RNA Arrays - The Next Generation – RNArrays TNG

RNArrays - The Next Generation

There is a need for a method that allows for a large-scale, yet precise mutational analysis of functional RNA molecules. Having control over the actual sequences to be synthesized also offers the prospect of the preparation of RNA libraries for third-generation sequencing approaches. Random combinatorial synthesis of RNA cannot meet these criteria, but high-density photolithographic synthesis of RNA arrays provides a real solution. However, the currently only available method for in situ RNA array synthesis is limited in throughput due to long coupling and photodeprotection times as well as degradation during the deprotection steps.

Our objective is to develop two new sets of RNA phosphoramidites for RNA array fabrication with faster coupling times, increased photosensitivity and milder deprotection strategies, and thus to access long RNA oligonucleotides of high quality in a short amount of synthetic time. We then intend to prepare RNA microarrays for on and off-surface applications, from the study of the fluorogenic properties of a specific RNA aptamer to the direct sequencing of RNA libraries on a Nanopore instrument. The two sets of phosphoramidites having different sensitivity to base-mediated deprotection, 2'-OH protection may be maintained at particular locations to study the role of the 2' hydroxyl group in functional RNA.

We will transform the commercially available 2'-O pivaloyloxymethyl (PivOM) and propionyloxymethyl (PrOM) ribonucleosides , initially developed in our group, into their 5' highly photosensitive versions and isolate all eight corresponding RNA phosphoramidite building blocks. Incorporation of those phosphoramidites into RNA arrays will be done using in situ photolithography. The Mango III aptamer will be grown on-array and assayed against Thiazole Orange and sequence libraries will be read first on Illumina, then on a Nanopore GridION.

In so doing, we expect RNA synthesis to proceed 3-4× faster and to thus overcome the current limitation of ~30-nt, up to 100-nt long oligomers. With a low synthetic error-rate, this approach could then be regarded as an excellent source of controlled sequence diversity. Systematic and large-scale mutation will be extremely useful in aptamer and ribozyme research.

This is an international collaboration between the “Modified Oligonucleotides” group at IBMM in the University of Montpellier, France and the “Nucleic Acid Chemistry” group at the University of Vienna, Austria, with Drs Françoise Debart (France) and Jory Liétard (Austria) as joint PIs and supervisors. The synthesis of all eight novel RNA phosphoramidites will be carried out in France. RNA microarray synthesis and all following applications will be performed in Austria.