Method for generating proximity probes
Inventors
Fredriksson, Johan Erik Simon • LUNDBERG, Klas Martin
Assignees
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Publication Number
US-10781473-B2
Publication Date
2020-09-22
Expiration Date
Abstract
The present invention provides a plurality of pairs of proximity probes, each pair being capable of binding to a different target analyte, wherein the first and second proximity probes of each pair of probes comprise universal oligonucleotides conjugated to their analyte binding moieties, and hybridised to the universal oligonucleotides are different tag oligonucleotides comprising universal complement domains common to all tag oligonucleotides and unique domains unique to each tag oligonucleotide, as well as methods for their production.
Core Innovation
The invention relates to manufacturing a plurality of pairs of proximity probes for different target analytes, where each pair of proximity probes comprises a first and a second proximity probe that can simultaneously bind to a target analyte. Each proximity probe includes an analyte binding domain and a partially double-stranded nucleic acid domain, and the nucleic acid domains of the pair are able to interact directly or indirectly upon binding of the pair to their target analyte.
The manufacturing approach uses universal oligonucleotides conjugated to analyte binding moieties, followed by hybridising tag oligonucleotides to the universal oligonucleotides to form proximity probes. A plurality of different first tag oligonucleotides hybridises to a set of first universal conjugates, each tag including a first universal complement domain common to all first tag oligonucleotides and a first unique domain that is not capable of hybridising to a first or second universal oligonucleotide. Likewise, a plurality of different second cognate tag oligonucleotides hybridise to second universal conjugates using a second universal complement domain and a second unique domain.
The method enables selection of multiple first proximity probes and multiple cognate second proximity probes to provide a plurality of pairs of proximity probes, thereby enabling multiplex detection by pairing cognate tags. The nucleic acid domains in the resulting pairs are configured to interact upon target binding, including interactions compatible with proximity extension, proximity ligation, and in situ PLA concepts. The described strategy is modular through the use of universal complement regions and unique domains on tag oligonucleotides, and it supports direct or indirect nucleic-acid domain interactions.
Claims Coverage
The independent claim describes a manufacturing method for paired proximity probes with a universal/unique tag architecture. The main inventive features concentrate on using first and second universal oligonucleotides conjugated to analyte binding moieties, hybridising universal-complement/common regions with tag oligonucleotides that contain unique domains, and selecting cognate pairs whose nucleic acid domains can interact upon target binding.
Universal conjugates to form first proximity probes
Conjugating a first universal oligonucleotide to each of a plurality of first analyte binding moieties, to form a set of first universal conjugates; and hybridising to each first universal oligonucleotide of the set of first universal conjugates one of a plurality of different first tag oligonucleotides, each different first tag oligonucleotide comprising a first universal complement domain which is common to all first tag oligonucleotides and which is complementary to the first universal oligonucleotide, and a first unique domain which is unique to each different first tag oligonucleotide and which is not capable of hybridising to a first or second universal oligonucleotide, thereby to form a plurality of first proximity probes.
Universal conjugates to form second proximity probes
Conjugating a second universal oligonucleotide to each of a plurality of second analyte binding moieties, to form a set of second universal conjugates; and hybridising to each second universal oligonucleotide of the set of second universal conjugates one of a plurality of different second cognate tag oligonucleotides, each different second tag oligonucleotide comprising a second universal complement domain which is common to all second tag oligonucleotides and which is complementary to the second universal oligonucleotide and a second unique domain which is unique to each different second tag oligonucleotide, and which is not capable of hybridising to a first or second universal oligonucleotide, thereby to form a plurality of second proximity probes.
Cognate selection of proximity probe pairs
Selecting multiple first proximity probes from said plurality of first proximity probes and multiple cognate second proximity probes from said plurality of second proximity probes, thereby to provide a plurality of pairs of proximity probes, wherein each pair of proximity probes comprises a first and a second proximity probe capable of simultaneously binding to a different target analyte and wherein the nucleic acid domains of a pair of proximity probes are able to interact directly or indirectly upon binding of the pair of proximity probes to their target analyte.
The claim set coverage centers on manufacturing paired proximity probes by conjugating first and second universal oligonucleotides to analyte binding moieties, hybridising different tag oligonucleotides containing common universal-complement domains and unique domains to form first and second proximity probes, and selecting cognate pairs so that the partially double-stranded nucleic acid domains of the pair can interact directly or indirectly when both probes bind the target analyte.
Stated Advantages
Supports multiplex detection by providing a plurality of pairs of proximity probes using universal oligonucleotides and selectable cognate tag domains.
Enables modular switching of tag sequence specificities through universal complement regions and unique domains on tag oligonucleotides.
Documented Applications
Compatible with proximity extension assay (PEA), proximity ligation assay (PLA), and in situ PLA concepts via direct or indirect nucleic-acid domain interactions including duplexing, splints, and templated ligation/extension concepts.
Interested in licensing this patent?