Exonuclease enabled proximity extension assays

Inventors

Fredriksson, Simon • Lundberg, Martin • Eriksson, Anna • Rennel-Dickens, Emma

Assignees

Olink Proteomics AB

Abstract

The present invention relates to a proximity probe based detection assay (“proximity assay”) for an analyte in a sample, specifically a proximity probe extension assay (PEA), an in particular to an improvement in the method to reduce non-specific “background” signals, wherein the improvement comprises the use in such assays of a component comprising 3′ exonuclease activity, said method comprising: (a) contacting said sample with 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; (b) allowing the nucleic acid domains of the proximity probes to interact with each other upon binding of said proximity probes to said analyte, wherein said interaction comprises the formation of a duplex; (c) contacting said sample with a component comprising 3′ exonuclease activity; (d) extending the 3′ end of at least one nucleic acid domain of said duplex to generate an extension product, wherein the step may occur contemporaneously with or after step (c); and (e) amplifying and detecting the extension product.

Core Innovation

The invention provides a method for detecting an analyte in a sample using proximity extension assays in which at least first and second proximity probes each comprise an analyte-binding domain and a nucleic acid domain. The proximity probes are allowed to simultaneously bind to the analyte, and the nucleic acid domains interact to form a duplex. The method then extends a 3′ end of the duplex to generate an extension product, followed by amplification and detection of the amplified extension product.

A central feature is the introduction of a component comprising 3′ exonuclease activity during and/or in conjunction with duplex extension. The component comprises a polymerase enzyme having 3′ exonuclease activity and/or an exonuclease enzyme other than the polymerase enzyme. This enables 3′ exonuclease activity to act on nucleic acid structures associated with the assay workflow, thereby preventing non-specific extension products and increasing signal-to-noise, specificity, and sensitivity, including in exonuclease-rich samples such as plasma.

The invention further supports multiplex analysis by providing multiple sets of proximity probes for two or more analytes, where duplex formation and extension are performed to generate unique extension products for each set. Amplification is performed for each unique extension product, with amplification reagents selected from a primer for amplification, a padlock probe or circular oligonucleotide, or a template oligonucleotide that acts as a ligation template for circularization of an oligonucleotide comprising an extended part of the extension product. Detection is then performed on the amplified unique extension products in the multiplex format.

Claims Coverage

The document includes two independent claims that share a common core: proximity probe duplex formation, 3′ exonuclease activity during or alongside 3′ end extension, generation of an extension product, amplification using specified amplification reagents, and detection. Across the independent claims, three main inventive aspects are multiplex-capable proximity probe duplex extension with 3′ exonuclease activity, and constrained amplification reagent selection.

Across the independent claims, the coverage centers on proximity probes that form duplexes upon analyte binding, a component providing 3′ exonuclease activity in relation to 3′ end extension, and amplification performed using amplification reagents selected from primer or padlock/circular oligonucleotide or template oligonucleotide configured as a ligation template for circularization. The multiplex independent claim further requires unique extension products per analyte set, followed by amplification and detection of each unique product.

Stated Advantages

Documented Applications