Self-Assembly of Ultrasmall 3D Architectures of (l)‑Acyclic Threoninol Nucleic Acids with High Thermal and Serum Stability

The primary challenge of implementing DNA nanostructures in biomedical applications lies in their vulnerability to nuclease degradation and variations in ionic strength. Furthermore, the size minimization of DNA and RNA nanostructures is limited by the stability of the DNA and RNA duplexes. This study presents a solution to these problems through the use of acyclic (l)-threoninol nucleic acid (aTNA), an artificial acyclic nucleic acid, which offers enhanced resilience under physiological conditions. The high stability of homo aTNA duplexes enables the design of durable nanostructures with dimensions below 5 nm, previously unattainable due to the inherent instability of DNA structures. The assembly of a stable aTNA-based 3D cube and pyramid that involves an i-motif formation is demonstrated. In particular, the cube outperforms its DNA-based counterparts in terms of stability. We furthermore demonstrate the successful attachment of a nanobody to the aTNA cube using the favorable triplex formation of aTNA with ssDNA. The selective in vitro binding capability to human epidermal growth factor receptor 2 is demonstrated. The presented research presents the use of aTNA for the creation of smaller durable nanostructures for future medical applications. It also introduces a new method for attaching payloads to these structures, enhancing their utility in targeted therapies.