Electroporation and microinjection successfully deliver single-stranded and duplex DNA into live cells as detected by FRET measurements

Förster resonance energy transfer (FRET) technology relies on the close proximity of two compatible fluorophores for energy transfer. Tagged (Cy3 and Cy5) complementary DNA strands forming a stable duplex and a doubly-tagged single strand were shown to demonstrate FRET outside of a cellular environm...

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Bibliographic Details
Published in:PloS one 2014-04, Vol.9 (4), p.e95097-e95097
Main Authors: Bamford, Rosemary A, Zhao, Zheng-yun, Hotchin, Neil A, Styles, Iain B, Nash, Gerard B, Tucker, James H R, Bicknell, Roy
Format: Article
Language:English
Subjects:
Acidification
Aerospace technology transfer
Animals
Biology and life sciences
Carbocyanines - metabolism
Cell Survival
Chemical compounds
CHO Cells
Complementary DNA
Cricetinae
Cricetulus
Deoxyribonucleic acid
DNA
DNA probes
DNA Probes - metabolism
DNA, Single-Stranded - metabolism
Efficiency
Electroporation
Endocytosis
Energy transfer
Fluorescence
Fluorescence resonance energy transfer
Fluorescence Resonance Energy Transfer - methods
Fluorophores
Genetic research
Hybridization
Methods
Microinjection
Microinjections
Microscopy, Confocal
Nucleic acids
Physical Sciences
Proteins
Reagents
Real time
Single-stranded DNA
Strands
Technology transfer
Transfection
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Summary:Förster resonance energy transfer (FRET) technology relies on the close proximity of two compatible fluorophores for energy transfer. Tagged (Cy3 and Cy5) complementary DNA strands forming a stable duplex and a doubly-tagged single strand were shown to demonstrate FRET outside of a cellular environment. FRET was also observed after transfecting these DNA strands into fixed and live cells using methods such as microinjection and electroporation, but not when using lipid based transfection reagents, unless in the presence of the endosomal acidification inhibitor bafilomycin. Avoiding the endocytosis pathway is essential for efficient delivery of intact DNA probes into cells.
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0095097