RESUMO
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.
Assuntos
DNA Catalítico/química , RNA/química , Ribose/química , Térbio/química , Adenosina/química , Biotinilação , Catálise , Reagentes de Ligações Cruzadas , DNA/química , Guanosina/química , Processamento de Proteína Pós-Traducional , Ribonucleoproteína Nuclear Pequena U4-U6/química , RiboswitchRESUMO
Chemically stable nitroxide radicals that can be monitored by electron paramagnetic resonance (EPR) spectroscopy can provide information on structural and dynamic properties of functional RNA such as riboswitches. The convertible nucleoside approach is used to install 2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPO) and 2,2,5,5-tetramethylpyrrolidin-1-oxyl (proxyl) labels at the exocyclic N(4)-amino group of cytidine and 2'-O-methylcytidine nucleotides in RNA. To obtain site-specifically labeled long riboswitch RNAs beyond the limit of solid-phase synthesis, we report the ligation of spin-labeled RNA using an in vitro selected deoxyribozyme as catalyst, and demonstrate the synthesis of TEMPO-labeled 53 nt SAM-III and 118 nt SAM-I riboswitch domains (SAM=S-adenosylmethionine).
Assuntos
DNA/química , Nucleosídeos/química , RNA/síntese química , Riboswitch , Marcadores de Spin/síntese química , Sequência de Bases , Catálise , DNA/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Modelos Moleculares , Conformação de Ácido Nucleico , RNA/químicaRESUMO
Palladium-catalyzed intramolecular dehydrogenative direct arylations of 1,2,3-triazoles were accomplished under ambient pressure of air, which set the stage for a modular synthesis of annulated phenanthrenes through a reaction sequence comprising two distinct catalytic C-H bond functionalization reactions.