Abstract:
:While RNA structures have been extensively characterized in vitro, very few techniques exist to probe RNA structures inside cells. Here, we have exploited mechanisms of post-transcriptional regulation to synthesize fluorescence-based probes that assay RNA structures in vivo. Our probing system involves the co-expression of two constructs: (i) a target RNA and (ii) a reporter containing a probe complementary to a region in the target RNA attached to an RBS-sequestering hairpin and fused to a sequence encoding the green fluorescent protein (GFP). When a region of the target RNA is accessible, the area can interact with its complementary probe, resulting in fluorescence. By using this system, we observed varied patterns of structural accessibility along the length of the Tetrahymena group I intron. We performed in vivo DMS footprinting which, along with previous footprinting studies, helped to explain our probing results. Additionally, this novel approach represents a valuable tool to differentiate between RNA variants and to detect structural changes caused by subtle mutations. Our results capture some differences from traditional footprinting assays that could suggest that probing in vivo via oligonucleotide hybridization facilitates the detection of folding intermediates. Importantly, our data indicate that intracellular oligonucleotide probing can be a powerful complement to existing RNA structural probing methods.
journal_name
Nucleic Acids Resjournal_title
Nucleic acids researchauthors
Sowa SW,Vazquez-Anderson J,Clark CA,De La Peña R,Dunn K,Fung EK,Khoury MJ,Contreras LMdoi
10.1093/nar/gku1191subject
Has Abstractpub_date
2015-01-01 00:00:00pages
e13issue
2eissn
0305-1048issn
1362-4962pii
gku1191journal_volume
43pub_type
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