Abstract: The present invention relates to improvements in the use of padlock probes whereby the ligation of the padlock probe may be controlled, and in particular to a method of detecting a target nucleic acid in a sample which comprises the use of a padlock probe complexed with a blocking oligonucleotide. The blocking oligonucleotide binds the target-binding regions of the padlock probe and holds them apart in a manner which prevents their ligation, until the padlock probe is in the vicinity of the target nucleic acid molecule, at which point the padlock probe is released from the blocking probe so that it can bind its target, thereby reducing background signal. Also provided is a kit comprising a padlock probe and blocking oligonucleotide, which can be used in the methods of the invention.

Abstract: The present invention provides a method for detecting two target molecules in a sample, both as individual molecules and in proximity, or interaction with one another. The method involves performing three separate assay reactions to detect the first and second target molecules, and their interaction, using proximity probes which are common between the three assay reactions, and nucleic acid reagents which interact with the probes. In particular the methods use unique nucleic acid substrate molecules, such as padlock probes, to detect probes bound to their target and to determine when probes for the two targets are in close proximity, indicating an interaction between the targets.

Abstract: Nucleic acid probes for detection of a target nucleic acid molecule by an RCA reaction in the presence of the target nucleic acid molecule, comprise a first circular template strand which is capable of acting as a template for RCA, and is protected from RCA in the absence of the target nucleic acid molecule by at least a second protector strand which comprises a region of complementarity to the first template strand and is hybridised thereto to form a double-stranded circular structure containing the first template strand inside the protector strand(s). At least one of the second and/or any further protector strands comprises a target binding site, such that upon binding of the probe to the target nucleic acid molecule the probe is able to undergo a strand displacement reaction which allows RCA of the first template strand. Methods of detecting target analytes use such probes.

Abstract: The present invention relates to a method for selecting a target region of interest (ROI) in a target nucleic acid molecule using a nucleic acid probe comprising a 3? sequence capable of hybridising to a target nucleic acid molecule and acting as a primer for the production of a complement of the target ROI (i.e. by target templated extension of the primer), and a sequence capable of templating the circularisation and ligation of the extended probe comprising the reverse complement of the target ROI and a portion of the probe. The circularised molecule thus obtained contains the reverse complement of the target ROI and may be subjected to further analysis and/or amplification etc. The probe may be provided as an oligonucleotide comprising a stem-loop structure or as a partially double-stranded construct and comprises a single-stranded 3? end region containing the target-binding site.

Abstract: The present invention provides a method for detecting an analyte in a sample, said method comprising a) contacting said sample with a set of proximity probes comprising at least first and second proximity probes, which probes each comprise an analyte-binding domain capable of binding directly or indirectly to said analyte and a nucleic acid domain, such that the proximity probes can simultaneously bind, directly or indirectly, to the analyte, wherein i) the nucleic acid domains of said first and second proximity probes comprise regions capable of mediating an interaction involving said domains when under permissive conditions; and ii) the nucleic acid domain of one of said first and second probes comprises an HCR initiator region comprised within a metastable secondary structure such that it is unable to initiate an HCR reaction until released from said metastable secondary structure; b) introducing permissive conditions to allow the nucleic acid domains of said first and second probes to interact with each o

Abstract: Methods for detecting and quantifying an analyte employ a pair of proximity probes, each comprising a proteinaceous target-binding domain coupled to a nucleic acid domain (NAD), which NADs interact when the proximity probes have bound in proximity to their respective target; and a set of markers, wherein each marker is a nucleic acid molecule comprising a binding domain and a reporter domain giving a detectable signal, can interact with said NADs to form a nucleic acid molecule from which a detectable signal is generated, or with a nucleic acid molecule generated by interaction of said NADs, cannot interact with said NADs simultaneously with another marker in the set, generates a signal that is distinguishable from another marker signal, and is present in an amount capable of detecting analyte at a range of concentrations differing from the range of concentrations detectable by other markers.

Abstract: The present invention relates to a method of selecting a target region of interest (ROI) in a target nucleic acid molecule using a nucleic acid probe comprising sequences capable of directing the cleavage of a target nucleic acid molecule to release a fragment comprising the ROI and sequences capable of templating the circularisation and ligation of the target fragment. The circularised molecule thus obtained contains the selected ROI and may be subjected to further analysis and/or amplification etc. Also provided are probes and kits for use in such methods.

Abstract: 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.

Abstract: The present invention provides 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 subst