NEW YORK – A research team led by investigators at the University of Vienna has developed a new approach for fabricating high-density RNA microarrays, with potential applications in RNA library synthesis and multiplex assays.
The researchers discussed their technology in a paper published last week in Science Advances. While they have no plans to sell RNA chips based on the technology, they do intend to commercialize a device for making them, according to corresponding author Jory Lietard.
Lietard is an assistant professor in the department of inorganic chemistry at the University of Vienna. He said his interest in designing RNA microarrays dates back a decade or more, when he began working on methods to fabricate RNA arrays to complement DNA microarrays.
"The overwhelming majority of academics and industry that have and still are working with microarrays are only making DNA microarrays," he said, a situation he found "peculiar and unfortunate," as the same chemistry used to make DNA arrays is used to synthesize nucleic acids, meaning it could be fashioned to make RNA arrays, too. "My involvement in this technology was to exactly prove that you can make microarrays of any kind of nucleic acids, not necessarily just DNA," he said.
In 2018, Lietard and colleagues reported on a new method for chemically synthesizing RNA arrays at high density in Angewandte Chemie, a journal of the German Chemical Society. As detailed in the paper, the approach relied on a combination of photolithography and light-sensitive RNA phosphoramidites, a chemical compound used in the synthesis of oligonucleotides. Using modified phosphoramidite chemistry, they were able to synthesize RNA oligos of at least 30 nucleotides and to make a complex RNA permutation library with 262,144 unique sequences.
Even though it was a breakthrough for the team, the technology was not yet mature. While the 2018 paper showed that it was "in principle feasible to make high-density RNA chips," Lietard said, the synthesis time was too long and the resulting RNA was too fragile during post-synthesis chemical treatment. "The fabrication process was cumbersome, time-consuming, and it was limited to short RNA sequences," he said.
The technology described in the new Science Advances paper builds upon the work outlined in the 2018 paper but addresses these earlier shortcomings. Lietard's group developed a new series of RNA phosphoramidites that reduce the coupling and photolysis times during the synthesis process. Sequencing libraries that would take six hours to synthesize now take about three hours, according to the paper. The degradation of the RNA was also minimized, while hybridization signals were seven times stronger than those described previously. Using the approach, high-density RNA arrays and RNA libraries can thus be synthesized at a faster rate, the researchers claim. As part of their work, they demonstrated the synthesis of fluorogenic RNA Mango aptamers on microarrays and assessed the effect of sequence mutations on their fluorogenic properties. RNA Mango aptamers are a kind of high-affinity RNA aptamers with an orange fluorophore.
The work was accomplished in cooperation with partners at the University of Montpellier in France who synthesized the new RNA building blocks, which were originally developed for solid-phase RNA fabrication.
"They are highly reactive and are fully compatible with microarray photolithography," Lietard said, adding that his French colleagues "helped in making these building blocks photosensitive, which is a prerequisite for photolithography."
Through these innovations, the researchers were able to synthesize RNA much faster than before. "This is important because it not only makes the overall array fabrication time much faster, as well, but the quality of the chip is also increased because the surface is less affected [due to] the shorter contact time with the synthesis reagents," he said.
He noted that the researchers also modified chemical treatments post-synthesis, so the resulting RNA does not experience as much degradation as before. "Overall, this means greater signals that can be recorded on the surface of the chip, and longer sequences" of up to 60 nucleotides, he said, double the previous size.
Microarrays have been ubiquitous in genomics applications since the early 2000s, he acknowledged, though the chemical synthesis of RNA and DNA preexisted their appearance in the 1990s by at least a decade. Still, he said the usefulness of arrays has "gone way beyond and above their original purpose," for example for making DNA libraries for use in next-generation sequencing. DNA arrays are also used to run massively parallel assays against a given target.
According to Lietard, being able to carry out a single binding assay on the chip can return results on how the binding can be sequence-dependent, which positions are important for binding, and whether it can be improved upon. He said these qualities are "extremely important" in the field of aptamers, as well as in order to understand the mechanisms of interactions between proteins and nucleic acids.
RNA is also of increased interest as therapeutics and vaccines. "There is consequently an ever-growing need for synthetic RNA to probe its properties, and methodologies that can also accommodate chemical modifications are particularly essential," the authors wrote in their paper.
Lietard added that RNA arrays could be used to test the binding specificity of RNA-binding proteins, as they could support large-scale binding assays. Also of interest are proteins that bind base modifications, like methylated bases. "We can in principle incorporate these modified bases into our RNA chips and see how the presence of a modification affects the binding, relative to an unmodified RNA," he said. "This could help shed some light onto the function of these ubiquitous base modifications."
Lietard and colleagues are planning to set up a company to commercialize a device that can synthesize such arrays. He described the platform as a "new and improved photolithography device" that includes an optical circuit and an automated nucleic acid synthesizer. The optical circuit is "tabletop sized," while the nucleic acid synthesizer is a "standard apparatus that fits under a table." A computer is needed to carry out the synthesis, as well as standard nucleic acid reagents and microscope slides for the chips. The researchers described the device in a preprint published in ChemRxiv earlier this year.
In the meantime, Lietard's group is further developing, optimizing, and refining the technology, compressing fabrication time without sacrificing quality and introducing RNA modifications that are naturally present in messenger and transfer RNA. Such modifications "would be of great interest to researchers working in the field of genetics, epigenetics and transcriptomics," he said.