Unusual stability exhibited by (AT)XN12(AT)Y motif associated with high fetal hemoglobin levels

Abstract

Quasi-palindromic sequences (AT)XN12(AT)Y present in HS2 (hypersensitive site 2) of the human β-globin locus are known to be significantly associated with increased fetal hemoglobin (HbF) levels. High HbF levels in some adults arise due to pathological conditions such as sickle cell disease and β-thalassemia. However, elevated levels of HbF are also associated with a reducing morbidity and mortality in patients with β-thalassemia and thus ameliorate the severity of the disease. Using gel-electrophoresis, ultraviolet (UV)-thermal denaturation, and circular dichroism (CD) techniques, we demonstrated that it exhibits a hairpin-duplex equilibrium. Intramolecular species (hairpin) were observed in both low and high salt concentrations in gel assay studies displaying the unusual stability of intramolecular species even at the high counter-ion concentration. The unusual stability of hairpin secondary structures was also demonstrated by the monophasic nature of the melting profiles for the oligonucleotides which persisted at low as well as high salt and oligomer concentrations. Change in CD spectra as a function of oligomer concentration indicates that the bimolecular duplex formation is selectively favored over monomolecular hairpin formation at and above 9 µM oligomer concentration. Thus, we hypothesize that imperfect inverted repeat sequence (AT)XN12(AT)Y of HS2 of β-globin gene LCR forms the unusually stable hairpins which may result in the formation of a cruciform structure that may be recruited for binding by various nuclear proteins that could result in elevated HbF levels.Communicated by Ramaswamy H. Sarma.