title: Studies on the formation and stability of triplex DNA using fluorescence correlation spectroscopy.
abstract: Triplex DNA has become one of the most useful recognition motifs in the design of new molecular biology tools, therapeutic agents and sophisticated DNA-based nanomaterials because of its direct recognition of natural double-stranded DNA. In this paper, we developed a sensitive and microscale method to study the formation and stability characterization of triplex DNA using fluorescence correlation spectroscopy (FCS). The principle of this method is mainly based on the excellent capacity of FCS for sensitively distinguishing between free single-strand DNA (ssDNA) fluorescent probes and fluorescent probe-double-strand DNA (dsDNA) hybridized complexes. First, we systematically investigated the experimental conditions of triplex DNA formation. Then, we evaluated the equilibrium association constants (K(a)) under different ssDNA probe lengths, composition and pH. Finally, we used FCS to measure the hybridization fraction of a 20-mer perfectly matched ssDNA probe and three single-base mismatched ssDNA probes with 146-mer dsDNA. Our data illustrated that FCS is a useful tool for the direct determination of the thermodynamic parameters of triplex DNA formation and discrimination of a single-base mismatch of triplex DNA without denaturation. Compared with current methods, our method is characterized by high sensitivity, good universality and small sample and reagent requirements. More importantly, our method has the potential to become a platform for triplex DNA research in vitro.
authors.affiliation: College of Chemistry & Chemical Engineering, Shanghai Jiao Tong University, Shanghai, People's Republic of China.
authors.forename: Hongyan
authors.initials: H
authors.lastname: Hu
authors.affiliation: College of Chemistry & Chemical Engineering, Shanghai Jiao Tong University, Shanghai, People's Republic of China.
authors.forename: Xiangyi
authors.initials: X
authors.lastname: Huang
authors.affiliation: College of Chemistry & Chemical Engineering, Shanghai Jiao Tong University, Shanghai, People's Republic of China.
authors.forename: Jicun
authors.initials: J
authors.lastname: Ren
mesh_terms: D004247:DNA / Q000138:chemical synthesis* / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry; D013050:Spectrometry, Fluorescence
chemical_list: D005456:Fluorescent Dyes; C051802:triplex DNA; D004247:DNA
keywords: association constants; fluorescence correlation spectroscopy; formation; stability; triplex DNA