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.