Citation: WANG Xiao-Qian, Ghulam Murtaza, ZHU Chao, QU Feng. Online Capillary Electrophoresis Reaction for Interaction Study of Amino Acid Modified Peptide Nucleic Acid and Proteins[J]. Chinese Journal of Analytical Chemistry, ;2018, 46(12): 1895-1903. doi: 10.11895/j.issn.0253-3820.181407 shu

Online Capillary Electrophoresis Reaction for Interaction Study of Amino Acid Modified Peptide Nucleic Acid and Proteins

School of Life Science, Beijing Institute of Technology, Beijing 100081, China

Received Date: 19 June 2018
Revised Date: 31 August 2018

Fund Project: This work was supported by the National Natural Science Foundation of China (Nos. 21375008, 21675012).

Peptide nucleic acid (PNA) is a kind of nucleic acid analog which consists of purines, pyrimidines bases, and a neutrally charged peptide backbone. PNA has the potential to be a very useful biological probe for proteins 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 PNA backbone is used to improve its water-solubility and cell-permeability, but these modifications may affect the interaction between PNA and proteins. In this research, 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 units 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 interactions of (Lys)₂-PNA/(Glu)₂-PNA and three proteins including thrombin (THB), single-stranded 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 K_b of (Glu)₂-PNA and three proteins was determined by affinity capillary electrophoresis (ACE). The K_b was calculated as 8.39×10⁶ L/mol for thrombin and 5.91×10⁵ L/mol for SSB, while the binding affinity of (Glu)₂-PNA with HSA was too weak to be calculated. The quantitative results of ACE showed agreement with the online CE reaction results, verifying the reliability of online CE reaction method. Besides, the online CE reaction eliminated the requirement of incubation and thus it was faster in detection, simpler in operation with low testing cost. The presented method was particularly suitable for the interaction studies of expensive modified PNAs and proteins, and would assist the design of PNA probe that binds to proteins.
- Online capillary electrophoresis reaction,
- Interaction,
- Peptide nucleic acid,
- Protein,
- Binding constant
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