Exonuclease enabled proximity extension assays
Inventors
Fredriksson, Simon • Lundberg, Martin • Larsson, Anna • Rennel-Dickens, Emma
Assignees
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Publication Number
US-10731206-B2
Publication Date
2020-08-04
Expiration Date
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 an improvement to proximity probe extension assays (PEA) for detecting an analyte in a sample. Proximity probes comprise an analyte-binding domain and a nucleic acid domain, and the probes simultaneously bind to the analyte. After binding, the nucleic acid domains interact to form a duplex, and the 3′ end of at least one nucleic acid domain is extended to generate an extension product.
A component comprising 3′ exonuclease activity is contacted with the sample during or in connection with the extension workflow. The component comprises a polymerase enzyme having 3′ exonuclease activity and/or an exonuclease enzyme other than the polymerase enzyme. This 3′ exonuclease activity degrades free and unprotected 3′ ends on proximity probes to reduce non-specific extension products and increase specificity and/or sensitivity, including in exonuclease-rich samples such as plasma.
The assay includes amplification and detection of the extension product, with at least one amplification reaction using a separate polymerase enzyme different from the 3′ exonuclease-active polymerase used for extension. The duplex may be formed via direct interaction of single-stranded nucleic acid domains via complementarity, or via a partially double-stranded nucleic acid with a single-stranded domain hybridised to a splint oligonucleotide.
Claims Coverage
The document contains two independent claims and covers PEA-based analyte detection that incorporates 3′ exonuclease activity in connection with forming a duplex and extending the 3′ end, followed by amplifying and detecting an extension product. Across the independent claims, there are 5 main inventive feature clusters.
Proximity probes form a duplex on analyte binding
Contacting the sample with at least first and second proximity probes that each comprise an analyte-binding domain and a nucleic acid domain and simultaneously bind to the analyte; allowing the nucleic acid domains to interact upon binding to form a duplex.
3′ exonuclease activity is contacted during or with extension workflow
Contacting the sample with a component comprising 3′ exonuclease activity, wherein the component comprises a polymerase enzyme having 3′ exonuclease activity and/or an exonuclease enzyme other than the polymerase enzyme.
3′ end extension generates an extension product
Extending the 3′ end of at least one nucleic acid domain of the duplex to generate an extension product.
Amplifying and detecting using a polymerase different from the 3′ exonuclease-active polymerase
Amplifying and detecting the extension product, wherein the amplifying and detecting comprise at least one amplification reaction using a separate polymerase enzyme different from the polymerase having 3′ exonuclease activity used in the extension.
Duplex formation by single-stranded complementarity or splint-mediated partially double-stranded nucleic acid
Wherein either the nucleic acid domains of the proximity probes are single-stranded and interact directly via regions of complementarity to each other, or the nucleic acid domain of at least one proximity probe is partially double-stranded and comprises a single-stranded domain hybridised to a splint oligonucleotide.
Non-polymerase 3′ exonuclease component
Contacting the sample with a 3′ exonuclease enzyme, wherein the 3′ exonuclease enzyme is not a polymerase enzyme or a part thereof.
The independent claims require proximity probes binding to an analyte and forming a duplex through nucleic-acid interaction, followed by extension from a duplex 3′ end to generate an extension product, and then amplification and detection of that extension product. The claims further distinguish the 3′ exonuclease component and polymerase relationship.
Stated Advantages
Reduces non-specific extension products by degrading free and unprotected 3′ ends on proximity probes.
Increases specificity and/or sensitivity, including in exonuclease-rich samples such as plasma.
Documented Applications
Detecting an analyte in a sample using a proximity probe extension assay (PEA), including detection in plasma and assays described with IL-8 (interleukin-8) detection.
Multiplex detection of two or more analytes by contacting the sample with multiple proximity-probe sets and detecting unique extension products per set.
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