Commentary

A pilgrim's guide to G-quadruplex nucleic acid folding

Current state-of-the art explicit-solvent atomistic molecular dynamics (MD) simulations are reviewed. We describe the basic principles and limitations of MD methodology. We also explain how to compare MD simulations with experiments and how to correctly interpret MD data. Different types of simulation approaches are introduced. We start with standard simulations and then continue with techniques which allow one to a certain extent overcome sampling limitations and derive free energies: replica-exchange methods, collective-variable based methods such as metadynamics, alchemical methods, Markov state models and continuum solvent approach. We emphasize explaining the limitations of the methods, in order to avoid over-interpretation of the simulation data. Then we introduce recent progress in the force fields that are used for simulations of quadruplexes. Finally, two specific areas are reviewed. First, we explain how MD simulations are essential to unravel the uniqueness and complexity of quadruplex folding landscape, what are the basic consequences and hallmarks of kinetic partitioning of the landscape and why low-dimensionality descriptions (both experimental and computational) can easily lead to major oversimplifications. Second, MD simulations-based recent approaches to probe quadruplex-ligand interactions are discussed. The review is primarily written for readers who are not advanced experts in MD simulations, with the aim to openly expose their limitations.

Guanine quadruplexes (GQs) play vital roles in many cellular processes and are of much interest as drug targets. In contrast to the availability of many structural studies, there is still limited knowledge on GQ folding.

We review recent molecular dynamics (MD) simulation studies of the folding of GQs, with an emphasis paid to the human telomeric DNA GQ. We explain the basic principles and limitations of all types of MD methods used to study unfolding and folding in a way accessible to non-specialists. We discuss the potential role of G-hairpin, G-triplex and alternative GQ intermediates in the folding process. We argue that, in general, folding of GQs is fundamentally different from funneled folding of small fast-folding proteins, and can be best described by a kinetic partitioning (KP) mechanism. KP is a competition between at least two (but often many) well-separated and structurally different conformational ensembles.

The KP mechanism is the only plausible way to explain experiments reporting long time-scales of GQ folding and the existence of long-lived sub-states. A significant part of the natural partitioning of the free energy landscape of GQs comes from the ability of the GQ-forming sequences to populate a large number of syn-anti patterns in their G-tracts. The extreme complexity of the KP of GQs typically prevents an appropriate description of the folding landscape using just a few order parameters or collective variables.

We reconcile available computational and experimental studies of GQ folding and formulate basic principles characterizing GQ folding landscapes. This article is part of a Special Issue entitled "G-quadruplex" Guest Editor: Dr. Concetta Giancola and Dr. Daniela Montesarchio.

The AT-rich genome of P. falciparum has uniquely localized G-rich stretches that have propensity to form G-quadruplexes. However, their global occurrence and potential biological roles in the parasite are poorly explored. Our genome-wide analysis revealed unique enrichment of quadruplexes in P. falciparum genome which was remarkably different from other Plasmodium species. A distinct predominance of quadruplexes was observed in nuclear and organellar genes that participate in antigenic variation, pathogenesis, DNA/RNA regulation, metabolic and protein quality control processes. Data also suggested association of quadruplexes with SNPs and DNA methylation. Furthermore, analysis of steady state mRNA (RNA-seq) and polysome-associated mRNA (Ribosome profiling) data revealed stage-specific differences in translational efficiency of quadruplex harboring genes. Taken together, our findings hint towards existence of regulatory dynamics associated with quadruplexes that may modulate translational efficiency of quadruplex harboring genes to provide survival advantage to the parasite against host immune response and antimalarial drug pressure.