Proteomic and Transcriptome Profiling of G-Quadruplex Aptamers Developed for Cell Internalization

Area of Science:

• Biotechnology
• Molecular Biology
• Drug Delivery

Background:

• Nucleic acid medicine holds promise for next-generation therapies.
• Direct *in vivo* cell penetration of nucleic acids remains a significant challenge.
• External assistance is often required for effective cellular delivery of nucleic acids.

Purpose of the Study:

• To develop cell-penetrating aptamers for enhanced therapeutic nucleic acid delivery.
• To discover and characterize novel aptamers with superior cell-penetrating capabilities.
• To elucidate the molecular mechanisms underlying aptamer-mediated cellular internalization.

Main Methods:

• Systematic Evolution of Ligands by EXponential enrichment (SELEX) for aptamer discovery.
• Circular Dichroism (CD) spectroscopy to analyze G-quadruplex folding topologies.
• Aptamer pull-down assays combined with mass spectrometry for protein interaction profiling.
• Whole transcriptome analysis to assess the impact of aptamers on gene expression.

Main Results:

• A G-quadruplex-forming DNA aptamer, C20-4 min, exhibited enhanced cell-penetrating capacity compared to controls like AS1411.
• CD spectral analysis confirmed the formation of parallel G-quadruplex structures for C20-4 min.
• Mass spectrometry identified helicases and GTPase proteins as interacting partners during aptamer internalization.
• Transcriptome analysis revealed differential gene expression related to GTPase functions, indicating a role in internalization pathways.

Conclusions:

• C20-4 min is a promising G-quadruplex aptamer for facilitating nucleic acid delivery.
• Helicase and GTPase proteins are key cellular interactors in the G-quadruplex aptamer internalization process.
• Integrative proteomic and transcriptomic analyses provide insights into the molecular mechanisms of aptamer internalization.
• These findings can inform the development of novel nucleic acid delivery systems.