An international research team led by the University of Vienna has developed a new version of RNA building blocks with enhanced chemical reactivity and photosensitivity. This significant innovation reduces the production time of RNA chips used in biotechnological and medical research, making the chemical synthesis process twice as fast and seven times more efficient. The findings were recently published in Science Advances.
The Evolution of RNA-Based Medical Products
The emergence and approval of RNA-based medical products, particularly mRNA vaccines during the COVID-19 pandemic, have spotlighted the RNA molecule. Ribonucleic acid is an information-carrying polymer with greater structural and functional diversity than DNA. Approximately 40 years ago, phosphoramidite chemistry was developed for the chemical synthesis of DNA and RNA, allowing any sequence to be assembled from RNA or DNA building blocks. These special chemical building blocks, or phosphoramidites, carry chemical protecting groups that prevent unwanted reactions and ensure the formation of a natural link in the nucleic acid chain.
Addressing Challenges in RNA Microarray Production
This chemical method is also employed in producing microchips (microarrays), where millions of unique sequences can be synthesized and analyzed simultaneously on a solid surface. DNA microarrays are still widely used; however, amending the technology to RNA microarrays has been challenging due to RNA's lower stability.
In their previous work, the University of Vienna revealed the production of high-density RNA chips through photolithography. This technique uses a beam of light to prepare surface areas for the attachment of the next building block through a photochemical reaction. Despite being a pioneering method, it had the disadvantages of long production times, low yields, and poor stability. The current study has substantially improved this approach.
New Generation of RNA Building Blocks
The research team from the Institute of Inorganic Chemistry at the University of Vienna, in collaboration with the Max Mousseron Institute for Biomolecules at the University of Montpellier, developed a new version of RNA building blocks that boasts higher chemical reactivity and photosensitivity. This development significantly reduces the production time of RNA chips and enhances synthesis efficiency.
“Making RNA microarrays containing functional RNA molecules was simply out of reach with our earlier setup, but it is now a reality with this improved process using the propionyloxymethyl (PrOM) protecting group,” said Jory Lietard, Assistant Professor at the Institute of Inorganic Chemistry.
Applications in RNA Aptamer Research
As a direct application of these enhanced RNA chips, the research focused on RNA aptamers, which are small oligonucleotides designed to bind specifically to target molecules. The study selected two "light-up" aptamers that fluoresce upon binding to a dye. Thousands of these aptamer variants were synthesized on the chip, and a single binding experiment was sufficient to collect data on all the variants simultaneously. This method enabled the identification of aptamers with superior diagnostic capabilities.
“High-quality RNA chips could be especially valuable in the rapidly growing field of non-invasive molecular diagnostics. New and improved RNA aptamers are critically sought after, such as those that can track hormone levels in real-time or monitor other biological markers directly from sweat or saliva,” explained Tadija Kekić, a Ph.D. candidate in Lietard's group.
Publication Details
Tadija Kekić et al., Accelerated, high-quality photolithographic synthesis of RNA microarrays in situ. Sci. Adv. 10, eado6762 (2024). DOI:10.1126/sciadv.ado6762