Advancing Applications of the Expanded Genetic Alphabet: Monitoring Expanded Genetic Letters in Complex DNA Context...

Genetic alphabet expansion by creating unnatural base pairs (UBPs) could renovate biological systems and next-generation biotechnologies. Among the expanded genetic letters, TPT3-NaM is one of the most advanced UBPs. It has been utilized in semi-synthetic organisms (SSOs) to recode therapeutic proteins. This article describes the synthesis of isoTAT and demonstrates that isoTAT can convert NaM to G, simultaneously pairing with both NaM and G as a bridging base. Additionally, the inherent base' preference for NaM leads to its transformation into T. Consequently, TPT3-NaM can be converted to C-G or A-T base pairs through simple PCR assays, enabling the first method to locate multiple sites of TPT3-NaM pairs dually. Taken together, this work presents the first general and convenient approach capable of locating, tracing, and sequencing site- and number-unlimited TPT3-NaM pairs. The data presented in this article are based on our previously published reports. © 2025 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of isoTAT triphosphate Basic Protocol 2: Incorporation and extension reaction of isoTAT for a template containing NaM Basic Protocol 3: Monitor DNA containing multiple UBPs using simple PCR assays with isoTAT through Sanger sequencing.