Interference with MCP-1 gene expression by vector generated triple helix-forming RNA oligonucleotides.
Triple helix-forming oligonucleotides (TFOs) that specifically bind to double-stranded DNA sequences can be rationally designed, while intracellular delivery of single stranded RNA TFOs has not yet been studied in detail. In this report, we demonstrate gene and sequence-specific inhibition of MCP-1 gene expression due to interference of intracellular-generated single-stranded RNA (CU-TFO) with an overlapping SP-1/AP-1 target. Binding of synthetic 19-nucleotide (19-nt) CU-TFO to the MCP-1 promoter duplex was verified by triplex blotting. Furthermore, we confirmed binding of a 1.1-kb fusion transcript containing the 19-nt pyrimidine CU sequence to a plasmid-encoded MCP-1 promoter target duplex at pH 7.0. In tumour necrosis factor-alpha-stimulated HEK cells, CU-TFOs inhibited MCP-1 protein release by 76 +/- 10.2% compared to intracellular-generated control oligonucleotides. Interleukin-8 as a control target gene was not affected by CU-TFO, confirming both highly specific and effective chemokine gene repression by transfectable TFO-shuttle vectors.
Kautz
K
K
Pharmazentrum Frankfurt, Dr.-Hans-Schleussner-Foundation, Immune Pharmacology, Clinic of the Goethe University, 60590 Frankfurt, Germany.
Schwarz
M
M
Radeke
H H
HH
D001483:Base Sequence; D002460:Cell Line; D018932:Chemokine CCL2 / Q000235:genetics*; D005786:Gene Expression Regulation; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009693:Nucleic Acid Hybridization; D009841:Oligonucleotides / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D012313:RNA / Q000235:genetics* / Q000378:metabolism; D034622:RNA Interference / Q000502:physiology*
C111835:CCL2 protein, human; D018932:Chemokine CCL2; D009841:Oligonucleotides; D012313:RNA
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