What Is NanoLuc Luciferase and How Is It Used?

Bioluminescence, the production of light by a living organism, is driven by enzymes called luciferases. These proteins catalyze chemical reactions that release energy as light. In biological research, scientists have harnessed these light-producing systems for various applications. One such tool is NanoLuc luciferase, an engineered enzyme designed for high performance in laboratory settings.

Unveiling NanoLuc: Origin and Mechanism

NanoLuc luciferase originates from the deep-sea shrimp Oplophorus gracilirostris. Scientists engineered the shrimp’s luciferase through directed evolution to enhance its properties for research. This resulted in a small, monomeric protein of approximately 19 kilodaltons (kDa), making it significantly smaller than other common luciferases. Its compact structure is designed to minimize interference with the biological processes it is used to study.

The light-producing mechanism of NanoLuc is efficient. It catalyzes the oxidation of a specific substrate called furimazine. When the NanoLuc enzyme binds to furimazine with oxygen, it initiates a chemical reaction that results in the emission of a bright, steady, blue light with a peak wavelength around 460 nanometers. This process is independent of adenosine triphosphate (ATP).

Furimazine is a substrate developed alongside the enzyme. It was synthesized for enhanced stability and a low tendency to produce light on its own, which improves measurement quality. The combination of the engineered enzyme and its optimized substrate creates an effective bioluminescent system. This ATP-independent nature allows the system to function effectively in cellular environments where ATP levels might be low.

Distinctive Features of NanoLuc

The engineered properties of NanoLuc give it several advantages for research. Its primary feature is its brightness; the enzyme produces a luminescent signal around 100 times brighter than that of traditional luciferases like Firefly or Renilla. This allows for sensitive detection, enabling researchers to measure low levels of biological activity or use smaller quantities of material.

Beyond its brightness, NanoLuc also exhibits high physical stability, remaining functional across a broad range of temperatures and pH levels. The small 19.1 kDa size of the enzyme is another benefit. When fused to a protein being studied, its small stature is less likely to disrupt the protein’s normal function or location within the cell. This size also makes it well-suited for delivery into cells using viral vectors.

The biochemical reaction it catalyzes offers further advantages. As the reaction does not require ATP, its light output is not affected by the cell’s energy status, leading to more consistent data. The furimazine substrate also contributes to a high signal-to-background ratio. This results in a “glow-type” luminescence with a long half-life, meaning the light signal is stable for extended periods, which is beneficial for high-throughput screening.

Applications in Scientific Discovery

The unique characteristics of NanoLuc luciferase have made it a versatile tool in biological research. One of its primary uses is in reporter gene assays, where the enzyme is used as a sensitive indicator of gene expression. By linking the NanoLuc gene to a specific gene promoter, scientists can measure the promoter’s activity by quantifying the amount of light produced.

The technology is also integral to Bioluminescence Resonance Energy Transfer (BRET) assays. In a BRET experiment, NanoLuc serves as an energy donor. If a protein fused to NanoLuc comes into close proximity with another protein fused to a fluorescent acceptor molecule, energy is transferred from NanoLuc to the acceptor. This allows researchers to study protein-protein interactions, conformational changes, and the binding of drugs to their targets in living cells.

Its intense brightness makes it an excellent tool for in vivo imaging. Researchers can use NanoLuc to label cells, viruses, or other pathogens to track their movement and proliferation within a living organism in real time. This capability is valuable for studying infection processes, cancer metastasis, and the effectiveness of therapeutic interventions. The small size of the enzyme is an advantage here, as it allows for the creation of reporter viruses without disrupting their ability to infect cells.

The bright and stable signal is advantageous for high-throughput screening (HTS) in drug discovery. The consistent light output facilitates the rapid testing of thousands of chemical compounds to identify potential new drugs. Scientists have also adapted the technology to develop biosensors that can detect specific molecules or changes in the cellular environment. From virology to immunology and cell signaling, the applications of NanoLuc continue to expand.