Laser-Induced Fluorescence Detection of ATP, cAMP, and GlcN6P by way of Capillary Electrophoresis Using Aptazymes and their Cleavage Products

Abstract

The detection of ATP, cAMP, GlcN6P, and other small biomolecules are of Interest due to their prevalence in a variety of cellular and biological processes. ATP is a carrier of chemical energy, cyclic AMP is a second messenger that transduces intracellular signals that otherwise could not get through the cell membrane, and glucosamine-6-phosphate is the biochemical precursor of all nitrogen-containing sugars. In the case of this research, aptazymes were used to detect the presence of these three biomolecules in solution. Aptazymes, also known as allosteric ribozymes, are a specific type of ribozyme that can catalyze chemical reactions in response to effector binding. Most often, ribozymes are used to catalyze the hydrolysis of a phosphodiester bond in RNA. Due to this, these aptazymes can have both recognitive and transductive properties in bioanalytical assays. They can be designed to specifically recognize a target analyte and transduce that signal into cleavage ofthe RNA into two fragments. The cleavage product can be separated by capillary electrophoresis with detection of the fluoresceinlabeled substrate by laser induced fluorescence. Capillary electrophoresis is used over slab gels due to its quick separation times and low limit of detection (LOD). To an analytical scientist aptazymes are significant because it is possible for each individual aptazyme to be designed to respond to one unique analyte. Even a difference of one nucleotide can mean a different analyte to which the ribozyme will respond. This leads to a high specificity in detection of the target analyte. The importance of this is that it allows for multiplexing, which means that multiple biomolecules can be detected simultaneously in one assay.