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Direct structural analysis of modified RNA by fluorescent in-line probing.

Nucleic acids research (2011-09-16)
Benjamin Strauss, Alexander Nierth, Marco Singer, Andres Jäschke
ABSTRACT

Chemical probing is a common method for the structural characterization of RNA. Typically, RNA is radioactively end-labelled, subjected to probing conditions, and the cleavage fragment pattern is analysed by gel electrophoresis. In recent years, many chemical modifications, like fluorophores, were introduced into RNA, but methods are lacking that detect the influence of the modification on the RNA structure with single-nucleotide resolution. Here, we first demonstrate that a 5'-terminal (32)P label can be replaced by a dye label for in-line probing of riboswitch RNAs. Next, we show that small, highly structured FRET-labelled Diels-Alderase ribozymes can be directly probed, using the internal or terminal FRET dyes as reporters. The probing patterns indeed reveal whether or not the attachment of the dyes influences the structure. The existence of two dye labels in typical FRET constructs is found to be beneficial, as 'duplexing' allows observation of the complete RNA on a single gel. Structural information can be derived from the probing gels by deconvolution of the superimposed band patterns. Finally, we use fluorescent in-line probing to experimentally validate the structural consequences of photocaging, unambiguously demonstrating the intentional destruction of selected elements of secondary or tertiary structure.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
GenElute Bacterial Genomic DNA Kits, sufficient for 10 purifications
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Atto 633, BioReagent, suitable for fluorescence
Supelco
Atto 633 NHS ester, BioReagent, suitable for fluorescence, ≥90.0% (coupling to amines)
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Atto 633 amine, suitable for fluorescence
Sigma-Aldrich
Atto 633 iodoacetamide, suitable for fluorescence
Sigma-Aldrich
Atto 633 azide, suitable for fluorescence, ≥80.0% (HPCE)

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