Fluorescence Correlation Spectroscopy Studies The Thermodynamics Of Triple-stranded DNA Formation

Triple-stranded DNA is one of the most useful tools for the direct identification of specific-sequences of double-stranded DNA, which can be used to target DNA cleavage or crosslinking reagent, a transcription factor or a nucleic acid to a selected site on the DNA.They are not only as biotechnological tools to control gene expression but also as anti-gene drugs in vivo.In this paper, fluorescence correlation spectroscopy(FCS) was applied for characterizing the formation and stabilityof triplex DNA. The principle of this method is mainly based on the excellent capacity of FCS for sensitively distinguishing the free single strand DNA(ss DNA) fluorescent probes and the fluorescent probe-double strand DNA(ds DNA) complexes. Formation of triplex DNA was monitored by FCS of increased characterized diffusion time, due to the increase of molecular dimensions, upon Cy5-labeled ss DNA probe binding to the target duplex DNA.In this study, the Cy5-labeled ss DNA probe reaction with 70-mer ds DNA was used as a model. We firstly systematically investigated the experimental conditions of triplex DNA formation. And then, the equilibrium association constants(Ka) were achieved under different ss DNA probe length and composition, p Hs, and temperature. In the presence of 0.1 m M spermine and 150 m M KCl, the association constants were 2.2 × 106, 1.5 × 109 and 2.8 × 109 M-1 for 10-mer T, 15-mer T and 20-mer T ss DNA, respectively. Finally, FCS was applied to measure the hybridization fraction of a 20-mer perfectly matched ss DNA probe and three single-base mismatched ss DNA probes with 146 bp ds DNA(from human methylenetetrahydrofolate reductase(MTHFR) gene). We investigated the role of Ag+ specific stabilizing C? GC+ base triplex DNA,and selected spermine as the most suitable stabilizer.Our data illustrated that FCS was a useful tool for the direct determination of the thermodynamic parameters of triplex DNA formation and discrimination of single-base mismatch of triplex DNA. Compared to current methods, our method is characterized as high sensitivity, good universality and small sample and reagent requirements. We believe that this method will become an effective platform for researching of triplex DNA both in vitro and in vivo.