NDLI: Real time detection of DNA⋅RNA hybridization inliving cells

Content Provider	PubMed Central
Author	Sokol, Deborah L. Zhang, Xiaolin Lu, Ponzy Gewirtz, Alan M.
Copyright Year	1998
Abstract	Demonstrating hybridization between an antisenseoligodeoxynucleotide and its mRNA target has proven to be extremelydifficult in living cells. To address this fundamental problem inantisense research, we synthesized “molecular beacon” (MB)reporter oligodeoxynucleotides with matched fluorescent donor andacceptor chromophores on their 5′ and 3′ ends. In the absence of acomplementary nucleic acid strand, the MB remains in a stem–loopconformation where fluorescence resonance energy transfer preventssignal emission. On hybridization with a complementary sequence, thestem–loop structure opens increasing the physical distance between thedonor and acceptor moieties thereby reducing fluorescence resonanceenergy transfer and allowing a detectable signal to be emitted when thebeacon is excited by light of the appropriate wavelength. Solutionhybridization studies revealed that in the presence of a complementarystrand targeted MB could yield up to a 60-fold increase in fluorescenceintensity in comparison to control MB. By using a fluorescencemicroscope fitted with UV fluoride lenses, the detection limit ofpreformed MB/target sequence duplexes microinjected into cells wasfound to be ≥1 × 10−1 ag of MB, or ∼10 moleculesof mRNA. On the basis of this exquisite sensitivity, real-timedetection of MB/target mRNA hybridization in living cells wasattempted by microinjecting MB targeted to the vavprotooncogene, or control MB, into K562 human leukemia cells. Within 15min, confocal microscopy revealed fluorescence in cells injected withtargeted, but not control, MB. These studies suggest that real-timevisualization and localization of oligonucleotide/mRNA interactionsis now possible. MB could find utility in studying RNA processing,trafficking, and folding in living cells. We hypothesize that MB mayalso prove useful for finding targetable mRNA sequence underphysiologic conditions.
Starting Page	11538
File Format	PDF
ISSN	10916490
e-ISSN	10916490
Journal	Proceedings of the National Academy of Sciences of the United States of America
Issue Number	20
Volume Number	95
Language	English
Publisher	National Academy of Sciences
Publisher Date	1998-09-29
Access Restriction	Open
Rights Holder	National Academy of Sciences
Subject Keyword	Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Multidisciplinary

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