Deoxyribonucleic acid (DNA) ligases are essential enzymes that catalyze the formation of phosphodiester bonds to repair nicks in DNA during replication and repair processes. Their ability to ligate synthetic non-canonical nucleic acids (NCNAs), such as unnatural base pairs (UBPs) and backbone-modified DNA/RNA, has significant implications for synthetic biology, biotechnology, and therapeutic applications. This study aimed to evaluate the activity of various DNA ligases on UBP and backbone-modified DNA/RNA substrates, optimize ligation conditions, and explore the relationship between ligase structure and substrate preference. The study focused on three main objectives: (1) establishing baseline ligation activity for UBP and backbone-modified substrates across multiple ligases, (2) investigating the impact of ligase binding domain size on substrate preference, and (3) optimizing ligation conditions to enhance enzyme activity. A panel of ligases, including Human DNA ligase I, Escherichia coli ligase A, Neisseria gonorrhoeae ligase A, and several ATP-dependent ligases (Lig 3, Lig 4, Lig 5, Lig 12, Lig 15), were tested on substrates containing UBPs (P-Z and S-B systems) and backbone modifications (phosphorothioate (PPT) and 2’-Methoxyethoxy (MOE) RNA). Results revealed that ligases exhibited varying levels of activity depending on the substrate type and modification position. H-Lig I and Lig 4 demonstrated the highest ligation activity on UBP substrates, particularly those with modifications at the 5’ end of the nick. In contrast, substrates with UBPs on both ends of the nick were poorly tolerated. Gel shift assays confirmed that low ligation activity was not due to binding failure but rather steric hindrance during the adenyl group transfer step. For backbone-modified substrates, Lig 3 and Lig 5 emerged as the most effective, with PPT modifications being better tolerated than MOE modifications. The addition of 10% polyethylene glycol (PEG) 3350 as a crowding agent significantly improved ligation efficiency, particularly for MOE substrates, but inhibited the activity of larger ligases like Lig 12 and H-Lig I. In conclusion, this study provides valuable insights into the activity of DNA ligases on non-canonical substrates, highlighting the importance of ligase structure and reaction conditions in determining ligation efficiency. The findings contribute to the growing toolbox of synthetic biology and pave the way for further exploration of ligase-substrate interactions, enzyme engineering, and the development of novel biotechnological applications.