Transcriptome In Vivo Analysis (TIVA) and Transcriptome In Situ Analysis (TISA)
Inventors
Eberwine, James • Dmochowski, Ivan J. • Yeldell, Sean B. • Griepenburg, Julianne C. • Rapp, Teresa L. • Singh, Jennifer M. • Sul, Jai-Yoon • Lee, Jaehee
Assignees
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Publication Number
US-12662501-B2
Publication Date
2026-06-23
Expiration Date
Abstract
Compositions and methods of capturing one or more nucleic acid molecules of a cell or subcellular compartment are described. In certain aspects, the compositions comprise a caged molecule comprising one or more photolinkers and an antisense oligonucleotide, which when uncaged hybridizes to a target nucleic acid molecule.
Core Innovation
The invention provides Transcriptome in Vivo Analysis (TIVA) and Transcriptome in situ Analysis (TISA) approaches for detecting a nucleic acid molecule from a cell by introducing a hybrid nucleic acid molecule into the cell. The hybrid nucleic acid molecule comprises a first, second, and third oligonucleotide linked through photocleavable linkers, and the first oligonucleotide comprises one or more terminal deoxynucleotides and an antisense oligonucleotide complementary to a target nucleic acid molecule.
The disclosed workflow activates the hybrid nucleic acid molecule under conditions suitable for binding of the first oligonucleotide to the target nucleic acid molecule, where activation cleaves the photocleavable linkers. This allows formation of a complex comprising the target nucleic acid molecule hybridized to the first oligonucleotide, followed by synthesizing cDNA from the target nucleic acid molecule using the first oligonucleotide and the one or more terminal deoxynucleotides as a primer.
The target nucleic acid molecule comprises RNA, and the one or more terminal deoxynucleotides comprise one or more thymidines at the 3′ end of the first oligonucleotide. The first oligonucleotide comprises 2′-fluoro uridine, and the document further describes Ru-photochemistry and ruthenium-based photolinkers as enabling activation in biological samples using light and, in some forms, two-photon excitation.
Beyond single-target detection, the document describes optional multiplexing and longitudinal transcriptome analysis through indexing and promoter elements associated with the synthesized cDNA. It also describes optional complex isolation and labeling via affinity tags and optional fluorophore/FRET pairs, including applications to fixed tissues with staining to distinguish cell types or subcellular compartments.
Claims Coverage
The independent claim recites three core inventive features, with dependent claims refining activation conditions, downstream processing, and tissue context.
Photocleavable three-oligonucleotide hybrid construct with terminal deoxynucleotides
Introducing a hybrid nucleic acid molecule into the cell, the hybrid nucleic acid molecule comprising a first, second, and third oligonucleotide, wherein the first oligonucleotide is linked to the second oligonucleotide through a first photocleavable linker and the second oligonucleotide is linked to the third oligonucleotide through a second photocleavable linker, and wherein the first oligonucleotide comprises one or more terminal deoxynucleotides and an antisense oligonucleotide complementary to a target nucleic acid molecule of the cell.
Light activation that cleaves photocleavable linkers to form a target hybrid complex
Activating the hybrid nucleic acid molecule under conditions suitable for binding of the first oligonucleotide to the target nucleic acid molecule of the cell, wherein the activation cleaves the photocleavable linkers and allows formation of a complex comprising the target nucleic acid molecule hybridized to the first oligonucleotide.
In situ cDNA synthesis from the target RNA using the first oligonucleotide and terminal deoxynucleotides as a primer
Synthesizing cDNA from the target nucleic acid molecule using the first oligonucleotide and the one or more terminal deoxynucleotides as a primer, wherein the target nucleic acid molecule comprises RNA, wherein the one or more terminal deoxynucleotides comprise one or more thymidines, wherein the first oligonucleotide comprises 2′-fluoro uridine, and wherein the one or more thymidines are at the 3′ end of the first oligonucleotide.
Overall, the claims cover RNA detection in cells using a three-oligonucleotide photocleavable hybrid construct, light activation to cleave linkers and enable target hybridization, and cDNA synthesis using 3′-end thymidines with a first oligonucleotide comprising 2′-fluoro uridine.
Stated Advantages
Ru-photochemistry and ruthenium-based photolinkers enable activation in biological samples using light and, in some forms, two-photon excitation.
Documented Applications
Transcriptome in vivo analysis (TIVA) and transcriptome in situ analysis (TISA) by detecting a nucleic acid molecule from a cell using the described hybrid nucleic acid molecule and cDNA synthesis workflow.
Detection of RNA targets in fixed cells or tissue contexts, including immunostaining to differentiate between two or more cell types.
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