# Efficacy of DNA Intercalator‐Conjugated Triplex‐Forming Oligonucleotide as Anticancer Agent

## TL;DR

Researchers improved the effectiveness of a cancer-targeting molecule by attaching a DNA intercalator, leading to stronger cell death in breast cancer cells with a specific gene amplification.

## Contribution

A DNA intercalator-conjugated TFO was developed to enhance binding affinity and anticancer activity against HER2-amplified breast cancer cells.

## Key findings

- DNA intercalator-conjugated TFOs showed higher binding affinity for the HER2 gene than unmodified TFOs.
- Increased binding affinity correlated with stronger cell death in HER2-positive breast cancer cells.
- The TFOs selectively targeted amplified gene loci, inducing double-strand breaks and cancer cell death.

## Abstract

A triplex‐forming oligonucleotide (TFO) can form a sequence‐specific triple helix via Hoogsteen hydrogen bonding to polypurine tracts within a major groove side of a DNA duplex. Triplex formation can induce a double‐strand break, and this phenomenon at the amplified gene loci can selectively induce the cell death of cancer cells with specific gene amplification. However, the relationship between the binding affinity of TFO for target gene loci and the cell death response remains unclear. In this study, it is aimed to develop DNA intercalator‐conjugated TFOs with higher affinity for the human epidermal growth factor receptor type2 gene, which is often amplified in breast cancer cells, than the unmodified TFO. The binding affinity of the TFOs for the target DNA duplex is analyzed using nondenaturing polyacrylamide gel electrophoresis, and one of the DNA intercalator‐conjugated TFOs show a higher binding affinity for the target duplex than the unmodified TFO. The cell death responses induced by these TFOs using the WST‐8 assay is also evaluated suggesting that the higher binding affinity of the TFO for amplified gene loci can lead to a stronger cell death response of cancer cells with specific gene amplification.

The binding affinity of triplex‐forming oligonucleotide (TFO) is successfully improved for human epidermal growth factor receptor type2 (HER2) gene sequence by using DNA intercalator. The higher binding affinity of TFO for HER2 gene sequence are correlated with the anticancer activity induced by the TFO and resulted in stronger cell death response of HER2‐positive breast cancer cells.© 2025 WILEY‐VCH GmbH