Bioorthogonal labeling of live dissociated and organotypic slice culture neurons

Over the past couple decades, the explosion in the development of high-resolution and super-resolution microscopy techniques has led to the need for the development of new protein labeling techniques. Click-labeling via genetic code expansion (GCE) has received particular attention given its potential has the ultimately small labelling probe for proteins. Click-labeling via GCE offers a reliable and sterically minimally demanding capacity to label proteins, but its application in non dividing cells such as neurons remains poorly exploited due to its low efficiency. Here, we describe a simple, efficient and reproducible protocol that allows to fluorescently label transmembrane proteins in live neurons using click-labeling via GCE, both in dissociated culture and organotypic brain slices.

Bioorthogonal labeling of transmembrane proteins with non-canonical amino acids unveils masked epitopes in live neurons

Progress in biological imaging is intrinsically linked to advances in labeling methods. The explosion in the development of high-resolution and super-resolution imaging calls for new approaches to label targets with small probes. These should allow to faithfully report the localization of the target within the imaging resolution – typically nowadays a few nanometers - and allow access to any epitope of the target, in the native cellular and tissue environment. We report here the development of a complete labeling and imaging pipeline using genetic code expansion and non-canonical amino acids in neurons that allows to fluorescently label masked epitopes in target transmembrane proteins in live neurons, both in dissociated culture and organotypic brain slices. This allows us to image the differential localization of two AMPA receptor (AMPAR) auxiliary subunits of the transmembrane AMPAR regulatory protein family in complex with their partner with a variety of methods including widefield, confocal, and dSTORM super-resolution microscopy.

Isocyanides: Promising Functionalities in Bioorthogonal Labeling of Biomolecules

Isocyanides have drawn increasing attention in biological applications due to their attractive properties and unique reactivities, which can undergo various reactions, such as multicomponent reactions, α-addition reactions, [4 + 1] cycloaddition reactions, and the reaction scope keeps expanding. In addition to acting as reactants for the preparation of structurally interesting and diverse N-heterocycles or peptidomimetics, this type of functionality may be a good choice in the labeling and modulation of biomolecules due to the high biocompatibility and small size to minimize modifications on the parent molecule. It has been demonstrated that isocyanides can participate in biomolecule labeling through three strategies, including the two-component bioorthogonal reaction, multicomponent reaction, and metal chelation. Among them, the isocyanide-tetrazine reaction has been better studied recently, augmenting the potency of isocyanide as a bioorthogonal handle. This review will focus on the recent progress in isocyanide chemistry for labeling of biomolecules. Meanwhile, methods to introduce isocyano groups into biomacromolecules are also described to facilitate wider applications of this unique functionality.

Bioorthogonal labeling of transmembrane proteins with non-canonical amino acids allows access to masked epitopes in live neurons

ABSTRACTProgress in biological imaging is intrinsically linked to advances in labeling methods. The explosion in the development of high-resolution and super-resolution imaging calls for new approaches to label targets with small probes. These should allow to faithfully report the localization of the target within the imaging resolution – typically nowadays a few nanometers - and allow access to any epitope of the target, in the native cellular and tissue environment. We report here the development of a complete labeling and imaging pipeline using genetic code expansion and non-canonical amino acids in primary neurons that allows to fluorescently label masked epitopes in target transmembrane proteins in live neurons, both in dissociated culture and organotypic brain slices. This allowed us to image the differential localization of two glutamate receptor auxiliary proteins in complex with their partner with a variety of methods including widefield, confocal, and dSTORM super-resolution microscopy.

An enzyme-mediated bioorthogonal labeling method for genome-wide mapping of 5-hydroxymethyluracil

We developed an enzyme-mediated bioorthogonal labeling strategy for the enrichment and genome-wide mapping of 5hmU. With this strategy, we provided the first map of 5hmU in the whole Trypanosoma brucei genome.