Fluorophore-NanoLuc BRET Reporters Enable Sensitive In Vivo Optical Imaging and Flow Cytometry for Monitoring Tumorigenesis

Abstract

Fluorescent proteins are widely used to study molecular and cellular events, yet this traditionally relies on delivery of excitation light, which can trigger autofluorescence, photoxicity, and photobleaching, impairing their use in vivo. Accordingly, chemiluminescent light sources such as those generated by luciferases have emerged, as they do not require excitation light. However, current luciferase reporters lack the brightness needed to visualize events in deep tissues. We report the creation of chimeric eGFP-NanoLuc (GpNLuc) and LSSmOrange-NanoLuc (OgNLuc) fusion reporter proteins coined LumiFluors, which combine the benefits of eGFP or LSSmOrange fluorescent proteins with the bright, glow-type bioluminescent light generated by an enhanced small luciferase subunit (NanoLuc) of the deep sea shrimp Oplophorus gracilirostris. The intramolecular bioluminescence resonance energy transfer (BRET) that occurs between NanoLuc and the fused fluorophore generates the brightest bioluminescent signal known to date, including improved intensity, sensitivity and durable spectral properties, thereby dramatically reducing image acquisition times and permitting highly sensitive in vivo imaging. Notably, the self-illuminating and bi-functional nature of these LumiFluor reporters enables greatly improved spatio-temporal monitoring of very small numbers of tumor cells via in vivo optical imaging and also allows the isolation and analyses of single cells by flow cytometry. Thus, LumiFluor reporters are inexpensive, robust, non-invasive tools that allow for markedly improved in vivo optical imaging of tumorigenic processes.

Near-infrared bioluminescent proteins for two-color multimodal imaging

“…Using novel coelenterazine-based substrates for violet light emission of RLuc8 and optimizing iRFPs—RLuc8 fusions, we have developed two-component NIR chimeric luciferases for multicolor multimodal BLI and FLI techniques. Due to high contrast and narrow emission peaks of the engineered NIR luciferases, they can be multiplexed with other fluorescent proteins and luciferases, such as FLuc, NanoLuc623, Lumifluors6 and Nano-Lanterns5, greatly expanding the palette of in vivo colors.…”

“…The resulting chimeric constructs can have a significantly increased brightness due to bioluminescence resonance energy transfer (BRET)56. However, currently used constructs are limited by emission spectra of donor luciferases that are commonly selected for their development (e.g., Renilla luciferase (RLuc) and Firefly luciferase (FLuc)).…”

NanoLuc: A Small Luciferase Is Brightening Up the Field of Bioluminescence

“…led to the development of a novel class of BRET imaging reporters for in vivo use, called LumiFluors. 49 Two LumiFluors were constructed, eGFP-NLuc (GpNLuc) and LSSmOrange-NanoLuc (OgNLuc) that were connected together by a flexible peptide linker between the C-terminus of the fluorophore and N-terminus of NLuc. The intramolecular BRET signal gained from these two LumiFluors resulted in the brightest bioluminescent signal known to date in A549 (non-small cell lung cancer) subcutaneous and orthotopic mice injected with furimazine.…”

A Bioluminescent Ca2+ Indicator Based on a Topological Variant of GCaMP6s

“…Based on the crystal structure of GCaMP6m (PDB ID: 3WLD, the closest crystal structure to that of GCaMP6s), the distance from the chromophore, which is located near the geometric center of the protein, to the N and C termini are 42 and 30 Å, respectively. It has been previously reported that fusion of NanoLuc to the C terminus of enhanced GFP (EGFP) results in highly efficient BRET and an emission ratio (as defined above) of 2.97, thus indicating that the normal EGFP termini are closer to the chromophore. Based on the crystal structure of EGFP (PDB ID: 2Y0G), the distance from the N and C termini of EGFP to the chromophore are 22 and 25 Å, respectively.…”