Red-shifted bioluminescence reporters are desirable for biological imaging. We describe the development of red-shifted luciferins based on synthetic coelenterazine analogs and corresponding mutants of NanoLuc that enable bright bioluminescence. One pair in particular shows superior sensitivity over other commonly used bioluminescence reporters in vitro and in vivo. This pair was adapted to develop a bioluminescence resonance energy-based Antares reporter called Antares2, which offers improved signal from deep tissues.

Coelenterazine (CTZ)-utilizing marine luciferases and their derivatives have attracted significant attention because of their ATP-independency, fast enzymatic turnover, and high bioluminescence brightness. However, marine luciferases typically emit blue photons and their substrates, including CTZ and the recently developed diphenylterazine (DTZ), have poor water solubility, hindering their in vivo applications. Herein, we report a family of pyridyl CTZ and DTZ analogs that exhibit spectrally shifted emission and improved water solubility. Through directed evolution, we engineered a LumiLuc luciferase with broad substrate specificity. In the presence of corresponding pyridyl substrates (i.e., pyCTZ, 6pyDTZ, or 8pyDTZ), LumiLuc generates highly bright blue, teal, or yellow bioluminescence. We compared our LumiLuc-8pyDTZ pair with several benchmark reporters in a tumor xenograft mouse model. Our new pair, which does not need organic cosolvents for in vivo administration, surpasses other reporters by detecting early tumors. We further fused LumiLuc to a red fluorescent protein, resulting in a LumiScarlet reporter with further red-shifted emission and enhanced tissue penetration. LumiScarlet-8pyDTZ was comparable to Akaluc-AkaLumine, the brightest ATP-dependent luciferase-luciferin pair, for detecting cells in deep tissues of mice. In summary, we have engineered a new family of ATP-independent bioluminescent reporters, which will have broad applications because of their ATP-independency, excellent biocompatibility, and superior in vivo sensitivity.

Boronic acid and esters have been extensively utilized for molecular recognition and chemical sensing. We recently reported a genetically encoded peroxynitrite (ONOO−)-specific fluorescent sensor, pnGFP, based on the incorporation of a boronic acid moiety into a circularly permuted green fluorescent protein (cpGFP) followed by directed protein evolution. Different from typical arylboronic acids and esters, the chromophore of pnGFP is unreactive to millimolar concentrations of hydrogen peroxide (H2O2). The focus of this study is to explore the mechanism for the observed unusual chemoselectivity of pnGFP toward peroxynitrite over hydrogen peroxide by using site-directed mutagenesis, X-ray crystallography, 11B NMR, and computational analysis. Our data collectively support that a His residue on the protein scaffold polarizes a water molecule to induce the formation of an sp3-hybridized boron in the chromophore, thereby tuning the reactivity of pnGFP with various reactive oxygen and nitrogen species (ROS/RNS). Our study demonstrates the first example of tunable boron chemistry in a folded nonnative protein, which offers wide implications in designing selective chemical probes.

Intein-mediated protein splicing has found broad biotechnological applications. Herein, we describe our recent result in engineering a photoactivatable intein compatible with living mammalian cells. A photocaged cysteine amino acid residue was genetically introduced into a highly efficient Nostoc punctiforme (Npu) DnaE intein. The resulting photocaged intein was inserted into a red fluorescent protein (RFP) mCherry and a human Src tyrosine kinase to create inactive chimeric proteins. A light-induced photochemical reaction was able to reactivate the intein and trigger protein splicing. Active mCherry and Src were formed as observed by direct fluorescence imaging or imaging of an Src kinase sensor in mammalian cells. The genetically encoded photocaged intein is a general optogenetic tool, allowing effective photocontrol of primary structures and functions of proteins.

Single-electron oxidation and α-deprotonation of tertiary anilines using Fe­(phen)3(PF6)3 afford α-aminoalkyl radicals, which can be coupled with electrophilic partners to afford various tetrahydroquinolines. Mechanistically, the Fe­(phen) n 2+/3+ catalytic cycle is maintained by O2 or a TBHP oxidant, and the presence of the oxygen bound iron complex, Fe­(III)–OO­(H), was elucidated by electron paramagnetic resonance and electrospray ionization mass spectrometry. This redox-selective nonheme iron catalyst behaves similarly to bioinspired heme iron catalysts.