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.