A global research group led by the University of Vienna has created a new type of RNA building blocks that possess enhanced chemical reactivity and photosensitivity. This groundbreaking advancement is set to considerably shorten the production time of RNA chips utilized in biotechnology and medical studies. The process of chemically synthesizing these chips is now twice as rapid and seven times more effective. The findings have recently been shared in the journal Science Advances.
The rise of RNA-based medical products, like the mRNA vaccines developed during the COVID-19 pandemic, has highlighted the significance of RNA. RNA (ribonucleic acid) is a polymer that carries information, comprised of similar subunits, and it exhibits much greater structural and functional diversity than DNA. Approximately 40 years ago, a technique was established to chemically synthesize DNA and RNA, allowing for the assembly of any sequence from DNA or RNA building blocks through phosphoramidite chemistry. This process builds a nucleic acid chain incrementally using specific chemical building blocks known as phosphoramidites. Each building block includes chemical ‘protecting groups’ that prevent undesired reactions, ensuring the formation of a natural link in the nucleic acid chain.
Addressing Challenges
This chemical approach is also applied in creating microchips (microarrays), enabling the simultaneous synthesis and analysis of millions of distinct sequences on a solid surface about the size of a fingernail. While DNA microarrays are extensively utilized, adapting this technology for RNA microarrays has been challenging due to RNA’s lower stability.
In 2018, the University of Vienna pioneered the production of high-density RNA chips through photolithography: by accurately directing a light beam, specific areas on a surface can be prepared for the addition of the next building block via a photochemical reaction. Although this initial development was groundbreaking and unparalleled, it faced issues such as lengthy production times, low yields, and stability concerns. This method has now seen substantial enhancements.
Creation of a New Generation of RNA Building Blocks
A collaborative effort between the Institute of Inorganic Chemistry at the University of Vienna and the Max Mousseron Institute for Biomolecules at the University of Montpellier (France) led to the development of upgraded RNA building blocks that offer greater chemical reactivity and photosensitivity. This innovation drastically reduces RNA chip production times, achieving synthesis that is twice as quick and seven times more efficient. The new RNA chips can screen millions of potential RNAs for valuable sequences across various applications.
“Producing RNA microarrays with functional RNA molecules was previously unattainable with our earlier methods, but this upgraded process utilizing the propionyloxymethyl (PrOM) protecting group has made it feasible,” expresses Jory Lietard, Assistant Professor at the Institute of Inorganic Chemistry.
As an immediate application of these enhanced RNA chips, the publication includes a study on RNA aptamers, which are small oligonucleotides that bind specifically to target molecules. Two “light-up” aptamers, which emit fluorescence when binding to a dye, were selected, resulting in the synthesis of thousands of variants of these aptamers on the chip. A single binding experiment can collect data on all variants at once, paving the way for identifying better aptamers with enhanced diagnostic capabilities.
“High-quality RNA chips could prove particularly beneficial in the burgeoning field of non-invasive molecular diagnostics. There is a vital demand for new and improved RNA aptamers, especially those capable of tracking hormone levels in real-time or monitoring other biological markers directly from sweat or saliva,” notes Tadija Keki?, a PhD candidate working with Jory Lietard.
This project received financial support through a collaborative grant from the Agence Nationale pour la Recherche and the Austrian Science Fund (FWF International Program I4923).
