Chemical synthesis of selenium-modified oligoribonucleotides and their enzymatic ligation leading to an U6 SnRNA stem-loop segment.
The derivatization of nucleic acids with selenium is highly promising to facilitate nucleic acids structure determination by X-ray crystallography using the multiwavelength anomalous dispersion (MAD) technique. The foundation for such an approach has been laid by Huang, Egli, and co-workers and was exemplified on small DNA duplexes. Here, we present a comprehensive study on the preparation of RNAs containing 2'-Se-methylpyrimidine nucleoside labels. This includes the synthesis of a novel 2'-Se-methylcytidine phosphoramidite 11 and its incorporation into oligoribonucleotides by solid-phase synthesis. Deprotection of the oligonucleotides is achieved in the presence of millimolar amounts of threo-1,4-dimercapto-2,3-butandiol (DTT). With this additive, oxidation products and follow-up side-products are suppressed and acceptable HPLC traces of the crude material are obtained, so far tested for sequences of up to 22-mers. Moreover, an extensive investigation on the enzymatic ligation of the selenium-containing oligoribonucleotides demonstrates the high flexibility of the selenium approach. Our target sequence, an U6 snRNA stem-loop motif comprising all naturally occurring nucleoside modifications beside the Se-label is achieved by ligation using T4 RNA ligase.
Höbartner
Claudia
C
Institute of Organic Chemistry, Leopold Franzens University, Innrain 52a, A-6020 Innsbruck, Austria.
Micura
Ronald
R
D001483:Base Sequence; D018360:Crystallography, X-Ray; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009843:Oligoribonucleotides / Q000138:chemical synthesis* / Q000737:chemistry; D009943:Organophosphorus Compounds / Q000138:chemical synthesis / Q000737:chemistry; D016566:Organoselenium Compounds / Q000138:chemical synthesis* / Q000737:chemistry; D012313:RNA / Q000138:chemical synthesis* / Q000737:chemistry; D012342:RNA, Small Nuclear / Q000737:chemistry*
D009843:Oligoribonucleotides; D009943:Organophosphorus Compounds; D016566:Organoselenium Compounds; D012342:RNA, Small Nuclear; C405486:U6 small nuclear RNA; C434331:phosphoramidite; D012313:RNA