Surveillance of siRNA integrity by FRET imaging.
Techniques for investigation of exogenous small interfering RNA (siRNA) after penetration of the cell are of substantial interest to the development of efficient transfection methods as well as to potential medical formulations of siRNA. A FRET-based visualization method including the commonplace dye labels fluorescein and tetramethylrhodamin (TMR) on opposing strands of siRNA was found compatible with RNA interference (RNAi). Investigation of spectral properties of three labelled siRNAs with differential FRET efficiencies in the cuvette, including pH dependence and FRET efficiency in lipophilic environments, identified the ratio of red and green fluorescence (R/G-ratio) as a sensitive parameter, which reliably identifies samples containing >90% un-degraded siRNA. Spectral imaging of siRNAs microinjected into cells showed emission spectra indistinguishable from those measured in the cuvette. These were used to establish a calibration curve for assessing the degradation state of siRNA in volume elements inside cells. An algorithm, applied to fluorescence images recorded in standard green and red fluorescence channels, produces R/G-ratio images of high spatial resolution, identifying volume elements in the cell with high populations of intact siRNA with high fidelity. To demonstrate the usefulness of this technique, the movement of intact siRNA molecules are observed after introduction into the cytosol by microinjection, standard transfection and lipofection with liposomes.
Järve
Anne
A
Department of Chemistry, Institute for Pharmacy and Molecular Biotechnology (IPMB), University of Heidelberg, Im Neuenheimer Feld 364, D-69120 Heidelberg, Germany.
Müller
Julius
J
Kim
Il-Han
IH
Rohr
Karl
K
MacLean
Caroline
C
Fricker
Gert
G
Massing
Ulrich
U
Eberle
Florian
F
Dalpke
Alexander
A
Fischer
Roger
R
Trendelenburg
Michael F
MF
Helm
Mark
M
D000465:Algorithms; D000818:Animals; D002460:Cell Line; D019793:Fluorescein / Q000737:chemistry; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry; D008055:Lipids; D008845:Microinjections; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D034622:RNA Interference; D020871:RNA Stability; D034741:RNA, Small Interfering / Q000008:administration & dosage / Q000032:analysis / Q000737:chemistry*; D051381:Rats; D012235:Rhodamines / Q000737:chemistry; D014162:Transfection
D005456:Fluorescent Dyes; D008055:Lipids; D034741:RNA, Small Interfering; D012235:Rhodamines; C484027:oligofectamine; C005358:tetramethylrhodamine; D019793:Fluorescein