Mr. Oliver Zhao Profile

Oliver Zhao

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Proceedings Article | 7 March 2019 Presentation + Paper

High-throughput activator sequence selection for silver nanocluster beacons

Yu-An Kuo, Cheulhee Jung, Yu-An Chen, James Rybarski, Trung Nguyen, Yin-An Chen, Hung-Che Kuo, Oliver Zhao, Victor Madrid, Yuan-I Chen, Yen-Liang Liu, John Hawkins, Jeffrey Petty, Ilya Finkelstein, Hsin-Chih Yeh

Proceedings Volume 10893, 108930G (2019) https://doi.org/10.1117/12.2510649

KEYWORDS: Silver, Luminescence, Fluorometers, Chemistry, Target detection, Fluorescence resonance energy transfer, Image acquisition

Read Abstract +

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.

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