As a newly developed assay for the detection of endogenous enzyme activity at the single-catalytic-event level Rolling Circle Enhanced Enzyme Activity Detection (REEAD) has been used to measure enzyme activity in both single human cells and malaria-causing parasites parasites. assays rely on organic dye-tagged linear probes to report the rolling circle amplification products (RCPs).5 While these organic dye-tagged reporters are easy to use they are not cost-effective and can only provide modest target-to-background (T/B) ratio even with the intervention of anti- fading agents. Among all the fluorescent reporters available activatable probes 6 7 in particular offer a high T/B ratio in molecular detection – they fluoresce only upon binding with specific target molecules but otherwise remain dark. While a high T/B ratio makes quantification easier and elimination of the need to remove the unbound probes simplifies the assay 7 activatable probes COG 133 are often much more expensive than the organic dye-tagged reporter probes and difficult to prepare. For instance molecular beacons 8 the most widely used activatable probes for DNA detection need to be dually labeled (i.e. with an organic dye at one end of the hairpin and a quencher at the other end). Removal of extra dyes and singly labeled impurities during the manufacturing process is necessary adding preparation complexity and cost to the molecular beacons. Both semiconductor quantum dots and fluorescent proteins have been converted to activatable COG 133 probes for DNA detection 9 but again the material costs are high and the preparation processes are not straightforward. Here we present a versatile strategy to design activatable probes for REEAD assays that are not only simple but cost-effective. Our probes use few-atom silver nanoclusters (Ag NCs) as fluorescent reporters that can be prepared at room temperature sequential mixing of three inexpensive components in a buffer: a cytosine-rich oligonucleotide a silver salt and a reducing reagent (see ESI for detailed processes).12-14 There is no need to remove any excess reactants as they are essentially non-fluorescent thus eliminating any purification cost. COG 133 Moreover upon conversation with a nearby DNA sequence (called COG 133 an enhancer sequence15) silver clusters exhibit fluorescence activatability15-17 and tunability17 18 that cannot be obtained with organic dyes luminescent nanocrystals and fluorescent proteins.12 These COG 133 properties have led to the development of NanoCluster Beacon (NCB an activatable probe) for DNA detection16 and chameleon NanoCluster Beacon (cNCB a multicolor probe) for single-nucleotide polymorphism identification.18 In particular not relying on resonance energy transfer as activation or color-switching mechanism NCBs have achieved a T/B ratio that is five times better than that of a conventional molecular beacon in DNA sensing.16 While being explored by researchers in the detection of proteins19 20 and metabolites 21 NCBs have not been used in surface-based DNA detection. Due to their low cost and high performance (high T/B ratio and good photostability) NCBs are perfect reporter probes for REEAD assays. Results and discussion In REEAD assays (Fig. 1A) 1 the enzyme (e.g. human topoisomerase I hTopI) under detection first converts a custom-designed linear DNA substrate (the dumbbell structure) into Rabbit Polyclonal to Caspase 14 (p10, Cleaved-Lys222). a circularized product through cleavage and ligation. The circularized substrate is usually then used as the template for isothermal rolling circle amplification (RCA) on a glass surface (see ESI for detailed processes) leading to multiple (~103) tandem copies of the circularized substrate called rolling circle amplification products (RCPs). Traditionally these RCPs are visualized under a fluorescence microscope by labeling them with organic dye-tagged DNA probes.5 This organic fluorophore labeling allows direct quantification of single enzymatic events by simply counting the resulting fluorescent RCP dots. Fig. 1 (A) NCB/REEAD detection scheme (not drawn to scale). The detection of enzymatic activity involves three actions: (1) enzyme-mediated (i.e. human topoisomerase I hTopI) circularization of a synthetic DNA dumbbell hTopI substrate (2) signal enhancement … By replacing organic dye-tagged reporter probes with NCBs we take advantage of the low cost and the “fluorescing-upon-hybridization” nature of NCBs.12 16 17 The conventional NCB has a binary probe configuration (Fig. 1B) 6 having two oligonucleotide strands (an NC probe.