Modulation of nucleic acid structure by ligand binding: Induction of a DNA·RNA·DNA hybrid tripler by DAPI intercalation

The aromatic diamidine, DAPI (4',6-diamidino-2-phenylindole), is used as an important biological and cytological tool since it forms highly fluorescent complexes with nucleic acid duplexes via minor groove-directed/intercalative modes of interaction. In this study, we find that DAPI binding can induce the formation of an RNA-DNA hybrid tripler that would not otherwise form. More specifically, through application of a broad range of spectroscopic, viscometric, and molecular modeling techniques, we demonstrate that DAPI intercalation induces the formation of the poly(dT)·poly(rA)·poly(dT) hybrid triple helix, a structure which does not form in the absence of the ligand. Using UV mixing studies, we demonstrate that, in the presence of DAPI, the poly(rA)·poly(dT) duplex and the poly(dT) single strand form a 1:1 complex (a triplex) that does not form in the absence of DAPI. Through temperature-dependent absorbance measurements, we show that the poly(dT)poly(rA)poly(dT) tripler melts via two distinct transitions: initial conversion of the tripler to the duplex state, with the DAPI remaining bound, followed by denaturation of the duplex-DAPI complex to its component single strands and free DAPI. Using optical melting profiles, we show that DAPI binding enhances the thermal stability of the poly(dT)·poly·(rA)·poly(dT) tripler, an observation consistent with the preferential binding of the ligand to the tripler versus the duplex and single-stranded states. Our differential scanning calorimetric measurements reveal melting of the DAPI-saturated poly·(dT)poly(rA)·poly(dT) tripler to be associated with a lower enthalpy but greater cooperativity than melting of the corresponding DAPI-saturated poly(rA)·poly(dT) duplex. Our now linear dichroism and viscometric data are consistent with an intercaIative mode of binding when DAPI interacts with both the poly(dT)·poly(rA) poly(dT) tripler and the poly(rA)·poly·(dT) duplex. Finally, computer modeling studies suggest that a combination of both stacking and electrostatic interactions between the intercalated ligand and the host nucleic acid play important roles in the DAPI-induced stabilization of the poly(dT)·poly(rA)·poly(dT) triplex. In the aggregate, our results demonstrate that ligand binding can be used to induce the formation of tripler structures that do not form in the absence of the ligand. This tripler-inducing capacity has potentially important implications in the design of novel antisense, antigene, antiviral, and diagnostic strategies.