Complementary assays are required to comprehensively map complex biological entities such as genomes, proteomes and interactome networks. However, how various assays can be optimally combined to approach completeness while maintaining high precision often remains unclear. Here, we propose a framework for binary protein-protein interaction (PPI) mapping based on optimally combining assays and/or assay versions to maximize detection of true positive interactions, while avoiding detection of random protein pairs. We have engineered a novel NanoLuc two-hybrid (N2H) system that integrates 12 different versions, differing by protein expression systems and tagging configurations. The resulting union of N2H versions recovers as many PPIs as 10 distinct assays combined. Thus, to further improve PPI mapping, developing alternative versions of existing assays might be as productive as designing completely new assays. Our findings should be applicable to systematic mapping of other biological landscapes.

We describeh ere an extensive structure-bioluminescence relationship studyo fachemical library of analogues of coelenterazine, using nanoKAZ/NanoLuc, am utated luciferase originated from the catalytic subunit of the deep-sea shrimp Oplophorus gracilirostris. Out of the 135 Oacetylated precursors that were prepared by using our recently reporteds ynthesis and following their hydrolysis to give solutions of the corresponding luciferins,n otable bioluminescence improvements were achieved in comparison with furimazine, whichi sc urrently amongst the best substrates of nanoKAZ/NanoLuc.F or instance, the ratherm ore lipophilic analogue 8-(2,3-difluorobenzyl)-2-((5-methylfuran-2-yl)methyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one provid-ed a1 .5-fold improvement of the total light outputo ver a 2h period, ac lose to threefold increase of the initial signal intensity and as ignal-to-background ratio five timesg reater than furimazine. The kinetic parameters for the enzymatic reactionw ere obtainedf or as electiono fl uciferina nalogues and provided unexpected insights into the luciferase activity. Most prominently,a long with ag eneral substrate-dependent and irreversible inactivation of this enzyme,i nt he case of the optimized luciferin mentioned above,t he consumption of 2664 molecules was foundt ob er equired for the detection of as ingleR elative Light Unit (RLU;aluminometer-dependentfraction of ap hoton).Scheme1.Mechanism for coelenterazine (1)b ioluminescence.[a] Dr.Supporting information and the ORCID identification number(s) for the author(s) of this articlecan be found under: https://doi.

An original three component synthetic access to coelenterazine and analogues can lead to grams of marine luciferins which are extensively used in bioluminescence-based assays.