Interference with MCP-1 gene expression by vector generated triple helix-forming RNA oligonucleotides

Abstract

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.

Monitoring DNA triplex formation using multicolor fluorescence and application to insulin‐like growth factor I promoter downregulation

“…Our fluorescent gel-shift assay could be used to confirm binding of known triplex-binding proteins (for review see [8]), but also allow the identification of new proteins that recognized particular triplex structures, e.g. purine-or pyrimidine-rich RNA third-strand bound to DNA duplex [10][11][12]. Isolation of these proteins using our method would be facilitated through the use of covalent triple helical structure.…”

“…TFOs were initially designed to interfere with the binding of transcription factors or RNA polymerase to targeted gene promoters and thereby to inhibit transcription [9]. TFOs can be transfected or produced by cells to inhibit gene expression [10][11][12]. Blocking of translation through triplex formation on the RNA strand was recently proposed [5].…”

Effects of protein kinase Cδ and phospholipase C-γ1 on monocyte chemoattractant protein-1 expression in taxol-induced breast cancer cell death

“…Chemokines are broadly classified into four distinct groups, CXC(·), CC(ß), C(Á), and CX 3 C(‰), based on the number and location of N-terminal cysteine residues (2,3). A wide variety of cells, including leukocytes, muscle cells, endothelial cells, fibroblasts, and cancer cells produce chemokines in response to various stimuli, such as tumor necrosis factor-· (TNF-·), lipopolysaccharide (LPS), interferon-Á (IFN-Á), and phorbol 12-myristate 13-acetate (PMA) (4)(5)(6).…”