NanoCluster Beacon - A new molecular probe for homogeneous detection of nucleic acid targets

Oligonucleotide-templated nanoclusters consisting of a few atoms of silver (DNA/Ag NCs) have been made into a new molecular probe that “lights up” upon target DNA binding, termed a NanoCluster Beacon (NCB). We discovered that interactions between silver nanoclusters and a proximal, guanine-rich DNA strand can lead to tremendous red fluorescence enhancement [1]. Here we show that dark silver nanoclusters templated on an ssDNA can be lit up into a palette of colors (green, yellow/orange, and red) by employing different proximal sequences, potentially enabling the use of NCBs in multiplexed assays. We tested a suite of nanocluster-nucleation sequences and found the sequences that created strong red fluorescence enhancement share a common 5´-C₃NNNNNC₄ motif, where N is either a thymine (T) or an adenine (A) base. We optimized the design of NCBs by testing the effect of different lengths of an interaction stem. The highest signal-to-background (S/B) ratio, 175, was achieved when the stem length was 3 base pairs long.