High-Throughput Optimization Of Nanocluster Beacons Using An Ngs Platform

The key to future fluorescence sensing lies in the discovery of new methods for fluorescence modulation, beyond what is currently available. DNA-templated silver nanoclusters (DNA/AgNCs) are fascinating fluorescent nanomaterials whose fluorescence emission color can be tuned by their DNA hosts or activated by nearby DNA strands termed the activators. Whereas a variety of activator designs have been explored and reported, further diversification and optimization of the activated fluorescence of DNA/AgNCs require a full scan of the activator nucleobase composition space. Previously, we reported a new approach to study the fluorescence activation behavior of Ag clusters by screening ∼104 activator sequences on a single next-generation sequencing (NGS) chip (Illumina MiSeq). The NGS platform enabled us to improve the selection throughput by 100-folds and successfully tuned the activation color of an originally dark Ag cluster from yellow (590 nm) to red (665 nm) with 2-fold enhancement of its red emission. By investigating 104 activator sequences, we have identified the nucleobases that are the most critical in determining the activation intensity and color of Ag clusters. By gaining insights into the silver cluster activation behaviors in NanoCluster Beacons, eight guanine-rich 6-mer motifs, which often appeared at the center of the activators and favored the activation of Ag clusters, were discovered. Built upon the NGS selection results, we created a workflow to generate new activator designs using random sampling and a support vector machine. Moreover, an autoencoder was employed to visualize the distribution of screened activators in latent space. Our preliminary results indicated that the NGS chips are a great platform to predictively design NanoCluster Beacons.