Folding intermediate states of the parallel human telomeric G-quadruplex DNA explored using Well-Tempered Metadynamics

Abstract

An increasingly comprehension of the folding intermediate states of DNA G-quadruplexes (G4s) iscurrently an important scientific challenge, especially for the human telomeric (h-tel) G4s-forming sequences, characterized by a highly polymorphic nature. Despite the G-triplex conformation was proposed as one of the possible folding intermediates for the antiparallel and hybrid h-tel G4s, for the parallel h-tel topology with an all-anti guanine orientation, a vertical strand-slippage involving the G-triplets was proposed in previous works through microseconds-long standard molecular dynamics simulations (MDs). Here, in order to get further insights into the vertical strand-slippage and the folding intermediate states of the parallel h-tel G4s, we have carried out a Well-Tempered Metadynamics simulation (WT-MetaD), which allowed us to retrieve an ensemble of six G4s having two/G-tetrad conformations derived by the G-triplets vertical slippage. The insights highlighted in this work are aimed at rationalizing the mechanistic characterisation of the parallel h-tel G4 folding process.G-quadruplexes 1,2 (G4s) are non canonical DNA structures present at the telomere ends and involved in the genome stability and in the inhibition of the telomerase enzyme, a reverse transcriptase responsible for the cellular immortalization 3,4 and up-regulated in ~85% of human tumors 3 . The evidence that G4s formation at the 3′-end of telomere DNA can effectively hamper telomerase from adding further repeats 3 suggests G4s as a promising target for anticancer therapy 5-7 . In order to identify new compounds as potential anticancer drugs, the structure-based drug design targeting G4s could be a crucial step in gaining accurate information about the structural features of G4s 8-13 . Unfortunately, the rational design of novel G4 binders is hampered both by the structural polymorphism of these non-canonical conformations and by the lack of a well-defined ligand binding site. Therefore, enhanced sampling methods, like Metadynamics, have been required in order to deeply define the ligand binding mode 14,15 . Monomolecular G4s of the human telomeric sequence d[AG 3 (TTAGGG) 3 ] (h-tel) can adopt different folding topologies according to the relative orientations of the four strands 16 . In fact, while the arrangement of the h-tel monomolecular G4s has been well characterized by NMR in Na + to adopt a basket-type conformation with the single strands assuming an antiparallel orientation 17 , in presence of K + ions the folding topology of the h-tel monomolecular G4s includes both parallel-stranded and hybrid (mixed parallel/antiparallel) conformations, raising the question of which conformation is biologically more favoured. In 2006, the NMR hybrid syn/anti conformation was proposed as the most favoured one under K + physiological conditions 18 , whose folding organization is different from that reported in K + ions by crystallography consisting, instead, in the parallel-stranded topology with three G-tetrads and three symmetrical external d(TTA) l...

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

“…Slip-stranded structures were suggested to act as late-stage folding/formation intermediates of parallel-stranded all-anti G4s. [51][52][53] The schemes in panels (B) and (C) and in next figures are drawn as follows: guanines are yellow; a pair of H-bonds between two guanines is depicted by a red line; the sugar-phosphate backbone is depicted by a black arrow showing the 5'→ 3' progression; loop nucleotides and channel cations are not shown; bound hemin will be shown as a cyan rectangle (not present in this figure).…”

“…To simplify the calculations we used the minimal d[GGG] 4 construct, however, the studied strand-slippage movements are fully relevant to complete intramolecular G4s. [52][53] We used three structures and two positions for hemin, which together with simulations lacking hemin resulted into nine starting states, sufficiently though not completely representing the part of folding landscape represented by slip-stranded structures. Black font denotes structures used as starting structures while the red ones occurred spontaneously in simulations.…”

“…In the case of the parallel-stranded G4s, substantial involvement of on-pathway structures with slipped strands has been proposed. [51][52][53] Flat ligands may promote their formation compared to off-pathway intermediates, reducing the kinetic partitioning, speeding up reduction of strand slippage and stabilizing the complete parallel-stranded G4. Although the full folding is far beyond the simulation time scale, our results provide indirect support to the above considerations: comparison of simulations with and without hemin showed that the ligand exerts the aforementioned effects within the folding funnel pertinent to the parallel-stranded all-anti G4 state ( Figure 5).…”

“…Key folding intermediates of parallel-stranded all-anti G4s may correspond to transiently-populated slipped G4s with a reduced number of quartets. [51][52][53] Transitions between such structures can occur without any unfolding of the G-stem, through vertical strand-slipping movements. Although this process appears straightforward, experiments indicate that some slip-stranded structures can have long lifetimes for G4s with GGGG or longer G-tracts.…”

Time-resolved NMR-spectroscopic studies of conformational dynamics in DNA G-quadruplexes

“…174 In general, folding of G4s is multiphasic and is supposed to involve intermediates like anti-parallel hairpins 165,170,171 , triplexes [193][194][195] and (n-1)-tetrad conformations. 167,[196][197][198] Folding of the human telomeric sequence (TTAGGG) n and its sequence variants have been studied most extensively, both experimentally 144,159,167,169,171,[173][174][175]188,[199][200][201] and theoretically [202][203][204] . The telomeric G4 adopts at least two different hybrid conformations under physiological conditions.…”