Computational Design and Synthesis of Novel Nucleotides to Expand Nucleic Acid Synthetic Biology

Unnatural base pairs (UBPs) have advanced from scarcely incorporable constructs to systems supporting >99% incorporation fidelities and compatibility with transcription and reverse transcription, enabling DNA SELEX with six-letter alphabets. Moreover, semi-synthetic organisms incorporating unnatural bases have been established, advancing the site-specific incorporation of non-canonical amino acids into proteins. Despite this progress, extending robustness and functionality beyond optimized settings remains challenging. Building on this progress, this work addresses three complementary research lines: (i) halogen-bonding UBPs, (ii) the development of photoresponsive nucleosides enabling light-controlled behavior, and (iii) supporting studies on modified nucleosides and computational assessment. An in silico screen of 280 candidate UBPs prioritized halogen-bonding motifs based on C1′–C1′ distance. Two nucleosides, PPMO and PTMO, were synthesized. Halogen-bonding nucleosides (Br-TPT1, I-TPT1, IIPO, and oIPP) were prepared in the research group. Primer-extension assays showed full-length products for the IIPO:oIPP combination, whereas IIPO opposite PTMO displayed poor incorporation, consistent with computational predictions. Second, a synthetic route to a diarylethene photoswitch embedded in a TPT2 nucleoside was established. TPT2 provides a scaffold due to its high enzymatic compatibility. The route included bromide installation, improved glycosylation, and a successful Lawesson conversion, resulting in a Suzuki-ready intermediate. The photoswitch is intended to enable light-controlled modulation of base-pairing behavior. Complementary efforts provided 5-deaza-adenosine for twister-ribozyme studies and computational support: DFT-based spectroscopic predictions guided structure screening prior to synthesis, short GFN-FF MD simulations estimated distance distributions for spin labeling, and ESP analyses of a C-glycosidic TPT3 analogue assessed charge distribution and pairing compatibility.