Aptamers for proteomics

• Main Author: Le, Thao Thi
• Other Authors: Cass, Tony ; Miller, Andrew
• Published: Imperial College London 2008
• Subjects: 572.6
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.484778

Changes in post-translation modifications are very important in the regulation of biological processes. Many modifications occur at very low levels, resulting in a low-abundance of the modified proteins in cells, and therefore assessing those modifications is not an easy task. Modern proteomics needs improved methods for identifying such changes. In this thesis, we focus on generating aptamers that can bind phosphoproteins with high affinities and therefore would be able to detect even low-abundance proteins. Aptamers are short sequences of nucleic acids that can be selected from libraries through a process called SELEX to bind targets of interest with high affinity and specificity. In this work, a phosphotyrosine (pY) peptide in a consensus sequence, commonly found in a class of phosphoproteins recognised by SH2 domains of signalling cascades in cells, was chosen as the target. By choosing this peptide target, we aim to create aptamers that can bind a class of proteins that carry this peptide sequence, mimicking the action of the intracellular SH2 domains. An RNA library with 7×1014 molecules with 30 nucleotides in the random region was employed for the selection and aptamers that bind the pY peptide were selected. Using surface plasmon resonance (SPR), binding affinities of these aptamers with their peptide target were determined (Kd values in high nanomolar (nM) range). In addition, aptamers that bind streptavidin tightly (Kd values in low nM range) were also isolated, as streptavidin was used as the matrix in partitioning step during the selection. Affinities of these aptamers were also determined by SPR. Moreover, fluorescence quenching suggested that the streptavidin binding aptamers bound in or near the biotin binding site. These aptamers can be used as affinity tags for RNA molecules. The secondary structures of both types of the aptamers were predicted based on their random-region sequences using the Mfold program.