Triplex forming Triplex forming oligonucleotidesoligonucleotides
(TFO) (TFO)
Dr. Derakhshandeh, PhD
IntroductionIntroduction
Agents for modifying gene function
In most instances they are utilized for repression of transcription
TFOsTFOs
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 The antigene and antisense applicationantisense application
of TFOof TFO
promise of therapeutic utility
Triplex formationTriplex formation
Triplex formation has been shown to inhibit transcription in mammalian cells
TFOTFOThey 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: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
TFOTFO
The up regulation of the The up regulation of the gene (gene (induced mutagenesis ))
TriplexTriplexformationformation
Is known to induce mutagenesisi.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 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
The sequence of the hairpin-TFO and a potential interaction The sequence of the hairpin-TFO and a potential interaction of the hairpin TFO, with the target duplex and GAL4 proteinof 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 The potential use of chimaericchimaeric
hairpin-TFO to promote hairpin-TFO to promote transcription activationtranscription activation
Transcriptional activationTranscriptional activationTriplex forming oligonucleotides +The cognate binding site for transcription
activator Could be targeted to the upstream
poly(pu/py) region of specific genes in vivoLeading 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 Effect of hairpin-TFO on transcriptiontranscription
The hairpin-TFO on transcription:
– of STE6 and CBT1
– An over producer of GAL4 protein was used
The cellsThe cellsgrown in medium were induced with galactosetransfected 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 The sequence of the hairpin-TFO and a potential interaction
of the hairpin TFO, with the target duplex and GAL4 proteinof the hairpin TFO, with the target duplex and GAL4 protein
The 65mer hairpin-TFO
Optimization of RT-PCR 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.
ACT1 geneACT1 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:Optimization of RT-PCR: Conditions Concentration of the primers for Conditions Concentration of the primers for
ACT1 and STE6 are variedACT1 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 reasonThe reasonfor the lower level of for the lower level of activation of STE6 activation of STE6
genegene
The sequence of the hairpin-TFO and a potential interaction The sequence of the hairpin-TFO and a potential interaction
of the hairpin TFO, with the target duplex and GAL4 proteinof the hairpin TFO, with the target duplex and GAL4 protein
The 65mer hairpin-TFO
The reasonThe reasonfor the lower level of activation of for the lower level of activation of
STE6 gene 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 The lack of activation in a GAL4 mutant: mutant: Down activation of gene expressionDown activation of gene expression
Activation through hairpin-TFO is Activation through hairpin-TFO is specifically mediated by GAL4specifically mediated by GAL4 proteinprotein
Effect of transfection of hairpin-TFO on transcription Effect of transfection of hairpin-TFO on transcription of targeted genes in the yeast strain HF7c (GAL4−)of targeted genes in the yeast strain HF7c (GAL4−)
TFOTFO as an anti tumor polycyclic as an anti tumor polycyclic acridinesTriplex DNAacridinesTriplex DNA
A target for DNA-binding polycyclic A target for DNA-binding polycyclic acridine derivativesacridine derivatives
promise of therapeutic utility
Antigene therapiesAntigene therapies
It’s based on the recognition and binding of a single oligonucleotide strand
To a double-stranded sequenceForming a triple helix
Triplex DNA formationTriplex 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 acridinesCompounds: 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 Only purinepurine TFOs have been TFOs have been shown to mediate genome shown to mediate genome
modification without the need for a modification without the need for a targeted DNA-adducttargeted DNA-adduct
TFOsTFOs For altering gene function
By either repressing transcription
Inhibiting DNA replication
Inducing site-specific mutagenesis and recombination
DNA:RNA:DNA Triplex DNA:RNA:DNA Triplex FormationFormation
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
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