Triplex forming oligonucleotides (TFO)

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Triplex forming oligonucleotides (TFO)

• Dr. Derakhshandeh, PhD

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Introduction

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

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TFOs

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

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(No Transcript)
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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

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The antigene and antisense applicationof TFO

• promise of therapeutic utility

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Triplex formation

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

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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

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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

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Therapeutic applications of TFO

• To silence gene expression
• Through antigene or antisense approach
  have been reported in the literature

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TFO

• The up regulation of the gene (induced
  mutagenesis )

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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.

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• The up regulation of ? -globin
• The symptoms of sickle cell anemia and
  thalassemia
• TFO-directed mutagenesis of the upstream
  sequences
• Xu XS et al. Gene 242 219228, 2000

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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 147155,
2005
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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

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

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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 147155, 2005.

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Effect of hairpin-TFO on transcription

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

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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

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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
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Optimization of RT-PCR
ACT1 gene contains two stretches of poly(pu/py)
sequenceButnone of these have any
complementarity to the poly(pu/py)sequence
present upstream of STE6 and CBT1 genes.
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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

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Optimization of RT-PCR Conditions Concentration
of the primers for ACT1 and STE6 are varied
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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
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The possible transcription complex recruited by
the hairpin-TFODNA binding domain/ Activating
domain of Gal4 Protein
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The reasonfor the lower level of activation of
STE6 gene
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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
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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.

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The lack of activation in a GAL4 mutant Down
activation of gene expression Activation
through hairpin-TFO is specifically mediated by
GAL4 protein
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Effect of transfection of hairpin-TFO on
transcription of targeted genes in the yeast
strain HF7c (GAL4-)
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TFO as an anti tumor polycyclic acridinesTriplex
DNA A target for DNA-binding polycyclic acridine
derivatives

• promise of therapeutic utility

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Antigene therapies

• Its based on the recognition and binding of a
  single oligonucleotide strand
• To a double-stranded sequence
• Forming a triple helix

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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.

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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

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

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TFOs

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

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DNARNADNA Triplex Formation

• Their potential as tools in molecular biology
• Therapeutic agents
• Unstable DNARNA triplexes play key roles in
  many biological processes
• Inhibition of RNAse, DNAse I, and RNA polymerase

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