Surface Functionalizations towards Nucleic Acid Purification: a nanoscale study

Protein byosynthesis is performed by ribosomes, that translate the genetic information contained in a strand of mRNA and assemble the peptide chain. During translation, several ribosomes associate to a single strand of mRNA, forming supramolecular complexes known as polyribosomes (polysomes).This project is aimed at developing and studying a miniaturized purification system able to isolate and extract polysome-associated mRNA, namely mRNA under active translation. The resulting microdevice will constitute a faster, simpler and low-cost alternative to the time-consuming traditional laboratory procedures for polysome purification and mRNA extraction (sucrose gradient centrifugation and phenol/ethanol RNA extraction). Polysome purification on microdevice will be based on the immobilization of polysomes to the device surfaces, opportunely treated to enhance polysome adhesion. Surface funtionalization will be achieved by formation of Self-Assembled Monolayers (SAM) of organic molecules. In particular, since both ribosomes and nucleic acids expose an high quantity of electrical charged moieties towards the environment [Anger et al., 2013], organic molecules containing charged functional groups will be used as SAM constituents. In this thesis a characterization of gold and silicon oxide plane samples functionalized with different alkanethiols and alkylsilanes SAMs will be presented as well as a quantitative and qualitative evaluation of polysome adhesion performed mainly by Atomic Force Microscopy (AFM). A proof of principle of the purification and extraction of RNA from polysomes using a silicon/Pyrex microdevice will be also reported. [Anger et al., 2013] Anger A.M., Armache J.P., Berninghausen O., Habeck M., Subklewe M., Wilson D.N. and Beckmann R. (2013) Structures of the human and drosophila 80S ribosome. Nature, 497(7447):80-85