Molecular Association Assay Systems for Imaging Protein–Protein Interactions in Mammalian Cells DOI Creative Commons
Sung Bae Kim, Tadaomi Furuta, Suresh Thangudu

et al.

Biosensors, Journal Year: 2025, Volume and Issue: 15(5), P. 299 - 299

Published: May 8, 2025

Molecular imaging probes play a pivotal role in assaying molecular events various physiological systems. In this study, we demonstrate new genre of bioluminescent for protein–protein interactions (PPIs) mammalian cells, named the association assay (MAA) probe. The MAA probe is designed to be as simple full-length marine luciferase fused protein interest with flexible linker. This fusion alone surprisingly works by recognizing specific ligand, interacting counterpart PPI, and developing bioluminescence (BL) cells. We made use an artificial intelligence (AI) tool simulate binding modes working mechanisms. Our AlphaFold-based analysis on mode suggests that hinge region before ligand but becomes stiff after association. sensorial properties representative probes, FRB-ALuc23 FRB-R86SG, are characterized respect quantitative feature, BL spectrum, vivo tumor using xenografted mice. AI-based simulation mechanisms reveals other proteins way facilitate substrate’s access active sites (ALuc23 or R86SG). prove concept generally applicable examples, such ALuc16- R86SG-fused estrogen receptor ligand-binding domain (ER LBD). Considering versatility conceptionally unique distinctive compared conventional ones, expecting widespread application these repertoire determine PPIs living organisms.

Language: Английский

Luciferase complementation for cellular assays beyond protein–protein interactions DOI Open Access
Genki Kawamura,

Takeaki Ozawa

Analytical Sciences, Journal Year: 2025, Volume and Issue: unknown

Published: Feb. 18, 2025

Abstract Luciferase complementation assays have emerged in 2001 as a useful tool to analyze biological processes through diverse such cellular studies and vivo imaging. The assay has an advantage of wide dynamic ranges, high signal-to-noise ratios, capability for real-time monitoring events with readout bioluminescence. While it was initially harnessed detecting protein–protein interactions, biosensors based on luciferase-fragment achieved significant advancements their designs, expanding versatility applicability beyond the initial scope. This review aims provide comprehensive overview designing strategies employed split luciferase highlight bioanalytical applications. Because simple bi-molecular detection interactions by this approach is well-established, will focus introducing sensor designs using concept complementation. Graphical abstract

Language: Английский

Citations

0
Coelenterazine Analogs for Bioassays and Molecular Imaging DOI Creative Commons
Sung Bae Kim,

Genta Kamiya,

Tadaomi Furuta

et al.

Sensors, Journal Year: 2025, Volume and Issue: 25(6), P. 1651 - 1651

Published: March 7, 2025

Coelenterazine (CTZ) is a common substrate of marine luciferases upon emission bioluminescence (BL) in living organisms. Because CTZ works as “luminophore” the process BL emission, chemical modification has been centered for improving optical properties BL. In this review, we showcase recent advances designs with unique functionalities. We first elucidate light-emitting mechanisms CTZ, and then focus on how rational analogs developed years are connected to development functionalities even without luciferases, which include color tunability covering visible region, specificity various proteins (e.g., luciferase, albumin, virus protein), activatability ions or reactive oxygen species (ROS) anticancer drugs. This review provides new insights into broad utilities designed bioassays molecular imaging.

Language: Английский

Citations

0
Noninvasive Bioluminescence Imaging of Serum Albumins in Living Mice DOI
Sung Bae Kim,

Genta Kamiya,

Tadaomi Furuta

et al.

ACS Chemical Biology, Journal Year: 2025, Volume and Issue: unknown

Published: April 2, 2025

Bioluminescence (BL) is an emerging optical readout that has been extensively used in various bioassays and molecular imaging systems. In this study, we present the bioanalytical application of marine luciferins as excellent indicator for noninvasive serum albumins. We synthesized 30 kinds regioisomeric coelenterazine (CTZ) analogs investigated their specificities major proteins from species. The results found some CTZ exhibited surprisingly specific signals upon binding with These showed diverse emission spectra ranging 495 to 558 nm according albumin species acting pseudoluciferases. selective indicators, TS1 TS2, long linear dose-response curves were sensitive enough determine clinically normal abnormal (microalbuminuria) ranges albumins saliva urine. sensitivity assay superior conventional Bromocresol purple (BCP) method. further demonstrated advantages indicators through liver-albumin vivo living mice. ex confirmed analog TS2 can sensitively image high signal-to-background ratio. This study paves a new way make use conceptualizes pseudoluciferase-based imaging. distinct potentially aid clinicians providing insight into patients' liver function other vital factors needed whole-body homeostasis.

Language: Английский

Citations

0
Molecular Association Assay Systems for Imaging Protein–Protein Interactions in Mammalian Cells DOI Creative Commons
Sung Bae Kim, Tadaomi Furuta, Suresh Thangudu

et al.

Biosensors, Journal Year: 2025, Volume and Issue: 15(5), P. 299 - 299

Published: May 8, 2025

Molecular imaging probes play a pivotal role in assaying molecular events various physiological systems. In this study, we demonstrate new genre of bioluminescent for protein–protein interactions (PPIs) mammalian cells, named the association assay (MAA) probe. The MAA probe is designed to be as simple full-length marine luciferase fused protein interest with flexible linker. This fusion alone surprisingly works by recognizing specific ligand, interacting counterpart PPI, and developing bioluminescence (BL) cells. We made use an artificial intelligence (AI) tool simulate binding modes working mechanisms. Our AlphaFold-based analysis on mode suggests that hinge region before ligand but becomes stiff after association. sensorial properties representative probes, FRB-ALuc23 FRB-R86SG, are characterized respect quantitative feature, BL spectrum, vivo tumor using xenografted mice. AI-based simulation mechanisms reveals other proteins way facilitate substrate’s access active sites (ALuc23 or R86SG). prove concept generally applicable examples, such ALuc16- R86SG-fused estrogen receptor ligand-binding domain (ER LBD). Considering versatility conceptionally unique distinctive compared conventional ones, expecting widespread application these repertoire determine PPIs living organisms.

Language: Английский

Citations

0