Targeted inhibition of transcription elongation in cells mediated by triplex-forming oligonucleotides.

Triple-helix-forming oligonucleotides (TFOs) bind in the major groove of double-stranded DNA at oligopyrimidine small middle dotoligopurine sequences and therefore are candidate molecules for artificial gene regulation, in vitro and in vivo. We recently have described oligonucleotide analogues containing N3'-P5' phosphoramidate (np) linkages that exhibited efficient inhibition of transcription elongation in vitro. In the present work we provide conclusive evidence that np-modified TFOs targeted to the HIV-1 polypurine tract (PPT) sequence can inhibit transcriptional elongation in cells, either in transient or stable expression systems. The same constructs were used in transient expression assays (target sequence on transfected plasmid) and in the generation of stable cell lines (target sequence integrated into cellular chromosomes). In both cases the only distinguishable feature between the cellular systems is the presence of an insert containing the wild-type PPT/HIV-1 sequence, a mutated version with two mismatches, or the absence of the insert altogether. The inhibitory action induced by np-TFOs was restricted to the cellular systems containing the complementary wild-type PPT/HIV-1 target, and consequently can be attributed only to a triple-helix-mediated mechanism. As a part of this study we also have applied an imaging technique to quantitatively investigate the dynamics of TFO-mediated specific gene silencing in single cells.

[1]  K. Fearon,et al.  Comparison of binding of N3'-->P5' phosphoramidate and phosphorothioate oligonucleotides to cell surface proteins of cultured cells. , 1999, Antisense & nucleic acid drug development.

[2]  D. Lloyd,et al.  Stable triple helices formed by oligonucleotide N3'-->P5' phosphoramidates inhibit transcription elongation. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[3]  C. Giovannangeli,et al.  Progress in developments of triplex-based strategies. , 1997, Antisense & nucleic acid drug development.

[4]  P. Charneau,et al.  A second origin of DNA plus-strand synthesis is required for optimal human immunodeficiency virus replication , 1992, Journal of virology.

[5]  S. Gryaznov,et al.  Oligodeoxyribonucleotide N3'.fwdarw.P5' Phosphoramidates: synthesis and Hybridization Properties , 1994 .

[6]  P. Glazer,et al.  Targeted gene knockout mediated by triple helix forming oligonucleotides , 1998, Nature Genetics.

[7]  Julian R. E. Davis,et al.  Dynamic changes in prolactin promoter activation in individual living lactotrophic cells. , 1998, Endocrinology.

[8]  C. Giovannangeli,et al.  Efficient inhibition of transcription elongation in vitro by oligonucleotide phosphoramidates targeted to proviral HIV DNA. , 1996, Journal of molecular biology.

[9]  D. Spiller,et al.  The uptake kinetics of chimeric oligodeoxynucleotide analogues in human leukaemia MOLT-4 cells. , 1992, Anti-cancer drug design.

[10]  Z. Wang,et al.  DNA damage-dependent transcriptional arrest and termination of RNA polymerase II elongation complexes in DNA template containing HIV-1 promoter. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[11]  S. Mccurdy,et al.  N3'-->P5' Oligodeoxyribonucleotide Phosphoramidates: A New Method of Synthesis Based on a Phosphoramidite Amine-Exchange Reaction. , 1997, The Journal of organic chemistry.

[12]  H. Gamper,et al.  Triplex targeting of a native gene in permeabilized intact cells: covalent modification of the gene for the chemokine receptor CCR5. , 1998, Nucleic acids research.

[13]  D. Scherman,et al.  Efficient purification of plasmid DNA for gene transfer using triple-helix affinity chromatography , 1997, Gene Therapy.

[14]  D. Spiller,et al.  Enhanced Antisense Effects Resulting from an Improved Streptolysin-O Protocol for Oligodeoxynucleotide Delivery into Human Leukaemia Cells , 1997 .

[15]  S. Mccurdy,et al.  An improved method for the synthesis of N3′→P5′ phosphoramidate oligonucleotides , 1997 .

[16]  D. Spiller,et al.  Improving the intracellular delivery and molecular efficacy of antisense oligonucleotides in chronic myeloid leukemia cells: a comparison of streptolysin-O permeabilization, electroporation, and lipophilic conjugation. , 1998 .

[17]  C. Giovannangeli,et al.  Accessibility of nuclear DNA to triplex-forming oligonucleotides: the integrated HIV-1 provirus as a target. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[18]  M. Rougée,et al.  Triple-helix formation by oligonucleotides containing the three bases thymine, cytosine, and guanine. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[19]  P. Glazer,et al.  Potassium-resistant triple helix formation and improved intracellular gene targeting by oligodeoxyribonucleotides containing 7-deazaxanthine. , 1997, Nucleic acids research.

[20]  M. Braddock,et al.  Real-time analysis of the transcriptional regulation of HIV and hCMV promoters in single mammalian cells. , 1995, Journal of cell science.

[21]  S. Mccurdy,et al.  An improved synthesis of oligodeoxynucleotide N3'-->P5' phosphoramidates and their chimera using hindered phosphoramidite monomers and a novel handle for reverse phase purification. , 1998, Nucleic acids research.

[22]  J. Francois,et al.  Triple-helix formation by alpha oligodeoxynucleotides and alpha oligodeoxynucleotide-intercalator conjugates. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[23]  J. Nelson,et al.  Acridine- and cholesterol-derivatized solid supports for improved synthesis of 3'-modified oligonucleotides. , 1991, Bioconjugate chemistry.

[24]  N. Thuong,et al.  Sequence‐Specific Recognition and Modification of Double‐Helical DNA by Oligonucleotides , 1993 .

[25]  S. Tsao,et al.  Rat mammary preadipocytes in culture produce a trophic agent for mammary epithelia—prostaglandin E2 , 1984, Journal of cellular physiology.

[26]  P. Glazer,et al.  Targeted mutagenesis in mammalian cells mediated by intracellular triple helix formation , 1995, Molecular and cellular biology.