RESUMO
Base stacking is fundamentally important to the stability of double-stranded DNA. However, few experiments can directly probe the local conformations and conformational fluctuations of the DNA bases. Here we report a new spectroscopic approach to study the local conformations of DNA bases using the UV-absorbing fluorescent guanine analogue, 6-methyl isoxanthopterin (6-MI), which can be used as a site-specific probe to label DNA. In these experiments, we apply a two-photon excitation (2PE) approach to two-dimensional fluorescence spectroscopy (2DFS), which is a fluorescence-detected nonlinear Fourier transform spectroscopy. In 2DFS, a repeating sequence of four collinear laser pulses (with center wavelength ~ 675 nm and relative phases swept at radio frequencies) is used to excite the lowest energy electronic-vibrational (vibronic) transitions of 6-MI (with center wavelength ~ 340 nm). The ensuing low flux fluorescence is phase-synchronously detected at the level of individual photons and as a function of inter-pulse delay. The 2PE transition pathways that give rise to electronically excited state populations include optical coherences between electronic ground and excited states and non-resonant (one-photon-excited) virtual states. Our results indicate that 2PE-2DFS experiments can provide information about the electronic-vibrational spectrum of the 6-MI monomer, in addition to the conformation-dependent exciton coupling between adjacent 6-MI monomers within a (6-MI)2 dimer. In principle, this approach can be used to determine the local base-stacking conformations of (6-MI)2 dimer-substituted DNA constructs.
RESUMO
Fatty acids are of particular interest for industrial applications of microalgal feedstock, as these have a wide array of different uses such as pharmaceuticals and biofuels. Fourier transform infrared (FTIR) spectroscopic techniques used in combination with multivariate prediction modeling are showing great potential as analytical methods for characterizing microalgal biomass. The present study investigated the use of diffuse reflectance Fourier transform infrared spectroscopy (DRIFTS) coupled with partial least squares regression (PLSR) to estimate fatty acid contents in microalgae. A prediction model for microalgal samples was developed using algae cultivated in both Bold's basal medium (BBM) and sterilized municipal wastewater under axenic conditions, as well as algal polycultures cultivated in open raceway ponds using untreated municipal wastewater influent. This universal prediction model was able to accurately predict microalgal samples of either type with high accuracy (RMSEP = 1.38, relative error = 0.14) and reliability (R2 > 0.92). DRIFTS in combination with PLSR is a rapid method for determining fatty acid contents in a wide variety of different microalgal samples with high accuracy. The use of spectral characterization techniques offers a reliable and environmentally friendly alternative to traditional labor intensive techniques based on the use of toxic chemicals.
RESUMO
DNA regulation and repair processes require direct interactions between proteins and DNA at specific sites. Local fluctuations of the sugar-phosphate backbones and bases of DNA (a form of DNA 'breathing') play a central role in such processes. Here we review the development and application of novel spectroscopic methods and analyses - both at the ensemble and single-molecule levels - to study structural and dynamic properties of exciton-coupled cyanine and fluorescent nucleobase analogue dimer-labeled DNA constructs at key positions involved in protein-DNA complex assembly and function. The exciton-coupled dimer probes act as 'sensors' of the local conformations adopted by the sugar-phosphate backbones and bases immediately surrounding the dimer probes. These methods can be used to study the mechanisms of protein binding and function at these sites.