DNA secondary structures, such as dimers and hairpins, are important for the synthesis of DNA templateembedded silver nanoclusters (DNA/AgNCs). However, the arrangement of AgNCs within a given DNA template and how the AgNC influences the secondary structure of the DNA template are still unclear. Here, we introduce a noncanonical head-to-head hairpin DNA nanostructure that is driven by orange-emissive AgNCs. Through detailed in-gel analysis, sugar backbone switching, inductively coupled plasma mass spectrometry, small-angle X-ray scattering, and small angle neutron scattering, we show that the orange-emissive AgNCs mediate cytosine-Ag-cytosine bridging between two six-cytosine loop (6C-loop) hairpin DNA templates. Unlike green, red, or far-red emissive AgNCs, which are embedded inside a hairpin and duplex DNA template, the orange-emissive AgNCs are localized on the interface between the two 6C-loop hairpin DNA templates, thereby linking them. Moreover, we found that deoxyribose in the backbone of the 6C-loop at the third and fourth cytosines is crucial for the formation of the orange-emissive AgNCs and the head-to-head hairpin DNA structure. Taken together, we suggest that the specific wavelength of AgNCs fluorescence is determined by the mutual interaction between the secondary or tertiary structures of DNA-and AgNC-mediated intermolecular DNA cross-linking.

Greater understanding of the mutual influence between DNA and the associated nanomaterial on the properties of each other can provide alternative strategies for designing and developing DNA nanomachines. DNA secondary structures are essential for encapsulating highly emissive silver nanoclusters (DNA/AgNCs). Likewise, AgNCs stabilize secondary DNA structures, such as hairpin DNA, duplex DNA, and parallel-motif DNA triplex. In this study, we found that the fluorescence of AgNCs encapsulated within a Hoogsteen triplex DNA structure can be turned on and off in response to pH changes. We also show that AgNCs can act as nanoscale rivets, linking two functionally distinctive DNA nanostructures. For instance, we found that a Hoogsteen triplex DNA structure with a seven-cytosine loop encapsulates red fluorescent AgNCs. The red fluorescence faded under alkaline conditions, whereas the fluorescence was restored in a near-neutral environment. Hairpin DNA and random DNA structures did not exhibit this pH-dependent AgNCs fluorescence. A fluorescence lifetime measurement and a small-angle X-ray scattering analysis showed that the triplex DNA-encapsulated AgNCs were photophysically convertible between bright and dark states. An in-gel electrophoresis analysis indicated that bright and dark convertibility depended on the AgNCs-riveted dimerization of the triplex DNAs. Moreover, we found that AgNCs rivet the triplex DNA and hairpin DNA to form a heterodimer, emitting orange fluorescence. Our findings suggest that AgNCs between two cytosine-rich loops can be used as nanorivets in designing noncanonical DNA origami beyond Watson–Crick base pairing.

DNA-encapsulated Silver Nanoclusters (DNA/AgNCs) based sensors have gained increasing attention in past years due to their diverse applications in bioimaging, biosensing, and enzymatic assays. Given the potential of DNA/AgNCs for practical applications, the systematic studies of the fluorescent stability over an extended period is necessary. However, the correlation between nucleic acid properties and the long-term stability of DNA/AgNCs is less known. With locking-to-unlocking sensors, in which the secondary structure of DNA template is standardized, we investigated the correlation between the DNA structure and the fluorescence stability of AgNCs. Post-synthesis of DNA/AgNCs, the fluorescence, and structures of templates were monitored over three weeks. By combining the fluorescence spectroscopy with the in-gel fluorescent assay, we found that AgNCs encapsulated by dimer-structured DNA/AgNCs templates were more stable than those of hairpin-structured DNA/AgNCs templates. While the orange fluorescence from the dimer templates increased over three weeks, the red fluorescence from the hairpin templates was diminished by >80% within two days at room temperature. Further tests revealed that hairpin-encapsulated red-emissive AgNCs is more sensitive to oxidation by atmospheric oxygen compared to dimer encapsulated orange AgNCs. Our observations may provide an important clue in encapsulating photophysically more stable AgNCs by tuning the DNA secondary structures. The proposed strategy here can be essential for pragmatic applications of DNA/AgNCs templates.