Abstract
(Abstract) This protocol describes the methodology for the determination of the secondary structure of an RNA fragment in solution using Selective 2´ -Hydroxyl Acylation analyzed by Primer Extension, abbreviation SHAPE. It consists in the very fast chemical modification of flexible and therefore possibly single-stranded nucleotides in a sequence-independent manner using benzoyl cyanide (BzCN), forming 2´ -O-adducts. The modifications in the RNA are then analyzed by primer extension. Reverse transcriptase is blocked by the 2´ -O-adducts formed. The advantage of the method is, first, that not each RNA molecule studied but the primer used in the extension reaction is labelled and, second, that the resulting cDNA analyzed in sequencing gels is much more stable than the modified RNA.
Highlights
We have design a structure cassette that contains flanking sequences that allow evaluate all positions within the RNA of interest
The primer binding site of this cassette efficiently binds to a cDNA primer
Purification was made by phenol: chloroform extraction and alcohol precipitation as described in protocol
Summary
A. Cloning of RNA to be analyzed in SHAPE cassette RNA segment was cloned into SHAPE cassette (Figure 1) (Wang et al, 2010). SHAPE cassette has been checked to ensure that it is not prone to forming stable base pairing interactions with the internal sequence. We have design a structure cassette that contains flanking sequences that allow evaluate all positions within the RNA of interest. The primer binding site of this cassette efficiently binds to a cDNA primer. 2. After transcription, purification was made by phenol: chloroform extraction and alcohol precipitation as described in protocol. Alcohol precipitation was carried out overnight at 20 °C with 3 M sodium acetate (pH 5.2) instead ammonium acetate. 3. RNA recovery by centrifugation, followed by wash with 70% ethanol and dissolve pellet in 20 μl nuclease-free water
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