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Silver nanoclusters (AgNCs) are the next-generation nanomaterials representing supra-atomic structures where silver atoms are organized in a particular geometry. DNA can effectively template and stabilize these novel fluorescent AgNCs. Only a few atoms in size – the properties of nanoclusters can be tuned using only single nucleobase replacement of C-rich templating DNA sequences. A high degree of control over the structure of AgNC could greatly contribute to the ability to fine-tune the properties of silver nanoclusters. In this study, we explore the properties of AgNCs formed on a short DNA sequence with a C 12 hairpin loop structure (AgNC@hpC 12 ). We identify three types of cytosines based on their involvement in the stabilization of AgNCs. Computational and experimental results suggest an elongated cluster shape with 10 silver atoms. We found that the properties of the AgNCs depend on the overall structure and relative position of the silver atoms. The emission pattern of the AgNCs depends strongly on the charge distribution, while all silver atoms and some DNA bases are involved in optical transitions based on molecular orbital (MO) visualization. We also characterize the antibacterial properties of silver nanoclusters and propose a possible mechanism of action based on the interactions of AgNCs with molecular oxygen.