Property Characterization of Leucine-Integrated Oligonucleotide

The introduction of peptide bonds into oligonucleotides as a backbone has led to the development of powerful peptide nucleic acids. However, the integration of amino acid residues (R groups) as nucleobase surrogates in oligonucleotides remains largely unexplored, presenting opportunities for creating chimeras with unique properties. In this study, we describe the design and synthesis of oligonucleotides (ONs: molecular beacons, aptamers, and ASOs) functionalized with leucine-mimicking elements (LEs), specifically isobutyl, neopentyl, and trimethylsilyl ethyl. By design and synthesis of corresponding phosphoramidites, oligonucleotide chimeras containing mono-, di-, and poly-LEs were constructed by solid phase synthesis, including the PTK7 protein-targeted aptamer Sgc8c, STAT3 (signal transducer and activator of transcription 3), and HER2 mRNA-targeted antisense oligonucleotide (ASO). Through fluorescence assays, we analyzed the stability of poly-LE-induced helical structures and investigated the assembly of molecular beacons via hydrophobic interactions. The introduction of poly-LE helices into aptamers and the incorporation of a dileucine-like moiety into antisense oligonucleotides demonstrated the functional versatility of these chimeras in improved internalization properties while adding hydrophobic elements. Notably, poly-LE incorporation significantly improved the target binding affinity and <i>in vivo</i> tumor-targeting efficiency of the aptamer. This work presents a novel approach to oligonucleotide engineering, offering new insights into protein-like functions in DNA and expanding the scope for molecular design in biological and therapeutic applications.