The effect of replacing guanosines with inosines in the two stabilizing strands (5 '-CACCTAGCGA-3 ') of the NIR emissive DNA-Ag16NC was investigated. The spectroscopic behavior of the inosine mutants is position-dependent: when the guanosine in position 7 was exchanged, the nanosecond fluorescence decay time shortened, while having the inosine in position 9 made the decay time longer. Thanks to structural information gained from single crystal X-ray diffraction measurements, it was possible to propose a mechanistic origin for the observed changes.