Development of Analytical Assays for Detection of Small Molecules Using Aptazymes.

[摘要] Advances in understanding the complexity of cellular mechanisms depend upon methods that can quantify affinity interactions between biological molecules. Additionally, fields such as chemical biology and drug discovery require methods for identifying molecules that can probe these affinity interactions. Aptazymes are RNA-based enzymes that catalyze chemical reactions in response to binding interactions with targets of interest, making them suitable for use in quantifying affinity interactions and screening for bioactive molecules. Much of the promise of aptazymes comes from the ability to design or select them for chosen target molecules, and remains unexplored as relatively few of the known aptamers have been appended to ribozymes to create aptazymes. The analytical potential, which arises from the unique and tunable ability of these molecules to translate effector binding into a detectable signal in the form of an RNA cleavage reaction, is also still in early stages of development.We have developed glucosamine-6-phosphate (GlcN6P) sensitive assays that detect substrate cleavage by the naturally occurring GlcN6P-dependent glmS ribozyme via either fluorescence resonance energy transfer (FRET) or capillary electrophoresis with laser induced fluorescence (CE-LIF). We achieve a dynamic range extending from 0.5 to 500 ìM GlcN6P when turning over a single substrate. Signal amplification and 13-fold increased sensitivity are achieved under multiple-turnover reaction conditions. We have analyzed aptazymes that are activated by ATP and cAMP for compatibility in multiplexed assays and developed a qualitative assay for these molecules based on polyacrylamide gel electrophoresis (PAGE) with fluorescence detection. We also developed a multiplexed assay for simultaneous detection of GlcN6P and ATP using the glmS ribozyme and ATP aptazyme. This assay detects GlcN6P with a limit of detection (LOD) of 40 nM and ATP with an LOD of 1 ìM. Our assays demonstrate the potential of aptazymes, in conjunction with FRET, CE-LIF, and PAGE detection, to serve as recognition and transduction elements for assays of small molecules.

[发布机构] University of Michigan

[效力级别] Capillary Electrophoresis

[关键词] Aptazyme;Capillary Electrophoresis;FRET;Chemistry;Science;Chemistry