Transfection of cells with aplasmid encoding for the first ten strands of the GFP protein (GFP1-10) provides the means to detect cytosolic peptide import at low micromolar concentrations.C ytosolic import of the eleventh strand of the GFP protein either by electroporation or by cell-penetrating peptide-mediated import leads to formation of the full-length GFP protein and fluorescence.A ni ncrease in sensitivity is achieved through structural modifications of the peptide and the expression of GFP1-10 as af usion protein with mCherry.

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Arguably, fluorescence-based assays are on the rise and, stepby-step, will replace alternative radioactive or absorptionbased assays on a laboratory microscale as well in highthroughput screening (HTS) for the pharmaceutical industry. [1][2][3][4][5][6] One of the more recent developments is the use of time-resolved fluorescence (TRF) assays, in which a long fluorescence lifetime is utilized to distinguish the probe fluorescence from any other short-lived background emission. [7][8][9][10] Another independent trend is the use of fluorescent peptide substrates, which are based on intramolecular probe/quencher contact, [11][12][13][14] rather than on quenching by conventional fluorescence resonance energy transfer (FRET). [15][16][17][18] Herein, we introduce an assay type that combines the assets of time-resolved fluorescence detection with the advantages of collision-induced quenching and the use of a highly biocompatible fluorophore:We have previously introduced DBO derivatives as an unconventional class of fluorescent probes; they have been termed fluorazophores.[19] The most remarkable feature is their exceedingly long fluorescence lifetime (up to 1 ms, l max = 430 nm, f f = 0.26), [20] which we have previously exploited for mechanistic investigations in the area of supramolecular and biomolecular chemistry, namely for the determination of the association kinetics of water-soluble host molecules, [21][22][23][24][25] the diffusional properties of antioxidants in membrane models, [26][27][28] and the kinetics of oligonucleotide and polypeptide folding. [29][30][31] This study describes an implementation of these basic research efforts for fluorescence-based assays with immediate biochemical relevance for studies on enzymatic activity and drug discovery.TRF assays have become popular for lanthanide emitters. Their fluorescence, which should be more generally referred to as luminescence due to the nature of the electronic transitions involved, has lifetimes in the ms range that can be easily detected, given the presence of suitable enhancement agents or chelation, and differentiated from background emission. [7][8][9][10] This background is ubiquitous and originates from unconverted substrate, scattered excitation light, sample container materials, enzyme cofactors, impurities, and, most relevant for inhibitor screening and HTS, library compounds to be screened for biological activity. [5] When employing genuine fluorescence of organic chromophores, fluorescence lifetimes above 100 ns and time gates in the nanosecond range are required to allow the same measurement mode, which we refer to as Nano-TRF. Resting on the possibility of laser excitation, highly sensitive detection, and electronic time gating, the pertinent lifetime regime is sufficiently long to allow an accurate discrimination from unconverted substrate as well as background. DBO presents the first organic fluorophore to be utilized for Nano-TRF assays, and DBO-labeled asparagine ("Dbo"), which was previously synthesized in Fmoc-protected form, [29] c...

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A GA-based evolutionary selection is a valuable combinatorial optimization strategy to discover compounds with desired properties without needing to synthesize and test all possible combinations (i.e. all molecules). GAs are especially powerful when dealing with very large combinatorial libraries for which synthesis and screening of all members is not possible and/or when only a small number of compounds compared with the library size can be synthesized or tested. The optimization gradient or 'learning' per individual increases when using smaller population sizes and decreases for higher mutation rates.