Unfolding proximity probes and methods for the use thereof

Inventors

Landegren, Ulf • Nong, Rachel Yuan • Söderberg, Ola • Weibrecht, Irene

Assignees

Olink Proteomics AB

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.

Core Innovation

The disclosure provides an improved proximity-probe detection assay in which a set of at least first and second proximity probes each comprise an analyte-binding domain and a nucleic acid domain. The proximity probes simultaneously bind to the analyte directly or indirectly, and at least one nucleic acid domain comprises a hairpin structure that can be unfolded by cleavage of the nucleic acid domain.

Cleavage of the hairpin nucleic acid domain generates at least one ligatable free end or region of complementarity to another nucleic acid molecule in the sample, so that the nucleic acid domains of the at least first and second proximity probes can interact directly or indirectly. The interaction forms a ligation product or a nucleic acid extension product, and detecting the ligation product or the nucleic acid extension product indicates the presence of the analyte.

The invention further addresses sequential activation of proximity probes by using unfolding and cleavage to permit nucleic acid interaction after analyte binding, thereby reducing non-specific background and increasing specificity, sensitivity, efficiency, and enabling simultaneous component addition without premature probe-probe interactions. The disclosed architectures and readouts include ligation-template/splint mediated ligation and template-directed extension, and the disclosure covers multiplexing and in situ detection formats as well as kit compositions including probe sets, unfolding means, ligase/template, optional detection means, and optional blocking or gap oligonucleotides.

Claims Coverage

Two independent claims are identified: a method claim and a kit claim. Together they specify core inventive features comprising analyte-dependent simultaneous binding by first and second proximity probes, cleavage-unfoldable hairpin nucleic-acid domains producing ligatable free ends or complementary regions, and post-unfolding interaction that yields a detectable ligation or extension product, with corresponding kit components for unfolding and optionally detection.

The independent claims cover analyte-dependent proximity probe assays where hairpin nucleic acid domains are unfolded by cleavage to generate ligatable free ends or complementary regions, enabling post-unfolding interaction between nucleic acids of different proximity probes to form a detectable ligation product or nucleic acid extension product. The kit claim corresponds by requiring a probe set plus ligation-template and/or ligase components, hairpin unfolding means, and optionally detection means.

Stated Advantages

Documented Applications