Fundamentals of Quadruplex Structures

GARY NIGEL PARKINSON

The School of Pharmacy, University of London, 29–39 Brunswick Square, Bloomsbury, WC1N 1AX, London, UK

1.1 Background and Introduction to Quadruplexes

Self-association of guanosine at millimolar concentrations has been observed in solution since the 19th century as characterized by the ready formation of polycrystalline gels. In the 1960s Gellert et al. determined the associated guanine bases to be in a tetrameric arrangement by crystallographic methods, described simply as a G-quartet arrangement. The four guanine bases form a square co-planar array where each base is both a hydrogen bond donor and hydrogen bond acceptor. Utilization of both the N1 and N2 of one face with the O6 and N7 of the second face on guanosine yields eight hydrogen bonds per planar G-quartet [Figure 1(a-b)]. With the development of chemical synthesis of extended polyguanine oligonucleotides strands additional associations were observed in the laboratory environment. CD and IR spectroscopy confirmed the same self-assembly and association of the guanines into G-quartets, while X-ray fiber diffraction studies demonstrated a four-stranded motif with stacked tetrad planes, termed quadruplexes. These stacked tetrads align themselves to give a similar appearance to that of duplex DNA [Figure 2(a)], characterized by a regular rise and twist between the tetrad planes and generating a right-handed helical twist [Figure 2(c)]. In this case the phosphate backbones, linking the nucleosides together, generate four grooves of variable width, instead of two, giving the quadruplex DNA motif a characteristic duplex DNA feel.

Interest in the structural arrangements of G-quadruplexes was ignited in the early 1990s by the identification of G-rich repetitive sequences located at the end of chromosomes and a protein, with a reverse transcriptase activity, involved in their maintenance. This ground-breaking work was carried out by Blackburn et al. in Joe Gall's research group. They applied sequencing techniques developed previously in Sanger's research laboratory in the late 1970s, a group that was conducting the first comprehensive sequencing experiments on genomic material. It was quickly realized that these guanine-rich repetitive telomeric DNA sequences could form higher order structures and were likely to be involved in chromosomal maintenance. Structural studies on the telomeric sequences revealed both parallel and anti-parallel strand orientations, as well as mixed anti and syn glycosidic torsion angles with the specific features of the quadruplex structural motif dependent upon sequence. The structures determined for the telomeric 3' overhang were of particular interest in terms of chromosomal DNA packaging, and molecular self-assembly, particularly as these G-rich sequences can form compact, well-defined and stable structural motifs.

1.2 Fundamental Components of DNA and RNA

The following is a very basic overview of nucleic acids and their associated properties, focused in areas that relate to quadruplex structure. For a more comprehensive description references 15–19 provide excellent reading. Nucleic acids are polymers of nucleotide units. Each nucleotide unit is composed of three important building blocks: the bases, sugars, and phosphate groups. The nucleoside consists of a base attached to a pentose sugar ring [Figure 3(a)]. In RNA the sugar is a ribose, and in DNA a deoxyribose. The unmodified bases utilized in DNA comprise guanine, cytosine, adenine, and thymine, while for RNA the thymine base is substituted by uracil. The phosphate groups are attached at the 5' side of the nucleoside serving as the linking element between the nucleosides to form a nucleotide. Polymers formed from these three basic components have particular properties that make them ideal for the long-term storage of genetic information in living cells. The nucleotide units are chemically stable and the individual strands have the ability to associate together via complementary bases to form a stacked duplex structure. These polymers are then able to store complimentary copies of genetic information in a compact form that can both disassociate and re-associate.

1.2.1 Building Blocks

The bases are the key components that confer chemical variability to DNA/ RNA. The bases have complementary hydrogen bond donors and acceptors that generate specific associations between bases. There are two faces involved in hydrogen bond formation, the Watson–Crick and the Hoogsteen face, as shown in Figure 3(b). Association via base pairing is normally seen between purines (Guanine, Adenine) and pyrimidines, (Thymine, Cytosine, and Uracil) bases utilizing the basic Watson–Crick base-pairing motif. The G:C base pairing, with its three hydrogen bonds is more stable than the A:T/A:U base pairing with only two hydrogen bonds. This is partially reflected in the higher melting temperatures for GC rich sequences. Other hydrogen bonding arrangements are possible between base pairs and these include a reversed Watson–Crick, a GT wobble pair as well as the use of the Hoogsteen face to give Hoogsteen pairings and reversed Hoogsteen pairings. It is the additional use of the Hoogsteen faces that is critical in the formation and stabilization of tetrads [Figure 3(b)]. The bases with their nitrogen atoms have the added capability to change protonation states based on pH. At neutral pH standard base pairing occurs but at elevated (pH >9.2) or reduced pH (pH <4.2) additional base associations have been observed. The bases are covalently linked to the sugar via the glycosylic bond. The energy minima conformations available to this linkage, within an extended nucleic acid structure, are important in determining how DNA folds and the stability of these folded structures. This dihedral angle linkage χ defined as O4'-C1'-N9-C4 for purines and O4'-C1'-N1-C2 for pyrimidine bases, Figure 3(c). Glycosidic...