Time-Resolved Luminescence Biosensor for Continuous Activity Detection of Protein Acetylation-Related Enzymes Based on DNA-Sensitized Terbium(III) Probes

Abstract

Protein acetylation of histone is an essential post-translational modification (PTM) mechanism in epigenetic gene regulation, and its status is reversibly controlled by histone acetyltransferases (HATs) and histone deacetylases (HDACs). Herein, we have developed a sensitive and label-free time-resolved luminescence (TRL) biosensor for continuous detection of enzymatic activity of HATs and HDACs, respectively, based on acetylation-mediated peptide/DNA interaction and Tb(3+)/DNA luminescent probes. Using guanine (G)-rich DNA-sensitized Tb(3+) luminescence as the output signal, the polycationic substrate peptides interact with DNA with high affinity and subsequently replace Tb(3+), eliminating the luminescent signal. HAT-catalyzed acetylation remarkably reduces the positive charge of the peptides and diminishes the peptide/DNA interaction, resulting in the signal on detection via recovery of DNA-sensitized Tb(3+) luminescence. With this TRL sensor, HAT (p300) can be sensitively detected with a wide linear range from 0.2 to 100 nM and a low detection limit of 0.05 nM. The proposed sensor was further used to continuously monitor the HAT activity in real time. Additionally, the TRL biosensor was successfully applied to evaluating HAT inhibition by two specific inhibitors, anacardic acid and C464, and satisfactory Z'-factors above 0.73 were obtained. Moreover, this sensor is feasible to continuously monitor the HDAC (Sirt1)-catalyzed deacetylation with a linear range from 0.5 to 500 nM and a detection limit of 0.5 nM. The proposed sensor is a convenient, sensitive, and mix-and-read assay, presenting a promising platform for protein acetylation-targeted epigenetic research and drug discovery.

Chemical Biology Approaches for Investigating the Functions of Lysine Acetyltransferases

“…Recently,N ie et al developed aT b 3+ -mediated timeresolved luminescence (TRL) biosensor for the detection of KATand KDAC activity ( Figure 11). [105] Acetylation neutralizes the positive charges on lysine residue and thus weakens histone interaction with guanine-rich ssDNA( G-rich ssDNA), the latter of which has been reported as an antenna to boost the luminescence of Tb 3+ . [106] G-rich ssDNAw as paired with peptide substrates to mimic the DNA/histone interaction.…”

“…Although most methods are sensitive,r apid, and highly quantitative,a nd some of them have the potential for use in KATi nhibitor screening in ah igh-throughput format, concerns such as inner filter effect and false-positives have to be carefully calibrated when applying these methods for KAT activity study.New materials such as nanoparticle/nanocrystal and organometallic polymers have been integrated with spectrometry-based strategies for the exploration of KAT activities. [101,[103][104][105] These probes possess advantageous characteristics such as superior optical properties and high photostability.B ioorthogonal probes [112a,c] and chemoproteo-mic profiling methods [52a, 117] offer more functional information about intracellular acetylated proteins and corresponding KATenzymes.…”

Untersuchung der epigenetischen Funktionen von Lysin‐Acetyltransferasen mit Methoden der chemischen Biologie

“…[105] Die Acetylierung neutralisiert die positiven Ladungen am Lysinrest und schwächt somit die Wechselwirkung von Histon mit Guanin-reicher ssDNA(G-reicher ssDNA), die als Antenne zur Verbesserung der Lumineszenz von Tb 3+ bekannt ist. [105] Die Acetylierung neutralisiert die positiven Ladungen am Lysinrest und schwächt somit die Wechselwirkung von Histon mit Guanin-reicher ssDNA(G-reicher ssDNA), die als Antenne zur Verbesserung der Lumineszenz von Tb 3+ bekannt ist.…”

“…Kürzlich entwickelten Nie et al einen Biosensor fürd ie Detektion von KAT-und KDAC-Aktivitäten, der auf Tb 3+vermittelter zeitaufgelçster Lumineszenz (TRL) beruht (Abbildung 11). [105] Die Acetylierung neutralisiert die positiven Ladungen am Lysinrest und schwächt somit die Wechselwirkung von Histon mit Guanin-reicher ssDNA(G-reicher ssDNA), die als Antenne zur Verbesserung der Lumineszenz von Tb 3+ bekannt ist. [106] G-reiche ssDNAwurde an Peptidsubstraten angelagert, um die DNA-Histon-Wechselwirkung nachzuahmen.…”

Precise Construction and Tuning of an Aerolysin Single-Biomolecule Interface for Single-Molecule Sensing

“…Nanopore measurements illustrate that the substitution of Q212 with arginine hinders the assembly of the nanopore, whereas replacement of positively charged R282 with glycine enhances the selectivity. Compared with wild-type (WT) aerolysin, N226Q prolongs the translocation time of poly(dA) 4 by almost a factor of two while enhancing the blockage amplitude by 25%. Therefore, the authors highlight three critical regions on the structure of aerolysin, which are responsible for pore forming, sensing selectivity, and sensitivity.…”