NanoLuc-Driven Multiplex Imaging Platforms Incorporating Novel G-series Coelenterazine Variants.

Area of Science:

• Biochemistry
• Molecular Biology
• Bioimaging

Background:

• Bioluminescence (BL) is a crucial optical readout in bioassays and molecular imaging.
• Developing novel substrates is key to enhancing BL imaging capabilities.

Purpose of the Study:

• To systematically develop advanced multiplex imaging systems using novel coelenterazine (CTZ) analogs.
• To create a versatile and tunable molecular imaging platform with superior optical properties.

Main Methods:

• Synthesis of 20 CTZ analogs (G-series) with diverse functional groups.
• Characterization of analogs for luciferase specificity, BL stability, emission spectra, kinetics, and binding modes.
• Evaluation of G-series analogs in single-chain BL probes and in vivo xenograft imaging.

Main Results:

• Most G-series CTZ analogs showed water-solubility and high specificity for NanoLuc luciferase.
• G1 analog exhibited high specificity for ALuc16 luciferase with unique blue, green, and orange emissions.
• Substrate G6 facilitated specific red BL signals in response to estrogen antagonists.
• In vivo imaging of xenografts demonstrated excellent signal-to-background ratios using G1 and G20.

Conclusions:

• The G-series CTZ analogs represent a versatile and tunable molecular imaging platform.
• These analogs possess superior optical properties suitable for both in vitro and in vivo applications.
• The developed system advances multiplex imaging capabilities in biological research.