Practical Synthesis of Cap‐4 RNA. (8th November 2019)

• Record Type: Journal Article
• Authors: Leiter, Josef; Reichert, Dennis; Rentmeister, Andrea; Micura, Ronald
• Is Part Of: Chembiochem. Volume 21:Number 1/2(2020)
• Page Start: 265
• Page End: 271
• Publication Date: 2019-11-08
• Subjects: chemoenzymatic synthesis -- oligonucleotides -- mRNA caps -- RNA modifications -- solid-phase synthesis; Biochemistry -- Periodicals; Molecular biology -- Periodicals; Pharmaceutical chemistry -- Periodicals; 572
• DOI: 10.1002/cbic.201900590
• Languages: English
• ISSNs: 1439-4227

Abstract

Abstract: Eukaryotic mRNAs possess 5′ caps that are determinants for their function. A structural characteristic of 5′ caps is methylation, with this feature already present in early eukaryotes such as Trypanosoma . While the common cap‐0 (m 7 GpppN) shows a rather simple methylation pattern, the Trypanosoma cap‐4 displays seven distinguished additional methylations within the first four nucleotides. The study of essential biological functions mediated by these unique structural features of the cap‐4 and thereby of the metabolism of an important class of human pathogenic parasites is hindered by the lack of reliable preparation methods. Herein we describe the synthesis of custom‐made nucleoside phosphoramidite building blocks for m 6 2 Am and m 3 Um, their incorporation into short RNAs, the efficient construction of the 5′‐to‐5′ triphosphate bridge to guanosine by using a solid‐phase approach, the selective enzymatic methylation at position N7 of the inverted guanosine, and enzymatic ligation to generate trypanosomatid mRNAs of up to 40 nucleotides in length. This study introduces a reliable synthetic strategy to the much‐needed cap‐4 RNA probes for integrated structural biology studies, using a combination of chemical and enzymatic steps. Abstract : Highly methylated mRNA caps are encountered in Trypanosoma . A solid‐phase‐based chemoenzymatic approach makes these RNAs synthetically accessible, thereby facilitating the study of the metabolism of, and essential biological functions mediated by these unique structural features found in this important class of human pathogenic parasites.