The Benefits of Unnatural Amino Acid Incorporation as Protein Labels for Single Molecule Localization Microscopy

Abstract

Single Molecule Localization Microscopy (SMLM) is an imaging method that allows for the visualization of structures smaller than the diffraction limit of light (~200 nm). This is achieved through techniques such as stochastic optical reconstruction microscopy (STORM) and photoactivated localization microscopy (PALM). A large part of obtaining ideal imaging of single molecules is the choice of the right fluorescent label. An upcoming field of protein labeling is incorporating unnatural amino acids (UAAs) with an attached fluorescent dye for precise localization and visualization of individual molecules. For this technique, fluorescent probes are conjugated to UAAs and are introduced into the protein of interest (POI) as a label. Here we contrast this labeling method with other commonly used protein-based labeling methods such as fluorescent proteins (FPs) or self-labeling tags such as Halotag, SNAP-tags, and CLIP-tags, and highlight the benefits and shortcomings of the site-specific incorporation of UAAs coupled with fluorescent dyes in SMLM.

Fluorescence-based sensing of the bioenergetic and physicochemical status of the cell

“…In particular, it is possible to perform in vitro bioconjugation reactions of natural amino acids on a protein surface, specifically targeting exposed cysteine (Kim et al, 2008), lysine (Larda, Pichugin, & Prosser, 2015), tyrosine (Dorta, Deniaud, M evel, & Gouin, 2020) and tryptophan (Ladner, Turner, & Edwards, 2007) residues. Several in vivo techniques are available for labeling molecules, such as the SNAP-tag method, in which a protein of interest (POI) is functionalized with an enzyme tag that allows the covalent labeling of the protein (Cole, 2013), the incorporation of unnatural amino acids in the sequence of a POI (Laxman, Ansari, Gaus, & Goyette, 2021), allowing their direct chemical modification, and the Staudinger-Bertozzi ligation reaction (Saxon, Armstrong, & Bertozzi, 2000), which consists in the ligation of a triarylphosphine conjugate reporter to an azide-functionalized biomolecular analogue that can be incorporated in cell structures, allowing for the detection of many different macromolecules, such as glycans, lipids, DNA and proteins (van Berkel, van Eldijk, & van Hest, 2011).…”

“…If the POI needs to be localized immediately after translation, or if the conditions in which the POI needs to be expressed require absence of oxygen (such as in obligated anaerobes), the preferred imaging tool would be FAST (Plamont et al, 2016) or CTPE (Iyer et al, 2021), for which the fluorescence development is not dependent of oxygen and for which there is no delay between protein expression and fluorescence emission. Another way to visualize a POI is through the incorporation into proteins of fluorophores conjugated to unnatural amino acids (Laxman et al, 2021), but the number of dyes available for direct protein labeling is limited.…”

Nucleic Acid Probes for Single-Molecule Localization Imaging of Cellular Biomolecules

“…3,4 Single-molecule localization microscopy (SMLM) randomly activates fluorescent molecules in batches so that only a few fluorescent molecules emit light in the imaging area at a time. 5 Subsequently, Gaussian fitting is used to localize the center position of the single fluorescent molecule (point diffusion function), thus cleverly bypassing the diffraction limit to achieve high-precision space localization. 6 Owing to their excellent photophysical properties, fluorescent proteins are often used as imaging probes for SMLM to obtain singlemolecule localization information.…”

In Vivo Behavior of the Antibacterial Peptide Cyclo[RRRWFW], Explored Using a 3-Hydroxychromone-Derived Fluorescent Amino Acid

“…Unnatural fluorescent amino acids have been successfully used for labeling large proteins and elongated polypeptides and for their consecutive observation in cells and tissues by fluorescent microscopy ( Lee, 2019 ; Cheng et al, 2020 ; Laxman et al, 2021 ). However, in the case of short peptides which possess less than 20 amino acid residues, the applicability of this potent exploration tool is hampered by severe structural and functional perturbations often caused by the label.…”