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

Strack, Rita; Hein, Birka; Bhattacharyya, Dibyendu; Hell, Stefan W.; Keenan, Robert J.; Glick, Benjamin S.

doi:10.1021/bi900870u

Cited by 174 publications

(177 citation statements)

References 15 publications

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“…TagRFP657, a further optimized mKate mutant with an λ em of 657 nm, is currently the most red-shifted monomeric RFP (31). Recently, Strack et al (28) developed E2-Crimson, a very red-shifted DsRed-derived RFP. E2-Crimson was isolated after screening more than 1,000,000 colonies in three rounds of targeted combinatorial mutagenesis and two rounds of random mutagenesis.…”

Section: Contains An Additional Undesired Mutation (R125h)mentioning

confidence: 99%

Generation of longer emission wavelength red fluorescent proteins using computationally designed libraries

Chica

Moore

Allen

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

156

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The longer emission wavelengths of red fluorescent proteins (RFPs) make them attractive for whole-animal imaging because cells are more transparent to red light. Although several useful RFPs have been developed using directed evolution, the quest for further red-shifted and improved RFPs continues. Herein, we report a structure-based rational design approach to red-shift the fluorescence emission of RFPs. We applied a combined computational and experimental approach that uses computational protein design as an in silico prescreen to generate focused combinatorial libraries of mCherry mutants. The computational procedure helped us identify residues that could fulfill interactions hypothesized to cause red-shifts without destabilizing the protein fold. These interactions include stabilization of the excited state through H-bonding to the acylimine oxygen atom, destabilization of the ground state by hydrophobic packing around the charged phenolate, and stabilization of the excited state by a π-stacking interaction. Our methodology allowed us to identify three mCherry mutants (mRojoA, mRojoB, and mRouge) that display emission wavelengths >630 nm, representing red-shifts of 20-26 nm. Moreover, our approach required the experimental screening of a total of ∼5,000 clones, a number several orders of magnitude smaller than those previously used to achieve comparable red-shifts. Additionally, crystal structures of mRojoA and mRouge allowed us to verify fulfillment of the interactions hypothesized to cause red-shifts, supporting their contribution to the observed red-shifts. The red fluorescence displayed by these proteins arises from the presence of an acylimine group conjugated with the standard p-hydroxybenzylideneimidazolinone GFP chromophore (6). The additional double bond extends the size of the chromophore conjugated system leading to an increase in emission wavelength. The longer emission wavelength of RFPs makes them attractive for whole-animal imaging because cells are more transparent to red light. For imaging applications, higher emission wavelengths (650-900 nm) are desirable because they tend to minimize background absorption and light scattering by tissue components and are less damaging to cells, enabling longer acquisition times.Naturally-occurring Anthozoa RFPs, such as zRFP574 (7), eqFP578 (8), DsRed (9), and eqFP611 (10), are obligate oligomers that display emission wavelengths ranging from 574 nm to 611 nm. Significant effort has been made to monomerize and red-shift the emission wavelength of these RFPs using directed evolution. Starting from various wild-type precursors, these procedures have produced several far-red (λ em > 630 nm) monomeric RFPs such as mPlum (11), mKate2 (12), and mNeptune (13). Each of these useful monomeric RFPs was developed using random mutagenesis (5,13,14). Although directed evolution has successfully yielded red-shifted monomeric RFPs, a strictly rational methodology to red-shift Anthozoa class FPs has not yet been described. Aside from the T203Y mutation in Aequorea victor...

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Section: Contains An Additional Undesired Mutation (R125h)mentioning

confidence: 99%

Generation of longer emission wavelength red fluorescent proteins using computationally designed libraries

Chica

Moore

Allen

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

156

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“…One of these, tdTomato, is the only fluorescent protein brighter than Ypet, 9) but it has a serious limitation in that it is cytotoxic. 11) Researchers have developed non-toxic DsRed derivatives, [12][13][14] but their brightness is not comparable to that of Ypet. Hence Ypet is currently the best choice as fluorescent protein reporter.…”

Section: Fig 1 Performance Of Ypet As Reporter Proteinmentioning

confidence: 99%

A Comparison of Ypet and Firefly Luciferase as Reporter Proteins for High-Throughput Screening

Yoon

Namkung

Lee

2013

Bioscience, Biotechnology, and Biochemistry

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“…Abbreviations: DMSO 5 dimethyl sulfoxide. Adapted from Steenbergen et al, 2011 with permission from Elsevier B.V. (Strack et al, 2009), providing the opportunity to simultaneously monitor four colors. In addition to autofluorescent protein genes, research in zebrafish also makes use of small molecular fluorescent probes.…”

Section: Fluorescent Readout Technologiesmentioning

confidence: 99%

Zebrafish embryos and larvae: A new generation of disease models and drug screens

Ali

Champagne

Spaink

et al. 2011

Birth Defects Research Part C: Embryo Today: Reviews

216

135

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Technological innovation has helped the zebrafish embryo gain ground as a disease model and an assay system for drug screening. Here, we review the use of zebrafish embryos and early larvae in applied biomedical research, using selected cases. We look at the use of zebrafish embryos as disease models, taking fetal alcohol syndrome and tuberculosis as examples. We discuss advances in imaging, in culture techniques (including microfluidics), and in drug delivery (including new techniques for the robotic injection of compounds into the egg). The use of zebrafish embryos in early stages of drug safety-screening is discussed. So too are the new behavioral assays that are being adapted from rodent research for use in zebrafish embryos, and which may become relevant in validating the effects of neuroactive compounds such as anxiolytics and antidepressants. Readouts, such as morphological screening and cardiac function, are examined. There are several drawbacks in the zebrafish model. One is its very rapid development, which means that screening with zebrafish is analogous to "screening on a run-away train." Therefore, we argue that zebrafish embryos need to be precisely staged when used in acute assays, so as to ensure a consistent window of developmental exposure. We believe that zebrafish embryo screens can be used in the pre-regulatory phases of drug development, although more validation studies are needed to overcome industry scepticism. Finally, the zebrafish poses no challenge to the position of rodent models: it is complementary to them, especially in early stages of drug research.

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

Cited by 174 publications

References 15 publications

Generation of longer emission wavelength red fluorescent proteins using computationally designed libraries

Generation of longer emission wavelength red fluorescent proteins using computationally designed libraries

A Comparison of Ypet and Firefly Luciferase as Reporter Proteins for High-Throughput Screening

Zebrafish embryos and larvae: A new generation of disease models and drug screens

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