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.

Dual-Targeting of the HER2 Cancer Receptor with an Antibody-Directed Enzyme and a Nanobody-Guided MMAE Prodrug Scaffold.

Antibody-enzyme conjugates have shown potential as tissue-specific prodrug activators by antibody-directed enzyme prodrug therapy (ADEPT), but the approach met challenges clinically due to systemic drug release. Here, we report a novel dual-targeting ADEPT system (DuADEPT) which is based on active cancer receptor targeting of both a trastuzumab-sialidase conjugate (Tz-Sia) and a highly potent sialidase-activated monomethyl auristatin E (MMAE) prodrug scaffold. The scaffold is based on a four-way junction of the artificial nucleic acid analog acyclic (L)-threoninol nucleic acid ((L)-aTNA) which at the ends of its four arms carries one nanobody targeting HER2 and three copies of the prodrug. Dual-targeting of the constructs to two proximal epitopes of HER2 was shown by flow cytometry, and a dual-targeted enzymatic drug release assay revealed cytotoxicity upon prodrug activation specifically for HER2-positive cancer cells. The specific delivery and activation of prodrugs in this way could potentially be used to decrease systemic side effects and increase drug efficacy, and utilization of Tz-Sia provides an opportunity to combine the local chemotherapeutic effect of the DuADEPT with an anticancer immune response.

Synthesis of peptide-siRNA conjugates via internal sulfonylphosphoramidate modifications and evaluation of their in vitro activity.

Conjugates of therapeutic oligonucleotides (ONs) including peptide conjugates, provide a potential solution to the major challenge of specific tissue delivery faced by this class of drugs. Conjugations are often positioned terminal at the ONs, although internal placement of other chemical modifications are known to be of critical importance. The introduction of internal conjugation handles in chemically modified ONs require highly specialized and expensive nucleoside phosphoramidites. Here, we present a method for synthesizing a library of peptide-siRNA conjugates by conjugation at internal phosphorous positions via sulfonylphosphoramidate modifications incorporated into the sense strand. The sulfonylphosphoramidate modification offers benefits as it can be directly incorporated into chemically modified ONs by simply changing the oxidation step during synthesis, and furthermore holds the potential to create multifunctionalized therapeutic ONs. We have developed a workflow using a novel pH-controlled amine-to-amine linker that yields peptide-siRNA conjugates linked via amide bonds, and we have synthesized conjugates between GLP1 peptides and a HPRT1 siRNA as a model system. The in vitro activity of the conjugates was tested by GLP1R activity and knockdown of the HPRT1 gene. We found that conjugation near the 3'-end is more favorable than certain central internal positions and different internal conjugation strategies were compared.