Edwin Quiñones

Structure and dynamics of single DNA molecules manipulated by magnetic tweezers and or flow

Here we describe the use of magnetic tweezers and or microfluidics to manipulate single DNA molecules. We describe experiment which employ magnetic tweezers coupled to an inverted microscope as well as the use of a magnetic tweezers setup with an upright microscope. Using a chamber prepared via soft lithography, we also describe a microfluidic device for the manipulation of individual DNA molecules. Finally, we present some past successful examples of using these approaches to elucidate unique information about protein-nucleic acid interactions.

Collisional nature of the magnetic field quenching of the acetylene state

Abstract Laser fluorescence excitation from the V2Kn(n=0–2) levels of acetylene was measured as a function of acetylene pressure in the absence and in the presence of an external magnetic field. A magnetic field (H) accelerates the rate of intersystem crossing, directly affecting the dynamics of the A 1 A u state. Studies carried out in the collisionless environment of a supersonic free-jet indicate that (1) the integrated fluorescence intensity remains the same upon applying H, (2) the fluorescence decay times increase with H, (3) the fluorescence decay amplitudes decrease with H. Therefore, molecules transferred to the triplet manifold can return to the singlet manifold and emit. In contrast, faster fluorescence decays are measured when the excited molecules are allowed to undergo collisions in the presence of H. The fluorescence is quenched because the system spends more time in the triplet manifold, which increases the chance of collisional deactivation. A discussion is presented in the context of the acetylene level structure, non-radiative processes, level coupling induced by H, and collisional relaxation.

A spectroscopic method to determine the activity of the restriction endonuclease EcoRV that involves a single reaction.

A one-step protocol is presented to determine the activity of EcoRV as a model of restriction enzymes. The protocol involved a molecular beacon as DNA substrate, with the target sequence recognized by EcoRV in the stem. EcoRV cleaved the stem forming two fragments, one of which contained the fluorophore and quencher, initially bound by 3 bp. This shorter fragment rapidly dissociated at 37 °C, causing an increase of fluorescence intensity that was used to gauge the reaction kinetics. The reaction can be described using the Michaelis-Menten mechanism, and the kinetic parameters obtained were compared with literature values involving other protocols.

Relaxation of individual rotational levels of the electronic state of acetylene excited to the 2ν3′ and (ν1′+ν3′+ν6′) vibrational modes

Abstract Collisionless and collisional (self-quenching) rate constants were measured for several rotational states of the V 2 1 K 0 1 and V 2 1 K 2 1 subbands of acetylene under bulk conditions. For jet-cooled acetylene, the collisionless decay rates measured for the 1 1 6 1 V 1 6 K 1 0 subband are larger than those for the V 2 1 K 0 1 and V 2 1 K 2 1 subbands. The present results are compared with the kinetic data available from the literature. The differences observed among the collisionless rate constants as a function of vibro-rotational energy are explained in terms of non-radiative processes in acetylene.

Assays for the determination of the activity of DNA nucleases based on the fluorometric properties of the YOYO dye.

Here we characterize the fluorescence of the YOYO dye as a tool for studying DNA-protein interactions in real time and present two continuous YOYO-based assays for sensitively monitoring the kinetics of DNA digestion by λ-exonuclease and the endonuclease EcoRV. The described assays rely on the different fluorescence intensities between single- and double-stranded DNA-YOYO complexes, allowing straightforward determination of nuclease activity and quantitative determination of reaction products. The assays were also employed to assess the effect of single-stranded DNA-binding proteins on the λ-exonuclease reaction kinetics, showing that the extreme thermostable single-stranded DNA-binding protein (ET-SSB) significantly reduced the reaction rate, while the recombination protein A (RecA) displayed no effect.

Rotational structure of a super-excited state of the NO molecule revealed by OODR-multiphoton laser spectroscopy.

The optical-optical double resonance time of flight (OODR-TOF) spectroscopy technique was employed to examine the 65,000-66,500 cm(-1) region of the nitric oxide spectrum. In this region, we detected the following three electronic states: E (2)Sigma(+) (nu = 2) (Rydberg state), B (2)Pi (nu = 23) (valence state), and L (2)Pi (nu = 4) (valence state). The rotational structure analysis of an unexpected band in the red part of the spectra revealed the presence of a new super-excited (2)Sigma(+) Rydberg state at approximately 13.3 eV, which was populated through a three-photon transition from the intermediate A (2)Sigma(+) (nu = 0) state. This super-excited state converges to the NO (a(3)Sigma(+)) ionic state with electronic configuration (1sigma)(2)(2sigma)(2)(3sigma)(2)(4sigma)(2)(5sigma)(2)(1pi)(3)(2pi)(1)(3ssigma)(1).