Conformational Dynamics of DNA G‐Quadruplex in Solution Studied by Kinetic Capillary Electrophoresis Coupled On‐line with Mass Spectrometry

ChemistryOpen

Gleb G. Mironov¹  Dr. Victor Okhonin¹  Dr. Nasrin Khan¹  Dr. Christopher M. Clouthier¹ 

[1] Department of Chemistry, University of Ottawa, 10 Marie Curie, Ottawa K1N 6N5 (Canada)

关键词: capillary electrophoresis;    DNA folding;    G-quadruplexes;    kinetics;    mass spectrometry;    structure characterization of biomolecules;   

DOI  :  10.1002/open.201400002

来源: Wiley

【 摘 要 】

Abstract

G-quadruplex-forming DNA/RNA sequences play an important role in the regulation of biological functions and development of new anticancer and anti-aging drugs. In this work, we couple on-line kinetic capillary electrophoresis with mass spectrometry (KCE-MS) to study conformational dynamics of DNA G-quadruplexes in solution. We show that peaks shift and its widening in KCE can be used for measuring rate and equilibrium constants for DNA–metal affinity interactions and G-quadruplex formation; and ion mobility mass spectrometry (IM-MS) provides information about relative sizes, absolute molecular masses and stoichiometry of DNA complexes. KCE-MS separates a thrombin-binding aptamer d[GGTTGGTGTGGTTGG] from mutated sequences based on affinity to potassium, and reveals the apparent equilibrium folding constant (K_F≈150 μm), folding rate constant (k_on≈1.70×10³ s⁻¹ m⁻¹), unfolding rate constant (k_off≈0.25 s⁻¹), half-life time of the G-quadruplex (t_1/2≈2.8 s), and relaxation time (τ≈3.9 ms at physiological 150 mm [K⁺]). In addition, KCE-MS screens for a GQ-stabilizing/-destabilizing effect of DNA binding dyes and an anticancer drug, cisplatin.

【 授权许可】

CC BY-NC-ND   
© 2014 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.