Methods and/or use of oligonucleotide conjugates for assays and flow cytometry detections
Inventors
Schwartz, David A. • Williams, Jimmy • Zhao, Xinfang • Zhao, Chunfang • Busa, William B. • Kron, Stephen J. • Flor, Amy Catherine
Assignees
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Publication Number
US-12291738-B2
Publication Date
2025-05-06
Expiration Date
Abstract
The present disclosure is directed to methods and/or uses of oligonucleotide conjugates for assays and flow cytometry detections and related systems and/or kits. Certain methods are directed to a method for detecting one or more biological targets of a sample in a detection assay, comprising: providing a molecular probe, comprising a binding moiety and an oligonucleotide sequence, to a sample comprising one or more biological targets; binding the one or more biological targets with the binding moiety; providing a detectable component to the sample, wherein the detectable component comprises a signal generating moiety conjugated to an oligonucleotide sequence complementary to the oligonucleotide sequence of the molecular probe; hydridizing the oligonucleotide sequence of the target-bound molecular probe to the detectable component; and detecting a signal generated from the hydridized detectable component. Various other embodiments, applications etc. are disclosed herein.
Core Innovation
The invention relates to oligonucleotide-conjugate assay technology for live cell flow cytometry detection of a target in a sample comprising living cells. A molecular probe includes a binding moiety conjugated to a first oligonucleotide sequence, and a detectable component includes a signal generator conjugated to a second oligonucleotide sequence complementary to the first oligonucleotide sequence. The molecular probe and detectable component are mixed with living cells, the first and second oligonucleotides are hybridized, and a signal generated from the hybridized, target-bound detectable component is detected using flow cytometry.
The detectable component is not immobilized on a solid support during signal generation. The signal generator includes a scaffold molecule conjugated to more than one signal generating moiety, where the scaffold molecule comprises a dendrimer, a polysaccharide, or combinations or derivatives thereof, and the second oligonucleotide is directly conjugated to the scaffold via one or more covalent bond linkages selected from hydrazine, oxime, and triazine. The binding moiety is conjugated to the first oligonucleotide via one or more of the same covalent bond linkages.
The invention addresses potential cross-talk in oligonucleotide-based multiplex detection by adding additional oligonucleotides to the molecular probe and the detectable component. The added oligonucleotide comprises at least one of an unconjugated oligonucleotide complementary to the first oligonucleotide sequence and an unconjugated oligonucleotide complementary to the second oligonucleotide sequence. After detecting the signal, the hybridized detectable component is removed from the bound target by stripping and washing.
Claims Coverage
The independent claim content describes one live cell flow cytometry method with four main inventive features: complementary oligonucleotide hybridization between a molecular probe and a detectable component, covalent conjugation chemistry using hydrazine, oxime, or triazine, a scaffold-based signal generator with more than one signal generating moiety, and cross-talk reduction using unconjugated complementary oligonucleotides.
Live cell flow cytometry with hybridized complementary oligonucleotides
Detecting a target in a sample comprising living cells by mixing the sample with a molecular probe and a detectable component, binding the target with the binding moiety, hybridizing a first oligonucleotide on the molecular probe with a second complementary oligonucleotide on the detectable component, detecting a signal generated from the detectable component hybridized to the target-bound molecular probe using flow cytometry, and removing the hybridized detectable component from the bound target by stripping and washing.
Binding moiety conjugated to first oligonucleotide via hydrazine, oxime, or triazine
Conjugating a binding moiety to a first oligonucleotide sequence through one or more covalent bond linkages selected from hydrazine, oxime, and triazine.
Detectable component not immobilized on a solid support
Using a detectable component that is not immobilized on a solid support and comprises a signal generator conjugated to a second oligonucleotide having a sequence complementary to the first oligonucleotide sequence.
Scaffolded signal generator with multiple signal generating moieties
Including in the signal generator a scaffold molecule further conjugated to more than one signal generating moiety, where the scaffold molecule comprises a dendrimer, a polysaccharide, or combinations or derivatives thereof.
Direct scaffold conjugation to the second oligonucleotide via hydrazine, oxime, or triazine
Directly conjugating the second oligonucleotide to the scaffold molecule of the signal generator via one or more covalent bond linkages selected from hydrazine, oxime, and triazine.
Cross-talk reduction using unconjugated complementary oligonucleotides
Reducing potential for cross-talk by adding to the molecular probe and the detectable component an oligonucleotide comprising at least one of an unconjugated oligonucleotide complementary to the first oligonucleotide sequence and an unconjugated oligonucleotide complementary to the second oligonucleotide sequence.
The claim coverage is directed to live cell flow cytometry detection of a target using an oligonucleotide-bridge formed by a binding-moiety/first-oligonucleotide molecular probe and a non-solid-support scaffold-based signal-generator/second-oligonucleotide detectable component, with hydrazine/oxime/triazine conjugation, cross-talk reduction using complementary unconjugated oligonucleotides, signal detection by flow cytometry, and stripping/washing removal after detection.
Stated Advantages
Reduced false positives versus secondary-antibody detection.
Reduces potential for cross-talk.
Enables signal detection from a detectable component that is not immobilized on a solid support.
Allows removal of the hybridized detectable component from the bound target by stripping and washing.
Enables substantially less time than conventional conjugations for producing customizable molecular probes and detectable components.
High conjugation efficiency and high purity are described.
Documented Applications
Live cell flow cytometry for detecting a target in a sample comprising living cells using molecular probes and detectable components with complementary oligonucleotides.
Detection of a target using a molecular probe hybridized to a non-solid-support detectable component, with detection and subsequent stripping and washing removal.
Immunodetection formats including immunohistochemistry, ELISA, ELISpot, arrays, bead arrays, and HCS.
Multiplexing architectures for simultaneous detection aligned to flow cytometer channels.
Multiplexed detection is supported by dependent claims that refine the method for multiplexing.
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