A New Cationic Porphyrin Derivative (TMPipEOPP) with Large Side Arm Substituents: A Highly Selective G-Quadruplex Optical Probe

The discovery of uncommon DNA structures and speculation about their potential functions in genes has brought attention to specific DNA structure recognition. G-quadruplexes are four-stranded nucleic acid structures formed by G-rich DNA (or RNA) sequences. G-rich sequences with a high potential to form G-quadruplexes have been found in many important genomic regions. Porphyrin derivatives with cationic side arm substituents are important G-quadruplex-binding ligands. For example, 5,10,15,20-Tetrakis(N-methylpyridinium-4-yl)-21H,23H-porphyrin (TMPyP4), interacts strongly with G-quadruplexes, but has poor selectivity for G-quadruplex versus duplex DNA. To increase the G-quadruplex recognition specificity, a new cationic porphyrin derivative, 5,10,15,20-tetra-{4-[2-(1-methyl-1- piperidinyl)ethoxy]phenyl} porphyrin (TMPipEOPP), with large side arm substituents was synthesized, and the interactions between TMPipEOPP and different DNA structures were compared. The results show that G-quadruplexes cause large changes in the UV-Vis absorption and fluorescence spectra of TMPipEOPP, but duplex and single-stranded DNAs do not, indicating that TMPipEOPP can be developed as a highly specific optical probe for discriminating G-quadruplex from duplex and single-stranded DNA. Visual discrimination is also possible. Job plot and Scatchard analysis suggest that a complicated binding interaction occurs between TMPipEOPP and G-quadruplexes. At a low [G-quadruplex]/[TMPipEOPP] ratio, one G-quadruplex binds two TMPipEOPP molecules by end-stacking and outside binding modes. At a high [G-quadruplex]/[TMPipEOPP] ratio, two G-quadruplexes bind to one TMPipEOPP molecule in a sandwich-like end-stacking mode

Binding of Gemini Bisbenzimidazole Drugs with Human Telomeric G-Quadruplex Dimers: Effect of the Spacer in the Design of Potent Telomerase Inhibitors

The study of anticancer agents that act via stabilization of telomeric G-quadruplex DNA (G4DNA) is important because such agents often inhibit telomerase activity. Several types of G4DNA binding ligands are known. In these studies, the target structures often involve a single G4 DNA unit formed by short DNA telomeric sequences. However, the 3′-terminal single-stranded human telomeric DNA can form higher-order structures by clustering consecutive quadruplex units (dimers or n-mers). Herein, we present new synthetic gemini (twin) bisbenzimidazole ligands, in which the oligo-oxyethylene spacers join the two bisbenzimidazole units for the recognition of both monomeric and dimeric G4DNA, derived from d(T2AG3)4 and d(T2AG3)8 human telomeric DNA, respectively. The spacer between the two bisbenzimidazoles in the geminis plays a critical role in the G4DNA stability. We report here (i) synthesis of new effective gemini anticancer agents that are selectively more toxic towards the cancer cells than the corresponding normal cells; (ii) formation and characterization of G4DNA dimers in solution as well as computational construction of the dimeric G4DNA structures. The gemini ligands direct the folding of the single-stranded DNA into an unusually stable parallel-stranded G4DNA when it was formed in presence of the ligands in KCl solution and the gemini ligands show spacer length dependent potent telomerase inhibition properties

Fluorescence Enhancement upon G-Quadruplex Folding: Synthesis, Structure and Biophysical Characterization of a Dansyl/Cyclodextrin-tagged Thrombin Binding Aptamer

A novel fluorescent thrombin binding aptamer (TBA), conjugated with the environment-sensitive dansyl probe at the 3'-end and a beta-cyclodextrin residue at the 5'-end, has been efficiently synthesized exploiting Cu(I)-catalyzed azide-alkyne cycloaddition procedures. Its conformation and stability in solution have been studied by an integrated approach, combining in-depth NMR, CD, fluorescence and DSC studies. ITC measurements have allowed to analyze in detail its interaction with human thrombin. All the collected data show that this bis-conjugated aptamer fully retains its G-quadruplex formation ability and thrombin recognition properties, with the terminal appendages only marginally interfering with the conformational behavior of TBA. Folding of this modified aptamer into the chair-like, antiparallel G-quadruplex structure, promoted by K+ and/or thrombin binding, typical of TBA, is associated with a net fluorescence enhancement, due to encapsulation of dansyl, attached at the 3'-end, into the apolar cavity of the beta-cyclodextrin at the 5'-end. Overall, the structural characterization of this novel, bis-conjugated TBA fully demonstrates its potential as a diagnostic tool for thrombin recognition, also providing a useful basis for the design of suitable aptamer-based devices for theranostic applications, allowing simultaneously both detection and inhibition or modulation of the thrombin activit