1. Triplex forming oligonucleotides (TFO) Dr Pupak Derakhshandeh, PhD Ass Prof of Medical Science of Tehran University

2. 2 Introduction DNA structure is a critical element in determining its function Agents for modifying gene function In most instances they are utilized for repression of transcription

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

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6. 6 Classification of DNA triple helices Intermolecular triplexes Interamolecular triplexes

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9. 9 DNA triplex structures Either intermolecular triplexes formed by binding of an exogenously applied (TFO, therapeutic ) Or naturally occurring intra molecular triplexes (H-DNA) –specific alteration of the genome –site-specific mutagenesis, for stimulat- –Ing DNA repair or recombination

10. 10 Such that elements of the H- DNA structure may be pharmacologically exploitable

11. 11 An example of triplex formation with a poly purine TFO sequence specific for the human c-MYC P2 promoter The TFO is placed in an anti parallel orientation relative to the target duplex

12. 12 Canonical base triplets formed in purine and pyrimidine triplex motifs Watson-Crick base pairing is illustrated by dotted lines

13. 13 In the anti parallel, purine motif: –G:G-C –A:A-T –In the parallel, pyrimidine motif: –T:A-T –C:G-C

14. 14 triplex motifs Triplex formation is kinetically slow compared to duplex annealing However, once formed, triplexes are very Stable

15. 15 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

16. 16 The anti-gene and antisense application of TFO promise of therapeutic utility

17. 17 Triplex formation Triplex formation has been shown to inhibit transcription in mammalian cells

18. 18 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

19. 19 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

20. 20 Potential applications of DNA triplex formation in therapeutics Allowing the covalent attachment of DNA damaging agents A potential advantage of targeting DNA rather than RNA or protein: –Limited number of copies to be targeted

21. 21 Another advantage of targeting DNA rather than RNA or protein: Facile synthesis of the reagents The availability of a variety of chemical modifications: –To the bases, sugar-phosphate backbone, and the 5’ and 3’ ends)

22. 22 Therapeutic applications of TFO  To silence gene expression  Through antigene approach have been reported in the literature

23. 23 Modulating gene expression via triplex formation TFOs: –Act as ‘‘decoy’’ oligonucleotides –Bind transcription factors, they are not available to bind their duplex consensus sequences for transcription activaion !

24. 24 TFO The up regulation of the gene () The up regulation of the gene ( induced mutagenesis )

25. 25 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.

26. 26  The up regulation of  -globin  reduce the symptoms of sickle cell anemia and thalassemia  TFO-directed mutagenesis of the upstream sequences Xu XS et al. Gene 242: 219–228, 2000

27. 27 the presence of high levels of fetal and  -thalassemia, are very common genetic diseases when  -globin genes are Human  - globin disorders, such as sickle cell anemia highly expressed hemoglobin (HbF,  ) in erythrocytes (~20–30%) that affect over a million people worldwide and cause: –can compensate for the defective  - globin gene product

28. 28 when  -globin genes are highly expressed: –the presence of high levels of fetal hemoglobin (HbF,  ) in erythrocytes (~20–30%)can compensate for the defective  -globin gene product –and such patients have a much improved disease condition (Stamatoyannopoulos et al., 1994).

29. 29 In adults, the β-globin gene is predominantly expressed (98%) while the γ-globin gene is expressed at very low levels (<1%). To increase the levels of HbF in patients with sickle cell / Beta thalassmia disease: –many drugs have been developed: Butyric acid and its analogs have been found to increase the levels of HbF Hydroxyurea –However, many patients cannot achieve increased HbF with these treatments! –With hydroxyurea treatment, for example, only about 60% of patients were found to have increased HbF in their erythrocytes

30. 30 Hereditary Persistence of Fetal Hemoglobin (HPFH) is a human genetic condition in which the γ- globin genes continue to be expressed at very high levels in the adult life of individuals. single base mutations or a small deletion in the 5′ flanking region of the γ-globin gene

31. 31 Molecular biology studies suggest that most of these mutations are located in binding motifs for transcription regulators such as Sp1, GATA-1, and CP1 site. Some mutations also create new binding sites for transcription regulators

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33. 33 Octamer-binding transcription factor-1 gene activating the  -globin gene expression by triplex-forming oligonucleotide (TFO)- directed targeted mutagenesis TFO designed to bind to a site overlapping with an Oct-1 binding site (at the −280 region of the  -globin gene) targeted mutagenesis of the Oct-1 binding site has been achieved by: –transfecting the in-vitro-formed plasmid-oligo complex into human normal fibroblast (NF) cells Xu XS et al. Gene 242: 219–228, 2000

34. 34 The mutation frequency at the target site was estimated to be 20% by direct DNA sequencing analysis Xu XS et al. Gene 242: 219–228, 2000

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36. 36 In-vivo gene expression assays: –activation of  -globin gene expression from these mutations in mouse erythroleukemia (MEL) cells –The levels of the  -globin gene expression increased by as much as fourfold in mutants with single base changes Xu XS et al. Gene 242: 219–228, 2000

37. 37 mutations at the Oct-1 binding site can lead to activation of the  -globin gene and generate the hereditary persistence of fetal hemoglobin (HPFH) condition Xu XS et al. Gene 242: 219–228, 2000

38. 38 Using TFOs : –to bind to the γ-globin gene −280 region region Oct-1 binding site was achieved in a plasmid construct upon in-vitro triplex formation transfection into human cells the mutation frequency of the target site was found to be in the range of 20% The mutations were found to result in reduced binding of Oct-1 transcription factor to the site The mutations also led to γ-globin gene expression in MEL cells

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40. 40 triplex-mediated targeted mutagenesis (TFO), oligo Gamma 2, directed targeted mutagenesis of pUSAG9 plasmid was studied The pUSAG9 plasmid DNA was incubated with oligo Gamma 2 (10 μM) in triplex binding buffer for 2 h at 37°C to induce triplex formation The plasmid-oligo complex was transfected into human normal fibroblast (NF) cells

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42. 42 Plasmid DNA was isolated from individual colonies and the sequence analyzed A total of one hundred plasmids were directly sequence-analyzed and 20 of them were found to contain mutations )−287 to −285 of Aγ-globin gene)

43. 43 Oct-1 binding site at the −280 region of the γ-globin gene negatively regulates γ-globin gene expression mutations in the Oct-1 binding site lead to activation of the γ-globin gene expression Since TFO-directed targeted mutagenesis has already been demonstrated in mammalian cells

44. 44 The sequence of the hairpin-TFO and a potential interaction of the hairpin TFO, with the target duplex and GAL4 protein 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

45. 45 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

46. 46 The potential use of chimaeric hairpin-TFO to promote transcription activation

47. 47 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.

48. 48 Effect of hairpin-TFO on transcription  The hairpin-TFO on transcription: –of STE6 and CBT1 – An over producer of GAL4 protein was used

49. 49 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

50. 50 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

51. 51 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.

52. 52 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

53. 53 Optimization of RT-PCR: Conditions Concentration of the primers for ACT1 and STE6 are varied

54. 54 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

55. 55 The possible transcription complex recruited by the hairpin-TFO: DNA binding domain/ Activating domain of Gal4 Protein

56. 56 The reason for the lower level of activation of STE6 gene

57. 57 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

58. 58 The reason for 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.

59. 59  The lack of activation in a GAL4 mutant: Down activation of gene expression Activation through hairpin-TFO is specifically mediated by GAL4 protein

60. 60 Effect of transfection of hairpin-TFO on transcription of targeted genes in the yeast strain HF7c (GAL4−)

61. 61 TFO as an anti tumor Triplex DNA A target for DNA-binding polycyclic acridine derivatives promise of therapeutic utility

62. 62 Antigene therapies  It’s based on the recognition and binding of a single oligonucleotide strand  To a double-stranded sequence  Forming a triple helix

63. 63 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.

64. 64 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

65. 65 Only purine TFOs have been shown to mediate genome modification without the need for a targeted DNA-adduct Only purine TFOs have been shown to mediate genome modification without the need for a targeted DNA-adduct

66. 66 TFOs  For altering gene function  By either repressing transcription  Inhibiting DNA replication  Inducing site-specific mutagenesis and recombination

67. 67 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

68. 68 Models of structures that may mediate mRNA synthesis and DNA replication inhibition by Triplex