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

Many fluorescence-based in situ hybridization (FISH) methods have been developed to spatially resolve DNA (genes) and RNA (transcripts) in tissues. Signal amplification is achieved in a variety of ways, including branched DNA (bDNA) methods that create multiple fluorescent probe binding sites on the target nucleic acid. To avoid spectral overlap, high levels of multiplexing are achieved by extensive cycling, using a few nonoverlapping fluorophores per cycle. However, these methods can be slow, cause accumulating tissue damage, and are negatively impacted by autofluorescence. In addition, FISH-based methods alone do not provide a comprehensive multiomic picture of the complex biological contributions from the different molecular species in a tissue, including metabolites, nucleic acids, proteins, and xenobiotics. We report the development of novel photocleavable mass-tagged oligonucleotide probes generated by copper-free Click chemistry for use with amplified and multiplexed MALDI mass spectrometric imaging-based in situ hybridization (MALDI-ISH). These probes were successfully substituted for fluorescent detector probes using RNAscope but required no cycling. We also demonstrate a fully mass spectrometric workflow that enables multiomic imaging of label-free metabolites (lipids) and targeted transcripts from a single Alzheimer’s mouse brain tissue section. Furthermore, we demonstrate a triomic workflow where, in addition to label-free lipids, adding MALDI-ISH combined with MALDI-immunohistochemistry (MALDI-IHC) enables imaging of targeted transcripts and proteins on the same tissue section. K-means cluster analysis of multiomic biomarkers reveals spatial correlations of these various molecular species with Alzheimer’s plaques.