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Practical Synthesis of Cap‐4 RNA
Author(s) -
Leiter Josef,
Reichert Dennis,
Rentmeister Andrea,
Micura Ronald
Publication year - 2020
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.201900590
Subject(s) - guanosine , nucleotide , phosphoramidite , methylation , rna , biology , nucleoside , biochemistry , guanosine triphosphate , computational biology , trypanosoma brucei , oligonucleotide , dna , gene
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.