Functional and Structural Analyses of Diverse G-Quadruplex and Non-G-Quadruplex Structures Formed by Guanine-Rich Nucleic Acids: A Study on the Insulin Aptamer

Abstract

Guanine-rich sequences in nucleic acids play a critical role in various biological processes owing to their ability to form G-quadruplexes. In addition, biotechnology tools exploiting the unique functionalities of G-quadruplex structures have garnered considerable attention. However, the potential roles of non-G-quadruplex structures have not been explored. Herein, a guanine-rich oligonucleotide, which forms non-G-quadruplexes, is found to still demonstrate an affinity for proteins, specifically in the case of an aptamer. The insulin-binding aptamer IGA3, known for its guanine-rich sequence, forms various structures depending on the environmental conditions. At low cation concentrations, IGA3 adopts a distinct monomeric structure that is not G-quadruplex but retains its insulin-binding capacity. These findings suggest an interaction mechanism that extends beyond G-quadruplex structures. To accurately characterize the non-G-quadruplex structures of a guanine-rich oligonucleotide, this study integrates nuclear magnetic resonance, thermal difference spectroscopy, and circular dichroism (CD) spectroscopy with principal component analysis. In addition, the multivariate curve resolution of the CD spectra is used to analyze the structural mixture and identify the specific chemical species responsible for protein affinity. Results indicate that guanine-rich sequences bind to proteins through non-G-quadruplex formation, offering new insights into the functionality of guanine-rich DNA fragments.