Abstract
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.
Highlights
Technologies based on the exponential amplification of nucleic acids are widely used in molecular diagnostics and biosensing
The self-replicating RNA enzyme is the only known macromolecule that can produce additional copies of itself and undergo exponential amplification in a self-sustained manner. This enzyme has been used to study processes of molecular evolution relevant to the origins of life [6,8,16]. It has been investigated for potential applications in molecular diagnostics and biosensing [3,4]
The latter make use of the ability of the enzyme to operate in a ligand-dependent manner, where the rate of exponential amplification is dependent on the concentration of the target ligand
Summary
Technologies based on the exponential amplification of nucleic acids are widely used in molecular diagnostics and biosensing. The quantitative polymerase chain reaction (qPCR) is a sensitive and precise method for detecting target nucleic acids [1,2]. Other methods can be used to detect non-nucleic-acid targets based on the binding of the target to an antibody, aptamer, or other capture reagent. In such cases the binding event must usually be coupled to a means for signal amplification, such as enzyme turnover or nucleic acid amplification. A different approach is to employ a capture reagent that itself undergoes exponential amplification upon binding the target. Such a system has been demonstrated based on aptamer-containing, self-replicating
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