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 explicit-solvent molecular dynamics simulations to scrutinize the G4/hemin interaction. We found 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. Importantly, the simulations do not support several mechanistic possibilities (i.e., the wobbling guanine and the iron-bound water molecule) but rather suggest tentative mechanisms in which the external G-quartet itself is responsible for the unique H2O2-promoted biocatalytic properties of the G4/hemin complexes. Our simulations show that 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 simulation technique are also briefly discussed.Graphical AbstractHemin can bind to G-quadruplex via loop-assisted delivery. The catalytically active structure does not seem to contain an iron-bound water molecule sandwiched in between hemin and G-quadruplex or wobbling guanine.

Efficient Biocatalytic System for Biosensing by Combining Metal–Organic Framework (MOF)-Based Nanozymes and G-Quadruplex (G4)-DNAzymes

“…We also looked at the mechanism of this novel nanozyme, which is still a matter of debate. 47 To this end, we used various radical scavengers such as ascorbic acid (AA) for •OH and O2 •-, 48 thiourea (TH) for •OH, 49 p-benzoquinone (p-BQ) for O2 •-50 and NaN3 for 1 O2. 51 The catalytic activity of MIL-53(Fe)/G4-hemin was strongly decreased upon addition of AA and TH, implying the generation of •OH and O2 •during the catalytic process (Figure 4A), which is fully in line with the most recent mechanism.…”