Transcriptional programs specify and elaborate cell identity during animal development, as a single cell gives rise to the hundreds of cell types that comprise the adult animal. The time, place, level and molecular context in which a gene is expressed are therefore critical for its function. Imaging techniques are currently in a unique position to capture all of these features simultaneously with sub-cellular resolution. In situ hybridization, where a target mRNA is hybridized to a complementary nucleic acid probe, is the primary method used to visualize mRNA expression in intact embryos and tissue samples. However, current detection methods for mRNA in situ hybridization do not take full advantage of the potential of imaging to capture quantitative information about the expression of many genes over space and time. Here, we propose to develop a fluorescent detection method for mRNA in situ hybridization based on the concept of a """"""""metafluorophore"""""""": a programmable DNA scaffold to target defined numbers of fluorophores to mRNA. Compared to current methods, this approach will improve signal to noise characteristics, will be quantitative, will be able to detecting many genes simultaneously and will be more feasible - in terms of cost, scale and time to usable data.
All cells in the body have the same genome, but acquire different capabilities by expressing subsets of their genes. This proposal develops technology to measure the level of expression of many genes in individual cells using fluorescent imaging, and applies it to understand how gene expression varies during development.
|Jungmann, Ralf; AvendaÃ±o, Maier S; Dai, Mingjie et al. (2016) Quantitative super-resolution imaging with qPAINT. Nat Methods 13:439-42|
|Dai, Mingjie; Jungmann, Ralf; Yin, Peng (2016) Optical imaging of individual biomolecules in densely packed clusters. Nat Nanotechnol 11:798-807|
|Beliveau, Brian J; Boettiger, Alistair N; AvendaÃ±o, Maier S et al. (2015) Single-molecule super-resolution imaging of chromosomes and in situ haplotype visualization using Oligopaint FISH probes. Nat Commun 6:7147|
|Iinuma, Ryosuke; Ke, Yonggang; Jungmann, Ralf et al. (2014) Polyhedra self-assembled from DNA tripods and characterized with 3D DNA-PAINT. Science 344:65-9|
|Jungmann, Ralf; AvendaÃ±o, Maier S; Woehrstein, Johannes B et al. (2014) Multiplexed 3D cellular super-resolution imaging with DNA-PAINT and Exchange-PAINT. Nat Methods 11:313-8|
|Wunderlich, Zeba; Bragdon, Meghan D; DePace, Angela H (2014) Comparing mRNA levels using in situ hybridization of a target gene and co-stain. Methods 68:233-41|
|Wei, Bryan; Dai, Mingjie; Myhrvold, Cameron et al. (2013) Design space for complex DNA structures. J Am Chem Soc 135:18080-8|