Combinatorial selection of thioaptamers targeting viral and cellular proteins

This dissertation is about the study of the development of drugs. In order to develop drugs targeting viral proteins and a human cellular protein, a "combinatorial selection of aptamers" method was employed. An aptamer is a nucleic acid molecule which specifically binds to a target molecule and possibly modulates the functions of the target molecule. In employing this technique, methods which modify aptamers so that they contain phosphorothioates in the backbones were used. The phosphorothioate aptamers (thioaptamers) have been shown to have a higher affinity to target molecules and to be more stable in biological environments, so that they should be more suitable as candidates for drugs.; The target proteins in this dissertation were two viral proteins, VEE capsid protein and domain III of West Nile envelope protein, and one human cellular protein, hnRNP C1. In order to isolate thioaptamers against these target proteins, several techniques were used such as: transcription, PCR, RT-PCR, nucleic acid labeling, single-stranded DNA isolation, protein overexpression and purification, cloning, electrophoretic mobility shift assay, CCD camera-based gel image analysis, footprinting, and bioinformatics tools available from the world wide web. Using these methods, an RNA thioaptamer for the VEE capsid protein and single-stranded DNA thioaptamers for domain III of the West Nile envelope protein and the hnRNP C1 protein were isolated. Their affinities to the target proteins were 7 nM (VEE capsid RNA thioaptamer), 2.7 muM (hnRNP C1 single-stranded DNA thioaptamer), and 4 muM (domain III of West Nile envelope protein).; These thioaptamers were shown to bind specifically to the target proteins and are expected to be therapeutically promising molecules.