Transcriptome In Vivo Analysis (TIVA) and Transcriptome In Situ Analysis (TISA)
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.
1 . A method of detecting a nucleic acid molecule from a cell, the method comprising the steps of:
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, wherein the first oligonucleotide comprises one or more terminal deoxynucleotides and an antisense oligonucleotide comprising a nucleic acid sequence that is complementary to a target nucleic acid molecule of the cell;
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 in the molecule;
allowing the formation of a complex comprising the target nucleic acid molecule hybridized to the first oligonucleotide; and
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 comprises 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.
2 . The method of claim 1 , wherein the antisense oligonucleotide comprises a nucleic acid sequence that is complementary to the polyA tail of an mRNA, wherein the RNA comprises mRNA, and where the method detects the mRNA transcriptome of the cell.
3 . The method of claim 1 , wherein the hybrid nucleic acid molecule further comprises an index oligonucleotide.
4 . The method of claim 1 , further comprising processing the synthesized cDNA to generate a double-stranded cDNA comprising an index oligonucleotide and a promoter region.
5 . The method of claim 1 , wherein the method comprises administering to the cell a plurality of hybrid nucleic acid molecules, each hybrid nucleic acid molecule comprising a different index oligonucleotide.
6 . The method of claim 1 , wherein the cell is fixed.
7 . The method of claim 6 , wherein the cell is in a fixed tissue sample.
8 . The method of claim 7 , wherein the fixed tissue sample is immunostained to distinguish between two or more cell types.
9 . The method of claim 7 , wherein the fixed tissue sample is stained to distinguish between two or more subcellular compartments.
10 . The method of claim 1 , wherein activation of the hybrid nucleic acid molecule comprises administering ultraviolet light to the cell.
11 . The method of claim 1 , wherein activation of the hybrid nucleic acid molecule comprises administering light to the cell, wherein the light has a wavelength of about 350 nm-1500 nm.
12 . The method of claim 1 , wherein activation of the hybrid nucleic acid molecule comprises exposing the cell to two photon excitation of near-infrared or infrared light.