Bioluminescence, photophysical, computational and molecular docking studies of fully conformationally restricted enamine infraluciferin

A new rationally designed fully rotationally restricted luciferin has been synthesised. This synthetic luciferin, based upon the structure of infraluciferin, has two intramolecular H-bonds to reduce degrees of freedom, an amine group to enhance ICT process, and an alkenyl group to increase π-conjugation. In the spectroscopic measurements and computational calculations, enamine luciferin showed more red-shifted absorption and fluorescence emission than LH2 and iLH2. With PpyWT luciferase enamine luciferin gave bioluminescence at 564 nm which is similar to LH2 at 561 nm. Further investigation by docking studies revealed that the emission wavelength of enamine luciferin might be attributed to the unwanted twisted structure caused by Asp531 within the enzyme. With mutant luciferase FlucRed, the major emission peak was shifted to 606 nm, a distinct shoulder above 700 nm, and 21% of its spectrum located in the nIR range

Smartphone-based multicolor bioluminescent 3D spheroid biosensors for monitoring inflammatory activity

Whole-cell biosensors present many advantages, including being able to monitor the toxicity and bioavailability of chemicals; cells grown in traditional 2D cultures, however, do not reproduce the complexity of in vivo physiology. In the last years, 3D cell-culture models have garnered great attention due to their capability to better mimic in vivo cellular responses to external stimuli, providing excellent model living organisms. In order to obtain a predictive, sensitive, and robust yet low-cost 3D cell biosensor, we developed a smartphone-based bioluminescent 3D cell biosensor platform for effect-based analysis. We exploited the Nuclear Factor-kappa B (NF-kB) signal transduction pathway, which is induced by several types of stressors and is involved in the regulation of cell-cycle/growth, inflammation, apoptosis, and immunity. The smartphone-based biosensor relies on immobilized HEK293 spheroids genetically engineered with powerful red- and green-emitting luciferases utilized as inflammation and viability reporters. It provides a limit of detection for Tumor Necrosis Factor (TNF\u3b1) of 0.15\u202f\ub1\u202f0.05\u202fng/mL and could be a useful tool to initially screen environmental samples or other compounds on-site, especially for additional more accurate chemical analyses

A higher spectral range of beetle bioluminescence with infraluciferin

Coleopteran bioluminescence is unique in that beetle luciferases emit colors ranging between green (ca.550 nm) and red (ca.600 nm), including intermediate colors such as yellow and orange, allowing up to 3 simultaneous parameters to be resolved in vitro with natural luciferin (D-LH2). Here, we report a more than doubling of the maximum bioluminescence wavelength range using a single synthetic substrate, infraluciferin (iLH2). We report that different luciferases can emit colors ranging from visible green to near-infrared (nIR) with iLH2, including in human cells. iLH2 was designed for dual color far-red to nIR bioluminescence imaging (BLI) in small animals and has been utilized in different mouse models of cancer (including a metastatic hepatic model showing detailed hepatic morphology) and for robust dual parameter imaging in vivo (including in systemic hematological models). Here, we report the properties of different enzymes with iLH2: Lampyrid wild-type (WT) Photinus pyralis (Ppy) firefly luciferase, Ppy-based derivatives previously engineered to be thermostable with D-LH2, and also color-shifted Elaterid-based enzymes: blue-shifted Pyrearinus termitilluminans derivative Eluc (reported D-LH2 λmax = 538 nm) and red-shifted Pyrophorus plagiopthalamus derivative click beetle red (CBR) luciferase (D-LH2 λmax = 618 nm). As purified enzyme, in bacteria or in human cells, Eluc emitted green light (λmax = 536 nm) with DL-iLH2 whereas Ppy Fluc (λmax = 689 nm), x2 Fluc (λmax = 704 nm), x5 Fluc (λmax = 694 nm), x11 Fluc (λmax = 694 nm) and CBR (λmax = 721 nm) produced far-red to nIR peak wavelengths. Therefore, with iLH2, enzyme λmaxes can be separated by ca.185nm, giving almost non-overlapping spectra. This is the first report of single-substrate bioluminescence color emission ranging from visible green to nIR in cells and may help shed light on the color tuning mechanism of beetle luciferases. We also report on the reason for the improvement in activity of x11 Fluc with iLH2 and engineer an improved infraluciferase (iluc) based on this mutant

Bioheterojunction Effect on Fluorescence Origin and Efficiency Improvement of Firefly Chromophores

We propose the heterojunction effect in the analysis of the fluorescence mechanism of the firefly chromophore. Following this analysis, and with respect to the HOMO-LUMO gap alignment between the chromophore's functional fragments, three main heterojunction types (I, II, and I*) are identified. Time-dependent density-functional theory optical absorption calculations for the firefly chromophore show that the strongest excitation appears in the deprotonated anion state of the keto form. This can be explained by its high HOMO-LUMO overlap due to strong bio-heterojunction confinement. It is also found that the nitrogen atom in the thiazolyl rings, due to its larger electronegativity, plays a key role in the emission process, its importance growing when HOMO and LUMO overlap at its location. This principle is applied to enhance the chromophore's fluorescence efficiency and to guide the functionalization of molecular optoelectronic devices.Comment: 7 pages, 6 figure

Systematic Comparison of Beetle Luciferase-Luciferin Pairs as Sources of Near-Infrared Light for In Vitro and In Vivo Applications

Luciferases catalyze light-emitting reactions that produce a rainbow of colors from their substrates (luciferins), molecular oxygen, and often additional cofactors. These bioluminescence (BL) systems have afforded an incredible variety of basic research and medical applications. Driven by the importance of BL-based non-invasive animal imaging (BLI) applications, especially in support of cancer research, new BL systems have been developed by engineering beetle luciferase (Luc) variants and synthetic substrate combinations to produce red to near-infrared (nIR) light to improve imaging sensitivity and resolution. To stimulate the application of BLI research and advance the development of improved reagents for BLI, we undertook a systematic comparison of the spectroscopic and BL properties of seven beetle Lucs with LH2 and nine substrates, which included two new quinoline ring-containing analogs. The results of these experiments with purified Luc enzymes in vitro and in live HEK293T cells transfected with luc genes have enabled us to identify Luc/analog combinations with improved properties compared to those previously reported and to provide live cell BL data that may be relevant to in vivo imaging applications. Additionally, we found strong candidate enzyme/substrate pairs for in vitro biomarker applications requiring nIR sources with minimal visible light components. Notably, one of our new substrates paired with a previously developed Luc variant was demonstrated to be an excellent in vitro source of nIR and a potentially useful BL system for improved resolution in BLI

Chapter 13 - Biotechnological Improvements of Bioluminescent Systems

Genetic and biomolecular engineering are two of the leading disciplines in biotechnology that have lead to great advancements in protein engineering. Applications of analytical bioluminescence, such as genetic reporter assays, optical in vivo imaging, and cell viability assays can often be improved by enhancing wild-type bioluminescent systems. The ability to rationally or randomly modify proteins has expanded their employment in various bioanalytical applications. Specifically, a wide range of bioluminescent proteins and photoproteins have been engineered that can be utilized in many detection and diagnostic applications. Herein, we focus on the improvements of two of the most commonly studied photoproteins, aequorin and obelin, and their uses in a variety of bioanalytical applications. Techniques such as random mutagenesis, site-directed mutagenesis, bioluminescence resonance energy transfer, and the incorporation of coelenterazine analogues are discussed as ways that have expanded the palette of these designer proteins by altering their emission wavelengths and/or half-lifes. Strategic amino acid substitutions and insertions have been also used to improve luciferase stability in high temperature, extreme pH, and harsh chemical environments, and to customize their kinetic properties and bioluminescence colors. As researchers advance engineering techniques to expand the array of photoproteins, luciferases from fireflies, click beetles, marine organisms and bacteria, their use in bioanalytical applications will continue to grow and it is envisioned that photoproteins and bioluminescent proteins will become as diverse as their fluorescence counterparts