Modeling and validation of multiplex proteo-nucleic self-assemblies monitored by surface plasmon resonance imagery

摘要

Hybridization based stepwise assembly of proteo-nucleic blocks to form hybrid linear structures was monitored by surface plasmon resonance imagery (SPRI). The multiplex approach involved the parallel extension on the chip of primers giving rise to assemblies differing by sequence, order and orientation of building blocks. Statistical analyses of reflectivity profiles following data filtering, normalization and corrections, illustrated that automated sorting of structures is feasible and that reproducible building of complex assemblies was achieved onto the dextran matrix. In order to characterize the influence of matrix environment, relations between kinetics of reflectivity changes and hybridization parameters measured into solution were investigated. A global model taking into account of transport and diffusion mechanisms into the aqueous phase and accessibility and hybridization mechanisms into the dextran layer, was set and numerically solved. The model was used to define critical assembly parameters and to derive the relations between microscopic and observable events in the experimental SPRI conditions. Different computing approaches for extracting rate constants from more or less noisy and truncated experimental traces were tested and validated. Finally the best processing methods were exploited, in association with simulation, to extract kinetic parameters. ANCOVA analysis of data was confirmatory of observations in solution concerning the influence of proteo-nucleic block orientation and the hybridization of DNA segments. However, interaction of protein domain with matrix within the dextran micro-environment was found to enhance hybridization rates of PDNAs as compared to DNA. In contrast, vicinity of hybridizing segments to dextran attachment decreased it.