biostudies-literatureKuo YATexas 4000Welch FoundationNational Institutes of HealthUT-Austin Catalyst GrantNIGMS NIH HHSNIH HHSNational Science Foundatione2204957https://www.ebi.ac.uk/biostudies/studies/S-EPMC9588665biostudies-literatureUnknown34(41)NanoCluster Beacons (NCBs) are multicolor silver nanocluster probes whose fluorescence can be activated or tuned by a proximal DNA strand called the activator. While a single-nucleotide difference in a pair of activators can lead to drastically different activation outcomes, termed polar opposite twins (POTs), it is difficult to discover new POT-NCBs using the conventional low-throughput characterization approaches. Here, a high-throughput selection method is reported that takes advantage of repurposed next-generation-sequencing chips to screen the activation fluorescence of ≈40 000 activator sequences. It is found that the nucleobases at positions 7-12 of the 18-nucleotide-long activator are critical to creating bright NCBs and positions 4-6 and 2-4 are hotspots to generate yellow-orange Advanced materials (Deerfield Beach, Fla.)Massively Parallel Selection of NanoCluster Beacons.PMC9588665F‐1155F‐1808F-1808R21 GM129617F-1155CBET2029266GM129617CBET2041345F-1833F‐1833Wylie DCWalker JNChen YAYeh HCNguyen TDKuo HCFinkelstein IJJung CRybarski JRBrodbelt JSChen YIPetty JTZhao OSHawkins JAKuo YAShields SWJHong SfalseMassively Parallel Selection of NanoCluster Beacons.NanoCluster Beacons (NCBs) are multicolor silver nanocluster probes whose fluorescence can be activated or tuned by a proximal DNA strand called the activator. While a single-nucleotide difference in a pair of activators can lead to drastically different activation outcomes, termed polar opposite twins (POTs), it is difficult to discover new POT-NCBs using the conventional low-throughput characterization approaches. Here, a high-throughput selection method is reported that takes advantage of repurposed next-generation-sequencing chips to screen the activation fluorescence of ≈40 000 activator sequences. It is found that the nucleobases at positions 7-12 of the 18-nucleotide-long activator are critical to creating bright NCBs and positions 4-6 and 2-4 are hotspots to generate yellow-orange 2022-01-01T00:00:00Z2022 Oct2025-04-25T21:32:10.021Z2025-04-06T08:43:06.321ZS-EPMC95886653594515910.1002/adma.202204957