Chemical synthesis and NMR spectroscopy of long stable isotope labelled RNA

Abstract

We showcase the high potential of the 2 0 -cyanoethoxymethyl (CEM) methodology to synthesize RNAs with naturally occurring modified residues carrying stable isotope (SI) labels for NMR spectro- Solution and solid state nuclear magnetic resonance (NMR) spectroscopy have proven to be highly suitable to address structural and dynamic features of RNA. 1-4 A prerequisite to apply state-of-the-art NMR experiments is the introduction of a stable isotope (SI) labelling pattern using 13 C/ 15 N labelled RNA or DNA precursors. [5][6][7][8] The most wide-spread method uses labelled (2 0 -deoxy)-ribonucleotide triphosphates and enzymes to produce the desired RNA or DNA sequence enriched with 13 C and 15 N nuclei. 1,5 This approach enables to produce sufficient amounts of RNA and DNA for NMR spectroscopic applications. This well-established method allows nucleotide specific labeling by mixing a SI-labeled with unlabeled d/rNTPs. Especially in larger RNAs (460 nt) such nucleotide specific SI-labeling can still lead to significant resonance overlap. That is why, the PLOR (position-selective labelling of RNA) method was recently introduced, which holds the promise to site-specifically label RNA using SI-labelled ribonucleotide triphosphates and T7 RNA polymerase. 9 An alternative method was concurrently developed making use of the synthesis of 2 0 -O-tri-iso-propylsilyloxymethylphosphoramidites and solid phase synthesis. 10-13The approach works well for medium sized RNAs up to 50 nts and the synthetic access to the SI-labelled building blocks is well established.10,12 Thus, the fully chemical SI-labelling protocol can be regarded as an expedient expansion to the settled enzymatic procedures to freely chose the number and positioning of SI-labeled residues into a target RNA. In our hands, however, the standard solid phase synthesis methods are not that well suited to produce larger amounts (450 nmol) and purities higher than 95% for RNAs exceeding 60 nts. Due to this restriction, large RNAs are only accessible via enzymatic ligation strategies using T4 RNA/DNA ligase making extra optimization steps necessary or introducing new problems, such as finding the optimal ligation site or issues regarding up-scaling and yield of the ligation product. 14-16 Thus, an improved synthetic procedure to directly address SI-labelling of larger RNAs (460 nt) at amounts suitable for NMR would be highly desirable. We report the synthesis of SI-labelled RNAs ranging in size between 60 to 80 nts capitalizing on the 2 0 -cyanoethoxymethyl (CEM) RNA synthesis method. 17,18 As these CEM building blocks are not commercially available all phosphoramidites were produced in-house and we further synthesized 13 C-/ 15 N-labelled unmodified and naturally occurring modified RNA phosphoramidites (Fig. 1a and b). In detail, we focused on the synthesis of 8- We used these monomer units to produce SI-labelled RNAs exceeding the size limitation of 60 nucleotides for NMR up to 20 nucleotides. The RNAs reported here were synthesized on a 1.3 mmol scale and on a 1000...

Translation of non-standard codon nucleotides reveals minimal requirements for codon-anticodon interactions

“…Standard RNA nucleoside building blocks were used in form of labile base- and 2′-cyanoethoxymethyl (CEM)-protected phosphoramidites, synthesized according to published procedures 64 . Modified nucleosides were either self-synthesized (DHU, s 4 U [will be published elsewhere], m 5 U) or purchased as 2′-TBDMS-protected phosphoramidite (Pseudouridine, Glen Research) (Supplementary Fig.…”

Solid-Phase Chemical Synthesis of Stable Isotope-Labeled RNA to Aid Structure and Dynamics Studies by NMR Spectroscopy

“…The CEM group has a high coupling efficiency (~99%), short coupling time (2–4 min), and minimal steric hindrance that allows synthesis of RNAs as large as 170 nts [57,58]. In terms of labeled RNA, sequences up to 76 nts have been successfully prepared for NMR analysis with 2′- O -CEM phosphoramidites [38]. Two other 2′-OH protecting groups that have not yet been applied to RNA labeling are the photolabile 2-nitrobenzyloxymethyl (NBOM) group and the acid-labile bis(acetoxyethoxy)methyl ether (ACE) group [47,53,54].…”

“…The latter technique is complicated by the acid-labile nature of the phosphoramidites, which requires specialized electro-spray ionization techniques in non-aqueous buffers with lithium chloride for accurate mass determination [63,64]. Several groups have developed synthetic strategies to obtain 13 C/ 15 N-labeled RNA phosphoramidites with a variety of 2′-OH protecting groups [38,39,55,56,59,60,61,62,65,66,67,68]. This section will highlight the many efforts employed to synthesize labeled RNA phosphoramidites.…”

“…Furthermore, solid-phase synthesis has no sequence requirements, unlike T7 RNAP and PLOR. Despite these advantages, solid-phase synthesis is currently limited to labeling RNAs less than 76 nts [38]. Fortunately, many biologically relevant RNAs fall within this size limit [6].…”

Self‐Assembly of DNA and RNA Building Blocks Explored by Nitrogen‐14 NMR Crystallography: Structure and Dynamics

“…However, the isotopic 15 N/ 13 C enrichment of nucleosides incorporated into oligonucleotides by solid‐phase synthesis is complex, labor‐intensive and expensive, particularly in longer sequences. Indeed, the introduction of these isotopes into nucleosides requires either modified phosphoramidite building blocks for solid phase synthesis or nucleoside triphosphate analogs for enzymatic DNA or RNA production . Since the preparation of these isotope‐enriched building blocks requires multistep synthetic protocols, the possibility of characterizing nitrogen environments by exploiting the naturally abundant 99.9 % 14 N isotope without recourse to isotopic enrichment is particularly appealing.…”