Systems, compositions, and methods for target entity detection
Inventors
Sedlak, Joseph Charles • Bortolin, Laura Teresa • Salem, Daniel Parker
Assignees
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Publication Number
US-11085089-B2
Publication Date
2021-08-10
Expiration Date
Abstract
The present disclosure provides technologies for target entity detection. One aspect of the present disclosure provides technologies for detection (e.g., early detection) of a disease, disorder, or condition (e.g., cancer). In another aspect, technologies provided herein are useful for selecting and/or monitoring and/or evaluating efficacy of, a treatment administered to a subject in need thereof, e.g., a subject determined to have or susceptible to cancer. In some embodiments, technologies provided herein are useful for development of companion diagnostics, e.g., by measuring tumor burdens and changes in tumor burdens in conjunction with therapeutics.
Core Innovation
The invention provides a method for detecting a biological entity of interest using a set of detection probes directed to targets. Each detection probe comprises a target-binding moiety coupled to an oligonucleotide domain that includes a double-stranded portion and a single-stranded overhang extended from one end. The overhangs are configured to hybridize when the first and second targets are simultaneously present on the entity of interest, forming a double-stranded complex under conditions that permit binding to their respective targets.
The double-stranded complex is contacted with a nucleic acid ligase to generate a ligated template comprising a strand of the first double-stranded portion and a strand of the second double-stranded portion. Presence of the ligated template is indicative of presence in the sample of the entity of interest comprising the first target and the second target. The approach is extended to multiplex target entity detection systems with probe combinations and overhang structures that permit ligation only when multiple probes bind the same entity.
The document further describes control and inhibitor probes to reduce false positives, background, or cross-reactive signal, together with amplification and qPCR or PCR detection of the ligated template. Optional capture or immobilization on solid substrates is also described, and the biological entity of interest includes embodiments where the entity of interest is a population of extracellular vesicles for cancer detection.
Claims Coverage
Independent claim coverage centers on a multi-target detection method using probe pairs with complementary single-stranded overhangs that hybridize only when both targets are present, followed by ligated-template detection. The inventive features comprise the probe structure, simultaneous target binding, ligase-mediated generation of a ligated template, and detection of that ligated template as an indicator of dual-target presence.
Complementary overhang detection probes for dual-target assembly
A first detection probe and a second detection probe each comprise a target-binding moiety coupled to an oligonucleotide domain with a double-stranded portion and a single-stranded overhang, and the overhangs are complementary so that they hybridize together when the first and second targets are simultaneously present on the entity of interest.
Ligase-mediated generation of a ligated template
The method maintains the combination under conditions permitting binding of the probes to their respective targets, forms a double-stranded complex, and contacts the complex with a nucleic acid ligase to generate a ligated template comprising a strand of the first double-stranded portion and a strand of the second double-stranded portion.
Detection of ligated template as indicative dual-target presence
Detecting the ligated template, where presence of the ligated template is indicative of presence in the sample of the entity of interest comprising the first target and the second target.
Probe set size constraint for multiple targets
The detection probes include 2–20 probes, each having a target-binding moiety linked to an oligonucleotide domain with a double-stranded portion and a single-stranded overhang extending from at least one end.
Amplification and detection of an amplification product
The method further includes amplifying the ligated template and detecting whether an amplification product is present.
Control detection probe that inhibits ligated-template generation and/or amplification
The method includes a control detection probe that binds the entity of interest and inhibits generation of a ligated template and/or inhibits amplification of a ligated template from a non-target biological entity.
Extracellular vesicle population as the biological entity of interest
The biological entity of interest is or comprises a population of extracellular vesicles.
Cancer embodiments for treating specified cancer types
The method is for treating cancer selected from an enumerated list that includes bladder cancer, brain cancer, breast cancer, cervical cancer, and other specified cancer types.
Overall, the claim coverage centers on complementary overhang hybridization enabled by simultaneous target presence, ligase-mediated formation of a ligated template from bound probe domains, and detection of the ligated template as an indicator that the entity of interest comprises both targets. Dependent claims further narrow probe quantity, add amplification and detection, specify inhibitory control probe behavior, and specify biological contexts including extracellular vesicles and enumerated cancer embodiments.
Stated Advantages
specificity
sensitivity
LOD for cancer diagnostics using extracellular vesicle (EV) biomarker signatures
High sensitivity and specificity for early-stage cancer screening versus bulk assays, as described.
Reduction of false positives, background, or cross-reactive signal using control and inhibitor probes, as described.
Ultrahigh specificity for early-stage ovarian cancer, as described in the disclosed patient plasma pilot study.
Improved signal-to-noise using inhibitor probes, as described in the disclosed examples.
Oligonucleotide domain length optimization improves signal with minimal background change, as described in the disclosed examples.
Documented Applications
cancer diagnostics using extracellular vesicle (EV) biomarker signatures
capture/immobilization on solid substrates
direct detection
amplified detection including PCR
Detection in a sample for presence of an entity of interest comprising first and second targets, including embodiments where the entity of interest is a population of extracellular vesicles.
Cancer diagnostics and companion diagnostics, including cancer-related embodiments and ovarian cancer cohorts.
Early-stage cancer screening and detection in asymptomatic individuals.
Target detection of cancer using extracellular vesicles, including patient plasma and cell-line derived EVs.
Use with blood- or plasma-derived samples and EV biomarker signatures, including membrane-bound polypeptides, surface or intravesicular proteins, and intravesicular RNA biomarkers.
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