Development and characterization of novel reversibly switchable red fluorescent proteins with opposing switching modes

Abstract

Since the discovery of the green fluorescent protein (GFP) from the jellyfish Aequorea victoria, a multitude of fluorescent proteins have been derived from the original GFP and its homologs by protein engineering. Their characteristics were especially adapted to the requirements of different types of live-cell fluorescence microscopy. This includes super-resolution microscopy techniques, which overcome the diffraction barrier by distinguishing fluorophores based on different molecular states. Fluorescent proteins in the subclass of reversibly switchable fluorescent proteins (RSFPs) can be switched from a fluorescent to a non-fluorescent state and vice versa by light of certain wavelengths. These RSFPs can be utilized as fusion tags in reversible saturable optical fluorescence transition (RESOLFT) microscopy to achieve diffraction-unlimited resolution. Compared to the green-emitting RSFPs, the application of red-emitting RSFPs benefits from the longer excitation and switching wavelengths utilized and the associated less phototoxicity. However, the number of red RSFPs applicable for live-cell RESOLFT microscopy is limited until now and the available red RSFPs exhibit undesirable characteristics like dimerization tendency. This work presents the development of novel red RSFPs for RESOLFT microscopy. In order to identify superior red RSFPs, a microscopic screening of mutant libraries expressed in E. coli colonies was employed. To additionally facilitate the screening for improved fluorescent proteins in a mammalian expression system, a monoclonal Bxb1 landing pad cell line was generated using CRISPR/Cas9 genome engineering. Since the application of these Bxb1 landing pad cells enables the expression of only a single mutant from a transfected library in each cell, this strategy was suitable to screen for red RSFPs with higher brightness and offers a large potential to establish further screening approaches. By semi-rational design and screening, novel red RSFPs with opposing switching modes were generated from bright conventional red fluorescent proteins. Three positive-switching RSFPs were developed on the basis of mRuby2 and two negative-switching RSFPs were derived from mScarlet. All of these five RSFPs are monomeric, feature low residual fluorescence in the off-state (below 5 %), and are among the red RSFPs with the highest molecular brightness known today. The characterization of their performance as fusion tag in transient and stable expressions suggest that the novel red RSFPs are suitable for live-cell microscopy. The quantum yields of the switchable mScarlet variants are the highest determined for red RSFPs to date and the switching kinetics of the new negative-switching RSFPs exceeded those of the recently published rsFusionRed2 and rsFusionRed3. In addition, the generated RSFPs based on mRuby2 outperformed rsCherry in all analyzed switching characteristics and represent the only red fluorescent and positive-switching candidates for live-cell RESOLFT microscopy. A resolution beyond the diffraction limit was demonstrated utilizing one of the switchable mRuby2 variants in a proof of concept experiment.