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