An 11-nt long G-rich DNA oligonucleotide, 5′-d(GTGTGGGTGTG)-3′, corresponding to the most abundant sequence motif in irregular telomeric DNA from Saccharomyces cerevisiae (yeast) has been shown to fold into a G-hairpin. The first atomic resolution structure of a stable G-hairpin formed by a natively occurring DNA sequence demonstrates a novel type of mixed parallel/antiparallel fold-back DNA structure, which is stabilized by dynamic G:G base pairs that transit between N1-carbonyl symmetric and N1-carbonyl, N7-amino base-pair arrangements. G-hairpin is thermodynamically stable structure with a rather complex topology that includes a chain reversal arrangement of the backbone in the center of the continuous G-tract and 3′-to-5′ stacking of the terminal residues. The structure reveals previously unknown principles of the folding of G-rich oligonucleotides that could be applied to the prediction of natural and/or the design of artificial recognition DNA elements. The structure also demonstrates that the folding landscapes of short DNA single strands is much more complex than previously assumed.
Oligoethylene glycols are used in cell-mimicking systems designed to assess DNA characteristics. In addition, DNAs with covalently attached oligoethylene glycols are used as cargo carriers for drug delivery systems. We investigated DNA incorporating thymidine residues with covalently attached tetraethylene glycol (TEG), whereby the modified residues were incorporated into loop regions of antiparallel G-quadruplex or hairpin structures. By performing thermodynamic analysis we showed that the modified residues stabilize G-quadruplexes, whereas destabilize the hairpin structures. Furthermore, the NMR-based structural characterization and molecular dynamics calculations revealed that the TEG interacts with bases in the G-quartet and loop via CH-pi and lone pair-pi interactions. These results suggest that numerous cellular co-solutes likely affect DNA function through interactions, which were previously overlooked.
Structural studies with the use of solution-state nuclear magnetic resonance (NMR) spectroscopy have shown that oligonucleotides containing AGCGA repeats fold into structures that belong to a new structural family which we named AGCGA-quadruplexes. The structural core of the family is comprised out of four AGCGA repeats that form quartet planes. AGCGA-quadruplexes contain unusual structural elements such as GCGC and AGCGA quartets and are additionally stabilized with noncanonical base pairs. To the best of our knowledge, we have described GAGA-quartets formed by two G-A pairs in N1-N7 carbonyl amino geometry for the first time. G-G base pairs in N1-carbonyl symmetric geometry formally form loop regions and connect quartets inside AGCGA-quadruplexes. It is especially interesting that even though guanine residues are very common in oligonucleotides that form AGCGA-quadruplexes they in turn do not contain G-quartets and are insensitive to the presence of different cations such as Na+, K+ and NH4+. This property makes the AGCGA-quadruplex structural family unique compared to the related and more widely known family of G-quadruplexes. With bioinformatics studies we have shown that AGCGA rich sequences are found in regulatory regions of 39 human genes responsible for basic cellular processes that are related to neurological disorders, cancer and abnormalities in bone and cartilage development. With the use of NMR and CD spectroscopy we have confirmed that 46 oligonucleotides found in regulatory regions of the above mentioned 39 human genes fold into AGCGA-quadruplexes.
NMR structural study of the interaction between a small-molecule optical probe (DAOTA-M2) and a G-quadruplex from the promoter region of the c-myc oncogene revealed that they interact at 1:2 binding stoichiometry. NMR restrained structural calculations show that binding of DAOTA-M2 occurs mainly through pi–pi stacking between the polyaromatic core of the ligand and guanine residues of the outer G-quartets. High-resolution structure provides molecular guidelines for the design of triangulenium derivatives that can be used as optical probes for G-quadruplexes.
G-rich sequences found in human genome include hexanucleotide repeat TTAGGG in telomeric ends. Human telomere G-rich sequences have been shown to fold into diverse topologies in the presence of different cations, heterocyclic ligands, solution environments, and flanking sequences. A large role suggested for G-quadruplexes with antiparallel basket-type topology stimulated our study by NMR, CD, and UV spectroscopy focused on oligonucleotide d[TAGGG(TTAGGG)2TTAGG] (designated as htel1-G23), a truncated version of the human telomere sequence forming hybrid-1 G-quadruplex. htel1-G23 in the presence of KCl forms two distinct two G-quartet antiparallel basket-type G-quadruplexes. The topology of the KDH+ form exhibits unique structural features including orientations of loops and capping base pairs and base triple that effectively stack on two-quartet core. Its topology is distinctive to any reported human telomere G-quadruplexes with unique protonated T18·A20+·G5 base triple. Specific stacking interactions amongst two G-quartets flanking base triples and base pairs in TD and KDH+ forms are reflected in 10 K higher thermal stability of KDH+. Populations of TD and KDH+ forms are controlled by pH. The (de)protonation of A20 is the key for pH driven structural transformation of htel1-G23. Reversibility offers possibilities for its utilization as a conformational switch within different compartments of living cell enabling specific ligand and protein interactions.