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Triplex forming oligonucleotides (TFO)

Triplex forming oligonucleotides (TFO). Dr. Derakhshandeh, PhD. Introduction. Agents for modifying gene function In most instances they are utilized for repression of transcription. TFOs. TFOs can bind in the major groove of DNA: polypurine / polypyrimidine sequences

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Triplex forming oligonucleotides (TFO)

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  1. Triplex forming oligonucleotides (TFO) Dr. Derakhshandeh, PhD

  2. Introduction • Agents for modifying gene function • In most instances they are utilized for repression of transcription

  3. TFOs • TFOs can bind in the major groove of DNA: • polypurine / polypyrimidine sequences • forming specific Hoogsteen

  4. Triplex-forming oligonucleotides (TFOs) • Bind DNA in a sequence-specific manner at polypurine / polypyrimidine sites • Mediate targeted genome modification • Formation in cells, leading to mutagenesis or recombination

  5. The antigene and antisense applicationof TFO promise of therapeutic utility

  6. Triplex formation • Triplex formation has been shown to inhibit transcription in mammalian cells

  7. TFO • They have been used to deliver DNA reactive conjugates to specific target sites: • leading to site-directed mutagenesis in some cases • both in mammalian cells • in culture • in vitro even

  8. It is interesting to note: The hairpin-TFO is able to invade the duplex: that is present as nucleosome associated chromatin • mutagenesis or gene silencing

  9. Therapeutic applications of TFO • To silence gene expression • Through antigene or antisense approach have been reported in the literature

  10. TFO The up regulation of the gene (induced mutagenesis )

  11. Triplexformation • Is known to induce mutagenesis i.g.: • Activation of human gamma-globin gene expression via triplex-forming oligonucleotides • Mutations in the gamma-globin gene 5 flanking region.

  12. The up regulation of γ-globin • The symptoms of sickle cell anemia and thalassemia • TFO-directed mutagenesis of the upstream sequences Xu XS et al. Gene 242: 219–228, 2000

  13. The sequence of the hairpin-TFO and a potential interaction of the hairpin TFO, with the target duplex and GAL4 proteinGhosh,M K, et al. Molecular and Cellular Biochemistry 278: 147–155, 2005

  14. A bifunctional hairpin-TFO • including the targeting sequences • polypurine stretch • genes in Saccharomyces cerevisiae • could bind GAL4 protein with high affinity • stable triplex with target sequence

  15. The potential use of chimaeric hairpin-TFO to promote transcription activation

  16. Transcriptional activation • Triplex forming oligonucleotides + • The cognate binding site for transcription activator • Could be targeted to the upstream poly(pu/py) region of specific genes in vivo • Leading to transcriptional activation • By endogenously available transcription activator Ghosh,M K, et al. Molecular and Cellular Biochemistry 278: 147–155, 2005.

  17. Effect of hairpin-TFO on transcription • The hairpin-TFO on transcription: • of STE6 and CBT1 • An over producer of GAL4 protein was used

  18. The cells • grown in medium were induced with galactose • transfected with 1.5μM hairpin-TFO in the presence of 0.8nM PEI • PEI: • to aid in transfection • to increase the stability of the triplex structure in vitro • The efficiency of transfection under these conditions was measured: • using pGAD424 plasmid After transfection • The cells were harvested at different time • RNA was extracted • RNA: subjected to RT-PCR in multiplex

  19. The sequence of the hairpin-TFO and a potential interaction of the hairpin TFO, with the target duplex and GAL4 protein The 65mer hairpin-TFO

  20. Optimization of RT-PCR ACT1 genecontains two stretches of poly(pu/py) sequenceButnone of these have any complementarity to the poly(pu/py)sequence presentupstream ofSTE6andCBT1 genes.

  21. ACT1 gene • The gene should contain poly(pu/py) sequence • In the upstream region • But not similar to that in the upstream region of STE6 and CBT1

  22. Optimization of RT-PCR: Conditions Concentration of the primers for ACT1 and STE6 are varied

  23. Effect of transfection of hairpin-TFO on transcription of targeted genes of yeast strain Sc340(A) STE6 transcripts measured by RT-PCR at different time points after transfection (B) CBT1 transcript levels

  24. The possible transcription complex recruited by the hairpin-TFO:DNA binding domain/ Activating domain of Gal4 Protein

  25. The reasonfor the lower level of activation of STE6 gene

  26. The sequence of the hairpin-TFO and a potential interaction of the hairpin TFO, with the target duplex and GAL4 protein The 65mer hairpin-TFO

  27. The reasonfor the lower level of activation of STE6 gene • STE6 gene: • the criteria for optimum distance of GAL4 recruitment is fulfilled • In the case of CBT1: • the distance of the GAL4 recruitment site is more than what is suggested as the optimum distance.

  28. The lack of activation in a GAL4 mutant: Down activation of gene expressionActivation through hairpin-TFO is specifically mediated by GAL4 protein

  29. Effect of transfection of hairpin-TFO on transcription of targeted genes in the yeast strain HF7c (GAL4−)

  30. TFOas an anti tumor polycyclic acridinesTriplex DNA A target for DNA-binding polycyclic acridine derivatives promise of therapeutic utility

  31. Antigene therapies • It’s based on the recognition and binding of a single oligonucleotide strand • To a double-stranded sequence • Forming a triple helix

  32. Triplex DNA formation • A relatively weak and temporary phenomenon • Therefore, molecules that selectively bind to and stabilize triple helices may show a variety of novel biological effects.

  33. Compounds: Polycyclic acridines • A series of antitumor • That bind to triplex DNA • Whose synthesis has been previously reported • Have been tested for their interaction with both purine and pyrimidine type triple helices • As a pyrimidine triplex model • Antitumor activity

  34. Only purine TFOs have been shown to mediate genome modification without the need for a targeted DNA-adduct

  35. TFOs • For altering gene function • By either repressing transcription • Inhibiting DNA replication • Inducing site-specific mutagenesis and recombination

  36. DNA:RNA:DNA Triplex Formation • Their potential as tools in molecular biology • Therapeutic agents • Unstable DNA:RNA triplexes play key roles in many biological processes • Inhibition of RNAse, DNAse I, and RNA polymerase

  37. Models of structures that may mediate mRNA synthesis and DNA replication inhibition by Triplex

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