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 long-range (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.

Fig. 1

Method used to produce RNA with homogeneous 5′ and 3′ ends. a Hammerhead (HH) cleavage produces homogeneous 5′-end; b HDV/HH ribozyme, RNase P cleavage or 2′-O-Me of the last two nucleotides on the DNA template can produce 3′-homogeneous ends. c RNase H cleavage of the transcribed RNA can produce both homogeneous 5′ and 3′ ends at the cleaving point. For details see Hartmann et al. (2005)

Fig. 2

Reaction scheme for the enzymatic conversion of glucose to the four NTPs used to make RNA. Glucose and all intermediates are shown in boldface type, and enzymes denoted in italics. Reprinted with permission from Scott et al. (2000)

Fig. 3

Relief of spectral crowding by site-specific deuteration. a Secondary structure of a 30 KDa GAAA tetra loop–receptor RNA. The helical regions are shown in black, the tetra loop is shown in red and the receptor is in green. b, c: portions of the 2D NOESY spectra obtained for the fully-protonated b and selectively deuterated c tetra loop receptor RNA. Reprinted, with permission, from Davis et al. (2005)

Fig. 4

Simplification of the 2D NOESY spectrum obtained for nucleotide-specific protonated, ²H-labeled RNA. a Secondary structure of the 101-nt core encapsidation signal of the Moloneymurine leukemia virus. b Portion of the 2D NOESY spectrum of the G-protonated, C,U,A-perdeuterated RNA. Breakthrough peaks from a small amount of A-H8 substitution facilitated assignment of the NMR signals. Reprinted, with permission, from D’Souza et al. (2004)

Fig. 5

H8 enrichment of perdeuterated purines simplifies the NMR spectrum and enables assignment of larger RNAs. a Predicted secondary structure of stem loops C and D of the Moloneymurine leukemia virus 5′-UTR. b 2D NOESY spectrum of the fully protonated SL-CD dimer. c 2D NOESY spectrum of the SL-CD dimer synthesized using fully-protonated GTP, H8-protonated, perdeuterated ATP, perdeuterated CTP and perdeuterated UTP. Black and red lines denote inter-guanosine and adenosine-to guanosine-H1′ connectivities, respectively (Miyazaki and Summers, unpublished)

Fig. 6

Commonly used methods for large-scale RNA ligation. a DNA ligase mediated ligation. b RNA ligase mediated ligation. c Deoxyribozyme mediated RNA ligation. RNA ligase mediated ligation can occur with or without DNA splint templating. c is reproduced from Purtha et al. (2005) with permission

Fig. 7

Scheme for preparation of segmentally labeled RNAs from a single template. (a) Plasmid template, in which the 3′-hammerhead (HH) and 5′-HH were engineered into the donor and acceptor RNAs respectively. (b–d): Representative aromatic regions of ¹H,¹³C-TROSY spectra of BC1 DTE RNA recorded with (b) a uniformly 13C-/15 N-labeled sample, (c) a ¹³C-SI,¹⁵N-SII segmentally labeled sample and (d) a ¹⁵N-SI,¹³C-SII segmentally labeled sample. Reproduced from Tzakos et al. (2007) with permission

Fig. 8

Representative ¹H–¹³C HMQC NMR spectrum obtained for the AUG region of the intact, 712 nt dimeric HIV-1 5′-UTR. a One of several predicted secondary structures of HIV-1_NL4-3 5′-UTR; DIS, dimer initiation site; SD, major splice donor site; AUG, (green, bold), gag start codon. ¹³C-labeled residues are shown in green. b ¹H–¹³C HMQC spectrum obtained for the dimeric HIV-1 5′-UTR at low ionic strength (10 mM Tris–HCl, pH 7.0) (Lu and Summers, unpublished)