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Development of more efficient far red light emitting luciferases for bioanalytical purposes and bioimaging uses

Grant number: 22/03538-0
Support Opportunities:Scholarships in Brazil - Doctorate (Direct)
Effective date (Start): August 01, 2022
Effective date (End): July 31, 2026
Field of knowledge:Biological Sciences - Biophysics - Radiology and Photobiology
Principal Investigator:Vadim Viviani
Grantee:Gabriel Felder Pelentir
Host Institution: Centro de Ciências e Tecnologias para a Sustentabilidade (CCTS). Universidade Federal de São Carlos (UFSCAR). Sorocaba , SP, Brazil

Abstract

Bioluminescence, the emission of cold light by organisms for the purpose of biological communication, has been used for bioanalytical purposes for several decades. The beetle luciferin-luciferase system is certainly the most used, being used as bioanalytical reagents, and the genes that encode its luciferases as reporters of biological and pathological processes, such as gene expression, bioimaging of mammalian cells in processes such as cancer, screening for viral, bacterial and fungal infections and drug selection by the pharmaceutical industry. However, the absorption spectrum of mammalian tissues limits the effectiveness of using luciferases emitting green-yellow light, due to absorption of light in the blue to yellow range. For this purpose, brighter luciferases with spectra increasingly shifted towards the red region are needed. Phrixothrix hirtus luciferase is the only one that naturally presents a red emission peak (623 nm), however, this enzyme has a lower bioluminescent activity than traditional firefly luciferases, and the spectrum is not in the far red region, as would be desirable. Some luciferin analogs that emit far red light, including commercial ones, have been synthesized and are being used with modified luciferases, however, the bioluminescent signal of these systems is still low when compared to wild systems. Recently, using P.hirtus luciferase, we developed for the first time a combination of mutant luciferase and luciferin analogue with high brightness and far red emission (>650nm), with properties superior to the commercial Akalumine system. However, we believe that this system can still be improved, through the use of protein engineering and combination with 6'-amino-analogs. Thus, in this project we plan to develop by directed evolution and combinatorial chemistry with 6'amino-analogs, an even more efficient far red light emitting luciferase, in addition to deepening the understanding between structure and function in this luciferase. (AU)

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