The blueprint of life, DNA, is the master instruction manual for a cell. To carry out these instructions, a cell creates temporary copies of specific genes using a molecule called messenger RNA, or mRNA. This mRNA acts like a short-term work order, guiding the cell’s machinery to build a specific protein, after which the message is quickly degraded.
Scientists have explored ways to introduce their own custom-designed mRNA into cells, instructing them to produce proteins that could treat or prevent disease. A challenge, however, has been that these synthetic messages are often fragile and can be targeted by the body’s defense systems. Researchers discovered that making small changes to the chemical structure of these molecules can make them more robust and effective.
Understanding Pseudouridine
One of the four chemical bases that make up an RNA molecule is called uridine. Pseudouridine is a naturally occurring variant of this base. It is an isomer of uridine, meaning it is composed of the same atoms but arranged in a slightly different three-dimensional structure. This difference lies in the connection between the uracil base and the ribose sugar backbone; in pseudouridine, this bond is between two carbon atoms (C-C), whereas in uridine, it is between a carbon and a nitrogen atom (C-N).
This structural rearrangement gives pseudouridine unique properties. The C-C bond offers more rotational freedom, making the molecule more flexible. It also creates an extra spot for a hydrogen bond to form, which helps stabilize the local structure of an RNA strand.
This modification is not a human invention; it is the most common RNA modification found in nature. Cells use it to fine-tune the function of their own non-coding RNAs, such as transfer RNA (tRNA) and ribosomal RNA (rRNA). In these natural contexts, pseudouridine helps stabilize the intricate folds of the RNA, ensuring they hold their proper shape to function correctly.
How Pseudouridine Improves mRNA
When synthetic mRNA made with standard uridine is introduced into the body, it faces two immediate problems. First, human cells are equipped with sensors designed to detect foreign RNA, such as that from an invading virus. These sensors, known as Toll-like receptors (TLRs), recognize the unmodified synthetic mRNA and trigger an innate immune response. This inflammatory reaction can cause unwanted side effects and also leads to the rapid destruction of the mRNA.
The second issue is the instability of single-stranded mRNA. Cellular enzymes called ribonucleases quickly seek out and dismantle these molecules, sometimes within minutes. This short lifespan means that an mRNA molecule has a very limited window in which to be translated into its target protein. The combination of immune rejection and rapid degradation made early versions of therapeutic mRNA highly inefficient.
Substituting uridine with its isomer, pseudouridine, addresses both of these challenges. The slightly altered shape of pseudouridine helps the synthetic mRNA avoid recognition by immune sensors like TLR7 and TLR8. By going “under the radar” of these receptors, the modified mRNA does not provoke the same strong inflammatory cascade, reducing adverse reactions and allowing the molecule to persist.
This modification also leads to a dramatic increase in protein production. The structural stability conferred by pseudouridine makes the mRNA molecule more durable, slowing its degradation within the cell. Furthermore, the presence of pseudouridine makes the mRNA a more efficient template for the ribosome, the cellular machine that reads the mRNA code and assembles the protein. This enhanced efficiency means a single modified mRNA molecule can be read more times, resulting in a much higher yield of the desired protein.
The Development of Pseudouridine-Modified mRNA
The path to harnessing pseudouridine for therapeutic use was the result of decades of research by scientists Katalin Karikó and Drew Weissman. Throughout the 1990s and early 2000s, their work focused on overcoming the obstacles that prevented synthetic mRNA from being a viable platform for therapies. They systematically investigated how different nucleoside modifications could alter the way the immune system responded to foreign RNA.
Their breakthrough came in 2005, when they published a paper demonstrating that incorporating modified nucleosides, particularly pseudouridine, into synthetic mRNA could suppress its ability to activate Toll-like receptors. This discovery addressed the inflammatory problem that had plagued the field. A few years later, they showed that this modification not only dampened the immune response but also significantly increased the amount of protein produced from the mRNA.
For years, their findings were not widely recognized within the broader scientific community. The research, however, laid the groundwork for a technology that would later become globally significant. When the COVID-19 pandemic emerged, the challenge was to develop a vaccine with unprecedented speed. The pseudouridine-modified mRNA platform, developed by Karikó and Weissman, was suited for this task.
The technology allowed companies like Pfizer-BioNTech and Moderna to design and produce highly effective vaccines rapidly. The work of Karikó and Weissman, once a niche area of research, became the basis for a new class of vaccines that were instrumental in addressing a global health crisis.