Molecular Dynamics Simulation of a PNA·DNA·PNA Triple Helix in Aqueous Solution

Molecular dynamics simulations have been used to explore the conformational flexibility of a PNA·DNA·PNA triple helix in aqueous solution. Three 1.05 ns trajectories starting from different but reasonable conformations have been generated and analyzed in detail. All three trajectories converge within about 300 ps to produce stable and very similar conformational ensembles, which resemble the crystal structure conformation in many details. However, in contrast to the crystal structure, there is a tendency for the direct hydrogen-bonds observed between the amide hydrogens of the Hoogsteen-binding PNA strand and the phosphate oxygens of the DNA strand to be replaced by water-mediated hydrogen bonds, which also involve pyrimidine O2 atoms. This structural transition does not appear to weaken the triplex structure but alters groove widths and so may relate to the potential for recognition of such structures by other ligands (small molecules or proteins). Energetic analysis leads us to conclude that the reason that the hybrid PNA/DNA triplex has quite different helical characteristics from the all-DNA triplex is not because the additional flexibility imparted by the replacement of sugar−phosphate by PNA backbones allows motions to improve base-stacking but rather that base-stacking interactions are very similar in both types of triplex and the driving force comes from weak but definate conformational preferences of the PNA strands.

Understanding Electrophoretic Stacking Of In-Capillary Generated Reaction Products

The separation of small molecules by capillary electrophoresis is governed by a complex interplay among several physical effects. Until recently, a systematic understanding of how the influence of all of these effects is observed experimentally has remained unclear. The work presented in this thesis involves the use of transient isotachophoretic stacking (tITP) and computer simulation to improve and better understand an in-capillary chemical assay for creatinine. This assay involves the use of electrophoretically mediated micro-analysis (EMMA) to carry out the Jaffé reaction inside a capillary tube. The primary contribution of this work is the elucidation of the role of the length and concentration of the hydroxide plug used to achieve tITP stacking of the product formed by the in-capillary EMMA/Jaffé method. Computer simulation using SIMUL 5.0 predicts that a 3-4 fold gain in sensitivity can be recognized by timing the tITP stacking event such that the Jaffé product peak is at its maximum height as that peak is electrophoresing past the detection window. Overall, the length of the hydroxide plug alters the timing of the stacking event and lower concentration plugs of hydroxide lead to more rapidly occurring tITP stacking events. Also, the inclusion of intentional tITP stacking in the EMMA/Jaffé method improves the sensitivity of the assay, including creatinine concentrations within the normal biological range. Ultimately, improvement in assay sensitivity can be rationally designed by using the length and concentration of the hydroxide plug to engineer the timing of the tITP stacking event such that stacking occurs as the Jaffé product is passing the detection window.

A MICROFLUIDIC METHOD TO MEASURE DIFFUSION IN HYDROGELS

A novel microfluidic method is proposed for studying diffusion of small molecules in a hydrogel. Microfluidic devices were prepared with semi-permeable microchannels defined by crosslinked poly(ethylene glycol) (PEG). Uptake of dye molecules from aqueous solutions flowing through the microchannels was observedoptically and diffusion of the dye into the hydrogel was quantified. To complement the diffusion measurements from the microfluidic studies, nuclear magnetic resonance(NMR) characterization of the diffusion of dye in the PEG hydrogels was performed. The diffusion of small molecules in a hydrogel is relevant to applications such asdrug delivery and modeling transport for tissue-engineering applications. The diffusion of small molecules in a hydrogel is dependent on the extent of crosslinking within the gel, gel structure, and interactions between the diffusive species and the hydrogel network. These effects were studied in a model environment (semi-infinite slab) at the hydrogelfluid boundary in a microfluidic device. The microfluidic devices containing PEG microchannels were fabricated using photolithography. The unsteady diffusion of small molecules (dyes) within the microfluidic device was monitored and recorded using a digital microscope. The information was analyzed with techniques drawn from digital microscopy and image analysis to obtain concentration profiles with time. Using a diffusion model to fit this concentration vs. position data, a diffusion coefficient was obtained. This diffusion coefficient was compared to those from complementary NMR analysis. A pulsed field gradient (PFG) method was used to investigate and quantify small molecule diffusion in gradient (PFG) method was used to investigate and quantify small molecule diffusion in hydrogels. There is good agreement between the diffusion coefficients obtained from the microfluidic methods and those found from the NMR studies. The microfluidic approachused in this research enables the study of diffusion at length scales that approach those of vasculature, facilitating models for studying drug elution from hydrogels in blood-contacting applications.

Quantifying The Diffusion And Erosion Of Degradable Hydrogels Using A Microfluidic Technique

Hydrogels are composed of cross-linked networks of hydrophilic polymers that are biocompatible due to their high water content. Mass transfer through hydrogels has been suggested as an effective method of drug delivery, specifically in degradable polymers to minimize lasting effects within the body. Diffusion of small molecules in poly (ethylene glycol) diacrylate (PEG-DA) and dextran methacrylate (dex-MA) hydrogels was characterized in a microfluidic device and by complementary techniques. Microfluidic devices were prepared by crosslinking a formulation of hydrogel and photo-initiator, with and without visible dye, using photolithography to define a central microchannel. Channel sizes within the devices were approximately 600 ¿m to simulate vessels within the body. The microfluidic technique allows for both image and effluent analyses. To visualize the diffusive behavior within the dextran hydrogel, methylene blue and sulforhodamine 101 dyes were used in both elution and uptake experiments. Three analysis techniques for measuring diffusion coefficients were used to quantify the diffusion of solute in the hydrogel, including optical microscopy, characterization of device effluent, and NMR analyses. The optical microscopy technique analyzes images of the dye diffusion captured by a stereomicroscope to generate dye concentration v. position profiles. The data was fit to a diffusion model to determine diffusion coefficients and the dye release profile. In a typical elution experiment, aqueous solution is pumped through the microchannel and dye diffuses out of the hydrogel and into the aqueous phase. During elution, images are taken at regular time intervals and the effluent was collected. Analysis of the device effluent was performed using ultraviolet-visible (UV/Vis) spectroscopy to determine the effluent dye concentration and thus a short-time diffusion coefficient. Nuclear magnetic resonance (NMR) was used to determine a free diffusion coefficient of molecules in hydrogel without the effect of a concentration gradient. Diffusion coefficients for methylene blue and sulforhodamine 101 dyes in dex-MA hydrogel calculated using the three analysis methods all agree well. It was determined that utilizing a combination of the three techniques offers greater insight into molecular diffusion in hydrogels than employing each technique individually. The use of the same microfluidic devices used to measure diffusion is explored in the use of studying the degradation of dex-MA hydrogels. By combining what is known about the degradation rate in regards to the effect of pH and crosslinking and the ability to use a dye solution in contrast to establish the hydrogel boundaries could be a novel approach to studying hydrogel degradation.

Ability of the PM3 Quantum Mechanical Method to Model Intermolecular Hydrogen Bonding between Neutral Molecules

The PM3 semiempirical quantum-mechanical method was found to systematically describe intermolecular hydrogen bonding in small polar molecules. PM3 shows charge transfer from the donor to acceptor molecules on the order of 0.02-0.06 units of charge when strong hydrogen bonds are formed. The PM3 method is predictive; calculated hydrogen bond energies with an absolute magnitude greater than 2 kcal mol-' suggest that the global minimum is a hydrogen bonded complex; absolute energies less than 2 kcal mol-' imply that other van der Waals complexes are more stable. The geometries of the PM3 hydrogen bonded complexes agree with high-resolution spectroscopic observations, gas electron diffraction data, and high-level ab initio calculations. The main limitations in the PM3 method are the underestimation of hydrogen bond lengths by 0.1-0.2 for some systems and the underestimation of reliable experimental hydrogen bond energies by approximately 1-2 kcal mol-l. The PM3 method predicts that ammonia is a good hydrogen bond acceptor and a poor hydrogen donor when interacting with neutral molecules. Electronegativity differences between F, N, and 0 predict that donor strength follows the order F > 0 > N and acceptor strength follows the order N > 0 > F. In the calculations presented in this article, the PM3 method mirrors these electronegativity differences, predicting the F-H- - -N bond to be the strongest and the N-H- - -F bond the weakest. It appears that the PM3 Hamiltonian is able to model hydrogen bonding because of the reduction of two-center repulsive forces brought about by the parameterization of the Gaussian core-core interactions. The ability of the PM3 method to model intermolecular hydrogen bonding means reasonably accurate quantum-mechanical calculations can be applied to small biologic systems.