Download triplex-forming oligonucleotide (TFO)

Triplex forming
oligonucleotides
(TFO)
Dr Pupak Derakhshandeh, PhD
Ass Prof of Medical Science of
Tehran University
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
2
TFOs
TFOs can bind in the major groove of DNA:
polypurine / polypyrimidine sequences
forming specific Hoogsteen hydrogen bonds
3
4
5
Classification of DNA triple
helices
• Intermolecular triplexes
• Interamolecular triplexes
6
7
8
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
9
Such that elements of the HDNA structure may be
pharmacologically
exploitable
10
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
11
Canonical base triplets formed in
purine and pyrimidine triplex motifs
Watson-Crick base pairing is illustrated by dotted lines
12
• In the anti parallel, purine motif:
–G:G-C
–A:A-T
–In the parallel, pyrimidine motif:
– T:A-T
– C:G-C
13
triplex motifs
• Triplex formation is kinetically slow
compared to duplex annealing
• However, once formed, triplexes are
very Stable
14
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
15
The anti-gene and
antisense application
of TFO
promise of therapeutic utility
16
Triplex formation
Triplex formation has been shown to
inhibit transcription in mammalian cells
17
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
18
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
19
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
20
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)
21
Therapeutic applications of
TFO
 To silence gene expression
 Through antigene approach have
been reported in the literature
22
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 !
23
TFO
The up regulation of the
gene (induced mutagenesis )
24
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.
25
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
26
• the presence of high levels of fetal
and b-thalassemia, are very
common genetic diseases
• when g-globin genes are Human bglobin disorders, such as sickle cell
anemia highly expressed
• hemoglobin (HbF, a2g2) in erythrocytes
(~20–30%) that affect over a million
people worldwide and cause:
– can compensate for the defective b27
globin gene product
• when g-globin genes are highly
expressed:
– the presence of high levels of fetal
hemoglobin (HbF, a2g2) in
erythrocytes (~20–30%)can
compensate for the defective b-globin
gene product
– and such patients have a much
improved disease condition
(Stamatoyannopoulos et al., 1994).
28
• 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
29
have increased HbF in their erythrocytes
• Hereditary Persistence of Fetal
Hemoglobin (HPFH) is a human
genetic condition in which the γglobin 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
30
• 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
31
32
Octamer-binding transcription
factor-1 gene
• activating the g-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 g-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
33
• 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
34
35
• In-vivo gene expression assays:
–activation of g-globin gene
expression from these mutations in
mouse erythroleukemia (MEL) cells
–The levels of the g-globin gene
expression increased by as much
as fourfold in mutants with single
base changes
36
Xu XS et al. Gene 242: 219–228, 2000
• mutations at the Oct-1 binding site can
lead to activation of the g-globin gene
and generate the hereditary persistence
of fetal hemoglobin (HPFH) condition
Xu XS et al. Gene 242: 219–228, 2000
37
• 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
38
39
• 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
40
41
• 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)
42
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
43
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
44
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
45
The potential use of
chimaeric
hairpin-TFO to promote
transcription activation
46
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.
47
Effect of hairpin-TFO on
transcription
The hairpin-TFO on transcription:
– of STE6 and CBT1
– An over producer of GAL4 protein
was used
48
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
49
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
50
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.
51
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
52
Optimization of RT-PCR:
Conditions Concentration of the primers for
ACT1 and STE6 are varied
53
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
54
The possible transcription complex recruited by the
hairpin-TFO:
DNA binding domain/ Activating domain of Gal4 Protein
55
The reason
for the lower level of
activation of STE6
gene
56
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
57
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.
58
 The lack of activation in a GAL4
mutant:
Down activation of gene expression
Activation through hairpin-TFO is
specifically mediated by GAL4
protein
59
Effect of transfection of hairpin-TFO on transcription
of targeted genes in the yeast strain HF7c (GAL4−)
60
TFO as an anti tumor
Triplex DNA
A target for DNA-binding polycyclic
acridine derivatives
promise of therapeutic utility
61
Antigene therapies
 It’s based on the recognition and
binding of a single oligonucleotide
strand
 To a double-stranded sequence
Forming a triple helix
62
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.
63
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
64
Only purine TFOs have been
shown to mediate genome
modification without the need for a
targeted DNA-adduct
65
TFOs
 For altering gene function
 By either repressing transcription
 Inhibiting DNA replication
 Inducing site-specific mutagenesis
and recombination
66
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
67
Models of
structures
that may
mediate
mRNA
synthesis
and DNA
replication
inhibition
by Triplex
68