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.

Quantitative Determination of Telomerase Activity by Combining Fluorescence Correlation Spectroscopy with Telomerase Repeat Amplification Protocol

“…FCS is a sensitive single molecule method, and its principle is based on measuring the fluorescence fluctuations in a small detection volume due to Brownian motion of single molecule . FCS is widely used to determine molecular concentration and study molecular diffusion, chemical kinetics, and molecular interactions like DNA–DNA, mRNA–protein, protein–protein, enzyme activity, and drug–protein interaction in vitro or in vivo. − In this new method, FCS is used to quantitatively analyze DNA fragments in the TRAP reaction. The principle of FCS-TRAP is based on measurement of the changes in characteristic diffusion time and molecule number of TRAP products by FCS.…”