Abstract
A comparative study on human telomeric DNA G-quadruplex binding of meso-5,10,15,20-tetrakis(N-methyl-4-pyridyl)porphyrin (TMPyP4) between its two salt forms, i.e., tetratosylate and tetrachloride, was conducted by using ESI-TOF-MS, UV-melting measurement, and molecular modeling methods. Besides cation TMPyP4, the tosyl anion was found to bind to human telomeric DNA G-quadruplex with multiple binding stoichiometries from 1:1 to 3:1 observed in ESI-TOF-MS spectra, indicating that the stabilization activity of TMPyP4 tetratosylate on G-quadruplex is derived from a synergetic effect of both TMPyP4 cation and tosyl anion. A molecular modeling study suggests that a tosyl anion fills up the vacant space between TMPyP4 cation and DNA G-quadruplex and thus stabilizes the complex by 3.8 kcal/mol. Therefore, it is estimated that TMPyP4 tetratosylate’s activity might not reflect the real effect of TMPyP4 cation in some bioassays related to G-quadruplex stabilization. This was verified by the results of less binding affinity of TMPyP4 tetrachloride with DNA G-quadruplex obtained from ESI-TOF-MS measurement, and of 2.27 °C less thermal stabilization of TMPyP4 tetrachloride for DNA G-quadruplex, compared to its tetratosylate under the same conditions. Our study demonstrated the influence of counter ions of TMPyP4 on G-quadruplex binding, which sheds light on the proper usage of TMPyP4 salt in the chemical and biological research associated with G-quadruplex binding. Subsequently, the binding of TMPyP4 tetrachloride to human telomeric RNA G-quadruplexes was studied with ESI-TOF-MS technique. The binding constants of TMPyP4 with human telomeric G-quadruplexes indicated that TMPyP4 binds to human telomeric RNA G-quadruplex one order of magnitude stronger than DNA counterpart. This is a comprehensive mass spectrometric report on binding study of TMPyP4 with human telomeric DNA/RNA G-quadruplexes. ᅟ Electronic supplementary materialThe online version of this article (doi:10.1007/s00216-014-7943-0) contains supplementary material, which is available to authorized users.
Highlights
In order to directly observe the influence of tosyl group on the G-quadruplex binding of TMPyP4, ESI-TOF-MS was utilized to examine the binding of TMPyP4 tetratosylate with human telomeric DNA G-quadruplex d[(TTAGGG)4TTA]
Our study demonstrated the influence of counter ions of TMPyP4 on G-quadruplex binding, which shed light on the proper usage of TMPyP4 salt in the chemical and biological research associated with G-quadruplex binding
Based on the results obtained from ESI-TOF-MS, ESI-QTOFMS/MS, UV-melting measurement, and molecular modeling, it was concluded that the tosyl anion of commercial TMPyP4 tetratosylate contributes to the G-quadruplex stabilizing effect
Summary
As a wide spectrum of G-quadruplex binder, meso5,10,15,20-tetrakis(N-methyl-4-pyridyl)porphyrin (TMPyP4, see Electronic Supplementary Material Fig. S1) has been extensively studied in the aspects of G-quadruplex binding [1,2,3,4,5,6] and inducing activities [7, 8], telomerase inhibition [1, 3, 9,10,11,12], and anticancer effects [10,11,12,13,14]. The antitumor activity of TMPyP4 tetratosylate (see Electronic Supplementary Material Fig. S1) has been examined in K562 leukemic cells, retinoblastoma cell lines, and osteosarcoma cell lines. The G-quadruplex binding affinities of these two salt forms of TMPyP4 with human telomeric DNA were compared. G-quadruplex binding activity of TMPyP4 tetrachloride was quantitatively compared between two types of human telomeric G-quadruplexes, i.e., DNA and its RNA anologue, for a purpose of exploring its binding specificity with nucleic acid G-quadruplexes
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