New form of guanine-rich DNA binding elucidated by TUAT

Professor Kazunori Ikebukuro and Professor Yasumoto Nakazawa from the Department of Biotechnology and Life Science, Graduate School of Engineering at Tokyo University of Agriculture and Technology (TUAT), and Distinguished Professor Koji Sode from the University of North Carolina at Chapel Hill, in collaboration with Toray Research Center and JASCO Corporation, have announced the discovery that guanine-rich DNA (DNA with high guanine content), which is believed to be involved in various biological functions, can specifically bind to proteins without forming G-quadruplex structures (G4 structures). This was revealed through analysis of the interaction between insulin and the insulin aptamer "IGA3," which forms G4 structures and binds to insulin. This development is expected to lead to the creation of biosensors capable of real-time measurement of insulin in blood. The results were published in the journal Small on June 16.

DNA and RNA (nucleic acids) form specific non-canonical structures in living organisms in addition to the commonly known double helix structure (canonical structure). One of these non-canonical structures is the G4 structure formed by guanine-rich nucleic acids, and human DNA contains approximately 700,000 sequences capable of forming G4 structures.

Aptamers are nucleic acids that specifically bind to target molecules. They have high molecular recognition capabilities and are more practically useful than antibodies due to easier chemical synthesis. G4 structures are known to bind easily to proteins, making aptamers that form G4 structures promising for biosensor applications.

The research group had previously developed IGA3, an aptamer that specifically binds to insulin and forms G4 structures. However, there were also reports of no binding, and the reason was unclear.

Therefore, the research group first investigated whether promoting G4 structure formation in IGA3 would cause changes in its affinity for insulin. Previously, it was believed that binding to insulin occurred while maintaining the G4 structure.

The results showed that promoting G4 structure formation decreased insulin binding ability, suggesting that binding does not occur through G4 structures.

The group then varied conditions and examined IGA3's structure using CD (circular dichroism) spectroscopy, NMR (nuclear magnetic resonance), and TDS (thermal difference spectroscopy) using absorbance. Furthermore, by applying JASCO Corporation's newly developed patented technology, thermodynamic model-constrained multivariate curve resolution alternating least squares (MCR-ALS), to CD spectral data, they successfully extracted the specific structure that binds to insulin from a mixed state of multiple structures, discovering a new structure that binds to insulin.

It was also confirmed that when Na⁺ and K⁺ concentrations are low in the extremely minute regions around IGA3, G4 structure formation is suppressed and specific binding to insulin occurs.

Guanine-rich DNA abundant in living organisms may, like IGA3, flexibly change its three-dimensional structures according to the surrounding environment and interact with surrounding proteins.

Ikebukuro commented: "In the future, we want to investigate whether guanine-rich DNA actually forms non-G4 structures in living organisms. Also, since environments with low Na⁺ and K⁺ concentrations are difficult to imagine in living organisms, we expect that IGA3 will not function as an aptamer. We are currently working to overcome this challenge and design aptamers that function in living organisms. Diabetes diagnosis and condition assessment require measurement not only of blood glucose levels but also insulin concentrations. If aptamers that function in living organisms can be developed, we expect this will lead to the development of biosensors capable of real-time insulin measurement."

Journal Information
Publication: Small
Title: 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
DOI: 10.1002/smll.202501336