title: Real-time imaging of single engineered RNA transcripts in living cells using ratiometric bimolecular beacons.
abstract: The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3'-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.
authors.affiliation: Department of Bioengineering, University of Pennsylvania.
authors.forename: Yang
authors.initials: Y
authors.lastname: Song
authors.affiliation: Department of Bioengineering, University of Pennsylvania.
authors.forename: Xuemei
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Integrated DNA Technologies, Inc.
authors.forename: Lingyan
authors.initials: L
authors.lastname: Huang
authors.affiliation: Integrated DNA Technologies, Inc.
authors.forename: Mark A
authors.initials: MA
authors.lastname: Behlke
authors.affiliation: Department of Bioengineering, University of Pennsylvania; atsourk@seas.upenn.edu.
authors.forename: Andrew
authors.initials: A
authors.lastname: Tsourkas
mesh_terms: D020413:3' Untranslated Regions; D045744:Cell Line, Tumor; D018274:Electroporation / Q000379:methods; D006801:Humans; D008856:Microscopy, Fluorescence / Q000379:methods*; D057054:Molecular Imaging / Q000379:methods*; D015345:Oligonucleotide Probes / Q000737:chemistry* / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics; D020080:Tandem Repeat Sequences
chemical_list: D020413:3' Untranslated Regions; D015345:Oligonucleotide Probes; D012333:RNA, Messenger