US 2024/0254545 A1Published application

Spatiotemporally resolved transcriptomics at subcellular resolution

AssigneesThe Broad Institute, Inc., Massachusetts Institute of Technology

InventorsXiao Wang, Jingyi Ren, Hu Zeng

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Abstract

The present disclosure provides methods for profiling spatiotemporal gene expression, including methods for profiling spatiotemporal gene expression in vivo in a subject. The present disclosure also provides methods for profiling the role of post-transcriptional modification in spatiotemporal gene expression, methods for studying the role of spatiotemporal gene expression in the development or progression of a disease or disorder, methods for screening for an agent capable of modulating spatiotemporal gene expression, methods for diagnosing a disease or disorder in a subject, and methods for treating a disease or disorder in a subject. Oligonucleotide probes useful in the methods described herein are also provided by the present disclosure. The present disclosure also provide kits comprising the oligonucleotide probes disclosed herein. Systems for profiling spatiotemporal gene expression are also provided by the present disclosure.

Claims207

Dependent claims

A method for profiling spatiotemporal gene expression in a cell, the method comprising:

a) incubating a cell in the presence of a pool of nucleoside analogs for an amount of time t1 to metabolically label nucleic acids synthesized by the cell, wherein each nucleoside analog in the pool of nucleoside analogs comprises a reactive chemical moiety;
b) contacting the metabolically labeled nucleic acids with a population of first oligonucleotide probes, wherein each first oligonucleotide probe in the population of first oligonucleotide probes comprises a chemical moiety that reacts with the reactive chemical moiety of the nucleoside analogs;
c) contacting the metabolically labeled nucleic acids with one or more pairs of oligonucleotide probes comprising a second oligonucleotide probe and a third oligonucleotide probe, wherein:
i) the second oligonucleotide probe comprises a barcode sequence and a portion that is complementary to a metabolically labeled nucleic acid of interest;
and
ii) the third oligonucleotide probe comprises a portion that is complementary to the metabolically labeled nucleic acid of interest, a first barcode sequence, a portion that is complementary to a first oligonucleotide probe in the population of first oligonucleotide probes, and a second barcode sequence, wherein the first barcode sequence of the third oligonucleotide probe is complementary to the barcode sequence of the second oligonucleotide probe;
d) ligating the 5′ end and the 3′ end of the third oligonucleotide probe together to produce a circular oligonucleotide;
e) performing rolling circle amplification to amplify the circular oligonucleotide using the second oligonucleotide probe as a primer to produce one or more concatenated amplicons;
f) embedding the one or more concatenated amplicons in a polymer matrix;
g) contacting the one or more concatenated amplicons embedded in the polymer matrix with a fourth oligonucleotide probe comprising a sequence that is complementary to the second barcode sequence of the third oligonucleotide probe;
and
h) imaging the fourth oligonucleotide probe to determine the location of the one or more concatenated amplicons embedded in the polymer matrix.

9 more elements

A method for profiling the role of post-transcriptional modification in spatiotemporal gene expression in a cell, the method comprising:

a) incubating a cell comprising a knockdown of a gene involved in post-transcriptional modification in the presence of a pool of nucleoside analogs for an amount of time t1 to metabolically label nucleic acids synthesized by the cell, wherein each nucleoside analog in the pool of nucleoside analogs comprises a reactive chemical moiety;
b) contacting the metabolically labeled nucleic acids with a population of first oligonucleotide probes, wherein each first oligonucleotide probe in the population of first oligonucleotide probes comprises a chemical moiety that reacts with the reactive chemical moiety of the nucleoside analogs;
c) contacting the metabolically labeled nucleic acids with one or more pairs of oligonucleotide probes comprising a second oligonucleotide probe and a third oligonucleotide probe, wherein:
i) the second oligonucleotide probe comprises a barcode sequence and a portion that is complementary to a metabolically labeled nucleic acid of interest;
and
ii) the third oligonucleotide probe comprises a portion that is complementary to the metabolically labeled nucleic acid of interest, a first barcode sequence, a portion that is complementary to a first oligonucleotide probe in the population of first oligonucleotide probes, and a second barcode sequence, wherein the first barcode sequence of the third oligonucleotide probe is complementary to the barcode sequence of the second oligonucleotide probe;
d) ligating the 5′ end and the 3′ end of the third oligonucleotide probe together to produce a circular oligonucleotide;
e) performing rolling circle amplification to amplify the circular oligonucleotide using the second oligonucleotide probe as a primer to produce one or more concatenated amplicons;
f) embedding the one or more concatenated amplicons in a polymer matrix;
g) contacting the one or more concatenated amplicons embedded in the polymer matrix with a fourth oligonucleotide probe comprising a sequence that is complementary to the second barcode sequence of the third oligonucleotide probe;
and
h) imaging the fourth oligonucleotide probe to determine the location of the one or more concatenated amplicons embedded in the polymer matrix,
wherein an alteration in the expression of the metabolically labeled nucleic acid of interest relative to expression in a wild-type cell indicates that the post-transcriptional modification is involved in regulating spatiotemporal expression of the metabolically labeled nucleic acid of interest.

10 more elements

A method for studying the role of spatiotemporal gene expression in the development or progression of a disease or disorder, the method comprising:

a) incubating a cell from a diseased tissue in the presence of a pool of nucleoside analogs for an amount of time t1 to metabolically label nucleic acids synthesized by the cell, wherein each nucleoside analog in the pool of nucleoside analogs comprises a reactive chemical moiety;
b) contacting the metabolically labeled nucleic acids with a population of first oligonucleotide probes, wherein each first oligonucleotide probe in the population of first oligonucleotide probes comprises a chemical moiety that reacts with the reactive chemical moiety of the nucleoside analogs;
c) contacting the metabolically labeled nucleic acids with one or more pairs of oligonucleotide probes comprising a second oligonucleotide probe and a third oligonucleotide probe, wherein:
i) the second oligonucleotide probe comprises a barcode sequence and a portion that is complementary to a metabolically labeled nucleic acid of interest;
and
ii) the third oligonucleotide probe comprises a portion that is complementary to the metabolically labeled nucleic acid of interest, a first barcode sequence, a portion that is complementary to a first oligonucleotide probe in the population of first oligonucleotide probes, and a second barcode sequence, wherein the first barcode sequence of the third oligonucleotide probe is complementary to the barcode sequence of the second oligonucleotide probe;
d) ligating the 5′ end and the 3′ end of the third oligonucleotide probe together to produce a circular oligonucleotide;
e) performing rolling circle amplification to amplify the circular oligonucleotide using the second oligonucleotide probe as a primer to produce one or more concatenated amplicons;
f) embedding the one or more concatenated amplicons in a polymer matrix;
g) contacting the one or more concatenated amplicons embedded in the polymer matrix with a fourth oligonucleotide probe comprising a sequence that is complementary to the second barcode sequence of the third oligonucleotide probe;
and
h) imaging the fourth oligonucleotide probe to determine the location of the one or more concatenated amplicons embedded in the polymer matrix,
wherein an alteration in the expression of the metabolically labeled nucleic acid of interest relative to expression in a non-diseased cell or a cell from a non-diseased tissue indicates that the alteration in spatiotemporal expression of the metabolically labeled nucleic acid of interest may be involved in the development or progression of the disease or disorder.

10 more elements

A method for screening for an agent capable of modulating spatiotemporal gene expression, the method comprising:

a) incubating a cell in the presence of one or more candidate agents and a pool of nucleoside analogs for an amount of time t1 to metabolically label nucleic acids synthesized by the cell, wherein each nucleoside analog in the pool of nucleoside analogs comprises a reactive chemical moiety;
b) contacting the metabolically labeled nucleic acids with a population of first oligonucleotide probes, wherein each first oligonucleotide probe in the population of first oligonucleotide probes comprises a chemical moiety that reacts with the reactive chemical moiety of the nucleoside analogs;
c) contacting the metabolically labeled nucleic acids with one or more pairs of oligonucleotide probes comprising a second oligonucleotide probe and a third oligonucleotide probe, wherein:
i) the second oligonucleotide probe comprises a barcode sequence and a portion that is complementary to a metabolically labeled nucleic acid of interest;
and
ii) the third oligonucleotide probe comprises a portion that is complementary to the metabolically labeled nucleic acid of interest, a first barcode sequence, a portion that is complementary to a first oligonucleotide probe in the population of first oligonucleotide probes, and a second barcode sequence, wherein the first barcode sequence of the third oligonucleotide probe is complementary to the barcode sequence of the second oligonucleotide probe;
d) ligating the 5′ end and the 3′ end of the third oligonucleotide probe together to produce a circular oligonucleotide;
e) performing rolling circle amplification to amplify the circular oligonucleotide using the second oligonucleotide probe as a primer to produce one or more concatenated amplicons;
f) embedding the one or more concatenated amplicons in a polymer matrix;
g) contacting the one or more concatenated amplicons embedded in the polymer matrix with a fourth oligonucleotide probe comprising a sequence that is complementary to the second barcode sequence of the third oligonucleotide probe;
and
h) imaging the fourth oligonucleotide probe to determine the location of the one or more concatenated amplicons embedded in the polymer matrix,
wherein an alteration in the expression of the metabolically labeled nucleic acid of interest in the presence of the one or more candidate agents relative to expression in the absence of the one or more candidate agents indicates that the one or more candidate agents modulate spatiotemporal gene expression.

10 more elements

A method for diagnosing a disease or disorder in a subject, the method comprising:

a) incubating a cell from a sample taken from a subject in the presence of a pool of nucleoside analogs for an amount of time t1 to metabolically label nucleic acids synthesized by the cell, wherein each nucleoside analog in the pool of nucleoside analogs comprises a reactive chemical moiety;
b) contacting the metabolically labeled nucleic acids with a population of first oligonucleotide probes, wherein each first oligonucleotide probe in the population of first oligonucleotide probes comprises a chemical moiety that reacts with the reactive chemical moiety of the nucleoside analogs;
c) contacting the metabolically labeled nucleic acids with one or more pairs of oligonucleotide probes comprising a second oligonucleotide probe and a third oligonucleotide probe, wherein:
i) the second oligonucleotide probe comprises a barcode sequence and a portion that is complementary to a metabolically labeled nucleic acid of interest;
and
ii) the third oligonucleotide probe comprises a portion that is complementary to the metabolically labeled nucleic acid of interest, a first barcode sequence, a portion that is complementary to a first oligonucleotide probe in the population of first oligonucleotide probes, and a second barcode sequence, wherein the first barcode sequence of the third oligonucleotide probe is complementary to the barcode sequence of the second oligonucleotide probe;
d) ligating the 5′ end and the 3′ end of the third oligonucleotide probe together to produce a circular oligonucleotide;
e) performing rolling circle amplification to amplify the circular oligonucleotide using the second oligonucleotide probe as a primer to produce one or more concatenated amplicons;
f) embedding the one or more concatenated amplicons in a polymer matrix;
g) contacting the one or more concatenated amplicons embedded in the polymer matrix with a fourth oligonucleotide probe comprising a sequence that is complementary to the second barcode sequence of the third oligonucleotide probe;
and
h) imaging the fourth oligonucleotide probe to determine the location of the one or more concatenated amplicons embedded in the polymer matrix,
wherein an alteration in the expression of the metabolically labeled nucleic acid of interest relative to expression in a non-diseased cell or a cell from a non-diseased tissue sample indicates that the subject has the disease or disorder.

10 more elements

A method for treating a disease or disorder in a subject, the method comprising:

a) incubating a cell from a sample taken from a subject in the presence of a pool of nucleoside analogs for an amount of time t1 to metabolically label nucleic acids synthesized by the cell, wherein each nucleoside analog in the pool of nucleoside analogs comprises a reactive chemical moiety;
b) contacting the metabolically labeled nucleic acids with a population of first oligonucleotide probes, wherein each first oligonucleotide probe in the population of first oligonucleotide probes comprises a chemical moiety that reacts with the reactive chemical moiety of the nucleoside analogs;
c) contacting the metabolically labeled nucleic acids with one or more pairs of oligonucleotide probes comprising a second oligonucleotide probe and a third oligonucleotide probe, wherein:
i) the second oligonucleotide probe comprises a barcode sequence and a portion that is complementary to a metabolically labeled nucleic acid of interest;
and
ii) the third oligonucleotide probe comprises a portion that is complementary to the metabolically labeled nucleic acid of interest, a first barcode sequence, a portion that is complementary to a first oligonucleotide probe in the population of first oligonucleotide probes, and a second barcode sequence, wherein the first barcode sequence of the third oligonucleotide probe is complementary to the barcode sequence of the second oligonucleotide probe;
d) ligating the 5′ end and the 3′ end of the third oligonucleotide probe together to produce a circular oligonucleotide;
e) performing rolling circle amplification to amplify the circular oligonucleotide using the second oligonucleotide probe as a primer to produce one or more concatenated amplicons;
f) embedding the one or more concatenated amplicons in a polymer matrix;
g) contacting the one or more concatenated amplicons embedded in the polymer matrix with a fourth oligonucleotide probe comprising a sequence that is complementary to the second barcode sequence of the third oligonucleotide probe;
h) imaging the fourth oligonucleotide probe to determine the location of the one or more concatenated amplicons embedded in the polymer matrix;
and
i) administering a treatment for the disease or disorder to the subject if an alteration in spatiotemporal gene expression relative to expression in a non-diseased cell or a cell from a non-diseased tissue sample is observed.

10 more elements

A method for profiling spatiotemporal gene expression in a subject, the method comprising:

a) administering a pool of nucleoside analogs to a subject in vivo for an amount of time t1 to metabolically label nucleic acids synthesized by one or more cells in the subject, wherein each nucleoside analog in the pool of nucleoside analogs comprises a reactive chemical moiety;
b) harvesting a tissue sample from the subject;
c) contacting the metabolically labeled nucleic acids in the harvested tissue sample with a population of first oligonucleotide probes, wherein each first oligonucleotide probe in the population of first oligonucleotide probes comprises a chemical moiety that reacts with the reactive chemical moiety of the nucleoside analogs;
d) contacting the metabolically labeled nucleic acids with one or more pairs of oligonucleotide probes comprising a second oligonucleotide probe and a third oligonucleotide probe, wherein:
i) the second oligonucleotide probe comprises a barcode sequence and a portion that is complementary to a metabolically labeled nucleic acid of interest;
and
ii) the third oligonucleotide probe comprises a portion that is complementary to the metabolically labeled nucleic acid of interest, a first barcode sequence, a portion that is complementary to a first oligonucleotide probe in the population of first oligonucleotide probes, and a second barcode sequence, wherein the first barcode sequence of the third oligonucleotide probe is complementary to the barcode sequence of the second oligonucleotide probe;
e) ligating the 5′ end and the 3′ end of the third oligonucleotide probe together to produce a circular oligonucleotide;
f) performing rolling circle amplification to amplify the circular oligonucleotide using the second oligonucleotide probe as a primer to produce one or more concatenated amplicons;
g) embedding the one or more concatenated amplicons in a polymer matrix;
h) contacting the one or more concatenated amplicons embedded in the polymer matrix with a fourth oligonucleotide probe comprising a sequence that is complementary to the second barcode sequence of the third oligonucleotide probe;
and
i) imaging the fourth oligonucleotide probe to determine the location of the one or more concatenated amplicons embedded in the polymer matrix.

10 more elements

Dependencies are inferred from the claim text.

Facts

Application: 18/559,322Filed 6 May 2022
Priority: 7 May 2021
Published: 1 Aug 2024
Term ends: Not estimatedThe source records don’t support an estimate.

Classification10 codes

C12Q 1/6841
Hybridisation assays: in situ hybridisation
C12N 15/1093
Isolating an individual clone by screening libraries: general methods of preparing gene libraries, not provided for in other subgroups
C12Q 1/6876
Involving nucleic acids: nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
G01N 21/6458
Spatial resolved fluorescence measurements; Imaging: fluorescence microscopy