A recent development enables researchers to examine the function of thousands of RNA enzymes, known as ribozymes, all in one experiment. The “RNA world” theory suggests that the initial forms of life on Earth may have relied on RNA, a single-stranded molecule that resembles DNA and shares characteristics with some modern viruses. Like DNA, RNA conveys genetic information; however, it can also function as an enzyme, facilitating or speeding up chemical reactions. Although a handful of RNA enzymes, or ribozymes, have been studied individually, there is a vast number that researchers have theorized exists based on computational models, spanning various organisms from bacteria to higher plants and animals. Now, researchers from Penn State have created a new method that allows them to evaluate the activity of numerous predicted ribozymes simultaneously.
The research team explored over 2,600 RNA sequences identified as belonging to a type of RNA enzyme known as “twister ribozymes,” which can cleave themselves into two parts. Remarkably, around 94% of these tested ribozymes showed activity, demonstrating that they can function effectively even when their structures contain minor flaws. The researchers also made a groundbreaking discovery by identifying the first instance of a twister ribozyme in mammals, located in the genome of a bottlenose dolphin.
A paper explaining this research was published online today, November 5, in the journal Nucleic Acids Research.
Phil Bevilacqua, a distinguished professor of chemistry and biochemistry and molecular biology in the Eberly College of Science at Penn State, who led the research, stated, “While DNA forms a double-stranded helical structure, RNA is single-stranded and capable of folding onto itself to form a range of structures, such as loops and helixes. The functioning of RNA enzymes is dependent on these structures, and they can be categorized into various groups. We focused on ‘twister ribozymes’ because they can cleave themselves, which we can detect by analyzing their genetic sequences.”
Before this study, approximately 1,600 twister ribozymes had been suggested based on genomic sequences and structure predictions, but only a few had undergone experimental validation. The team created an experimental process that enabled them to evaluate the self-cleaving actions of thousands of these ribozymes at once, known as the “Cleavage High-Throughput Assay,” or CHiTA. They also found nearly 1,000 additional ribozyme candidates by meticulously examining genomic regions surrounding a short sequence conserved across many organisms.
CHiTA hinges on two crucial components. The first is a novel technology called “massively parallel oligonucleotide synthesis” (MPOS), which allows the team to design and obtain thousands of diverse ribozyme sequences packaged in a single vial. Each sequence has one of the 2,600 predicted ribozyme sequences at its core, with additional short DNA segments attached to help amplify the DNA and convert it to RNA for activity testing.
Lauren McKinley, a graduate student at Penn State and the study’s first author, explained, “With MPOS, I can easily put together a list of desired sequences, send it to a vendor, and receive a solution containing a small amount of each. For CHiTA, we require numerous copies of each sequence, so we attach DNA segments at both ends that enable us to replicate millions of copies using a technique called PCR; though there is a risk that these segments might affect the functionality of the ribozymes.”
The second key element of CHiTA addresses this risk by using a restriction enzyme that selectively cuts the DNA near the recognition sites without leaving behind any additional DNA that might influence the riboze’s structure and function. Nevertheless, most restriction enzymes tend to cut within their recognition sites, potentially retaining parts of the recognition site that could affect the ribozyme.
“We discovered a restriction enzyme that can slice the DNA at a distance from its recognition site, allowing us to design sequences that would cut without leaving remnants,” McKinley said. “This ensures that we are evaluating the exact sequences of ribozymes.”
In the laboratory, the team initially produced more copies of the sequences designed through MPOS, removed any extra DNA using the restriction enzyme, and then transcribed RNA from the DNA sequences. If any of the sequences encoded active ribozymes, they would quickly fold into their functional forms and self-cleave as the RNA was produced. The researchers could then collect the RNA and convert it back to DNA (cDNA) to analyze its sequence for completeness or signs of cleavage.
“By sequencing the cDNA, we can determine how much RNA, if any, has been cleaved, an indication of ribozyme activity,” McKinley noted. “Of the sequences we evaluated, a significant portion showed signs of cleavage, with approximately 94% demonstrating activity. The rate of cleavage for active ribozymes aligns closely with earlier studies focusing on individually tested ribozymes.”
Upon reviewing the predicted structures of the sequences tested, the team found that many had minor differences or flaws compared to the standard twister ribozyme structure, yet they still demonstrated self-cleavage. This led the researchers to conclude that ribozymes are quite tolerant of minor structural variations, allowing them to function effectively despite these imperfections.
This tolerance to structural discrepancies suggests the potential existence of more twister ribozymes in nature that may not be uncovered using traditional searching methods. Indeed, the new descriptors developed from this study led to the discovery of the first mammalian twister ribozyme in the bottlenose dolphin’s genome.
“Gaining insight into how ribozymes tolerate sequence and structural variations will enable us to devise more effective strategies for identifying them,” Bevilacqua remarked. “Our present understanding of ribozyme function largely stems from chemistry, and we are just beginning to unravel their biological roles. Utilizing large-scale assays like CHiTA can significantly enhance our ability to uncover new ribozymes and deepen our comprehension of their cellular functions. Additionally, this may provide insights into the evolutionary history of RNA and its critical role in the early development of life on Earth.”
The research team included McCauley O. Meyer, Aswathy Sebastian, and Kyle J. Messina, all of whom were graduate students at the time and have since earned their doctorates; former undergraduate student Benjamin K. Chang; and Research Professor of Bioinformatics Istvan Albert. Funding for this research was provided by the National Institutes of Health and the Penn State Huck Institutes of the Life Sciences.
