US 2024/0254475 A1 · PROTEOMIC ANALYSIS WITH NUCLEIC ACID IDENTIFIERS

Abstract

The disclosure provides methods and compositions useful for labeling of target molecules with origin-specific nucleic acid identifiers (for example, barcodes), which can be used subsequently to identify, quantify, or otherwise characterize a feature or activity of target molecules originating from a particular discreet volume. Such target molecules can include polypeptides expressed by cells, in which nucleic acid molecules encoding the polypeptides are labeled with the same, or matched, origin-specific nucleic acid identifiers.

Claims21

Dependent claims

1-42. (canceled)

A method of determining whether a previously compartmentalized target mRNA and target polypeptide were from the same compartment after being pooled with target mRNA and target polypeptides from other compartments, the method comprising:

(A) labelling target polypeptides expressed by at least a portion of a cell population with first oligonucleotides, each of the first oligonucleotides comprising:
(i) a binding sequence;
(ii) a sequence unique to each type of the expressed target polypeptides;
and,
(iii) a molecule for capturing each type of the expressed target polypeptides,
wherein the cells with labelled target polypeptides further comprise target mRNA;
(B) generating subsamples by segregating each cell into compartments, wherein each of the compartments further comprise:
(i) a second oligonucleotide comprising:
(a) an origin specific barcode sequence;
and,
(b) a sequence complementary to the binding sequence of at least one of the first oligonucleotides, or a portion thereof;
(ii) a third oligonucleotide comprising:
(a) the origin specific barcode sequence;
and,
(b) a sequence for binding the target mRNA in the compartment;
wherein the origin specific barcode sequences differ among the individual compartments;
(C) lysing the cells in each of the compartments;
(D) annealing, in each of the compartments:
(i) the first oligonucleotide or a portion thereof to the second oligonucleotide or a portion thereof, to produce barcoded DNA molecules identifying the type and compartment of the expressed target polypeptides;
and,
(ii) the third oligonucleotide to the target mRNA, to produce barcoded cDNA products identifying the compartment associated with the target mRNA;
(E) releasing and pooling the barcoded DNA molecules and barcoded cDNA products from the compartments;
(F) generating amplicons by:
(i) amplifying the pooled barcoded DNA molecules;
and,
(ii) amplifying the pooled barcoded cDNA products;
(G) sequencing the amplified pooled barcoded DNA molecules and barcoded cDNA products;
(H) determining whether a target polypeptide and a target mRNA were from the same compartment by identifying the sequenced pooled barcoded DNA molecules and sequenced barcoded cDNA products having an identical barcode sequence.

25 more elements

A method for labeling a target polypeptide and a target nucleic acid located in the same compartment with the same origin specific barcode sequence, the method comprising:

contacting the target polypeptide expressed by at least a portion of a cell population with a nucleic acid tag sequence covalently attached to a molecule capable of binding the expressed target polypeptide, wherein the nucleic acid tag sequence comprises an annealing sequence and a sequence unique to the expressed target polypeptide, thereby generating a nucleic acid-tagged target polypeptide, wherein the cells with the nucleic acid-tagged expressed target polypeptide further comprise target mRNA;
generating subsamples by segregating each cell into compartments, wherein each of the compartments further comprise:
a first oligonucleotide comprising a sequence complementary to the target mRNA, or a portion thereof;
and a second oligonucleotide comprising the same origin specific barcode sequence identifying the individual compartment and a sequence capable of binding the annealing sequence of the nucleic acid tag of the nucleic acid-tagged target polypeptide, under conditions sufficient to allow for binding of the first oligonucleotide to the target mRNA and binding of the second oligonucleotide to the annealing sequence of the nucleic acid-tagged target polypeptide;
and
performing polymerase extension upon each of the first oligonucleotide and the second oligonucleotide,
thereby labeling the target polypeptide and the target nucleic acid located in the same compartment with the same origin specific barcode sequence.

4 more elements

Dependencies are inferred from the claim text.

Proteomic analysis with nucleic acid identifiers

Life of this patent

Proteomic analysis with nucleic acid identifiers

Life of this patent

Abstract

Claims21