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by claude@2026-06, 2026-06-24
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The paper presents PASTA (Protein and nucleic Acid Serial Tyramine Amplification), an HRP-based signal amplification method that uses tyramine radicals to deposit oligonucleotides for highly multiplexed spatial imaging across many biomarker modalities. In demonstrations, the authors report up to 100-fold signal enhancement for markers with minimal background in blank controls and show compatibility with in situ hybridization, proximity ligation assays, sequential antibody staining, and modular combinations, including antibody rescue for targets with suboptimal signal-to-noise and multi-round RNA probe systems. A major caveat highlighted is that performance and applicability depend on integration with existing spatial profiling workflows and on controlling background under blank controls. This paper does not explicitly discuss endometriosis or adenomyosis; it was included in the corpus via a keyword match in the upstream search index.
Abstract
Spatial proteomics techniques have revolutionized our understanding of tissue architecture, but are frequently limited by detection sensitivity, bioconjugation limitations, multiplexing capacity, and multi-modal integration. Here we present P rotein and nucleic A cid S erial T yramine A mplification (PASTA), a novel signal amplification approach that significantly enhances detection sensitivity while maintaining compatibility with diverse spatial profiling methodologies. PASTA utilizes horseradish peroxidase (HRP) recruitment pathways to generate tyramine radicals that deposit oligonucleotides, enabling adaptable signal amplification across multiple biomarkers at high-plex via cyclical imaging using complementary fluorophore-labeled oligonucleotides. We demonstrate that PASTA achieves up to 100-fold signal enhancement for markers with minimal background in blank controls. The method is compatible with in situ hybridization for DNA/RNA detection, proximity ligation assays for protein-protein interactions, sequential antibody staining protocols, or any modular combination thereof. PASTA enables antibody rescue of markers with suboptimal signal-to-noise ratios and is versatile in its applications to unconjugated antibodies, and multi-round probe-based RNA detection systems beyond current capabilities. This technique addresses key limitations in spatial-omics by enhancing sensitivity for challenging targets while maintaining compatibility with established multiplexing strategies, providing a versatile, cost-efficient, and valuable tool for comprehensive spatial tissue analysis in both research and clinical applications.
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Abstract
Spatial proteomics techniques have revolutionized our understanding of tissue architecture, but are frequently limited by detection sensitivity, bioconjugation limitations, multiplexing capacity, and multi-modal integration. Here we present Protein and nucleic Acid Serial Tyramine Amplification (PASTA), a novel signal amplification approach that significantly enhances detection sensitivity while maintaining compatibility with diverse spatial profiling methodologies. PASTA utilizes horseradish peroxidase (HRP) recruitment pathways to generate tyramine radicals that deposit oligonucleotides, enabling adaptable signal amplification across multiple biomarkers at high-plex via cyclical imaging using complementary fluorophore-labeled oligonucleotides. We demonstrate that PASTA achieves up to 100-fold signal enhancement for markers with minimal background in blank controls. The method is compatible with in situ hybridization for DNA/RNA detection, proximity ligation assays for protein-protein interactions, sequential antibody staining protocols, or any modular combination thereof. PASTA enables antibody rescue of markers with suboptimal signal-to-noise ratios and is versatile in its applications to unconjugated antibodies, and multi-round probe-based RNA detection systems beyond current capabilities. This technique addresses key limitations in spatial-omics by enhancing sensitivity for challenging targets while maintaining compatibility with established multiplexing strategies, providing a versatile, cost-efficient, and valuable tool for comprehensive spatial tissue analysis in both research and clinical applications.
Competing Interest Statement
S.J. is a co-founder of Elucidate Bio Inc, has received speaking honorariums from Cell Signaling Technology, and has received research support from Roche unrelated to this work. S.J. and Y.B. are listed as inventors on Patent WO2020176534A1 on multiplexed signal amplification methods using enzymatic-based chemical deposition. S.J. and H.A.M. are listed as inventors on a patent application based on the work presented in this manuscript has been filed by Beth Israel Deaconess Medical Center (BIDMC). S.S. reports receiving commercial research grants from Bristol-Myers Squibb, AstraZeneca, Exelixis, Merck, NiKang Therapeutics, and Arsenal Biosciences; is a consultant/advisory board member for Merck, AstraZeneca, Bristol Myers Squibb, NextPoint Therapeutics, AACR, and NCI; receives royalties from Biogenex; and mentored several non-US citizens on research projects with potential funding (in part) from non-US sources/Foreign Components.
Footnotes
↵+ Lead contact: sjiang3{at}bidmc.harvard.edu
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