Abstracts

Beetle luciferases are very useful for bioimaging purposes, such as monitoring metastasis, bacterial and viral infections in cell cultures or animal models. In mammalian tissues, red-emitting luciferases are preferred, due to low absorption by hemoglobin, myoglobin or melanin. Among beetle luciferases, red bioluminescence is produced naturally only by P. hirtus railroadworm luciferase (PxRE) and also by some engineered luciferases. Recently, new red-emitting luciferin analogues have been synthesized, which in combination of engineered luciferases, emit far-red (FR) or near-infrared (NIR) light. Departing from the principle that PxRE luciferase is a better starting point to produce more efficient red emitting luciferases, our group has previously developed a system combining an engineered luciferase (RE-R215K) and the analogue 6′-(1-pyrrolidinyl)luciferin (N5), which emits FR light (650 nm) with very high efficiency. The aim of this work was to further improve this FR emitting system by engineering RE-R215K luciferase in combination with N5. Site-directed mutagenesis using PhusionTM High-Fidelity DNA Polymerase (Thermo Fisher) was performed, the luciferase mutants were expressed in E. coli BL-21 and purified by affinity chromatography using Ni-NTA-Agarose, and their kinetics, bioluminescence and thermostability properties were characterized. Double mutants were produced with improved properties, including higher thermostability (4 times increase of half-life at 37 ºC); ~10 nm red-shifted BL spectra (658 nm) using the N5 luciferin analogue in relation to the original mutant (650 nm), and 28 nm red-shifted in relation to D-luciferin, and a much higher bioluminescent activity with N5 analogue in relation to D-luciferin. The K_M value for N5 luciferin analogue were also very low (≤ 1 μM), whereas the KM values ​​for D-luciferin and ATP were quite high (300 and 250 μM, respectively). Compared to firefly luciferases and wild-type PxRE luciferase, this new FR mutant showed improved in vivo bioluminescence signal when expressed in monkey COS-1 fibroblasts. Considering that intracellular concentrations of ATP range from 2-8 mM depending on the cell, the higher K_M for ATP makes this luciferase an interesting option to measure physiological intracellular changes of this molecule. Altogether, these results show that this new FR emitting luciferase combination constitutes a promising reporter gene option for bioimaging purposes in mammalian tissues.