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...

Complete analysis! Combinatorial mutation interference analysis (CoMA) is a highly efficient method to identify catalytically essential nucleotides in deoxyribozymes by the simultaneous assessment of all possible mutations in the active site of the catalyst. The application of CoMA for two different deoxyribozymes revealed indispensable guanosine nucleotides for DNA‐catalyzed RNA ligation.

Most deoxyribozymes (DNA catalysts) require metal ions as cofactors for catalytic activity, with Mg(2+), Mn(2+), and Zn(2+) being the most represented activators. Trivalent transition-metal ions have been less frequently considered. Rare earth ions offer attractive properties for studying metal ion binding by biochemical and spectroscopic methods. Here we report the effect of lanthanide cofactors, in particular terbium (Tb(3+)), for DNA-catalyzed synthesis of 2',5'-branched RNA. We found up to 10(4)-fold increased ligation rates for the 9F7 deoxribozyme using 100 μM Tb(3+) and 7 mM Mg(2+), compared to performing the reaction with 7 mM Mg(2+) alone. Combinatorial mutation interference analysis (CoMA) was used to identify nucleotides in the catalytic region of 9F7 that are essential for ligation activity with different metal ion combinations. A minimized version of the DNA enzyme sustained high levels of Tb(3+)-assisted activity. Sensitized luminescence of Tb(3+) bound to DNA in combination with DMS probing and DNase I footprinting results supported the CoMA data. The accelerating effect of Tb(3+) was confirmed for related RNA-ligating deoxyribozymes, pointing toward favorable activation of internal 2'-OH nucleophiles. The results of this study offer fundamental insights into nucleotide requirements for DNA-catalyzed RNA ligation and will be beneficial for practical applications that utilize 2',5'-branched RNA.

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