In Vitro Synthesis and RNA Structure Probing of CUG Triplet Repeat RNA
Aberrant RNA structure plays a central role in the molecular mechanisms guided by repeat RNAs in diseases like myotonic dystrophy (DM), C9orf72-linked amyotrophic lateral sclerosis (ALS) and fragile X tremor/ataxia syndrome (FXTAS). Much knowledge remains to be gained about how these repeat-expanded transcripts are actually folded, especially regarding the properties specific to very long and interrupted repeats. RNA structure can be interrogated by chemical as well as enzymatic probes. These probes usually bind or cleave single-stranded nucleotides, which can subsequently be detected using reverse transcriptase primer extension. In this chapter, we describe methodology for in vitro synthesis and structure probing of expanded CUG repeat RNAs associated with DM type 1 and approaches for the associated data analysis.
KeywordsCUG repeat DMPK In vitro transcription Myotonic dystrophy RNA folding RNA structure Hairpin SHAPE RNase T1 DMS
We wish to thank Prof. Bé Wieringa for his contribution to supervision of this study as part of R.T.P. van Cruchten’s Ph.D. project and for critical reading of the manuscript.