Detection of single and multimodal analytes
View Patent ↗A method for detecting analyte in a sample comprises: (a) contacting said sample with at least one set comprising a cassette oligonucleotide and first, second, third and fourth proximity probes, which probes each comprise an analyte-binding domain and a nucleic acid domain and can simultaneously bind to the analyte, the nucleic acid domains of said first and third proximity probes being complementary in a first overlap region and the nucleic acid domains of said second and fourth proximity probes being complementary in a second overlap region; and said cassette oligonucleotide being complementary to the nucleic acid domain of said third proximity probe in a third overlap region and to the nucleic acid domains of said fourth proximity probe in a fourth overlap region; (b) allowing the overlap regions of said proximity probes and said cassette oligonucleotide to hybridise; and (c) detecting said hybridisation. A kit is for performing the method.
1. A method for detecting an analyte in a sample, wherein the analyte can simultaneously bind four analyte-binding domains, comprising:
(a) contacting said sample with at least one set comprising a cassette oligonucleotide and first, second, third and fourth proximity probes, which probes each comprise an analyte-binding domain and a nucleic acid domain, wherein the respective analyte-binding domains simultaneously bind to the analyte;
wherein the nucleic acid domains of said first and said third proximity probes include respective first overlapping regions which are complementary and can hybridize and the nucleic acid domains of said second and said fourth proximity probes include respective second overlapping regions which are complementary and can hybridize; and
wherein said cassette oligonucleotide and the nucleic acid domain of said third proximity probe include respective third overlapping regions which are complementary and can hybridize, and said cassette oligonucleotide and the nucleic acid domain of said fourth proximity probe include respective fourth overlapping regions which are complementary and can hybridize;
(b) hybridising the respective overlapping regions of said proximity probes and said cassette oligonucleotide; and
(c) detecting the analyte in the sample based on detecting a hybridization product of step (b).
2. A method according to claim 1 , wherein said sample is further contacted with an analyte-binding moiety bound to a solid support and the analyte binds to the solid support via the analyte binding moiety.
3. A method according to claim 2 , wherein analyte-binding moiety is an antibody which binds to a different epitope of the analyte than the analyte-binding domains.
4. A method according to claim 1 , wherein said first and second overlapping regions on the first and second proximity probes are respectively located at the ends of the nucleic acid domains of said first and second proximity probes.
5. A method according to claim 1 , further comprising ligating the ends of the nucleic acid domains of said first and second proximity probes to the ends of said cassette oligonucleotide.
6. A method according to claim 1 , wherein said overlapping regions independently are 5-20 basepairs in length.
7. A method according to claim 1 , wherein said first and said third overlapping regions on the third proximity probe are immediately adjacent each other or spaced 1, 2, 3, 4, 5, 7, 10, 15, 20 or 30 nucleotides apart along the nucleic acid domain of the third proximity probe; and, independently, said second and said fourth overlapping regions on the fourth proximity probe are immediately adjacent each other or spaced 1, 2, 3, 4, 5, 7, 10, 15, 20 or 30 nucleotides apart along the nucleic acid domain of the fourth proximity probe.
8. A method according to claim 7 , further comprising filling a gap between the respective overlapping regions by means of enzymatic nucleic acid extension and/or ligation.
9. A method according to claim 1 , wherein the detection of the hybridisation product is done by nucleic acid amplification.
10. A method according to claim 1 , wherein the respective analyte-binding domains of said proximity probes bind to different epitopes of said analyte.
11. A method according to claim 1 , wherein said analyte is selected from the group consisting of proteins, peptides, endosomes, exosomes, organelles, cells, unicellular organisms, viruses, protein aggregates and multi-epitope proteins.
12. A method according to claim 1 , wherein each analyte-binding domain is an antibody or fragment thereof, a Protein A-derived affinity molecule, a molecularly imprinted polymer or any affinity binder that can be conjugated to DNA oligonucleotides.
13. A method according to claim 1 , wherein the overlapping region of the nucleic acid domain of at least one proximity probe is initially protected by a blocking DNA oligonucleotide or other blocking moiety capable of being released to allow the nucleic acid domain of the proximity probe to hybridise to the nucleic acid domain of another proximity probe to which it is complementary.
14. A method according to claim 1 , wherein said overlapping regions independently are 7-13 basepairs in length.
15. A method according to claim 1 , wherein said overlapping regions independently are about one helical turn in length.
16. A method according to claim 1 , wherein the detection of the hybridization product is done by PCR or by RCA after the hybridisation product is converted to a circularized DNA.
17. A method according to claim 1 , wherein the analyte-binding domains are antibodies and the respective antibodies bind to different epitopes of the analyte.
18. A method according to claim 17 , wherein the sample is further contacted with an antibody bound to a solid support, wherein the antibody bound to the solid support binds to a different epitope of the analyte than the analyte-binding domain antibodies, and wherein the analyte binds to the solid support via the antibody bound to the solid support.
19. A method according to claim 17 , wherein the hybridisation product is converted to a circularized DNA and the circularized DNA is detected by nucleic acid amplification.