Novel strategy for activating gene expression through triplex DNA formation targeting epigenetically suppressed genes

Area of Science:

• Molecular Biology
• Genomics
• Epigenetics

Background:

• Triplex DNA formation is a promising genomic targeting tool but faces sequence limitations.
• DNA methylation, particularly at 5mCG sites, epigenetically silences genes, posing challenges for gene regulation strategies.

Purpose of the Study:

• To investigate the effect of triplex DNA formation on methylated DNA sequences at the RASSF1A gene promoter.
• To assess if targeting methylated DNA with modified oligonucleotides can restore gene expression and impact cancer cell behavior.

Main Methods:

• Development of guanidino-dN modified oligonucleotides (TFOs) for high-affinity recognition of 5mCG base pairs.
• Application of these TFOs to the RASSF1A gene promoter in MCF-7 breast cancer cells.
• Analysis of RASSF1A gene expression at transcript and protein levels, and assessment of cell proliferation and apoptosis-related gene expression.

Main Results:

• Triplex DNA formation using TFOs with guanidino-dN significantly increased RASSF1A gene expression at both transcript and protein levels.
• Activation of RASSF1A in MCF-7 cells resulted in suppressed cell growth and antiproliferative activity.
• Gene expression changes indicated promotion of apoptosis and reduced breast cancer survival markers.

Conclusions:

• Modified triplex DNA formation targeting methylated sequences is a viable strategy to reactivate epigenetically silenced genes.
• This approach holds potential as a novel genomic targeting tool for gene expression modulation with therapeutic implications, particularly in cancer.
• The study demonstrates the feasibility of using TFOs with guanidino-dN to overcome limitations in targeting methylated DNA for gene activation.