Use of anion-exchange HPLC to study DNA conformational polymorphism

Abstract

Anion-exchange chromatography carried out under non-denaturing conditions is a versatile tool to differentiate DNA conformations. In this work, the utility of this form of HPLC was demonstrated in four examples. The hairpin and duplex forms of d(CG)9 were readily resolved, which allowed for the studies of the influence of salt on the equilibrium of these two forms of secondary structures. Similarly, the minimum size of Tn in the loop region required for the sequence 5′-d(CCCAA-(T)n-TTGGG)-3′ to form hairpin was established to be two nucleotides using anion-exchange HPLC and fluorescence resonance energy transfer. Furthermore, the efficiency of hybridization of partially self-complementary sequences d[(CG)6Nx] was readily monitored by non-denaturing anion-exchange HPLC. Finally, different structures adopted by quadruplex-forming sequences were resolved in the same manner.