Site-specific labeling of RNA at internal ribose hydroxyl groups: terbium-assisted deoxyribozymes at work.
A general and efficient single-step method was established for site-specific post-transcriptional labeling of RNA. Using Tb(3+) as accelerating cofactor for deoxyribozymes, various labeled guanosines were site-specifically attached to 2'-OH groups of internal adenosines in in vitro transcribed RNA. The DNA-catalyzed 2',5'-phosphodiester bond formation proceeded efficiently with fluorescent, spin-labeled, biotinylated, or cross-linker-modified guanosine triphosphates. The sequence context of the labeling site was systematically analyzed by mutating the nucleotides flanking the targeted adenosine. Labeling of adenosines in a purine-rich environment showed the fastest reactions and highest yields. Overall, practically useful yields >70% were obtained for 13 out of 16 possible nucleotide (nt) combinations. Using this approach, we demonstrate preparative labeling under mild conditions for up to ~160-nt-long RNAs, including spliceosomal U6 small nuclear RNA and a cyclic-di-AMP binding riboswitch RNA.
Research Group Nucleic Acid Chemistry, Max Planck Institute for Biophysical Chemistry , Am Fassberg 11, 37077 Göttingen, Germany.
Lea
L
Büttner
Fatemeh
F
Javadi-Zarnaghi
Claudia
C
Höbartner
D000241:Adenosine / Q000737:chemistry; D019921:Biotinylation; D002384:Catalysis; D003432:Cross-Linking Reagents; D004247:DNA / Q000737:chemistry; D021881:DNA, Catalytic / Q000737:chemistry*; D006151:Guanosine / Q000737:chemistry; D011499:Protein Processing, Post-Translational; D012313:RNA / Q000737:chemistry*; D017414:Ribonucleoprotein, U4-U6 Small Nuclear / Q000737:chemistry; D012266:Ribose / Q000737:chemistry*; D058928:Riboswitch; D013725:Terbium / Q000737:chemistry*
D003432:Cross-Linking Reagents; D021881:DNA, Catalytic; D017414:Ribonucleoprotein, U4-U6 Small Nuclear; D058928:Riboswitch; D013725:Terbium; D006151:Guanosine; D012313:RNA; D012266:Ribose; D004247:DNA; D000241:Adenosine