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  1. Article ; Online: A novel molecular rotor facilitates detection of p53-DNA interactions using the Fluorescent Intercalator Displacement Assay

    Walter L. Goh / Min Yen Lee / Ting Xiang Lim / Joy S. Chua / Sydney Brenner / Farid J. Ghadessy / Yin Nah Teo

    Scientific Reports, Vol 8, Iss 1, Pp 1-

    2018  Volume 13

    Abstract: Abstract We have investigated the use of fluorescent molecular rotors as probes for detection of p53 binding to DNA. These are a class of fluorophores that undergo twisted intramolecular charge transfer (TICT). They are non-fluorescent in a freely ... ...

    Abstract Abstract We have investigated the use of fluorescent molecular rotors as probes for detection of p53 binding to DNA. These are a class of fluorophores that undergo twisted intramolecular charge transfer (TICT). They are non-fluorescent in a freely rotating conformation and experience a fluorescence increase when restricted in the planar conformation. We hypothesized that intercalation of a molecular rotor between DNA base pairs would result in a fluorescence turn-on signal. Upon displacement by a DNA binding protein, measurable loss of signal would facilitate use of the molecular rotor in the fluorescent intercalator displacement (FID) assay. A panel of probes was interrogated using the well-established p53 model system across various DNA response elements. A novel, readily synthesizable molecular rotor incorporating an acridine orange DNA intercalating group (AO-R) outperformed other conventional dyes in the FID assay. It enabled relative measurement of p53 sequence-specific DNA interactions and study of the dominant-negative effects of cancer-associated p53 mutants. In a further application, AO-R also proved useful for staining apoptotic cells in live zebrafish embryos.
    Keywords Medicine ; R ; Science ; Q
    Subject code 500
    Language English
    Publishing date 2018-08-01T00:00:00Z
    Publisher Nature Publishing Group
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  2. Article: Rapid colorimetric detection of p53 protein function using DNA-gold nanoconjugates with applications for drug discovery and cancer diagnostics

    Assah, Enock / David Lane / Farid Ghadessy / Jun Li / Ting Xiang Lim / Walter Goh / Xin Ting Zheng / Yen Nee Tan

    Colloids and surfaces. 2018 Sept. 01, v. 169

    2018  

    Abstract: The tumor suppressor protein p53 plays a central role in preventing cancer through interaction with DNA response elements (REs) to regulate target gene expression in cells. Due to its significance in cancer biology, relentless efforts have been directed ... ...

    Abstract The tumor suppressor protein p53 plays a central role in preventing cancer through interaction with DNA response elements (REs) to regulate target gene expression in cells. Due to its significance in cancer biology, relentless efforts have been directed toward understanding p53-DNA interactions for the development of cancer therapeutics and diagnostics. In this paper, we report a rapid, label-free and versatile colorimetric assay to detect wildtype p53 DNA-binding function in complex solutions. The assay design is based on a concept that alters interparticle-distances between RE-AuNPs from a crosslinking effect induced through tetramerization of wildtype p53 protein (p53-WT) upon binding to canonical DNA motifs modified on gold nanoparticles (RE-AuNPs). This leads to a visible solution color change from red to blue, which is quantifiable by the UV- visible absorption spectra with a detection limit of 5 nM. Contrastingly, no color change was observed for the binding-deficient p53 mutants and non-specific proteins due to their inability to crosslink RE-AuNPs. Based on this sensing principle, we further demonstrate its utility for fast detection of drug-induced DNA binding function to cancer-associated Y220C mutant p53 protein using well-established reactivating compounds. By exploiting the dominant-negative property of mutant p53 over p53-WT and interactions with RE-AuNPs, this assay is configurable to detect low numbers of mutant p53 expressing cells in miniscule sample fractions obtained from typical core needle biopsy-sized tissues without signal attrition, alluding to the potential for biopsy sampling in cancer diagnostics or for defining cancer margins. This nanogold enabled colorimetric assay provides a facile yet robust method for studying important parameters influencing p53-DNA interactions with great promises for clinically pertinent applications.
    Keywords biopsy ; colloids ; color ; colorimetry ; crosslinking ; diagnostic techniques ; DNA ; drugs ; gene expression ; mutants ; nanogold ; neoplasms ; nucleotide motifs ; response elements ; spectral analysis ; tumor suppressor protein p53
    Language English
    Dates of publication 2018-0901
    Size p. 214-221.
    Publishing place Elsevier B.V.
    Document type Article
    ZDB-ID 1500523-9
    ISSN 1873-4367 ; 0927-7765
    ISSN (online) 1873-4367
    ISSN 0927-7765
    DOI 10.1016/j.colsurfb.2018.05.007
    Database NAL-Catalogue (AGRICOLA)

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