Self-Association and Membrane-Binding Behavior of Melittins Containing Trifluoroleucine

We have investigated the effect of trifluoroleucine substitution on the membrane-binding and tetramerization behavior of melittin. Analogues were synthesized in which Leu 9, Leu 13, and all four intrinsic leucine residues of melittin were replaced by 5,5,5-trifluoroleucine. Both the mono- and tetra-substituted melittins were found to exhibit stronger self-association and enhanced affinity for lipid bilayer membranes, compared to the wild-type peptide. The extent of the observed effects depends on the site of introduction of trifluoroleucine and, in the case of substitution at position 13, on the stereochemistry of the trifluoroleucine side chain. Analysis of the membrane association isotherms is consistent with aggregation of fluorinated melittins within the lipid bilayer. These results suggest that fluorocarbon−hydrocarbon separation, in addition to an increase in hydrophobic character, contributes to enhanced membrane binding

Sequence dependence of isothermal DNA amplification via EXPAR

Isothermal nucleic acid amplification is becoming increasingly important for molecular diagnostics. Therefore, new computational tools are needed to facilitate assay design. In the isothermal EXPonential Amplification Reaction (EXPAR), template sequences with similar thermodynamic characteristics perform very differently. To understand what causes this variability, we characterized the performance of 384 template sequences, and used this data to develop two computational methods to predict EXPAR template performance based on sequence: a position weight matrix approach with support vector machine classifier, and RELIEF attribute evaluation with Naïve Bayes classification. The methods identified well and poorly performing EXPAR templates with 67–70% sensitivity and 77–80% specificity. We combined these methods into a computational tool that can accelerate new assay design by ruling out likely poor performers. Furthermore, our data suggest that variability in template performance is linked to specific sequence motifs. Cytidine, a pyrimidine base, is over-represented in certain positions of well-performing templates. Guanosine and adenosine, both purine bases, are over-represented in similar regions of poorly performing templates, frequently as GA or AG dimers. Since polymerases have a higher affinity for purine oligonucleotides, polymerase binding to GA-rich regions of a single-stranded DNA template may promote non-specific amplification in EXPAR and other nucleic acid amplification reactions

Redox and molecular recognition in biological and man-made systems

Understanding the fundamental principles governing the interplay of redox and molecular recognition is invaluable in the context of biochemistry and material science. Enzymes containing redox-active organic molecules such as flavins and quinones use specific enzyme-cofactor interactions to regulate the reactivity of the prosthetic group. Due to the complexity of biological systems, the effects of hydrogen bonding, π-stacking and dipolar interactions are difficult to quantify individually, and subtle interactions may go unnoticed. We have utilized synthetic model systems to parametrically probe the effect of molecular recognition on the redox behavior and physical properties of redox active organic molecules. The results are applied both to explain effects observed in biological systems and in the design of novel man-made molecular devices. We used synthetic receptors to reproduce specific hydrogen bond patterns to the flavin imide moiety present in flavoenzymes. Hydrogen bonding was found to stabilize the flavin radical by decreasing its reduction potential and suppressing proton transfer responsible for two electron reduction, analogous to the effect seen in flavoenzymes. We further observed a significant increase in the association constant to the hydrogen bonding receptor upon reduction of flavin from the oxidized form to the radical anion. Specific hydrogen bond interactions were found to redistribute the spin density of the naphthalimide radical anion, which undergoes molecular recognition analogous to flavin. Experimental hyperfine coupling constants and DFT-B3LYP calculations show an overall increase in spin polarization upon hydrogen bonding and a distortion of spin density away from the binding site. We are currently investigating a similar effect predicted for hydrogen bound flavin using electron nuclear double resonance spectroscopy. The complimentarity dependence of hydrogen bonding and π-stacking on the redox-state of a bound molecule enabled us to design a molecular device, in which the redox state of the guest switches the preference between two competing hosts, rendering the system a three component molecular switch. We developed a synthetic strategy to attach molecular recognition elements to gold surfaces through reaction of primary amines with SAMs displaying acid fluoride-headgroups, which will enable the creation of solid-supported analogs of such devices

DNA Adsorption to and Elution from Silica Surfaces: Influence of Amino Acid Buffers

Solid phase extraction and purification of DNA from complex samples typically requires chaotropic salts that can inhibit downstream polymerase amplification if carried into the elution buffer. Amino acid buffers may serve as a more compatible alternative for modulating the interaction between DNA and silica surfaces. We characterized DNA binding to silica surfaces, facilitated by representative amino acid buffers, and the subsequent elution of DNA from the silica surfaces. Through bulk depletion experiments, we found that more DNA adsorbs to silica particles out of positively compared to negatively charged amino acid buffers. Additionally, the type of the silica surface greatly influences the amount of DNA adsorbed and the final elution yield. Quartz crystal microbalance experiments with dissipation monitoring (QCM-D) revealed multiphasic DNA adsorption out of stronger adsorbing conditions such as arginine, glycine, and glutamine, with DNA more rigidly bound during the early stages of the adsorption process. The DNA film adsorbed out of glutamate was more flexible and uniform throughout the adsorption process. QCM-D characterization of DNA elution from the silica surface indicates an uptake in water mass during the initial stage of DNA elution for the stronger adsorbing conditions, which suggests that for these conditions the DNA film is partly dehydrated during the prior adsorption process. Overall, several positively charged and polar neutral amino acid buffers show promise as an alternative to methods based on chaotropic salts for solid phase DNA extraction