Computer modelling studies on guanine-rich quadruplexes

Abstract

On the basis of these MD studies, it can be concluded that parallel G-quadruplexes are very stable structures, both energetically as well as structurally. Base interaction energy within the G-tetrad, as well as guanine tetrad stacking energy in PA structure, are more favourable than in PA-Na and PA-K structures. However, inclusion of the contribution due to the coordinated ion in energy calculations makes PA-Na and PA-K structures more favourable than the PA structure with water molecules in the central cavity. Base interaction energy within the G-tetrad primarily depends on O6 atom orientation and hydrogen bonding scheme within the G-tetrad. In-plane base rotation in the middle G-tetrads in the PA-Na and PA-K structures brings O6 atoms closer together while also distorting the hydrogen bonds and consequently, base interaction energies in middle G-tetrads are less favourable than the terminal tetrads, which show standard Hoogsteen type hydrogen bonds. In the absence of any coordinated ion, due to O6–O6 repulsion, adjacent guanine bases in the PA structure rotate in opposite directions to that found in the PA-K structure, leading to additional N2–H2–O6 hydrogen bonds and more favourable base-base interaction energy within the G-tetrads. Quadruplex structures with coordinated ions (Na+ or K+) are less flexible than the quadruplex with water molecules in the cavity. In contrast to the recently reported MD studies on 4-mer quadruplexes (Spackova et al., 1999), we find that the 7-mer quadruplex structure with waters in the cavity remains intact during the simulation. In this structure, the central cavity is occupied by water molecules, during the heating phase itself, but subsequently one Na+ ion from the solvent also enters through the 3'-end of the pre-formed quadruplex and coordinates with O6 atoms of the two terminal G-tetrads. Analysis of the 7-mer PA and PA-Na structures clearly shows that Na+ ions can enter and exit through the quadruplex ends within the present molecular dynamics time scale, while coordinated K+ ions are static within their respective coordination sites. Na+ counter ions in the solvent bind most often to the phosphate groups, in all the three structures. However, a few counter ions are also located within the quadruplex grooves. In all the three structures, several hydrogen-bonded water molecules are observed within the shallow quadruplex grooves. Water molecules are localized around the exposed 2-amino group and N3 atom of guanine bases in the G-tetrads. A few of the exposed N7 atoms in the PA structure also act as good binding sites for water molecules. In conclusion, the G-quadruplex is a very stable structure, which can undergo small variations in the G-tetrad geometry, depending on the presence of ions or solvent molecules in the central cavity, while retaining its overall structural integrity. The slightly shorter ion–O6 distances observed in the Na+ ion coordinated MD structures, as compared to the high-resolution crystal structure, probably suggests that the ion parameters need further refinement. Our MD studies demonstrated that base interaction energy within a G-tetrad depends on the tetrad geometry, in particular on the position of the charged groups. Similarly, base-stacking energy between two successive guanine tetrads depends on orientation of O6 atoms and it is close to zero in both the PA-Na and PA-K structures. However, interaction energy between coordinated ion and G-tetrad is favourable for both the structures. In the MD structures, strong attractive force between the coordinated Na+ ions and guanine O6 atoms rotates the guanine bases in G-tetrads, leading to smaller O6–O6 separation and a network of bifurcated hydrogen bonds between neighbouring guanine bases, rather than the two standard Hoogsteen type hydrogen bonds, seen in the crystal structures. However, four tetrads at the two terminals in PA-K structure are stabilized by standard Hoogsteen hydrogen bonds while the middle tetrad in K+ ion coordinated structure is stabilized only by a cyclic single hydrogen bond. The effect of the size of the coordinated ion on the quadruplex structures appears to be more pronounced for the longer fragment used in our simulation, perhaps due to the cumulative effect of the larger number of ions present in the central cavity. The smaller size of the coordinated Na+ ions allows them greater mobility than the K+ ions. The present analysis along with our earlier results on parallel quadruplexes indicates that the Na+ ions can move through the quadruplex channel without disturbing the tetrad geometry, as well as enter or exit the quadruplex core through the quadruplex ends, indicating that not all sites in the quadruplex cavity need to be occupied for structural integrity. The coordinated K+ ions, due to their larger size, cannot easily move within the central channel and are forced out of the quadruplex cavity at higher temperature only by disrupting the G-tetrad geometry. However, the essential quadruplex nature of the structure is retained for both these larger quadruplexes, even at this higher (400K) temperature. Na+ counter ions, present in the solvent, are most often located near the phosphate groups of quadruplex structures. However, some counter ions are also bound inside the quadruplex grooves. The two smaller grooves in Greek key type antiparallel structures are better binding sites for counter ions than the two wider grooves. In both the antiparallel structures, quite a few water molecules stay close to the guanine base atoms and form hydrogen bonds, with the exposed 2-amino groups and N3 atoms of guanines being the preferred binding sites for water molecules. Hence, Na and K ions coordinated antiparallel G-quadruplexes are structurally very stable and can retain quadruplex structure even at 400K. However, hydration free energy of antiparallel structure is noticeably less favourable than for the parallel quadruplex structures, which can explain the preference for parallel strand arrangement in guanine oligomers. Among the two antiparallel structures, hydration free energy for the K+ ion coordinated structure is more favourable than the hydration free energy for the Na+ ion coordinated structure. This along with the higher desolvation energy for a Na+ ion could account for the observed preference for K+ ions, as indicated by NMR experiments. An extended molecular dynamics simulation of parallel four-stranded d(G)7 structure indicates that the guanine quadruplex is stable due to favourable stacking energy between successive guanine base tetrads, even in the absence of coordinated cations. Water molecules can occupy the empty coordination sites in this situation. However, some Na counter ions from the surrounding solvent medium enter the quadruplex channel, by replacing the bound water molecules, thus indicating that the monovalent Na+ ions are the preferred ligands at these coordination sites. The total interaction energy of a cation in the central channel of a partially coordinated G-quadruplex is more favourable than the interaction energy of cation in the surrounding solvent. Thus, in the presence of a few channel-bound cations, the 7-mer quadruplex structure retains its tetrameric form even during MD at 400K. In the absence of any coordinated ion, due to strong O6–O6 repulsion, adjacent guanines in the tetrad undergo rotation, so as to move the O6 atoms further apart and the G-tetrad is stabilized by bifurcated hydrogen bonds similar to that reported in an ab initio study. However, after the entry of Na+ ions, strong attractive force between O6 atoms and the ion restores the standard Hoogsteen type hydrogen bonded G-tetrads. The sodium ions enter the quadruplex through the ends and travel within the quadruplex channel without significantly distorting the G-tetrad geometry. However, the movement of the Na+ ion within the quadruplex channel is slower than that in the surrounding medium. Quadruplex grooves are well hydrated during the entire simulation and water molecules are tightly bound inside the grooves, through hydrogen bond formation with the exposed 2-amino groups and N3 atoms of guanine bases. While the 7-mer quadruplex structure does not dissociate in the absence of coordinated cations, the free energy of hydration for the partially coordinated quadruplex structure is more favourable than the structure without any coordinated ion and comparable to that of a fully coordinated quadruplex, indicating that all coordination sites need not be occupied for the formation of a stable G-quadruplex structure. Several human genetic diseases are associated with expansion of triplet repeats. Genetic analysis of triplet repeat diseases loci in patients' DNA indicate that there is a close relationship between the number of repeats and the genetic stability, the age of onset, and the severity of the disease symptoms (Pearson and Sinden, 1998; Mahadevan et al., 1992). A mechanism of triplet repeat expansion was involved in blockage of DNA replication by unknown higher-order structures. Several gel electrophoresis studies indicate that triplet repeats containing DNA oligonucleotides readily and specifically adopt unusual compact structures that migrate abnormally fast on native but not denaturing gels (Chastain et al., 1995; Mitchell et al., 1995). Hairpin and quadruplex structures are the most common structures proposed for these sequences, by various groups. Present model building and molecular mechanics studies indicate that hairpin as well as hairpin dimer structures with triplet repeat sequences are as favourable as corresponding B-DNA type structures. Furthermore, Greek key type antiparallel structures could also be formed at high salt conditions. It is also observed that non-guanine tetrads (A, C, and T-tetrads) can be nicely accommodated in the quadruplex structures and provide favourable stacking energy. Interestingly, in the minimized hairpin dimer and quadruplex structures, adenine (A), cytosine (C), and thymine (T) bases form hydrogen bonded A, C, and T-tetrads, even though they were not hydrogen bonded in the starting structures. Recently, several NMR studies report the formation of A, C, and T tetrads (Patel et al., 1999; Patel et al., 2000; Patel and Hosur, 1999). The hydrogen bonding scheme within these tetrads is similar to that suggested by the present model building and molecular mechanics studies. It is possible to have energetically favourable hairpin as well as hairpin dimer structures with stacked and intra as well as inter-loop hydrogen-bonded loop regions. At low salt conditions (normal charge minimization) hairpin structures with these triplet repeats are energetically comparable with corresponding B-DNA duplex, whereas hairpin dimer structures are marginally less favourable and quadruplex structures are clearly less favourable. However, at high salt conditions (reduced charge minimization) both hairpin dimer and quadruplex structures are energetically more favourable than corresponding B-DNA structures. Thus, these sequences are highly polymorphic and depending on the environmental condition, they can take up hairpin or quadruplex structures. Our MD study on the d(GGC)5 hairpin dimer structure demonstrated that in the presence of coordinated ion, the structure is stable. The stem part of hairpin dimer structure is stabilized by both G-C-G-C and G-G-G-G type tetrads. The G-tetrads are stabilized by cyclic Hoogsteen type hydrogen bonds while G-C-G-C tetrads are stabilized by a pair of Watson-Crick G:C base pairs and these two G:C pairs are hydrogen bonded to each other via N7 of the guanine residues and N4 of the cytosine residues. With both loops occurring on the same side, the guanines and cytosines in the loops can also form a G-C-G-C tetrad by formation of intra-loop WC base pairs as well as inter-loop base pairing, which remain intact throughout the course of dynamics. Middle guanine bases in both the loops can also interact through hydrogen bonds. Coordinated Na+ ions produce favourable interaction energy with both G-C-G-C and G-G-G-G tetrads. However, interaction energy between Na+ ion and G-tetrad is better than the interaction energy between ion and the G-C-G-C tetrad. Interaction energy between the bases within a G-C-G-C tetrad is more favourable than that of the G-G-G-G tetrad. Tetrad stacking energies are largely influenced by the presence of coordinated ions. However, van der Waals (vdW) components of stacking energies involving G-tetrads are more favourable. Na+ counter ions are frequently bound to phosphate groups of hairpin dimer structure but counter ions are not seen in the vicinity of base atoms of quadruplex stem. During the entire course of dynamics, quite a few water molecules stay close to the base atoms and form hydrogen bonds. Exposed 2-amino groups of guanine, N3 atoms of guanine, and O2 atoms of cytosine are the most favourable binding sites for water molecules, as in all guanine quadruplex structures. Model building and molecular mechanics study of RNA G-quadruplex structures indicates that all-anti parallel structures are more favorable than antiparallel structures. Thus, single stretches of r(G)n would favor an all-anti parallel structure as deduced from earlier fiber diffraction studies on poly r(G) and also observed in the NMR analysis of r(UG4U)4 RNA quadruplex structure (Cheong and Moore, 1992). It is observed that at high salt conditions, all-anti parallel quadruplex structure with C2-endo sugar pucker is noticeably more favorable than the parallel all-anti structure with C1'-endo sugar pucker. However, at low salt conditions, both structures are energetically comparable. It is interesting to notice that syn orientation of ribo(O) does not prefer C3'-endo sugar pucker. Therefore, the parallel alternating anti-syn structure with C3'-endo sugar pucker is less favorable than similar parallel structures with C2-endo or C1'-endo sugar pucker, while Indian and Greek key type of antiparallel quadruplex structures are energetically intact in the structures minimized with both normal as well as reduced phosphate charge. All guanine tetrads remain intact in the structures. In the RNA quadruplex structures, 2'-hydroxyl groups form intra- and inter-strand hydrogen bonds, which can provide favorable electrostatic energy.