3.1. Chemical Solid-Phase RNA Synthesis

Originally developed for DNA synthesis by Beaucage and Caruthers [76], the amidite method has since been adapted to RNA [32,33,34,35,36]. This approach has been further adapted to incorporate isotope-labeled amidites [36]. SPS is carried out in an automated synthesizer, requires amidite building blocks, and occurs in four steps (Figure 4). First, the 4,4′-dimethoxytrityl (DMTr) protecting group at the 5′-hydroxyl (OH) of the solid-support bound 3′-nucleoside is removed. Second, the deprotected 5′-OH then attacks the activated amidite to couple the two nucleosides. Synthesis continues to grow the RNA molecule 3′-to-5′ by repeating the first two steps following oxidation of the phosphite-triester to the phosphotriester and subsequent deprotection. Cleavage from the solid-support terminates the cycle.

Chemical solid-phase synthesis of position-specifically-labeled RNA. Amidite building blocks (unlabeled and isotope-labeled) are used in an automated synthesizer to make pre-programmed RNA sequences with position-specific labels. Magenta nucleotides harbor stable isotope labels. Magenta circles and D represent ¹³C and ²H atoms, respectively. This example shows a [6-¹³C-5-²H]-UTP-labeled RNA at nucleotide U7.

SPS efficiency depends on the protecting group (PG) choice. RNA amidites are 5′-O-DMTr-protected, and the nucleobase exocyclic amino groups are protected with acetyl (Ac), phenoxyacetyl (Pac), benzoyl (Bz), or isobutyryl (iBu) groups. The choice of the various 2′-OH PGs requires careful deliberation. These PGs can be classified as acid- [77], photo- [78], and fluoride-labile [33,79,80]. While RNAs have been synthesized with a variety of 2′-OH PGs, only [(triisopropylsilyl)oxy]methyl (TOM) [79], tert-butyldimethylsilyl (tBDMS) [33], and 2′-cyanoethoxymethyl (CEM) [80] amidites have been widely used in NMR studies. Even though the CEM is the only PG that enables synthesis of RNAs >60 nts, it is commercially unavailable, which requires researchers to synthesize unlabeled and labeled CEM amidites in-house [69]. On the other hand, unlabeled TOM and tBDMS amidites and isotope-labeled tBDMS amidites are commercially available (Table 1) and therefore more accessible to a larger group of researchers. In practice, chemical SPS is rarely employed to make RNAs >60 nt for NMR analysis, and therefore TOM and tBDMS amidites are mainly used. For example, the Kreutz research group synthesized [6-¹³C]- and [6-¹³C-5-²H-]-uridine and -cytidine, and [2,8-¹³C₂]- and [8-¹³C]-adenosine and -guanosine TOM and tBDMS amidites for use in ¹H- [70] and ¹³C-relaxation dispersion [17,71] NMR experiments to study conformational dynamics of RNAs up to 53 nts.