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Synthesis of modified RNA for structure-function studies

corresponding

JIACUI XU, DANILO SPRINGSTUBBE, NICO RUBLACK, BETTINA APPEL, SABINE MÜLLER*
*Corresponding author
Ernst-Moritz-Arndt Universität Greifswald, Institut für Biochemie, Felix-Hausdorff-Str. 4, Greifswald, D-17487, Germany

Abstract

Over the past two decades, RNA synthesis has become a very active field. Synthetic RNAs are required for a large number of applications. Beyond synthetic RNA for antisense, aptamer, ribozyme and siRNA technologies, oligoribonucleotides carrying site-specific modifications for structure and function studies are needed. We have synthesized specifically modified RNA molecules by (i) direct coupling of modified monomer units, (ii) by post-synthetic conjugation of a specific functionality to the synthesized RNA, or (iii) by action of a ribozyme.
Herein, we review our work on the preparation of RNA labelled with dyes, spin probes and other functionalities.


INTRODUCTION

Driven by the exciting developments in the field of RNA biology and oligonucleotide therapeutics, the chemical synthesis of natural and modified RNA has become a very active field. Investigation into antisense RNA, aptamers, ribozymes and small interfering RNAs for therapeutic and diagnostic application along with RNA research in molecular biology and nanosciences has provided a strong impetus for the development of efficient strategies for the preparation of RNA fragments of defined length and sequence, eventually conjugated to specific functionalities. Several strategies for the synthesis of RNA are available, all of them based on the phosphoramidite approach, but differing in the nature of the 2’-O-protecting group (1). Thus, the chemical synthesis of RNA oligonucleotides from the microgram to multi-gram scale is well possible today with fragment lengths up to about 80 nucleotides. Alternatively, RNA can be also prepared enzymatically by run-off transcription from linearized plasmid templates or chemically synthesized DNA templates. This is a very efficient procedure as long as the RNA to be synthesized contains only the natural nucleot ...



 

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