Testing of thousands of RNA enzymes discovers first ‘Twister ribozyme’ in mammals

A novel cleavage high-throughput assay (CHiTA) developed at Penn State University provides a flawless method to characterize thousands of diverse small self-cleaving RNA enzymes called Twister ribozymes in a single experiment. This image shows 2D models of some of the ribozymes tested that had defects in the helical and loop elements but still remained active, demonstrating that the Twister ribozyme’s self-cleavage ability can overcome these slight structural defects. shows that it is resistant to Credit: Lauren McKinley and Philip Bevilacqua, Penn State University
The “RNA world” hypothesis proposes that the earliest life on Earth, like modern viruses, may have been based on RNA (a single-stranded molecule similar to DNA in many ways). Masu. This is because RNA, like DNA, can carry genetic information, but like proteins, it can also act as enzymes that initiate or accelerate reactions. The activity of a few RNA enzymes, called ribozymes, is tested on a case-by-case basis, but computers predict that thousands more exist in organisms ranging from bacteria to plants and animals. Now, a new method developed by researchers at Penn State University can now test the activity of thousands of these predicted ribozymes in a single experiment.
The researchers tested the activity of more than 2,600 different RNA sequences predicted to belong to a class of RNA enzymes called twister ribozymes that have the ability to cut themselves in half. Studies have shown that about 94% of the ribozymes tested are active and can continue to function even if their structures contain small defects. The research team also identified the first example of a twister ribozyme in the genome of a mammal, specifically a bottlenose dolphin.
A paper describing the research was published online today, November 5, in the journal Nucleic Acids Research.
“DNA is a double-stranded molecule that usually forms a simple helical structure, whereas RNA is single-stranded and can fold back on itself to form diverse structures such as loops, bulges, and helices. ” said Phil Bevilacqua, distinguished professor of chemistry and medicine. majoring in biochemistry and molecular biology at Penn State Eberly College of Science and is a research team leader. “The functions of RNA enzymes are based on these structures, and they are divided into several different classes. We decided to focus on the so-called ‘Twister ribozymes’ because one of their functions is Because one is to cut itself in two. To determine their genetic sequence. ”
Prior to this study, approximately 1,600 Twister ribozymes had been proposed based on genome sequence and structure predictions, but only a small number had been experimentally validated. The research team developed an experimental pipeline that can assess the self-cleavage activity of thousands of ribozymes in a single experiment. We call this a “cleavage high-throughput assay” (CHiTA). They also discovered about 1,000 more by meticulously hand searching the genomic context around short, highly conserved sequences shared by many of the ribozymes in the genomes of thousands of organisms. We identified a Twister ribozyme candidate.
CHiTA relies on two key elements. One is a recently developed technique called “Massively Parallel Oligonucleotide Synthesis” (MPOS). MPOS allows research teams to design thousands of diverse ribozyme sequences in the form of small pieces of DNA, all available for purchase in one vial. Each sequence they designed has one of 2,600 predicted ribozyme sequences as its core. The researchers then add short bits of DNA to each end so they can make a copy of the DNA and transcribe it into RNA and test its activity.
“With MPOS, you just create a spreadsheet with the sequences you’re interested in, send it to a commercial vendor, and they send you back a tube with a small amount of each sequence,” says Lauren, a graduate student.・Mr. McKinley said. He was enrolled at Penn State University at the time of the research, recently completed his Ph.D., and is also the lead author of the paper. “For CHiTA, each sequence is needed in large quantities, so we add DNA bits to each end of the sequence and use a technique called PCR to make millions of copies of each. But these Additional bits may affect the ability to test the ribozyme’s functionality.
The second key element of CHiTA overcomes this hurdle by removing these added sequence bits using proteins called restriction enzymes that cut DNA at specific short sequences called recognition sites. will help you. However, most restriction enzymes cut DNA near the middle of the recognition site, leaving part of the recognition site sequence bound to the two DNA fragments, which can affect the structure and function of the ribozyme. .
“Because we discovered a restriction enzyme that cuts DNA at a short distance from the recognition site, we were able to design the sequence to cut without leaving any trace of additional DNA,” McKinley said. “In this way, we were able to reliably assess the function of the exact sequence of the ribozyme.”
In the lab, the team first makes additional copies of the sequence ordered through MPOS, trims the additional DNA with restriction enzymes, and then transcribes RNA from the DNA sequence. If any of the sequences encode active ribozymes, they will quickly fold into a functional structure and cleave themselves when the RNA is produced. Researchers can then collect the RNA and convert it back into DNA, called cDNA, so they can read its sequence to see if it’s full-length or if it’s been truncated.
“Sequencing the cDNA tells us how much, if any, RNA has been cleaved as an indicator of ribozyme activity,” McKinley said. “In about 94% of the sequences we tested, a significant portion of the RNA was cleaved. In fact, the proportion of each active ribozyme that was cleaved was very similar to previous efforts that tested ribozymes individually. I did.”
The team then looked at the predicted structures of the sequences they tested and found that although many of them had small variations or imperfections compared to the standard Twister ribozyme structure, they were still able to self-cleave. got it. This suggested to the researchers that ribozyme function is highly resistant to small structural changes and may function even when not fully formed.
The researchers say that allowing for imperfections suggests that there may be many more twisters hidden in nature that cannot be found using the original search parameters. In fact, this study’s new sequence-based descriptors led the research team to the discovery of the first mammalian twister ribozyme identified in the genome of a bottlenose dolphin.
“Understanding these types of resistance to ribozyme sequence and structural variations will help us design new and rigorous methods to identify ribozymes,” Bevilacqua said. “Current knowledge of ribozyme function is primarily based on chemistry, and we are only beginning to learn about their role in biology. The ability to test ribozyme activity in large-scale assays like CHiTA will help. We hope to accelerate our ability to discover new ribozymes.” All of this will help us better understand the role RNA plays in cells, and how RNA’s ability to do everything started life on early Earth. It also helps you go back in time and see what could have been possible when it could have been the key. ”
In addition to Bevilacqua and McKinley, the Penn State research team included McCauley O. Meyer, Aswathy Sebastian, and Kyle J. Messina. All of them were graduate students at the time of the study and have since completed their Ph.D. former undergraduate student Benjamin K. Chan; Istvan Albert Research Professor of Bioinformatics.
Further information: Lauren N McKinley et al, Direct testing of natural twister ribozymes from more than 1000 organisms reveals widespread resistance to structural defects, Nucleic Acids Research (2024). DOI: 10.1093/nar/gkae908
Provided by Pennsylvania State University
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