Fluorophore‐Promoted RNA Folding and Photostability Enables Imaging of Single Broccoli‐Tagged mRNAs in Live Mammalian Cells

Li, Xing; Kim, Hyaeyeong; Litke, Jacob L.; Wu, Jiahui; Jaffrey, Samie R.

doi:10.1002/ange.201914576

Cited by 14 publications

(17 citation statements)

References 30 publications

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“…In parallel, the suggestion that the accumulation of trans -conformers with short lifetimes is at least partially responsible for the discrepancy of absorption and excitation spectra should certainly be tested on its general validity in experiments spanning the role of preparative details and truncated vs full sequence of Spinach, sequence as well as on other miniaturized aptamers as Broccoli 10 – 12 and Corn 13 , 14 . Another attractive line of experiments this paper might stimulate is along the recent developments of modified DFHBI fluorophores that carry bulky substituents 25 for slowing down internal rotation and thus cis–trans isomerisation. Finally, addressing again the present issue of absorption vs. fluorescence excitation spectra.…”

Section: Discussionmentioning

confidence: 99%

Photophysics of DFHBI bound to RNA aptamer Baby Spinach

Dao

Haselsberger

Khuc

et al. 2021

Sci Rep

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The discovery of the GFP-type dye DFHBI that becomes fluorescent upon binding to an RNA aptamer, termed Spinach, led to the development of a variety of fluorogenic RNA systems that enable genetic encoding of living cells. In view of increasing interest in small RNA aptamers and the scarcity of their photophysical characterisation, this paper is a model study on Baby Spinach, a truncated Spinach aptamer with half its sequence. Fluorescence and fluorescence excitation spectra of DFHBI complexes of Spinach and Baby Spinach are known to be similar. Surprisingly, a significant divergence between absorption and fluorescence excitation spectra of the DFHBI/RNA complex was observed on conditions of saturation at large excess of RNA over DFHBI. Since absorption spectra were not reported for any Spinach-type aptamer, this effect is new. Quantitative modelling of the absorption spectrum based on competing dark and fluorescent binding sites could explain it. However, following reasoning of fluorescence lifetimes of bound DFHBI, femtosecond-fluorescence lifetime profiles would be more supportive of the notion that the abnormal absorption spectrum is largely caused by trans-isomers formed within the cis-bound DFHBI/RNA complex. Independent of the origin, the unexpected discrepancy between absorption and fluorescence excitation spectra allows for easily accessed screening and insight into the efficiency of a fluorogenic dye/RNA system.

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Section: Discussionmentioning

confidence: 99%

Photophysics of DFHBI bound to RNA aptamer Baby Spinach

Dao

Haselsberger

Khuc

et al. 2021

Sci Rep

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“…The class of Spinach, Broccoli, Corn, and Chili are RNA mimics of green fluorescent proteins (GFP) that bind and activate derivatives of 4-hydroxybenzylidene imidazolone (HBI), a small-molecule analog of the tripeptide chromophore in GFP 13 . Spinach and Broccoli bind the difluorinated ligand DFHBI and its derivatives 5 , 14 , 15 , and emulate the deprotonated chromophore and small Stokes shift of the enhanced GFP (eGFP) 16 . The dimeric Corn aptamer activates DFHO 7 , a spectrally shifted oxime derivative of DFHBI, which resembles the chromophore of the red fluorescent protein DsRed 17 .…”

Section: Introductionmentioning

confidence: 99%

Large Stokes shift fluorescence activation in an RNA aptamer by intermolecular proton transfer to guanine

et al. 2021

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Fluorogenic RNA aptamers are synthetic functional RNAs that specifically bind and activate conditional fluorophores. The Chili RNA aptamer mimics large Stokes shift fluorescent proteins and exhibits high affinity for 3,5-dimethoxy-4-hydroxybenzylidene imidazolone (DMHBI) derivatives to elicit green or red fluorescence emission. Here, we elucidate the structural and mechanistic basis of fluorescence activation by crystallography and time-resolved optical spectroscopy. Two co-crystal structures of the Chili RNA with positively charged DMHBO+ and DMHBI+ ligands revealed a G-quadruplex and a trans-sugar-sugar edge G:G base pair that immobilize the ligand by π-π stacking. A Watson-Crick G:C base pair in the fluorophore binding site establishes a short hydrogen bond between the N7 of guanine and the phenolic OH of the ligand. Ultrafast excited state proton transfer (ESPT) from the neutral chromophore to the RNA was found with a time constant of 130 fs and revealed the mode of action of the large Stokes shift fluorogenic RNA aptamer.

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“…However, the introduction of multiple extensively structured exogenous sequences into a given RNA molecule may alter the physiological behavior of the respective molecules. This question has been addressed by Xing et al and they determined no effect of the repeated Broccoli RNA aptamer on mRNA stability [10]; quite different to our experience with it.…”

Section: Introductionmentioning

confidence: 55%

“…Single aptamer tags so far have not been successful in the tracking or quantification of single molecules though, but reminiscent of the MS2-tag concatemers, tandem arrays could greatly empower the scope of the methodology. Recently, the tandem array approach has been successfully utilized, in bacteria [9], and, with a new substrate, to visualize overexpressed RFP carrying mRNA molecules in mammalian cells [10]. However, the introduction of multiple extensively structured exogenous sequences into a given RNA molecule may alter the physiological behavior of the respective molecules.…”

Section: Introductionmentioning

confidence: 99%

Concatemeric Broccoli reduces mRNA stability, aggregates and induces p-body formation

Rink

Baptista

Hennig

et al. 2020

Preprint

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Fluorogenic aptamers are an alternative to established methodology for real-time imaging of RNA transport and dynamics. We developed Broccoli-aptamer concatemers ranging from 4 to 128 substrate-binding site repeats and characterized their behavior fused to an mCherry-coding mRNA in transient transfection, stable expression, and in recombinant cytomegalovirus infection. Concatemerization of substrate-binding sites increased Broccoli fluorescence up to a concatemer length of 16 copies, upon which fluorescence did not increase and mCherry signals declined. This was due to the combined effects of RNA aptamer aggregation, a nuclear export defect and reduced RNA stability. Unfortunately, both cellular and cytomegalovirus genomes were unable to maintain and express high Broccoli concatemer copy numbers, possibly due to recombination events. Overexpression of Broccoli-tagged mRNA led to the formation of p-bodies. However, Broccoli RNAs did not localize to these sites. Interestingly, negative effects of Broccoli concatemers could be partially rescued by introducing linker sequences in between Broccoli repeats warranting further studies. Finally, we show that even though substrate-bound Broccoli is easily photobleached, it can still be utilized in live-cell imaging by adapting a time-lapse imaging protocol.

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Fluorophore‐Promoted RNA Folding and Photostability Enables Imaging of Single Broccoli‐Tagged mRNAs in Live Mammalian Cells

Cited by 14 publications

References 30 publications

Photophysics of DFHBI bound to RNA aptamer Baby Spinach

Photophysics of DFHBI bound to RNA aptamer Baby Spinach

Large Stokes shift fluorescence activation in an RNA aptamer by intermolecular proton transfer to guanine

Concatemeric Broccoli reduces mRNA stability, aggregates and induces p-body formation

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