RNA Arrays - The Next Generation – RNArrays TNG
Accelerated, high quality photolithographic synthesis of RNA microarrays in situ
Nucleic acid photolithography is the only microarray fabrication process that has demonstrated chemical versatility beyond DNA; accommodating RNA as well as nucleic acid analogs. The current approach to RNA microarray synthesis employs a set of building blocks that requires long coupling and photolysis times, and the procedure suffers from unavoidable degradation post-synthesis. We purpose to develop new RNA phosphoramidites with improved chemical and photochemical protection of 2' and 5'-OH.
Novel, highly photosensitive RNA building blocks to unlock the potential of RNA microarrays in the preparation of nucleic acid libraries for aptamer and sequencing purposes
The structural and functional domain of RNA is the subject of constant exploration, and its importance in medicine and biotechnology has recently skyrocketed with the advent and market approval of RNA-based therapeutics. As a result, the need for synthetic RNAs to study their properties continues to grow, and methodologies that can also take chemical modifications into account are particularly essential. Similarly, throughput-driven approaches are in high demand, as they can provide nucleic acid libraries of highly complex sequences and offer the possibility of parallel assays: over 780,000 unique sequences synthesized in parallel and on the same surface. <br />High-throughput nucleic acid synthesis has become commonplace for DNA since the development of microarrays, particularly those where DNA synthesis takes place in situ on the surface of the microarray, enabling up to hundreds of thousands of unique sequences to be obtained in a single run. RNA microarray synthesis has received much less attention due to technical challenges associated with RNA instability, lower synthesis efficiency and the protecting group strategy for 2'-OH function.<br />The chemistry of DNA and RNA microarray synthesis draws heavily on classical solid-phase synthesis, using phosphoramidite building blocks. <br />A few years ago, an initial method for RNA microarray synthesis was developed by the Austrian partner using photosensitive RNA phosphoramidites protected in the 2' position by an acetal levulinyl ester (ALE) group. However, this method suffers from a number of limitations: long ALE phosphoramidite coupling times and photolysis time to remove the nitrophenylpropyloxycarbonyl (NPPOC) group, resulting in long high-density RNA chip synthesis times with surface degradation affected by prolonged exposure to solvents and reagents. <br />The development of a new synthesis method therefore made sense to improve the efficiency and quality of RNA microarrays.<br />As part of an interdisciplinary and international collaboration, the 2 nucleic acid chemist partners propose to prepare new RNA phosphoramidites protected at 2'O by acetal ester groups used in solid-state synthesis, coupling faster than ALE units. The thiophenyl derivative of NPPOC (SPhNPPOC) as 5'OH protection will be evaluated, again with the intention of reducing fabrication times of microarrays.<br />These new phosphoramidites will be incorporated into RNA arrays using in situ photolithography. The Mango III aptamer will be prepared on chip and tested against Thiazole Orange, and sequence libraries will be read first on Illumina, then on a GridION Nanopore.
1. Phosphoramidite synthesis
A set of 4 ribonucleoside phosphoramidites 2'-O-PivOM 5'-O-NPPOC and
a set of 8 phosphoramidite ribonucleosides 2'-O-PrOM 5'-O-NPPOC or 5'-O-SPhNPPOC were prepared by a 5-step synthesis route from commercial ribonucleosides, already protected on the bases by acyl groups, in 2'O by a PivOM or PrOM group and in 5'O by a temporary dimethoxytrityl group. NPPOC or SPhNPPOC photosensitive protections were introduced regioselectively at 5'OH after transient protection of the 3'OH by a silyl group and deprotection of the 5'OH. The NPPOC-Cl reagent had to be prepared in advance in the laboratory, while its thiophenyl derivative was purchased. After releasing the 3'OH from the silyl groups, the phosphoramidite function was installed on the ribonucleosides in good yields. Several grams of each phosphoramidite ribonucleoside were obtained and supplied to the Austrian partner for incorporation into RNA chip sequences.
2. RNA microarray synthesis
-Evaluation of phosphoramidite coupling and RNA degradation
-Evaluation of photolysis of NPPOC and SPhNPPOC groups on RNA 25 nucleotides long.
Chemists from the Montpellier team have developed new building blocks to improve the RNA synthesis on microarrys by photolithography carried out by partners from the Vienna team. These consist of novel phosphoramidite ribonucleosides, protected at 2'OH by an acetalester group (pivaloyloxymethyl (PivOM) or propionyloxymethyl (PrOM), originally developed for solid-phase RNA synthesis, and at 5'OH by a photolabile gurpe (NPPOC or SPhNPPOC).
- the 2'-O-PivOM 5'-O-NPPOC synthons failed to produce good-quality RNAs efficiently, and were therefore discarded for the rest of the project.
- the 2'-O-PrOM 5'-O-SPhNPPOC phosphoramidites showed higher coupling efficiencies than the 2'-O-ALE phosphoramidites of 1st generation RNA microarrays. Less RNA degradation was observed.
The use of these PrOM and SPhNPPOC protections reduced the time taken to couple the units together by more than half compared with traditional protections, while photolysis time was cut by a factor of 4. Libraries of RNA sequences that would have taken over six hours to synthesize can now be prepared in half the time.
Thanks to faster, more efficient synthesis, RNA microarrays containing more than ten thousand fluorogenic aptamer variants on the same surface were produced. These microarrays were then subjected to a single fluorescent marker interaction test, which revealed how mutations and truncations in known aptamer sequences can greatly enhance or diminish their fluorogenic properties.
This optimization of RNA microarray synthesis by photolithography represents a major technological advance in terms of quality, efficiency and speed. With a low synthetic error rate, this method will enable the synthesis of long (>100 nucleotides) and complex RNA oligonucleotide libraries on high-density chips, for direct RNA sequencing and to study the error rate of RNA synthesis.
With the improved chemistry and photochemistry of RNA photolithography, it now becomes possible to envisage the preparation of decorated microarrays with larger functional RNA molecules, such as tRNA, guide RNAs and RNAzymes, which were simply out of reach with ALE chemistry. In addition, commercially available 5'-DMTr 2'-O-PrOM ribonucleoside precursors naturally occurring or with unusual bases (5-methyl-cytosine, hypoxanthine, 6-methyladenine) provide a useful gateway to the synthesis of base-modified phosphoramidite RNAs or 3'-photoprotected «reverse« 5'-phosphoramidite RNAs, which are set to become the next upgrade in the chemistry toolbox of Maskless Array Synthesis (MAS).
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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.
Project coordination
Françoise Debart (Institut des Biomolécules Max Mousseron)
The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.
Partnership
IBMM Institut des Biomolécules Max Mousseron
Institute of Inorganic Chemistry
Help of the ANR 215,403 euros
Beginning and duration of the scientific project:
December 2020
- 36 Months
