RNA arrays: The Next Generation

RNA is the intermediate link between genetic information (stored on DNA) and the product of the expression of genes (proteins) and, like DNA, is a nucleic acid, a biopolymer composed of four different building blocks (A, C, G and U) joined together in a very specific order called a sequence. But RNA does not only store information in the form of a code using the genetic alphabet, its structure and tridimensional shape allows it to become a chemically active molecule able to perform reactions and to bind to a wide ensemble of targets. The functional diversity of RNA therefore makes it an ideal nucleic acid to identify new binding motifs and to study how target recognition works. Typically, this requires preparing sequence variants which, if all RNA bases have to permuted at each position, very quickly amounts to tens and hundreds of thousands of RNA sequences. The only available method that can match these requirements is microarray synthesis. Microarray synthesis allows for very large libraries of nucleic acid sequences to be synthesized in parallel, on the same platform, each sequence being strictly confined to a specific spot on the surface of the microarray. In our lab, we use UV light to control the synthesis of DNA sequences, in a process called photolithography, and we recently expanded our protocol to RNA synthesis too. The goal of this project is to prepare an entirely new set of building blocks that would allow for RNA synthesis to proceed much faster and more efficiently, breaking our current length limitations. To do so, we will employ special RNA building blocks (phosphoramidites) which have previously been shown to yield RNA oligonucleotides of high purity on standard solid-phase synthesis and we will adapt their molecular structure so as to make them photosensitive. These new phosphoramidites will actually be equipped with the most photosensitive protecting group compatible with microarray photolithography, which is key to bring down the total RNA array synthesis time. With access to longer RNA oligonucleotides of higher quality, complex libraries of RNA sequences will be prepared on a single microarray to study the binding properties fluorogenic aptamers. RNA aptamers are structures whose 3D folding allow them to bind to target molecules, and in the case of fluorogenic aptamers, the binding event brings the RNAtarget complex to light up and display strong fluorescent properties, a particularly useful technique for in vivo RNA monitoring. RNA libraries synthesized on arrays will also be tested in direct RNA sequencing. The project is a collaboration between Dr. Jory Lietard, of the Institute of Inorganic Chemistry at the University of Vienna, and Dr. Francoise Debart of the University of Montpellier.

The project aimed to bring forward the process of fabricating microarrays of RNA. Microarrays are physical objects where a set of unique molecules are attached to a flat surface, each molecule being assigned a given location on that surface. DNA microarrays are commonplace and commercially available, but RNA microarrays are significantly harder to prepare. Because of their great potential in answering biologically relevant questions in a high-throughput manner, we wished to develop a fast and efficient process to make RNA microarrays. To do so, we teamed up with the group of Françoise Debart (University of Montpellier, France) who supplied the necessary reagents for the synthesis of RNA microarrays. Her group developed a set of novel RNA building blocks that were designed to accelerate the process of attaching those building blocks together in order to assemble an RNA molecule. They contained a photosensitive group that allowed for integration within our microarray fabrication process called photolithography. These new RNA building blocks, called PrOM phosphoramidites, were tested in microarray photolithography. We were able to produce RNA microarrays much faster than before, due to the assembly process ("coupling") now being twice as fast and photosensitivity four times greater. We also noticed that the overall quality was improved, with less degradation of the RNA molecules. This study on technical improvement culminated with the preparation of RNA microarrays containing hundreds of thousands of unique RNA sequences, all contained within a ~1.4 cm area, in a little over 3 hours of synthesis time and very close to how long it would take to synthesize a DNA version of the same microarray. We also explored applications for RNA microarrays. In one particular case, we studied so-called fluorogenic RNA aptamers. These are RNA sequences that can bind to small molecules and the resulting binding interaction generates a fluorescent signal. These aptamers have found use in the tracking of RNA inside cells. We were able to design a very complex library of fluorogenic aptamers on microarrays and to study how changes in the RNA sequence leads to a decreased ability to bind and generate a fluorescent signal. This work on systematic sequence modification highlights the great value of DNA and RNA microarrays: a library of RNA sequences synthesized at the same time and contained within the same object requiring a single experiment to interrogate each member of the library simultaneously with high precision and accuracy. Future work will focus on producing longer RNA sequences and exploring other binding interactions.