title: Binding and cleavage of DNA with the restriction enzyme EcoR1 using time-resolved second harmonic generation.
abstract: The binding of EcoR1 to a 90-bp DNA duplex attached to colloidal microparticles and the subsequent cleavage by the enzyme was observed in real time and label-free with time-resolved second harmonic (SH) spectroscopy. This method provides a unique way to investigate biomolecular interactions based on its sensitivity to changes in structure and electrical charge on formation of a complex and subsequent dynamics. The binding of EcoR1 to the recognition sequence in DNA appears as a rapid increase in the SH signal, which is attributed to the enzyme-induced change in the DNA conformation, going from a rod-like to a bent shape. In the presence of the cofactor Mg(2+), the subsequent decay in the SH signal was monitored in real time as the following processes occurred: cleavage of DNA, dissociation of the enzyme from the DNA, and diffusion of the 74-bp fragment into the bulk solution leaving the 16-bp fragment attached to the microparticle. The observed decay was dependent on the concentration of Mg(2+), which functions as a cofactor and as an electrolyte. With SH spectroscopy the rehybridization dynamics between the rehybridized microparticle bound and free cleaved DNA fragments was observed in real time and label-free following the cleavage of DNA. Collectively, the experiments reported here establish SH spectroscopy as a powerful method to investigate equilibrium and time-dependent biological processes in a noninvasive and label-free way.
authors: Department of Chemistry, Columbia University, 3000 Broadway, New York, NY 10027, USA.
authors: Benjamin
authors: B
authors: Doughty
authors: Samuel W
authors: SW
authors: Kazer
authors: Kenneth B
authors: KB
authors: Eisenthal
mesh_terms: D001483:Base Sequence; D004247:DNA / Q000378:metabolism*; D015246:Deoxyribonuclease EcoRI / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization / Q000187:drug effects; D011485:Protein Binding / Q000187:drug effects; D012965:Sodium Chloride / Q000494:pharmacology; D013057:Spectrum Analysis / Q000379:methods*; D013997:Time Factors
chemical_list: D012965:Sodium Chloride; D004247:DNA; D015246:Deoxyribonuclease EcoRI