Thesis or Dissertation In Vitro Selection of Fluoroscent Signaling Peptide Aptamers Using Ribosome Display

Manandhar, Yasodha

pp.1 - 98 , 2015-03-25
Description
Fluorescent signaling peptide aptamers are useful for biological science and diagnosis. In order to in vitro select the fluorescent signaling peptide aptamers, an environment sensitive fluorescent probe, 7-nitro-2,1,3-benzoxadiazole (NBD), was coupled with tRNA through aminophenylalanine and the tRNA was added during preparation of random sequence peptide library for ribosome display. By introduction of NBD as a side chain of non-natural amino acid into a library of random sequences, I successfully obtained fluorescent signaling peptide aptamers and investigated the interactions of selected peptides with the target molecule in detail. I selected an aptamer against a target verotoxin which is a shiga-like toxin protein that causes mild diarrhea to life threatening hemolytic uremic syndrome. In addition, I investigated the interaction between the previously selected peptides with calmodulin (CaM). The selected peptide aptamer against verotoxin showed change in fluorescence on interacting with verotoxin. This aptamer specifically interact with verotoxin and the dissociation constant (Kd) was 3.90±1.6μM. The fluorescence intensity of peptide aptamer was found to be decreased by 78% on increasing the verotoxin concentration. The selected peptides can be used for the detection of verotoxin. On the other hand, the peptide aptamers against CaM showed enhancement in fluorescence by interacting with CaM in presence of calcium ions. The binding interactions of the selected peptide aptamers with CaM were investigated by nuclear magnetic resonance measurements, in addition to the surface plasmon resonance and fluorescence titration measurements.
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