Engineering of a fluorescent chemogenetic reporter with tunable color for advanced live-cell imaging

Abstract

Fluorescent reporters are essential tools in cell biology for imaging the dynamics of proteins in living cells and organisms with high spatial and temporal resolution. Chemogenetic systems made of a genetically encoded protein tag acting as an anchor for synthetic fluorophores combine the targeting selectivity of genetic tags with the advantages of synthetic fluorophores. Here, we present the directed evolution of a small 14-kDa protein tag with extended chromophore promiscuity capable of efficiently forming non-covalent fluorescent assemblies with a collection of membrane-permeant and membrane-impermeant fluorogenic chromophores displaying spectral properties spanning the entire visible spectrum. The ability to adapt the fluorescence color by choosing a different live-cell compatible fluorogenic chromophore enables to genetically encode blue, cyan, green, yellow, orange and red fluorescence with a single tag, providing an unprecedent experimental versatility. The possibility to form dark assemblies using non-fluorescent chromophores provides moreover an innovative way for switching off fluorescence on-demand in cells and organisms with high temporal resolution by chromophore replacement. Selective wash-free fluorogenic labeling of fusion proteins could be achieved with high efficiency in live cells, including delicate cultured hippocampal neurons, and in multicellular organisms, allowing high contrast imaging with various advanced microscopy techniques. The remarkable labeling efficiency and fluorescence performance allowed the multicolor imaging of dynamic processes in multicellular systems. The ability to match the spectral properties to the imaging modalities and the high photostability enabled to achieve efficient stimulated emission depletion (STED) nanoscopy of fusion proteins in live cells and live primary cultured neurons.

A fluorogenic chemically induced dimerization technology for controlling, imaging and sensing protein proximity

“…FIRE tag and FIRE mate were generated by bisection of the fluorescent chemogenetic reporter pFAST 13 . pFAST is a promiscuous and improved version of FAST (Fluorescence-Activating and absorption-Shifiting Tag), a 14 kDa protein that binds and stabilizes the fluorescent state of fluorogenic chromophores.…”

Exchangeable HaloTag Ligands (xHTLs) for multi-modal super-resolution fluorescence microscopy

“…5 However, exchangeable probes based on DNA are incompatible with live-cell imaging of intracellular targets. 6 Other tools based on exchangeable probes, such as the fluorogen binder FAST 7 , are live-cell compatible but do not meet the demanding requirement of multiframe STED microscopy. Here we introduce exchangeable HaloTag7 Ligands (xHTLs, Fig 1A ) that open up application of HaloTag7 for PAINT, MINFLUX 8 and multi-frame, live-cell STED imaging.…”

High-throughput directed evolution: a golden era for protein science

“…Another often-used method for in vitro gene diversification is DNA shuffling (Box 1), wherein libraries are created by random fragmentation and recombination of homologous DNA sequences (Figure 2C) [29]. Since its invention, DNA shuffling has been widely used and adapted to engineer a range of properties, including improved thermostability [30] or catalytic activity [27], and, most recently, to develop chemogenetic fluorescent reporters with tuneable fluorescent properties [31].…”