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.
■ INTRODUCTIONThe site-specific attachment of labels onto RNA is of significant value in many areas of RNA chemical biology. The study of RNA folding and RNA−protein and RNA−ligand interactions by spectroscopic methods requires installation of observable reporter groups. Biochemical assays of RNA functions benefit from position-specific placement of reactive functional groups or probing agents. 1 Emissive nucleotide (nt) analogues or abiotic functional groups for bioorthogonal labeling can be sitespecifically installed by solid-phase synthesis. 2−4 Synthesis of RNAs longer than ∼40 nt often involves enzymatic manipulation or ligation using T4 ligases or deoxyribozymes. 5−9 As alternatives to the fragment-based approach, direct post-transcriptional labeling methods are gaining increasing attention. Recent developments include the use of specific methyltransferases together with functionalized Sadenosylmethionine (SAM) analogues, as exemplified by the alkylation of a specific guanosine in tRNA Phe with Trm1. 10 More generally applicable may be the reconstitution of C/D box small nucleolar ribonucleoprotein particles (snoRNPs), using guide RNAs responsible for the selection of the labeling site in the target RNA, as recently shown for the site-specific 2′-alkynylation of tRNA and mRNA. 11 A non-enzymatic example of oligonucleotide-guided post-transcriptional RNA modification was introduced with the functionality transfer reaction from 6-thioguanosine in a donor DNA to the exocyclic amino groups of cytosine and guanine residues in the target RNA. 12−14 Particularly attractive was the combination of a guide and an enzyme in a single strand of DNA, introduced as "DNA-catalyzed labeling of RNA". 15 In this approach, Baum and Silverman prepared a labeled tagging RNA by in vitro transcription using 5-aminoallyl-CTP and subsequent labeling by N-hydroxysuccinimide chemistry. This tagging RNA was then used as substrate for DNA-catalyzed ligation to the target RNA, forming labeled 2′,5′-branched RNA. Despite the elegance of t...
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.
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