To understand where and how much a gene is expressed or where a genetic element locates on chromosomes, in situ hybridization is often the technique of choice. In situ hybridization involves detecting nucleic acid sequences with complementary probes labeled with isotopes, fluorophores, or enzymes [Reviewed in 1]. Though in situ hybridization is powerful and enables characterization and quantification of gene expression in tissues with single cell resolution, several challenges remain.
It is challenging to examine more than a couple of genes simultaneously due to the limitation of available fluorescent channels or chromogens, and signal intensity is an issue for in situ hybridization, where low intensity leads to indistinguishable signal in complicated tissues or an environment with auto-fluorescence.
To overcome these challenges, several modifications have been developed. A number of simultaneously detected targets can be increased using a combination of fluorophores on each of the probes [2, 3] or by labeling removal and re-labeling. Signal intensity can be amplified using a probe with “branched” binding site for tagging  or in situ amplification of detectable concatemers . While these methods greatly broadened the application of in situ hybridization, the cost and complicated workflow can often limit the availability of these methods.
Kishi et al. aimed to improve fluorescence in situ hybridization (FISH) by establishing a simple but robust protocol to synthesize probes tagged by repeated sequences (or concatemers) in a programmable fashion, so that multiple fluorophore-labeled “imager” oligonucleotides complementary to the concatemer can specifically bind to and light up the probe.
Utilizing primer exchange reaction, which they previously developed , the authors demonstrated that concatemers can be synthesized efficiently with defined lengths, and different concatemer sequences can be synthesized on the same probes by using different combination of catalytic DNA hairpin with primer exchange reaction. This programmability of concatemer length enables precise control of the degree and variability of signal amplification in FISH (Figure 1).
The probes synthesized with this new technique, signal amplification by exchange reaction (SABER), were then tested on an MRC-5 cell model and retinal tissues. On MRC-5 cells, unbranched SABER provided 35 times stronger signal when detecting telomere DNA, compared to unextended FISH probes. When detecting Cbx5 mRNA, branched SABER provided even more powerful signal amplification: 464 times stronger than unextended FISH probes. On mouse retinal tissues, SABER was able to detect cell type markers specifically even with low expression level, and the signal intensity of each marker closely corresponded to relative abundance observed in single-cell RNA-seq.
In terms of the ability to multiplex, the authors showed that SABER is compatible with multiple rounds of labeling removal and relabeling and immunocytochemistry by simultaneously staining 7 transcripts with SABER along with two proteins with antibody. Furthermore, SABER is also compatible with using a combination of 3 fluorophores to perform 6-color labeling at the same time (Figure 2).
Finally, the authors examined potential enhancers for Grik1, a receptor enriched in OFF bipolar cells. Six enhancers were cloned into reporter constructs, driving expression of specific sequences detected with SABER. Two cell type-specific enhancers were identified, one driving expressing in Grik1-positive bipolar cells, and another driving expression in rod cells. Taking advantage of being able to detect DNA and RNA simultaneously, the authors also used SABER to quantify reporter transcripts and reporter plasmids at the same time to assess enhancer activity when accounting for plasmid number variation in each cell.
Why I like this preprint
The ease of manipulating nucleic acids makes in situ hybridization (ISH) time-efficient and powerful. Time from designing a new probe to experiment takes days instead of months. Nonetheless, the improvements to signal intensity and the ability to detect multiple targets require modifications that are not accessible to many labs, and thus the cost in time and resources to apply these improved ISH become similar to antibody-based detection methods.
In this preprint, Kishi et al. elegantly showed how strand displacing polymerase can be harnessed to simplify probe synthesis for signal-amplified FISH in a programmable and modular fashion. This technique allows precise control of concatemer length and a more defined degree of signal amplification. It is also compatible with fluorescence multiplexing and re-labeling, which enables potential high-throughput FISH on the same piece of tissue and the collection of expression profile with spatial resolution. FABER can potentially speed up and scale up research and clinical investigations relying on FISH and make the enhanced approach more accessible by lowering the cost of application.
Several methods were developed to amplify FISH signal, and some of them used similar approach to enhance signal intensity per probe. I was wondering how well the signal intensity and specificity of SABER looks when compared to those.
The authors showed that iterative branching concatemers enhance signal intensity by orders of magnitude in figure 2. This is a favorable feature when detecting lowly expressed transcripts, and since the probes used in figure 3 on are non-branching ones. I became curious about:
Whether the signal from probes with iterative branching concatemers still closely reflects transcript abundance
If probes with branching concatemers allow simultaneous detection of multiple probes
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