The human genome contains many G-quadruplex-forming sequences, including sequences containing long single-stranded loops that are believed to be unfavorable for G-quadruplex formation. The ...intracellular environment of biological cells is crowded with proteins with charged surfaces. Understanding the effects of protein-rich environments is important for understanding the formation of G-quadruplexes in an intracellular environment. In this study, we investigated the structural stability of DNA G-quadruplexes in the presence of several types of globular proteins (lysozyme, cytochrome c, bovine serum albumin, myoglobin, histone proteins, and serum proteins), unstructured polypeptides (protamine and poly-l-lysine), and oligopeptides (RGG/RG-domain peptides and short repeated peptides). Thermal melting studies of G-quadruplex-forming oligonucleotides derived from the human telomeric repeat sequence revealed that environments containing high concentrations of proteins and peptides differently affected the G-quadruplex stability according to their loop lengths. We found that weak electrostatic interactions of G-quadruplex loops with basic proteins and peptides improved the stability of long-loop G-quadruplexes and the interactions were strengthened under crowded conditions simulated by dextran. The comparison of the effects of different types of proteins and peptides indicated that excluded volume interactions and structural flexibility of both DNA and polypeptide chains influenced the efficiency of their interactions. This study provides insights into long-loop G-quadruplex stability in a crowded intracellular environment and the recognition of G-quadruplexes by arginine-rich domains of G-quadruplex-binding proteins.
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•Basic protein- and peptide-induced improvement of the stability of DNA G-quadruplex.•Weak electrostatic interactions between long-loop G-quadruplexes and basic proteins.•Enhanced interaction of G-quadruplexes with basic proteins under crowded conditions.•Different binding properties depending on flexibility of DNA and polypeptide chains.
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► Cytosine methylation induced a conformational transition of the promoter G-quadruplex in bcl-2. ► Cytosine methylation greatly increased the stability of the promoter G-quadruplex ...in bcl-2. ► Cytosine methylation enhanced the ability to arrest the polymerase in vitro.
The C-5-methylation of cytosine in the CpG islands is an important pattern for epigenetic modification of gene, which plays a key role in regulating gene transcription. G-quadruplex is an unusual DNA secondary structure formed in G-rich regions and is identified as a transcription repressor in some oncogenes, such as c-myc and bcl-2. In the present study, the results from CD spectrum and FRET assay showed that the methylation of cytosine in the CpG islands could induce a conformational change of the G-quadruplex in the P1 promoter of bcl-2, and greatly increase the thermal-stability of this DNA oligomer. Moreover, the methylation of cytosine in the G-quadruplex could protect the structure from the disruption by the complementary strand, showing with the increasing ability to arrest the polymerase in PCR stop assay. This data indicated that the stabilization of the G-quadruplex structure in the CpG islands might be involved in the epigenetical transcriptional regulation for specific genes through the C-5-methylation modification pattern.
The structural polymorphism of G-quadruplex nucleic acids is an important factor in their recognition by proteins and small-molecule ligands. However, it is not clear why the binding of several ...ligands alters G-quadruplex topology. We addressed this question by following the (un)folding and binding of the human telomeric fragment 5′-(GGGTTA)3GGGT-3′ (22GT) by calorimetry (DSC, ITC) and spectroscopy (CD). A thermodynamic analysis of the obtained data led to a detailed description of the topological phase space of stability (phase diagram) of 22GT and shows how it changes in the presence of a specific bisquinolinium ligand (360A). Various 1:1 and 2:1 ligand–quadruplex complexes were observed. With increasing temperature, the 1:1 complexes transformed into 2:1 complexes, which is attributed to the preferential binding of the ligand to the folding intermediates. Overall, the dissection of the thermodynamic parameters in combination with molecular modelling clarified the driving forces of the topological quadruplex transformations in a wide range of ligand concentrations and temperatures.
The
WT1
gene is an important oncogene, and its overexpression is considered as an effective target for anticancer therapy. Regulation of its gene transcription is one way for
WT1
-targeting drug ...design. Recently, in silico analysis of some oncogene promoters like
WT1
showed some guanine-rich regions with the ability to form G-quadruplex structures. Ligands like 5,10,15,20-tetra(
N
-methyl-4-pyridyl)-porphine (TMPyP4) have predominant effect on G-quadruplex stabilization. The aim of this study was to understand the effect of TMPyP4 on
WT1
gene transcription via stabilization of promoter G-quadruplexes. We examined the formation of new G-quadruplex motifs in
WT1
promoter in the presence of TMPyP4. In order to understand the nature of its interaction with
WT
1 promoter quadruplexes, differential pulse voltammetry (DPV), circular dichroism (CD), polyacrylamide gel electrophoresis, electrophoretic mobility shift assay (EMSA), polymerase chain reaction (PCR) stop assays, and quantitative RT-PCR were performed. According to the results, the
WT
1 promoter can form stable intramolecular parallel G-quadruplexes. In addition, after 48 and 96 h of incubation, 100 μM TMPyP4 reduced the
WT1
transcription to 9 and 0.4 %, respectively, compare to control. We report that ligand-mediated stabilization of G-quadruplexes within the
WT
1 promoter can silence
WT1
expression. This study might offer the basis for the reasonable design and improvement of new porphyrin derivatives as effective anti-leukemia agents for cancer therapy.
