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 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.
Department of Bioengineering, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America.
Xuemei
X
Zhang
Cell Biology and Physiology, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America.
Allison L
AL
Zajac
Integrated DNA Technologies, Inc., Coralville, Iowa, United States of America.
Lingyan
L
Huang
Integrated DNA Technologies, Inc., Coralville, Iowa, United States of America.
Mark A
MA
Behlke
Department of Bioengineering, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America.
Andrew
A
Tsourkas
D015374:Biosensing Techniques; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D006801:Humans; D008856:Microscopy, Fluorescence; D015345:Oligonucleotide Probes; D034443:RNA Transport; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D020080:Tandem Repeat Sequences; D059008:Time-Lapse Imaging
D015345:Oligonucleotide Probes; D012333:RNA, Messenger