Development of Credible Molecular Probes to Identify Pregnenolone-Binding Proteins and Site-Selectively Alkylate G-Quadruplex DNA
Date Issued
2015
Date
2015
Author(s)
Chen, Chien-Han
Abstract
The work presented here consists of two parts: Section I describes that pregnenolone (P5) was equipped with benzophenone photoreactive group and biotin tag at C7 position in ether linkage to explore P5-binding proteins in the stage of embryonic development of the zebrafish. Various spacer lengths and orientations of P5-photoaffinity probes had been employed to investigate the influences on the activity of in vitro tubulin polymerization. With the preservation of the biological functions as P5, P5-NBPN was used to label P5-binding proteins from zebrafish embryo lysates and the P5 binding protein (Figure 1), cytoplasmic linker protein 170 (CLIP-170), had ultimately been found by LC-MS/MS identification. The photolabeling experiments of CLIP-170 and/or its various depletion mutants showed that the binding region of P5 on CLIP-170 located in the region between aa 920-970 with remarkable labeling selectivity and specificity. Section II describes that a series of G-quadruplex (G-4)-directing alkylating agents, BMVC-CnM (n = 2, 3, and 6) and BMVC-SW, integrating BMVC with aniline mustard in spacers of various lengths or with longer bridge length to react with different G-4 structures (hybrid-2 type, antiparallel and parallel) (Figure 2). The intact alkylated adducts were elaborately characterized by electrospray ionization mass spectroscopy (ESI-MS), LC-MS, and chemical/enzymatic footprinting to determine precise alkylation sites and plausible binding profiles. These results indicated that alkylation selectivity, specificity, and reactivity are modulated by adjusting linker lengths, whereas intrastrand cross-link efficiency which showed higher cytotoxicity is determined by the distance between two reactive warheads. Our preliminary findings regarding the different distance effects on G-4-specific alkylation provide a structural foundation for the development of G-4-selective bifunctional alkylating agents.
Subjects
alkylation
G-quadruplex
DNA structure
molecular recognition
Type
thesis
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