Functional artificial luciferases as an optical readout for bioassays

Area of Science:

• Biochemistry
• Molecular Biology
• Biotechnology

Background:

• Development of novel bioluminescent reporters is crucial for sensitive bioassays and molecular imaging.
• Existing luciferases have limitations in stability, optical intensity, and applicability in various assays.
• Artificial enzymes offer a platform for engineering tailored functionalities.

Purpose of the Study:

• To design and synthesize novel functional artificial luciferases (ALucs) with enhanced properties.
• To validate the utility of ALucs in live-cell applications, purification, and immunological assays.
• To predict the structural basis for the enhanced performance of ALucs.

Main Methods:

• Sequence modification of previously reported ALucs based on multi-sequence alignment and hydrophobicity analysis.
• In vitro and live-cell stability assessments.
• Optical intensity measurements using a coelenterazine derivative (CTZ i).
• Bioinformatic prediction of supersecondary structure using X-ray crystallographic data.

Main Results:

• Synthesized ALucs demonstrated stability in live cells and culture media for 25 days post-secretion.
• Engineered ALucs exhibited 9400-fold higher optical intensity compared to Renilla reniformis luciferase 8.6-535 with CTZ i.
• Epitopes on ALucs were exposed upon secretion, enabling column purification and immunological assays.
• Structural prediction indicated ALuc30's capacity to accommodate the CTZ i iodide.

Conclusions:

• Novel functional artificial luciferases (ALucs) were successfully synthesized for advanced bioassays and molecular imaging.
• The engineered ALucs offer superior stability, optical signal, and assay compatibility.
• Bioinformatic approaches can guide the rational design and structural prediction of artificial enzymes.