Methods for investigating G-quadruplex DNA/ligand interactions

Abstract

DNA is considered an important target for drug design and development. Until recently, the focus was on double-stranded (duplex) DNA structures. However, it has now been shown that single stranded DNA can fold into hairpin, triplex, i-motif and G-quadruplex structures. The more interesting G-quadruplex DNA structures comprise four strands of stacked guanine (G)-tetrads formed by the coplanar arrangement of four guanines, held together by Hoogsteen bonds. The DNA sequences with potential to form G-quadruplex structures are found at the chromosomal extremities (i.e. the telomeres) and also at the intra-chromosomal region (i.e. oncogenic promoters) in several important oncogenes. The formation of G-quadruplex structures is considered to have important consequences at the cellular level and such structures have been evoked in the control of expression of certain genes involved in carcinogenesis (c-myc, c-kit, K-ras etc.) as well as in the perturbation of telomeric organization. It has been shown that the formation of quadruplexes inhibits the telomere extension by the telomerase enzyme, which is up-regulated in cancer cells. Therefore, G-quadruplex structures are an important target for drug design and development and there is a huge interest in design and development of small molecules (ligands) to target these structures. A large number of so-called G-quadruplex ligands, displaying varying degrees of affinity and more importantly selectivity (i.e. the ability to interact only with quadruplex-DNA and not duplex-DNA), have been reported. Access to efficient and robust in vitro assays is needed to effectively monitor and quantify the G-quadruplex DNA/ligand interactions. This tutorial review provides an overview of G-quadruplex ligands and biophysical techniques available to monitor such interactions.

An RNA-binding Protein, Lin28, Recognizes and Remodels G-quartets in the MicroRNAs (miRNAs) and mRNAs It Regulates

“…By one-dimensional NMR spectroscopy, we found that Lin28-binding RNAs contain imino resonances from 10 to 12.2 ppm, indicative of non-canonical hydrogen bonds (48,51,60). The imino resonance pattern is characteristic of the G:G:G:G hydrogen-bonding networks found in G4s (48,51,61).…”

“…The imino resonance pattern is characteristic of the G:G:G:G hydrogen-bonding networks found in G4s (48,51,61). We were unable to obtain high-resolution two-dimensional NMR spec- Lin28 Recognizes and Remodels G-quartets tra that could provide further structural support.…”

“…We were unable to obtain high-resolution two-dimensional NMR spec- Lin28 Recognizes and Remodels G-quartets tra that could provide further structural support. G4-containing RNAs are prone to aggregate (51,(62)(63)(64), which may have foiled our efforts.…”

“…TMPyP4, a porphyrin analogue of NMM that binds G4s and double-stranded DNA, blocks MYC transcription and decreases tumor growth and increases survival in tumor xenograft studies (57,58). By blocking transcription and translation of oncogenes, G4-intercalating agents have demonstrated anticancer activity (57,50,51). Similar agents could be designed to target the G4 feature of Lin28-binding RNAs, preventing Lin28 remodeling G4s and inhibiting Lin28 activity.…”

“…G4-intercalating agents have been assessed for anticancer ther-apy (57,50,51). TMPyP4, a porphyrin analogue of NMM that binds G4s and double-stranded DNA, blocks MYC transcription and decreases tumor growth and increases survival in tumor xenograft studies (57,58).…”