Engineering at Peking University published their latest research results online in Nature Chemistry, in which they developed a series of fluorescent membrane potential probes with high sensitivity and imaging signal-to-noise ratio, HVI (hybrid voltage indicator), using a combination of bioorthogonal reaction and membrane protein engineering modification strategy. voltage indicator) with high sensitivity and imaging signal-to-noise ratio. Do you know about the diels alder reaction rates?

As the "currency" of information exchange in the nervous system, neural electrical activity is the physical basis for the brain to process complex information. Compared with traditional electrophysiological techniques based on electrode materials such as membrane clamp and microelectrode array recording, fluorescence membrane potential imaging has obvious advantages in terms of spatial and temporal resolution and measurement throughput. Among them, fluorescent probes with emission wavelengths in the far red region (above 640 nm) are favored by researchers because their red-shifted spectra have stronger tissue penetration capabilities and can be suitable for multi-pathway imaging observations. However, the currently available far-red region membrane potential probes have serious shortcomings in terms of brightness and sensitivity, so there is an urgent need to develop high-performance fluorescent probes suitable for recording neuronal action potentials.  Scientists are studying the diels alder reaction rates.

On April 15, 2021, Peng Zou's group at the School of Chemical and Molecular Engineering, Peking University, and Peng Chen's group at the McGovern Institute for Brain Science, Peking University, published their latest research result "A far-red hybrid voltage indicator enabled by bioorthogonal engineering" online in Nature Chemistry. indicator enabled by bioorthogonal engineering of rhodopsin on live neurons", in which they developed a series of fluorescent membrane potential probes with high sensitivity and imaging signal-to-noise ratio using a combination of bioorthogonal reactions and membrane protein engineering modification strategies. HVI (hybrid voltage indicator). Depending on the imaging spectral requirements, the protein backbone of HVI can be combined with different fluorescent dye structures by bioorthogonal reactions to build a series of composite probes across the visible spectrum. Some scientific experiments on the diels alder reaction rates are being conducted now. Among them, the orange-red probe HVI-Cy3 has the highest sensitivity and can record neural action potential with a signal-to-noise ratio of up to 90; the far-red probe HVI-Cy5 has the most red-shifted spectrum and can be used simultaneously with green or red fluorescent probes to achieve parallel observation of membrane potential and important physiological signals such as calcium ions and neurotransmitters, and can also be used in conjunction with optogenetic tools to achieve all-optical neuroelectrophysiological detection.

Peng Zou's group has long been dedicated to the development and application of chemical probe technology to study the biomolecules involved in neural signal transduction processes, and the chemical and physical signals that govern neural activity. They have pioneered the concept of "composite membrane potential probe", which uses a fluorescent dye coupled with retinal proteins to achieve membrane potential imaging by using the electrochromic effect of the latter (Angew. Chem. Int. Ed. 2018, 57, 3949-3953). Peng Chen's group has long been working on the development of bioorthogonal reactions applicable to living cells and living animals, and has achieved specific labeling, activation and regulation of proteins through genetic encoding techniques (Nat. Chem. Biol. 2016, 12, 129-137). Some scientific progress has been made in the diels alder reaction rates. In the latest research, the two groups collaborated to combine chemical reaction strategies with protein backbone modification to optimize the in situ "bioorthogonal" engineering of neuronal membrane proteins. On the one hand, in view of the difficulty of most current bioorthogonal reactions to efficiently label membrane proteins and the toxicity of click chemistry (copper-catalyzed alkyne-azide cycloaddition) to neurons, they switched to a more biocompatible and efficient inverse electron-demanding Diels-Alder reaction (IEDDA) to introduce a high fluorescence quantum yield far-red region dye into the specific site of the engineered retinoid protein. sites. When the neuronal membrane potential is depolarized, the change in proton electrochemical potential promotes the protonation of retinoid Schiff base, which changes the absorption spectrum of retinol. This ultimately affects the quantum yield of its coupling fluorescent dye through FRET effect, leading to fluorescence signal changes.