FLEth RNA intercalating probe is a convenient reporter for small interfering RNAs.
Here we report that the phenanthridine derivative covalently linked to a fluorescein moiety (FLEth) can act as a fluorescence based probe for duplex short interfering RNA (siRNA) and that this probe can also be used to report on protein-RNA interactions. A fluorescence resonance energy transfer (FRET) signal that is observed at 600 nm occurs when FLEth is complexed with siRNA. At least 2 molecules of FLEth can bind to 21 nt duplex siRNA, and the dissociation constants for these interactions are reported. We find that FLEth can also report on the interaction of siRNAs with the Carnation Italian ringspot viral suppressor of RNA silencing p19. FLEth does not bind to the siRNA-p19 complex nor can p19 bind to the siRNA-FLEth complex; rather FLEth can report on the fraction of siRNA that is unbound. FLEth can also bind siRNA in delivery systems such as liposomes. Once the siRNA reaches the interior of Huh 7.5 cells, FLEth dissociates from the siRNA and is found in the nucleoli suggesting that FLEth cannot bind to siRNAs that are associated with the RNA silencing machinery.
van der Wiel
Ingrid M
IM
Steacie Institute for Molecular Sciences, National Research Council, 100 Sussex Drive, Ottawa, ON, Canada K1A 0R6.
Cheng
Jenny
J
Koukiekolo
Roger
R
Lyn
Rodney K
RK
Stevens
Nathan
N
O'Connor
Naphtali
N
Turro
Nicholas J
NJ
Pezacki
John Paul
JP
D000081247:CSK Tyrosine-Protein Kinase; D002460:Cell Line; D004996:Ethidium / Q000737:chemistry; D005452:Fluoresceins / Q000032:analysis / Q000737:chemistry; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D015335:Molecular Probes / Q000032:analysis / Q000737:chemistry*; D011505:Protein-Tyrosine Kinases / Q000235:genetics; D011518:Proto-Oncogene Proteins / Q000235:genetics; D034741:RNA, Small Interfering / Q000032:analysis / Q000737:chemistry* / Q000235:genetics; D013816:Thermodynamics; D019061:src-Family Kinases
D005452:Fluoresceins; D005456:Fluorescent Dyes; D015335:Molecular Probes; D011518:Proto-Oncogene Proteins; D034741:RNA, Small Interfering; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases; C000631346:CSK protein, human; D004996:Ethidium