Modified nucleotides enable the creation of more stable, precise, and effective genetic tools, such as CRISPR systems or mRNA vaccines. These compounds not only improve molecular performance but also reduce immune responses in the body, making them highly valuable in medicine and pharmaceuticals.
For university researchers working in this field, patenting helps protect the intellectual property generated during scientific research and encourages collaboration with the business sector. Patents allow universities to be more active in technology transfer, strengthen innovation ecosystems, and contribute to societal progress. A patent titled N4-modified cytidine nucleotides and their use was recently granted to Prof. Rolandas Meškys and his colleagues at Vilnius University (VU). We spoke with the scientist about this and other patents and their potential applications.
A System for Enzyme Selection
Prof. Meškys explains that nucleotides, broadly speaking, are found in every cell of our body. “Nucleotides are the building blocks of nucleic acids like RNA and DNA. These are naturally occurring molecules present in every cell,” says the researcher.
During scientific investigations, the amount of original DNA is often very low, especially if extracted from biological samples such as a drop of blood, a single cell, or aged tissues. Therefore, scientists need to amplify the DNA sequence. This is done using a method called polymerase chain reaction (PCR), which allows rapid and precise amplification of a specific DNA sequence millions of times. A polymerase is an enzyme that joins nucleotides into DNA or RNA strands.
“PCR allows us to isolate and amplify a specific gene or DNA fragment. To do this, we need a particular polymerase enzyme. In nature, polymerases recognize natural nucleotides. So, if we want to replicate a DNA fragment, we need natural nucleotides,” explains the professor.
Modified nucleotides differ from natural ones in that they have synthetic chemical groups attached. “We essentially place an imaginary cap on the nucleotide. Polymerase can no longer recognize or use this capped molecule – the cap gets in the way, like it can’t fit through the door. So we must find a way to knock the cap off,” says the scientist.
One way to remove the cap is to use an enzyme that can break or form chemical bonds, converting the modified nucleotide back into a natural one. This would trigger the polymerase reaction and enable the amplification of a DNA fragment that encodes the desired enzyme.
“This introduces another player into the system – an enzyme that can return the modification to its natural state. We start with a natural nucleotide and modify it, but unless we add a protein that removes the modification, the reaction won’t proceed. In this invention, we demonstrated that we can detect such enzymes. We patented several DNA sequences that make this possible – they prove the principle works. So we have a system suitable for discovering enzymes capable of removing specific modifications,” says Prof. Meškys.
A Time-Saving Method
Like many of us, the professor jokes that his goal is to work less and use systems with minimal human involvement. “Imagine a gene or DNA fragment that we want to amplify. Now imagine that the DNA fragment itself encodes the enzyme needed for amplification. Let’s say we have two isolated droplets: one contains a gene that produces an enzyme that removes the modification, and the other contains a gene variant that does not. In this case, the gene will only replicate in the first droplet. But what if we want to test a million gene variants? Then we need a million droplets or test tubes,” he explains.
Finding a single effective DNA variant among a million would be challenging if each variant needed its test tube. Fortunately, the scientific community has developed a clever solution: emulsion systems – a mixture of oil and water separated into millions of tiny droplets.
“Each droplet contains a different DNA variant. If a certain DNA fragment encodes an enzyme that allows its replication, the droplets containing the effective enzyme will produce more DNA copies. After the reaction, all droplets are pooled, and sequencing is performed to determine which DNA variant ‘won.’ This advanced method enables rapid and efficient discovery of biologically important enzymes or DNA variants. Although our team didn’t invent the emulsion technique, we successfully applied it with modified nucleotides,” says Prof. Meškys.
Patented DNA Sequences for Medical Progress
One application of modified nucleotides is to insert them into a DNA sequence, giving the DNA a new functional property. “In our patented invention, we attach a ‘tail’ of modified nucleotides to the nucleic acid, adding a modification that allows the DNA to bind to plastic when exposed to UV light. This method could be used to develop various biosensors,” explains the researcher.
Today, chemotherapy is commonly used to treat cancer, but it often harms not only cancer cells but also healthy ones. Increasingly, prodrugs – inactive drug forms that become active only in the presence of specific cancer-related triggers – are being explored. “Drugs work by targeting specific proteins they bind to. If we modify a drug’s structure, it can no longer bind to the protein and becomes inactive and non-toxic. However, if a cancer cell contains an enzyme that can remove that modification, then the drug becomes active only in that cell. Suppose you know which enzyme removes the modification and how to deliver the drug specifically to the cancer cell. In that case, you have a drug that works exactly where it should – without harming other cells,” says the professor.
The prodrug principle is already used in medications for hypertension, where drugs are activated only in the target tissue. The modified nucleotides patented by Prof. Meškys and his team could enhance prodrug activation. “We selected enzymes that do not exist in nature but can activate specific compounds. We can search for enzymes suitable for prodrug activation – and we decided to patent one of the enzymes we discovered,” the researcher adds.
It’s important to note that the most significant current challenge is delivering the right enzyme to the correct location. “Many labs worldwide are working on how to transport these enzymes to the right cells. Our contribution is a method to modify and select enzymes best suited for a specific task – essentially a pre-tuned tool that works efficiently where it’s needed most,” concludes Prof. Meškys.
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