Advances in fluorescence labeling strategies for dynamic cellular imaging

Abstract

Synergistic advances in optical physics, probe design, molecular biology, labeling techniques and computational analysis have propelled fluorescence imaging into new realms of spatiotemporal resolution and sensitivity. This review aims to discuss advances in fluorescent probes and live-cell labeling strategies, two areas that remain pivotal for future advances in imaging technology. Fluorescent protein- and bio-orthogonal-based methods for protein and RNA imaging are discussed as well as emerging bioengineering techniques that enable their expression at specific genomic loci (for example, CRISPR and TALENs). Important attributes that contribute to the success of each technique are emphasized, providing a guideline for future advances in dynamic live-cell imaging.

Figure 1. Spectral properties of chromophore classes found in FPs

(a) Top, fluorescence emission spectra represented in terms of brightness (ε × ϕ_Fl), normalized to the brightest FP available (mNeonGreen). Bottom, absorption spectra of select FPs. Minor dots represent a cross section of solid-state laser sources that are commercially available and major dots are those typically equipped on microscopy equipment. Colors approximate the emission wavelength of the FP. (b) Chromophores and cofactors responsible for fluorescence in select constitutive FPs shown.

Figure 2. Covalent bio-orthogonal labeling mechanisms

Each panel provides the approximate size of the fusion protein, shown as a crystal structure. Chemical reactions have been enlarged for clarity, and all of the rates are measured in M⁻¹ s⁻¹. For all panels, scale bar (at bottom) represents 1.5 nm. (a) Genetic fusion of SNAP-tag to a protein of interest and subsequent suicide inhibition with fluorescent O⁶-benzylguanine derivatives results in a fluorescently labeled protein (Protein Data Bank (PDB) code 3KYZ). The fluorophore shown is SiR-SNAP, a silicon-based near-infrared fluorophore. (b) In the same way as SNAP-tag, CLIP-tag reacts with fluorescent O²-benzylcytosine derivatives (PDB code 3KYZ). (c) HaloTag fusion proteins react with chlorinated haloalkanes to form an irreversible fluorophore-alkyl-enzyme tether (PDB code 4KAA). (d) TMP-tag binds trimethoprim with nanomolar affinity and accelerates thiol reactivity with an acrylamide appendage (right) through proximity effects, irreversibly labeling the protein of interest (PDB code 1RD7). (e) Lipoic acid ligase, expressed in trans to the protein of interest labeled with a 13-amino-acid peptide, catalyzes the site-specific incorporation of coumarin. (f) Biotin ligase, which is analogous to lipoic acid ligase, catalyzes the site-specific incorporation of biotin or biotin isomeres, which can be chemically modified or used as an antigen for subsequent detection.

Figure 3. Bio-orthogonal labeling mechanisms based on reversible binding equilibria

(a) FlAsH (top) and ReAsH (bottom) recognize a specific peptide motif (Cys-Cys-Pro-Gly-Cys-Cys) with high affinity (K_D ~4 pM) and undergo a large increase in fluorescence intensity upon binding. (b) Ligand-specific binding by a protein, RNA or generic chelating agent triggers proximity-induced reactivity between a nucleophile located on the chelating group and the reactive tosyl-conjugated fluorophore. (c) A multidomain RNA construct with a target-specific aptamer domain and a target-dependent ′Spinach′ domain. An endogenous protein serves as the ligand for the aptamer domain, and binding of the protein triggers a conformational rearrangement in the Spinach domain, allowing productive binding of DFHBI to yield a fluorescent state. In the absence of the antigen, Spinach cannot bind DFHBI and remains nonfluorescent.

Figure 4. Methods for labeling RNA biomolecules

(a,b) Initial efforts to label RNAs in vivo were performed on chemically fixed samples and included long antisense DNA and RNA oligonucleotides labeled with a single fluorophore (shown as a star) (a) or multiple shorter oligonucleotides, each labeled with a single fluorophore (b). (c) Branched DNA structures provide increased specificity through the use of two adaptor oligonucleotides that nucleate the formation of a branched and multiply labeled oligonucleotides analogous to DNA origami. (d) A ‘molecular beacon’ fluorophore-quencher conjugate remains nonfluorescent until hybridization with the sequence of interest. (e) For live-cell imaging, 24 tandem repeats of a hairpin structure are inserted into the 3′ untranslated region of a transcript. Two GFP-MS2 (or equivalent orthogonal phage protein) fusion proteins identify each hairpin motif and bind with high affinity, generating a transcript with 48 GFP molecules and enabling routine single-molecule imaging of RNA in live cells. (f) To eliminate nonspecific background, a multicomponent system (for example, Pum-HD) oligomerizes on a unique RNA hairpin structure, triggering bimolecular complementation of GFP. (g) DFHBI, which is a GFP chromophore analog that is nonfluorescent when free in solution, is recognized by a specific RNA aptamer sequence, ′Spinach′, that turns on fluorescence upon binding.