Genomic Science Program
U.S. Department of Energy | Office of Science | Biological and Environmental Research Program

2023 Abstracts

From Strand Design Principles to SNP Detection—Probing Oligonucleotide Hybridization at the Single Molecule Level


Johannes Stein1,2* ([email protected]), 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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Bouwman, B. A. M., N. Crosetto, and M. Bienko. 2022. “The Era of 3D and Spatial Genomics,” Trends in Genetics 38(10), 1062–75.

Boyle, S., et al. 2011. “Fluorescence In Situ Hybridization with High-Complexity Repeat-Free Oligonucleotide Probes Generated by Massively Parallel Synthesis,” Chromosome Research 19, 901–09.

Hickey, J. W., et al. 2022. “Spatial Mapping of Protein Composition and Tissue Organization: A Primer for Multiplexed Antibody-Based Imaging,” Nature Methods 19, 284–95.

Jerkovic´, I., and G. Cavalli. 2021. “Understanding 3D Genome Organization by Multidisciplinary Methods,” Nature Reviews Molecular Cell Biology 22, 511–28.

Kishi, J. Y., et al. 2019. “SABER Amplifies FISH: Enhanced Multiplexed Imaging of RNA and DNA in Cells and Tissues,” Nature Methods 16, 533–44.

Larsson, L., J. Frisén, and J. Lundeberg. 2021. “Spatially Resolved Transcriptomics Adds a New Dimension to Genomics,” Nature Methods 18, 15–18.

Lee, J. H., et al. 2014. “Highly Multiplexed Subcellular RNA Sequencing In Situ,” Science 343, 1360–63.

Porreca, G. J., et al. 2007. “Multiplex Amplification of Large Sets of Human Exons,” Nature Methods 4, 931–36.

Wang, S., et al. 2016. “Spatial Organization of Chromatin Domains and Compartments in Single Chromosomes,” Science 353, 598–602.

Zhuang, X. 2021. “Spatially Resolved Single-Cell Genomics and Transcriptomics by Imaging,” Nature Methods 18, 18–22.

Funding Information

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 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.