Noncytotoxic orange and red/green derivatives of DsRed-Express2 for whole-cell labeling

<p>Abstract</p> <p>Background</p> <p>Whole-cell labeling is a common application of fluorescent proteins (FPs), but many red and orange FPs exhibit cytotoxicity that limits their use as whole-cell labels. Recently, a tetrameric red FP called DsRed-Express2 was engineered for enhanced solubility and was shown to be noncytotoxic in bacterial and mammalian cells. Our goal was to create derivatives of this protein with different spectral properties.</p> <p>Results</p> <p>Building on previous studies of DsRed mutants, we created two DsRed-Express2 derivatives: E2-Orange, an orange FP, and E2-Red/Green, a dual-color FP with both red and green emission. We show that these new FPs retain the low cytotoxicity of DsRed-Express2. In addition, we show that these new FPs are useful as second or third colors for flow cytometry and fluorescence microscopy.</p> <p>Conclusion</p> <p>E2-Orange and E2-Red/Green will facilitate the production of healthy, stably fluorescent cell lines and transgenic organisms for multi-color labeling studies.</p

Plug-and-Play Fluorophores Extend the Spectral Properties of Spinach

Spinach and Spinach2 are RNA aptamers that can be used for the genetic encoding of fluorescent RNA. Spinach2 binds and activates the fluorescence of (<i>Z</i>)-4-(3,5-difluoro-4-hydroxybenzylidene)-1,2-dimethyl-1<i>H</i>-imidazol-5­(4<i>H</i>)-one (DFHBI), allowing the dynamic localizations of Spinach2-tagged RNAs to be imaged in live cells. The spectral properties of Spinach2 are limited by DFHBI, which produces fluorescence that is bluish-green and is not optimized for filters commonly used in fluorescence microscopes. Here we characterize the structural features that are required for fluorophore binding to Spinach2 and describe novel fluorophores that bind and are switched to a fluorescent state by Spinach2. These diverse Spinach2–fluorophore complexes exhibit fluorescence that is more compatible with existing microscopy filter sets and allows Spinach2-tagged constructs to be imaged with either GFP or YFP filter cubes. Thus, these “plug-and-play” fluorophores allow the spectral properties of Spinach2 to be altered on the basis of the specific spectral needs of the experiment

A Rapidly Maturing Far-Red Derivative of DsRed-Express2 for Whole-Cell Labeling

Fluorescent proteins (FPs) with far-red excitation and emission are desirable for multicolor labeling and live-animal imaging. We describe E2-Crimson, a far-red derivative of the tetrameric FP DsRed-Express2. Unlike other far-red FPs, E2-Crimson is noncytotoxic in bacterial and mammalian cells. E2-Crimson is brighter than other far-red FPs and matures substantially faster than other red and far-red FPs. Approximately 40% of the E2-Crimson fluorescence signal is remarkably photostable. With an excitation maximum at 611 nm, E2-Crimson is the first FP that is efficiently excited with standard far-red lasers. We show that E2-Crimson has unique applications for flow cytometry and stimulated emission depletion (STED) microscopy

A Rapidly Maturing Far-Red Derivative of DsRed-Express2 for Whole-Cell Labeling

Fluorescent proteins (FPs) with far-red excitation and emission are desirable for multicolor labeling and live-animal imaging. We describe E2-Crimson, a far-red derivative of the tetrameric FP DsRed-Express2. Unlike other far-red FPs, E2-Crimson is noncytotoxic in bacterial and mammalian cells. E2-Crimson is brighter than other far-red FPs and matures substantially faster than other red and far-red FPs. Approximately 40% of the E2-Crimson fluorescence signal is remarkably photostable. With an excitation maximum at 611 nm, E2-Crimson is the first FP that is efficiently excited with standard far-red lasers. We show that E2-Crimson has unique applications for flow cytometry and stimulated emission depletion (STED) microscopy

Small Molecule Recognition and Tools to Study Modulation of r(CGG)^exp in Fragile X‑Associated Tremor Ataxia Syndrome

RNA transcripts containing expanded nucleotide repeats cause many incurable diseases via various mechanisms. One such disorder, fragile X-associated tremor ataxia syndrome (FXTAS), is caused by a noncoding r­(CGG) repeat expansion (r­(CGG)^exp) that (i) sequesters proteins involved in RNA metabolism in nuclear foci, causing dysregulation of alternative pre-mRNA splicing, and (ii) undergoes repeat associated non-ATG translation (RANT), which produces toxic homopolymeric proteins without using a start codon. Here, we describe the design of two small molecules that inhibit both modes of toxicity and the implementation of various tools to study perturbation of these cellular events. <u>C</u>ompetitive <u>Chem</u>ical <u>C</u>ross <u>L</u>inking and <u>I</u>solation by <u>P</u>ull Down (C-Chem-CLIP) established that compounds bind r­(CGG)^exp and defined small molecule occupancy of r­(CGG)^exp in cells, the first approach to do so. Using an RNA GFP mimic, r­(CGG)^exp-Spinach2, we observe that our optimal designed compound binds r­(CGG)^exp and affects RNA localization by disrupting preformed RNA foci. These events correlate with an improvement of pre-mRNA splicing defects caused by RNA gain of function. In addition, the compounds reduced levels of toxic homopolymeric proteins formed via RANT. Polysome profiling studies showed that small molecules decreased loading of polysomes onto r­(CGG)^exp, explaining decreased translation