Ionic Microenvironmental effects on Triplex DNA Stabilization

Abstract

DNA structure and conformation are influenced by metal ions, polyamines, and the microenvironment. Positively charged ions stabilize DNA and RNA structures, and provoke conformational transitions. In poly(purine).poly(pyrimidine) sequences, triplex DNA formation is facilitated by metal ions, polyamines and several other ligands. We studied the effects of mono, and di-valent metal ions, and ammonium salts on the stability of triple- and double-stranded structures formed from poly(dA) and poly(dT) by measuring their respective melting temperatures. In the presence of metal ions, the absorbance versus temperature profile showed two transitions: Tm1 for triplex to duplex and single stranded DNA, and Tm2 for duplex DNA melting to single stranded DNA. Monovalent cations (Li+, Na+, K+, Rb+, Cs+ and NH4+) promoted triplex DNA at concentrations ≥ 150 mM. The triplex DNA melting temperature varied from 49.8 °C in the presence of 150 mM Li+ to 30.6 °C in the presence of 150 mM K+. Among ammonium compounds, NH4+ was the most effective ion in stabilizing triplex DNA and its efficacy decreased with increasing substitution of the hydrogen atoms with bulky alkyl groups. Divalent cations were 1000-fold more efficacious than monovalent ions in stabilizing triplex DNA. All positively charged cations increased the melting temperature of duplex DNA. using 1/Tm1/1/Tm2 versus ln [ion concentration] plots, we calculated the amount of cation release and appropriate thermodynamic parameters, on triple/duplex melting. Circular dichroism spectroscopic studies showed distinct conformational changes in triplex DNA stabilized by alkali metal and ammonium ions. The results suggest ion specific effects on triplex DNA stabilization. Our results might be useful in developing triplex forming oligonucleotide based gene silencing techniques.