Massively Parallel Selection of NanoCluster Beacons

Abstract

Whereas activatable probes have greatly simplified the assays by eliminating the need to remove unbound probes, the development of new activatable probes is largely constrained by the scarce activation mechanisms (e.g., fluorescence resonance energy transfer (FRET)), the limited activation colors (e.g., existing FRET pairs), and the poor enhancement ratios (e.g., 10-to 60-fold for a typical molecular beacon). [2] NanoCluster Beacons (NCBs) [3] are a unique class of activatable probes as they provide a palette of activation colors from the same dark origin [4] (not via FRET) and achieve fluorescence enhancement ratios as high as 1500- [5] to 2400-fold. [6] The core of an NCB is a few-atom silver nanocluster [7] (e.g., Ag 8 , Ag 10 , or Ag 16 ) whose fluorescence can be tuned by its surrounding nucleobases. [7b,c,8] To create an NCB, a dark silver nanocluster (AgNC) is first synthesized in a C-rich DNA host (termed the NC probe), and a G-rich overhang (termed the activator) is brought into close proximity of the AgNC (via target-probe hybridization, Figure S1, Supporting Information) to activate its fluorescence (Figure 1A,B). [3-5,8a,d] 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 and red POTs, respectively. Based on these findings, a "zipper-bag" model is proposed that can explain how these hotspots facilitate the formation of distinct silver cluster chromophores and alter their chemical yields. Combining high-throughput screening with machine-learning algorithms, a pipeline is established to design bright and multicolor NCBs in silico.

Massively Parallel Profiling of RNA-targeting CRISPR-Cas13d

“…Unlike earlier approaches, CHAMP does not modify any Illumina hardware and is compatible with modern sequencers and chip configurations 32,33,[42][43][44] . Imaging biomolecules on upcycled NGS chips can be adapted by any laboratory with a commercial fluorescence microscope that is capable of either TIR-or epi-illumination and a wide-field camera 36,38,52 . In addition to profiling protein-DNA and protein-RNA interactions, related methods have been adapted for peptide display and other imaging applications [53][54][55] .…”

A non-FRET DNA reporter that changes fluorescence color upon nuclease digestion

“…Subak reporters were prepared in hairpin DNA templates using the conventional nucleation-and-reduction process to form AgNCs in DNA 21,22 (Fig. 1a, Methods).…”

“…The stem was 7-bp long, which we believe holds the preferred nuclease cleavage sites. The overhangs, which are the 5'-arm and the 3'-arm, were designed based on our experience in fabricating bright and dark AgNCs in DNA (Supplementary Figure 4) 22 . In this report we focus on the two most interesting Subak reporters (termed Subak-1 and Subak-2), where Subak-1 was specifically designed for DNase I activity detection (Fig.…”

Beyond nature's base pairs: machine learning-enabled design of DNA-stabilized silver nanoclusters

“…79 Recently, Yeh and coauthors have used nextgeneration-sequencing chips to screen nearly 40 000 activator strands for NanoCluster Beacon (NCB) sensing schemes based on Ag N -DNAs. 108 NCBs are composed of a nonfluorescent or ''dark'' Ag N -DNA that becomes brightly emissive when bound to a target DNA strand, typically rich in guanines. 109 These NCBs have utility in biomedical sensing, such as single nucleotide polymorphism (SNP) detection, 80 nucleobase methylation detection, 110 and enzymatic activity detection.…”