Ligand-Dependent Exponential Amplification of Self-Replicating RNA Enzymes

Abstract

A general analytical method for the detection of target ligands has been developed, based on a special class of self-replicating aptazymes. These “autocatalytic aptazymes” are generated by linking an aptamer domain to the catalytic domain of a self-replicating RNA enzyme. Ligand-dependent self-replication of RNA proceeds in a self-sustained manner, undergoing exponential amplification at a constant temperature without the assistance of any proteins or other biological materials. The rate of exponential amplification is dependent on the concentration of the ligand, thus enabling quantitative ligand detection. This system has the potential to detect any ligand that can be recognized by an aptamer, including small molecules and proteins. The instability of RNA in biological samples due to the presence of ribonucleases can be overcome by employing the enantiomeric L-RNA form of the self-replicating enzyme. Methods for real-time fluorescence monitoring over the course of exponential amplification are currently being developed.

The Theophylline Aptamer: 25 Years as an Important Tool in Cellular Engineering Research

“…While not the focus of this paper, the aptamer has been used to create diagnostic tools through several approaches. Some of these approaches include electrochemical biosensors, − fluorescent reporters, ,,− surface plasmon resonance, − fluorescence resonance energy transfer (FRET), − and quartz crystal microbalances, , among others. − In one interesting set of studies, Lam and Joyce fused the aptamer to cross-replicating RNA enzymes and demonstrated exponential growth of RNA enzymes in the presence of theophylline, but there was no activity when caffeine, theobromine, or trimethyl uric acid were present. − Thus, the aptamer can be a powerful tool not only in cellular engineering, but also in detection and quantification of theophylline in biological and environmental samples.…”