Summary of Article
INTRODUCTION
Altered oligonucleotides, specifically, labeled RNA serve as important tools in biochemical and biomedical research and have been commonly utilized for therapeutic and analytical purposes . Although there are many studies on post-synthetic techniques on DNA, such as by Marx et al., very few reports have described the techniques related to RNA. An efficient automated RNA synthesis with an optimal yield is a prerequisite for post-synthetic derivatization. It is a commonly accepted truth that RNA production is much more troublesome and challenging than DNA synthesis, owing to its lesser coupling efficiency in the synthesis cycle along with the requirement to permanently 2’ hydroxyl activity making the development of the technique more complex. The most widely utilized masking group methods include the classical TBDMS (tert-butyldimethylsilyl) approach, which may also include substitute employment of 2’-O-TOM (triisopropylsilyloxymethyl) domains leading to decrease stearic impediment and consequently increasing coupling frequencies. These methods along with ACE (bis-acetoxyethyloxy)-methyl transfer enhance the outcome of RNA production.
Although successful, the use of ACE) chemistry has been found to be restricted, owing to the formation of side-products . Enzymatic degradation of ploy uridine sequences disclosed the presence of large amounts of N3-methylated uridine derivatives formed due to the movement of methyl as a phosphate- protecting moiety utilized in the ACE chemistry.
The researchers recorded NMR spectra on Bruker AV instruments at 300 MHz and 300 K. Chemical shifts were demonstrated in parts per million compared to the solvent signal. Thin layer chromatograms (TLC) were recorded on plates purchased from Merck. Column chromatography was conducted on a silica gel 60 with thickness 40-63 mm (Merck). High-resolution mass spectra (HRMS) was quantified using MALDI Orbitrap LTQ device obtained from Thermo Fisher. RNA’s were chemically synthesized using ACE, TC or TBDMS methods, post-synthetically altered, demasked, purified and characterized. Some RNA were treated with certain procedures, wherein the methyl groups were eliminated directly after production of RNA, followed by Stille coupling, with a final step of basic deprotection and breakdown from solid base. N-methyl uridine was used as a reference compound and synthesized similar to methylation of thymidine as demonstrated earlier. Finally, analytical enzyme processing and tracking of monomer model reactions were performed.
CONCLUSION
References
Wicke, L. (2014). An unexpected methyl group migration during on-column Stille derivatization of RNA. Tetrahedron, 70: 327-333.
