Dataset Information

Monitoring metal-amyloid-? complexation by a FRET-based probe: design, detection, and inhibitor screening.

ABSTRACT: Aggregation of amyloidogenic peptides could cause the onset and progression of neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease. These amyloidogenic peptides can coordinate to metal ions, including Zn(ii), which can subsequently affect the peptides' aggregation and toxicity, leading to neurodegeneration. Unfortunately, the detection of metal-amyloidogenic peptide complexation has been very challenging. Herein, we report the development and utilization of a probe (A-1) capable of monitoring metal-amyloid-? (A?) complexation based on Förster resonance energy transfer (FRET). Our probe, A-1, is composed of A?1-21 grafted with a pair of FRET donor and acceptor capable of providing a FRET signal upon Zn(ii) binding even at nanomolar concentrations. The FRET intensity of A-1 increases upon Zn(ii) binding and decreases when Zn(ii)-bound A-1 aggregates. Moreover, as the FRET intensity of Zn(ii)-added A-1 is drastically changed when their interaction is disrupted, A-1 can be used for screening a chemical library to determine effective inhibitors against metal-A? interaction. Eight natural products (out of 145 compounds; >80% inhibition) were identified as such inhibitors in vitro, and six of them could reduce Zn(ii)-A?-induced toxicity in living cells, suggesting structural moieties useful for inhibitor design. Overall, we demonstrate the design of a FRET-based probe for investigating metal-amyloidogenic peptide complexation as well as the feasibility of screening inhibitors against metal-bound amyloidogenic peptides, providing effective and efficient methods for understanding their pathology and finding therapeutic candidates against neurodegenerative disorders.

SUBMITTER: Lee HJ

PROVIDER: S-EPMC6349019 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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Publications

Monitoring metal-amyloid-β complexation by a FRET-based probe: design, detection, and inhibitor screening.

Lee Hyuck Jin HJ Lee Young Geun YG Kang Juhye J Yang Seung Hyun SH Kim Ju Hwan JH Ghisaidoobe Amar B T ABT Kang Hyo Jin HJ Lee Sang-Rae SR Lim Mi Hee MH Chung Sang J SJ

Chemical science 20181206 4

Aggregation of amyloidogenic peptides could cause the onset and progression of neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease. These amyloidogenic peptides can coordinate to metal ions, including Zn(ii), which can subsequently affect the peptides' aggregation and toxicity, leading to neurodegeneration. Unfortunately, the detection of metal-amyloidogenic peptide complexation has been very challenging. Herein, we report the development and utilization of a probe (< ...[more]

PMID: 30774894

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Project description:MicroRNAs (miRNAs) have been shown to play an important role in many different cellular, developmental, and physiological processes. Accordingly, numerous methods have been established to identify and quantify miRNAs. The shortness of miRNA sequence results in a high dynamic range of melting temperatures and, moreover, impedes a proper selection of detection probes or optimized PCR primers. While miRNA microarrays allow for massive parallel and accurate relative measurement of all known miRNAs, they have so far been less useful as an assay for absolute quantification. Here, we present a microarray based approach for global and absolute quantification of miRNAs. The method relies on an equimolar pool of about 1000 synthetic miRNAs of known concentration which is used as an universal reference and labeled and hybridized in a dual colour approach on the same array as the sample of interest. Each single miRNA is quantified with respect to the universal reference outbalancing bias related to sequence, labeling, hybridization or signal detection method. We demonstrate the accuracy of the method by various spike in experiments. Further, we quantified miRNA copy numbers in liver samples and CD34(+)CD133(-) hematopoietic stem cells. Different probe designs were investigated with respect to sensitivity and selectivity of microarray hybridization. An array with 11 different variants of oligonucleotides for miR-122a and miR-16 was produced. 5 µg of respective total RNA was fluorescently labelled by 3’ ligation. Total RNA was hybridized in a dual colour approach to microarrays versus a second labelled synthetic miRNA pool. The synthetic miRNA pool consisted of 5 fmol of each of 816 non redundant miRNAs sequences and miRControl 3 sequences. Local background was subtracted from the signal to obtain the net signal intensity and the mean of the net signal intensities of 4 corresponding spots representing the same miRNA was computed for those spots only which were unflagged (empty spots, poor spots, negative spots) and for which the fluorescent intensity of the miRNAs derived from the samples of interest was two-fold the mean background value (2bkg dataset).

Dataset Information

Monitoring metal-amyloid-? complexation by a FRET-based probe: design, detection, and inhibitor screening.

Publications

Monitoring metal-amyloid-β complexation by a FRET-based probe: design, detection, and inhibitor screening.

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