Utilization of circular dichroism and electrospray ionization mass spectrometry to understand the formation and conversion of G-quadruplex DNA at the human c-myb proto-oncogene.

G-quadruplex DNAs are involved in a number of key biological processes, including gene expression, transcription, and apoptosis. The c-myb oncogene contains a number of GGA repeats in its promoter which forms G-quadruplex, thus it could be used as a target in cancer therapeutics. Several in-vitro studies have used Circular Dichroism (CD) spectroscopy or electrospray ionization mass spectrometry (ESI-MS) to demonstrate formation and stability of G-quadruplex DNA structure in the promoter region of human c-myb oncogene. The factors affecting the c-myb G-quadruplex structures were investigated, such as cations (i.e. K+, NH4+ and Na+) and co-solutes (methanol and polyethylene glycol). The results indicated that the presence of cations and co-solutes could change the G-quadruplex structural population and promote its thermodynamic stabilization as indicated by CD melting curves. It indicated that the co-solutes preferentially stabilize the c-myb G-quadruplex structure containing both homo- and hetero-stacking. In addition, protopine was demonstrated as a binder of c-myb G-quadruplex as screened from a library of natural alkaloids using ESI-MS method. CD spectra showed that it could selectively stabilize the c-myb G-quadruplex structure compared to other six G-quadruplexes from tumor-related G-rich sequences and the duplex DNAs (both long and short-chain ones). The binding of protopine could induce the change in the G-quadruplex structural populations. Therefore, protopine with its high binding specificity could be considered as a precursor for the design of drugs to target and regulate c-myb oncogene transcription.

The formation and characteristics of the i-motif structure within the promoter of the c-myb proto-oncogene.

C-myb proto-oncogene is a potential therapeutic target for some human solid tumors and leukemias. A long cytosine-rich sequence, which locates the downstream of the transcription initiation site, is demonstrated to fold into an intramolecular i-motif DNA using electrospray ionization mass spectrometry (ESI-MS) and circular dichroism (CD) spectroscopy. Effects of factors, including the pH value, the number of C:C(+) dimers, the concentration of buffer, the molecular crowding condition, and the coexistence of the complementary DNA, on the formation and the structural stability of the i-motif DNA are systematically studied. We have demonstrated that the i-motif folding in the c-myb promoter could be accelerated upon synergistic physiological stimuli including intracellular molecular crowding and low pH values, as well as the large number of the i-motif C:C(+) dimers. Meanwhile, various inputs, such as acids/bases and metal ions, have exhibited their abilities in controlling the conformational switch of the c-myb GC-rich DNA. Acidic pH values and the presence of K(+) ions can induce the dissociation of the double helix. Our present strategy can greatly extend the potential usages of i-motif DNA molecules with specific sequences as conformational switch-controlled devices. Moreover, this work demonstrates the superiority of CD spectroscopy associated with ESI-MS as a rapid, more cost-effective and sensitive structural change responsive method in the research of DNA conformational switching.

Exploration of binding affinity and selectivity of brucine with G-quadruplex in the c-myb proto-oncogene by electrospray ionization mass spectrometry.

RATIONALE: The c-myb gene is a potential therapeutic target for human tumors and leukemias. Active ingredients from natural products may be used as drugs in chemotherapy for human cancers. Here, electrospray ionization mass spectrometry (ESI-MS) was used to probe the formation and recognition of the G-quadruplex structure from the G-rich sequence that is found in the c-myb gene promoter, 5'-GGGCTGGGCTGGGCGGGG-3'. The aim of our study is to evaluate a potential binder for the c-myb gene from natural products, and thereby to modulate c-myb gene expression. METHODS: ESI-MS, as an effective method, was utilized not only to characterize the formation of the G-quadruplex in the c-myb oncogene, but also as a tool to probe the binding characteristics of alkaloid molecules with the target G-quadruplex DNA. RESULTS: ESI-MS results with the support of circular dichroism (CD) spectra demonstrated the formation of an intramolecular parallel-stranded G-quadruplex in the c-myb oncogene promoter. A screening of six alkaloid molecules showed that brucine (P1) had a strong binding affinity to the c-myb G-quadruplex DNA. It is notable that P1 can bind selectively to the c-myb G-quadruplex with respect to duplex DNAs, as well as to G-quadruplexes in other types of gene sequences. According to ESI-MS results, in which the stability was tested by capillary heating and collision-induced dissociation, the binding of P1 could thermally stabilize the c-myb G-quadruplex DNA. CONCLUSIONS: In this work, brucine (P1), an alkaloid molecule, has been found to bind to the intramolecular parallel G-quadruplex in the c-myb oncogene promoter with high affinity and selectivity, and could thermally stabilize the c-myb G-quadruplex DNA, indicating that the binding of P1 has the potential to modulate c-myb gene expression. Copyright © 2015 John Wiley & Sons, Ltd.