Structural Features of G-Quadruplexes

Many different structures and topologies have been characterized by X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy in solution in the last years. To date (December 2014) 136 entries of DNA G-quadruplex structures have been deposited in the nucleic acid database (http://ndbserver.rutgers.edu/). G-quadruplexes can be formed by one G-rich oligonucleotide strand folding back on itself yielding an intramolecular G-quadruplex (Figure 5A).

In addition intermolecular G-quadruplexes can be formed by two or more different DNA strands that interact with each other. The number of the stacked G-tetrads as well as the orientation and length of the loops lead to a high heterogeneity of G-quadruplex topologies as reviewed in (44,73,75).

In case of intramolecular quadruplexes one generally distinguishes between four different types of loop and strand arrangements as illustrated at the example of a G-quadruplex with three tetrads in Figure 5A: In an antiparallel G-quadruplex the strands are arranged in pairwise antiparallel orientation (2+2). Two prominent conformations are the chair structure with only lateral loops (Figure 5A left) and the basket structure with two lateral and one diagonal loop (Figure 5A, 2^nd from left). In a parallel G-quadruplex all G-tracts are arranged with the same strand polarity; as result only external loops are found (Figure 5A right). An example of the 3D structure of an all parallel propeller structure determined by NMR is also shown in Figure 3I. In the so-called (3+1) hybrid structure three strands are found in parallel arrangement with the fourth being aligned in the antiparallel orientation. Lateral and external loops connect the different G-tracts in this case (Figure 5A 3^rd from left).

Examples of various intermolecular G-quadruplex are shown in Figure 5B. Depending on loop arrangement three different antiparallel G-quadruplexes with two lateral or two diagonal loops can be distinguished. Furthermore the topology of a (3+1) hybrid formed from two nucleotide strands of unequal length is shown. The NMR solution structure of such an asymmetric complex in Na⁺ solution was reported by Zhang et al. and the 3D structure is shown in Figure 3H (55). Most of the first reported G-quadruplex structures were tetramers formed from short oligonucleotide strands (Figure 5B bottom) (33,36). Again antiparallel as well as parallel topologies are possible.

Figure 5: G-Quadruplex Topologies

A: Examples of different intramolecular G-quadruplexes with three G-tetrads, guanines are depicted by gray squares.

The following types of G-quadruplexes are distinguished according to strand orientation, from left to right: antiparallel chair structure, antiparallel basket structure, (3+1) hybrid with three strands in parallel orientation and one strand in antiparallel orientation and the all parallel propeller structure with only external loops. The general oligonucleotide sequence GGGLnGGGLnGGGLnGGG is shown underneath; G-tracts with bottom-to-top orientation are marked green, the reverse orientation in blue, connecting loops are gray. B: Examples of different intermolecular G-quadruplexes with three G-tetrads. Dimeric G-quadruplexes are shown in the top row, left: three antiparallel G-quadruplexes formed by GGGLnGGG dimers, right: a dimeric (3+1) hybrid structure. Bottom row: tetrameric G-quadruplexes with antiparallel and all parallel topologies.

To gain an insight into the relationship between loop length and topology Neidle and co-workers studied the loop length-dependent folding of G-quadruplexes and noted a preference for quadruplexes with short loops (1-2 nt) to fold in the parallel conformation with lateral loops.

Molecular dynamics simulations showed that linker length of T1-2 was too short for diagonal crossing of a tetrad, increased instability and hence favoured the formation of a parallel topology (74,76). Using a combination of circular dichroism (CD), UV melting, molecular modelling and simulation techniques, they determined an all parallel intramolecular G-quadruplex as the only possible structure for nucleic acids with three loops consisting of single nucleotides, e.g.

d(TGGGTGGGTGGGTGGGT) (74).

While all of the above examples depict G-quadruplexes with perfect G-tracts also broken-strand structures and G-tracts with looped out nucleotides have been reported. For instance Chen et al.

recently discovered a novel broken-strand structure for a G-quadruplex found in the G-rich

Figure 6: Syn- and Anti-Conformations of the Guanine Bases in G-Tetrads

A: Rotation of the nucleobase around the glycosidic bond results in anti or syn glycosidic arrangements. The gray highlighted region represents the sugar phosphate backbone, in a G-tetrad the base in the anti arrangement would be standing out from the paper plane, while in the syn conformation the base would be located behind the paper plane.

Figure adapted from (75). B: Examples of arrangements of syn/anti glycosidic arrangements in the different G-quadruplex topologies. In G-quadruplexes with antiparallel oriented strands nucleobases are rotated around the glycosidic bond in order to retain tetrad formation. Guanines with syn conformation are shown in blue, anti conformation is shown in green. Figures from B are adapted from Chapter 1 in (78).

In G-quadruplexes containing antiparallelly oriented strands nucleobases need to be rotated around the glycosidic bond in order to retain the Hoogsteen base pairing within the tetrad, such quadruplexes show syn as well as anti glycosidic conformations (Figure 6A). This results in four possible types of glycosidic steps between two guanines from the same strand: syn-syn, anti-anti, anti-syn and syn-anti (79). Structures with any combination of arrangements have been observed, selected examples are shown in Figure 6B (80). Alternating syn/anti or anti/syn glycosidic arrangements along the G-strand are found in antiparallel G-quadruplexes with lateral and diagonal loops. In the hybrid structure one base per tetrad is flipped in relation to the others, while in an all parallel G-quadruplex with only external loops the same glycosidic arrangement can be adopted by all bases.