Elutrap system

Expression cassettes consisting of the promoter and 5′ untranslated region (UTR) of the polyubiquitin (Ubi) gene from Z. mays (Toki et al. 1992 (link)), followed by the entire coding sequence of PdCCR1-1 with a frame-shift created by deleting the seventh base-pair downstream of the start codon and the 3′ UTR comprising transcriptional terminator and polyadenylation site from the fructosyltransferase 4 (FT4) gene from L. perenne were synthesised (GeneArt, Life Technologies, Regensberg, Germany). A plant selectable marker cassette consisting of the promoter and 5′ UTR from the actin (act1) gene from O. sativa (McElroy et al. 1990 (link)) followed by the neomycin phosphotransferase (npt2) gene was also synthesised. This cassette was terminated with the 3′ UTR, comprising transcriptional terminator and polyadenylation site, from the octopine synthase (oct) gene from Agrobacterium tumefaciens. Cassettes were liberated from the vector backbones by restriction digestion and separated from the plasmid DNA fragment using the Elutrap system (Whatman, Maidstone, UK) according to the manufacturer’s instructions.

RNA was produced by in vitro transcription (IVT). DNA template concentrations were varied from 1–2 µg/mL and T7 polymerase varied from 0.1–0.2 mg/mL. Individual rNTP concentrations were kept constant at 5 mM. Full-scale transcription reactions (200 µL) were carried out in T7 buffer (40 mM Tris, pH 8.0, 25 mM MgCl₂, 0.01% Triton X-100, 0.2 mM Spermidine, and 10 mM DTT) and incubated at 37°C for 4 h, then DNA template was digested by Turbo DNaseI (Invitrogen), and the reactions were quenched with EDTA. RNA DNA templates for IVT were generated via ligation of two complementary DNA oligos encoding first the T7 polymerase promoter sequence (5′-GAAATTAATACGACTCACTATA) and then the sequence of the desired RNA. Ligation was achieved by mixing equimolar amounts of each oligo in Oligo Binding Buffer (10 mM Tris, pH 8.0, 50 mM NaCl, 1 mM EDTA), which was then heated at 95°C for 5 min on a heat block and left on the heat block to cool slowly to room temperature. Products of IVT reactions were purified on 8% polyacrylamide gels with 7.5 M urea. Bands containing RNA were visualized by UV-shadowing (254 nm) and excised. RNA was then recovered from the gel slices by overnight electroelution at 100 V in TBE buffer using an EluTrap System (Whatman).

Uniformly ¹³C- and ¹⁵N-labeled fluoride riboswitch aptamer samples were prepared as described previously [54 (link)]. Briefly, samples were transcribed in vitro using T7 polymerase based on DNA purchased from Integrated DNA Technologies (IDT), Inc., (Coralville, IA, USA). The transcribed samples were ethanol precipitated and gel-purified under denaturing conditions, eluted using the Elutrap system (Whatman), and subsequently purified using a Hi-Trap Q column (GE Healthcare). The purified samples were exchanged to 10 mM sodium phosphate (pH 6.4) (containing 50 mM KCl, 2 mM MgCl₂, 10 mM NaF, and 50 μM EDTA) and concentrated to ~0.5–1.0 mM using Amicon filtration systems (Millipore). For preparing samples for residual dipolar coupling (RDC) measurements, the lyophilized powder of ¹³C- and ¹⁵N-isotope-labeled RNA was first dissolved in 100 µL of a 10 mM potassium phosphate buffer (pH 7.4, containing 50 mM NaCl) and followed by the addition of 180 µL of polymer-nanodiscs solution (100 mg/mL of lipids). A total of 10% of ²H₂O was included in all NMR samples.