Native Purification and Analysis of Long RNAs
| Autor: | Fei Liu, Isabel Chillón, Srinivas Somarowthu, Michal Legiewicz, Marco Marcia, Anna Marie Pyle |
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| Rok vydání: | 2015 |
| Předmět: |
RNA Folding
Transcription Genetic Cations Divalent Acylation Genetic Vectors Size-exclusion chromatography In Vitro Techniques Sulfuric Acid Esters Article Primer extension Analytical Ultracentrifugation Humans Magnesium Protein secondary structure Chromatography High Pressure Liquid Hydroxyl Radical Sequence Analysis RNA Chemistry RNA Long non-coding RNA Biochemistry Molecular Probes Chromatography Gel Nucleic Acid Conformation RNA Long Noncoding Ultracentrifuge Molecular probe Ultracentrifugation Plasmids |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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