RESUMO
G-quadruplexes (G4s) are secondary structures formed by guanine-rich oligonucleotides involved in various biological processes. However, characterizing G4s is challenging, because of their structural polymorphism. Here, we establish how hydrogen-deuterium exchange native mass spectrometry (HDX/MS) can help to characterize the G4 structures and dynamics in solution. We correlated the time range of G4 exchange to the number of guanines involved in the inner and outer tetrads. We also established relationships among exchange rates, numbers of tetrads and bound cations, and stability. The use of HDX/native MS allows for the determination of tetrads formed and assessment of G4 stability at a constant temperature. A key finding is that stable G4s exchange through local fluctuations (EX2 exchange), whereas less stable G4s also undergo exchange through partial or complete unfolding (EX1 exchange). Deconvolution of the bimodal isotope distributions resulting from EX1 exchange provides valuable insight into the kinetics of folding and unfolding processes and allows one to detect and characterize transiently unfolded intermediates, even if scarcely populated. HDX/native MS thus represents a powerful tool for a more comprehensive exploration of the folding landscapes of G4s.
Assuntos
Quadruplex G , Hidrogênio , Hidrogênio/química , Deutério , Medição da Troca de Deutério/métodos , Espectrometria de Massas/métodos , DNARESUMO
Hydrogen-deuterium exchange mass spectrometry (HDX/MS) is increasingly used to study the dynamics of protein conformation. Coupled to native MS, HDX can also characterize the conformations of oligonucleotides and their binding to cations, small molecules, and proteins. Data processing and visualization of native HDX/MS of oligonucleotides requires dedicated software solutions. OligoR is a web-browser-based application that addresses the specific needs of DNA HDX/MS and native MS experiments from raw data in an open format to visualization and export of results. Whole experiments spanning many time points can be processed in minutes for several mass-separated species. To access valuable folding dynamics information, we have developed a simple and robust approach to deconvolute bimodal isotope distributions, even when they are highly overlapping. This approach is based on modeling physically possible isotope distributions determined from chemical formulae and could be extended to any type of analyte (proteins, peptides, sugars, and small molecules). All results are presented in interactive data tables, and publication-quality figures can be generated, customized, and exported.
Assuntos
Medição da Troca de Deutério , Oligonucleotídeos , Medição da Troca de Deutério/métodos , Espectrometria de Massa com Troca Hidrogênio-Deutério/métodos , Proteínas/química , Peptídeos/análise , Conformação ProteicaRESUMO
Current multiagent chemotherapy regimens have improved the cure rate in acute leukemia patients, but they are highly toxic and poorly efficient in relapsed patients. To improve the treatment approaches, new specific molecules are needed. The G-quadruplexes (G4s), which are noncanonical nucleic acid structures found in specific guanine-rich DNA or RNA, are involved in many cellular events, including control of gene expression. G4s are considered as targets for the development of anticancer agents. Heterocyclic molecules are well known to target and stabilize G4 structures. Thus, a new series of 2,9-bis[(substituted-aminomethyl)phenyl]-1,10-phenanthroline derivatives (1a-i) was designed, synthesized, and evaluated against five human myeloid leukemia cell lines (K562, KU812, MV4-11, HL60, and U937). Their ability to stabilize various oncogene promoter G4 structures (c-MYC, BCL-2, and K-RAS) as well as the telomeric G4 was also determined through the fluorescence resonance energy transfer melting assay and native mass spectrometry. In addition, the more bioactive ligands 1g-i were tested for telomerase activity in HuT78 and MV4-11 protein extracts.