DNA is a highly dynamic carrier of genetic information which can form a variety of secondary structures beyond the canonical double helix. G-quadruplexes are among the best studied noncanonical structures, which emerge as potent regulators of gene expression. Most research on G-quadruplexes has been considering only motifs, which comply with the commonly accepted consensus sequence, however the focus has recently shifted towards alternative G-quadruplex formations. Long-looped G-quadruplexes are especially intriguing for their potential to indulge in highly specific interactions both with cellular proteins and small molecules – ligands.
We have identified several potential long-looped G-quadruplexes in the guanine-rich BCL-2 promoter, which under experimental conditions used failed to fold in any relevant secondary structure due to the suboptimal motif selection. However, with specific modifications, we were able to determine crucial structural characteristics of long-looped G-quadruplexes. These include bulge-less G-tracts, which seem to be a predominant factor, and Watson-Crick base-pairing within the long loop, which gains significance with increasing loop length.
During the process of optimizing the wild-type sequences we managed to create novel model sequences BCL2-A1d and BCL2-S5b, which we analysed is greater detail by NMR and complementary spectroscopic methods. Using CD spectroscopy, we confirmed that both samples form G-quadruplexes of parallel topology. Additionally, melting temperatures of 67.1 and 76.8 °C were determined with UV-melting experiments. DOSY spectra indicated that BCL2-A1d folds into a single major structure, while BCL2-S5b forms many structures. Using various methods of NMR spectroscopy, we determined that BCL2-A1d folds into a three-quartet G-quadruplex with a 17-nucleotide central loop.
Titrating both samples with G-quadruplex specific ligand RHPS4 showed that it is not suitable for targeting these specific structures. On the other hand, ligand PhenDC3 at low concentrations formed a complex with both structures interacting only with the Gquadruplex core. This was especially evident with BCL2-S5b, where no changes of the Watson-Crick base-pairing of the central loop were observed, meaning that PhenDC3 could be used in dual-specific targeting approaches.
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