High-resolution melting analysis for rapid and cost-effective detection of unnatural base pairs.

Expansion of the genetic alphabet with unnatural base pairs (UBPs) enables new approaches in protein engineering, synthetic biology, and biocontainment. Such applications rely on faithful incorporation and retention of UBPs in semi-synthetic systems, making their detection and monitoring essential tools for xenobiology. Current methods for detecting UBPs in DNA frequently rely on labelling strategies, specialised reagents, or advanced analytical instrumentation. These approaches increase cost, technical complexity, and turnaround time, limiting accessibility and scalability of xenobiological experiments. Here, we establish high-resolution melting (HRM) analysis as a rapid and accessible method for detecting the UBP NaM-TPT3 based on differences in melting temperature of short amplicons. The approach can be directly appended to standard PCR workflows using commonly available qPCR instrumentation, enabling fast and cost-effective screening of UBP incorporation and stability in DNA. After optimisation of HRM-specific PCR conditions for efficient UBP incorporation, we demonstrate that HRM analysis produces clear and reproducible melting signatures that distinguish natural and unnatural sequences and detect defined variant mixtures. The method is validated using non-amplified DNA controls and LC-MS analysis. By enabling low-cost, workflow-integrated verification of UBP presence using standard laboratory infrastructures, HRM provides a practical screening tool that lowers barriers to experimentation and accelerated iterative development of expanded genetic systems.