Insights into G-Quadruplex–Hemin Dynamics Using Atomistic Simulations: Implications for Reactivity and Folding

Abstract

Guanine quadruplex nucleic acids (G4s) are involved in key biological processes such as replication or transcription. Beyond their biological relevance, G4s find applications as biotechnological tools since they readily bind hemin and enhance its peroxidase activity, creating a G4-DNAzyme. The biocatalytic properties of G4-DNAzymes have been thoroughly studied and used for biosensing purposes. Despite hundreds of applications and massive experimental efforts, the atomistic details of the reaction mechanism remain unclear. To help select between the different hypotheses currently under investigation, we use extended explicitsolvent molecular dynamics (MD) simulations to scrutinize the G4/hemin interaction. We find that besides the dominant conformation in which hemin is stacked atop the external G-quartets, hemin can also transiently bind to the loops and be brought to the external G-quartets through diverse delivery mechanisms. The simulations do not support the catalytic mechanism relying on a wobbling guanine. Similarly, catalytic role of the iron-bound water molecule is not in line with our results, however, given the simulation limitations, this observation should be considered with some caution. The simulations rather suggest tentative mechanisms in which the external G-quartet itself could be responsible for the unique H2O2-promoted biocatalytic properties of the G4/hemin complexes. Once stacked atop a terminal G-quartet, hemin rotates about its vertical axis while readily sampling shifted geometries where the iron transiently contacts oxygen atoms of the adjacent G-quartet. This dynamics is not apparent from the ensemble-averaged structure. We also visualize transient interactions between the stacked hemin and the G4 loops. Finally, we investigated interactions between hemin and on-pathway folding intermediates of the parallel-stranded G4 fold. The simulations suggest that hemin drives the folding of parallel-stranded G4s from slip-stranded intermediates, acting as a G4 chaperone. Limitations of the MD technique are briefly discussed. KEYWORDSDNAzyme; G-quartet; DNA folding; DNA ligand; Molecular dynamics simulations Guanine quadruplexes (G4s) are undoubtedly the most studied non-canonical nucleic acid structures. G4s are quadruple helices that fold from both DNA and RNA guanine (G)-rich sequences and adopt a variety of topologies depending on their nucleotide sequence and experimental conditions. [1][2] The basic unit of G4 is a quartet of guanines (G-quartet), in which guanines are H-bonded in a cyclic arrangement (Figure 1A). 3 G4s are created when at least two quartets stack upon each other to form a G-stem, which creates a channel running through its whole length, with inwardly pointing guanine O6 atoms that chelate cations (e.g., K + ) and further stabilize the overall G4 architecture. G4 topology is characterized by structural rules comprising syn/anti conformations of guanines, strand directionality and loop types, which lead to a large topological diversity when combined. [4][5][6] Potential G4-fo...

Targeting a G-quadruplex from let-7e pre-miRNA with small molecules and nucleolin

“…The residues belonging to the long loop appear to point inward, while that of the short loops outward. The long loop can facilitate the binding of the ligands (as recently demonstrated for porphyrinic ligands) [46], while end-stacking interactions can be observed since no endcapping residues are present. The binding mode of each ligand to the 3D structure of rG4-let-7e was also investigated by molecular docking and molecular dynamics (Figures S7-S8);…”

Spectroscopic and In Silico Studies on the Interaction of Substituted Pyrazolo[1,2-a]benzo[1,2,3,4]tetrazine-3-one Derivatives with c-Myc G4-DNA

“…As illustrated in Figure 10 b, the sliding between the two quartets can be facilitated by interactions with the bases in the loops. Interactions between the loops’ bases and a variety of ligands, such as hemin, pentacyclic acridine compound RHPS4, Telomestatin, porphyrins derivative TMPyP4, benzothiazole-based CX-5461, the tri-substituted acridine BRACO19, Thioflavin T, and other ligands, have already been observed in recent studies by Stadlbauer et al [ 38 ], Mulholland et al [ 39 ], Sullivan et al [ 40 , 41 ], Machireddy et al [ 42 , 43 ], and Luo et al [ 44 ].…”

Insights into How NH4+ Ions Enhance the Activity of Dimeric G-Quadruplex/Hemin DNAzyme

“…More recently, Yamamoto and co-workers have characterized that the axial coordination site of the Fe atom is occupied by H 2 O in the hydrophobic interface between the hemin and G-quartet planes by the 1 H NMR experiment. , The identification of water coordination is crucial to elucidate the molecular mechanisms for G4-hemin DNAzyme with H 2 O 2 activated. Besides, Šponer et al scrutinized the G4/hemin interaction for the activity and folding using MD simulations . All the above discussion inspired our rethinking how NH 4 + regulates the catalytic activity in dimeric DNAzyme in the molecular details about the mechanism.…”

“…Besides, S ̌poner et al scrutinized the G4/hemin interaction for the activity and folding using MD simulations. 35 All the above discussion inspired our rethinking how NH 4 + regulates the catalytic activity in dimeric DNAzyme in the molecular details about the mechanism. So, QM calculations are further performed to elucidate the catalytic mechanisms and the roles of NH 4 + in promoting the catalytic efficiency of the dimeric G4/hemin DNAzyme system.…”