Ribozymes exist in nature as catalytic RNA molecules that either aid the hydrolysis of their own phosphodiester bonds or cause the hydrolysis of bonds in other RNAs. They also catalyze the aminotransferase activity of the ribosome. Research into the function and structure of these naturally occurring molecules has led to the development of synthetic ribozymes that are made in the laboratory.
For example, Tang and Breaker used in vitro selection of RNAs from random sequence RNAs to isolate self-cleaving RNAs. These synthetic, self-cleaving RNAs possess a good enzymatic activity that makes them useful in several areas of genetics, genomics and molecular biology. Some of the synthetic ribozymes that have been prepared have unique structures, while others resemble the naturally occurring hammerhead ribozyme. Further examples of ribozymes include the hairpin ribozyme, the Leadzyme and the VS ribozyme.
The methods used to discover artificial ribozymes are based on Darwinian evolution. The approach uses the fact that RNA has the ability to act as both a catalytic molecule as well as source of genetic information (like DNA). This dual nature of RNA provides a huge advantage to the investigator, who finds it easy to produce large populations of RNA catalysts using polymerase enzymes.
Reverse transcriptase is used to mutate these ribozymes by reverse transcribing them into various cDNA which can then be amplified with mutagenic PCR. The selection parameters may vary in these experiments. Biotin tags covalently linked to the substrate are used to select a ligase ribozyme. If a molecule demonstrates the required ligase activity, the active molecule can be recovered using a streptaidin matrix.
Lincoln and Joyce went on to create an RNA enzyme system that could self replicate in around one hour. This system uses molecular competition or in vitro evolution to establish an RNA enzyme pair from a candidate enzyme mixture. Each of the RNA enzymes synthesizes the other from synthetic oligonucleotides, in the absence of any protein.
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