Transcriptome in vivo analysis
The invention provides compositions and methods that permit a hybrid nucleic acid molecule to enter a cell and when specifically activated within the cell, the molecule anneals to endogenous cellular RNA and permits the isolation of the RNA.
1. A hybrid nucleic acid molecule comprising:
a) a first, second, and third oligomer, wherein the first oligomer is linked to the second oligomer through a first photocleavable linker and the second oligomer is linked to the third oligomer through a second photocleavable linker, wherein the first, second, and third oligomers are arranged in a caged hairpin conformation
wherein the second oligomer comprises a poly-A nucleic acid sequence, comprising at least 7 consecutive adenosines, that is complementary to a first nucleic acid sequence of the first oligomer, such that the first oligomer is hybridized to the second oligomer, and
wherein the third oligomer comprises a poly-A nucleic acid sequence, comprising at least 7 consecutive adenosines, that is complementary to a second nucleic acid sequence of the first oligomer, such that the first oligomer is hybridized to the third oligomer;
b) a label for isolating nucleic acids, wherein the label is linked to the first oligomer;
c) a cell penetrating domain (CPD).
2. The molecule of claim 1 , wherein the first oligomer comprises a nucleic acid sequence comprising at least 18 consecutive 2′-fluoro nucleotides, wherein the second oligomer comprises a poly-A nucleic acid sequence comprising at least 7 consecutive adenosines, and the third oligomer comprises a poly-A nucleic acid sequence comprising at least 7 consecutive adenosines.
3. The molecule of claim 1 , wherein the first oligomer comprises nucleic acid sequence comprising at least 18 consecutive 2′-fluoro nucleotides, wherein the second oligomer comprises a poly-A nucleic acid sequence, comprising at least 14 consecutive adenosines, and the third oligomer comprises a poly-A nucleic acid sequence, comprising at least 14 consecutive adenosines.
4. The molecule of claim 1 , wherein the molecule is caged in the absence of cleavage of the photocleavable linker.
5. The molecule of claim 1 , wherein the label is biotin.
6. The molecule of claim 1 , wherein the CPD is linked to the third oligomer.
7. The molecule of claim 1 , wherein the CPD is a cell-penetrating peptide.
8. The molecule of claim 1 , wherein the CPD comprises folate.
9. The molecule of claim 1 , wherein the CPD is a compound that specifically directs the entry of the molecule into a specific cell population.
10. The molecule of claim 1 , wherein the CPD directs entry of the molecule into a subcellular compartment selected from the group consisting of nucleus, nucleosome, mitochondria, chloroplast, dendrite, soma, and lysosome.
11. A method of isolating nuclear RNA in a live cell, the method comprising the steps of: introducing the hybrid nucleic acid molecule of claim 1 into the nucleus of the cell,
activating the molecule in the nucleus under conditions suitable for binding of the nucleic acid portion of the hybrid molecule to nuclear RNA, wherein the activation cleaves the photocleavable linkers in the molecule;
allowing the formation of a hybridized nucleic acid molecule/nuclear RNA complex; and
isolating the hybridized nucleic acid molecule/nuclear RNA complex.
12. The method of claim 11 , wherein the first oligomer is at least an 18mer 2′-fluoro, wherein the second oligomer is at least a poly-A 7mer, and the third oligomer is at least a poly-A 7mer.
13. The method of claim 11 , wherein the first oligomer is at least an 18mer 2′-fluoro, wherein the second oligomer is at least a poly-A 14mer, and the third oligomer is at least a poly-A 14mer.
14. The method of claim 11 , wherein the molecule is caged in the absence of cleavage of the photocleavable linker.
15. The method of claim 11 , wherein the molecule is uncaged in the presence of cleavage of the photocleavable linker.
16. The method of claim 11 , wherein the label is linked to the first oligomer.
17. The method of claim 11 , wherein the label is biotin.
18. The method of claim 11 , wherein the CPD is linked to the third oligomer.
19. The method of claim 11 , wherein the CPD is a cell-penetrating peptide.
20. The method of claim 11 , wherein the CPD comprises folate.
21. The method of claim 11 , wherein the CPD directs the entry of the molecule into a specific cell population.
22. The method of claim 11 , wherein the CPD directs entry of the molecule into the nucleus of a cell.
23. The method of claim 11 , wherein activating the molecule comprises cleavage of the photocleavable linker.
24. The method of claim 23 , wherein cleavage of the photocleavable linker comprises administering ultraviolet light to the nucleus.
25. The method of claim 23 , wherein cleavage of the photocleavable linker comprises administering light to the nucleus, wherein the light has a wavelength of about 350 nm-1500 nm.
26. The method of claim 23 , wherein cleavage of the photocleavable linker comprises exposing the nucleus to two photon excitation of near-infrared or infrared light.