SABER amplifies FISH: enhanced multiplexed imaging of RNA and DNA in cells and tissues

Abstract

Fluorescence in situ hybridization (FISH) reveals the abundance and positioning of nucleic acid sequences in fixed samples. Despite recent advances in multiplexed amplification of FISH signals, it remains challenging to achieve high levels of simultaneous amplification and sequential detection with high sampling efficiency and simple workflows. Here, we introduce signal amplification by exchange reaction (SABER), which endows oligo-based FISH probes with long, single-stranded DNA concatemers that aggregate a multitude of short complementary fluorescent imager strands. We show that SABER amplifies RNA and DNA FISH signals (5 to 450-fold) in fixed cells and tissues, apply 17 orthogonal amplifiers against chromosomal targets simultaneously, and detect mRNAs with high efficiency. We further apply 10-plexSABER-FISH to identify in vivo introduced enhancers with cell type-specific activity in the mouse retina. SABER represents a simple and versatile molecular toolkit for rapid and cost-effective multiplexed imaging of nucleic acid targets.

Nanobarcodes with multidimensional optical information beyond diffraction limit

“…7,29 The developed geometrical nanobarcodes could be simultaneously used for tagging different cellular species and identi ed by the super-resolution nanoscopy, bypassing the need for the stepwise addition of dyes for multiplexed imaging of intracellular structures. 47,48 The speed of this super-resolution technique remains low, but this disadvantage can also be circumvented by employing parallelized scanning with beam arrays in the future. 49 These nanobarcodes are also readily applicable for high-security anticounterfeiting when different batches of them are blended with inks and can be readily printed on high-value products for authenti cation.…”

The Growing Impact of Micro/Nanomaterial‐Based Systems in Precision Oncology: Translating “Multiomics” Technologies

“…SABER involved the use of fluorescent in situ hybridization (FISH) probes with long, single-stranded DNA concatemers that aggregated a multitude of short complementary fluorescent imager strands (Figure 14d) to amplify DNA and RNA FISH signals (5-to 450-fold) in fixed cells and tissues. [146] As an effective and simple method to robustly detect RNA and DNA sequences in cells and tissue, SABER evidenced the use of DNA nanostructures as a form of nanomaterial for powerful multiplex interspecies biomarker analysis.…”

Exploring single cells in space and time during tissue development, homeostasis and regeneration

“…Similar to in situ sequencing, multiplexed smFISH is limited by factors such as auto-fluorescence and spatial crowding within cells, when transcripts are too close for simultaneous optical resolution. To overcome this, clearing (Moffitt et al, 2016a) and signal amplification (Choi et al, 2014;Shah et al, 2016a;Kishi et al, 2019) approaches have been developed. In HCR-seqFISH, for example, seqFISH is combined with single-molecule hybridization chain reaction (smHCR) to achieve signal amplification (Shah et al, 2016a); this approach was applied successfully to quantify single cell transcription profiles within the mouse hippocampus (Shah et al, 2016b).…”

“…Indeed, using the latter method, the profiling of 10,421 nascent transcripts (Shah et al, 2018) as well as the imaging of RNAs for 10,000 genes in single cells has been demonstrated (Eng et al, 2019). As alternatives to sequential barcoding, other methods have used sequential hybridization whereby each RNA molecule is directly encoded by a unique color in each round, and multiplexing is achieved by multiple rounds of hybridization (Codeluppi et al, 2018;Kishi et al, 2019;Shah et al, 2016b) (Fig. 3A).…”