Photocleavable Mass-Tagged Oligonucleotide Probes for Multiplexed and Multiomic Tissue Imaging of Targeted...

Area of Science:

• Biochemistry
• Molecular Biology
• Neuroscience

Background:

• Fluorescence-based in situ hybridization (FISH) methods spatially resolve DNA and RNA in tissues.
• Current FISH methods face limitations including slow speed, tissue damage, autofluorescence, and limited multiomic capabilities.
• FISH methods alone do not provide a comprehensive multiomic picture of metabolites, nucleic acids, proteins, and xenobiotics in tissues.

Purpose of the Study:

• To develop novel photocleavable mass-tagged oligonucleotide probes for amplified and multiplexed MALDI mass spectrometric imaging-based in situ hybridization (MALDI-ISH).
• To establish a fully mass spectrometric workflow enabling multiomic imaging of metabolites and transcripts.
• To demonstrate a triomic workflow for imaging transcripts, proteins, and metabolites simultaneously.

Main Methods:

• Development of photocleavable mass-tagged oligonucleotide probes using copper-free Click chemistry.
• Substitution of fluorescent detector probes with mass-tagged probes in RNAscope.
• Implementation of a mass spectrometric workflow for multiomic imaging (lipids, transcripts, proteins).
• Utilized MALDI-ISH and MALDI-immunohistochemistry (MALDI-IHC) for simultaneous molecular imaging.
• Applied K-means cluster analysis for spatial correlation of multiomic biomarkers.

Main Results:

• Novel mass-tagged probes were successfully generated and utilized in MALDI-ISH, replacing fluorescent probes without cycling.
• A mass spectrometric workflow enabled multiomic imaging of label-free lipids and targeted transcripts from Alzheimer's mouse brain tissue.
• A triomic workflow demonstrated simultaneous imaging of lipids, transcripts, and proteins on the same tissue section.
• K-means cluster analysis revealed spatial correlations between multiomic biomarkers and Alzheimer's plaques.

Conclusions:

• The developed mass-tagged probes and MALDI-ISH workflow offer a powerful alternative to FISH for spatial multiomic analysis.
• This approach overcomes key limitations of FISH, including speed and tissue damage, while enabling broader molecular profiling.
• The ability to simultaneously image transcripts, proteins, and metabolites provides a comprehensive understanding of tissue biology, particularly in diseases like Alzheimer's.