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Isabel Chillón1, Marco Marcia1,4, Michal Legiewicz1, Fei Liu1, Srinivas Somarowthu1, Anna Marie Pyle1,2,3

Introduction

The purification and analysis of long noncoding RNAs (lncRNAs) in vitro is a challenge, particularly if one wants to preserve elements of functional structure. Here, we describe a method for purifying lncRNAs that preserves the cotranscriptionally derived structure. The protocol avoids the misfolding that can occur during denaturation–renaturation protocols, thus facilitating the folding of long RNAs to a native-like state. This method is simple and does not require addition of tags to the RNA or the use of affinity columns. LncRNAs purified using this type of native purification protocol are amenable to biochemical and biophysical analysis. Here, we describe how to study lncRNA global compaction in the presence of divalent ions at equilibrium using sedimentation velocity analytical ultracentrifugation and analytical size-exclusion chromatography as well as how to use these uniform RNA species to determine robust lncRNA secondary structure maps by chemical probing techniques like selective 2′-hydroxyl acylation analyzed by primer extension and dimethyl sulfate probing.

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Isabel Chillón1, Marco Marcia1,4, Michal Legiewicz1, Fei Liu1, Srinivas Somarowthu1, Anna Marie Pyle1,2,3

1 Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut, USA
2 Howard Hughes Medical Institute, Chevy Chase, Maryland, USA
3 Department of Chemistry, Yale University, New Haven, Connecticut, USA
4 European Molecular Biology Laboratory, Grenoble Outstation, France

Corresponding author: Marco Marcia
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Isabel Chillón, Marco Marcia<sup>1,4</sup>, Michal Legiewicz, Fei Liu, Srinivas Somarowthu, Anna Marie Pyle<sup>1,2,3</sup>
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