Red-shifted aequorin variants incorporating non-canonical amino acids: Applications in in vivo imaging

Kristen M. Grinstead, Laura Rowe, Charles M. Ensor, Smita Joel, Pirouz Daftarian, Emre Dikici, Jean Marc Zingg, Sylvia Daunert

Research output: Contribution to journalArticle

16 Citations (Scopus)

Abstract

The increased importance of in vivo diagnostics has posed new demands for imaging technologies. In that regard, there is a need for imaging molecules capable of expanding the applications of current state-of-the-art imaging in vivo diagnostics. To that end, there is a desire for new reporter molecules capable of providing strong signals, are non-toxic, and can be tailored to diagnose or monitor the progression of a number of diseases. Aequorin is a non-toxic photoprotein that can be used as a sensitive marker for bioluminescence in vivo imaging. The sensitivity of aequorin is due to the fact that bioluminescence is a rare phenomenon in nature and, therefore, it does not suffer from autofluorescence, which contributes to background emission. Emission of bioluminescence in the blue-region of the spectrum by aequorin only occurs when calcium, and its luciferin coelenterazine, are bound to the protein and trigger a biochemical reaction that results in light generation. It is this reaction that endows aequorin with unique characteristics, making it ideally suited for a number of applications in bioanalysis and imaging. Herein we report the site-specific incorporation of non-canonical or non-natural amino acids and several coelenterazine analogues, resulting in a catalog of 72 cysteine-free, aequorin variants which expand the potential applications of these photoproteins by providing several red-shifted mutants better suited to use in vivo. In vivo studies in mouse models using the transparent tissue of the eye confirmed the activity of the aequorin variants incorporating L-4-iodophehylalanine and L-4-methoxyphenylalanine after injection into the eye and topical addition of coelenterazine. The signal also remained localized within the eye. This is the first time that aequorin variants incorporating non-canonical amino acids have shown to be active in vivo and useful as reporters in bioluminescence imaging.

Original languageEnglish (US)
Article numbere0158579
JournalPLoS One
Volume11
Issue number7
DOIs
StatePublished - Jul 1 2016

Fingerprint

Aequorin
Bioluminescence
image analysis
bioluminescence
Imaging techniques
Amino Acids
amino acids
Luminescent Proteins
eyes
luciferin
Molecules
chemical reactions
in vivo studies
aequorin
Cysteine
cysteine
animal models
Tissue
injection
Calcium

ASJC Scopus subject areas

  • Agricultural and Biological Sciences(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Medicine(all)

Cite this

Red-shifted aequorin variants incorporating non-canonical amino acids : Applications in in vivo imaging. / Grinstead, Kristen M.; Rowe, Laura; Ensor, Charles M.; Joel, Smita; Daftarian, Pirouz; Dikici, Emre; Zingg, Jean Marc; Daunert, Sylvia.

In: PLoS One, Vol. 11, No. 7, e0158579, 01.07.2016.

Research output: Contribution to journalArticle

Grinstead, KM, Rowe, L, Ensor, CM, Joel, S, Daftarian, P, Dikici, E, Zingg, JM & Daunert, S 2016, 'Red-shifted aequorin variants incorporating non-canonical amino acids: Applications in in vivo imaging', PLoS One, vol. 11, no. 7, e0158579. https://doi.org/10.1371/journal.pone.0158579
Grinstead, Kristen M. ; Rowe, Laura ; Ensor, Charles M. ; Joel, Smita ; Daftarian, Pirouz ; Dikici, Emre ; Zingg, Jean Marc ; Daunert, Sylvia. / Red-shifted aequorin variants incorporating non-canonical amino acids : Applications in in vivo imaging. In: PLoS One. 2016 ; Vol. 11, No. 7.
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