Isotope labeling strategies for NMR studies of RNA

Abstract

The known biological functions of RNA have expanded in recent years and now include gene regulation, maintenance of sub-cellular structure, and catalysis, in addition to propagation of genetic information. As for proteins, RNA function is tightly correlated with structure. Unlike proteins, structural information for larger, biologically functional RNAs is relatively limited. NMR signal degeneracy, relaxation problems, and a paucity of longrange 1 H-1 H dipolar contacts have limited the utility of traditional NMR approaches. Selective isotope labeling, including nucleotide-specific and segmental labeling strategies, may provide the best opportunities for obtaining structural information by NMR. Here we review methods that have been developed for preparing and purifying isotopically labeled RNAs, as well as NMR strategies that have been employed for signal assignment and structure determination.

Structure determination and dynamics of protein–RNA complexes by NMR spectroscopy

“…The problem of 3' and 5' inhomogeneity can be circumvented by incorporation of a ribozyme sequence in cis, which cleaves co-transcriptionally leading to an homogenous 5'-hydroxyl or a 2',3'-cyclic phosphate end [105,109]. Concerning 5'-inhomogeneity, hammerhead ribozymes are interesting because they have no sequence requirements [100].…”

“…As discussed in Section 2.3.1, RNAs obtained by in vitro transcription contain a 5'-termini with a triphosphate and an inhomogenous 3'-hydroxyl termini. Ribozymes engineered at the 3'-end producing homogenous 2',3'-cyclic phosphates can thus be used to generate 3'-ends of both acceptor and donor fragment, whereupon the acceptor 3'-end has to be further dephosphorylated using T4 polynucleotide kinase, which has 3'-phosphatase activity [105,141,142]. Hammerhead ribozymes located 5' to the acceptor fragment generate the correct 5'-hydroxyl end, whereas the 5' donor end generated by a hammerhead ribozyme has to be phosphorylated by T4 polynucleotide kinase (PNK).…”

NMR Spectroscopy in Bioinorganic Chemistry

“…This method can be even applied to larger RNA constructs if partially deuterated samples are used. [35] More details on individual coordinating atoms can also be gained: For example, shifts of the purine N7 resonance upon addition of increasing amounts of metal ions can be followed by recording (Fig. 2B).…”

Metal Ion–RNA Interactions Studied via Multinuclear NMR

“…They all have spin ½ and are straightforward to record and study. Moreover, the now easy and cost-affordable access to 13 C and 15 N isotope labeled RNAs (12) overcomes the difficulties intrinsically related to the low receptivity and abundance of these nuclei. Nevertheless, it is worth noting that most of the biologically relevant metal ions are NMR active, but show low sensitivity and spins larger than ½, resulting in broad signals not always easy to record.…”

“…In the past, such nucleotides had prohibitive prices and different routes for their synthesis were proposed (8,12). In the last few years more competitive prices made it possible to overcome the need of synthesizing them for most applications.…”