METHODS, KITS, AND SYSTEMS FOR MULTIPLEXED DETECTION OF TARGET MOLECULES AND USES THEREOF

• US 20170275669A1
• Filed: 06/03/2014
• Published: 09/28/2017
• Est. Priority Date: 06/12/2013
• Status: Active Grant

• Current Assignee: The General Hospital Corporation (Mass General Brigham, Inc.)
• Original Assignee: The General Hospital Corporation (Mass General Brigham, Inc.)
• Inventors: WEISSLEDER, Ralph, AGASTI, Sarit S., PETERSON, Vanessa M., ULLAL, Adeeti
• Granted Patent: US 10,266,874 B2
• Time in Patent Office: Days
• CPC Class Codes: C12Q 1/6804 Nucleic acid analysis using immunogens immunoassay G01N33/53
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6823 Release of bound markers
• CPC Class Codes: C12Q 2523/319 Photocleavage, photolysis, photoactivation
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid
• CPC Class Codes: C12Q 2563/185 Nucleic acid dedicated to use as a hidden marker/bar code, e.g. inclusion of nucleic acids to mark art objects or animals
• CPC Class Codes: G01N 2458/10 Oligonucleotides as tagging agents for labelling antibodies
• CPC Class Codes: G01N 33/53 Immunoassay; Biospecific binding assay; Materials therefor

Abstract

Described herein are methods, compositions, kits and systems for multiplexed detection of target molecules from a sample. In some embodiments, the methods, compositions, kits and systems can be used to perform multiplexed protein analysis of a sample (e.g., a sample comprising a small number of cells or a single-cell sample). In some embodiments, the same sample subjected to a multiplexed protein analysis using the methods, compositions, kits and systems described herein can also be subjected to a nucleic acid (e.g., RNAs, microRNAs, and/or DNA) analysis, thereby creating an integrated expression profiling from a limited amount of sample.

Claims

1. A method for detecting a plurality of target protein molecules in a sample comprising:

a. contacting a sample with a composition comprising a plurality of target probes, wherein each target probe in the plurality comprises;

i. a target-binding molecule that specifically binds to a distinct target molecule in the sample;

ii. an identification nucleotide sequence that identifies the target-binding molecule; and

iii. a cleavable linker between the target-binding molecule and the identification nucleotide sequence;

b. separating unbound target probes from a plurality of complexes in the sample, each complex having a target protein molecule and a single target probe bound thereto, wherein the complex does not have a second target probe binding to a different region of the target protein molecule;

c. releasing the identification nucleotide sequences from the plurality of complexes; and

d. detecting signals from the released identification nucleotide sequences based on a non-gel electrophoresis method, wherein the signals are distinguishable for the identification nucleotide sequences, thereby identifying the corresponding target-binding molecules and detecting a plurality of different target molecules in the sample.

67. The method of claim 1, wherein the target-binding molecule is an antibody.

68. The method of claim 1, wherein the cleavable linker is a cleavable non-hybridizable linker.

69. The method of claim 68, wherein the cleavable, non-hybridizable linker is sensitive to an enzyme, pH, temperature, light, shear stress, sonication, a chemical agent (e.g., dithiothreitol), or any combination thereof.

70. The method of any of claims 68, wherein the cleavable, non-hybridizable linker comprises a photocleavable linker.

71. The method of claim 70, wherein the photocleavable linker is selected from the group consisting of molecules (i)-(xiv) and any combinations thereof, wherein the chemical structures of the molecules (i)-(xiv) are shown as follows:

72. The method of claim 70, wherein the photocleavable linker comprises the molecule (xiv).

74. The method of claim 1, further comprising, prior to the detecting step (d), coupling the released identification nucleotide sequences from the releasing step (c) to a detection composition comprising a plurality of reporter probes, wherein each reporter probe in the plurality comprises:

a first target probe-specific region that is capable of binding a first portion of the identification nucleotide sequence; and

a detectable label that identifies the reporter probe.

75. The method of claim 74, wherein the detecting comprises detecting signals from the respective detectable labels of the reporter probes that are coupled to the released identification nucleotide sequences, wherein the signals are distinguishable for the respective reporter probes and bound the identification nucleotide sequences, thereby identifying the corresponding target-binding molecules and detecting a plurality of target molecules in the sample.

76. The method of any of claims 1, wherein the detecting step (d) comprises no amplification of the released identification nucleotide sequences.

77. The method of any of claims 74, wherein the detecting step (d) comprises no amplification of the first target probe-specific region.

78. The method of claim 1, wherein the identification nucleotide sequences are derived from a potato genome.

79. The method of any of claims 1, wherein the identification nucleotide sequences have a length of about 30-100 nucleotides.

80. The method of claims 1, wherein the identification nucleotide sequences have a length of about 70 nucleotides.

81. The method of claim 80, wherein the identification nucleotide sequences have a sequence selected from Table 2 (SEQ ID NO:

1 to SEQ ID NO;

     110).

82. The method of claim 1, wherein the sample comprises less than 500 cells.

83. The method of claim 1, wherein the cells are rare cells selected from the group consisting of circulating tumor cells, fetal cells, stem cells, immune cells, clonal cells, and any combination thereof.

84. The method of any of claims 1, wherein the composition further comprises a plurality of control probes, wherein each control probe in the plurality comprises:

i. a control-binding molecule that specifically binds to a control protein molecule in the sample;

ii. an identification control sequence that identifies the control-binding molecule; and

iii. a cleavable linker between the control-binding molecule and the identification control sequence.

and wherein the method, further comprises quantifying the signals by normalizing the signals associated with the target probes by the signals associated with the control probes.

85. The method of claim 1, further comprising extracting a nucleic acid molecule from the same sample and subjecting the nucleic acid molecule to a nucleic acid analysis thereby detecting proteins and nucleic acid molecules from the same sample.

2-66. -66. (canceled)

73. The method of claim wherein the releasing of the identification nucleotide sequences from the bound target probes comprises exposing the bound target probes to ultraviolet light.

86. A kit for multiplexed detection of a plurality of different target molecules from a sample comprising:

a. a plurality of target probes, wherein each target probe in the plurality comprises;

i. a target-binding molecule that specifically binds to a distinct target molecule in the sample;

ii. an identification nucleotide sequence that identifies the target-binding molecule; and

iii. a cleavable, non-hybridizable linker between the target-binding molecule and the identification nucleotide sequence;

b. a plurality of reporter probes, wherein each reporter probe comprises;

i. a first target probe-specific region that is capable of binding a first portion of the identification nucleotide sequence; and

ii. a detectable label that identifies the reporter probe.

87. The kit of claim 86, wherein the target-binding molecule is an antibody.