Real-Time Detection of a Self-Replicating RNA Enzyme

• Main Authors: Charles Olea, Gerald F. Joyce
• Format: Article
• Language: English
• Published: MDPI AG 2016-09-01
• Series: Molecules
• Subjects: aptazyme; exponential amplification; fluorescence detection; in vitro selection; RNA enzyme; RNA ligation; self-replication
• Online Access: http://www.mdpi.com/1420-3049/21/10/1310

A system was developed to detect the self-replication of an RNA enzyme in real time. The enzyme is an RNA ligase that undergoes exponential amplification at a constant temperature and can be made to operate in a ligand-dependent manner. The real-time system is based on a fluorimetric readout that directly couples the ligation event to an increase in florescence signal that can be monitored using standard instrumentation. The real-time system can also operate entirely with l-RNA, which is not susceptible to degradation by ribonucleases that are present in biological samples. The system is analogous to real-time PCR, but with the potential to detect small molecules, proteins, and other targets that can be recognized by a suitable aptamer. The ligand-dependent self-replication of RNA has potential applications in molecular diagnostics and biosensing that benefit from the rapid, precise, and real-time detection of various target molecules.