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Red-Shifted Aequorin Variants Incorporating Non-Canonical Amino Acids: Applications in In Vivo Imaging.

Grinstead KM, Rowe L, Ensor CM, Joel S, Daftarian P, Dikici E, Zingg JM, Daunert S - PLoS ONE (2016)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, R. Bunn Gautier Bldg., 1011 NW 15th Street, Miller School of Medicine, University of Miami, Miami, FL, 33136, United States of America.

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.

No MeSH data available.


Related in: MedlinePlus

Emission wavelengths and bioluminescence half-lives of selected aequorin variants.(A) Bioluminescence emission spectra of aequorin with L-4-aminophenylalanine at position 86 with coelenterazine cp (red), aequorin with native coelenterazine (black), and L-4-methoxyphenylalanine at position 82 and 86 with coelenterazine i (green), illustrating the range of emission wavelengths in this study. (B) Half-life bioluminescence decay of aequorin with native coelenterazine (black), L-4-methoxyphenylalanine with coelenterazine n (orange), and L-4-iodophenylalanine with coelenterazine i (blue), illustrating the range of emission half-lives in this study. Reprinted from [47] under a CC BY license, with permission from Open Access Dissertations, original copyright 2015.
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pone.0158579.g004: Emission wavelengths and bioluminescence half-lives of selected aequorin variants.(A) Bioluminescence emission spectra of aequorin with L-4-aminophenylalanine at position 86 with coelenterazine cp (red), aequorin with native coelenterazine (black), and L-4-methoxyphenylalanine at position 82 and 86 with coelenterazine i (green), illustrating the range of emission wavelengths in this study. (B) Half-life bioluminescence decay of aequorin with native coelenterazine (black), L-4-methoxyphenylalanine with coelenterazine n (orange), and L-4-iodophenylalanine with coelenterazine i (blue), illustrating the range of emission half-lives in this study. Reprinted from [47] under a CC BY license, with permission from Open Access Dissertations, original copyright 2015.

Mentions: While all novel non-natural amino acid-modified aequorin variants show an increase in the half-lives of the bioluminescence emission, the most notable half-life changes are associated with the variants containing coelenterazine i. These variants display the longest bioluminescence decay half-lives with an average of approximately 1 minute, while the half-life of aequorin with native coelenterazine is approximately a half second (Table 1). Notably, the aequorin variant with a double substitution of L-4-iodophenylalanine at positions 82 and 86 paired with coelenterazine i has the longest bioluminescence half-life emission time reported as well at approximately 60 s, compared to our own previously reported 14 s with a single L-4-methoxyphenylalanine at position 82 paired with coelenterazine i (Fig 4) [46].


Red-Shifted Aequorin Variants Incorporating Non-Canonical Amino Acids: Applications in In Vivo Imaging.

Grinstead KM, Rowe L, Ensor CM, Joel S, Daftarian P, Dikici E, Zingg JM, Daunert S - PLoS ONE (2016)

Emission wavelengths and bioluminescence half-lives of selected aequorin variants.(A) Bioluminescence emission spectra of aequorin with L-4-aminophenylalanine at position 86 with coelenterazine cp (red), aequorin with native coelenterazine (black), and L-4-methoxyphenylalanine at position 82 and 86 with coelenterazine i (green), illustrating the range of emission wavelengths in this study. (B) Half-life bioluminescence decay of aequorin with native coelenterazine (black), L-4-methoxyphenylalanine with coelenterazine n (orange), and L-4-iodophenylalanine with coelenterazine i (blue), illustrating the range of emission half-lives in this study. Reprinted from [47] under a CC BY license, with permission from Open Access Dissertations, original copyright 2015.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4930207&req=5

pone.0158579.g004: Emission wavelengths and bioluminescence half-lives of selected aequorin variants.(A) Bioluminescence emission spectra of aequorin with L-4-aminophenylalanine at position 86 with coelenterazine cp (red), aequorin with native coelenterazine (black), and L-4-methoxyphenylalanine at position 82 and 86 with coelenterazine i (green), illustrating the range of emission wavelengths in this study. (B) Half-life bioluminescence decay of aequorin with native coelenterazine (black), L-4-methoxyphenylalanine with coelenterazine n (orange), and L-4-iodophenylalanine with coelenterazine i (blue), illustrating the range of emission half-lives in this study. Reprinted from [47] under a CC BY license, with permission from Open Access Dissertations, original copyright 2015.
Mentions: While all novel non-natural amino acid-modified aequorin variants show an increase in the half-lives of the bioluminescence emission, the most notable half-life changes are associated with the variants containing coelenterazine i. These variants display the longest bioluminescence decay half-lives with an average of approximately 1 minute, while the half-life of aequorin with native coelenterazine is approximately a half second (Table 1). Notably, the aequorin variant with a double substitution of L-4-iodophenylalanine at positions 82 and 86 paired with coelenterazine i has the longest bioluminescence half-life emission time reported as well at approximately 60 s, compared to our own previously reported 14 s with a single L-4-methoxyphenylalanine at position 82 paired with coelenterazine i (Fig 4) [46].

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, R. Bunn Gautier Bldg., 1011 NW 15th Street, Miller School of Medicine, University of Miami, Miami, FL, 33136, United States of America.

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.

No MeSH data available.


Related in: MedlinePlus