%0 Journal Article
%T Synthesis of Specifically Modified Oligonucleotides for Application in Structural and Functional Analysis of RNA
%A Nico Rublack
%A Hien Nguyen
%A Bettina Appel
%A Danilo Springstubbe
%A Denise Strohbach
%A Sabine M¨¹ller
%J Journal of Nucleic Acids
%D 2011
%I Hindawi Publishing Corporation
%R 10.4061/2011/805253
%X Nowadays, RNA synthesis has become an essential tool not only in the field of molecular biology and medicine, but also in areas like molecular diagnostics and material sciences. Beyond synthetic RNAs for antisense, aptamer, ribozyme, and siRNA technologies, oligoribonucleotides carrying site-specific modifications for structure and function studies are needed. This often requires labeling of the RNA with a suitable spectroscopic reporter group. Herein, we describe the synthesis of functionalized monomer building blocks that upon incorporation in RNA allow for selective reaction with a specific reporter or functional entity. In particular, we report on the synthesis of 5¡ä-O-dimethoxytrityl-2¡ä-O-tert-butyldimethylsilyl protected 3¡ä-O-phosphoramidites of nucleosides that carry amino linkers of different lengths and flexibility at the heterocyclic base, their incorporation in a variety of RNAs, and postsynthetic conjugation with fluorescent dyes and nitroxide spin labels. Further, we show the synthesis of a flavine mononucleotide-N-hydroxy-succinimidyl ester and its conjugation to amino functionalized RNA. 1. Introduction Over the past two decades, RNA synthesis has become a very active field. Synthetic RNAs are required for a large number of applications. Antisense oligonucleotides, ribozymes, aptamers, and siRNAs are required for medicinal diagnostic and therapy as well as for a wide variety of biochemistry and molecular biology studies [1, 2]. Furthermore, aptamers and reporter ribozymes are designed and applied in environmental diagnostics [3]. A number of other disciplines make use of synthetic oligoribonucleotides, for example, the field of material science, where novel materials from nanoparticle oligonucleotide conjugates are generated [4]. In parallel, RNA synthesis has developed to a degree that allows the synthesis of RNA oligonucleotides of any desired sequence from microgram to multigram scale. In addition to 2¡ä-O-TBDMS chemistry, which may be considered as the standard procedure for RNA preparation, novel strategies mainly focussing on different 2¡ä-O-protecting groups such as 2¡ä-O-TOM, 2¡ä-O-ACE or 2¡ä-O-TC are available for laboratory use applying the specific monomer building blocks, or have been commercialized for custom RNA synthesis [5, 6]. Research in our laboratory is devoted to the chemistry and biochemistry of RNA with a strong focus on RNA aptamers and ribozymes. Therefore, we synthesize natural and modified RNA strands required for the design of functional RNAs that we want to investigate. Furthermore, we also prepare RNA molecules
%U http://www.hindawi.com/journals/jna/2011/805253/