Online Capillary Electrophoresis Reaction for Interaction Study of Amino Acid Modified Peptide Nucleic Acid and Proteins |
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| Authors: | Xiao-Qian WANG Murtaza Ghulam Chao ZHU Feng QU |
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| Institution: | School of Life Science, Beijing Institute of Technology, Beijing 100081, China |
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| Abstract: | Peptide nucleic acid (PNA) is a nucleic acid analog which consists of purines, pyrimidines bases, and a neutrally charged peptide backbone. The PNA has the potential as a very useful biological probe for protein analysis since it has more in vivo biological stability as compared to DNA- or RNA-based aptamers. Usually, the addition of amino acids or peptide to the PNA backbone is used to improve its water-solubility and cell-permeability, but these modifications may affect the interaction between PNA and proteins. To date, the investigation of the interaction between PNA and proteins is rare, and there is no reported study about the effects of modifications. In this work, we designed two types of amino acid modified PNAs, (Lys)2-PNA and (Glu)2-PNA, which kept the same base sequence with 15-mer thrombin aptamer and had two basic lysine and two acidic glutamic acid residues on N-terminal of the peptide backbone, respectively. To rapidly assess the binding affinity and specificity of modified PNA and proteins, the online CE reaction method was developed to analyze the interactions of (Lys)2-PNA/(Glu)2-PNA and three proteins: thrombin (THB), single-strand DNA-binding protein (SSB) and human serum albumin (HSA). Meanwhile, the interactions of (Lys)2-PNA/(Glu)2-PNA and thrombin were compared with that of the corresponding complementary base sequence (Lys)2-cPNA/(Glu)2-cPNA and thrombin. The online CE reaction results showed that the interaction of (Lys)2-PNA and (Glu)2-PNA with three proteins was in the order of THB > SSB > HSA. However, (Lys)2-PNA and (Lys)2-cPNA showed similar binding affinity with thrombin; while the binding affinity of (Glu)2-PNA with thrombin was stronger than that of (Glu)2-cPNA with thrombin. Moreover, the binding constant Kb of (Glu)2-PNA and three proteins was determined by affinity capillary electrophoresis (ACE). The online CE reaction eliminates the requirement of incubation, and thus it is fast in detection, and easy to operate with minimum cost. The method is particularly suitable for the interaction studies of expensive modified PNAs and proteins, and can assist the design of PNA probe that binds to proteins. |
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| Keywords: | Online capillary electrophoresis reaction Interaction Peptide nucleic acid Protein Binding constant |
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