A new approach to precise thermodynamic characterization of hybridization properties of modified oligonucleotides: Comparative studies of deoxyribo- and glycine morpholine pentaadenines

The development of new derivatives and analogues of nucleic acids for the purposes of molecular biology, biotechnology, gene diagnostics, and medicine has been a hotspot for the last two decades. Methylenecarboxamide (glycine) morpholine oligomer analogues (gM) seem to be promising therapeutic candidates because of the ability to form sequence specific complexes with DNA and RNA. In this paper we describe new approaches to the determination of thermodynamic parameters for hybridization of tandem oligonucleotide complexes with the complementary template. It makes possible to determine changes in enthalpy and entropy corresponding to the binding of an individual oligomer with the template, and to the formation of cooperative contact at the helix-helix interface of two neighboring duplex fragments (in the nick). We have experimentally analyzed the series of model tandem complexes of different length at various oligomer concentrations, ionic strength, and pH. The analysis of thermodynamic parameters of complex formation for native and modified oligomers revealed higher Gibbs free energy values of hybridization and cooperative interaction of morpholine-containing complexes at the helix-helix interface under standard conditions (1 M NaCl, pH 7.2). Further comparative analysis of the hybridization properties of modified oligomers at ionic strength and pH allows us to determine the charge state of the morpholine backbone and the thermodynamic origin of the effects observed. It was found that the decrease in pH to 5.5 led to the protonation of internal morpholine nitrogens. The obtained results prove the veracity of the proposed model and the possibility to evaluate thermodynamic parameters of short native and modified oligomers with high accuracy.