Quantification of single-stranded nucleic acid and oligonucleotide interactions with metal ions by affinity capillary electrophoresis--Part II.

The interactions between tetranucleotides or heptanucleotides and inorganic cations have been measured by affinity CZE. The variation in migration behavior with increasing concentrations of divalent cations (Ca(2+), Mg(2+) and Ni(2+)) in the running buffer was investigated and quantified by the calculation of binding constants for mononuclear and multinuclear interactions. In addition to these fundamental studies of binding equilibria, the effect of sequence and the position of the guanine transition metal-binding site in the oligonucleotide have been investigated.

Electrophoretic affinity measurements on microchip. Determination of binding affinities between diketopiperazine receptors and peptide ligands.

ACE on a microchip (MC-ACE) is introduced as a fast and reliable method to determine binding affinities. It is based on monitoring the change in the ionic mobility of a receptor upon binding to a ligand, or vice versa. The method is complementary to other standard methods for binding affinity determinations, like isothermal titration calorimetry (ITC), NMR, UV-Vis spectroscopy, etc. and allows for affinity studies of weak to strong binding interactions. The method is attractive since it principally allows for the analysis of the binding affinity of multiple receptors to a given ligand and requires comparatively small quantities of the binding partners (particularly in comparison to affinity measurements on capillary). We demonstrate the applicability of MC-ACE for the determination of the binding affinities between acid-rich diketopiperazine receptors and basic tripeptides in aqueous solution.

Quantification of single-stranded nucleic acid and oligonucleotide interactions with metal ions by affinity capillary electrophoresis: part I.

The interactions between oligonucleotides and inorganic cations have been measured by capillary zone electrophoresis. With increasing concentrations of divalent cations (Ca(2+), Mg(2+), Mn(2+) and Ni(2+)) in the running buffer, the migration behavior was evaluated by calculation of the binding constants. Besides these fundamental studies of binding equilibria, different buffer components, tris(hydroxymethyl)aminomethane and 3-(N-morpholino)propanesulfonic acid, have been investigated and their effects on metal ion binding quantified.

Affinity capillary electrophoresis on microchips.

The present study shows that the application of the method of affinity capillary electrophoresis (ACE) to investigate interactions between ligands and their substrates can be realized on microchips. With ACE it is possible to characterize non-covalent molecular interactions (complexation and partition equilibria). Binding constants (K(B)) provide a measured value of the affinity of a ligand molecule to a substrate, which is basic information for the understanding of hormones, drugs and their targets, e.g. receptors in the human body. A microchip electrophoresis instrument equipped with a UV-detector and a home-built chip-station with electrochemical detection were used. ACE could be achieved with model solutions of neurotransmitters using sulfated beta-cyclodextrin (sCD) as substrate in a background buffer. This paper describes the advantages of microchip-ACE (MC-ACE) to traditional affinity capillary electrophoresis on a capillary. The results show that MC-ACE has great potential as a tool for fast scanning of interactions and to calculate binding constants of ligands with their substrates.