Computational protocol: Targeted inhibition of oncogenic miR-21 maturation with designed RNA-binding proteins

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Protocol publication

[…] We performed SHAPE chemical probing on pre-miRNA-21 in both free and bound to engineered Fox-RRM*. The resulting chemical modifications were analyzed by primer extension with a radiolabeled DNA oligonucleotides and gel electrophoresis. Pre-miR-21 was transcribed with a 3′-end extension in order to accommodate primer binding. RNAs were denatured by heating at 95 °C for 3 mins, and snap-cooled on ice to refold prior to SHAPE analysis.Free precursor miRNAs or protein-RNA complexes were diluted to 100 nM with final buffer content of 100 mM HEPES (pH 8.0) and 100 mM NaCl. Three 9 μL aliquots of the RNA solution were distributed to individual Eppendorf tubes and combined with 1 μL of either DMSO (control), or 65 mM NMIA (dissolved in DMSO), or 130 mM NMIA. Reactions were incubated at 37°C for 40 minutes. Following modification, the RNA solution was diluted to 100 μL, adjusted to 0.2 M NaCl, 20 μg glycogen, and 2 mM EDTA and finally precipitated with 3.5× ice-cold absolute ethanol. Precipitation was carried out on dry ice for 15 minutes and finally pelleted at 4°C by centrifugation. The RNA pellet was allowed to dry in air before re-suspension in 10 μL of water.The DNA primer was 5′-end labeled with γ-[32P]-ATP using T4 polynucleotide kinase (PNK), then purified on a G50 resin spin column. NMIA-modified RNA (~ 0.5 pmol) was combined with 1.5 pmol 32P-labeled DNA primer and annealed by heating at 95°C for 5 minutes followed by 5 minutes on ice. Reverse transcription was performed using Superscript III (Invitrogen), followed by incubation at 52°C for 10 minutes. Each sequencing reaction was similarly prepared using 1 pmol unmodified RNA. The reaction was stopped by degrading the RNA with 200 mM NaOH at 95°C for 5 minutes. The cDNA mixture was then neutralized at 95°C for 5 minutes. Each sequencing and modification reaction was loaded onto a pre-warmed 8% denaturing polyacrylamide gel and separated by electrophoresis. The resulting gel was transferred to Whatman paper and fixed with a solution of 40% Methanol and 10% Acetic Acid. The fixed gel was dried and exposed overnight onto a storage phosphor screen.The autoradiograph was imaged with a Typhoon laser scanner and manipulated with Semi-Automated Footprinting Analysis (SAFA) software. Secondary structure analysis was conducted with MC-Fold. […]

Pipeline specifications

Software tools SAFA, MC-Fold
Application RNA structure analysis