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Electrospray ionization mass spectrometric exploration of the high-affinity binding of three natural alkaloids with the mRNA G-quadruplex in the BCL2 5′-untranslated region

Authors

  • Wei Tan,

    1. Beijing National Laboratory for Molecular Sciences, Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, Department of Chemical Biology, College of Chemistry and Molecular Engineering, Peking University, Beijing, China
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  • Gu Yuan

    Corresponding author
    • Beijing National Laboratory for Molecular Sciences, Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, Department of Chemical Biology, College of Chemistry and Molecular Engineering, Peking University, Beijing, China
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G. Yuan, Beijing National Laboratory for Molecular Sciences, Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, Department of Chemical Biology, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China.

E-mail: guyuan@pku.edu.cn

Abstract

RATIONALE

The BCL2 gene encodes an integral outer mitochondrial membrane protein (25 kDa) which regulates the apoptotic death of cells. There is a 25-nucleotide G-rich sequence in the 5'-untranslated region (5'-UTR) of the BCL2 mRNA, which can adopt a G-quadruplex structure. Small molecules which could tightly bind to this structure have a potential function in the regulation of the expression of the BCL2 mRNA.

METHODS

The 25-mer oligonucleotide (5'-G5CCGUG4UG3AGCUG4-3') was synthesized by TaKaRa Biotechnology Co., Ltd. (TaKaRa, Dalian) with high-performance liquid chromatography (HPLC) purification. Electrospray ionization (ESI) mass spectrometry (MS) was used to probe the binding properties of natural small molecules (P) with the mRNA G-quadruplex in the BCL2 5′-UTR (BCL2Q). Collision-induced dissociation (CID) mass spectrometry and circular dichroism (CD) spectroscopy were performed to evaluate the stabilization of the mRNA G-quadruplex and its complexes.

RESULTS

The results from ESI mass spectra showed that three natural alkaloids (nitidine, palmatine, and jatrorrizine) have high binding affinities to the mRNA G-quadruplex with the binding stoichiometry ranging from 1:1 to 3:1. CID mass spectrometry results revealed that the G-quadruplex-ligand complex lost bases first rather than losing the binding molecules. Increases in the Tm values of the complexes of the G-quadruplex with the natural alkaloids in the CD melting experiments demonstrated that the three small molecules can stabilize the G-quadruplex structure.

CONCLUSIONS

Three natural small molecules were found to have very high binding affinities to the mRNA G-quadruplex and stabilize this structure. The properties of these alkaloids revealed promising potentials to regulate the expression of the BCL2 protein from the posttranscriptional pathway. Copyright © 2013 John Wiley & Sons, Ltd.

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