Book of Abstracts: Albany 2007
June 19-23 2007
A Novel G-quartets-containing Fold of Microsatellite d(GT)n Sequences
Polymorphic microsatellite d(GT)n sequences are known to drastically affect regulation of gene expression. The most widespread repeats consist of 10 up to 20 dinucleotides. Unusual secondary structures easily formed by simple d(GT)n stretches could be relevant, however they have been studied relatively poorly. Here we report that d(GT)8 and d(GT)16 oligonucleotides can form intramolecular helices with guanines making up quartets and thymines bulging out. Formation of an intramolecular structure by d(GT)16 in 10 mM Na phosphate buffer containing 0.1 M KCl or 0.1 M NaCl was detected by spectral methods. The CD spectra support the presence of guanine quartets with anti-conformation of glycosidic bonds as in a parallel quadruplex structure (1). Hydrodynamic volume of the oligonucleotide estimated by fluorescence polarization of EtBr probe indicates the monomolecular character of the structure. Up to six EtBr molecules cooperatively bind to d(GT)16 molecule, two EtBr molecules intercalating in each of three gaps between adjacent guanine quartets.
The shorter oligonucleotide d(GT)8 was shown to be in the unfolded conformation in sodium-containing solution, while in the presence of potassium ions the oligonucleotide forms relatively unstable G-containing intramolecular folds. EtBr addition induces and stabilizes the structure of (GT)8 with guanines forming quartets similar to those of a parallel quadruplex structure. This was evidenced by CD and thermal denaturation measurements. The cooperative type of EtBr binding, stoichiometry and dissociation constants were determined.
A fragment of the proposed model of the novel structure is given in the figure. The two of the four quartets of the proposed d(GT)16 structure are shown.
The guanines form G4 quartets, while thymines are bulged out. The internal K+ is located in the center between two quartets. The backbone is displayed as a band with arrow pointing toward the C3?end. Importantly, the model suggests a possibility of the structure extension with increasing the length of the d(GT) repeating stretches.
Reference and Footnotes
Dmitry N. Kaluzhny*
Engelhardt Institute of Molecular Biology