Detection and Analysis of Molecular Interactions with Backscattering Interferometry

Back-scattering Interferometry (BSI) is an analytical technique that measures changes in refractive index (RI) as two species interact in solution. A high-contrast interference fringe pattern in generated, and the fringes shift spatially as molecular interactions such as binding occur. Experiments can be performed label-free in free solution, eliminating the potential perturbations that can arise with surface immobilization or fluorescent labeling. The source of this signal change derives from intrinsic property changes as a new species, the complex, is formed in solution, particularly changes in conformation and solvation. Recently an expression for the source of this signal, the Free Solution Response Function (FreeSRF) was developed in an effort to more fully describe and quantify it.1 A binding curve is generated from the data, from which dissociation constants can be determined for a measure of binding affinity. The work in this dissertation covers three different types of binding systems analyzed with BSI. First, a series of known and novel inhibitors of acetylcholinesterase (AChE) were screened, the former to benchmark the technique and the latter to evaluate potential anti-Alzheimer’s disease (AD) agents. By comparing KD results from BSI to IC50 values obtained using Ellman’s assay,2 insight into the inhibition mechanism for each inhibitor could be obtained based on the Cheng-Prusoff relationship.3 Specifically, BSI KD values that were equal to Ellman IC50 indicated the inhibitor was acting via noncompetitive inhibition, while inhibitors with KD \u3c IC50 acted as competitive or mixed inhibitors. These results make BSI an especially good technique for evaluating potential anti-AD agents that act as noncompetitive inhibitors and target the peripheral site of the enzyme, where beta amyloids are known to aggregate and accelerate plaque formation.4 Additionally, using a high-affinity inhibitor, BW284c51, we showed that BSI is capable of detecting less than 23,000 AChE molecules at the lowest limit, a level that matches or exceeds detection limits of other techniques.5,6 Next, we evaluated BSI’s ability to distinguish between several 20-base pair DNA oligomers: a perfect complement and a sequence containing a SNP. The SNP-containing sequences had either a mismatch or a deleted base located in the middle of the sequence or five base pairs from the 3’-end. By comparing the BSI KD values of each duplex and comparing those to melting transition temperatures obtained from hyperchromicity experiments, we confirmed our hypothesis that the complement duplex was significantly more stable than any of the duplexes containing a SNP, having a KD ca. 18-130% lower than the SNP duplexes. Additionally, SNPs located in the middle of the sequence were more destabilizing than the same SNP located closer to the end of the sequence, and when comparing two different SNPs at the same location, the A/G mismatch was more destabilizing than the T deletion. The complement duplex also had at least a 35% larger signal compared to the SNP duplexes. SNP research is an important field for personalized medicine and disease diagnostics, and this work showed BSI could play a role in advancing those fields. Lastly, BSI was used to detect selection ion recognition of 18-crown-6 and three ionophores to potassium. These small molecules in nonaqueous media were expected to produce a much smaller signal compared to the large biochemical systems typically studied with BSI due to a lack of major conformation and solvation changes as seen with protein binding or DNA hybridization. Experiments with ITC benchmarked the BSI results. The goal was to quantify the smallest possible signal BSI could detect and evaluate the FreeSRF expression for nonaqueous, small molecule systems. Formation of potassium complexes with potassium ionophores I and II generated signals more than an order of magnitude smaller than complement DNA hybridization. Potassium ionophore I, also known as valinomycin, folds into a “tennis ball seam” conformation upon binding with potassium while potassium ionophore II forms a “closed clamshell” structure; these complexes generated a signal over 30% larger than that of the potassium-18C6 complex, which undergoes only a minor shift in conformation from a S6 or Ci structure to D3d upon complexation.7,8 Conversely, Na+ with 18C6 experiments generated no readable signal above the noise, highlighting BSI’s ability to selectively detect ion recognition. Interesting observations into the direction of the signal were also made, particularly with ionophore III, which features a dodecyl tail in the middle of the linker between the two 15C5 ends and caused the signal to reverse direction as potassium was added. Signal direction is thought to be related to hydrodynamic radius, which this hydrocarbon tail would significantly alter as the ionophore forms the “closed clamshell” complex. Other experiments show BSI has potential to examine the stoichiometry of binding systems, and decomplexation, rather than complexation, of the ionophore I-potassium complex in water. BSI offers many advantages compared to other techniques, including its ability to run experiments label-free and in free solution, its small sample size (with a probe volume of just 7.52 nL), and high sensitivity even in small-molecule systems with very minor conformational changes. These experiments show it has potential for many uses, from diagnostics, drug screening, and metal ion detection. As research into the signal origin and FreeSRF evaluation progress, it will continue to be a valuable refractive index detector for various chemical fields