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Engineered red Opto-mGluR6 Opsins, a red-shifted optogenetic excitation tool, an in vitro study.

Authors :
Shamsnajafabadi, Hoda
Soheili, Zahra-Soheila
Sadeghi, Mehdi
Samiee, Shahram
Ghasemi, Pouria
Zibaii, Mohammad Ismail
Gholami Pourbadie, Hamid
Ahmadieh, Hamid
Ranaei Pirmardan, Ehsan
Salehi, Najmeh
Samiee, Dorsa
Kashanian, Ali
Source :
PLoS ONE. 10/24/2024, Vol. 19 Issue 10, p1-19. 19p.
Publication Year :
2024

Abstract

Degenerative eye diseases cause partial or complete blindness due to photoreceptor degeneration. Optogenetic gene therapy is a revolutionary technique combining genetics and optical methods to control the function of neurons. Due to the inherent risk of photochemical damage, the light intensity necessary to activate Opto-mGluR6 surpasses the safe threshold for retinal illumination. Conversely, red-shifted lights pose a significantly lower risk of inducing such damage compared to blue lights. We designed red-shifted Opto-mGluR6 photopigments with a wide, red-shifted working spectrum compared to Opto-mGluR6 and examined their excitation capability in vitro. ROM19, ROM18 and ROM17, red-shifted variants of Opto-mGluR6, were designed by careful bioinformatics/computational studies. The predicted molecules with the best scores were selected, synthesised and cloned into the pAAV-CMV-IRES-EGFP vector. Expression of constructs was confirmed by functional assessment in engineered HEK-GIRK cells. Spectrophotometry and patch clamp experiments demonstrated that the candidate molecules were sensitive to the desired wavelengths of the light and directly coupled light stimuli to G-protein signalling. Herein, we introduce ROM17, ROM18 and ROM19 as newly generated, red-shifted variants with maximum excitation red-shifted of ~ 40nm, 70 nm and 126 nm compared to Opto-mGluR6. [ABSTRACT FROM AUTHOR]

Details

Language :
English
ISSN :
19326203
Volume :
19
Issue :
10
Database :
Academic Search Index
Journal :
PLoS ONE
Publication Type :
Academic Journal
Accession number :
180472760
Full Text :
https://doi.org/10.1371/journal.pone.0311102