Cellular uptake and localization of a Cy3-labeled siRNA specific for the serine/threonine kinase Pim-1.
A highly efficient and specific small interfering (siRNA) (PsiR4) for the serine/threonine kinase Pim-1 has been generated that silences the expression of a Pim1-green fluorescent protein (GFP) fusion gene at low nanomolar concentrations (approximately 5 nM). Only one of four siRNAs tested against Pim-1 had high potency, whereas the three other siRNAs were completely inefficient up to a concentration of 100 nM. PsiR4 was labeled with Cy3 at the 5' -end of the sense strand to investigate cellular uptake and localization in living COS-7 and F-11 cells. This modification has only minor effects on the potency of PsiR4 to inhibit Pim1-GFP. Cellular uptake of the Cy3-labeled siRNA by lipofection was observed in more than 90% of the cells and reaches a plateau 4-6 hours after transfection. Cotransfection studies with low PsiR4-Cy3 concentrations demonstrated that most cells that still expressed Pim1-GFP did not show siRNA uptake. Localization studies with PsiR4-Cy3 in the neuronal hybridoma cell line F-11 displayed a dotted, perinuclear accumulation of siRNAs. Moreover, cells with neuritelike structures contain PsiR4 in this cellular compartment.
Freie Universität Berlin, Institut für Chemie-Biochemie, D-14195 Berlin, Germany.
Arnold
A
Grünweller
Clemens
C
Gillen
Volker A
VA
Erdmann
Jens
J
Kurreck
D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002232:Carbocyanines; D002522:Chlorocebus aethiops; D005456:Fluorescent Dyes; D020868:Gene Silencing; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000235:genetics* / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000235:genetics* / Q000378:metabolism; D051573:Proto-Oncogene Proteins c-pim-1; D034741:RNA, Small Interfering / Q000235:genetics / Q000493:pharmacokinetics*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014162:Transfection
D002232:Carbocyanines; D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D011518:Proto-Oncogene Proteins; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; C089187:cyanine dye 3; D049452:Green Fluorescent Proteins; D017346:Protein Serine-Threonine Kinases; D051573:Proto-Oncogene Proteins c-pim-1