Effects of G-quartet DNA stationary phase destabilization on fibrinogen peptide resolution in capillary electrochromatography.

The migration of fibrinogen peptides in capillaries coated with G-quartet-forming DNA oligonucleotides in open-tubular CEC (OTCEC) was studied, in order to investigate factors affecting the retention of peptides on G-quartet DNA stationary phases. At 25 degrees C, the peptides eluted in the same order in OTCEC using a two-plane G-quartet DNA stationary phase as in CZE, including two peptides that were completely overlapped. It was found that baseline resolution of the coeluting peptides could be achieved in the OTCEC experiment, but not in CZE, at run temperatures of 35-40 degrees C. A stationary phase formed by a scrambled-sequence oligonucleotide that does not form a G-quartet did not provide any resolution of the two coeluting peptides, even at the higher temperatures, indicating that some destabilization of the G-quartet enhances resolution but that some degree of G-quartet structure is necessary. The effects of destabilization were further explored through variation of the cations (sodium or potassium) used in attachment of the G-quartet oligonucleotide to the capillary surface and in the mobile-phase buffer. Resolution was lower when a more stable, four-plane G-quartet stationary phase was used, supporting the conclusion that some flexibility in the G-quartet structure facilitates differential interactions that resolve otherwise coeluting peptides. The increase in peptide resolution upon destabilization of the G-quartet structure could prove to be an important factor in the application of G-quartet DNA stationary phases for nonaffinity-based separation of native proteins and peptides.

Selectivity of quadruplex DNA stationary phases toward amino acids in homodipeptides and alanyl dipeptides.

Series of dipeptides, including homodipeptides and alanyl dipeptides, were separated using quadruplex (G-quartet) DNA stationary phases in open-tubular capillary electrochromatography (OTCEC). The stationary phases were constructed by covalently attaching the DNA oligonucleotides to the inner capillary surface. Three different G-quartet forming oligonucleotides were investigated: the two-plane G-quartet forming thrombin-binding aptamer, the four-plane analogue of the thrombin-binding aptamer, and a two-plane oligonucleotide identical to the thrombin-binding aptamer except for the replacement of the guanine by thymine in the central bridging loop of the G-quartet structure. Results were compared with results obtained using capillary electrophoresis on a bare capillary and OTCEC using an oligonucleotide with the same base composition as the thrombin-binding aptamer but in a different sequence that does not allow G-quartet formation as the stationary phase.