The “RNA world” hypothesis suggests that the earliest life on Earth may have been based on RNA, a molecule that can carry genetic information and act as an enzyme. Penn State researchers have developed a new method that allows the testing of thousands of predicted RNA enzymes called “twister ribozymes” in a single experiment. The team found that approximately 94% of the tested ribozymes were active, even when their structure contained slight imperfections. They also discovered the first example of a twister ribozyme in a mammal, specifically in the genome of the bottlenose dolphin.
The study, published in the journal Nucleic Acids Research, focused on twister ribozymes because they have the ability to cleave themselves in two, which can be determined by analyzing their genetic sequence. Prior to this study, only a handful of twister ribozymes had been experimentally validated. The researchers developed an experimental pipeline called Cleavage High-Throughput Assay (CHiTA) that allowed them to assess the self-cleaving activity of thousands of ribozymes in a single experiment. They identified approximately 1,000 additional twister ribozyme candidates by searching the genomic context of many organisms.
CHiTA relies on Massively Parallel Oligonucleotide Synthesis (MPOS) technology, which enables the design and synthesis of thousands of diverse ribozyme sequences in a single vial. The researchers can make millions of copies of each sequence using a technique called PCR. Another key factor of CHiTA is the use of a restriction enzyme that removes any additional sequences from the ribozymes, ensuring that the function of the ribozymes is accurately assessed. By sequencing the resulting cDNA, the team could determine the activity of each ribozyme.
The study found that many of the tested ribozymes had slight structural variations compared to the canonical twister ribozyme structure, yet they still exhibited self-cleaving activity. This suggests that the function of ribozymes is very tolerant to structural changes, allowing them to operate even if not perfectly formed. The researchers believe that there may be more hidden twister ribozymes in nature that were not found using the original search parameters. The ability to identify these ribozymes and understand their function can provide insights into the origins of life on Earth.
Understanding the tolerance of ribozymes to sequence and structural variations can help in the design of new methods to identify these molecules. The research team hopes that large-scale assays like CHiTA will accelerate the discovery of new ribozymes and improve our understanding of their role in biology. This knowledge can also provide insights into how RNA may have played a key role in sparking life on early Earth. The study was funded by the National Institutes of Health and the Penn State Huck Institutes of the Life Sciences and involved a team of researchers at Penn State led by Phil Bevilacqua.
