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

5. 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

6. The antigene and antisense application of TFO
promise of therapeutic utility

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

8. 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

9. 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

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

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

12. Triplex formation 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.

13. 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

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

15. 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

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

17. 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.

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

19. 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

20. 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

21. Optimization of RT-PCR
ACT1 gene contains two stretches of poly(pu/py) sequence But none of these have any complementarity to the poly(pu/py) sequence present upstream of STE6 and CBT1 genes.

22. 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

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

24. 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

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

26. The reason for the lower level of activation of STE6 gene

27. 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

28. The reason for the lower level of activation of 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.

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

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

31. TFO as an anti tumor polycyclic acridinesTriplex DNA A target for DNA-binding polycyclic acridine derivatives
promise of therapeutic utility

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

33. 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.

34. 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

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

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

37. 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

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