Imaging the directed transport of single engineered RNA transcripts in real-time using ratiometric bimolecular beacons

The relationship between RNA expression and cell function can often be difficult to decipher due to the presence of both temporal and sub-cellular processing of RNA. These intricacies of RNA regulation can often be overlooked when only acquiring global measurements of RNA expression. This has led to...

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Bibliographic Details
Published in:PloS one 2014-01, Vol.9 (1), p.e85813-e85813
Main Authors: Zhang, Xuemei, Zajac, Allison L, Huang, Lingyan, Behlke, Mark A, Tsourkas, Andrew
Format: Article
Language:English
Subjects:
Acids
Beacons
Binding sites
Bioengineering
Biology
Biosensing Techniques
Bright spots
Cell Line, Tumor
Chemical compounds
Cloning, Molecular
Cytoplasm
Drosophila
Engineering
Fluorescence
Fluorophores
Gene expression
Humans
Information processing
Insects
Localization
Microscopy, Fluorescence
Nucleotide sequence
Oligonucleotide Probes
Oligonucleotides
Proteins
Real time
Ribonucleic acid
RNA
RNA processing
RNA Transport
RNA, Messenger - genetics
RNA, Messenger - metabolism
Scaling
Tandem Repeat Sequences
Time-Lapse Imaging
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Summary:The relationship between RNA expression and cell function can often be difficult to decipher due to the presence of both temporal and sub-cellular processing of RNA. These intricacies of RNA regulation can often be overlooked when only acquiring global measurements of RNA expression. This has led to development of several tools that allow for the real-time imaging of individual engineered RNA transcripts in living cells. Here, we describe a new technique that utilizes an oligonucleotide-based probe, ratiometric bimolecular beacon (RBMB), to image RNA transcripts that were engineered to contain 96-tandem repeats of the RBMB target sequence in the 3'-untranslated region. Binding of RBMBs to the target RNA resulted in discrete bright fluorescent spots, representing individual transcripts, that could be imaged in real-time. Since RBMBs are a synthetic probe, the use of photostable, bright, and red-shifted fluorophores led to a high signal-to-background. RNA motion was readily characterized by both mean squared displacement and moment scaling spectrum analyses. These analyses revealed clear examples of directed, Brownian, and subdiffusive movements.
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0085813