Transcript Document

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
forming specific Hoogsteen
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
The antigene and
antisense application
of TFO
promise of therapeutic utility
Triplex formation
Triplex formation has been shown to
inhibit transcription in mammalian cells
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
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
Therapeutic applications of
TFO
 To silence gene expression
 Through antigene or antisense
approach have been reported in
the literature
TFO
The up regulation of the
gene (induced mutagenesis )
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.
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: 219–228, 2000
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
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
The potential use of
chimaeric
hairpin-TFO to promote
transcription activation
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.
Effect of hairpin-TFO on
transcription
The hairpin-TFO on transcription:
– of STE6 and CBT1
– An over producer of GAL4 protein
was used
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
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
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.
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
Optimization of RT-PCR:
Conditions Concentration of the primers for
ACT1 and STE6 are varied
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
The possible transcription complex recruited by the
hairpin-TFO:
DNA binding domain/ Activating domain of Gal4 Protein
The reason
for the lower level of
activation of STE6
gene
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
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.
 The lack of activation in a GAL4
mutant:
Down activation of gene expression
Activation through hairpin-TFO is
specifically mediated by GAL4
protein
Effect of transfection of hairpin-TFO on transcription
of targeted genes in the yeast strain HF7c (GAL4−)
TFO as an anti tumor polycyclic
acridinesTriplex DNA
A target for DNA-binding polycyclic
acridine derivatives
promise of therapeutic utility
Antigene therapies
 It’s based on the recognition and
binding of a single oligonucleotide
strand
 To a double-stranded sequence
Forming a triple helix
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.
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
Only purine TFOs have been
shown to mediate genome
modification without the need for a
targeted DNA-adduct
TFOs
 For altering gene function
 By either repressing transcription
 Inhibiting DNA replication
 Inducing site-specific mutagenesis
and recombination
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
Models of
structures
that may
mediate
mRNA
synthesis
and DNA
replication
inhibition
by Triplex