179P - Identification of guanine-quadruplex forming DNA aptamer: A promising therapeutic strategy for EGFR overexpressed non-small cell lung cancer

Background

Lung cancer, particularly Non-Small Cell Lung Cancer (NSCLC), remains a leading cause of cancer deaths. EGFR overexpression is a frequent culprit, making it a crucial therapeutic target. Current anti-EGFR drugs like cetuximab and gefitinib have limitations like drug resistance and high costs. Aptamers offer a promising alternative.

Methods

This study uses the Cell-SELEX approach to create G-quadruplex-forming DNA aptamer inhibitors to target epidermal growth factor receptor (EGFR). We made a biased Guanine-quadruplex forming DNA aptamer library, 57 nucleotides long, with N-30 random regions. Starting with a diverse library of single-stranded DNA sequences, we conducted 11 iterative rounds of selection using the EGFR-overexpressing A431 cell line for positive selection and the Jurkat cell line for negative selection. This process enriched the EGFR-specific aptamer pool, confirmed by flow cytometry. The pool with the highest fluorescence was subjected to Next Generation Sequencing (NGS) to identify sequences specifically binding to the A431 cell line. Sequence analysis revealed conserved motifs crucial for G-quadruplex formation and EGFR recognition. The binding affinity and specificity of the selected G-quadruplex DNA aptamers were evaluated using flow cytometry, confocal microscopy, and cell-binding assays.

Results

In our results, we assessed the enrichment of the selected pool using flow cytometry, focusing on the 9th, 10th, and 11th rounds, with the highest enrichment observed in the 11th round (Figure 2). Subsequently, we amplified the 11th round pool using unlabelled primers, purified it, and subjected it to Next Generation Sequencing (NGS) (Figure 3). From the NGS data, we identified the 20 most abundant sequences, selecting 10 for further synthesis. These aptamers were then evaluated for their binding affinity and specificity using flow cytometry, confocal microscopy, and cell-binding assays to identify the most effective candidate.

Conclusions

The identified aptamers hold promise for the development of novel cancer diagnostics and therapeutics, enabling more precise and effective targeting of EGFR-positive lung cancer.

Editorial acknowledgement

Clinical trial identification

Legal entity responsible for the study

All India Institute of Medical Sciences, New Delhi.

Funding

Has not received any funding.

Disclosure

The author has declared no conflicts of interest.