Yeast trna

NPCs were transfected with biotin-labeled Bio-NC, Bio-miR-431-WT and Bio-miR-431-MUT RNA or Bio-NRF2-WT and Bio-NRF2-MUT RNA (50 nM) for 48 h. After washing with PBS, NPCs were incubated with lysis buffer containing protease inhibitor (Roche) and ribonuclease inhibitor (Fermentas, St. Leon-Rot, Germany) (20 mM Tris, pH 7.5, 200 mM NaCl, 2.5 mM MgCl₂, 0.05% IGEPAL CA-630, and 1 mM dithiothreitol) on ice for 10 min. The lysate was cultured with magnetic beads coated by streptavidin (M-280, Invitrogen) at 4°C overnight. The RNase free BSA (Sigma-Aldrich) and yeast tRNA (Sigma-Aldrich) were used to precoat magnetic beads. Expression of circ_0072464 and miR-431 was detected by RT-qPCR.

Biotin-labeled miR-98-5p-WT and miR-98-5p-Mut were synthesized by GeneCreate (Wuhan, China) and were transfected into GC cells which were incubated with lysis buffer (Ambion, Austin, Texas, USA). Then, the GC cell lysates were incubated with the streptavidin Dynabeads (Invitgen, USA) precoated with RNase-free bovine serum albumin (BSA) and yeast tRNA (Sigma-Aldrich, USA) overnight at 4°C. After washed with washing buffer, the bound RNA was purified by using Trizol. Finally, the enrichment of PITPNA-AS1 was identified and estimated by performing qRT-PCR.

Biotin pull-down was performed as previously described (Wang et al., 2014 (link)). Briefly, after 48 h of HSCs transfected with Bio-miR-181b-5p-Wt, Bio-miR-181b-5p-Mut, or Bio-miR-NC, the cells were washed with PBS followed by incubation in a lysis buffer for 10 min. To exclude RNA and protein complexes, the beads were blocked in lysis buffer including RNase-free bovine serum albumin and yeast tRNA (Sigma). After the lysates were incubated with streptavidin-coated magnetic beads (Life Technologies) at 4°C for 4 h, they were washed twice with lysis buffer, three times with the low salt buffer, and once with the high salt buffer. The bound RNAs were isolated using TRIzol reagent (Life Technologies). cMTO1 expression was determined by qRT-PCR.

Whole-cell extracts of immortalized PBMCs were prepared by lysis with M-PER Mammalian Protein Extraction Reagent (Thermo Scientific) in the presence of Halt Protease Inhibitor (Thermo Scientific). Crude lysates were centrifuged at 14,000 × g for 10 min at 4°C. The supernatant containing total cell extracts was transferred to clean eppendorf tubes. Protein concentrations were measured using the Quick Start Bradford Protein Assay (Bio-Rad). An equal amount of protein (7.5 μg per reaction) from each sample was added to reaction mix containing 12.5 mg/mL yeast tRNA (Sigma-Aldrich), 300 μM L-Phe or L-Gly, 1.6 μM [³H]-Phe (American Radiolabeled Chemicals) or 6 μM [³H]-Gly (Perkin Elmer), 4 mM ATP, 10 mM MgCl₂, 2 mM DTT, 20 mM KCl, and 50 mM HEPES (pH 7.5). The reaction was stopped by addition of quench solution consisting of 0.5 mg/mL DNA, 100 mM EDTA, and 300 mM NaOAc (pH 3.0). [³H]-Phe- or [³H]-Gly-labeled tRNA was precipitated by addition of 20% TCA and captured using Multiscreen HTS filter plates (EMD Millipore). Filters were washed with a 5% TCA, 100 mM Phe solution and captured tRNA was solubilized with 0.1 M NaOH. Amount of [³H] was quantified by addition of Optiphase Supermix scintillation cocktail (Perkin Elmer) and counting by a MicroBeta scintillation counter (Perkin Elmer).

Intracellular RNA from transfected cells was extracted using the QIAGEN RNeasy kit as described by the manufacturer. For extracellular RNA extraction, 2 mL of purified vector supernatant was centrifuged at 22,000 × g for 3 h at 4°C in 8.4% Opti-Prep (STEMCELL Technologies). Pellets were resuspended in phosphate-buffered saline (PBS) (Thermo Fisher Scientific) and incubated in 200 μL of proteinase K extraction buffer (50 mM Tris-HCl [pH 7.5], 100 mM NaCl, 10 mM EDTA, 1% SDS, 100 μg/mL proteinase K [Ambion], and 100 μg/mL yeast tRNA [Sigma-Aldrich]) at 37°C for 30 min. vRNA was extracted with phenol-chloroform and chloroform:isoamyl alcohol, precipitated with 1/10^th volume of 3 M sodium acetate (pH 5.5) and 2.5 volumes of ethanol, washed with ice-cold 70% ethanol, and air-dried.

Chemically synthesized DNA strands were purchased from Integrated DNA Technologies. Each strand was purified by anion-exchange chromatography. To prepare DNA duplexes, complementary strands were annealed, and excess single-stranded DNA was removed through anion-exchange chromatography. Concentrations of double-stranded DNA were measured using UV absorbance at 260 nm along with extinction coefficients calculated from the nucleotide sequences using the method of Tataurov et al (45 (link)).The fluorescence-labeled 20-bp DNA with a cisplatin modification were prepared as previously described (13 (link)). Yeast tRNA was purchased from Sigma-Aldrich (cat# 10109517001). To measure tRNA concentrations, UV absorbance at 260 nm was used along with an extinction coefficient of 7 × 10⁵ M⁻¹cm⁻¹, which was estimated from the average length and hypochromicity (46 (link),47 (link)).

SAXS data were collected in batch mode on the EMBL/DESY synchrotron beamline P12 [52 (link)], using standard procedures [53 (link)]. In addition to LigT alone, yeast tRNA (Sigma) and a 1:1 molar mixture of LigT and tRNA were analyzed. Data were processed with the ATSAS package [54 (link)]. Distance distribution functions were analyzed using GNOM [55 ]. For modelling LigT alone, GASBOR [56 (link)] was used. For modelling the protein-RNA complex, we used the program MONSA [57 (link)] with the protein and RNA in different phases. The tRNA alone was modelled using DAMMIN [57 (link)]. Molecular weight was estimated through a comparison of the forward scattering intensity of the sample to that of a fresh sample of monomeric bovine serum albumin.