US 2024/0409983 A1 · METHODS FOR ANALYZING A SAMPLE

Abstract

Provided herein are methods for analyzing a sample for an analyte using proximity-based extension, such as proximity extension strand displacement. Also provided are multiplexed methods for analyzing a sample using proximity-based extension. Provided herein are methods for screening for pairwise combination of binding moieties, for use in a sandwich-type assay. Also provided are compositions that find use in the present methods.

Claims28

Dependent claims

A method of analyzing a sample for an analyte, comprising:

a) combining:
i) a solid support comprising:
a capture moiety attached to the solid support, wherein the capture moiety specifically binds an analyte;
and
a capture oligonucleotide attached to the solid support independently of the capture moiety, wherein the capture oligonucleotide comprises a 3′ hybridizing region;
ii) a detection conjugate comprising:
a detection moiety that specifically binds the analyte;
and
a detection oligonucleotide attached to the detection moiety, wherein the detection oligonucleotide comprises a 3′ hybridizing region complementary to the 3′ hybridizing region of the capture oligonucleotide;
and
iii) a sample;
wherein, if the analyte is present in the sample, it is bound to the solid support by the capture moiety and to the detection conjugate by the detection moiety to form a capture complex in which the capture oligonucleotide and the detection oligonucleotide are in proximity;
b) permitting the 3′ hybridizing region of the capture oligonucleotide and the 3′ hybridizing region of the detection oligonucleotide that are in proximity to hybridize to each other;
c) extending the hybridized capture oligonucleotide and/or the hybridized detection oligonucleotide to generate an on-target extension product;
d) releasing the on-target extension product from the solid support;
and
e) determining the presence or the absence of the released on-target extension product to determine the presence or the absence of the analyte in the sample.

14 more elements

A method of determining a pairwise combination of binding moieties that can simultaneously bind to a binding target, the method comprising:

a) combining
i) a plurality of different populations of solid supports, each solid support comprising:
a first binding moiety attached to the solid support;
and
a capture oligonucleotide independently attached to the solid support, wherein the capture oligonucleotide comprises a 3′ hybridizing region and a capture barcode region, wherein the 3′ hybridizing regions of the capture oligonucleotides are the same in the plurality of different populations of solid supports, and wherein the capture barcode regions of the capture oligonucleotides are different for each different population of solid supports and the capture barcode region identifies the binding moiety attached to the same solid support, and;
ii) a plurality of different populations of detection conjugates comprising:
a second binding moiety;
and
a detection oligonucleotide attached to the second binding moiety, wherein the detection oligonucleotide comprises a 3′ hybridizing region complementary to the 3′ hybridizing region of the capture oligonucleotide and a detector barcode region, wherein the detector barcode regions of the detection oligonucleotide are different for each different population of detection conjugates and identifies the second binding moiety to which it is attached;
and
iii) one or more binding targets;
wherein, if a binding target is present in the sample, and the binding target is specifically bound by the first binding moiety and the second binding moiety, a capture complex is formed in which the capture oligonucleotide and the detection oligonucleotide are in proximity;
b) permitting the 3′ hybridizing region of the capture oligonucleotide and the 3′ hybridizing region of the detection oligonucleotide to hybridize to each other;
e) extending at least one of the hybridized capture oligonucleotide and the hybridized detection oligonucleotide to generate an extension product that comprises i) an extended capture oligonucleotide and a complement of at least a part of the detection oligonucleotide, and/or ii) an extended detection oligonucleotide and a complement of at least a part of the capture oligonucleotide;
f) releasing the extension product from the solid support;
and
g) determining the capture barcode region and the detector barcode region of the released extension product to thereby determine a combination of binding moieties that can simultaneously bind to the binding target.

14 more elements

A composition comprising:

a) a first pool comprising at least 30 different populations of solid supports, each solid support comprising:
i) a capture moiety attached to the solid support, wherein the capture moiety specifically binds an analyte;
ii) a tether oligonucleotide attached to the solid support independent of the capture moiety;
and
iii) a capture oligonucleotide comprising from 5′ to 3′:
a tethering region, a capture barcode region, and a 3′ hybridizing region, wherein the capture oligonucleotide is attached to the solid support via hybridization of the tethering region to the tether oligonucleotide attached to the solid support;
and
b) a second pool comprising at least 30 different populations of detection conjugates, each detection conjugate comprising:
i) a detection moiety that specifically binds the analyte;
ii) a tether oligonucleotide attached to the detection moiety;
and
ii) a detection oligonucleotide comprising from 5′ to 3′:
a tethering region, a detection barcode region, and a 3′ hybridizing region, wherein the detection oligonucleotide is attached to the detection moiety via hybridization of the tethering region to the tether oligonucleotide attached to the detection moiety,
wherein each of the at least 30 different populations of solid supports forms a paired combination with a corresponding detection conjugate of the at least 30 different populations of detection conjugates, wherein the analyte bound by the capture moiety and detection moiety of each paired combination is the same, wherein the analytes bound by different paired combinations are different, wherein the 3′ hybridizing region of the capture oligonucleotide and detection oligonucleotide of each paired combination is not complementary to the 3′ hybridizing region of the detection oligonucleotide and the capture oligonucleotide, respectively, of any other paired combination, and wherein each paired combination can be distinguished from any other paired combination by one or both of the sequence of the capture barcode region and/or the sequence of the detection barcode region.

11 more elements

A method of analyzing a sample for an analyte, comprising:

a) combining:
i) a solid support comprising:
a capture moiety attached to the solid support, wherein the capture moiety specifically binds an analyte;
and
a capture oligonucleotide attached to the solid support independently of the capture moiety;
ii) a detection conjugate comprising:
a detection moiety that specifically binds the analyte;
and
a detection oligonucleotide attached to the detection moiety;
iii) a first splint oligonucleotide comprising:
a first portion complementary to A) a 3′ hybridization region of the capture oligonucleotide, or B) a 5′ hybridization region of the capture oligonucleotide;
and
a second portion complementary to A) a 5′ hybridization region of the detection oligonucleotide if the first portion is complementary to the 3′ hybridization region of the capture oligonucleotide, or B) a 3′ hybridization region of the detection oligonucleotide if the first portion is complementary to the 5′ hybridization region of the capture oligonucleotide;
and
iv) a sample;
wherein, if the analyte is present in the sample, it is bound to the solid support by the capture moiety and to the detection conjugate by the detection moiety to form a capture complex in which the capture oligonucleotide and the detection oligonucleotide are in proximity;
b) permitting i) the 3′ hybridizing region of the capture oligonucleotide and the 5′ hybridizing region of the detection oligonucleotide that are in proximity to hybridize to the first splint oligonucleotide, or ii) the 5′ hybridizing region of the capture oligonucleotide and the 3′ hybridizing region of the detection oligonucleotide that are in proximity to hybridize to the first splint oligonucleotide;
c) ligating the hybridized capture oligonucleotide and the hybridized detection oligonucleotide to generate an on-target ligation product that comprises the capture oligonucleotide and the detection oligonucleotide;
d) releasing the on-target ligation product from the solid support;
and
e) determining the presence or the absence of the released on-target ligation product to determine the presence or the absence of the analyte in the sample.

18 more elements

A composition comprising:

a) a first pool comprising at least 30 different populations of solid supports, each solid support comprising:
i) a capture moiety attached to the solid support, wherein the capture moiety specifically binds an analyte;
ii) a tether oligonucleotide attached to the solid support independent of the capture moiety;
and
iii) a capture oligonucleotide comprising A) from 5′ to 3′:
a tethering region, a capture barcode region, and a 3′ hybridizing region, wherein the capture oligonucleotide is attached to the solid support via hybridization of the tethering region to the tether oligonucleotide attached to the solid support, or B) from 3′ to 5′:
a tethering region, a capture barcode region, and a 5′ hybridizing region, wherein the capture oligonucleotide is attached to the solid support via hybridization of the tethering region to the tether oligonucleotide attached to the solid support;
and
b) a second pool comprising at least 30 different populations of detection conjugates, each detection conjugate comprising:
i) a detection moiety that specifically binds the analyte;
ii) a tether oligonucleotide attached to the detection moiety;
and
ii) a detection oligonucleotide comprising A) from 5′ to 3′:
a tethering region, a detection barcode region, and a 3′ hybridizing region, or B) A) from 3′ to 5′:
a tethering region, a detection barcode region, and a 5′ hybridizing region, wherein the detection oligonucleotide is attached to the detection moiety via hybridization of the tethering region to the tether oligonucleotide attached to the detection moiety,
c) a third pool comprising at least 30 different populations of first splint oligonucleotides, each first splint oligonucleotide comprising:
i) a first portion complementary to A) a 3′ hybridization region of the capture oligonucleotide, or B) a 5′ hybridization region of the capture oligonucleotide;
and
ii) a second portion complementary to A) a 5′ hybridization region of the detection oligonucleotide if the first portion is complementary to the 3′ hybridization region of the capture oligonucleotide, or B) a 3′ hybridization region of the detection oligonucleotide if the first portion is complementary to the 5′ hybridization region of the capture oligonucleotide;
and
wherein each of the at least 30 different populations of first splint oligonucleotides forms a triad combination with a corresponding solid support of the at least 30 different populations of solid supports and a corresponding detection conjugate of the at least 30 different populations of detection conjugates, wherein the analyte bound by the capture moiety and detection moiety of each triad combination is the same, wherein the analytes bound by different triad combinations are different, wherein the capture oligonucleotide and detection oligonucleotide of each paired combination are not complementary to the first splint oligonucleotide of any other triad combination, and wherein each triad combination can be distinguished from any other triad combination by one or both of the sequence of the capture barcode region and/or the sequence of the detection barcode region.

18 more elements

A method of analyzing a sample for an analyte, comprising:

a) combining:
i) a first conjugate comprising:
a first moiety that specifically binds an analyte;
a first tether region attached to the first moiety;
and
a first splint oligonucleotide comprising A) a 5′ tethering region, a first moiety barcode, and a 3′ hybridizing region, wherein the 5′ tethering region of the first splint oligonucleotide is hybridized to the first tether region attached to the first moiety, or B) a 3′ tethering region, a first moiety barcode, and a 5′ hybridizing region, wherein the 3′ tethering region of the first splint oligonucleotide is hybridized to the first tether region attached to the first moiety;
ii) a second conjugate comprising:
a second moiety that specifically binds the analyte;
a second tether region attached to the second moiety;
and
a second splint oligonucleotide comprising A) a 5′ tethering region, a second moiety barcode, and a 3′ hybridizing region, wherein the 5′ tethering region of the second splint oligonucleotide is hybridized to the second tether region attached to the second moiety, or B) a 3′ tethering region, a second moiety barcode, and a 5′ hybridizing region, wherein the 3′ tethering region of the second splint oligonucleotide is hybridized to the second tether region attached to the second moiety;
iii) a third splint oligonucleotide comprising:
a first portion complementary to A) a 3′ hybridization region of the capture oligonucleotide, or B) a 5′ hybridization region of the capture oligonucleotide;
and
a second portion complementary to A) a 5′ hybridization region of the detection oligonucleotide if the first portion is complementary to the 3′ hybridization region of the capture oligonucleotide, or B) a 3′ hybridization region of the detection oligonucleotide if the first portion is complementary to the 5′ hybridization region of the capture oligonucleotide;
and
iv) a sample;
b) permitting the first splint oligonucleotide and the second splint oligonucleotide that are in proximity to hybridize to the third splint oligonucleotide if the analyte is present in the sample;
c) ligating the hybridized first splint oligonucleotide and the hybridized second splint oligonucleotide to generate an on-target ligation product that comprises the first splint oligonucleotide and the extended second splint oligonucleotide;
d) releasing the on-target ligation product by treating the on-target ligation product with a restriction endonuclease and a strand-displacing DNA polymerase;
and
e) determining the presence or the absence of the on-target ligation product to thereby determine the presence or the absence of the analyte in the sample.

19 more elements

Dependencies are inferred from the claim text.

Facts

Application

18/734,455Filed 5 Jun 2024

Priority

6 Jun 2023

Published

12 Dec 2024

Term ends

Not estimatedThe source records don’t support an estimate.

Classification10 codes

C12Q 1/6804
Involving nucleic acids: nucleic acid analysis using immunogens
C12Q 1/6823
Characterised by the detection means: release of bound markers
C12Q 1/6806
Involving nucleic acids: preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
C12Q 1/6834
Hybridisation assays: enzymatic or biochemical coupling of nucleic acids to a solid phase

Abstract

Claims28

Dependent claims

A method of analyzing a sample for an analyte, comprising:

a) combining:
i) a solid support comprising:
a capture moiety attached to the solid support, wherein the capture moiety specifically binds an analyte;
and
a capture oligonucleotide attached to the solid support independently of the capture moiety, wherein the capture oligonucleotide comprises a 3′ hybridizing region;
ii) a detection conjugate comprising:
a detection moiety that specifically binds the analyte;
and
a detection oligonucleotide attached to the detection moiety, wherein the detection oligonucleotide comprises a 3′ hybridizing region complementary to the 3′ hybridizing region of the capture oligonucleotide;
and
iii) a sample;
wherein, if the analyte is present in the sample, it is bound to the solid support by the capture moiety and to the detection conjugate by the detection moiety to form a capture complex in which the capture oligonucleotide and the detection oligonucleotide are in proximity;
b) permitting the 3′ hybridizing region of the capture oligonucleotide and the 3′ hybridizing region of the detection oligonucleotide that are in proximity to hybridize to each other;
c) extending the hybridized capture oligonucleotide and/or the hybridized detection oligonucleotide to generate an on-target extension product;
d) releasing the on-target extension product from the solid support;
and
e) determining the presence or the absence of the released on-target extension product to determine the presence or the absence of the analyte in the sample.

14 more elements

A method of determining a pairwise combination of binding moieties that can simultaneously bind to a binding target, the method comprising:

a) combining
i) a plurality of different populations of solid supports, each solid support comprising:
a first binding moiety attached to the solid support;
and
a capture oligonucleotide independently attached to the solid support, wherein the capture oligonucleotide comprises a 3′ hybridizing region and a capture barcode region, wherein the 3′ hybridizing regions of the capture oligonucleotides are the same in the plurality of different populations of solid supports, and wherein the capture barcode regions of the capture oligonucleotides are different for each different population of solid supports and the capture barcode region identifies the binding moiety attached to the same solid support, and;
ii) a plurality of different populations of detection conjugates comprising:
a second binding moiety;
and
a detection oligonucleotide attached to the second binding moiety, wherein the detection oligonucleotide comprises a 3′ hybridizing region complementary to the 3′ hybridizing region of the capture oligonucleotide and a detector barcode region, wherein the detector barcode regions of the detection oligonucleotide are different for each different population of detection conjugates and identifies the second binding moiety to which it is attached;
and
iii) one or more binding targets;
wherein, if a binding target is present in the sample, and the binding target is specifically bound by the first binding moiety and the second binding moiety, a capture complex is formed in which the capture oligonucleotide and the detection oligonucleotide are in proximity;
b) permitting the 3′ hybridizing region of the capture oligonucleotide and the 3′ hybridizing region of the detection oligonucleotide to hybridize to each other;
e) extending at least one of the hybridized capture oligonucleotide and the hybridized detection oligonucleotide to generate an extension product that comprises i) an extended capture oligonucleotide and a complement of at least a part of the detection oligonucleotide, and/or ii) an extended detection oligonucleotide and a complement of at least a part of the capture oligonucleotide;
f) releasing the extension product from the solid support;
and
g) determining the capture barcode region and the detector barcode region of the released extension product to thereby determine a combination of binding moieties that can simultaneously bind to the binding target.

14 more elements

A composition comprising:

a) a first pool comprising at least 30 different populations of solid supports, each solid support comprising:
i) a capture moiety attached to the solid support, wherein the capture moiety specifically binds an analyte;
ii) a tether oligonucleotide attached to the solid support independent of the capture moiety;
and
iii) a capture oligonucleotide comprising from 5′ to 3′:
a tethering region, a capture barcode region, and a 3′ hybridizing region, wherein the capture oligonucleotide is attached to the solid support via hybridization of the tethering region to the tether oligonucleotide attached to the solid support;
and
b) a second pool comprising at least 30 different populations of detection conjugates, each detection conjugate comprising:
i) a detection moiety that specifically binds the analyte;
ii) a tether oligonucleotide attached to the detection moiety;
and
ii) a detection oligonucleotide comprising from 5′ to 3′:
a tethering region, a detection barcode region, and a 3′ hybridizing region, wherein the detection oligonucleotide is attached to the detection moiety via hybridization of the tethering region to the tether oligonucleotide attached to the detection moiety,
wherein each of the at least 30 different populations of solid supports forms a paired combination with a corresponding detection conjugate of the at least 30 different populations of detection conjugates, wherein the analyte bound by the capture moiety and detection moiety of each paired combination is the same, wherein the analytes bound by different paired combinations are different, wherein the 3′ hybridizing region of the capture oligonucleotide and detection oligonucleotide of each paired combination is not complementary to the 3′ hybridizing region of the detection oligonucleotide and the capture oligonucleotide, respectively, of any other paired combination, and wherein each paired combination can be distinguished from any other paired combination by one or both of the sequence of the capture barcode region and/or the sequence of the detection barcode region.

11 more elements

A method of analyzing a sample for an analyte, comprising:

a) combining:
i) a solid support comprising:
a capture moiety attached to the solid support, wherein the capture moiety specifically binds an analyte;
and
a capture oligonucleotide attached to the solid support independently of the capture moiety;
ii) a detection conjugate comprising:
a detection moiety that specifically binds the analyte;
and
a detection oligonucleotide attached to the detection moiety;
iii) a first splint oligonucleotide comprising:
a first portion complementary to A) a 3′ hybridization region of the capture oligonucleotide, or B) a 5′ hybridization region of the capture oligonucleotide;
and
a second portion complementary to A) a 5′ hybridization region of the detection oligonucleotide if the first portion is complementary to the 3′ hybridization region of the capture oligonucleotide, or B) a 3′ hybridization region of the detection oligonucleotide if the first portion is complementary to the 5′ hybridization region of the capture oligonucleotide;
and
iv) a sample;
wherein, if the analyte is present in the sample, it is bound to the solid support by the capture moiety and to the detection conjugate by the detection moiety to form a capture complex in which the capture oligonucleotide and the detection oligonucleotide are in proximity;
b) permitting i) the 3′ hybridizing region of the capture oligonucleotide and the 5′ hybridizing region of the detection oligonucleotide that are in proximity to hybridize to the first splint oligonucleotide, or ii) the 5′ hybridizing region of the capture oligonucleotide and the 3′ hybridizing region of the detection oligonucleotide that are in proximity to hybridize to the first splint oligonucleotide;
c) ligating the hybridized capture oligonucleotide and the hybridized detection oligonucleotide to generate an on-target ligation product that comprises the capture oligonucleotide and the detection oligonucleotide;
d) releasing the on-target ligation product from the solid support;
and
e) determining the presence or the absence of the released on-target ligation product to determine the presence or the absence of the analyte in the sample.

18 more elements

A composition comprising:

a) a first pool comprising at least 30 different populations of solid supports, each solid support comprising:
i) a capture moiety attached to the solid support, wherein the capture moiety specifically binds an analyte;
ii) a tether oligonucleotide attached to the solid support independent of the capture moiety;
and
iii) a capture oligonucleotide comprising A) from 5′ to 3′:
a tethering region, a capture barcode region, and a 3′ hybridizing region, wherein the capture oligonucleotide is attached to the solid support via hybridization of the tethering region to the tether oligonucleotide attached to the solid support, or B) from 3′ to 5′:
a tethering region, a capture barcode region, and a 5′ hybridizing region, wherein the capture oligonucleotide is attached to the solid support via hybridization of the tethering region to the tether oligonucleotide attached to the solid support;
and
b) a second pool comprising at least 30 different populations of detection conjugates, each detection conjugate comprising:
i) a detection moiety that specifically binds the analyte;
ii) a tether oligonucleotide attached to the detection moiety;
and
ii) a detection oligonucleotide comprising A) from 5′ to 3′:
a tethering region, a detection barcode region, and a 3′ hybridizing region, or B) A) from 3′ to 5′:
a tethering region, a detection barcode region, and a 5′ hybridizing region, wherein the detection oligonucleotide is attached to the detection moiety via hybridization of the tethering region to the tether oligonucleotide attached to the detection moiety,
c) a third pool comprising at least 30 different populations of first splint oligonucleotides, each first splint oligonucleotide comprising:
i) a first portion complementary to A) a 3′ hybridization region of the capture oligonucleotide, or B) a 5′ hybridization region of the capture oligonucleotide;
and
ii) a second portion complementary to A) a 5′ hybridization region of the detection oligonucleotide if the first portion is complementary to the 3′ hybridization region of the capture oligonucleotide, or B) a 3′ hybridization region of the detection oligonucleotide if the first portion is complementary to the 5′ hybridization region of the capture oligonucleotide;
and
wherein each of the at least 30 different populations of first splint oligonucleotides forms a triad combination with a corresponding solid support of the at least 30 different populations of solid supports and a corresponding detection conjugate of the at least 30 different populations of detection conjugates, wherein the analyte bound by the capture moiety and detection moiety of each triad combination is the same, wherein the analytes bound by different triad combinations are different, wherein the capture oligonucleotide and detection oligonucleotide of each paired combination are not complementary to the first splint oligonucleotide of any other triad combination, and wherein each triad combination can be distinguished from any other triad combination by one or both of the sequence of the capture barcode region and/or the sequence of the detection barcode region.

18 more elements

A method of analyzing a sample for an analyte, comprising:

a) combining:
i) a first conjugate comprising:
a first moiety that specifically binds an analyte;
a first tether region attached to the first moiety;
and
a first splint oligonucleotide comprising A) a 5′ tethering region, a first moiety barcode, and a 3′ hybridizing region, wherein the 5′ tethering region of the first splint oligonucleotide is hybridized to the first tether region attached to the first moiety, or B) a 3′ tethering region, a first moiety barcode, and a 5′ hybridizing region, wherein the 3′ tethering region of the first splint oligonucleotide is hybridized to the first tether region attached to the first moiety;
ii) a second conjugate comprising:
a second moiety that specifically binds the analyte;
a second tether region attached to the second moiety;
and
a second splint oligonucleotide comprising A) a 5′ tethering region, a second moiety barcode, and a 3′ hybridizing region, wherein the 5′ tethering region of the second splint oligonucleotide is hybridized to the second tether region attached to the second moiety, or B) a 3′ tethering region, a second moiety barcode, and a 5′ hybridizing region, wherein the 3′ tethering region of the second splint oligonucleotide is hybridized to the second tether region attached to the second moiety;
iii) a third splint oligonucleotide comprising:
a first portion complementary to A) a 3′ hybridization region of the capture oligonucleotide, or B) a 5′ hybridization region of the capture oligonucleotide;
and
a second portion complementary to A) a 5′ hybridization region of the detection oligonucleotide if the first portion is complementary to the 3′ hybridization region of the capture oligonucleotide, or B) a 3′ hybridization region of the detection oligonucleotide if the first portion is complementary to the 5′ hybridization region of the capture oligonucleotide;
and
iv) a sample;
b) permitting the first splint oligonucleotide and the second splint oligonucleotide that are in proximity to hybridize to the third splint oligonucleotide if the analyte is present in the sample;
c) ligating the hybridized first splint oligonucleotide and the hybridized second splint oligonucleotide to generate an on-target ligation product that comprises the first splint oligonucleotide and the extended second splint oligonucleotide;
d) releasing the on-target ligation product by treating the on-target ligation product with a restriction endonuclease and a strand-displacing DNA polymerase;
and
e) determining the presence or the absence of the on-target ligation product to thereby determine the presence or the absence of the analyte in the sample.

19 more elements

Dependencies are inferred from the claim text.

Methods for analyzing a sample

Life of this patent

Abstract

Claims28

Methods for analyzing a sample

Life of this patent

Abstract

Claims28