A fast, efficient and sequence-independent method for flexible multiple segmental isotope labeling of RNA using ribozyme and RNase H cleavage

Abstract

Structural information on RNA, emerging more and more as a major regulator in gene expression, dramatically lags behind compared with information on proteins. Although NMR spectroscopy has proven to be an excellent tool to solve RNA structures, it is hampered by the severe spectral resonances overlap found in RNA, limiting its use for large RNA molecules. Segmental isotope labeling of RNA or ligation of a chemically synthesized RNA containing modified nucleotides with an unmodified RNA fragment have proven to have high potential in overcoming current limitations in obtaining structural information on RNA. However, low yields, cumbersome preparations and sequence requirements have limited its broader application in structural biology. Here we present a fast and efficient approach to generate multiple segmentally labeled RNAs with virtually no sequence requirements with very high yields (up to 10-fold higher than previously reported). We expect this approach to open new avenues in structural biology of RNA.

Figure 1.

Principle of the method comprising three reaction steps. The isotope labeled material is highlighted in red, the unlabeled material in black. In the 2′-O-methyl RNA/DNA chimera, the DNA is in dark blue and the 2′-O-methyl RNA in light blue. The termini of both acceptor and donor fragments are encircled in green. Scissors indicate RNase H cleavage sites. P-2′/3′ stands for a 2′/3′-cyclic phosphate.

Figure 2.

Co-transcriptional ribozyme cleavage (Step 1) and its purification. (a) Denaturing anion-exchange HPLC profile of a 10 ml transcription mix (which corresponds to 200 nmol of the 72-nt RsmZ RNA product after purification) (top) and analytical 16% denaturing PAGE gel of the corresponding elution fractions (bottom). The different fragments obtained by co-transcriptional ribozyme cleavage are shown on the top of their corresponding peak (blue: target RsmZ RNA, red: hammerhead ribozyme, green: 24 nt VS stem-loop sequence required for VS ribozyme cleavage in trans, orange: VS ribozyme). The retention time of the HPLC profile is indicated on the x-axis. The purification conditions used are presented in the Materials and methods section. (b) ESI–MS spectrum of the homogenous unlabeled 72-nt RsmZ RNA with correct 5′- and 3′-termini. The measured mass is 23 414.8 daltons, whereas the calculated mass of the RsmZ RNA, which has a 5′-OH and 2′/3′-cyclic phosphate at its termini, is 23 415 daltons.

Figure 3.

RNase H cleavage (Step 2) and direct non-splinted cross-religation using T4 RNA ligase (Step 3). (a) Denaturing anion-exchange HPLC profile of fragments obtained by site-specific RNase H cleavage in SL2 between A29 and C30 of the RsmZ RNA (60 nmol reaction). The RNase H cleavage was performed with a chimera/RNA ratio of 0.75:1. The different fragments obtained by RNase H cleavage are shown on the top of their corresponding peak. Side-products occurring because of ‘unspecific’ cleavage in SL4 are marked by asterisks (see Supplementary Figure S3). The retention time of the HPLC profile is indicated on the y-axis. The purification conditions used are presented in the methods section. (b) Scheme of RNase H cleavage reaction and corresponding reaction yields. The yield of the cleavage reaction before HPLC purification is indicated, the values in brackets are expressing the yield after purification. The site of cleavage is shown by scissors. (c) Analytical 16% denaturing PAGE gel of the ligation reaction. Left lane: 400 pmol of each 5′-RNA (29 nt) and 3′-RNA (43 nt) before ligation, right lane: after ligation. (d) Reaction scheme and corresponding reaction yields for T4 RNA ligase mediated non-splinted cross-ligation of both a labeled (in red) and an unlabeled (in black) fragment, respectively. The ligation yield determined with a reaction using only unlabeled fragments is indicated.

Figure 4.

Principle, reaction efficiencies and NMR evidence for isotope labeling of each stem-loop of the RsmZ RNA separately. (a) Sequence-specific RNase H cleavages to obtain all four isotopically labeled stem-loop fragments. The yields of the cleavage reactions before HPLC purification are indicated, the values in brackets are expressing the yield after purification. The different stem-loops are colored (SL1: magenta, SL2: green, SL3: orange, SL4: cyan). (b) Splinted T4 DNA ligase mediated ligations of isotope labeled (in color) and unlabeled (in black) fragments. The unlabeled fragments were obtained in a similar way as the labeled fragments. (c) NMR evidence for the successful segmental isotope labeling of each stem-loop separately. ¹H-¹⁵N-HSQC NMR spectrum of the uniformly ¹⁵N-labeled RsmZ RNA (left) and overlay of the ¹H-¹⁵N-HSQC NMR spectra of the four segmentally labeled RsmZ RNAs with each stem-loop labeled separately (right). The spectra were recorded on a Bruker 600 MHz spectrometer at 10°C.