Systems, compositions, and methods for target entity detection
The present disclosure provides technologies for target entity detection. One aspect of the present disclosure provides technologies for detection (e.g., early detection) of a disease, disorder, or condition (e.g., cancer). In another aspect, technologies provided herein are useful for selecting and/or monitoring and/or evaluating efficacy of, a treatment administered to a subject in need thereof, e.g., a subject determined to have or susceptible to cancer. In some embodiments, technologies provided herein are useful for development of companion diagnostics, e.g., by measuring tumor burdens and changes in tumor burdens in conjunction with therapeutics.
1 . A method of detecting co-localization of proteins, the method comprising steps of:
capturing an extracellular vesicle (EV) with a capture agent that binds to a first protein on a surface of the EV;
contacting the captured EV with (a) a first probe comprising a first oligonucleotide linked to a first ligand that binds to a second protein on the EV surface, wherein the first oligonucleotide comprises a first double-stranded portion and a first single-stranded overhang, and (b) a second probe comprising a second oligonucleotide linked to a second ligand that binds to a third protein on the EV surface, wherein the second oligonucleotide comprises a second double-stranded portion and a second single-stranded overhang, and the overhangs are complementary to each other and can thereby hybridize when the probes are bound to their respective proteins and in proximity, wherein the second and third proteins are different;
ligating the hybridized first and the second oligonucleotides to form a ligated template; and
measuring co-localization of the second and third proteins on the EV surface by detecting levels of the ligated template.
2 . The method of claim 1 , wherein the first oligonucleotide comprises a first double-stranded portion and a first single-stranded overhang and
the second oligonucleotide comprises a second double-stranded portion and a second single-stranded overhang, and wherein the second single-stranded overhang comprises a nucleotide sequence complementary to at least a portion of the first single-stranded overhang and can thereby hybridize to the first single-stranded overhang.
3 . The method of claim 2 , wherein when the probes are bound to their respective proteins on the extracellular vesicles, the first single-stranded overhang and the second single-stranded overhang hybridize to form a double-stranded complex.
4 . The method of claim 3 , further comprising contacting the double-stranded complex with a nucleic acid ligase to generate the ligation product.
5 . The method of claim 4 , wherein the nucleic acid ligase is selected from the group comprising a T4 DNA ligase and a T7 DNA ligase.
6 . The method of claim 4 , wherein the step of detecting levels of a ligation product further comprises a step of amplifying the ligation product and detecting the presence of the ligation product.
7 . The method of claim 6 , wherein the step of amplifying is performed by quantitative polymerase chain reaction.
8 . The method of claim 1 , wherein the extracellular vesicles are immobilized on a solid substrate during the step of capturing.
9 . The method of claim 8 , wherein the solid substrate is a bead.
10 . The method of claim 8 , wherein the solid substrate is coated with the capture agent.