From Strand Design Principles to SNP Detection—Probing Oligonucleotide Hybridization at the Single Molecule Level
Johannes Stein1,2* (email@example.com), Lorenzo Magni1, Peng Yin1, Chao-ting Wu2, and George M. Church1,2
1Harvard University; and 2Harvard Medical School
Advance understanding of oligonucleotide hybridization at the single molecule level in order to maximize detection sensitivity and throughput in DNA-based highly multiplexed fluorescence microscopy applications.
Recent advances in DNA nanotechnology have become a major driver of highly multiplexed and super-resolution fluorescence microscopy (Beliveau et al. 2012; Porreca et al. 2007; Boyle et al. 2011). Clever sequential schemes of in situ sequencing and barcoding together with both libraries of Oligopaint (oligonucleotide-based primary hybridization) probes (targeting DNA and RNA) and with DNA-functionalized antibodies (targeting proteins) can provide visual access to a vast number of targets in the genome, transcriptome, or proteome with subcellular resolution (Beliveau et al. 2012; Larsson, Frisén, and Lundeberg 2021; Bouwman, Crosetto, and Bienko 2022; Zhuang 2021; Jerkovic´ and Cavalli 2021; Hickey et al. 2022). Signal amplification strategies such as rolling circle amplification (Lee et al. 2014), linear appending (Kishi et al. 2019), or a minimum number of hybridization probes per RNA/DNA (Wang et al. 2016) target allow robust detection yet come at the cost of resolution and limited throughput. This work sets out to establish a simple single-molecule approach to assay the absolute efficiency of successful 1:1 probe hybridization events, given a known number of surface-immobilized DNA origami each carrying just a single copy of the complementary target site. Researchers further quantify the influences of oligo purification, directionality, and length of single-stranded overhang and derive design principles that maximize target hybridization and, hence, minimize the need for signal amplification. In a final proof-of-concept, the team demonstrates the versatility of this approach quantifying single nucleotide polymorphism detection efficiencies.
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This project has been funded by DOE grant DE-FG02-02ER63445 (to GMC), NIH 5RM1HG011016-03 (to CTW) and by the European Molecular Biology Organization ALTF 816-2021 (to JS). Dr. Church is a founder of companies in which he has related financial interests: ReadCoor; EnEvolv; and 64-x. For a complete list of Dr. Church’s financial interests, see also v.ht/PHNc. Dr. Wu holds or has patent filings pertaining to imaging, and her laboratory has held a sponsored research agreement with Bruker Inc. Although non-equity holding, Dr. Wu is a co-founder of Acuity Spatial Genomics and, through personal connections to George Church, has equity in companies associated with him, including 10x Genomics and Twist.