9-Aminoacridine peptide derivatives as versatile reporter systems for use in fluorescence lifetime assays

Abstract

A novel long lifetime fluorescence reporter based on 9-aminoacridine was designed, the lifetime of which can be modulated in a defined manner when in proximity to a tryptophan residue enabling fluorescence lifetime based biochemical assays to be configured.

Fluorescence lifetime plate reader: Resolution and precision meet high-throughput

“…The plate reader acquires waveforms with high precision across a range of lifetimes, from short (1.6 ns, rhodamine B) to long (12.7 ns, EDANS). While there is great interest in developing long lifetime probes to improve the resolution of lifetime assays, 15 the high precision of DWR permits the use of short lifetime probes as well. The per-well precision of lifetime measurement is routinely near 10 ps allowing us to resolve small changes in lifetime with high precision.…”

“…9 Lifetime-based assays have been used to measure macromolecular interactions, 8,10 distances, 11,12 and forces 13 as well as enzyme activity and ligand binding. [14][15][16] High-precision lifetime measurements are typically performed with time-correlated single-photon counting (TCSPC), 17 a digital method that employs low-intensity excitation at the expense of long acquisition times (typically seconds or longer to obtain signal-to-noise ≥ 100). The alternative method of direct waveform recording (DWR) acquires analog fluorescence decays in response to high-intensity a) Author to whom correspondence should be addressed.…”

Enzymatic Assays of Histone Methyltransferase Enzymes

“…In principle, FLT is defined as the average time taken for the fluorescence intensity of a fluorophore to decay from the excited state to the ground state [70]. methylated residue [71]. 5-carboxylfluorescein tracer (5-FAM) is incorporated at the C-terminus of synthetic substrate peptide for fluorescent detection.…”

Fluorescence Lifetime–Based Competitive Binding Assays for Measuring the Binding Potency of Protease Inhibitors In Vitro

“…2 Assays based on monitoring enzyme-mediated peptide modifications that directly affect the FLT of a reporter dye have been successfully introduced for routine inhibitor profiling in automated processes within the hit-to-lead and lead optimization phases for enzyme classes such as proteases, kinases, and phosphatases. [3][4][5][6][7] The assay principle presented here employs changes in FLT, enabling determination of binding potency of compounds to the active site of an enzyme through the displacement of a conjugated binding probe. Using such an approach, compound screening in cases in which enzymes exhibit only low activity with known substrates is possible.…”