Metal-dependent electrochemical discrimination of DNA quadruplex sequences

Films of four different DNA quadruplex-forming (G4) sequences (c-KIT, c-MYC, HTelo, and BCL2) on gold surfaces were investigated by electrochemical impedance spectroscopy (EIS) to evaluate whether they evoke unique electrochemical responses that can be used for their identification. This could render EIS an alternative means for the determination of G4 sequences of unknown structure. Towards, this end, cation-dependent topology changes in the presence of either K+, K+ in combination with Li+, or Pb2+ in the presence of Li+ were first evaluated by circular dichroism (CD) spectroscopy, and electrochemical studies were performed subsequently. As a result, G4-sequence specific charge transfer resistance (RCT) patterns were in fact observed for each G4 sequence, allowing their discrimination by EIS. Graphic abstract

Synthesis, Biophysical Characterization, and Anti–HIV-1 Fusion Activity of DNA Quadruplex-based Inhibitors with Dipeptide MT Hook Conjugation

Background: Human immunodeficiency virus type-1 (HIV-1) infection is the reason for the epidemic of acquired immunodeficiency syndrome (AIDS). Developing HIV-1 fusion inhibitors gained increasing attention as they took effect in the early stage of HIV-1 infecting cells. DNA G-quadruplex-based inhibitors had been found to interact with HIV-1 envelope glycoprotein, showing anti–HIV-1 fusion activity. C-peptide derived molecules with Met-Thr terminal also showed potent anti-fusion activity, the Met-Thr dipeptide adopted a hook-like structure (termed MT hook) in the hydrophobic pocket to "anchor" inhibitors to the N-terminal heptad repeat (NHR) of HIV-1 envelope glycoprotein gp41. Objective: Our work was to conjugate MT hooks to the 5'-terminal ends of DNA quadruplex-based inhibitor and demonstrate its biophysical characterization and anti–HIV-1 fusion activity. Methods: A 6-aminohexanol phosphonamidite was utilized in solid synthesis for the conjunction of oligodeoxynucleotide and MT dipeptide. Hydrophobic groups were introduced by a nucleoside analogue from the base site. Circular dichroism spectrum and native polyacrylamide gel electrophoresis were used to confirm the helix formation. A cell-cell fusion assay was carried out to test the anti-fusion activity. Results: The conjugate G1 showed improved anti-cell-cell fusion activity than quadruplex without MT hook. The MT hook did not affect the oligodeoxynucleotide (ODN) G-quadruplex assembly. It was also proved that G1 could effectively interfere with endogenous 6-helical bundle (6HB) formation between the N-terminal heptad repeat N36 (NHR) and the C-terminal heptad repeat C34 (CHR) during virus fusion course. Conclusion: In this work, conjugate of DNA-oligopeptide were successfully synthesized. The conjugation of MT hook did improve the anti-fusion activity of DNA G-quadruplex-based inhibitors. Our results can add information regarding on structure-activity relationships of DNA helix-based inhibitors and provide a reference for the follow-up experimental studies.

Quadruplex folding promotes the condensation of linker histones and DNAs via liquid-liquid phase separation

Liquid-liquid phase separation (LLPS) of proteins and DNA has recently emerged as a possible mechanism underlying the dynamic organization of chromatin. We herein report the role of DNA quadruplex folding in liquid droplet formation via LLPS induced by interactions between DNA and linker histone H1 (H1), a key regulator of chromatin organization. <a>Fluidity measurements inside the droplets, binding assays using G-quadruplex-selective probes, and structural analyses based on circular dichroism demonstrated that quadruplex DNA structures, such as the G-quadruplex and i-motif, promote droplet formation with H1 and decrease molecular motility within droplets. </a><a></a><a></a><a>The dissolution of the droplets in the presence of additives and the LLPS of the DNA structural units indicated that in addition to electrostatic interactions between the DNA and the intrinsically disordered region of H1, π-π stacking between quadruplex DNAs could potentially drive droplet formation, unlike in the electrostatically driven LLPS of duplex DNA and H1. According to phase diagrams of anionic molecules with various conformations, the high LLPS ability associated with quadruplex folding arises from the formation of interfaces consisting of organized planes of guanine bases and the side surfaces with high charge density. </a>Given that DNA quadruplex structures are well documented in heterochromatin regions, it is imperative to understand the role of DNA quadruplex folding in the context of intranuclear LLPS.<br>

Quadruplex Folding of DNA Promotes the Condensation of Linker Histones via Liquid-Liquid Phase Separation

<p>Liquid-liquid phase separation (LLPS) of proteins and DNA has recently emerged as a possible mechanism underlying the dynamic organization of chromatin. We herein report the role of DNA quadruplex folding in liquid droplet formation via LLPS induced by interactions between DNA and linker histone H1 (H1), a key regulator of chromatin organization. Fluidity measurements inside the droplets and binding assays using G-quadruplex-selective probes demonstrated that quadruplex DNA structures, such as the G-quadruplex and i-motif, promote droplet formation with H1 and decrease molecular motility within droplets. The dissolution of the droplets in the presence of additives indicated that in addition to electrostatic interactions between the DNA and the intrinsically disordered region of H1, π-π stacking between quadruplex DNAs could potentially drive droplet formation. Given that DNA quadruplex structures are well documented in heterochromatin regions, it is imperative to understand the role of DNA quadruplex folding in the context of intranuclear LLPS.<b></b></p>

Quadruplex Folding of DNA Promotes the Condensation of Linker Histones via Liquid-Liquid Phase Separation

<p>Liquid-liquid phase separation (LLPS) of proteins and DNA has recently emerged as a possible mechanism underlying the dynamic organization of chromatin. We herein report the role of DNA quadruplex folding in liquid droplet formation via LLPS induced by interactions between DNA and linker histone H1 (H1), a key regulator of chromatin organization. Fluidity measurements inside the droplets and binding assays using G-quadruplex-selective probes demonstrated that quadruplex DNA structures, such as the G-quadruplex and i-motif, promote droplet formation with H1 and decrease molecular motility within droplets. The dissolution of the droplets in the presence of additives indicated that in addition to electrostatic interactions between the DNA and the intrinsically disordered region of H1, π-π stacking between quadruplex DNAs could potentially drive droplet formation. Given that DNA quadruplex structures are well documented in heterochromatin regions, it is imperative to understand the role of DNA quadruplex folding in the context of intranuclear LLPS.<b></b></p>

Quadruplex Folding of DNA Promotes the Condensation of Linker Histones via Liquid-Liquid Phase Separation

<p>Liquid-liquid phase separation (LLPS) of proteins and DNA has recently emerged as a possible mechanism underlying the dynamic organization of chromatin. We herein report the role of DNA quadruplex folding in liquid droplet formation via LLPS induced by interactions between DNA and linker histone H1 (H1), a key regulator of chromatin organization. Fluidity measurements inside the droplets and binding assays using G-quadruplex-selective probes demonstrated that quadruplex DNA structures, such as the G-quadruplex and i-motif, promote droplet formation with H1 and decrease molecular motility within droplets. The dissolution of the droplets in the presence of additives indicated that in addition to electrostatic interactions between the DNA and the intrinsically disordered region of H1, π-π stacking between quadruplex DNAs could potentially drive droplet formation. Given that DNA quadruplex structures are well documented in heterochromatin regions, it is imperative to understand the role of DNA quadruplex folding in the context of intranuclear LLPS.<b></b></p>

Application of DNA Quadruplex Hydrogels Prepared from Polyethylene Glycol-Oligodeoxynucleotide Conjugates to Cell Culture Media

Application of Na+-responsive DNA quadruplex hydrogels, which utilize G-quadruplexes as crosslinking points of poly(ethylene glycol) (PEG) network as cell culture substrate, has been examined. PEG-oligodeoxynucleotide (ODN) conjugate, in which four deoxyguanosine (dG4) residues are tethered to both ends of PEG, was prepared by modified high-efficiency liquid phase (HELP) synthesis of oligonucleotides and used as the macromonomer. When mixed with equal volume of cell culture media, the solution of PEG-ODN turned into stiff hydrogel (G-quadruplex hydrogel) as the result of G-quadruplex formation by the dG4 segments in the presence of Na+. PEG-ODN itself did not show cytotoxicity and the resulting hydrogel was stable enough under cell culture conditions. However, L929 fibroblast cells cultured in G-quadruplex hydrogel remained spherical for a week, yet alive, without proliferation. The cells gradually sedimented through the gel day by day, probably due to the reversible nature of G-quadruplex formation and the resulting slow rearrangement of the macromonomers. Once they reached the bottom glass surface, the cells started to spread and proliferate.