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.H ere,w ep resent the chemoenzymatic RNAs ynthesis containing photoactivatable or 13 C, 15 N-labelled nucleosides.A ll four ribonucleotides containing ortho-nitrophenylethyl (NPE) photocages,p hotoswitchable azobenzene C-nucleotides and 13 C, 15 N-labelled nucleotides were incorporated position-specifically in high yields.W e applied this approach for the synthesis of light-inducible 2'dGsensing riboswitch variants and detected ligand-induced structural reorganization upon irradiation by NMR spectroscopy. This chemo-enzymatic method opens the possibility to incorporate awide range of modifications at any desired position of RNAs of any lengths beyond the limits of solid-phase synthesis.Scheme 1. Schematic representation of the three-stepe nzymatic procedure for synthesis of RNA labelled with an azobenzene C-nucleoside, aphotocage, or a 13 C, 15 N-enriched nucleoside.

Isotope-Labeled RNA Building Blocks for NMR Structure and Dynamics Studies

“…In an attempt to make position-specifically-labeled RNA more accessible, Schwalbe and co-workers developed a chemo-enzymatic synthetic approach. Therein, a single modified nucleoside-3′,5′-bisphosphate is incorporated to the 3′-end of an RNA fragment followed by DNA-splinted ligation to complete the desired internally labeled RNA sequence ( Figure 7 ) [ 116 ]. This method was used to introduce photocaged, photoswitchable, and isotope-labeled ribonucleosides into RNAs of up to 392 nts.…”

“…Furthermore, this method uses standard laboratory equipment and the commercially available enzymes T4 RNA ligase 1, rSAP, and T4 RNA ligase 2, making it readily accessible to most research groups. However, the relatively low yields of bis-phosphorylation (6–22%) and ligation (9–49%) reactions are a major drawback of this approach [ 116 ]. Improvements in these reaction steps would encourage the widespread adoption of this methodology.…”

Strategic labelling approaches for RNA single-molecule spectroscopy

“…The method is relatively cost-and time-saving; it is however quite sensitive to reaction conditions, so in order to gain acceptable yields optimization steps are necessary. However, as either of the unmodified RNAs can be obtained from transcription or solid phase synthesis, this convergent approach offers a very broad range of modifications in RNAs and additionally renders long RNAs available for sitespecific modifications [35].…”

RNA Structural Differentiation: Opportunities with Pattern Recognition

“…For applications in which site-specific information about a large RNA is critical, ligation of a chemically synthesized fragment is under investigation. [224][225][226] PRRSM could also evolve to inform the recognition of RNA tertiary motifs. A wide range of tertiary structures is formed through secondary structure motif interactions, including pseudoknots, kissing loops, and G-quadruplexes.…”