Bioluminescence Assisted Switching and Fluorescence Imaging (BASFI)

Förster resonance energy transfer (FRET) and bioluminescence resonance energy transfer (BRET) are two major biophysical techniques for studying nanometer-scale motion dynamics within living cells. Both techniques read photoemission from the transient RET-excited acceptor, which makes RET and detection processes inseparable. We here report a novel hybrid strategy, bioluminescence assisted switching and fluorescence imaging (BASFI) using a bioluminescent <i>Renilla</i> luciferase RLuc8 as the donor and a photochromic fluorescent protein Dronpa as the acceptor. When in close proximity, RET from RLuc8 switches Dronpa from its original dark state to a stable bright state, whose fluorescence is imaged subsequently with an external laser. Such decoupling between RET and imaging processes in BASFI promises high photon flux as in FRET and minimal bleedthroughs as in BRET. We demonstrated BASFI with Dronpa-RLuc8 fusion constructs and drug-inducible intermolecular FKBP-FRB protein–protein interactions in live cells with high sensitivity, resolution, and specificity. Integrating the advantages of FRET and BRET, BASFI will be a valuable tool for various biophysical studies

Second-Generation Covalent TMP-Tag for Live Cell Imaging

Chemical tags are now viable alternatives to fluorescent proteins for labeling proteins in living cells with organic fluorophores that have improved brightness and other specialized properties. Recently, we successfully rendered our TMP-tag covalent with a proximity-induced reaction between the protein tag and the ligand-fluorophore label. This initial design, however, suffered from slow <i>in vitro</i> labeling kinetics and limited live cell protein labeling. Thus, here we report a second-generation covalent TMP-tag that has a fast labeling half-life and can readily label a variety of intracellular proteins in living cells. Specifically, we designed an acrylamide-trimethoprim-fluorophore (A-TMP-fluorophore v2.0) electrophile with an optimized linker for fast reaction with a cysteine (Cys) nucleophile engineered just outside the TMP-binding pocket of Escherichia coli dihydrofolate reductase (eDHFR) and developed an efficient chemical synthesis for routine production of a variety of A-TMP-probe v2.0 labels. We then screened a panel of eDHFR:Cys variants and identified eDHFR:L28C as having an 8-min half-life for reaction with A-TMP-biotin v2.0 <i>in vitro</i>. Finally, we demonstrated live cell imaging of various cellular protein targets with A-TMP-fluorescein, A-TMP-Dapoxyl, and A-TMP-Atto655. With its robustness, this second-generation covalent TMP-tag adds to the limited number of chemical tags that can be used to covalently label intracellular proteins efficiently in living cells. Moreover, the success of this second-generation design further validates proximity-induced reactivity and organic chemistry as tools not only for chemical tag engineering but also more broadly for synthetic biology

Multicolor Live-Cell Chemical Imaging by Isotopically Edited Alkyne Vibrational Palette

Vibrational imaging such as Raman microscopy is a powerful technique for visualizing a variety of molecules in live cells and tissues with chemical contrast. Going beyond the conventional label-free modality, recent advance of coupling alkyne vibrational tags with stimulated Raman scattering microscopy paves the way for imaging a wide spectrum of alkyne-labeled small biomolecules with superb sensitivity, specificity, resolution, biocompatibility, and minimal perturbation. Unfortunately, the currently available alkyne tag only processes a single vibrational “color”, which prohibits multiplex chemical imaging of small molecules in a way that is being routinely practiced in fluorescence microscopy. Herein we develop a three-color vibrational palette of alkyne tags using a ¹³C-based isotopic editing strategy. We first synthesized ¹³C isotopologues of EdU, a DNA metabolic reporter, by using the newly developed alkyne cross-metathesis reaction. Consistent with theoretical predictions, the mono-¹³C (¹³C¹²C) and bis-¹³C (¹³C¹³C) labeled alkyne isotopologues display Raman peaks that are red-shifted and spectrally resolved from the originally unlabeled (¹²C¹²C) alkynyl probe. We further demonstrated three-color chemical imaging of nascent DNA, RNA, and newly uptaken fatty-acid in live mammalian cells with a simultaneous treatment of three different isotopically edited alkynyl metabolic reporters. The alkyne vibrational palette presented here thus opens up multicolor imaging of small biomolecules, enlightening a new dimension of chemical imaging