Site-Specific Labeling of RNA at Internal Ribose Hydroxyl Groups: Terbium-Assisted Deoxyribozymes at Work

Abstract

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...

Structural Analysis of Protein–RNA Complexes in Solution Using NMR Paramagnetic Relaxation Enhancements

“…As with protein spin labels, the intrinsic flexibility of the spin label when attached to the RNA needs to be carefully considered. RNA spin labeling can be used to analyze the conformation and dynamics of larger RNA molecules, as has been shown with HIV-1 TAR RNA (Wunderlich et al, 2013), riboswitches, and spliceosomal U6 snRNA (Buttner, Javadi-Zarnaghi, & Hobartner, 2014;Buttner, Seikowski, Wawrzyniak, Ochmann, & Hobartner, 2013). Furthermore, the RNA-binding orientation with respect to an RNA-binding protein can be determined (Mackereth et al, 2011), and protein-RNA distances can be obtained and used for structure calculation and validation (Allen, Varani, & Varani, 2001).…”

Conformational dynamics of nucleic acid molecules studied by PELDOR spectroscopy with rigid spin labels

“…With the rigid spinlabel Ç, such motion can be suppressed if the carrying nucleic acid molecule is immobilized or large enough to tumble slowly. The possibility to incorporate spin labels into long RNA molecules by ligation techniques [88,89] as well as the investigation of RNA-protein complexes [90,88] has been already demonstrated. A rigid spin label will not only prolong the transversal relaxation time, but also allow collection of information on dynamics of biomolecules that occur on the nano-to microsecond timescale.…”

Combining NMR and EPR to Determine Structures of Large RNAs and Protein–RNA Complexes in Solution

“…While two elegant methods have been introduced by the Hobartner lab performing ligations of smaller RNA fragments using deoxyribozymes (Buttner, Javadi-Zarnaghi, & Hobartner, 2014;Buttner, Seikowski, Wawrzyniak, Ochmann, & Hobartner, 2013), these approaches are currently limited by strong sequence requirements at the sites of ligation.…”