Unfolding proximity probes and methods for the use thereof
The present invention relates to a proximity-probe based detection assay for detecting an analyte in a sample and in particular to a method that comprises the use of at least one set of at least first and second proximity probes, which probes each comprise an analyte-binding domain and a nucleic acid domain and can simultaneously bind to the analyte directly or indirectly, wherein the nucleic acid domain of at least one of said proximity probes comprises a hairpin structure that can be unfolded by cleavage of the nucleic acid domain to generate at least one ligatable free end or region of complementarity to another nucleic acid molecule in said sample, wherein when the probes bind to said analyte unfolding said hairpin structure allows the nucleic acid domains of said at least first and second proximity probes to interact directly or indirectly.
1. An unfolding proximity probe comprising an analyte-binding domain coupled to a nucleic acid domain, wherein said nucleic acid domain comprises:
(i) at least one region of complementarity to a target sequence, wherein said target sequence comprises part of a ligation template for the probe and is not the analyte or any part of the analyte; and
(ii) regions of self-complementarity forming a hairpin structure present in the probe in the absence of the analyte, wherein said hairpin structure has a stem region and a loop region and inhibits said at least one region of complementarity from hybridizing to the target sequence;
wherein the hairpin structure comprises a cleavable site which is cleavable to open the hairpin structure to result in a partially double stranded nucleic acid domain which comprises one strand having free 5′ and 3′ ends and hybridized to another strand in the stem region, thereby to release said at least one region of complementarity of the nucleic acid domain and enable it to hybridize to the target sequence.
2. The unfolding proximity probe of claim 1 , wherein the analyte-binding domain is other than a nucleic acid.
3. The unfolding proximity probe of claim 1 , wherein
(i) the site at which cleavage occurs is located in the hairpin structure of the nucleic acid domain; and/or
(ii) said cleavage is enzymatic cleavage; and/or
(iii) the hairpin structure comprises a cleavage recognition site that is recognised by one or more enzymes capable of cleaving nucleic acid molecules; and/or
(iv) said one or more enzymes is a nickase or a restriction endonuclease; and/or
(v) the nickase enzyme is removed from the assay or inactivated following unfolding of the nucleic acid domain of the proximity probe.
4. The unfolding proximity probe of claim 1 , wherein upon release, the at least one region of complementarity is capable of hybridizing to the target sequence comprising said ligation template in the form of a nucleic acid domain of a proximity probe.
5. The unfolding proximity probe of claim 1 , wherein upon release, the at least one region of complementarity is capable of hybridizing to the target sequence comprising said ligation template in the form of a free oligonucleotide.
6. The unfolding proximity probe of claim 1 , wherein the free 5′ and 3′ ends of the partially double stranded nucleic acid domain are ligatable directly or indirectly to form a circular oligonucleotide.
7. The unfolding proximity probe of claim 1 , wherein the 5′ end of the partially double stranded nucleic acid domain are ligatable directly or indirectly to the 3′ end of the nucleic acid domain of a further proximity probe.
8. The unfolding proximity probe of claim 1 , wherein the 3′ end of the partially double stranded nucleic acid domain are ligatable directly or indirectly to the 5′ end of the nucleic acid domain of a further proximity probe.
9. The unfolding proximity probe of claim 6 , wherein the free 5′ and 3′ ends of the partially double stranded nucleic acid domain hybridize to their respective targets such that
(i) the free 5′ and 3′ ends hybridise to one or more ligation templates which act to template the ligation of the free ends to each other directly or indirectly;
(ii) the free 5′ and 3′ ends hybridise to the ligation template(s) directly adjacent to each other; or
(iii) the free 5′ and 3′ ends hybridise to the ligation template with a space in between and optionally, (iiia) wherein the space in between the ends is filled by a gap oligonucleotide such that each ligatable end is ligated to one end of the gap oligonucleotide, or (iiib) wherein prior to the ligation reaction the space in between the ends of the ligatable nucleic acid domain(s) is filled-in by extension of the free 3′ end using the ligation template as an extension template.
10. A sample comprising an analyte, a target sequence, and an unfolding proximity probe, wherein:
the target sequence comprises part of a ligation template for the unfolding proximity probe and is not the analyte or any part of the analyte; and
the unfolding proximity probe comprises an analyte-binding domain coupled to a nucleic acid domain, wherein said nucleic acid domain comprises:
(i) at least one region of complementarity to the target sequence; and
(ii) regions of self-complementarity forming a hairpin structure present in the probe in the absence of the analyte, wherein said hairpin structure has a stem region and a loop region and inhibits said at least one region of complementarity from hybridizing to the target sequence;
wherein the hairpin structure comprises a cleavable site which is cleavable to open the hairpin structure to result in a partially double stranded nucleic acid domain which comprises one strand having free 5′ and 3′ ends and hybridized to another strand in the stem region, thereby to release said at least one region of complementarity of the nucleic acid domain and enable it to hybridize to the target sequence.