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.cleavage kinetics | DNA-endonuclease | rehybridization kinetics | nonspecific binding T he highly selective binding of proteins to specific nucleotide sequences in DNA is an essential step in many biological processes, including gene regulation, gene expression, and DNA hybridization (1). The family of type II restriction enzymes, which includes EcoR1, have been, and continue to be, of major importance in serving as model systems of protein-DNA interactions. The endonucleases, because of their capability to bind and cleave specific DNA sequences, also play a key role in DNA recombinant and cloning methods (1, 2). The EcoR1-DNA equilibria and reaction dynamics have been probed extensively by a number of established methods, such as, but not limited to: FRET (3-5), gel electrophoresis (6-15), surface plasmon resonance (16), microfluidic trapping (17), two-color cross-correlation spectroscopy (18), and isothermal calorimetry (19).The essential characteristic of the method used here is the application of second harmonic (SH) spectroscopy to selectively probe the interface of a polystyrene carboxylate (PSC) microparticle, to which target DNA molecules have been attached. Significant effort and progress has been made in hybridizing artificial colloidal nano-and micromaterials with naturally occurring biological reagents to build self-assembled superstructured materials (20-26), and to develop biologically coded probes for protein detection and disease treatment (27,28). The present work builds on these pioneering studies, and on studies of DNA supported on flat surfaces using SH and sum-frequ...

Plasmon-Enhanced Photocleaving Dynamics in Colloidal MicroRNA-Functionalized Silver Nanoparticles Monitored with Second Harmonic Generation

“…The higher SHG signal in the miRNA-functionalized SNPs is mainly attributed to the negatively charged phosphate groups of the oligonucleotides, which increase the surface charge density magnitude and the corresponding surface potential, resulting in a larger χ (3) term. This interpretation is consistent with previous work and with our earlier results on miRNA-functionalized GNPs. ,,, The 6-TAMRA fluorophore has negligible contribution to the SHG signal because it has negligible absorption near the 800 nm probe and 400 nm SHG wavelengths. Figure b shows representative SHG spectra from the miRNA-functionalized SNPs at different UV-irradiation times of 0, 90, and 800 s using 365 nm irradiation with a 20 mW average power.…”

“…This interpretation is consistent with previous work and with our earlier results on miRNA-functionalized GNPs. 10,33,58,62 The 6-TAMRA fluorophore has negligible contribution to the SHG signal because it has negligible absorption near the 800 nm probe and 400 nm SHG wavelengths. Figure 4b shows representative SHG spectra from the miRNA-functionalized SNPs at different UV-irradiation times of 0, 90, and 800 s using 365 nm irradiation with a 20 mW average power.…”

“…The SHG electric field can be described by the equation E SHG = A + B [ C ], where A is an offset, B is a proportionality constant, and [ C ] is the cumulative concentration of miRNA attached to the surface of nanoparticles. As noted earlier, the change in E SHG as a function of attached miRNA at the nanoparticle surface is attributed mainly to the surface charge density and the resulting χ (3) term. ,, Under the UV irradiation-induced photocleaving, the attached miRNA concentration should exponentially decay in time t according to the equation, [ C ]­( t ) = [ C ] 0 e – k ′ t , where [ C ] 0 is the initial concentration of miRNA attached to the nanoparticle surface and the pseudo-first-order rate constant k ′ is linearly proportional to the UV laser intensity, assuming that the photocleaving is a single-photon process.…”

Probing the relative orientation of molecules bound to DNA through controlled interference using second-harmonic generation

“…The experimental apparatus has been described previously (4,5). Briefly, a KM Labs Ti:sapphire oscillator running at 80 MHz, center wavelength of 840 nm, producing 300 mW of average power with a pulse width of ∼60 fs was focused into a 2-mm cuvette.…”

Unraveling the Molecular Weight Dependence of Interfacial Interactions in Poly(2-vinylpyridine)/Silica Nanocomposites

“…The amplitudes for the −OH bands are scaled by the estimated number density of particles on the surface as determined by XPS measurements (see SI). Here, we assume a linear relationship between the observed SFG intensity and the particle number density, as typically done in studies of NP samples using nonlinear spectroscopies. − The scaled amplitude of the −OH bands, determined from the fit of the spectra, indicates higher concentration of the free OH binding sites on the surfaces of the NPs in PNCs with higher MW (Figure b). The observed systematic changes in the XPS and SFG spectra (Figures and ) suggest a significant decrease in the number of interaction sites between the polymer and NPs with increasing MW.…”