The new models of the human telomere d[AGGG(TTAGGG)3] in K+ solution
Graphical abstract
We substituted the Gs in the sequence with 8-bromoguanine and examined the resultant structures and thermal stabilities by circular dichroism (CD) spectroscopy. The results suggest that the 22 nt in K+ solution exists as a mixture of mixed-parallel/antiparallel and chair-type G-quadruplex.
Introduction
The various conformations of DNA—the A, B, and Z forms, the protein-induced DNA kink, and the G-quartet form—are thought to play important biological roles in processes such as DNA replication, gene expression and regulation, and the repair of DNA damage.1, 2 In particular, G-quadruplex DNA formed from telomeric sequence repeats may be important for telomere maintenance,3, 4 and cell aging or death, which have become currently an attractive therapeutic target for the development of novel anticancer agents.5, 6, 7, 8, 9, 10, 11, 12
G-quadruplexes are highly polymorphic, and a large number of different structures have been observed13, 14. The different G-quadruplex topologies may be associated with some related aspects: the syn/anti conformation of guanine residues, the relative orientation of the G-quartet core, the types of linking loops, and the nature of associated metal cations.15, 16, 17, 18, 19, 20, 21, 22 Detailed structural studies have provided evidence for two distinct conformations of the human telomere G-quadruplex in the presence of Na+ and K+ ions.23, 24 The solution structure of the 22 nt sequence d[AGGG(TTAGGG)3] in the presence of Na+ ions has been elucidated by NMR analysis. This showed an antiparallel basket-type structure in which the opposing GGG columns are antiparallel, with one diagonal and two lateral TTA loops (Fig. 1a). On the other hand, the same 22 nt sequence adopts a completely different propeller-type structure in a crystal grown in the presence of K+ ions. In this structure, four core GGGs are parallel, with the three linking external loops positioned on the exterior of the G-quartet core (Fig. 1b).
As the intracellular K+ concentration is greater than that of Na+, the structure of this sequence in K+ solution has drawn intense interest. To investigate the solution structure of the 22 nt sequence, various studies have been performed using platinum cross-linking, FRET, 125I-radioprobing, covalent ligation, sedimentation, and NMR.25, 26, 27, 28, 29, 30 Our laboratory used photochemical methods to detect the diagonal loops in the antiparallel structure, suggesting that in K+ the sequence does not possess a diagonal loop.31 Although these approaches gave some structural information, the definitive structural characteristics of the human telomere G-quadruplex in K+ solution have not yet been determined. To define the structural features of 22 nt sequence in K+ solution, we substituted the Gs in the 22 nt sequence with 8-bromoguanine (BrG) and examined the resultant structures and their thermal stabilities in K+ solution by circular dichroism (CD) spectroscopy. Based on these results, we propose a novel mixed parallel/antiparallel G-quadruplex structure containing two lateral loops and one external loop, together with an interconvertible chair-type G-quadruplex structure. These structures suggest the possible formation of models from the extended G-quadruplex: (i) individual quadruplexes form higher-order telomeric DNA structure, (ii) telomere sequences form two loop structures including a t-loop.32 This may provide important information for understanding telomere structure and the development of telomere G-quadruplex-binding molecules as telomerase inhibitors.
Section snippets
CD spectrum of 22 nt sequence in K+ solution
The typical bands in the CD spectra show fundamental characteristics allowing G-quadruplex structures to be distinguished.34, 35, 36, 37, 38, 39 As shown in Figure 2a (blue), the CD spectrum of the human telomeric sequence d[AGGG(TTAGGG)3] (ODN 1) in the presence of 100 mM Na+ ions has a 295 nm positive band and a 265 nm negative band, which is characteristic of an antiparallel G-quartet structure consistent with the results of previous NMR studies23. In contrast, the CD spectrum of ODN 1 in 100 mM
Synthesis of oligonucleotides
The phosphoamidite of 8-bromo-2′-deoxyguanosine was purchased from Glen Research. Oligonucleotides containing BrG were prepared by the phosphoamidite method on controlled pore glass supports (1 μmol) using an Applied Biosystems 3400 DNA synthesizer. After automated synthesis, the oligomers were detached from the support, deprotected, and purified by HPLC. The oligomers were identified by electrospray ionization mass spectrometry (ESI-MS) on a Perkin Elmer SCIEX API 165 mass spectrometer
Acknowledgments
This study was partly supported by a Grant-in-Aid for Priority Research from the Ministry of Education, Science, Sports and Culture, Japan; and SORST of Japan Science and Technology (JST).
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