Interactions of in vitro selected fluorogenic peptide aptamers with calmodulin
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Characterization of G Protein-coupled Receptors by a Fluorescence-based Calcium Mobilization Assay
Published on: July 28, 2014
Yasodha Manandhar1,2, Wei Wang1, Jin Inoue3
1Nano Medical Engineering Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama, 351-0198, Japan.
Objectives:
We examined the importance of aptamer usage under the same condition as the selection process by employing the previously selected aptamers for calmodulin (CaM) which includes a non-natural fluorogenic amino acid, 7-nitro-2,1,3-benzoxadiazole.
Results:
We added five amino acids at the N-terminus which was employed for the selection and then we tested the affinity and selectivity for CaM binding. Surface plasmon resonance and fluorescence measurements showed that the additional amino acids for one of the aptamers drastically improved binding affinity to CaM, indicating the importance of aptamer use under the same conditions as the selection process. Such drastic improvement in affinity was not observed for the sequence which had been reported previously. Nuclear magnetic resonance data identified that the primary binding site is located in a C-terminal of CaM and the additional residues enhance interactions with CaM.
Conclusions:
We found that the addition of the common sequence, which was employed for ribosome display, makes the affinity of a selected peptide as strong as the previously reported peptide.
