Understanding the Photophysics of the Spinach–DFHBI RNA Aptamer–Fluorogen Complex To Improve Live-Cell RNA Imaging

Han, Kyu Young; Leslie, Benjamin J.; Fei, Jingyi; Zhang, Jiaheng; Ha, Taekjip

doi:10.1021/ja411060p

Cited by 129 publications

(156 citation statements)

References 45 publications

(81 reference statements)

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“…3). Therefore, DFHBI and its associated ions and waters can more readily exchange with bulk solvent, consistent with the photophysical properties of Spinach 6,26,27 .…”

Section: Resultssupporting

confidence: 66%

“…The two K + ions are separated by 3.8 Å, as previously seen in other G-quadruplexes 24,25 . The complex topology required for the Spinach G-quadruplex to connect to flanking duplexes on both sides may explain the folding difficulties exhibited by some Spinach sequences 8,26,27 .…”

Section: Resultsmentioning

confidence: 99%

“…Spinach is swiftly photobleached, but recovers rapidly in the presence of excess DFHBI (ref. 6,26,27). GFP does not photobleach as readily, but once bleached does not recover 27 .…”

Section: Resultsmentioning

confidence: 99%

See 2 more Smart Citations

Structural basis for activity of highly efficient RNA mimics of green fluorescent protein

Warner

Chen

Song

et al. 2014

Nat Struct Mol Biol

309

442

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Green fluorescent protein (GFP) and its derivatives revolutionized the study of proteins. Spinach is a recently reported in vitro evolved RNA mimic of GFP, which as genetically encoded fusions, makes possible live-cell, real-time imaging of biological RNAs, without resorting to large RNA-binding protein-GFP fusions. To elucidate the molecular basis of Spinach fluorescence, we have solved its co-crystal structure bound to its cognate exogenous chromophore, revealing that Spinach activates the small molecule by immobilizing it between a base triple, a G-quadruplex, and an unpaired guanine. Mutational and NMR analyses indicate that the G-quadruplex is essential for Spinach fluorescence, is also present in other fluorogenic RNAs, and may represent a general strategy for RNAs to induce fluorescence of chromophores. The structure has guided the design of a miniaturized 'Baby Spinach', and provides the foundation for structure-driven design and tuning of fluorescent RNAs.

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“…3). Therefore, DFHBI and its associated ions and waters can more readily exchange with bulk solvent, consistent with the photophysical properties of Spinach 6,26,27 .…”

Section: Resultssupporting

confidence: 66%

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Structural basis for activity of highly efficient RNA mimics of green fluorescent protein

Warner

Chen

Song

et al. 2014

Nat Struct Mol Biol

309

442

View full text Add to dashboard Cite

show abstract

“…2B). Moreover, we observed that crystals melted and cracked during the experiment, suggesting conformational changes consistent with a DFHBI photo-inactivation model in which the dye isomerizes upon illumination and is rapidly ejected from the aptamer (Han et al 2013). Altogether, these results suggest that the crystallized form of the complex is representative of its native state in solution.…”

Section: Molecular Engineering and Crystallogenesissupporting

confidence: 67%

Crystal structure and fluorescence properties of the iSpinach aptamer in complex with DFHBI

Fernández-Millán¹,

Autour²,

Ennifar³

et al. 2017

RNA

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Fluorogenic RNA aptamers are short nucleic acids able to specifically interact with small molecules and strongly enhance their fluorescence upon complex formation. Among the different systems recently introduced, Spinach, an aptamer forming a fluorescent complex with the 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI), is one of the most promising. Using random mutagenesis and ultrahigh-throughput screening, we recently developed iSpinach, an improved version of the aptamer, endowed with an increased folding efficiency and thermal stability. iSpinach is a shorter version of Spinach, comprising five mutations for which the exact role has not yet been deciphered. In this work, we cocrystallized a reengineered version of iSpinach in complex with the DFHBI and solved the X-ray structure of the complex at 2 Å resolution. Only a few mutations were required to optimize iSpinach production and crystallization, underlying the good folding capacity of the molecule. The measured fluorescence half-lives in the crystal were 60% higher than in solution. Comparisons with structures previously reported for Spinach sheds some light on the possible function of the different beneficial mutations carried by iSpinach.

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“…Continuous bathing also requires little to no background fluorescence for standard wide-field imaging techniques. Rapid excitation of the fluorescent complexes can result in isomerization, photoinduced unbinding of the fluorogenic probe and diminished signals, although fluorophore recycling is a useful property for minimizing photobleaching 72 . Lastly, lower dissociation constants ( K D ) will need to be demonstrated for low-copy RNA detection, and fluorophores with improved photophysics should enable truly single-molecule imaging.…”

Section: Methods For Labeling Of Rnamentioning

confidence: 99%

Advances in fluorescence labeling strategies for dynamic cellular imaging

2014

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Synergistic advances in optical physics, probe design, molecular biology, labeling techniques and computational analysis have propelled fluorescence imaging into new realms of spatiotemporal resolution and sensitivity. This review aims to discuss advances in fluorescent probes and live-cell labeling strategies, two areas that remain pivotal for future advances in imaging technology. Fluorescent protein– and bio-orthogonal–based methods for protein and RNA imaging are discussed as well as emerging bioengineering techniques that enable their expression at specific genomic loci (for example, CRISPR and TALENs). Important attributes that contribute to the success of each technique are emphasized, providing a guideline for future advances in dynamic live-cell imaging.

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Understanding the Photophysics of the Spinach–DFHBI RNA Aptamer–Fluorogen Complex To Improve Live-Cell RNA Imaging

Cited by 129 publications

References 45 publications

Structural basis for activity of highly efficient RNA mimics of green fluorescent protein

Structural basis for activity of highly efficient RNA mimics of green fluorescent protein

Crystal structure and fluorescence properties of the iSpinach aptamer in complex with DFHBI

Advances in fluorescence labeling strategies for dynamic cellular imaging

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