Multiple modes of capillary electrophoresis applied in peptide nucleic acid related study

Peptide Nucleic Acid (PNA) is a nucleic acid analogue, whose neutrally charged and hydrophobic backbone makes it more stable in vivo, so it might act as a potentially better protein probe as compared to aptamer. Currently the investigation of PNA and protein interaction is scarce. In this research, multiple modes of capillary electrophoresis were established and applied for PNA characterization and its interaction with ssDNA and protein. A 15-mer PNA having the same nucleobase sequence as 15-mer anti-thrombin DNA aptamer was chosen as PNA model for this study, its pI (7.71) was estimated by capillary isoelectric focusing (cIEF). Due to its neutral charge and strong hydrophobicity, three micellar electrokinetic chromatography (MEKC) modes containing (a) SDS, (b) Triton X-100 and (c) CTAB were compared for PNA related analysis. CTAB was not applicable for PNA analysis, while in 4mM SDS or 2mM Triton X-100, PNA and PNA-ssDNA complex can be identified directly. The significant peak of PNA-ssDNA complex helped in validating the two MEKC modes for PNA and target interaction study. Furthermore, the effect of SDS and Triton X-100 concentrations in the two MEKC modes on the protein target thrombin analysis was investigated by capillary zone electrophoresis (CZE). 4mM SDS caused thrombin denaturation. So in 2mM Triton X-100, interactions of PNA with thrombin, PNA with RNase A and a non-aptameric PNA (n-PNA) with thrombin were compared. PNA with thrombin exhibited strongest binding. In summary, cIEF mode for PNA pI determination, MECK mode for direct PNA, PNA-ssDNA and PNA-protein complex identification and CZE mode for the effect of surfactant in MEKC modes on protein target thrombin analysis were applied. Above results showed that multiple modes of CE provide rapid and very low-sample cost methods for PNA related studies.