Multiplexed Proximity Ligation Assay

A method for detecting interactions between or with any two of at least three target substrates, or any two of at least three features of a target substrate, or a combination of interactions and features of target substrates, by a multiplexed proximity ligation assay, said method comprising:

• a) for each of the at least three target substrates or features, providing a proximity probe comprising a binding moiety with affinity for the feature or binding site on said substrate, and a proximity probe oligonucleotide coupled on the binding moiety;
• wherein each of the proximity probe oligonucleotide carries a unique tag sequence;
• b) mixing the proximity probes with a sample, under a condition to allow binding of each proximity probe to its respective binding site or feature on each of said substrates through the binding moiety,
• c) simultaneous with, or following step b), forming circularized DNA molecules where any two proximity probes bind sufficiently close to each other on the substrate, wherein each of the circularized DNA molecules comprise complementary sequences to the unique tag sequences from the two proximity probes oligonucleotides;
• d) amplifying the circularized DNA;
• and
• e) characterizing the amplified DNA.

A method for detecting in a sample a target substrate or for detecting two or more substrates in proximity, by a proximity ligation assay, said target substrate(s) comprising at least two binding sites for at least two proximity probes, said method comprising:

• (a) providing at least two proximity probes, each proximity probe comprising a binding moiety capable of directly or indirectly binding to a target substrate and a proximity probe oligonucleotide coupled thereto, wherein said proximity probe oligonucleotide comprises a unique tag sequence which is complementary to, and provides a binding site for, a domain of a connector oligonucleotide;
• (b) contacting said sample with said proximity probes under conditions which allow binding of the binding moieties to their respective binding sites on the target analyte(s);
• (c) simultaneously or subsequently to step (b), contacting said sample with
• (i) at least two connector oligonucleotides, each connector oligonucleotide comprising an internal domain having a nucleotide sequence which is complementary to and capable of hybridising (in particular hybridising selectively) to a unique tag sequence of a proximity probe oligonucleotide, and a detection tag sequence;
• and
• (ii) at least two bridging (i.e. ligation template) oligonucleotides which, when the proximity probes have each bound to their respective target substrate(s) and the connectors have each bound to their respective proximity probe oligonucleotides, are each capable of hybridising to one of each of the respective ends of two connector oligonucleotides, so as to bring the respective ends of two connectors into juxtaposition for ligation directly or indirectly to one another;
• (d) ligating said connector ends directly or indirectly to one another to form a circle, (i.e. optionally with a preceding gap filling step if the connector ends are not hybridised immediately adjacent to one another on the bridging oligonucleotide);
• and
• (e) detecting and characterising the circle.