The Novel Optical Biosensors For Detection Of Protein Acetylation-related Enzymes

Protein acetylationis 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）.Moreover, lysine acetylation is a prevalent modification in metabolic enzymes to regulate carbon source utilization and metabolic flux for the cellular response to environmental changes. The dysfunction of HATs causes the manifestation of several diseases, such as cancers, metabolic syndromes, and neurological disorders. Therefore, the potent bio-analytical method for detecting activity of acetylation-related enzymes and screening of their inhibitors will benefit biochemical research and pharmaceutical development.Traditional methods of probing HAT/HDAC activity relied on the autoradiography and radio isotopes, which suffer from hazards of radioactive materials. Heterogeneous detection of HAT activity has been achieved relied on enzyme-linked immunosorbant assay（ELISA）.Although the antibody recognition remains a dominant tool for the analysis of HAT/HDAC activity, some inherent shortcomings exist, such as the high batch-to-batch variability of antibodies, high cost of labeled antibodies, and the sophisticated probe preparation by modification of nanomaterials. Hence, in order to overcome these obstacles, it is highly desirable to design a new antibody-free strategy for detection of acetylation-related enzymes activity.Based on the above considerations and previousreports in the literature taking advantage of the unique characteristics of nanomaterials（gold nanoparticles, carbon nanotubes, graphene, lanthanide ion complexes, etc）, we developed several methods for rapidly antibody-free detecting HAT/HDAC. The details are summarized as follows:（1） Wedeveloped an antibody-free fluorescent method for facile detection of HAT activity based on carboxypeptidase Y（CPY） cleavage and nano-quenching ability of nanomaterials. We have firstly validated the effective suppression of exopeptidase digestion by peptide acetylation. This new finding serves as the antibody-free acetylation recognition mechanism in our biosensor design, in which lysine acetylation by HAT protects the peptide against enzymatic cleavage and retains the peptide fragment capable of binding on nano-quencher surface. Moreover, three kinds of nano-quenchers, including graphene oxide（GO）, carbon nanotubes（CNTs）, and gold nanoparticles（Au NPs）, were comprehensively compared. GO showed the best ability to selectively recognize the acetylated peptide from un-acetylated ones. With this GO-based nanosensor, HAT p300 has been sensitively detected down to 1 n M with wide linear range from 2.5 to 100 n M. This sensor was also feasible to assess HAT inhibition by anacradic acid. The proposed sensor was simple, sensitive, and cost-effective for HAT assay, presenting a promising toolkit for HAT-related biochemical research and inhibitor screening.（2） The core-shell magnetic graphitic nanocapsules（MGN） material was the graphite-coated, highly magnetic Fe Co core-shell nanoparticles. Because of its excellent quenching efficiency and magnetic enrichment property, MGN showed the best ability to selectively recognize the acetylated peptide from un-acetylated ones. By integration of CPY-based identification and MGN-derived nano-platform, a highly sensitive and selective HAT biosensor was developed.The lowest detectable concentrationof the MGN-based sensor was ten-fold lower than that using GO. Compared with the existing assays for HAT based on acetylation-specific antibodies, our system for acetylation recognition was more cost-effective, sensitive, time-saving and conquered the defect of the inconsistent batch-to-batch quality. Moreover, our work presents a first application of MGN material in peptide biosensor, implying the great potential of MGN in bio-analysis. We expect this biosensor could form a generic biosensing platform for HATs with an high promise in clinical diagnosis and anti-carcinogenic drug discovery.（3） We developed a facile fluorescent method based on FITC-peptide/ss DNA system to probe HAT activity, which mimics theacetylation-regulated histone/DNA interaction in cell nucleus. The highly positively charged FITC-peptide interacted with ss DNA to generate polyionic complex with quenching fluorescence due to the proximity-induced energy transfer between FITCs.HAT-catalyzed acetylation remarkably reduced the peptide charges and dissociated the peptide/ss DNA complex, leading to enhancement of fluorescence. For detection of HAT p300, there was a linear range from 0.5 to 100 n Mwith the detection limitof 0.1 n M（S/N =3）. For detection of HDAC Sirt 1, the linear range was from 5 to 500 n M and the detection limit was 1 n M（S/N=3）. Moreover, the biggest merit of this proposed sensor was that it can to continuously monitor the HAT and HDAC activity in real-time, presenting a promising platform for protein acetylation-targeted epigenetic research and drug discovery.（4） We have developeda sensitive and label-freetime-resolved luminescence（TRL） biosensor for continuous detection of enzymatic activity of HATs and HDACs, respectively, based on acetylation-mediated peptide/DNA interaction and Tb³⁺/DNA luminescent probe. The acetylation of the peptide probe significantly altered its intrinsic charges and subsequently regulated its capability to bind with G-rich ss DNA and replace Tb³⁺, resulting in the significant change of ss DNA-sensitized Tb³⁺ luminescence signals to reflect enzymatic activity of HAT or HDAC.With this TRL sensor, HAT（p300） can be sensitively detected with a wide linear range from 0.2 to 100 n M and a low detection limit of 0.05 n M. The proposed sensor was further used to continuously monitor the HAT activity in real-time. Additionally, the TRL biosensor was successfully applied to evaluate 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 n M and a detection limit of 0.5 n M. Moreover, the longer lifetime of the DNA-sensitized luminescence of Tb³⁺ provided an opportunity to eliminate any interference from autofluorescence and other background within the sample, increasing the specificity and signal-to-noise for detection.We expect this TRL sensing system could form a generic bio-sensing platform for HATs and HDACs with a high promise for clinical diagnosis and anti-carcinogenic drug discovery.