Invented in 2010, NanoCluster Beacons (NCBs) (1) are an emerging class of turn-on probes that show unprecedented capabilities in single-nucleotide polymorphism (2) and DNA methylation (3) detection. As the activation colors of NCBs can be tuned by a near-by, guanine-rich activator strand, NCBs are versatile, multicolor probes suitable for multiplexed detection at low cost. Whereas a variety of NCB designs have been explored and reported, further diversification and optimization of NCBs require a full scan of the ligand composition space. However, the current methods rely on microarray and multi-well plate selection, which only screen tens to hundreds of activator sequences (4, 5). Here we take advantage of the next-generation-sequencing (NGS) platform for high-throughput, large-scale selection of activator strands. We first generated a ∼104 activator sequence library on the Illumina MiSeq chip. Hybridizing this activator sequence library with a common nucleation sequence (which carried a nonfluorescent silver cluster) resulted in hundreds of MiSeq chip images with millions of bright spots (i.e. light-up polonies) of various intensities and colors. With a method termed Chip- Hybridized Associated Mapping Platform (CHAMP) (6), we were able to map these bright spots to the original DNA sequencing map, thus recovering the activator sequence behind each bright spot. After assigning an "activation score" to each "light-up polony", we used a computational algorithm to select the best activator strands and validate these strands using the traditional in-solution preparation and fluorometer measurement method. By exploring a vast ligand composition space and observing the corresponding activation behaviors of silver clusters, we aim to elucidate the design rules of NCBs.
|Title of host publication||Reporters, Markers, Dyes, Nanoparticles, and Molecular Probes for Biomedical Applications XI|
|Editors||Samuel Achilefu, Ramesh Raghavachari|
|Publication status||Published - 2019|
|Event||Reporters, Markers, Dyes, Nanoparticles, and Molecular Probes for Biomedical Applications XI 2019 - San Francisco, United States|
Duration: 2019 Feb 4 → 2019 Feb 5
|Name||Progress in Biomedical Optics and Imaging - Proceedings of SPIE|
|Conference||Reporters, Markers, Dyes, Nanoparticles, and Molecular Probes for Biomedical Applications XI 2019|
|Period||19/2/4 → 19/2/5|
Bibliographical noteFunding Information:
This research is financially supported by Robert A. Welch Foundation (F-1833 to H.-C. Y.), National Science Foundation (Grant 1611451), and Associate Professor Experimental (APX) collaborative research fund at UT Austin (to H.-C. Y.). Jeff Petty thanks the National Institutes of Health (1R15GM102818) and the National Science Foundation EPSCoR Program under NSF Award #OIA-1655740. Special thanks for Jessica Podnar who helped us processing NGS chip production in GSAF and solve problem encountered during library preparation.
© 2019 SPIE.
- Fluorescence microscopy
- Next-generation sequencing
- Silver nanoclusters
- Turn-on probes
ASJC Scopus subject areas
- Electronic, Optical and Magnetic Materials
- Atomic and Molecular Physics, and Optics
- Radiology Nuclear Medicine and imaging