Experts comment on Nat Chemistry | Zou Peng/Chen Peng cooperate to develop membrane potential probes in the far red region based on "bio-orthogonal engineering"

Comments | Jiang Hualiang (Academician of Chinese Academy of Sciences), Dong Hui, Li Yulong (Peking University), editor in charge | Xi As the "currency" of information exchange in the nervous system, neuroelectric activity is the physical basis for the brain to process complex information.

Compared with traditional electrophysiological techniques based on electrode materials such as patch clamp and microelectrode array recording, fluorescent membrane potential imaging has obvious advantages in terms of temporal and spatial resolution and measurement flux.

Among them, fluorescent probes with emission wavelengths in the far red region (above 640 nm) are favored by researchers because of their red-shifted spectrum, which has stronger tissue penetration and can be suitable for multi-channel imaging observations.

However, the currently available membrane potential probes in the far red region have serious defects in brightness and sensitivity.
Therefore, there is an urgent need to develop high-performance fluorescent probes suitable for recording neuronal action potentials.

On April 15, 2021, Peng Zou's group from the School of Chemistry and Molecular Engineering of Peking University and Peng Chen's group jointly published their latest research results "A far-red hybrid voltage indicator enabled by bioorthogonal engineering of rhodopsin" in Nature Chemistry.
on live neurons", they comprehensively used bio-orthogonal reactions and membrane protein engineering strategies to develop a series of fluorescent membrane potential probes HVI (hybrid voltage indicator) with high sensitivity and imaging signal-to-noise ratio.

According to the requirements of imaging spectroscopy, the protein backbone of HVI can be combined with different fluorescent dye structures through bioorthogonal reactions to construct a series of composite probes that span the visible spectrum.

Among them, the orange-red zone probe HVI-Cy3 has the highest sensitivity and can record nerve action potentials with a signal-to-noise ratio of up to 90; the far-red zone probe HVI-Cy5 has the most red-shifted spectrum, not only compatible with green or red fluorescence The probe is used at the same time to realize the parallel observation of membrane potential and important physiological signals such as calcium ions and neurotransmitters.
It can also be used in conjunction with optogenetic tools to realize all-optical neuroelectrophysiological detection.

Figure 1 Conceptual diagram of compound membrane potential probe HVI to detect neuron membrane potential.
Zou Peng’s research group has been committed to the development and application of chemical probe technology for a long time to study the biological macromolecules involved in the process of neural signal transduction, and the chemistry and physics that innervate neural activities signal.

They took the lead in proposing the concept of "composite membrane potential probe", using chemical means to couple fluorescent dyes with rhodopsin protein, and using the electrochromic effect of the latter to achieve membrane potential imaging (Angew.
Chem.
Int.
Ed.
2018, 57, 3949-3953).

Chen Peng’s research group has long been committed to the development of bio-orthogonal reactions suitable for living cells and living animals, and through genetic coding technology, it has achieved specific labeling, activation and regulation of proteins (Nat.
Chem.
Biol.
2016, 12, 129- 137).

In the latest research results, the two research groups collaborated to combine chemical reaction strategies with protein backbone modification to optimize in-situ "bio-orthogonal" engineering optimization of neuronal membrane proteins.

On the one hand, given that most of the current bioorthogonal reactions are difficult to efficiently label membrane proteins, and the click chemistry reaction (copper-catalyzed alkynyl-azide cycloaddition reaction) is toxic to neurons, they switched to biocompatibility.
Good and more efficient reverse electrons require the Diels-Alder reaction (IEDDA) to introduce high fluorescence quantum yield far-red dyes into specific sites of engineered rhodopsin proteins.

On the other hand, they screened for mutations in the key proton receptor amino acid residues in the rhodopsin protein Ace2, and finally obtained the far-red region composite probe HVI-Cy5, which eliminates the steady-state photocurrent and significantly improves the brightness and sensitivity.

When the neuron membrane potential is depolarized, the change of the proton electrochemical potential promotes the protonation of the retinal Schiff base, thereby changing the absorption spectrum of rhodopsin, and finally affecting the quantum yield of its coupled fluorescent dye through the FRET effect.
This results in a change in the fluorescence signal (Figure 2).

 Figure 2 Schematic diagram of the HVI fluorescent labeling method of the composite membrane potential probe and the principle diagram of the probe responding to changes in cell membrane potential.
Experiments show that HVI-Cy5 can be used in conjunction with optogenetic tools (Figure 3).

The researchers used short-wavelength light to unidirectionally or bidirectionally regulate neuron excitability, and at the same time record membrane potential changes in the fluorescence channel of the far red zone, expanding the all-optical electrophysiology toolbox (Figure 3a-c).

Compared with the traditional multi-electrode patch clamp stimulation and recording, this technique significantly reduces the technical difficulty.

HVI-Cy5 can also perform dual-color imaging with other fluorescent probes to monitor cell membrane potential and physiological signals such as calcium ions, transmitters, and pH without crosstalk (Figure 3d-f).

Multicolor imaging will help researchers better understand the relationship and difference between membrane potential and other physiological signal dynamics.

 Figure 3 HVI-Cy5 with deep red fluorescence spectrum can be combined with optogenetic tools and calcium probes to achieve full optical electrophysiological detection.
In addition, the experiment expressed HVI-Cy5 and optogenetic tools in the hippocampus of cultured rats.
In neurons, membrane potential imaging was used to evaluate the effects of APV and NBQX on neuronal synaptic connections, and the contribution of NMDAR and AMPAR glutamate receptors to synaptic signal transmission was analyzed (Figure 4).

In the future, HVI-Cy5 is expected to screen agonists or antagonists acting on receptor proteins in vitro.

Figure 4 Expressing HVI-Cy5 and optogenetic tools in different neurons can study the effects of drugs on synapses.
In short, this article has developed a "bio-orthogonal" engineering optimization for rhodopsin membrane proteins to develop The high-performance fluorescent probe HVI-Cy5 in the far red zone for neuronal electrical signal recording can realize multi-color imaging and all-optical electrophysiological applications.
It is expected that this probe can help researchers interpret more complex neuronal electrophysiological signals. Doctoral candidates Liu Shuzhang and Lin Chang from the School of Chemistry and Molecular Engineering of Peking University, and Xu Yongxian (now a postdoctoral fellow at Zhejiang University Medical Center), a doctoral graduate of Peking University-Tsinghua Life Center Joint Center, are the co-first authors of the paper.

Researcher Zou Peng and Professor Chen Peng of Peking University are the co-corresponding authors of the paper.

 Expert comment Jiang Hualiang (Academician of the Chinese Academy of Sciences, Shanghai Institute of Materia Medica, Chinese Academy of Sciences) bioorthogonal reaction refers to a type of chemical reaction that can be carried out in a physiological environment such as living cells and does not interfere with life processes.

As one of the key technologies developed by chemists for life science research, this type of reaction has been widely used in fields ranging from basic research to drug development and clinical testing, changing the research process of life science and medicine.

For example, bio-orthogonal reaction-based biomacromolecule-specific markers can be used to observe the dynamic behavior and functional changes of biomacromolecules in living cells in real time.

Especially through the combined use of bio-orthogonal reaction and fluorescence imaging technology, many fluorescent dyes with excellent properties can be used for living, dynamic, and super-resolution observation of biological macromolecules, which promotes the advancement of bioimaging technology.

However, because nerve cells are more fragile than ordinary cells, many bio-orthogonal reactions still show toxicity on nerve cells, and their biocompatibility is poor.

In addition, bio-orthogonal reactions generally have the problem of low efficiency of labeling membrane proteins.

Zou Peng's research group at Peking University has been committed to constructing a "fluorescent dye-sensing protein" composite membrane potential probe, which is used to observe chemical and physical signals in neural activity.

Chen Peng's research group has long been committed to the development and type expansion of bio-orthogonal reactions.

In this work, the two research groups collaborated to use the "inverse electron demand Diels-Alder reaction" to point and specifically label the "proton pump" membrane protein-rhodopsin on the surface of neurons, and then construct a A series of fluorescent membrane potential probes with high sensitivity and imaging signal-to-noise ratio can detect neuronal action potentials in the far red area.

The Diels-Alder reaction was reported by a pair of teachers and students, Diels and Alder in 1927, between an electron-rich dienophile and an electron-deficient dienophile.
The [4+2] cycloaddition reaction attracted the interest of many researchers as soon as it was reported, and the two discoverers won the Nobel Prize in Chemistry in 1950.

In contrast, the [4+2] cycloaddition reaction between the electron-deficient dienes and the electron-rich dienophiles is called the "Inverse Electron Demand Diels-Alder reaction".
Alder reaction, IEDDA)".

Since electron-deficient dienes usually contain heteroatoms and the reaction product is a six-membered ring, IEDDA is often used to synthesize natural products containing heteroatomic six-membered rings.

In recent years, with the rise of bio-orthogonal reactions, the Diels-Alder reaction has once again attracted people’s attention, especially trans-cyclooctene dienophiles and tetrazine compounds (dienomers) based on ring tension.
Very fast and efficient coupling reactions can occur under biocompatible conditions, which opened up the research craze of IEDDA as a bio-orthogonal reaction.

Chen Peng's research group expanded the IEDDA reaction into a chemical decagging reaction in 2014, which realized the in-situ activation of proteins and promoted the rise and development of the "biological orthogonal shear reaction".

In this latest development, the two research groups have systematically compared to prove that the IEDDA reaction is the most compatible bio-orthogonal reaction with nerve cells, which can achieve efficient, specific and interference-free labeling of membrane proteins; in contrast, Other bio-orthogonal reactions, including copper-catalyzed and non-copper-catalyzed click chemistry reactions, exhibit problems such as high toxicity and poor signal-to-noise ratio.

Finally, through IEDDA's "bio-orthogonal" optimization and transformation, a series of composite fluorescent probes HVI (hybrid voltage indicator) that span the visible spectrum were constructed, providing a powerful tool for "all-optical electrophysiological detection".

For a long time, chemists have been continuously developing and using mild, highly efficient and specific chemical reactions in order to modify and label biological macromolecules in a living environment to detect and regulate life processes.

The above-mentioned work is another important progress of this unremitting endeavor.
It provides key technologies for the development of life sciences, especially neuroscience research in living cells and animals, and opens up new frontiers for the application of bioorthogonal reactions.

Experts comment on Dong Hui (postdoctoral) and Li Yulong (professor) (Peking University) membrane potential is a vital biophysical signal in life activities, and the technology for recording brain neuron membrane potential changes with high spatiotemporal resolution and low damage Means can help us deeply understand the working mechanism of the brain.

Recently, the research group of Peking University Zou Peng and Chen Peng collaborated to develop a new generation of composite fluorescent membrane potential probes (Hybrid Voltage Indicators, HVIs) combined with chemical biology technology and protein engineering, which achieved high-temporal and spatial monitoring of neuronal activity.
Resolution imaging records, and related results are published in "Nature Chemistry".

Zou Peng's research group has been deeply involved in the field of membrane potential probes for many years.
In 2014, they developed a series of genetically encoded voltage indicators (GEVIs) based on Rhodopsin Archaerhodopsin.

However, currently commonly used GEVIs are generally darker in brightness and require high-intensity excitation light, which can easily cause photobleaching and phototoxicity.

Compared with GEVIs, chemical membrane potential dyes have stronger fluorescence brightness and light stability, but they lack genetic coding characteristics, making it difficult to selectively detect the activity of specific types of neurons.

In 2018, Zou Peng's research group combined the advantages of GEVIs and chemical membrane potential dyes, and developed a composite membrane potential probe by covalently modifying chemical fluorophores on rhodopsin Ace2 through bio-orthogonal and site-specific labeling methods.
.

In recent work, Zou Peng's group and Chen Peng's group have joined forces to apply the most efficient bio-orthogonal reaction-the "inverse electron demand Diels-Alder reaction" to the specific chemistry of rhodopsin protein on nerve cells.
Marking further improves the performance of HVIs. The new generation of HVIs directly connect the fluorescent group to the Ace2 protein backbone, avoiding the cytotoxicity caused by heavy metal reagents in the copper-catalyzed click reaction in the past.

Compared with GEVIs, the new generation of HVIs not only has a higher signal-to-noise ratio, but can also easily expand the imaging spectrum of the probe by connecting different fluorophores.

HVI-Cy5, which emits light in the far red region, can be used in combination with other molecular tools that use short-wavelength excitation light, such as optogenetic elements such as CheRiff, neurotransmitter probes such as iGluSnFR, and calcium probes such as GCaMP6s and R-GECO , Which reflects the good spectral compatibility of HVI-Cy5.

The probe is suitable for all-optical electrophysiology and can promote the research of neurobiological problems.

It is hoped that the new generation of HVIs can further realize in vivo applications in the future, combined with the tools including our developed neurotransmitter fluorescent probes, to better promote our decoding of the secrets of the brain's work.

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