Chemo‐Enzymatic Synthesis of Position‐Specifically Modified RNA for Biophysical Studies including Light Control and NMR Spectroscopy

Abstract

The investigation of non-coding RNAs requires RNAs containing modifications at every possible position within the oligonucleotide. Here, we present the chemo-enzymatic RNA synthesis containing photoactivatable or C, N-labelled nucleosides. All four ribonucleotides containing ortho-nitrophenylethyl (NPE) photocages, photoswitchable azobenzene C-nucleotides and C, N-labelled nucleotides were incorporated position-specifically in high yields. We applied this approach for the synthesis of light-inducible 2'dG-sensing riboswitch variants and detected ligand-induced structural reorganization upon irradiation by NMR spectroscopy. This chemo-enzymatic method opens the possibility to incorporate a wide range of modifications at any desired position of RNAs of any lengths beyond the limits of solid-phase synthesis.

NMR Spectroscopy of Large Functional RNAs: From Sample Preparation to Low‐Gamma Detection

“…Herein, we provide a protocol for the chemo-enzymatic synthesis of RNAs that contain a single modified/labeled nucleotide at a desired position without any size limitation. By the site-specific incorporation of 13 C, 15 N labeled or unnatural modified nucleosides, such as 19 F or 13 C-methoxy groups, the signal abundance is dramatically reduced and spectra with single signals can be obtained (Keyhani et al, 2018). Using this method, it is even possible to incorporate modified nucleosides carrying sterically demanding modifications such as photoremovable protecting groups (e.g., ortho-nitrophenylethyl) or photoswitches (e.g., azobenzene).…”

“…Our description within this protocol covers the synthesis of the DNA template (Support Protocols 1 and 2), the optimization of the transcription reaction (Basic Protocol 1), the purification of the RNA (Basic Protocol 1, Alternate Protocols 1 and 2) as well as the expression and purification of several required enzymes within this process (Support Protocols 3 and 4). Furthermore, we provide a general guide on the ligation-based chemo-enzymatic synthesis of an RNA that is labeled or modified at a single position (Basic Protocol 2; Keyhani, Goldau, Blümler, Heckel, & Schwalbe, 2018). Here, the chemical synthesis of the required 3 ,5 -nucleoside bisphosphate (Support Protocol 5), the ligation reactions with T4 RNA Ligases 1 and 2 as well as the expression and purification of T4 RNA Ligase 2 (Support Protocol 6) are described.…”

Chemo‐enzymatic treatment of RNA to facilitate analyses

“…A more detailed discussion of chemical incorporation of non‐natural nucleotides is subject of a recent review (Holstein & Rentmeister, ). While chemical synthesis allows for high flexibility regarding the position and nature of the non‐natural nucleotide, synthesis of RNAs longer than 50 nt remains challenging, necessitating enzymatic ligation (Keyhani, Goldau, Blümler, Heckel, & Schwalbe, ). Enzymatic production of modified RNA does not face length restrictions (Figure b).…”

More than Proton Detection—New Avenues for NMR Spectroscopy of RNA

“…Such RNAs carrying a hydroxyl group and a modified nucleotide at the 3′‐end undergo ligation with a 5′‐phosphorylated RNA in presence of T4 RNA ligase 2 in an ATP‐dependent reaction. With this method, shown in Figure , RNAs up to a length of 390 nts containing a single position‐specific modification have been synthesized …”

“…With this method, shown in Figure 2, RNAs up to a length of 390 nts containing a single positionspecific modification have been synthesized. [32] 3. 13 C-detection NMR spectroscopic experiments for RNA…”