A New Platform for Efficient Signal Amplification of Nucleic Acid Targets in Tissue and Clinical Research: PSABER

Background and Related Knowledge

Since its first introduction by Pardue and Gall in the 1960s, in situ hybridization (ISH) has been widely adopted in research and clinical applications for its ability to spatially map nucleic acid targets in fixed samples. ISH is based on the hybridization of target RNA or DNA with complementary probes, which can be analyzed using fluorescence or transmitted light microscopy. ISH serves diverse functions including the localization of genome regions, human karyotype analysis, quantification of single-cell RNA profiling, and research on the spatial organization of chromatin within the nucleus. Among these, Fluorescent In Situ Hybridization (FISH) stands out particularly for its multiplexing capabilities, which enable the visualization of multiple RNA transcripts in a single sample via fluorescence microscopy.

Despite continuous progress in ISH, improving signal sensitivity and broadening its applicability remain persistent challenges. Signal amplification becomes crucial, especially in complex sample types such as formalin-fixed paraffin-embedded (FFPE) tissue sections. Existing amplification strategies are generally categorized into two approaches: assembly-based techniques (e.g., branched DNA amplification, hybridization chain reaction) and enzymatic approaches (e.g., horseradish peroxidase (HRP)-catalyzed signal deposition at target sites). However, these methods often exhibit challenges such as operational complexity, high costs, or limited compatibility with clinical workflows. Recently, Kishi and colleagues introduced Signal Amplification by Exchange Reaction (SABER), which incorporates tandem arrays of single-stranded DNA sequence binding sites into probes via a primer exchange reaction (PER). While SABER is cost-effective and highly sensitive, its signal amplification is modest and has limited support for colorimetric detection.

To address these limitations, the research team developed “Peroxidase SABER” (PSABER), building upon and optimizing SABER. The PSABER method aims to overcome sensitivity and applicability deficits by coupling HRP-mediated deposition of fluorescent or colorimetric substrates to achieve localized signal amplification, extending its utility to histopathological studies.

Article Origin and Authors

The study was led by Sahar Attar, Valentino E. Browning, Mary Krebs, and other researchers from multiple institutions affiliated with the University of Washington, including the Department of Genome Sciences, Institute of Stem Cell and Regenerative Medicine, and Brotman Baty Institute for Precision Medicine. The paper was published in the January 2025 issue of Nature Methods (Volume 22, pages 156–165), with DOI: https://doi.org/10.1038/s41592-024-02512-2.

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Detailed Description of the PSABER Study

Experimental Workflow Design and Technological Innovation

1. Overview of Workflow

At the heart of the PSABER study is a methodology to construct tandem arrays of target-binding sites in situ, enabling the binding of HRP-conjugated oligonucleotides that subsequently catalyze the deposition of fluorescent or colorimetric signals. The overall process comprises the following steps:

• Probe Extension: Using Primer Exchange Reaction (PER), probes are extended into concatemeric (long tandem array) oligonucleotides containing specific repeat sequences.
• Sample Hybridization: The extended probes hybridize to target DNA/RNA in fixed cells or FFPE samples.
• HRP Signal Amplification: HRP-conjugated oligonucleotides hybridize to the probes, followed by the addition of H₂O₂ and signal substrates to locally amplify fluorescence or colorimetric detection.
• Signal Amplification and Multiplexed Detection: Multiplexed detection is achieved via solution exchange, with PSABER facilitating the visualization of multiple targets in sequential rounds.

2. Validation Across Different Target Types

The team conducted experiments targeting diverse nucleic acid species: 1. RNA hybridization targeting ADAMTS1 mRNA. 2. DNA hybridization targeting genomic alpha satellite repeats. 3. Detection of a 200-kb single-copy region on the human X chromosome (Xp22.32). 4. Study of RNA targets such as the UMOD transcript and 47S pre-rRNA sequence in FFPE kidney tissue sections.

3. Solution Exchange Multiplexing

Employing solution exchange techniques, PSABER was shown to support the iterative visualization of multiple molecular targets. The study successfully visualized the spatial patterns of CBX5 mRNA, 7SK snRNA, and pre-47S rRNA in fixed cells across multiple signal development rounds.

4. Compatibility with Optical Systems

Experiments demonstrated that PSABER is not limited to high-end fluorescence microscopy. Signals remain distinguishable even under low numerical aperture (NA) systems, such as objectives with 10× or 20× magnification.

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Experimental Results and Supporting Data

Key findings from the study include:

1. Enhanced Signal Amplification: Compared to conventional FISH, PSABER provided ~25× signal amplification for RNA detection. Under extended reaction and development conditions, amplification increased to ~70×.
2. Robust Applicability: The method proved reliable across various sample types, including high-sensitivity localization of RNA in complex FFPE tissue regions.
3. Distinct Multiplexed Imaging: Data showed that PSABER signals remain distinguishable with sufficient spatial resolution and analytical accuracy using even standard, cost-effective optical setups.

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Research Significance and Scientific Value

The broad applicability of PSABER highlights its significant potential in both basic research and clinical diagnostics:

1. Scientific Value: By substantially enhancing fixed-sample signal intensity, PSABER effectively addresses ISH’s limitations under challenging experimental conditions. Its cost-effectiveness and multiplexing capabilities make it particularly valuable for resource-constrained research labs and large-scale clinical pathology detection.
2. Clinical Value: For FFPE tissue, PSABER offers a sensitive and precise alternative for the localization of RNA/DNA targets. This creates opportunities for its application in pathogenic gene diagnostics, cancer detection, and pathology.

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Key Research Highlights

1. Technological Innovation: PSABER expands the capabilities of existing SABER technology, including its ability to support colorimetric detection.
2. Highly Sensitive Signal Amplification: With signal amplification ranging from several-fold to dozens of times, PSABER is amenable to low-sensitivity imaging setups.
3. Potential for Multiplexed Detection: Solution exchange enables multi-round dye deposition, allowing more targets to be visualized with limited microscope channels.
4. Cost Efficiency: PSABER provides an economical solution compared to high-cost commercial ISH amplification methods.

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PSABER technology significantly enhances the reliability of nucleic acid detection while expanding the applicability of ISH techniques in fixed samples. This breakthrough not only addresses existing technological gaps but establishes an accessible, flexible, and efficient approach that promises transformative potential in molecular biology and pathological diagnostics.