Methods and compositions for simultaneous single cell transcriptome and translatome profiling

Abstract

The present disclosure provides methods and systems for the simultaneous profiling of the RNA transcriptome and translatome in a single cell. Also provided by the present disclosure are methods for diagnosing a disease or disorder in a subject based on a profile of the RNAs being translated in a cell, including cells within an intact tissue. Methods of screening for or testing a candidate agent capable of modulating translation of one or more RNAs are also provided by the present disclosure. The present disclosure also provides methods for treating a disease or disorder in a subject in need thereof. Sets of probes comprising oligonucleotide portions, which may be useful for performing the methods described herein, are also described by the present disclosure. The present disclosure further provides non-naturally- occurring assembled ribosome complexes comprising ribosome subunits and oligonucleotide probes. Additionally, the present disclosure provides kits comprising any of the probes described herein.

Description

Atty. Docket No. 114203-1551METHODS AND COMPOSITIONS FOR SIMULTANEOUS SINGLE CELL TRANSCRIPTOME AND TRANSLATOME PROFILINGCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application No. 63 / 761,263, filed February 21, 2025, the entire contents of which are incorporated herein by reference in their entireties.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under Grant No. 1DP2GM146245-01 awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUND

[0003] Transcriptional profiles in single cells do not consistently correlate with proteomic profiles from the same cells. This suggests that various mechanisms exist at the post-transcriptional level to regulate protein production and degradation, including some that have yet to be characterized. Therefore, mRNA levels are an imperfect proxy for estimating protein production and are likely biased in defining cellular states. Additional methods for directly quantifying single-cell proteomes could provide a more functionally relevant base to interpret cell types and cell-type-specific responses to environments. An accurate determination of protein production in a cell could be useful in studying an array of physiological states or even in treating various diseases, as well as in drug development. Accordingly, additional and better systems for accurately quantifying protein production in a single cell are needed.SUMMARY

[0004] Methods, compositions, kits, and systems for in situ transcriptome and translatome profiling ( / .< ., profiling of RNAs of interest that are being actively translated) are described herein. Such systems represent a crucial technology to bridge the gap between the transcriptome and the proteome of a cell by quantifying / profiling simultaneously the transcriptome and translatome of a single cell. A strategy based on proximity ligation is described herein to achieve specific and high-throughput characterization of the RNA14934-6856-6158.1Atty. Docket No. 114203-1551transcriptome and translatome in situ as an estimation of single-cell proteomes. The present system utilizes agents that recognize a fully-assembled ribosome conjugated to an oligonucleotide probe that recognizes the sequences of corresponding probes annealed to RNA molecules that are bound by ribosomes.

[0005] In one aspect, the present disclosure provides a method for simultaneous transcriptome and translatome profiling in a cell, the method comprising: (a) contacting the cell with one or more sets of probes, wherein each set of probes comprises a first probe, a second probe, a third probe, a fourth probe, a fifth probe, and a sixth probe, wherein (i) the first probe comprises (1) a first oligonucleotide barcode sequence; (2) an oligonucleotide portion that is complementary to a portion of the fifth probe; (3) an oligonucleotide portion that is complementary to a portion of an RNA of interest; (4) a second oligonucleotide barcode sequence; and (5) an oligonucleotide portion that is complementary to a portion of the sixth probe; (ii) the second probe comprises (1) a first oligonucleotide barcode sequence; (2) an oligonucleotide portion that is complementary to a portion of the fifth probe; (3) an oligonucleotide portion that is complementary to a portion of an RNA of interest; and (4) a second oligonucleotide barcode sequence; (iii) the third probe comprises an oligonucleotide portion that recognizes the 40S subunit of a ribosome and an oligonucleotide portion that is complementary to a portion of the sixth probe; (iv) the fourth probe comprises an oligonucleotide portion that recognizes the 60S subunit of a ribosome and an oligonucleotide portion that is complementary to a portion of the sixth probe; (v) the fifth probe comprises (1) an oligonucleotide portion that is complementary to the RNA of interest; (2) an oligonucleotide portion that is complementary to a portion of the first probe; (3) an oligonucleotide portion that is complementary to a portion of the second probe; and (4) an oligonucleotide barcode sequence, wherein the oligonucleotide barcode sequence of the fifth probe is complementary to the first oligonucleotide barcode sequence of the first probe and the first oligonucleotide barcode sequence of the second probe; and (vi) the sixth probe comprises (1) an oligonucleotide portion that is complementary to a portion of the first probe; (2) an oligonucleotide portion that is complementary to a portion of the third probe; and (3) an oligonucleotide portion that is complementary to a portion of the fourth probe; (b) ligating the 5' end and the 3' end of the first probe together to produce a first circular oligonucleotide, and ligating the 5' end and the 3' end of the second probe together to produce a second circular oligonucleotide; (c) performing rolling circle amplification to amplify the first circular oligonucleotide and the second circular oligonucleotide, wherein the fifth probe is a primer for the amplification of each of the first circular oligonucleotide and the second 24934-6856-6158.1Atty. Docket No. 114203-1551circular oligonucleotide, thereby producing one or more concatenated amplicons from the first circular oligonucleotide and one or more concatenated amplicons from the second circular oligonucleotide; (d) embedding the one or more concatenated amplicons from the first circular oligonucleotide and one or more concatenated amplicons from the second circular oligonucleotide in a polymeric matrix; and (e) sequencing the concatenated amplicons embedded in the polymeric matrix to determine the identity, location, and translation status of each RNA of interest in the cell.

[0006] In one aspect, the present disclosure provides a method for diagnosing a disease or disorder in a subject, the method comprising: (a) contacting a cell obtained from the subject with one or more sets of probes, wherein each set of probes comprises a first probe, a second probe, a third probe, a fourth probe, a fifth probe, and a sixth probe, wherein (i) the first probe comprises; a first oligonucleotide barcode sequence; an oligonucleotide portion that is complementary to a portion of the fifth probe; an oligonucleotide portion that is complementary to a portion of an RNA of interest; a second oligonucleotide barcode sequence; and an oligonucleotide portion that is complementary to a portion of the sixth probe; (ii) the second probe comprises a first oligonucleotide barcode sequence; an oligonucleotide portion that is complementary to a portion of the fifth probe; an oligonucleotide portion that is complementary to a portion of an RNA of interest; and a second oligonucleotide barcode sequence; (iii) the third probe comprises a portion that recognizes the 40S subunit of a ribosome and an oligonucleotide portion that is complementary to a portion of the sixth probe; (iv) the fourth probe comprises a portion that recognizes the 60S subunit of a ribosome and an oligonucleotide portion that is complementary to a portion of the sixth probe; (v) the fifth probe comprises an oligonucleotide portion that is complementary to the RNA of interest; an oligonucleotide portion that is complementary to a portion of the first probe; an oligonucleotide portion that is complementary to a portion of the second probe; and an oligonucleotide barcode sequence, wherein the oligonucleotide barcode sequence of the fifth probe is complementary to the first oligonucleotide barcode sequence of the first probe and the first oligonucleotide barcode sequence of the second probe; and (vi) the sixth probe comprises an oligonucleotide portion that is complementary to a portion of the first probe; an oligonucleotide portion that is complementary to a portion of the third probe; and an oligonucleotide portion that is complementary to a portion of the fourth probe; (b) ligating the 5' end and the 3' end of the first probe together to produce a first circular oligonucleotide, and ligating the 5' end and the 3' end of the second probe together to produce a second circular oligonucleotide; (c)34934-6856-6158.1Atty. Docket No. 114203-1551performing rolling circle amplification to amplify the first circular oligonucleotide and the second circular oligonucleotide, wherein the fifth probe is a primer for the amplification of each of the first circular oligonucleotide and the second circular oligonucleotide, thereby producing one or more concatenated amplicons from the first circular oligonucleotide and one or more concatenated amplicons from the second circular oligonucleotide; (d) embedding the one or more concatenated amplicons from the first circular oligonucleotide and one or more concatenated amplicons from the second circular oligonucleotide in a polymeric matrix; and (e) sequencing the concatenated amplicons embedded in the polymeric matrix to determine the identity, location, and translation status of each RNA of interest in the cell, wherein a difference in the RNA translation profile of the cell relative to one or more nondiseased cells indicates that the subject has the disease or disorder.

[0007] In one aspect, the present disclosure provides a method for screening for an agent capable of modulating translation of one or more RNAs comprises the steps of: (a) contacting a cell that is being treated with or has been treated with a candidate agent with one or more sets of probes, wherein each set of probes comprises a first probe, a second probe, a third probe, a fourth probe, a fifth probe, and a sixth probe, wherein (i) the first probe comprises; a first oligonucleotide barcode sequence; an oligonucleotide portion that is complementary to a portion of the fifth probe; an oligonucleotide portion that is complementary to a portion of an RNA of interest; a second oligonucleotide barcode sequence; and an oligonucleotide portion that is complementary to a portion of the sixth probe; (ii) the second probe comprises a first oligonucleotide barcode sequence; an oligonucleotide portion that is complementary to a portion of the fifth probe; an oligonucleotide portion that is complementary to a portion of an RNA of interest; and a second oligonucleotide barcode sequence; (iii) the third probe comprises a portion that recognizes the 40S subunit of a ribosome and an oligonucleotide portion that is complementary to a portion of the sixth probe; (iv) the fourth probe comprises a portion that recognizes the 60S subunit of a ribosome and an oligonucleotide portion that is complementary to a portion of the sixth probe; (v) the fifth probe comprises an oligonucleotide portion that is complementary to the RNA of interest; an oligonucleotide portion that is complementary to a portion of the first probe; an oligonucleotide portion that is complementary to a portion of the second probe; and an oligonucleotide barcode sequence, wherein the oligonucleotide barcode sequence of the fifth probe is complementary to the first oligonucleotide barcode sequence of the first probe and the first oligonucleotide barcode sequence of the second probe; and (vi) the sixth probe comprises an oligonucleotide portion that is complementary to a portion of the first probe; an oligonucleotide portion that is44934-6856-6158.1Atty. Docket No. 114203-1551complementary to a portion of the third probe; and an oligonucleotide portion that is complementary to a portion of the fourth probe; (b) ligating the 5' end and the 3' end of the first probe together to produce a first circular oligonucleotide, and ligating the 5' end and the 3' end of the second probe together to produce a second circular oligonucleotide; (c) performing rolling circle amplification to amplify the first circular oligonucleotide and the second circular oligonucleotide, wherein the fifth probe is a primer for the amplification of each of the first circular oligonucleotide and the second circular oligonucleotide, thereby producing one or more concatenated amplicons from the first circular oligonucleotide and one or more concatenated amplicons from the second circular oligonucleotide; (d) embedding the one or more concatenated amplicons from the first circular oligonucleotide and one or more concatenated amplicons from the second circular oligonucleotide in a polymeric matrix; and (e) sequencing the concatenated amplicons embedded in the polymeric matrix to determine the identity, location, and translation status of each RNA of interest in the cell, wherein a difference in the profile of RNAs being translated in the presence of the candidate agent relative to in the absence of the candidate agent indicates that the candidate agent modulates translation of one or more RNAs.

[0008] In one aspect, the present disclosure provides a method for treating a disease or disorder in a subject comprising the steps of: (a) contacting a cell obtained from the subject with one or more sets of probes, wherein each set of probes comprises a first probe, a second probe, a third probe, a fourth probe, a fifth probe, and a sixth probe, wherein (i) the first probe comprises; a first oligonucleotide barcode sequence; an oligonucleotide portion that is complementary to a portion of the fifth probe; an oligonucleotide portion that is complementary to a portion of an RNA of interest; a second oligonucleotide barcode sequence; and an oligonucleotide portion that is complementary to a portion of the sixth probe; (ii) the second probe comprises a first oligonucleotide barcode sequence; an oligonucleotide portion that is complementary to a portion of the fifth probe; an oligonucleotide portion that is complementary to a portion of an RNA of interest; and a second oligonucleotide barcode sequence; (iii) the third probe comprises a portion that recognizes the 40S subunit of a ribosome and an oligonucleotide portion that is complementary to a portion of the sixth probe; (iv) the fourth probe comprises a portion that recognizes the 60S subunit of a ribosome and an oligonucleotide portion that is complementary to a portion of the sixth probe; (v) the fifth probe comprises an oligonucleotide portion that is complementary to the RNA of interest; an oligonucleotide portion that is complementary to a portion of the first probe; an oligonucleotide portion that is 54934-6856-6158.1Atty. Docket No. 114203-1551complementary to a portion of the second probe; and an oligonucleotide barcode sequence, wherein the oligonucleotide barcode sequence of the fifth probe is complementary to the first oligonucleotide barcode sequence of the first probe and the first oligonucleotide barcode sequence of the second probe; and (vi) the sixth probe comprises an oligonucleotide portion that is complementary to a portion of the first probe; an oligonucleotide portion that is complementary to a portion of the third probe; and an oligonucleotide portion that is complementary to a portion of the fourth probe; (b) ligating the 5' end and the 3' end of the first probe together to produce a first circular oligonucleotide, and ligating the 5' end and the 3' end of the second probe together to produce a second circular oligonucleotide; (c) performing rolling circle amplification to amplify the first circular oligonucleotide and the second circular oligonucleotide, wherein the fifth probe is a primer for the amplification of each of the first circular oligonucleotide and the second circular oligonucleotide, thereby producing one or more concatenated amplicons from the first circular oligonucleotide and one or more concatenated amplicons from the second circular oligonucleotide; (d) embedding the one or more concatenated amplicons from the first circular oligonucleotide and one or more concatenated amplicons from the second circular oligonucleotide in a polymeric matrix; and (e) sequencing the concatenated amplicons embedded in the polymeric matrix to determine the identity, location, and translation status of each RNA of interest in the cell; and (f) administering a treatment for the disease or disorder to the subject if a difference in the profile of RNAs being translated in the cell relative to one or more non-diseased cells is observed.

[0009] In one aspect, the present disclosure provides a method for simultaneous transcriptome and translatome profiling in a cell, the method comprising: (a) contacting the cell with one or more sets of probes, wherein each set of probes comprises a first probe, a second probe, a third probe, a fourth probe, a fifth probe, and a sixth probe, wherein (i) the first probe comprises (1) a first oligonucleotide barcode sequence; (2) an oligonucleotide portion that is complementary to a portion of the fifth probe; (3) an oligonucleotide portion that is complementary to a portion of an RNA of interest; (4) a second oligonucleotide barcode sequence; and (5) an oligonucleotide portion that is complementary to a portion of the sixth probe; (ii) the second probe comprises (1) a first oligonucleotide barcode sequence; (2) an oligonucleotide portion that is complementary to a portion of the fifth probe; (3) an oligonucleotide portion that is complementary to a portion of an RNA of interest; and (4) a second oligonucleotide barcode sequence; (iii) the third probe comprises an oligonucleotide portion that recognizes a first subunit of a ribosome and an oligonucleotide portion that is 64934-6856-6158.1Atty. Docket No. 114203-1551complementary to a portion of the sixth probe; (iv) the fourth probe comprises an oligonucleotide portion that recognizes a second subunit of a ribosome and an oligonucleotide portion that is complementary to a portion of the sixth probe; (v) the fifth probe comprises (1) an oligonucleotide portion that is complementary to the RNA of interest; (2) an oligonucleotide portion that is complementary to a portion of the first probe; (3) an oligonucleotide portion that is complementary to a portion of the second probe; and (4) an oligonucleotide barcode sequence, wherein the oligonucleotide barcode sequence of the fifth probe is complementary to the first oligonucleotide barcode sequence of the first probe and the first oligonucleotide barcode sequence of the second probe; and (vi) the sixth probe comprises (1) an oligonucleotide portion that is complementary to a portion of the first probe; (2) an oligonucleotide portion that is complementary to a portion of the third probe; and (3) an oligonucleotide portion that is complementary to a portion of the fourth probe; (b) ligating the 5' end and the 3' end of the first probe together to produce a first circular oligonucleotide, and ligating the 5' end and the 3' end of the second probe together to produce a second circular oligonucleotide; (c) performing rolling circle amplification to amplify the first circular oligonucleotide and the second circular oligonucleotide, wherein the fifth probe is a primer for the amplification of each of the first circular oligonucleotide and the second circular oligonucleotide, thereby producing one or more concatenated amplicons from the first circular oligonucleotide and one or more concatenated amplicons from the second circular oligonucleotide; (d) embedding the one or more concatenated amplicons from the first circular oligonucleotide and one or more concatenated amplicons from the second circular oligonucleotide in a polymeric matrix; and (e) sequencing the concatenated amplicons embedded in the polymeric matrix to determine the identity, location, and translation status of each RNA of interest in the cell.

[0010] In one aspect, the present disclosure provides a method for diagnosing a disease or disorder in a subject, the method comprising: (a) contacting a cell obtained from the subject with one or more sets of probes, wherein each set of probes comprises a first probe, a second probe, a third probe, a fourth probe, a fifth probe, and a sixth probe, wherein (i) the first probe comprises; a first oligonucleotide barcode sequence; an oligonucleotide portion that is complementary to a portion of the fifth probe; an oligonucleotide portion that is complementary to a portion of an RNA of interest; a second oligonucleotide barcode sequence; and an oligonucleotide portion that is complementary to a portion of the sixth probe; (ii) the second probe comprises a first oligonucleotide barcode sequence; an oligonucleotide portion that is complementary to a portion of the fifth probe; an74934-6856-6158.1Atty. Docket No. 114203-1551oligonucleotide portion that is complementary to a portion of an RNA of interest; and a second oligonucleotide barcode sequence; (iii) the third probe comprises a portion that recognizes a first subunit of a ribosome and an oligonucleotide portion that is complementary to a portion of the sixth probe; (iv) the fourth probe comprises a portion that recognizes a second subunit of a ribosome and an oligonucleotide portion that is complementary to a portion of the sixth probe; (v) the fifth probe comprises an oligonucleotide portion that is complementary to the RNA of interest; an oligonucleotide portion that is complementary to a portion of the first probe; an oligonucleotide portion that is complementary to a portion of the second probe; and an oligonucleotide barcode sequence, wherein the oligonucleotide barcode sequence of the fifth probe is complementary to the first oligonucleotide barcode sequence of the first probe and the first oligonucleotide barcode sequence of the second probe; and (vi) the sixth probe comprises an oligonucleotide portion that is complementary to a portion of the first probe; an oligonucleotide portion that is complementary to a portion of the third probe; and an oligonucleotide portion that is complementary to a portion of the fourth probe; (b) ligating the 5' end and the 3' end of the first probe together to produce a first circular oligonucleotide, and ligating the 5' end and the 3' end of the second probe together to produce a second circular oligonucleotide; (c) performing rolling circle amplification to amplify the first circular oligonucleotide and the second circular oligonucleotide, wherein the fifth probe is a primer for the amplification of each of the first circular oligonucleotide and the second circular oligonucleotide, thereby producing one or more concatenated amplicons from the first circular oligonucleotide and one or more concatenated amplicons from the second circular oligonucleotide; (d) embedding the one or more concatenated amplicons from the first circular oligonucleotide and one or more concatenated amplicons from the second circular oligonucleotide in a polymeric matrix; and (e) sequencing the concatenated amplicons embedded in the polymeric matrix to determine the identity, location, and translation status of each RNA of interest in the cell, wherein a difference in the RNA translation profile of the cell relative to one or more non-diseased cells indicates that the subject has the disease or disorder.

[0011] In one aspect, the present disclosure provides a method for screening for an agent capable of modulating translation of one or more RNAs comprises the steps of: (a) contacting a cell that is being treated with or has been treated with a candidate agent with one or more sets of probes, wherein each set of probes comprises a first probe, a second probe, a third probe, a fourth probe, a fifth probe, and a sixth probe, wherein (i) the first probe comprises; a first oligonucleotide barcode sequence; an oligonucleotide portion that is complementary to a 84934-6856-6158.1Atty. Docket No. 114203-1551portion of the fifth probe; an oligonucleotide portion that is complementary to a portion of an RNA of interest; a second oligonucleotide barcode sequence; and an oligonucleotide portion that is complementary to a portion of the sixth probe; (ii) the second probe comprises a first oligonucleotide barcode sequence; an oligonucleotide portion that is complementary to a portion of the fifth probe; an oligonucleotide portion that is complementary to a portion of an RNA of interest; and a second oligonucleotide barcode sequence; (iii) the third probe comprises a portion that recognizes a first subunit of a ribosome and an oligonucleotide portion that is complementary to a portion of the sixth probe; (iv) the fourth probe comprises a portion that recognizes a second subunit of a ribosome and an oligonucleotide portion that is complementary to a portion of the sixth probe; (v) the fifth probe comprises an oligonucleotide portion that is complementary to the RNA of interest; an oligonucleotide portion that is complementary to a portion of the first probe; an oligonucleotide portion that is complementary to a portion of the second probe; and an oligonucleotide barcode sequence, wherein the oligonucleotide barcode sequence of the fifth probe is complementary to the first oligonucleotide barcode sequence of the first probe and the first oligonucleotide barcode sequence of the second probe; and (vi) the sixth probe comprises an oligonucleotide portion that is complementary to a portion of the first probe; an oligonucleotide portion that is complementary to a portion of the third probe; and an oligonucleotide portion that is complementary to a portion of the fourth probe; (b) ligating the 5' end and the 3' end of the first probe together to produce a first circular oligonucleotide, and ligating the 5' end and the 3' end of the second probe together to produce a second circular oligonucleotide; (c) performing rolling circle amplification to amplify the first circular oligonucleotide and the second circular oligonucleotide, wherein the fifth probe is a primer for the amplification of each of the first circular oligonucleotide and the second circular oligonucleotide, thereby producing one or more concatenated amplicons from the first circular oligonucleotide and one or more concatenated amplicons from the second circular oligonucleotide; (d) embedding the one or more concatenated amplicons from the first circular oligonucleotide and one or more concatenated amplicons from the second circular oligonucleotide in a polymeric matrix; and (e) sequencing the concatenated amplicons embedded in the polymeric matrix to determine the identity, location, and translation status of each RNA of interest in the cell, wherein a difference in the profile of RNAs being translated in the presence of the candidate agent relative to in the absence of the candidate agent indicates that the candidate agent modulates translation of one or more RNAs.94934-6856-6158.1Atty. Docket No. 114203-1551

[0012] In one aspect, the present disclosure provides a method for treating a disease or disorder in a subject comprising the steps of: (a) contacting a cell obtained from the subject with one or more sets of probes, wherein each set of probes comprises a first probe, a second probe, a third probe, a fourth probe, a fifth probe, and a sixth probe, wherein (i) the first probe comprises; a first oligonucleotide barcode sequence; an oligonucleotide portion that is complementary to a portion of the fifth probe; an oligonucleotide portion that is complementary to a portion of an RNA of interest; a second oligonucleotide barcode sequence; and an oligonucleotide portion that is complementary to a portion of the sixth probe; (ii) the second probe comprises a first oligonucleotide barcode sequence; an oligonucleotide portion that is complementary to a portion of the fifth probe; an oligonucleotide portion that is complementary to a portion of an RNA of interest; and a second oligonucleotide barcode sequence; (iii) the third probe comprises a portion that recognizes a first subunit of a ribosome and an oligonucleotide portion that is complementary to a portion of the sixth probe; (iv) the fourth probe comprises a portion that recognizes a second subunit of a ribosome and an oligonucleotide portion that is complementary to a portion of the sixth probe; (v) the fifth probe comprises an oligonucleotide portion that is complementary to the RNA of interest; an oligonucleotide portion that is complementary to a portion of the first probe; an oligonucleotide portion that is complementary to a portion of the second probe; and an oligonucleotide barcode sequence, wherein the oligonucleotide barcode sequence of the fifth probe is complementary to the first oligonucleotide barcode sequence of the first probe and the first oligonucleotide barcode sequence of the second probe; and (vi) the sixth probe comprises an oligonucleotide portion that is complementary to a portion of the first probe; an oligonucleotide portion that is complementary to a portion of the third probe; and an oligonucleotide portion that is complementary to a portion of the fourth probe; (b) ligating the 5' end and the 3' end of the first probe together to produce a first circular oligonucleotide, and ligating the 5' end and the 3' end of the second probe together to produce a second circular oligonucleotide; (c) performing rolling circle amplification to amplify the first circular oligonucleotide and the second circular oligonucleotide, wherein the fifth probe is a primer for the amplification of each of the first circular oligonucleotide and the second circular oligonucleotide, thereby producing one or more concatenated amplicons from the first circular oligonucleotide and one or more concatenated amplicons from the second circular oligonucleotide; (d) embedding the one or more concatenated amplicons from the first circular oligonucleotide and one or more concatenated amplicons from the second circular oligonucleotide in a polymeric matrix; and (e) sequencing the concatenated104934-6856-6158.1Atty. Docket No. 114203-1551amplicons embedded in the polymeric matrix to determine the identity, location, and translation status of each RNA of interest in the cell; and (f) administering a treatment for the disease or disorder to the subject if a difference in the profile of RNAs being translated in the cell relative to one or more non-diseased cells is observed.

[0013] In one aspect, the present disclosure provides a set of probes comprising a first probe, a second probe, a third probe, a fourth probe, a fifth probe, and a sixth probe, wherein: (i) the first probe comprises a first oligonucleotide barcode sequence; an oligonucleotide portion that is complementary to a portion of the fifth probe; an oligonucleotide portion that is complementary to a portion of an RNA of interest; a second oligonucleotide barcode sequence; and an oligonucleotide portion that is complementary to a portion of the sixth probe; (ii) the second probe comprises a first oligonucleotide barcode sequence; an oligonucleotide portion that is complementary to a portion of the fifth probe; an oligonucleotide portion that is complementary to a portion of an RNA of interest; and a second oligonucleotide barcode sequence; (iii) the third probe comprises a portion that recognizes the 40S subunit of a ribosome and an oligonucleotide portion that is complementary to a portion of the sixth probe; (iv) the fourth probe comprises a portion that recognizes the 60S subunit of a ribosome and an oligonucleotide portion that is complementary to a portion of the sixth probe; (v) the fifth probe comprises an oligonucleotide portion that is complementary to the RNA of interest; an oligonucleotide portion that is complementary to a portion of the first probe; an oligonucleotide portion that is complementary to a portion of the second probe; and an oligonucleotide barcode sequence, wherein the oligonucleotide barcode sequence of the fifth probe is complementary to the first oligonucleotide barcode sequence of the first probe and the first oligonucleotide barcode sequence of the second probe; and (vi) the sixth probe comprises an oligonucleotide portion that is complementary to a portion of the first probe; an oligonucleotide portion that is complementary to a portion of the third probe; and an oligonucleotide portion that is complementary to a portion of the fourth probe.

[0014] In one aspect, the present disclosure provides systems for profiling RNAs being translated in a cell, such a system comprising: (a) a cell; (b) one or more sets of probes comprising a first probe, a second probe, a third probe, a fourth probe, a fifth probe, and a sixth probe, wherein (i) the first probe comprises a first oligonucleotide barcode sequence; an oligonucleotide portion that is complementary to a portion of the fifth probe; an oligonucleotide portion that is complementary to a portion of an RNA of interest; a second oligonucleotide barcode sequence; and an oligonucleotide portion that is complementary to a 114934-6856-6158.1Atty. Docket No. 114203-1551portion of the sixth probe; (ii) the second probe comprises a first oligonucleotide barcode sequence; an oligonucleotide portion that is complementary to a portion of the fifth probe; an oligonucleotide portion that is complementary to a portion of an RNA of interest; and a second oligonucleotide barcode sequence; (iii) the third probe comprises a portion that recognizes the 40S subunit of a ribosome and an oligonucleotide portion that is complementary to a portion of the sixth probe; (iv) the fourth probe comprises a portion that recognizes the 60S subunit of a ribosome and an oligonucleotide portion that is complementary to a portion of the sixth probe; (v) the fifth probe comprises an oligonucleotide portion that is complementary to the RNA of interest; an oligonucleotide portion that is complementary to a portion of the first probe; an oligonucleotide portion that is complementary to a portion of the second probe; and an oligonucleotide barcode sequence, wherein the oligonucleotide barcode sequence of the fifth probe is complementary to the first oligonucleotide barcode sequence of the first probe and the first oligonucleotide barcode sequence of the second probe; and (vi) the sixth probe comprises an oligonucleotide portion that is complementary to a portion of the first probe; an oligonucleotide portion that is complementary to a portion of the third probe; and an oligonucleotide portion that is complementary to a portion of the fourth probe; (c) a microscope; and (d) a computer.

[0015] In one aspect, the present disclosure provides an assembled ribosome complex comprising: (a) a first oligonucleotide probe, a second oligonucleotide probe, and a third oligonucleotide probe, wherein (i) the first probe comprises an oligonucleotide portion that recognizes the 40S subunit of a ribosome and an oligonucleotide portion that is complementary to a portion of the third probe; (ii) the second probe comprises an oligonucleotide portion that recognizes the 60S subunit of a ribosome and an oligonucleotide portion that is complementary to a portion of the third probe; and (iii) the third probe comprises an oligonucleotide portion that is complementary to a portion of the first probe and an oligonucleotide portion that is complementary to a portion of the second probe; (b) a 40S subunit of a ribosome; and (c) a 60S subunit of a ribosome.

[0016] In one aspect, the present disclosure provides a kit comprising any one or more of the probes described above.

[0017] Any of the probes ( / .< ., sets of probes) described herein may be used in the methods, composition, systems, and kits contemplated by the present disclosure.

[0018] It should be appreciated that the foregoing concepts, and additional concepts discussed below, may be arranged in any suitable combination, as the present disclosure is not limited in this respect. Further, other advantages and novel features of the present124934-6856-6158.1Atty. Docket No. 114203-1551disclosure will become apparent from the following detailed description of various nonlimiting embodiments when considered in conjunction with the accompanying figures.BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0020] FIG. 1 is a schematic illustrating an overview of 3D spatial multi-omics on RNA. FIG. 1A is a schematic showing traditional single modality spatial omics vs. all-in-one spatial multi-omics. Left: Traditional spatial technologies usually profile only a single modality of RNAs in each sample, including transcriptome, translatome, temporal transcriptome, epitranscriptome, and RNA-protein interactions. Right: various modalities of RNA are co-profiled in one single sample, including transcriptome, translatome, and other modalities (temporal transcriptome, epitranscriptome, RNA-protein interactions, protein immunostaining, etc.) at subcellular and single-cell level. FIG. IB is a schematic showing applications enabled by 3D spatial multi-omics on RNA. Top-left and top-right: Applicant’s technology described herein can reveal the interplay of each step of the RNA life cycle in single cells, and elucidate the heterogeneous RNA fate regulations across different cell types and at different subcellular locations. Bottom-left: Applicant’s technology described herein facilitates multi-modality RNA mapping in thick tissue blocks and circumvents the limitations of computational alignment and reconstruction. Bottom-right: Applicant’s technology described herein can be applied to samples perturbed by CRISPR libraries where different single cells receive distinct perturbations. In this way, novel cell-type-specific RNA regulators may be discovered using Applicant’s technology described herein.

[0021] FIG. 2 is a set of schematics illustrating the design of 80S qRIBOmap. FIG. 2A shows schematics of the 80S qRIBOmap: actively translating RNA is associated with the fully-assembled 80S ribosome while non-translating RNA is not. RNAs are hybridized with multiple primers as well as RIBO and STAR padlocks that compete with each other. The RIBO padlock and the STAR padlock share the same hybridization region, so each primer probe can pair with either the RIBO padlock probe or the STAR padlock probe. When both the 18s arm and the 28s arm are present, the 80S splint probe can be recruited and RIBO reads (translating RNA reads, magenta on amplicon and in cell) can be generated. Otherwise, STAR reads (RNA reads, green on amplicon and in cell) are expected to be generated.134934-6856-6158.1Atty. Docket No. 114203-1551Amplicon generation and sequencing are similar to previous methods (Wang et al. 2018). 5’ P: 5’ phosphorylation; 3’ invdT: 3’ inverted dT. FIG. 2B is a schematic showing the working principles and logic of 80S qRIBOmap signal generation. To obtain STAR and RIBO reads, the probes must pass through multiple AND gates, significantly enhancing probe specificity.FIG. 2C is a schematic showing potential applications of 80S qRIBOmap in tissue mapping. Single-cell translation efficiency (TE) can be quantified for different cell types.

[0022] FIG. 3 shows a panel of images and graphs describing experimental data related to 80S qRIBOmap analysis of ACTB RNA. FIG. 3A is a representative image of 80S qRIBOmap on ACTB mRNA in HeLa cells. FIG. 3B is a set of representative images of 80S qRIBOmap on ACTB mRNA in HeLa cells under partial hybridization conditions. FIG. 3C is a graph showing a statistical analysis of the decrease of ACTB 80S qRIBOmap signals under partial hybridization conditions. As expected, RIBO reads significantly decreased but STAR reads remained the same. Student’ s t-test, ***: p < 0.001; ns: non-significant

[0023] FIG. 4 shows a panel of images and graphs describing experimental data related to 80S qRIBOmap analysis oiMALATl and vtRNAl-1 non-coding RNA. FIG. 4A is a set of representative images of 80S qRIBOmap on non-coding RNAs in HeLa cells. FIG. 4B is a graph showing a statistical analysis of the difference in TE of ACTB RNA and non-coding RNAs. Student’s t-test, ****: p < 0.0001.

[0024] FIG. 5 shows a panel of images and graphs describing experimental data related to 80S qRIBOmap analysis in the presence of translation inhibitors. FIG. 5A is a panel of representative images of 80S qRIBOmap on ACTB mRNA in 3T3 cells after translation inhibitor treatment. FIG. 5B is a graph showing a statistical analysis of the change in 80S qRIBOmap signals upon translation inhibition. FIG. 5C is a graph showing a statistical analysis of the change in 40S qRIBOmap signals upon translation inhibition. FIG. 5D is a graph showing a statistical analysis of the change in 60S qRIBOmap signals upon translation inhibition. Student’s t-test, ****■. p < 0.0001; *** p < 0.001; **: p < 0.01; ns: non-significant.

[0025] FIG. 6 shows a panel of images and graphs describing experimental data related to 80S qRIBOmap analysis of a mouse midbrain sample. FIG. 6A is a representative image of 80S qRIBOmap in a part of the mouse midbrain. FIG. 6B is a graph showing a quantification of RIBO and STAR reads in the mouse brain sample.

[0026] FIG. 7 is a panel of images showing representative fields of view of qRIBOmap in a 127-gene in situ sequencing experiment on HeLa cells. RIBO reads are shown in green and magenta, while STAR reads are shown in cyan and yellow. Scale bar: 20 pm.144934-6856-6158.1Atty. Docket No. 114203-1551

[0027] FIG. 8 is a set of schematics showing alternative schemes of 80S qRIBOmap probe design. FIG. 8A is a schematic showing probe design of a top-performing Scheme 1 (also shown in FIG. 2A). The 28s arms have a 5’ phosphate and a 11-nt complementary region with the 80S splint probe, while the 18s arms have a 9-nt complementary region with the 80S splint probe. The sequences of the complementary regions and linkers are shown in detail. FIG. 8B is a schematic showing the probe design of Scheme 2. On top of the design of Scheme 1, inspired by the proximity probe design from Schulte et al(Schulte et al. 2024), a proximity probe hybridizes to both the 18s arms and 28s arms, thereby stabilizing the whole complex. FIG. 8C is a schematic showing the probe design of Scheme 3. On top of the design of Scheme 1, the 18s arms have an additional handle at their 3’ end and the 28s arms have an additional handle at their 5’ end. The handles are complementary to each other, thereby stabilizing the ternary complex. FIG. 8D is a schematic showing the probe design of Scheme 4. There are complementary regions between the 18s arms and the 80S probe, and between the 18s arms and the 28s arms. The 80S probe is pre-adenylated on its 5’ end. When both the 18s arms and 28s arms are present, the 18s arms will serve as a splint, and the 80S probe can be ligated to the 28s arms in the absence of ATP to prevent concurrent ligation of SNAIL probes. Hence, the complex is stabilized covalently. FIG. 8E is a schematic showing the probe design of Scheme 5. On top of the design of Scheme 4, an eraser strand is added. The eraser strand will eliminate partial hybridization but will not affect the signals from full hybridization. 5’App: 5’ adenylation.

[0028] FIG. 9 shows a schematic and experimental data illustrating amplicon compaction.FIG. 9A is a schematic showing amplicon compaction using azide-PEG4-dUTP and DBCO-PEG4-dUTP (abbreviated as azide-dUTP and DBCO-dUTP, respectively). On-amplicon SPAAC reaction limits the degree of freedom of the amplicon and leads to amplicon compaction. FIG. 9B is a panel of images showing DNA-PAINT reconstruction results of amplicons in different conditions. Normal condition denotes traditional RCA condition where no clickable-dUTPs are added. Scale bar: 1 pm. FIG. 9C is a panel of images showing representative fields of views of in situ sequencing of a 21 -gene STARmap on HeLa cells.FIG. 9D is a graph showing image signal intensity across conditions. Detector gains were differently tuned to avoid over-exposure of amplicons and maintain a similar image signal intensity across conditions. FIG. 9E is a graph showing a quantification of the number of amplicons per cell in each condition. Student’s t-test, ***: p < 0.001; ns: non-significant.

[0029] FIG. 10 is a set of schematics and experimental data illustrating the optimization of 80S splint probe hybridization. FIG. 10A is a schematic showing 80S splint probe full- 154934-6856-6158.1Atty. Docket No. 114203-1551hybridization scheme. FIG. 10B is a schematic showing 80S splint probe partial hybridization scheme, in which the 80S splint probe hybridizes only to an 18s arm probe. FIG. IOC is a schematic showing 80S splint probe partial hybridization scheme, in which the 80S splint probe hybridizes only to an 28s arm probe. FIG. 10D is a schematic showing 80S splint probe alternative full-hybridization scheme. FIG. 10E is a graph showing a quantification of ACTB RIBO reads normalized to STAR reads as a function of various lengths of the complementary regions between the 80S splint probe and the 18s RNA probe / 28s RNA probe in HeLa cells. Full hybridization (full arms) and partial hybridization (18s only and 28s only conditions) schemes were tested. x_y splint indicates an x nt complementary region between the 18s arm probe and 80S splint probe, and a y nt complementary region between the 28s arm probe and 80S splint probe. Probe configurations follow the scheme in FIG. 10A, except for the 11 11 splint, which uses the configuration in FIG. 10D

[0030] FIG. 11 is a schematic illustrating dU-enabled qRIBOmap and (thick-tissue) Deep-qRIBOmap. For thin tissue or cell culture, samples can be fixed, permeabilized, and hybridized with primers, RIBO padlocks, STAR padlocks, 18S arms, 28S arms, and the 80S splint probe. RIBO padlock and STAR padlock share the same hybridization target sequence and compete. The deoxythymidine nucleotides in 18S arms, 28S arms, and the 80S splint probe can be fully or partially replaced by deoxyuridine nucleotides. RIBO padlocks can be ligated where 80S ribosomes are present. Otherwise, STAR padlocks are expected to be ligated. Then, USER II enzyme digestion removes the 18S arms, 28S arms, and the 80S splint probe. Rolling circle amplification (RCA) creates amplicons for in situ sequencing that identifies RIBO and STAR reads. For thick tissue, samples can then be fixed, permeabilized, and hybridized with probes similarly with additional modifications that enable probe embedding into a polyacrylamide hydrogel. The 80S splint probe contains a 5’ Acrydite modification, and the primers have a universal 5’ flap sequences that can anneal with a 5’ Acrydite-modified adapter oligo. The adapter oligo contains aCNVK base that photocrosslinks with the primer at 368 nm UV light. After hydrogel embedding and tissue clearing via proteinase digestion, the sample can then undergo ligation, USER II digestion, and rolling circle amplification with Acrydite-modified dUTP. Then the amplicons can be re-embedded into the hydrogel before in situ sequencing.

[0031] FIG. 12 is a set of schematics and experimental data illustrating that 80S qRIBOmap with deoxyuridine arm and splint probes exhibit higher sensitivity and specificity. FIG. 12A is a schematic showing 80S qRIBOmap using conventional deoxythymidine-containing arm 164934-6856-6158.1Atty. Docket No. 114203-1551and splint probes (dT scheme) vs. deoxyuridine-containing arm and splint probes (dU scheme). Steps from after ligation to RCA were shown. In the dT scheme, the activity of Phi29 DNA polymerase may be scavenged by excessive oligonucleotides nearby, which makes the RCA inefficient. In the dU scheme, after the ligation step, arm and splint probes are digested by USER II enzyme, which clears up the oligos that may inhibit Phi29 activity.FIG. 12B is a graph showing quantification of RIBO reads and STAR reads per cell in HeLa cell 80S qRIBOmap experiment targeting ACTB, showing different combinations of dT or dU-containing arms and splint. FIG. 12C is a graph showing quantification of RIBO reads normalized by STAR reads in HeLa cell 80S qRIBOmap experiment targeting ACTB and MALAT1. “ACTB full” indicates full hybridization involving both 18S and 28S arms.“Partial” indicates (for arm probes) only adding 18S or 28S arms during hybridization. FIG.12D is a panel of representative images of HeLa cell 80S qRIBOmap experiment, showing the difference in sensitivity between the dT scheme and the dU scheme. Scale bar: 20 pm. FIG. 12E is a graph showing quantification of RIBO reads and STAR reads per cell in mouse brain tissue 80S qRIBOmap experiment targeting Actb, showing higher sensitivity of 80S qRIBOmap (dU scheme) than traditional 40S and 60S qRIBOmap. FIG. 12F is a graph showing quantification of RIBO reads normalized by STAR reads in mouse brain tissue 80S qRIBOmap experiment targeting Actb and Mt-Col . Welch’s Ltest. ns, non-significant; *P < 0.05; 0.0001.

[0032] FIG. 13 is a set of representative images and graphs illustrating the specificity of SOS-targeting Deep-RIBOmap. FIG. 13A is a panel of images showing a comparison between 80S Deep-qRIBOmap, 40S Deep-qRIBOmap, and Deep-STARmap. FIG. 13B is a graph showing a quantification of a comparison between 80S Deep-RIBOmap and 40S Deep-RIBOmap in RIBO reads per cell for genes Actb and Mt-Col. FIG. 13C is a graph showing a quantification of Actb and Mt-Col RIBO reads normalized by STAR using 80S Deep-qRIBOmap. FIG. 13D is a panel of images showing Actb and Mt-Col STAR and RIBO reads using 80S Deep-qRIBOmap in a 150 pm thick tissue block. Mann-Whitney U-test. ns, nonsignificant; *P < 0.05.

[0033] FIG. 14 is a set of schematics showing compatibility of the disclosed methods with other transcriptome and translatome analysis modalities. FIG. 14A shows extension to spatial multi-omic profiling of transcriptome with temporal information. Nascent RNA is labeled with 5-ethynyluridine and clicked on TEMPOmap splint probe while non-nascent RNA is not. TEMPO padlock probes compete with STAR padlock probes for the same hybridization sequence, resulting in simultaneous readout of both nascent and total transcriptome. FIG.174934-6856-6158.1Atty. Docket No. 114203-155114B shows extension to spatial multi-omic profiling of epitranscriptome with RNA-binding protein (RBP) or RNA modification information. RBP-bound or modified RNAs are targeted by splint-probe-conjugated antibodies. RBPmap padlock probes compete with STAR padlock probes for the same hybridization sequence, resulting in simultaneous readout of both RBP-bound and total transcriptome. FIG. 14C shows extension to spatial multi-omic profiling of transcriptome, translatome, temporal transcriptome, and epitranscriptome. Different modalities are targeted via splint probes with different ligation handle sequences and padlock probes that compete for the same binding sequence, resulting in simultaneous readout of multiple modalities.DETAILED DESCRIPTIONIntroduction

[0034] Translational control plays a fundamental role in the regulation of gene expression in cellular organisms. Modulating translational efficiency (i.e. the protein synthesis output per mRNA per unit time) allows the cell to fine-tune the expression of genes, spatially control protein localization, and trigger fast responses to environmental stresses. Different mRNAs exhibit varying levels of translational efficiencies and localized translation in different cell types, which contributes to specialized cellular functions. However, it remains a challenge to spatially profile transcriptome-wide translational efficiency at single-cell level. Current methods do not have high multiplexity or the capability of co-mapping both the transcriptome and the translatome in the same single cell, limiting the collective understanding of how translational efficiency is modulated in different cell states and cell types. Here, Applicant has developed 80S quantitative RIBOmap (80S qRIBOmap), a multi-modal spatial technology that enables simultaneous measurement of the transcriptome and translatome on the same sample, facilitating more accurate quantification of single-cell translational efficiency across thousands of genes. Additionally, 80S qRIBOmap utilizes probe sets that selectively target the actively translating 80S ribosome complex instead of individual subunits, thus achieving significantly improved specificity for translating RNAs compared to Applicant’s previous spatial translatomics approach. 80S qRIBOmap can be utilized as a platform technology to incorporate even more modalities, such as the profiling of temporal information of RNA, onto the same sample. Overall, Applicant’s method will help to decouple transcriptional regulation and translational regulation at the transcriptome scale and uncover single-cell translational efficiency with subcellular resolution.184934-6856-6158.1Atty. Docket No. 114203-1551Definitions

[0035] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton el aL, Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them unless specified otherwise.

[0036] The terms “administer,” “administering,” and “administration” refer to implanting, absorbing, ingesting, injecting, inhaling, or otherwise introducing a treatment or therapeutic agent, or a composition of treatments or therapeutic agents, in or on a subject.

[0037] The term “amplicon” as used herein refers to a nucleic acid (e.g., RNA) that is the product of an amplification reaction ( / .< ., the production of one or more copies of a genetic fragment or target sequence) or replication reaction. Amplicons can be formed artificially using, for example, PCR or other polymerization reactions. The term “concatenated amplicons” refers to multiple amplicons that are joined together to form a single nucleic acid molecule. Concatenated amplicons can be formed, for example, by rolling circle amplification (RCA), in which a circular oligonucleotide is amplified to produce multiple linear copies of the oligonucleotide as a single nucleic acid molecule comprising multiple amplicons that are concatenated.

[0038] An “antibody” refers to a glycoprotein belonging to the immunoglobulin superfamily. The terms antibody and immunoglobulin are used interchangeably. With some exceptions, mammalian antibodies are typically made of basic structural units each with two large heavy chains and two small light chains. There are several different types of antibody heavy chains, and several different kinds of antibodies, which are grouped together into different isotypes based on which heavy chain they possess. Five different antibody isotypes are known in mammals (IgG, IgA, IgE, IgD, and IgM, which perform different roles, and help direct the appropriate immune response for each different type of foreign object they encounter. The term “antibody” as used herein also encompasses antibody fragments and nanobodies, as well as variants of antibodies and variants of antibody fragments and nanobodies. In some embodiments, an antibody is administered as a treatment for a disease or disorder (e.g., one that is associated with a change in the profile of RNAs being translated in a cell taken from a194934-6856-6158.1Atty. Docket No. 114203-1551subject). In some embodiments, an antibody is conjugated to an oligonucleotide probe as described herein. In certain embodiments, the antibody binds to a ribosome (e.g., the antibody is an anti-40S ribosomal protein S3 (RPS3) antibody or an anti-60S ribosomal protein L4 (RPL4) antibody).

[0039] The term “cancer” (including cancers that may be studied, characterized, diagnosed, and / or treated using the methods described herein) refers to a class of diseases characterized by the development of abnormal cells that proliferate uncontrollably and have the ability to infiltrate and destroy normal body tissues. See e.g., Stedman ’s Medical Dictionary, 25th ed.; Hensyl ed.; Williams & Wilkins: Philadelphia, 1990. Cancer is one example of a proliferative disease. Exemplary cancers include, but are not limited to, acoustic neuroma; adenocarcinoma; adrenal gland cancer; anal cancer; angiosarcoma (e.g., lymphangiosarcoma, lymphangioendotheliosarcoma, hemangiosarcoma); appendix cancer; benign monoclonal gammopathy; biliary cancer (e.g., cholangiocarcinoma); bladder cancer; breast cancer (e.g., adenocarcinoma of the breast, papillary carcinoma of the breast, mammary cancer, medullary carcinoma of the breast); brain cancer (e.g., meningioma, glioblastomas, glioma (e.g., astrocytoma, oligodendroglioma), medulloblastoma); bronchus cancer; carcinoid tumor; cervical cancer (e.g., cervical adenocarcinoma); choriocarcinoma; chordoma; craniopharyngioma; colorectal cancer (e.g., colon cancer, rectal cancer, colorectal adenocarcinoma); connective tissue cancer; epithelial carcinoma; ependymoma; endotheliosarcoma (e.g., Kaposi’s sarcoma, multiple idiopathic hemorrhagic sarcoma); endometrial cancer (e.g., uterine cancer, uterine sarcoma); esophageal cancer (e.g., adenocarcinoma of the esophagus, Barrett’s adenocarcinoma); Ewing’s sarcoma; ocular cancer (e.g., intraocular melanoma, retinoblastoma); familiar hypereosinophilia; gall bladder cancer; gastric cancer (e.g., stomach adenocarcinoma); gastrointestinal stromal tumor (GIST); germ cell cancer; head and neck cancer (e.g. , head and neck squamous cell carcinoma, oral cancer (e.g., oral squamous cell carcinoma), throat cancer (e.g., laryngeal cancer, pharyngeal cancer, nasopharyngeal cancer, oropharyngeal cancer)); hematopoietic cancers (e.g., leukemia such as acute lymphocytic leukemia (ALL) (e.g., B-cell ALL, T-cell ALL), acute myelocytic leukemia (AML) (e.g., B-cell AML, T-cell AML), chronic myelocytic leukemia (CML) (e.g., B-cell CML, T-cell CML), and chronic lymphocytic leukemia (CLL) (e.g., B-cell CLL, T-cell CLL)); lymphoma such as Hodgkin lymphoma (HL) (e.g., B-cell HL, T-cell HL) and non-Hodgkin lymphoma (NHL) (e.g., B-cell NHL such as diffuse large cell lymphoma (DLCL) (e.g., diffuse large B-cell lymphoma), follicular lymphoma, chronic lymphocytic leukemia / small lymphocytic lymphoma (CLL / SLL), mantle cell lymphoma 204934-6856-6158.1Atty. Docket No. 114203-1551(MCL), marginal zone B-cell lymphomas (e.g., mucosa-associated lymphoid tissue (MALT) lymphomas, nodal marginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma), primary mediastinal B-cell lymphoma, Burkitt lymphoma, lymphoplasmacytic lymphoma ( / .< ., Waldenstrom’s macroglobulinemia), hairy cell leukemia (HCL), immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma and primary central nervous system (CNS) lymphoma; and T-cell NHL such as precursor T-lymphoblastic lymphoma / leukemia, peripheral T-cell lymphoma (PTCL) (e.g., cutaneous T-cell lymphoma (CTCL) (e.g., mycosis fungoides, Sezary syndrome), angioimmunoblastic T-cell lymphoma, extranodal natural killer T-cell lymphoma, enteropathy type T-cell lymphoma, subcutaneous panniculitis-like T-cell lymphoma, and anaplastic large cell lymphoma); a mixture of one or more leukemia / lymphoma as described above; and multiple myeloma (MM)), heavy chain disease (e.g., alpha chain disease, gamma chain disease, mu chain disease); hemangioblastoma; hypopharynx cancer; inflammatory myofibroblastic tumors; immunocytic amyloidosis; kidney cancer (e.g., nephroblastoma a. k.a. Wilms’ tumor, renal cell carcinoma); liver cancer (e.g., hepatocellular cancer (HCC), malignant hepatoma); lung cancer (e.g., bronchogenic carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), adenocarcinoma of the lung); leiomyosarcoma (LMS); mastocytosis (e.g., systemic mastocytosis); muscle cancer; myelodysplastic syndrome (MDS); mesothelioma; myeloproliferative disorder (MPD) (e.g., polycythemia vera (PV), essential thrombocytosis (ET), agnogenic myeloid metaplasia (AMM) a.k.a. myelofibrosis (MF), chronic idiopathic myelofibrosis, chronic myelocytic leukemia (CML), chronic neutrophilic leukemia (CNL), hypereosinophilic syndrome (HES)); neuroblastoma; neurofibroma (e.g., neurofibromatosis (NF) type 1 or type 2, schwannomatosis); neuroendocrine cancer (e.g., gastroenteropancreatic neuroendoctrine tumor (GEP-NET), carcinoid tumor); osteosarcoma e.g., bone cancer); ovarian cancer e.g., cystadenocarcinoma, ovarian embryonal carcinoma, ovarian adenocarcinoma); papillary adenocarcinoma; pancreatic cancer e.g., pancreatic adenocarcinoma, intraductal papillary mucinous neoplasm (IPMN), Islet cell tumors); penile cancer e.g., Paget’s disease of the penis and scrotum); pineal oma; primitive neuroectodermal tumor (PNT); plasma cell neoplasia; paraneoplastic syndromes; intraepithelial neoplasms; prostate cancer e.g., prostate adenocarcinoma); rectal cancer; rhabdomyosarcoma; salivary gland cancer; skin cancer e.g., squamous cell carcinoma (SCC), keratoacanthoma (KA), melanoma, basal cell carcinoma (BCC)); small bowel cancer e.g., appendix cancer); soft tissue sarcoma e.g., malignant fibrous histiocytoma (MFH), liposarcoma, malignant peripheral nerve sheath tumor (MPNST), chondrosarcoma, fibrosarcoma, myosarcoma);214934-6856-6158.1Atty. Docket No. 114203-1551sebaceous gland carcinoma; small intestine cancer; sweat gland carcinoma; synovioma; testicular cancer (e.g., seminoma, testicular embryonal carcinoma); thyroid cancer (e.g., papillary carcinoma of the thyroid, papillary thyroid carcinoma (PTC), medullary thyroid cancer); urethral cancer; vaginal cancer; and vulvar cancer (e.g., Paget’s disease of the vulva).

[0040] A “cell,” as used herein, may be present in a population of cells e.g., in a tissue, a sample, a biopsy, an organ, or an organoid). In some embodiments, a population of cells is composed of a plurality of different cell types. Cells for use in the methods of the present disclosure can be present within an organism, a single cell type derived from an organism, or a mixture of cell types. Included are naturally occurring cells and cell populations, genetically engineered cell lines, cells derived from transgenic animals, cells from a subject, etc. Virtually any cell type and size can be accommodated in the methods and systems described herein. In some embodiments, the cells are mammalian cells (e.g., complex cell populations such as naturally occurring tissues). In some embodiments, the cells are from a human. In certain embodiments, the cells are collected from a subject (e.g., a human) through a medical procedure such as a biopsy. Alternatively, the cells may be a cultured population (e.g., a culture derived from a complex population, or a culture derived from a single cell type where the cells have differentiated into multiple lineages). The cells may also be provided in situ in a tissue sample.

[0041] Cell types contemplated for use in the methods of the present disclosure include, but are not limited to, stem and progenitor cells (e.g., embryonic stem cells, hematopoietic stem cells, mesenchymal stem cells, neural crest cells, efc.), endothelial cells, muscle cells, myocardial cells, smooth and skeletal muscle cells, mesenchymal cells, epithelial cells, hematopoietic cells, lymphocytes such as T-cells (e.g., Thl T cells, Th2 T cells, ThO T cells, cytotoxic T cells) and B cells (e.g., pre-B cells), monocytes, dendritic cells, neutrophils, macrophages, natural killer cells, mast cells, adipocytes, immune cells, neurons, hepatocytes, and cells involved with particular organs (e.g., thymus, endocrine glands, pancreas, brain, neurons, glia, astrocytes, dendrocytes, and genetically modified cells thereof). The cells may also be transformed or neoplastic cells of different types (e.g., carcinomas of different cell origins, lymphomas of different cell types, efc.) or cancerous cells of any kind (e.g., from any of the cancers disclosed herein). Cells of different origins (e.g., ectodermal, mesodermal, and endodermal) are also contemplated for use in the methods of the present disclosure. In some embodiments, the cells are microglia, astrocytes, oligodendrocytes, excitatory neurons, or224934-6856-6158.1Atty. Docket No. 114203-1551inhibitory neurons. In some embodiments, cells of multiple cell types are present within the same sample.

[0042] The term “ribosome” is used herein to refer to a cellular macromolecular complex responsible for translating messenger RNA (mRNA) into polypeptides or proteins, and includes both naturally occurring and engineered ribosomes from any organism or artificial system. Unless otherwise specified, “ribosome” encompasses all structural and functional forms thereof, including cytosolic eukaryotic ribosomes (e.g., 80S ribosomes comprising 40S and 60S subunits), prokaryotic ribosomes (e.g., 70S ribosomes comprising 30S and 50S subunits), archaeal ribosomes, organellar ribosomes such as mitochondrial ribosomes (i.e., mitoribosomes, e.g., 55S mitoribosomes comprising 28S and 39S subunits), chloroplast ribosomes, and ribosomes from bacteria, fungi, plants, animals, and other non-eukaryotic and eukaryotic organisms.

[0043] The term “complementary” is used herein to refer to two oligonucleotide sequences (e.g., DNA or RNA) comprising bases that hydrogen bond to one another. The degree of complementarity between two oligonucleotide sequences can vary, from complete complementarity to no complementarity. For example, two oligonucleotide sequences may be only partially complementary to one another (e.g., in the probes described herein, wherein only a portion of the probe is complementary to another probe, or to an RNA of interest). Two oligonucleotide sequences may be, e.g., 70% or more complementary to one another, 75% or more complementary to one another, 80% or more complementary to one another, 85% or more complementary to one another, 90% or more complementary to one another, 95% or more complementary to one another, 96% or more complementary to one another, 97% or more complementary to one another, 98% or more complementary to one another, 99% or more complementary to one another, or 100% complementary to one another.

[0044] The terms “polynucleotide”, “nucleotide sequence”, “nucleic acid”, “nucleic acid molecule”, “nucleic acid sequence”, and “oligonucleotide” refer to a series of nucleotide bases (also called “nucleotides”) in DNA and RNA and mean any chain of two or more nucleotides. The polynucleotides can be chimeric mixtures or derivatives or modified versions thereof, and single-stranded or double-stranded. The oligonucleotide can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule, its hybridization parameters, etc.

[0045] A “protein,” “peptide,” or “polypeptide” comprises a polymer of amino acid residues linked together by peptide bonds. The term refers to proteins, polypeptides, and peptides of any size, structure, or function. Typically, a protein will be at least three amino acids long.234934-6856-6158.1Atty. Docket No. 114203-1551A protein may refer to an individual protein or a collection of proteins. Inventive proteins preferably contain only natural amino acids, although non-natural amino acids ( / .< ., compounds that do not occur in nature but that can be incorporated into a polypeptide chain) and / or amino acid analogs as are known in the art may alternatively be employed. Also, one or more of the amino acids in a protein may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a hydroxyl group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a linker for conjugation or functionalization, or other modification. A protein may also be a single molecule or may be a multi-molecular complex. A protein may be a fragment of a naturally occurring protein or peptide. A protein may be naturally occurring, recombinant, synthetic, or any combination of these. A protein may also be a therapeutic protein administered as a treatment for a disease or disorder (e.g., one that is associated with a change in the profile of RNAs being translated in a cell taken from a subject). In certain embodiments, the protein is an antibody.

[0046] A “transcript” or “RNA transcript” is the product resulting from RNA polymerase-catalyzed transcription of a DNA sequence. When the RNA transcript is a complimentary copy of the DNA sequence, it is referred to as the primary transcript, or it may be an RNA sequence derived from post-transcriptional processing of the primary transcript and is referred to as the mature RNA. “Messenger RNA (mRNA)” refers to the RNA that is without introns and can be translated into polypeptides by the cell. “cRNA” refers to complementary RNA, transcribed from a recombinant cDNA template. “cDNA” refers to DNA that is complementary to and derived from an mRNA template.

[0047] The term “sample” or “biological sample” refers to any sample including tissue samples (such as tissue sections, surgical biopsies, and needle biopsies of a tissue); cell samples (e.g., cytological smears (such as Pap or blood smears) or samples of cells obtained by microdissection); or cell fractions, fragments, or organelles (such as obtained by lysing cells and separating the components thereof by centrifugation or otherwise). Other examples of biological samples include, but are not limited to, blood, serum, urine, semen, fecal matter, cerebrospinal fluid, interstitial fluid, mucous, tears, sweat, pus, biopsied tissue (e.g., obtained by a surgical biopsy or needle biopsy), nipple aspirates, milk, vaginal fluid, saliva, swabs (such as buccal swabs), or any material containing biomolecules that is derived from a first biological sample. In some embodiments, a biological sample is a surgical biopsy taken from a subject, for example, a biopsy of any of the tissues described herein. In certain embodiments, a biological sample is a tumor biopsy (e.g., from a subject diagnosed with, suspected of having, or thought to have cancer). In some embodiments, the tumor biopsy 244934-6856-6158.1Atty. Docket No. 114203-1551captures primary tumor cells and tissue for direct study. In some embodiments, liquid biopsy can be used on tumor cells that have been captured and / or sorted via traditional methods (e.g., FACS, affinity capture, etc.) from blood or other bodily fluids (CNS fluid, lymph, etc.). In some embodiments, the sample is brain tissue. In some embodiments, the tissue is cardiac tissue. In some embodiments, the tissue is muscle tissue.

[0048] The term “small molecule” refers to molecules, whether naturally-occurring or artificially created (e.g., via chemical synthesis) that have a relatively low molecular weight. Typically, a small molecule is an organic compound (e.g., it contains carbon). The small molecule may contain multiple carbon-carbon bonds, stereocenters, and other functional groups (e.g., amines, hydroxyl, carbonyls, and heterocyclic rings, etc.). In certain embodiments, the molecular weight of a small molecule is not more than about 1,000 g / mol, not more than about 900 g / mol, not more than about 800 g / mol, not more than about 700 g / mol, not more than about 600 g / mol, not more than about 500 g / mol, not more than about 400 g / mol, not more than about 300 g / mol, not more than about 200 g / mol, or not more than about 100 g / mol. In certain embodiments, the molecular weight of a small molecule is at least about 100 g / mol, at least about 200 g / mol, at least about 300 g / mol, at least about 400 g / mol, at least about 500 g / mol, at least about 600 g / mol, at least about 700 g / mol, at least about 800 g / mol, at least about 900 g / mol, or at least about 1,000 g / mol. Combinations of the above ranges (e.g, at least about 200 g / mol and not more than about 500 g / mol) are also possible. In certain embodiments, the small molecule is a therapeutically active agent such as a drug (e.g, a molecule approved by the U.S. Food and Drug Administration. The small molecule may also be complexed with one or more metal atoms and / or metal ions. Preferred small molecules are biologically active in that they produce a biological effect in animals, preferably mammals, and more preferably humans. In certain embodiments, the small molecule is a drug. Preferably, though not necessarily, the drug is one that has already been deemed safe and effective for use in humans or animals by the appropriate governmental agency or regulatory body.

[0049] A “subject” to which administration is contemplated refers to a human ( / .< ., male or female of any age group, e.g, pediatric subject (e.g., infant, child, or adolescent) or adult subject (e.g., young adult, middle-aged adult, or senior adult)) or non-human animal. In some embodiments, the non-human animal is a mammal (e.g., primate (e.g., cynomolgus monkey or rhesus monkey) or mouse). The term “patient” refers to a subject in need of treatment of a disease. In some embodiments, the subject is human. In some embodiments, the patient is human. The human may be a male or female at any stage of development. A subject or 254934-6856-6158.1Atty. Docket No. 114203-1551patient “in need” of treatment of a disease or disorder includes, without limitation, those who exhibit any risk factors or symptoms of a disease or disorder. In some embodiments, a subject is a non-human experimental animal (e.g., a mouse, rat, dog, or non-human primate).

[0050] A “therapeutically effective amount” of a treatment or therapeutic agent is an amount sufficient to provide a therapeutic benefit in the treatment of a condition or to delay or minimize one or more symptoms associated with the condition. A therapeutically effective amount of a treatment or therapeutic agent means an amount of the therapy, alone or in combination with other therapies, that provides a therapeutic benefit in the treatment of the condition. The term “therapeutically effective amount” can encompass an amount that improves overall therapy, reduces or avoids symptoms, signs, or causes of the condition, and / or enhances the therapeutic efficacy of another therapeutic agent.

[0051] As used herein, a “tissue” is a group of cells and their extracellular matrix from the same origin. Together, the cells carry out a specific function. The association of multiple tissue types together forms an organ. The cells may be of different cell types. In some embodiments, a tissue is an epithelial tissue. Epithelial tissues are formed by cells that cover an organ surface (e.g., the surface of the skin, airways, soft organs, reproductive tract, and inner lining of the digestive tract). Epithelial tissues perform protective functions and are also involved in secretion, excretion, and absorption. Examples of epithelial tissues include, but are not limited to, simple squamous epithelium, stratified squamous epithelium, simple cuboidal epithelium, transitional epithelium, pseudostratified epithelium, columnar epithelium, and glandular epithelium. In some embodiments, a tissue is a connective tissue. Connective tissues are fibrous tissues made up of cells separated by non-living material (e.g., an extracellular matrix). Connective tissues provide shape to organs and hold organs in place. Connective tissues include fibrous connective tissue, skeletal connective tissue, and fluid connective tissue. Examples of connective tissues include, but are not limited to, blood, bone, tendon, ligament, adipose, and areolar tissues. In some embodiments, a tissue is a muscular tissue. Muscular tissue is an active contractile tissue formed from muscle cells. Muscle tissue functions to produce force and cause motion. Muscle tissue includes smooth muscle (e.g., as found in the inner linings of organs), skeletal muscle (e.g., as typically attached to bones), and cardiac muscle (e.g., as found in the heart, where it contracts to pump blood throughout an organism). In some embodiments, a tissue is a nervous tissue. Nervous tissue includes cells comprising the central nervous system and peripheral nervous system. Nervous tissue forms the brain, spinal cord, cranial nerves, and spinal nerves (e.g., motor264934-6856-6158.1Atty. Docket No. 114203-1551neurons). In certain embodiments, a tissue is brain tissue. In certain embodiments, a tissue is placental tissue. In some embodiments, a tissue is heart tissue.

[0052] The term “translatome” refers to all of the open reading frames that are being actively translated ( / .< ., DNA sequences that fall between start and stop codons) in a particular cell or organism. In some embodiments, the methods, probes, and kits provided herein are useful for studying and / or profiling the translatome.

[0053] The terms “treatment,” “treat,” and “treating” refer to reversing, alleviating, delaying the onset of, or inhibiting the progress of a disease described herein (e.g., cancer). In some embodiments, treatment may be administered after one or more signs or symptoms of the disease have developed or have been observed (e.g., prophylactically (as may be further described herein) or upon suspicion or risk of disease). In other embodiments, treatment may be administered in the absence of signs or symptoms of the disease. For example, treatment may be administered to a susceptible subject prior to the onset of symptoms (e.g., in light of a history of symptoms in the subject, or family members of the subject). Treatment may also be continued after symptoms have resolved, for example, to delay or prevent recurrence. In some embodiments, treatment may be administered after using the methods disclosed herein and observing a change in the profile of RNAs being expressed in a cell or tissue in comparison to a healthy cell or tissue.

[0054] The aspects described herein are not limited to specific embodiments, systems, compositions, methods, or configurations, and as such can, of course, vary. The terminology used herein is for the purpose of describing particular aspects only and, unless specifically defined herein, is not intended to be limiting.

[0055] The present disclosure provides methods, compositions, and systems for profiling RNAs being translated in a cell. The present disclosure also provides methods for diagnosing a disease or disorder in a subject based on a profile of the RNAs being translated in a cell, including cells within an intact tissue. Methods of screening for or testing a candidate agent capable of modulating translation of one or more RNAs are also provided by the present disclosure. The present disclosure also provides methods for treating a disease or disorder in a subject in need thereof. Pairs and sets of oligonucleotide probes, which may be useful for performing the methods described herein, are also described by the present disclosure, as well as kits comprising any of the oligonucleotide probes described herein.274934-6856-6158.1Atty. Docket No. 114203-1551Methods for simultaneous transcriptome and translatome profding in a cell

[0056] In one aspect, the present disclosure provides methods for simultaneous transcriptome and translatome profiling in a cell. In the methods disclosed herein, a cell may be contacted with one or more sets of probes, which are described further herein and may be used to amplify RNAs that are actively being translated by a ribosome to produce one or more concatenated amplicons. The one or more concatenated amplicons may then be embedded in a polymeric matrix and sequenced to determine the identity of the transcripts and their location within the polymeric matrix (e.g., through SEDAL sequencing as described further herein). Advantageously, the disclosed methods enable a direct measurement of translation efficiency (TE) by allowing for the measurement of a ratio between actively-translated ribosome-associated RNAs, and RNAs that are not undergoing active translation and are nonribosome-associated. Using the locations and ribosome-association status of the transcripts of interest, the transcriptome and translatome can be profiled simultaneously in a single cell. The methods provided herein have several advantages over previously disclosed methods and systems, including, but not limited to, the ability to profile the transcriptome and translatome in a sample without disrupting the spatial information of subcellular structures, cell morphology, and / or tissue organization in the sample. The method provided herein are also compatible with the co-profiling of additional modalities, including but not limited to temporal dynamics of RNA expression.

[0057] In some embodiments, the present disclosure provides a method for simultaneous transcriptome and translatome profiling in a cell, the method comprising: (a) contacting the cell with one or more sets of probes, wherein each set of probes comprises a first probe, a second probe, a third probe, a fourth probe, a fifth probe, and a sixth probe, wherein (i) the first probe comprises (1) a first oligonucleotide barcode sequence; (2) an oligonucleotide portion that is complementary to a portion of the fifth probe; (3) an oligonucleotide portion that is complementary to a portion of an RNA of interest; (4) a second oligonucleotide barcode sequence; and (5) an oligonucleotide portion that is complementary to a portion of the sixth probe; (ii) the second probe comprises (1) a first oligonucleotide barcode sequence; (2) an oligonucleotide portion that is complementary to a portion of the fifth probe; (3) an oligonucleotide portion that is complementary to a portion of an RNA of interest; and (4) a second oligonucleotide barcode sequence; (iii) the third probe comprises an oligonucleotide portion that recognizes the 40S subunit of a ribosome and an oligonucleotide portion that is complementary to a portion of the sixth probe; (iv) the fourth probe comprises an oligonucleotide portion that recognizes the 60S subunit of a ribosome and an oligonucleotide 284934-6856-6158.1Atty. Docket No. 114203-1551portion that is complementary to a portion of the sixth probe; (v) the fifth probe comprises (1) an oligonucleotide portion that is complementary to the RNA of interest; (2) an oligonucleotide portion that is complementary to a portion of the first probe; (3) an oligonucleotide portion that is complementary to a portion of the second probe; and (4) an oligonucleotide barcode sequence, wherein the oligonucleotide barcode sequence of the fifth probe is complementary to the first oligonucleotide barcode sequence of the first probe and the first oligonucleotide barcode sequence of the second probe; and (vi) the sixth probe comprises (1) an oligonucleotide portion that is complementary to a portion of the first probe; (2) an oligonucleotide portion that is complementary to a portion of the third probe; and (3) an oligonucleotide portion that is complementary to a portion of the fourth probe; (b) ligating the 5' end and the 3' end of the first probe together to produce a first circular oligonucleotide, and ligating the 5' end and the 3' end of the second probe together to produce a second circular oligonucleotide; (c) performing rolling circle amplification to amplify the first circular oligonucleotide and the second circular oligonucleotide, wherein the fifth probe is a primer for the amplification of each of the first circular oligonucleotide and the second circular oligonucleotide, thereby producing one or more concatenated amplicons from the first circular oligonucleotide and one or more concatenated amplicons from the second circular oligonucleotide; (d) embedding the one or more concatenated amplicons from the first circular oligonucleotide and one or more concatenated amplicons from the second circular oligonucleotide in a polymeric matrix; and (e) sequencing the concatenated amplicons embedded in the polymeric matrix to determine the identity, location, and translation status of each RNA of interest in the cell.

[0058] The methods disclosed herein contemplate the use of a set probes comprising a first probe, a second probe, a third probe, a fourth probe, a fifth probe, and a sixth probe. The third probe and fourth probe each comprises a portion that recognizes a ribosome. The ribosome can be but is not limited to eukaryotic ribosomes. Rather, it is to be understood that while the present disclosure shows a reduction to practice in an exemplified eukaryotic ribosome (e.g., 80S ribosomes comprising 40S and 60S subunits), the same general concept could be applied to other ribosomes, including but not limited to, prokaryotic ribosomes (e.g., 70S ribosomes comprising 30S and 50S subunits), archaeal ribosomes, organellar ribosomes such as mitochondrial ribosomes (i.e., mitoribosomes, e.g., 55S mitoribosomes comprising 28S and 39S subunits), chloroplast ribosomes, and ribosomes from bacteria, fungi, plants, animals, and other non-eukaryotic and eukaryotic organisms. The portion of the probe that recognizes a ribosome may be a protein, peptide, nucleic acid, or small molecule. In some 294934-6856-6158.1Atty. Docket No. 114203-1551embodiments, the portion of the third probe or fourth probe that recognizes a ribosome is an agent that binds an antibody, or an antibody variant or fragment. In certain embodiments, the portion of the third probe or fourth probe that recognizes the ribosome comprises an antibody (e.g., a secondary antibody), or an antibody variant or fragment. When the portion of the third probe or fourth probe that recognizes the ribosome is a secondary antibody, the method may optionally further comprise contacting the cell with a primary antibody that recognizes a ribosome and is recognized by the secondary antibody of the second probe. The primary antibody may recognize any portion of the ribosome, for example, any protein, nucleic acid (e.g., rRNA), or combination thereof of the ribosome. In some embodiments, the primary antibody is an anti-40S ribosomal protein S3 (RPS3) antibody (e.g., an anti-RPS3 monoclonal antibody), for the third probe. In some embodiments, the antibody is an anti-60S ribosomal protein L4 (RPL4) antibody (e.g., an anti-RPL4 polyclonal antibody), for the fourth probe. In place of an antibody, the present disclosure also contemplates the use of any agent capable of recognizing the ribosome on the probes described herein. In some embodiments, the portion of the third probe or fourth probe that recognizes the ribosome comprises an oligonucleotide that is complementary to a portion of the ribosomal RNA (rRNA) within the ribosome. In some embodiments, the portion of the third probe or fourth probe that recognizes the ribosome comprises an oligonucleotide that is complementary to a portion of the 40S small ribosomal subunit (including, e.g., the 18S rRNA) (for the third probe) or to a portion of the 60S large ribosomal subunit (including, e.g., the 5S rRNA, the 28S rRNA, and the 5.8S rRNA) (for the fourth probe). In some embodiments, the portion of the third probe that recognizes the 40S subunit of a ribosome comprises an oligonucleotide that is complementary to a portion of the 18S rRNA. In some embodiments, the portion of the fourth probe that recognizes the 60S subunit of a ribosome comprises an oligonucleotide that is complementary to a portion of the 28S rRNA. In certain embodiments, the oligonucleotide complementary to a portion of rRNA is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more than 30 nucleotides in length. In certain embodiments, the oligonucleotide complementary to a portion of rRNA is about 25 nucleotides in length.

[0059] In addition to the portion that recognizes the ribosome, the each of the third probe and the fourth probe also comprises a portion that is complementary to a portion of the sixth probe.

[0060] In some embodiments, the portion of the third probe that is complementary to a portion of the sixth probe is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides in length. In some embodiments, the portion of the third probe that is complementary to a portion of the 304934-6856-6158.1Atty. Docket No. 114203-1551sixth probe is 9-15 nucleotides in length. In some embodiments, the portion of the third probe that is complementary to a portion of the sixth probe is 9-11 nucleotides in length. The present disclosure contemplates any arrangement of the portions of the third probe. In some embodiments, the third probe used in the methods described herein comprises the structure:5 '-[portion recognizing the 40S subunit of the ribosome]-[portion complementary to the sixth probe]-3'; or5 '-[portion recognizing the 40S subunit of the ribosome]-[poly-A linker]-[portion complementary to the sixth probe]-3'wherein ]-[ comprises an optional linker (e.g., nucleotide linker, e.g., a poly-A linker). In some embodiments, ]-[ represents a direct linkage between two portions of the third oligonucleotide probe ( / .< ., a phosphodiester bond).

[0061] In some embodiments, the portion of the fourth probe that is complementary to a portion of the sixth probe is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides in length. In some embodiments, the portion of the fourth probe that is complementary to a portion of the sixth probe is 9-15 nucleotides in length. In some embodiments, the portion of the fourth probe that is complementary to a portion of the sixth probe is 9-11 nucleotides in length. The present disclosure contemplates any arrangement of the portions of the fourth probe. In some embodiments, the fourth probe used in the methods described herein comprises the structure:5 '-[portion complementary to the sixth probe]-[portion recognizing the 60S subunit of the ribosome]-3'; or5 '-[portion complementary to the sixth probe]-[poly-A linker]-[portion recognizing the 60S subunit of the ribosome]-3'wherein ]-[ comprises an optional linker (e.g., nucleotide linker, e.g., a poly-A linker). In some embodiments, ]-[ represents a direct linkage between two portions of the third oligonucleotide probe ( / .< ., a phosphodiester bond).

[0062] In some embodiments, the portion of the sixth probe that is complementary to a portion of the third probe is 9-11 nucleotides in length, and the portion of the sixth probe that is complementary to a portion of the fourth probe is 9-11 nucleotides in length. In some embodiments, the portion of the sixth probe that is complementary to a portion of the third probe is 9 nucleotides in length, and the portion of the sixth probe that is complementary to a portion of the fourth probe is 11 nucleotides in length.

[0063] The first probe used in the methods described herein (also referred to herein as the “RIBO padlock” probe) includes a first oligonucleotide barcode sequence and a second oligonucleotide barcode sequence made up of a specific sequence of nucleotides. In some 314934-6856-6158.1Atty. Docket No. 114203-1551embodiments, the first and second oligonucleotide barcode sequences on the first probe comprise the same nucleotide sequence. The presence of the second barcode sequence of the first oligonucleotide probe may increase the specificity of the detection of the RNA of interest in the methods described herein (z.e., as compared to if the method were performed using a first oligonucleotide probe that did not comprise a second barcode sequence). The use of an additional oligonucleotide barcode sequence of the first oligonucleotide probe may also play a role in reducing non-specific amplification of the RNA of interest in the methods described herein. This is accomplished because the oligonucleotide barcode sequence of the fifth probe used in the methods described herein is complementary to the first oligonucleotide barcode sequence on the first probe, adding an additional layer of specificity that is required before amplification can occur using the fifth probe as a primer. In some embodiments, the portion of the first probe that is complementary to the fifth probe is 5-15, 6-14, 7-13, 8-12, or 9-11 nucleotides in length. In some embodiments, the portion of the first probe that is complementary to the fifth probe is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides in length.

[0064] In some embodiments, the oligonucleotide barcode sequences of the first probe are about 3 to about 13, about 4 to about 12, about 5 to about 11, about 6 to about 10, or about 7 to about 9 nucleotides in length. In some embodiments, the oligonucleotide barcode sequences of the first probe are 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 nucleotides in length.

[0065] The second probe used in the methods described herein (also referred to herein as the “STAR padlock” probe) includes an oligonucleotide barcode sequence made up of a specific sequence of nucleotides. In some embodiments, the oligonucleotide barcode sequences of the second probe are about 3 to about 13, about 4 to about 12, about 5 to about 11, about 6 to about 10, or about 7 to about 9 nucleotides in length. In some embodiments, the oligonucleotide barcode sequences of the second probe are 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 nucleotides in length.

[0066] The barcodes of the oligonucleotide probes described herein may comprise genespecific sequences used to identify RNAs of interest that are being translated (z.e., each barcode sequence is associated with a specific gene or transcript). The barcodes of the oligonucleotide probes described herein may comprise gene-specific sequences used to identify RNAs of interest that are being translated (z.e., each barcode sequence is associated with a specific gene or transcript). In some embodiments, one or more barcodes of the oligonucleotide probes described herein may comprise a sequence used to identify RNAs of interest as ribosome-associated (e.g., detected as part of a fully-assembled ribosome complex)324934-6856-6158.1Atty. Docket No. 114203-1551or non-ribosome-associated (e.g., detected separately from a fully-assembled ribosome complex). The use of the barcodes on probes analogous to those described herein is further described in, for example, International Patent Application Publication No. WO 2019 / 199579, published October 17, 2019, and Wang etal., Science 2018, 361, 380, both of which are incorporated herein by reference in their entireties.

[0067] The first probe also comprises an oligonucleotide portion that is complementary to a portion of the sixth probe and a portion that is complementary to an RNA of interest. In some embodiments, the portion of the first probe that is complementary to a portion of the sixth probe is 4-20, 5-19, 6-18, 7-17, 8-16, 9-15, 10-14, or 11-13 nucleotides in length. In some embodiments, the portion of the first probe that is complementary to a portion of the sixth probe is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length. In certain embodiments, the oligonucleotide portion of the first probe that is complementary to a portion of the sixth probe is split between the 5' end and the 3 ' end of the first probe. In some embodiments, the portion of the first probe that is complementary to the RNA of interest is 10-30, 11-29, 12-28, 13-27, 14-26, 15-25, 16-24, 17-23, 18-22, or 19-21 nucleotides in length. In some embodiments, the portion of the first probe that is complementary to an RNA of interest is about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, or about 30 nucleotides long. In some embodiments, the first probe is about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, or about 40 nucleotides long or longer.

[0068] The arrangement of the portions of the first oligonucleotide probe in any order is contemplated by the present disclosure. In some embodiments, the first probe comprises the structure:5 '-[portion complementary to sixth probe]-[portion complementary to portion of fifth probe]-[portion complementary to RNA of interest]-[second oligonucleotide barcode sequence]-[portion complementary to sixth probe]-3'wherein ]-[ comprises an optional linker (e.g., a nucleotide linker). In some embodiments, ]-[ represents a direct linkage between two portions of the first probe (i.e., a phosphodiester bond). In some embodiments, the first oligonucleotide barcode sequence is within the portion complementary to portion of fifth probe.334934-6856-6158.1Atty. Docket No. 114203-1551

[0069] The second probe also comprises an oligonucleotide portion that is complementary to an RNA of interest. In some embodiments, the portion of the second probe that is complementary to the RNA of interest is 10-30, 11-29, 12-28, 13-27, 14-26, 15-25, 16-24, 17-23, 18-22, or 19-21 nucleotides in length. In some embodiments, the portion of the second probe that is complementary to an RNA of interest is about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, or about 30 nucleotides long. In some embodiments, the first probe is about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, or about 40 nucleotides long or longer. The arrangement of the portions of the second oligonucleotide probe in any order is contemplated by the present disclosure. In some embodiments, the second probe comprises the structure:5 '-[portion complementary to portion of fifth probe]-[portion complementary to RNA of interest] -[second oligonucleotide barcode sequence]- 3'wherein ]-[ comprises an optional linker (e.g., a nucleotide linker). In some embodiments, ]-[ represents a direct linkage between two portions of the first probe ( / .< ., a phosphodiester bond). In some embodiments, the first oligonucleotide barcode sequence is within the portion complementary to portion of fifth probe.

[0070] As described herein, the sixth probe (also referred to herein as a “splint probe” or as a “blocked probe”) comprises a portion that is complementary to a portion of the third probe and a portion that is complementary to a portion of the fourth probe, thereby recognizing a ribosome ( / .< ., a ribosome that is bound to and is actively translating the RNA of interest). In some embodiments, the sixth probe further comprises a polymerization blocker. The addition of a polymerization blocker prevents the sixth probe from being used as a primer in the amplification of step (c), ensuring that the fifth probe will be used as the primer during amplification. The polymerization blocker on the sixth probe can be any moiety capable of preventing the use of the sixth probe as a primer in the amplification of step (c) of the methods described herein. In some embodiments, the polymerization blocker is at the 3' end of the sixth probe. The polymerization blocker can be, for example, any chemical moiety that prevents a polymerase from using the sixth probe as a primer for polymerization. In some embodiments, the polymerization blocker is a nucleic acid residue comprising a blocked 3' hydroxyl group (e.g., comprising an oxygen protecting group on the 3' hydroxyl group). In some embodiments, the polymerization blocker comprises a hydrogen in place of the 3' 344934-6856-6158.1Atty. Docket No. 114203-1551hydroxyl group. In some embodiments, the polymerization blocker comprises any chemical moiety in place of the 3' hydroxyl group that prevents an additional nucleotide from being added. In some embodiments, the polymerization blocker comprises an inverted nucleic acid residue. In some embodiments, the polymerization blocker is an inverted adenosine, thymine, cytosine, guanosine, or uridine residue. In certain embodiments, the polymerization blocker is an inverted thymine residue.

[0071] In addition to the portion that recognizes the ribosome, the sixth probe also comprises a portion that is complementary to a portion of the first probe. In some embodiments, the portion of the sixth probe that is complementary to a portion of the first probe is 4-20, 5-19, 6-18, 7-17, 8-16, 9-15, 10-14, or 11-13 nucleotides in length. In some embodiments, the portion of the sixth probe that is complementary to a portion of the first probe is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length. In certain embodiments, the portion of the sixth probe that is complementary to a portion of the third probe and the portion of the sixth probe that is complementary to a portion of the fourth probe are joined to the portion of the sixth probe that is complementary to a portion of the first probe by a poly-A nucleotide linker. In some embodiments, the poly-A nucleotide linker is about 4-60, 5-50, or 10-40 nucleotides in length. In some embodiments, the poly-A nucleotide linker of the sixth probe is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 nucleotides in length.

[0072] The present disclosure contemplates any arrangement of the portions of the sixth probe. In some embodiments, the sixth probe used in the methods described herein comprises the structure:5 '-[portion complementary to portion of fourth probe]-[portion complementary to portion of third probe]-[portion complementary to portion of first probe]-3'; or 5 '-[portion complementary to portion of fourth probe]-[portion complementary to portion of third probe]-[poly-A linker]-[portion complementary to portion of first probe]-3'; or5 '-[portion complementary to portion of third probe]-[portion complementary to portion of fourth probe]-[portion complementary to portion of first probe]-3'; or 5 '-[portion complementary to portion of third probe]-[portion complementary to portion of fourth probe]-[poly-A linker]-[portion complementary to portion of first probe]-3'354934-6856-6158.1Atty. Docket No. 114203-1551wherein ]-[ comprises an optional linker (e.g., nucleotide linker). In some embodiments, ]-[ represents a direct linkage between two portions of the sixth oligonucleotide probe.

[0073] The fifth probe used in the methods disclosed herein (also referred to herein as the “primer” probe) includes a barcode sequence made up of a specific sequence of nucleotides. In some embodiments, the barcode sequence of the fifth probe is about 3 to about 20, about 5 to about 15, about 6 to about 14, about 7 to about 13, about 8 to about 12, or about 9 to about 11 nucleotides in length. In some embodiments, the barcode sequence of the fifth probe is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides in length. In certain embodiments, the barcode sequence of the fifth probe is 10 nucleotides in length.

[0074] The fifth probe also comprises a portion that is complementary to an RNA of interest and a portion that is complementary to a portion of the first probe or the second probe. In some embodiments, the first and the fifth probes are complementary to and bind different portions of the RNA of interest. In some embodiments, the second and the fifth probes are complementary to and bind different portions of the RNA of interest. In some embodiments, the portion of the fifth probe that is complementary to the RNA of interest is 10-30, 11-29, 12-28, 13-27, 14-26, 15-25, 16-24, 17-23, 18-22, or 19-21 nucleotides in length. In some embodiments, the portion of the fifth probe that is complementary to an RNA of interest is about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, or about 30 nucleotides long. In some embodiments, the third probe is about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, or about 40 nucleotides long or longer. In some embodiments, the portion of the fifth probe that is complementary to the first probe is 5-15, 6-14, 7-13, 8-12, or 9-11 nucleotides in length. In some embodiments, the portion of the fifth probe that is complementary to the first probe is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides in length. In some embodiments, the portion of the fifth probe that is complementary to the second probe is 5-15, 6-14, 7-13, 8-12, or 9-11 nucleotides in length. In some embodiments, the portion of the fifth probe that is complementary to the second probe is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides in length.

[0075] Any arrangement of the portions of the fifth probe is contemplated by the present disclosure. In some embodiments, the fifth probe comprises the structure:5'-[portion complementary to RNA of interest]-[portion complementary to first probe]-3'; or364934-6856-6158.1Atty. Docket No. 114203-15515'-[portion complementary to RNA of interest]-[portion complementary to second probe]-3'wherein ]-[ comprises an optional linker (e.g., nucleotide linker). In some embodiments, ]-[ represents a direct linkage between two portions of the fifth probe. In some embodiments, the oligonucleotide barcode sequence is within the portion complementary to first probe or the second probe.

[0076] In some embodiments, one or more of the probes comprises one or more deoxyuridine (dU) nucleotide. In some embodiments, one or more of the third probe, the fourth probe, and the sixth probe comprises one or more deoxyuridine (dU) nucleotide. In some embodiments, one or more of the third probe, the fourth probe, and the sixth probe comprises dU nucleotides and does not comprise dT nucleotides. In some embodiments, the third probe and the fourth probe each comprise dU nucleotides in the oligonucleotide portion that is complementary to a portion of the sixth probe. In some embodiments, the sixth probe comprises dU nucleotides in the oligonucleotide portion that is complementary to a portion of the third probe and the oligonucleotide portion that is complementary to a portion of the fourth probe. In some embodiments, the third probe, the fourth probe, and the sixth probe each comprise dT nucleotides outside of the complementary portions.

[0077] In some embodiments, the method further comprises digesting one or more of the third probe, the fourth probe, and the sixth probe. In some embodiments, digesting comprises contacting the one or more probes with an endonuclease enzyme. In some embodiments, the endonuclease enzyme is a USER II enzyme (i.e., a mixture of Antarctic uracil DNA glycosylase and Endonuclease III).

[0078] In some embodiments, the fifth probe comprises an adapter sequence capable of binding to an adapter probe. In some embodiments, the adapter sequence is about 20 nucleotides in length. In some embodiments, the adapter sequence is separated from an RNA-complementary region of the fifth probe by an adenosine linker. In some embodiments, the adenosine linker comprises about 5 adenosine nucleotides. In some embodiments, the adapter probe comprises one or more acryl functional groups. In some embodiments, the sixth probe comprises one or more acryl functional groups. In some embodiments, the method further comprises contacting the fifth probe with the adapter probe. In some embodiments, the method further comprises photocrosslinking the adapter probe to the fifth probe. In some embodiments, the adapter probe comprises an acryl functional group on its 5’-end or 3’-end, wherein the method further comprises incubating the cell with methyl-acrylate NHS ester (MA-NHS) under conditions sufficient to permit the conversion of a remaining terminal 374934-6856-6158.1Atty. Docket No. 114203-1551amine group of the adapter probe to an acryl functional group, thereby generating an adapter probe comprising a 5’ acryl functional group and a 3’ acryl functional group.

[0079] In some embodiments, the method further comprises embedding the cell in a hydrogel. In some embodiments, the method further comprises tissue clearing. In some embodiments, ligation is performed after embedding and tissue clearing.

[0080] The use of any type of cell in the methods disclosed herein is contemplated by the present disclosure (e.g., any of the cell types described herein). In some embodiments, the cell is a mammalian cell. In certain embodiments, the cell is a human cell. The present disclosure also contemplates performing the methods described herein on multiple cells simultaneously (e.g., more than 100 cells, more than 200 cells, more than 300 cells, more than 400 cells, more than 500 cells, more than 1000 cells, more than 10,000 cells, more than 20,000 cells, more than 30,000 cells, more than 40,000 cells, or more than 50,000 cells simultaneously). In some embodiments, the method is performed on multiple cells of the same cell type. In some embodiments, the method is performed on multiple cells comprising cells of different cell types. The cell types in which RNAs being translated may be profiled using the methods disclosed herein include, but are not limited to, stem cells, progenitor cells, neuronal cells, astrocytes, dendritic cells, endothelial cells, microglia, oligodendrocytes, muscle cells, myocardial cells, mesenchymal cells, epithelial cells, immune cells, hepatic cells, smooth and skeletal muscle cells, hematopoietic cells, lymphocytes, monocytes, neutrophils, macrophages, natural killer cells, mast cells, adipocytes, and neurons. In certain embodiments, the cell or cells are present within an intact tissue (e.g., of any of the tissue types described herein). In certain embodiments, the intact tissue is a fixed tissue sample. In some embodiments, the intact tissue comprises multiple cell types. In certain embodiments, the tissue is cardiac tissue, lymph node tissue, liver tissue, muscle tissue, bone tissue, eye tissue, or ear tissue. In certain embodiments, the tissue is brain tissue.

[0081] The RNAs of interest for which gene expression is profiled in the methods described herein may be transcripts that have been expressed from the genomic DNA of the cell. In some embodiments, the RNAs of interest are mRNA. The methods described herein may be used to profile one RNA being translated in a cell at a time, or multiple RNAs of interest simultaneously. In some embodiments, RNA translation in a cell, or multiple cells, is profiled for more than 1, more than 2, more than 3, more than 4, more than 5, more than 10, more than 20, more than 30, more than 40, more than 50, more than 100, more than 200, more than 500, more than 1000, more than 2000, more than 3000 RNAs, more than 4000 RNAs, or more than 5000 RNAs simultaneously.384934-6856-6158.1Atty. Docket No. 114203-1551

[0082] A polymeric matrix is used in the methods described herein following rolling circle amplification (RCA) to facilitate sequencing and imaging of the RNAs of interest being translated in the cell. The use of various polymeric matrices is contemplated by the present disclosure, and any polymeric matrix in which the one or more concatenated amplicons can be embedded is suitable for use in the methods described herein. In some embodiments, the polymeric matrix is a hydrogel ( / .< ., a network of crosslinked polymers that are hydrophilic). In some embodiments, the hydrogel is a polyvinyl alcohol hydrogel, a polyethylene glycol hydrogel, a sodium polyacrylate hydrogel, an acrylate polymer hydrogel, or a polyacrylamide hydrogel. In certain embodiments, the hydrogel is a polyacrylamide hydrogel. Such a hydrogel may be prepared, for example, by incubating the sample in a buffer comprising acrylamide and bis-acrylamide, removing the buffer, and incubating the sample in a polymerization mixture (comprising, e.g., ammonium persulfate andtetramethyl ethyl enedi amine) .

[0083] In some embodiments, the step of performing rolling circle amplification (RCA) to amplify the circular oligonucleotide to produce one or more concatenated amplicons further comprises providing nucleotides modified with reactive chemical groups (e.g., amine modified nucleotides such as 5 -(3 -aminoally l)-dUTP). In some embodiments, the nucleotides modified with reactive chemical groups make up about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% of the nucleotides used in the amplification reaction. For example, the step of performing rolling circle amplification to amplify the circular oligonucleotide to produce one or more concatenated amplicons may further comprise providing amine-modified nucleotides such as 5-(3-aminoallyl)-dUTP. During the amplification process, the amine-modified nucleotides are incorporated into the one or more concatenated amplicons as they are produced. The resulting amplicons are functionalized with primary amines, which can be further reacted with another compatible chemical moiety (e.g., A-hydroxysuccinimide) to facilitate the step of embedding the concatenated amplicons in the polymeric matrix. In some embodiments, the step of embedding the one or more concatenated amplicons in a polymeric matrix comprises reacting the amine-modified nucleotides of the one or more concatenated amplicons with acrylic acid A-hydroxysuccinimide ester and co-polymerizing the one or more concatenated amplicons and the polymer matrix.

[0084] The methods disclosed herein also include a step of sequencing the concatenated amplicons embedded in the polymeric matrix. In some embodiments, the step of sequencing comprises performing “sequencing with error-reduction by dynamic annealing and ligation” (SEDAL sequencing). In some embodiments, SEDAL is performed two, three, four, five, or 394934-6856-6158.1Atty. Docket No. 114203-1551more than five times on a particular sample. SEDAL sequencing is described further in Wang, X. etal., Three-dimensional intact-tissue sequencing of single-cell transcriptional states. Science 2018, 361, 380, and International Patent Application Publication No. WO 2019 / 199579, published October 17, 2019, each of which is incorporated herein by reference. In brief, an oligonucleotide probe comprising a detectable label (i.e., any label that can be used to visualize the location of the additional oligonucleotide probe, for example, through imaging) is provided to the cell. In certain embodiments, the detectable label is fluorescent (e.g., a fluorophore). The additional oligonucleotide probe is complementary to the oligonucleotide barcode sequence of the first probe and is thus linked to the identity of an RNA of interest and can be used to identify the location of an RNA of interest within the cell.

[0085] The additional oligonucleotide probe used in the methods described herein (e.g., as used in SEDAL sequencing) may be read out using a suitable imaging technique. For example, in embodiments where the additional oligonucleotide probe comprises a fluorophore, the fluorophore may be read out using imaging to identify the RNA of interest. As discussed above, the additional oligonucleotide probe comprises a sequence complementary to a barcode sequence on the first oligonucleotide probe, which is used to detect a specific RNA of interest. By imaging the location of the additional oligonucleotide probe comprising a fluorophore, the location of that specific RNA of interest within the sample can be determined. In some embodiments, the step of imaging comprises fluorescent imaging. In certain embodiments, the step of imaging comprises confocal microscopy. In certain embodiments, the step of imaging comprises epifluorescence microscopy. In certain embodiments, two rounds of imaging are performed. In certain embodiments, three rounds of imaging are performed. In certain embodiments, four rounds of imaging are performed. In certain embodiments, five or more rounds of imaging are performed.

[0086] In some embodiments, the methods for simultaneous profiling of the transcriptome and translatome in a single cell described herein may be combined with methods for profiling additional molecules within the cell. For example, expression of non-translating RNAs may be profiled alongside RNA translation using probes that do not comprise a portion that recognizes a ribosome. RNAs in other subcellular locations that are not actively being translated may be profiled alongside RNA translation as well. Profiling of protein expression (e.g., using traditional proteomics methods) may also be combined with the methods described herein, as well as profiling of the transcriptome, lipids, and / or small molecules. In some embodiments, any of the methods provided herein further comprise a step of404934-6856-6158.1Atty. Docket No. 114203-1551overexpressing or knocking out one or more genes in the cell to determine whether the one or more genes are involved in regulating the translation of the RNA of interest.

[0087] Any of the methods described herein may also be used to determine the cell type and / or cell state of one or more cells. In some embodiments, any of the methods provided herein further comprise a step of determining the cell type of the profiled cell, or the cell types of multiple profiled cells, by comparing the RNA transcriptome and translatome profile of the cell or cells to reference data comprising RNA transcriptome and translatome profiles of cells of various cell types. In some embodiments, any of the methods provided herein further comprise a step of determining the cell state of the profiled cell, or the cell states of multiple profiled cells, by comparing the RNA translation profile of the cell or cells to reference data comprising RNA translation profiles of cells of various cell states.Methods for diagnosing a disease or disorder in a subject

[0088] In another aspect, the present disclosure provides methods for diagnosing a disease or disorder in a subject. For example, the methods for simultaneous profiling the transcriptome and translatome described herein may be performed on a cell or multiple cells taken from a subject (e.g., a subject who is thought to have or is at risk of having a disease or disorder, or a subject who is healthy or thought to be healthy). The expression of various RNAs of interest in the cell can then be compared to the expression of the same RNAs of interest in a nondiseased cell or a cell from a non-diseased tissue sample (e.g., a cell from a healthy individual, or multiple cells from a population of healthy individuals). Any difference in the RNA translation profile of the cell (including of a single RNA or of multiple RNAs of interest, e.g., a specific disease signature) relative to one or more non-diseased cells may indicate that the subject has the disease or disorder. RNA translation in one or more nondiseased cells may be profiled alongside expression in a diseased cell as a control experiment. RNA translation in one or more non-diseased cells may have also been profiled previously, and expression in a diseased cell may be compared to this reference data for a non-diseased cell.

[0089] In some embodiments, a method for diagnosing a disease or disorder in a subject, the method comprising: (a) contacting a cell obtained from the subject with one or more sets of probes, wherein each set of probes comprises a first probe, a second probe, a third probe, a fourth probe, a fifth probe, and a sixth probe, wherein (i) the first probe comprises; a first oligonucleotide barcode sequence; an oligonucleotide portion that is complementary to a portion of the fifth probe; an oligonucleotide portion that is complementary to a portion of an 414934-6856-6158.1Atty. Docket No. 114203-1551RNA of interest; a second oligonucleotide barcode sequence; and an oligonucleotide portion that is complementary to a portion of the sixth probe; (ii) the second probe comprises a first oligonucleotide barcode sequence; an oligonucleotide portion that is complementary to a portion of the fifth probe; an oligonucleotide portion that is complementary to a portion of an RNA of interest; and a second oligonucleotide barcode sequence; (iii) the third probe comprises a portion that recognizes the 40S subunit of a ribosome and an oligonucleotide portion that is complementary to a portion of the sixth probe; (iv) the fourth probe comprises a portion that recognizes the 60S subunit of a ribosome and an oligonucleotide portion that is complementary to a portion of the sixth probe; (v) the fifth probe comprises an oligonucleotide portion that is complementary to the RNA of interest; an oligonucleotide portion that is complementary to a portion of the first probe; an oligonucleotide portion that is complementary to a portion of the second probe; and an oligonucleotide barcode sequence, wherein the oligonucleotide barcode sequence of the fifth probe is complementary to the first oligonucleotide barcode sequence of the first probe and the first oligonucleotide barcode sequence of the second probe; and (vi) the sixth probe comprises an oligonucleotide portion that is complementary to a portion of the first probe; an oligonucleotide portion that is complementary to a portion of the third probe; and an oligonucleotide portion that is complementary to a portion of the fourth probe; (b) ligating the 5' end and the 3' end of the first probe together to produce a first circular oligonucleotide, and ligating the 5' end and the 3' end of the second probe together to produce a second circular oligonucleotide; (c) performing rolling circle amplification to amplify the first circular oligonucleotide and the second circular oligonucleotide, wherein the fifth probe is a primer for the amplification of each of the first circular oligonucleotide and the second circular oligonucleotide, thereby producing one or more concatenated amplicons from the first circular oligonucleotide and one or more concatenated amplicons from the second circular oligonucleotide; (d) embedding the one or more concatenated amplicons from the first circular oligonucleotide and one or more concatenated amplicons from the second circular oligonucleotide in a polymeric matrix; and (e) sequencing the concatenated amplicons embedded in the polymeric matrix to determine the identity, location, and translation status of each RNA of interest in the cell, wherein a difference in the RNA translation profile of the cell relative to one or more nondiseased cells indicates that the subject has the disease or disorder.

[0090] In some embodiments, RNAs being profiled in one or more non-diseased cells are profiled simultaneously alongside the cell taken from a subject using the methods disclosed herein as a control experiment. In some embodiments, the profile of RNAs being translated 424934-6856-6158.1Atty. Docket No. 114203-1551in one or more non-diseased cells that is compared to expression in a diseased cell comprises reference data from when the method was performed on one or more non-diseased cells previously.

[0091] Diagnosis of any disease or disorder is contemplated by the methods described herein. In some embodiments, the disease or disorder is a genetic disease, a proliferative disease, an inflammatory disease, an autoimmune disease, a liver disease, a spleen disease, a lung disease, a hematological disease, a neurological disease, a psychiatric disease, a gastrointestinal (GI) tract disease, a genitourinary disease, an infectious disease, a musculoskeletal disease, an endocrine disease, a metabolic disorder, an immune disorder, a central nervous system (CNS) disorder, or a cardiovascular disease. In certain embodiments, the disease is cancer.

[0092] In some embodiments, the cell is present in a tissue (e.g., epithelial tissue, connective tissue, muscular tissue, or nervous tissue). In some embodiments, the tissue is a tissue sample from a subject. In some embodiments, the subject is a non-human experimental animal (e.g., a mouse, a rat, a dog, a pig, or a non-human primate). In some embodiments, the subject is a domesticated animal. In some embodiments, the subject is a human. In some embodiments, the tissue sample comprises a fixed tissue sample. In certain embodiments, the tissue sample is a biopsy (e.g., bone, bone marrow, breast, gastrointestinal tract, lung, liver, pancreas, prostate, brain, nerve, renal, endometrial, cervical, lymph node, muscle, or skin biopsy). In certain embodiments, the biopsy is a tumor biopsy. In certain embodiments, the tissue is brain tissue. In certain embodiments, the tissue is from the central nervous system.Methods of screening for an agent capable of modulating translation of one or more RNAs

[0093] In another aspect, the present disclosure provides methods for screening for an agent capable of modulating translation of one or more RNAs of interest. For example, the methods for profiling RNAs being translated described herein may be performed in a cell in the presence of one or more candidate agents. The expression of various RNAs of interest being translated in the cell (e.g, a normal cell, or a diseased cell) can then be compared to the expression of the same RNAs of interest in a cell that was not exposed to the one or more candidate agents. Any difference in the RNA translation profile relative to translation in the cell that was not exposed to the candidate agent(s) may indicate that translation of the one or more RNAs of interest is modulated by the candidate agent(s). In some embodiments, a particular signature (e.g, of multiple RNAs of interest being translated) that is known to be associated with treatment of the disease may be used to identify a candidate agent capable of 434934-6856-6158.1Atty. Docket No. 114203-1551modulating translation in a desired manner. The methods described herein may also be used to identify drugs that have certain side effects, for example, by looking for specific RNA translation signatures when one or more cells is treated with a candidate agent or known drug.

[0094] In some embodiments, the present disclosure provides a method for screening for an agent capable of modulating translation of one or more RNAs comprises the steps of: (a) contacting a cell that is being treated with or has been treated with a candidate agent with one or more sets of probes, wherein each set of probes comprises a first probe, a second probe, a third probe, a fourth probe, a fifth probe, and a sixth probe, wherein (i) the first probe comprises; a first oligonucleotide barcode sequence; an oligonucleotide portion that is complementary to a portion of the fifth probe; an oligonucleotide portion that is complementary to a portion of an RNA of interest; a second oligonucleotide barcode sequence; and an oligonucleotide portion that is complementary to a portion of the sixth probe; (ii) the second probe comprises a first oligonucleotide barcode sequence; an oligonucleotide portion that is complementary to a portion of the fifth probe; an oligonucleotide portion that is complementary to a portion of an RNA of interest; and a second oligonucleotide barcode sequence; (iii) the third probe comprises a portion that recognizes the 40S subunit of a ribosome and an oligonucleotide portion that is complementary to a portion of the sixth probe; (iv) the fourth probe comprises a portion that recognizes the 60S subunit of a ribosome and an oligonucleotide portion that is complementary to a portion of the sixth probe; (v) the fifth probe comprises an oligonucleotide portion that is complementary to the RNA of interest; an oligonucleotide portion that is complementary to a portion of the first probe; an oligonucleotide portion that is complementary to a portion of the second probe; and an oligonucleotide barcode sequence, wherein the oligonucleotide barcode sequence of the fifth probe is complementary to the first oligonucleotide barcode sequence of the first probe and the first oligonucleotide barcode sequence of the second probe; and (vi) the sixth probe comprises an oligonucleotide portion that is complementary to a portion of the first probe; an oligonucleotide portion that is complementary to a portion of the third probe; and an oligonucleotide portion that is complementary to a portion of the fourth probe; (b) ligating the 5' end and the 3' end of the first probe together to produce a first circular oligonucleotide, and ligating the 5' end and the 3' end of the second probe together to produce a second circular oligonucleotide; (c) performing rolling circle amplification to amplify the first circular oligonucleotide and the second circular oligonucleotide, wherein the fifth probe is a primer for the amplification of each of the first circular oligonucleotide and the second circular oligonucleotide, thereby 444934-6856-6158.1Atty. Docket No. 114203-1551producing one or more concatenated amplicons from the first circular oligonucleotide and one or more concatenated amplicons from the second circular oligonucleotide; (d) embedding the one or more concatenated amplicons from the first circular oligonucleotide and one or more concatenated amplicons from the second circular oligonucleotide in a polymeric matrix; and (e) sequencing the concatenated amplicons embedded in the polymeric matrix to determine the identity, location, and translation status of each RNA of interest in the cell, wherein a difference in the profile of RNAs being translated in the presence of the candidate agent relative to in the absence of the candidate agent indicates that the candidate agent modulates translation of one or more RNAs.

[0095] In some embodiments, the candidate agent is a small molecule, a protein, a peptide, a nucleic acid, a lipid, or a carbohydrate. In some embodiments, the candidate agent comprises a known drug or an FDA-approved drug. In certain embodiments, the protein is an antibody. In certain embodiments, the protein is an antibody fragment or an antibody variant. In certain embodiments, the protein is a receptor. In certain embodiments, the protein is a cytokine. In certain embodiments, the nucleic acid is an mRNA, an antisense RNA, an miRNA, an siRNA, an RNA aptamer, a double stranded RNA (dsRNA), a short hairpin RNA (shRNA), or an antisense oligonucleotide (ASO). Any candidate agent may be screened using the methods described herein. In particular, any candidate agents thought to be capable of modulating translation of one or more RNAs may be screened using the methods described herein. In some embodiments, modulation of translation of one or more RNAs of interest by the candidate agent is associated with reducing, relieving, or eliminating the symptoms of a disease or disorder, or preventing the development or progression of the disease or disorder. In some embodiments, the disease or disorder modulated by the candidate agent is a genetic disease, a proliferative disease, an inflammatory disease, an autoimmune disease, a liver disease, a spleen disease, a lung disease, a hematological disease, a neurological disease, a psychiatric disease, a gastrointestinal (GI) tract disease, a genitourinary disease, an infectious disease, a musculoskeletal disease, an endocrine disease, a metabolic disorder, an immune disorder, a central nervous system (CNS) disorder, or a cardiovascular disease. In certain embodiments, the disease or disorder modulated by the candidate agent is cancer.Methods for treating a disease or disorder in a subject

[0096] In another aspect, the present disclosure provides methods for treating a disease or disorder in a subject. For example, the methods for profiling RNAs being translated described herein may be performed in a cell from a sample taken from a subject (e.g., a 454934-6856-6158.1Atty. Docket No. 114203-1551subject who is thought to have or is at risk of having a disease or disorder). The profile of one or more RNAs being translated in the cell can then be compared to the translation status of the same RNAs of interest in a cell from a non-diseased tissue sample. A treatment for the disease or disorder may then be administered to the subject if any difference in the RNA translation profile relative to a non-diseased cell is observed. RNA translation in one or more non-diseased cells may be profiled alongside RNA translation in a diseased cell as a control experiment. RNA translation in one or more non-diseased cells may have also been profiled previously, and translation in a diseased cell may be compared to this reference data for a non-diseased cell.

[0097] In some embodiments, the present disclosure provides a method for treating a disease or disorder in a subject comprising the steps of: (a) contacting a cell obtained from the subject with one or more sets of probes, wherein each set of probes comprises a first probe, a second probe, a third probe, a fourth probe, a fifth probe, and a sixth probe, wherein (i) the first probe comprises; a first oligonucleotide barcode sequence; an oligonucleotide portion that is complementary to a portion of the fifth probe; an oligonucleotide portion that is complementary to a portion of an RNA of interest; a second oligonucleotide barcode sequence; and an oligonucleotide portion that is complementary to a portion of the sixth probe; (ii) the second probe comprises a first oligonucleotide barcode sequence; an oligonucleotide portion that is complementary to a portion of the fifth probe; an oligonucleotide portion that is complementary to a portion of an RNA of interest; and a second oligonucleotide barcode sequence; (iii) the third probe comprises a portion that recognizes the 40S subunit of a ribosome and an oligonucleotide portion that is complementary to a portion of the sixth probe; (iv) the fourth probe comprises a portion that recognizes the 60S subunit of a ribosome and an oligonucleotide portion that is complementary to a portion of the sixth probe; (v) the fifth probe comprises an oligonucleotide portion that is complementary to the RNA of interest; an oligonucleotide portion that is complementary to a portion of the first probe; an oligonucleotide portion that is complementary to a portion of the second probe; and an oligonucleotide barcode sequence, wherein the oligonucleotide barcode sequence of the fifth probe is complementary to the first oligonucleotide barcode sequence of the first probe and the first oligonucleotide barcode sequence of the second probe; and (vi) the sixth probe comprises an oligonucleotide portion that is complementary to a portion of the first probe; an oligonucleotide portion that is complementary to a portion of the third probe; and an oligonucleotide portion that is complementary to a portion of the fourth probe; (b) ligating the 5' end and the 3' end of the 464934-6856-6158.1Atty. Docket No. 114203-1551first probe together to produce a first circular oligonucleotide, and ligating the 5' end and the 3' end of the second probe together to produce a second circular oligonucleotide; (c) performing rolling circle amplification to amplify the first circular oligonucleotide and the second circular oligonucleotide, wherein the fifth probe is a primer for the amplification of each of the first circular oligonucleotide and the second circular oligonucleotide, thereby producing one or more concatenated amplicons from the first circular oligonucleotide and one or more concatenated amplicons from the second circular oligonucleotide; (d) embedding the one or more concatenated amplicons from the first circular oligonucleotide and one or more concatenated amplicons from the second circular oligonucleotide in a polymeric matrix; and (e) sequencing the concatenated amplicons embedded in the polymeric matrix to determine the identity, location, and translation status of each RNA of interest in the cell; and (f) administering a treatment for the disease or disorder to the subject if a difference in the profile of RNAs being translated in the cell relative to one or more non-diseased cells is observed.

[0098] In some embodiments, RNA translation in one or more non-diseased cells is profiled simultaneously using the methods disclosed herein as a control experiment. In some embodiments, the RNA translation data in one or more non-diseased cells that is compared to translation in a diseased cell comprises reference data from a time the method was performed on a non-diseased cell previously.

[0099] In some embodiments, the treatment comprises administering a therapeutic agent. In some embodiments, the treatment comprises surgery. In some embodiments, the treatment comprises imaging. In some embodiments, the treatment comprises performing further diagnostic methods. In some embodiments, the treatment comprises radiation therapy. In some embodiments, the therapeutic agent is a small molecule, a protein, a peptide, a nucleic acid, a lipid, or a carbohydrate. In some embodiments, the therapeutic agent is a known drug and / or an FDA-approved drug. In certain embodiments, the protein is an antibody. In certain embodiments, the protein is an antibody fragment or antibody variant. In certain embodiments, the protein is a receptor, or a fragment or variant thereof. In certain embodiments, the protein is a cytokine. In certain embodiments, the nucleic acid is an mRNA, an antisense RNA, an miRNA, an siRNA, an RNA aptamer, a double stranded RNA (dsRNA), a short hairpin RNA (shRNA), or an antisense oligonucleotide (ASO).

[0100] Treatment of any disease or disorder is contemplated by the methods described herein. In some embodiments, the disease or disorder is a genetic disease, a proliferative disease, an inflammatory disease, an autoimmune disease, a liver disease, a spleen disease, a 474934-6856-6158.1Atty. Docket No. 114203-1551lung disease, a hematological disease, a neurological disease, a gastrointestinal (GI) tract disease, a genitourinary disease, an infectious disease, a musculoskeletal disease, an endocrine disease, a metabolic disorder, an immune disorder, a central nervous system (CNS) disorder, a neurological disorder, an ophthalmic disease, or a cardiovascular disease. In certain embodiments, the disease is cancer.

[0101] In some embodiments, the subject is a human. In some embodiments, the sample comprises a biological sample. In some embodiments, the sample comprises a tissue sample. In certain embodiments, the tissue sample is a biopsy (e.g., bone, bone marrow, breast, gastrointestinal tract, lung, liver, pancreas, prostate, brain, nerve, renal, endometrial, cervical, lymph node, muscle, or skin biopsy). In certain embodiments, the biopsy is a tumor biopsy. In certain embodiments, the biopsy is a solid tumor biopsy. In some embodiments, the tissue sample is a brain tissue sample. In certain embodiments, the tissue sample is a central nervous system tissue sample.Probes

[0102] The present disclosure also provides sets of probes for use in the methods and systems described herein. In one aspect, the present disclosure provides a set of probes comprising a first probe, a second probe, a third probe, a fourth probe, a fifth probe, and a sixth probe, wherein: (i) the first probe comprises a first oligonucleotide barcode sequence; an oligonucleotide portion that is complementary to a portion of the fifth probe; an oligonucleotide portion that is complementary to a portion of an RNA of interest; a second oligonucleotide barcode sequence; and an oligonucleotide portion that is complementary to a portion of the sixth probe; the second probe comprises a first oligonucleotide barcode sequence; an oligonucleotide portion that is complementary to a portion of the fifth probe; an oligonucleotide portion that is complementary to a portion of an RNA of interest; and a second oligonucleotide barcode sequence; (iii) the third probe comprises a portion that recognizes the 40S subunit of a ribosome and an oligonucleotide portion that is complementary to a portion of the sixth probe; (iv) the fourth probe comprises a portion that recognizes the 60S subunit of a ribosome and an oligonucleotide portion that is complementary to a portion of the sixth probe; (v) the fifth probe comprises an oligonucleotide portion that is complementary to the RNA of interest; an oligonucleotide portion that is complementary to a portion of the first probe; an oligonucleotide portion that is complementary to a portion of the second probe; and an oligonucleotide barcode sequence, wherein the oligonucleotide barcode sequence of the fifth probe is complementary to the first 484934-6856-6158.1Atty. Docket No. 114203-1551oligonucleotide barcode sequence of the first probe and the first oligonucleotide barcode sequence of the second probe; and (vi) the sixth probe comprises an oligonucleotide portion that is complementary to a portion of the first probe; an oligonucleotide portion that is complementary to a portion of the third probe; and an oligonucleotide portion that is complementary to a portion of the fourth probe.

[0103] All of the probes described herein may optionally have spacers or linkers of various nucleotide lengths in between each of the recited components, or the components of the oligonucleotide probes may be joined directly to one another ( / .< ., by a phosphodiester bond). All of the probes described herein may comprise standard nucleotides, or some of the standard nucleotides may be substituted for any modified nucleotides known in the art.

[0104] The set of probes described herein comprises a first probe, a second probe, a third probe, a fourth probe, a fifth probe, and a sixth probe. The third probe and fourth probe each comprises a portion that recognizes a ribosome. The portion of the probe that recognizes a ribosome may be a protein, peptide, nucleic acid, or small molecule. In some embodiments, the portion of the third probe or fourth probe that recognizes a ribosome is an agent that binds an antibody, or an antibody variant or fragment. In certain embodiments, the portion of the third probe or fourth probe that recognizes the ribosome comprises an antibody (e.g., a secondary antibody), or an antibody variant or fragment. When the portion of the third probe or fourth probe that recognizes the ribosome is a secondary antibody, the method may optionally further comprise contacting the cell with a primary antibody that recognizes a ribosome and is recognized by the secondary antibody of the second probe. The primary antibody may recognize any portion of the ribosome, for example, any protein, nucleic acid (e.g, rRNA), or combination thereof of the ribosome. In some embodiments, the primary antibody is an anti-40S ribosomal protein S3 (RPS3) antibody (e.g, an anti-RPS3 monoclonal antibody), for the third probe. In some embodiments, the antibody is an anti-60S ribosomal protein L4 (RPL4) antibody (e.g., an anti-RPL4 polyclonal antibody), for the fourth probe. In place of an antibody, the present disclosure also contemplates the use of any agent capable of recognizing the ribosome on the probes described herein. In some embodiments, the portion of the third probe or fourth probe that recognizes the ribosome comprises an oligonucleotide that is complementary to a portion of the ribosomal RNA (rRNA) within the ribosome. In some embodiments, the portion of the third probe or fourth probe that recognizes the ribosome comprises an oligonucleotide that is complementary to a portion of the 40S small ribosomal subunit (including, e.g., the 18S rRNA) (for the third probe) or to a portion of the 60S large ribosomal subunit (including, e.g., the 5S rRNA, the 494934-6856-6158.1Atty. Docket No. 114203-155128S rRNA, and the 5.8S rRNA) (for the fourth probe). In some embodiments, the portion of the third probe that recognizes the 40S subunit of a ribosome comprises an oligonucleotide that is complementary to a portion of the 18S rRNA. In some embodiments, the portion of the fourth probe that recognizes the 60S subunit of a ribosome comprises an oligonucleotide that is complementary to a portion of the 28S rRNA. In certain embodiments, the oligonucleotide complementary to a portion of rRNA is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more than 30 nucleotides in length. In certain embodiments, the oligonucleotide complementary to a portion of rRNA is about 25 nucleotides in length.

[0105] In addition to the portion that recognizes the ribosome, the each of the third probe and the fourth probe also comprises a portion that is complementary to a portion of the sixth probe.

[0106] In some embodiments, the portion of the third probe that is complementary to a portion of the sixth probe is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides in length. In some embodiments, the portion of the third probe that is complementary to a portion of the sixth probe is 9-15 nucleotides in length. In some embodiments, the portion of the third probe that is complementary to a portion of the sixth probe is 9-11 nucleotides in length. The present disclosure contemplates any arrangement of the portions of the third probe. In some embodiments, the third probe used in the methods described herein comprises the structure:5 '-[portion recognizing the 40S subunit of the ribosome]-[portion complementary to the sixth probe]-3'; or5 '-[portion recognizing the 40S subunit of the ribosome]-[poly-A linker]-[portion complementary to the sixth probe]-3'wherein ]-[ comprises an optional linker (e.g., nucleotide linker, e.g., a poly-A linker). In some embodiments, ]-[ represents a direct linkage between two portions of the third oligonucleotide probe ( / .< ., a phosphodiester bond).

[0107] In some embodiments, the portion of the fourth probe that is complementary to a portion of the sixth probe is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides in length. In some embodiments, the portion of the fourth probe that is complementary to a portion of the sixth probe is 9-15 nucleotides in length. In some embodiments, the portion of the fourth probe that is complementary to a portion of the sixth probe is 9-11 nucleotides in length. The present disclosure contemplates any arrangement of the portions of the fourth probe. In some embodiments, the fourth probe used in the methods described herein comprises the structure:5 '-[portion complementary to the sixth probe]-[portion recognizing the 60S subunit of the ribosome]-3'; or504934-6856-6158.1Atty. Docket No. 114203-15515 '-[portion complementary to the sixth probe]-[poly-A linker]-[portion recognizing the 60S subunit of the ribosome]-3'wherein ]-[ comprises an optional linker (e.g., nucleotide linker, e.g, a poly-A linker). In some embodiments, ]-[ represents a direct linkage between two portions of the third oligonucleotide probe (z.e., a phosphodiester bond).

[0108] In some embodiments, the portion of the sixth probe that is complementary to a portion of the third probe is 9-11 nucleotides in length, and the portion of the sixth probe that is complementary to a portion of the fourth probe is 9-11 nucleotides in length. In some embodiments, the portion of the sixth probe that is complementary to a portion of the third probe is 9 nucleotides in length, and the portion of the sixth probe that is complementary to a portion of the fourth probe is 11 nucleotides in length.

[0109] The first probe used in the methods described herein (also referred to herein as the “RIBO padlock” probe) includes a first oligonucleotide barcode sequence and a second oligonucleotide barcode sequence made up of a specific sequence of nucleotides. In some embodiments, the first and second oligonucleotide barcode sequences on the first probe comprise the same nucleotide sequence. The presence of the second barcode sequence of the first oligonucleotide probe may increase the specificity of the detection of the RNA of interest in the methods described herein (z.e., as compared to if the method were performed using a first oligonucleotide probe that did not comprise a second barcode sequence). The use of an additional oligonucleotide barcode sequence of the first oligonucleotide probe may also play a role in reducing non-specific amplification of the RNA of interest in the methods described herein. This is accomplished because the oligonucleotide barcode sequence of the fifth probe used in the methods described herein is complementary to the first oligonucleotide barcode sequence on the first probe, adding an additional layer of specificity that is required before amplification can occur using the fifth probe as a primer. In some embodiments, the portion of the first probe that is complementary to the fifth probe is 5-15, 6-14, 7-13, 8-12, or 9-11 nucleotides in length. In some embodiments, the portion of the first probe that is complementary to the fifth probe is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides in length.

[0110] In some embodiments, the oligonucleotide barcode sequences of the first probe are about 3 to about 13, about 4 to about 12, about 5 to about 11, about 6 to about 10, or about 7 to about 9 nucleotides in length. In some embodiments, the oligonucleotide barcode sequences of the first probe are 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 nucleotides in length.514934-6856-6158.1Atty. Docket No. 114203-1551

[0111] The second probe used in the methods described herein (also referred to herein as the “STAR padlock” probe) includes an oligonucleotide barcode sequence made up of a specific sequence of nucleotides. In some embodiments, the oligonucleotide barcode sequences of the second probe are about 3 to about 13, about 4 to about 12, about 5 to about 11, about 6 to about 10, or about 7 to about 9 nucleotides in length. In some embodiments, the oligonucleotide barcode sequences of the second probe are 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 nucleotides in length.

[0112] The barcodes of the oligonucleotide probes described herein may comprise genespecific sequences used to identify RNAs of interest that are being translated ( / .< ., each barcode sequence is associated with a specific gene or transcript). The barcodes of the oligonucleotide probes described herein may comprise gene-specific sequences used to identify RNAs of interest that are being translated (i.e., each barcode sequence is associated with a specific gene or transcript). In some embodiments, one or more barcodes of the oligonucleotide probes described herein may comprise a sequence used to identify RNAs of interest as ribosome-associated (e.g., detected as part of a fully-assembled ribosome complex) or non-ribosome-associated (e.g., detected separately from a fully-assembled ribosome complex). The use of the barcodes on probes analogous to those described herein is further described in, for example, International Patent Application Publication No. WO 2019 / 199579, published October 17, 2019, and Wang etal., Science 2018, 361, 380, both of which are incorporated herein by reference in their entireties.

[0113] The first probe also comprises an oligonucleotide portion that is complementary to a portion of the sixth probe and a portion that is complementary to an RNA of interest. In some embodiments, the portion of the first probe that is complementary to a portion of the sixth probe is 4-20, 5-19, 6-18, 7-17, 8-16, 9-15, 10-14, or 11-13 nucleotides in length. In some embodiments, the portion of the first probe that is complementary to a portion of the sixth probe is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length. In certain embodiments, the oligonucleotide portion of the first probe that is complementary to a portion of the sixth probe is split between the 5' end and the 3 ' end of the first probe. In some embodiments, the portion of the first probe that is complementary to the RNA of interest is 10-30, 11-29, 12-28, 13-27, 14-26, 15-25, 16-24, 17-23, 18-22, or 19-21 nucleotides in length. In some embodiments, the portion of the first probe that is complementary to an RNA of interest is about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28,524934-6856-6158.1Atty. Docket No. 114203-1551about 29, or about 30 nucleotides long. In some embodiments, the first probe is about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, or about 40 nucleotides long or longer.

[0114] The arrangement of the portions of the first oligonucleotide probe in any order is contemplated by the present disclosure. In some embodiments, the first probe comprises the structure:5 '-[portion complementary to sixth probe]-[portion complementary to portion of fifth probe]-[portion complementary to RNA of interest]-[second oligonucleotide barcode sequence]-[portion complementary to sixth probe]-3'wherein ]-[ comprises an optional linker (e.g., a nucleotide linker). In some embodiments, ]-[ represents a direct linkage between two portions of the first probe ( / .< ., a phosphodiester bond). In some embodiments, the first oligonucleotide barcode sequence is within the portion complementary to portion of fifth probe.

[0115] The second probe also comprises an oligonucleotide portion that is complementary to an RNA of interest. In some embodiments, the portion of the second probe that is complementary to the RNA of interest is 10-30, 11-29, 12-28, 13-27, 14-26, 15-25, 16-24, 17-23, 18-22, or 19-21 nucleotides in length. In some embodiments, the portion of the second probe that is complementary to an RNA of interest is about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, or about 30 nucleotides long. In some embodiments, the first probe is about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, or about 40 nucleotides long or longer. The arrangement of the portions of the second oligonucleotide probe in any order is contemplated by the present disclosure. In some embodiments, the second probe comprises the structure:5 '-[portion complementary to portion of fifth probe]-[portion complementary to RNA of interest] -[second oligonucleotide barcode sequence]- 3'wherein ]-[ comprises an optional linker (e.g., a nucleotide linker). In some embodiments, ]-[ represents a direct linkage between two portions of the first probe ( / .< ., a phosphodiester bond). In some embodiments, the first oligonucleotide barcode sequence is within the portion complementary to portion of fifth probe.534934-6856-6158.1Atty. Docket No. 114203-1551

[0116] As described herein, the sixth probe (also referred to herein as a “splint probe” or as a “blocked probe”) comprises a portion that is complementary to a portion of the third probe and a portion that is complementary to a portion of the fourth probe, thereby recognizing a ribosome ( / .< ., a ribosome that is bound to and is actively translating the RNA of interest). In some embodiments, the sixth probe further comprises a polymerization blocker. The addition of a polymerization blocker prevents the sixth probe from being used as a primer in the amplification of step (c), ensuring that the fifth probe will be used as the primer during amplification. The polymerization blocker on the sixth probe can be any moiety capable of preventing the use of the sixth probe as a primer in the amplification of step (c) of the methods described herein. In some embodiments, the polymerization blocker is at the 3' end of the sixth probe. The polymerization blocker can be, for example, any chemical moiety that prevents a polymerase from using the sixth probe as a primer for polymerization. In some embodiments, the polymerization blocker is a nucleic acid residue comprising a blocked 3' hydroxyl group (e.g., comprising an oxygen protecting group on the 3' hydroxyl group). In some embodiments, the polymerization blocker comprises a hydrogen in place of the 3' hydroxyl group. In some embodiments, the polymerization blocker comprises any chemical moiety in place of the 3' hydroxyl group that prevents an additional nucleotide from being added. In some embodiments, the polymerization blocker comprises an inverted nucleic acid residue. In some embodiments, the polymerization blocker is an inverted adenosine, thymine, cytosine, guanosine, or uridine residue. In certain embodiments, the polymerization blocker is an inverted thymine residue.

[0117] In addition to the portion that recognizes the ribosome, the sixth probe also comprises a portion that is complementary to a portion of the first probe. In some embodiments, the portion of the sixth probe that is complementary to a portion of the first probe is 4-20, 5-19, 6-18, 7-17, 8-16, 9-15, 10-14, or 11-13 nucleotides in length. In some embodiments, the portion of the sixth probe that is complementary to a portion of the first probe is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length. In certain embodiments, the portion of the sixth probe that is complementary to a portion of the third probe and the portion of the sixth probe that is complementary to a portion of the fourth probe are joined to the portion of the sixth probe that is complementary to a portion of the first probe by a poly-A nucleotide linker. In some embodiments, the poly-A nucleotide linker is about 4-60, 5-50, or 10-40 nucleotides in length. In some embodiments, the poly-A nucleotide linker of the sixth probe is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,544934-6856-6158.1Atty. Docket No. 114203-155128, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 nucleotides in length.

[0118] The present disclosure contemplates any arrangement of the portions of the sixth probe. In some embodiments, the sixth probe used in the methods described herein comprises the structure:5 '-[portion complementary to portion of fourth probe]-[portion complementary to portion of third probe]-[portion complementary to portion of first probe]-3'; or 5 '-[portion complementary to portion of fourth probe]-[portion complementary to portion of third probe]-[poly-A linker]-[portion complementary to portion of first probe]-3'; or5 '-[portion complementary to portion of third probe]-[portion complementary to portion of fourth probe]-[portion complementary to portion of first probe]-3'; or 5 '-[portion complementary to portion of third probe]-[portion complementary to portion of fourth probe]-[poly-A linker]-[portion complementary to portion of first probe]-3'wherein ]-[ comprises an optional linker (e.g., nucleotide linker). In some embodiments, ]-[ represents a direct linkage between two portions of the sixth oligonucleotide probe.

[0119] The fifth probe used in the methods disclosed herein (also referred to herein as the “primer” probe) includes a barcode sequence made up of a specific sequence of nucleotides. In some embodiments, the barcode sequence of the fifth probe is about 3 to about 20, about 5 to about 15, about 6 to about 14, about 7 to about 13, about 8 to about 12, or about 9 to about 11 nucleotides in length. In some embodiments, the barcode sequence of the fifth probe is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides in length. In certain embodiments, the barcode sequence of the fifth probe is 10 nucleotides in length.

[0120] The fifth probe also comprises a portion that is complementary to an RNA of interest and a portion that is complementary to a portion of the first probe or the second probe. In some embodiments, the first and the fifth probes are complementary to and bind different portions of the RNA of interest. In some embodiments, the second and the fifth probes are complementary to and bind different portions of the RNA of interest. In some embodiments, the portion of the fifth probe that is complementary to the RNA of interest is 10-30, 11-29, 12-28, 13-27, 14-26, 15-25, 16-24, 17-23, 18-22, or 19-21 nucleotides in length. In some embodiments, the portion of the fifth probe that is complementary to an RNA of interest is about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23,554934-6856-6158.1Atty. Docket No. 114203-1551about 24, about 25, about 26, about 27, about 28, about 29, or about 30 nucleotides long. In some embodiments, the third probe is about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, or about 40 nucleotides long or longer. In some embodiments, the portion of the fifth probe that is complementary to the first probe is 5-15, 6-14, 7-13, 8-12, or 9-11 nucleotides in length. In some embodiments, the portion of the fifth probe that is complementary to the first probe is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides in length. In some embodiments, the portion of the fifth probe that is complementary to the second probe is 5-15, 6-14, 7-13, 8-12, or 9-11 nucleotides in length. In some embodiments, the portion of the fifth probe that is complementary to the second probe is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides in length.

[0121] Any arrangement of the portions of the fifth probe is contemplated by the present disclosure. In some embodiments, the fifth probe comprises the structure:5'-[portion complementary to RNA of interest]-[portion complementary to first probe]-3'; or5'-[portion complementary to RNA of interest]-[portion complementary to second probe]-3'wherein ]-[ comprises an optional linker (e.g., nucleotide linker). In some embodiments, ]-[ represents a direct linkage between two portions of the fifth probe. In some embodiments, the oligonucleotide barcode sequence is within the portion complementary to first probe or the second probe.

[0122] In some embodiments, one or more of the third probe, the fourth probe, and the sixth probe comprises one or more deoxyuridine (dU) nucleotide. In some embodiments, one or more of the third probe, the fourth probe, and the sixth probe comprises dU nucleotides and does not comprise dT nucleotides. In some embodiments, the third probe and the fourth probe each comprise dU nucleotides in the oligonucleotide portion that is complementary to a portion of the sixth probe. In some embodiments, the sixth probe comprises dU nucleotides in the oligonucleotide portion that is complementary to a portion of the third probe and the oligonucleotide portion that is complementary to a portion of the fourth probe. In some embodiments, the third probe, the fourth probe, and the sixth probe each comprise dT nucleotides outside of the complementary portions. In some embodiments, the set of probes further comprises an adapter probe comprising a portion that is capable of hybridizing to the fifth probe. In some embodiments, the fifth probe comprises an adapter sequence capable of binding to the adapter probe. In some embodiments, the adapter sequence is about 20564934-6856-6158.1Atty. Docket No. 114203-1551nucleotides in length. In some embodiments, the adapter sequence is separated from a RNA-complementary region of the fifth probe by an adenosine linker. In some embodiments, the adenosine linker comprises about 5 adenosine nucleotides. In some embodiments, the adapter probe comprises one or more acryl functional groups. In some embodiments, the sixth probe comprises one or more acryl functional groups. In some embodiments, the fifth probe comprises a photocrosslinking moiety. In some embodiments, the adapter probe comprises an acryl functional group on its 5 ’-end or 3 ’-end, wherein The set of probes further comprises incubating the cell with methyl-acrylate NHS ester (MA-NHS) under conditions sufficient to permit the conversion of a remaining terminal amine group of the adapter probe to an acryl functional group, thereby generating an adapter probe comprising a 5’ acryl functional group and a 3’ acryl functional group.

[0123] In some embodiments, the present disclosure provides a plurality of probes comprising multiple sets of probes as described herein. In certain embodiments, each set of probes in the plurality of probes comprises oligonucleotide portions that are complementary to a different RNA of interest.Kits

[0124] Also provided by the disclosure are kits. In one aspect, the kits provided may comprise one or more of the probes as described herein. In some embodiments, the kits comprise any of the pairs of probes described herein, or multiple pairs of probes. In some embodiments, the kits comprise any of the sets of probes described herein, or multiple sets of probes. In some embodiments, the kits may further comprise a container (e.g., a vial, ampule, bottle, and / or dispenser package, or other suitable container). The kits may also comprise cells for performing control experiments. In some embodiments, the kits may further comprise other reagents for performing the methods disclosed herein (e.g., enzymes such as a ligase or a polymerase, amine-modified nucleotides as described herein, primary antibodies, secondary antibodies, buffers, and / or reagents and monomers for making a polymeric matrix (e.g., a polyacrylamide matrix)). In some embodiments, the kits further comprise an endonuclease enzyme. In some embodiments, the endonuclease enzyme is thermolabile. In some embodiments, the endonuclease enzyme is thermostable. In some embodiments, the endonuclease enzyme generates a single nucleotide gap at the location of a uracil residue (e.g., a Uracil-Specific Excision Reagent). In some embodiments, the endonuclease enzyme is a USER II enzyme. In some embodiments, the endonuclease enzyme is a574934-6856-6158.1Atty. Docket No. 114203-1551mixture / combination of a uracil glycosylase (e.g., a uracil DNA glycosylase or UDG) that can catalyze excision of a uracil base and an endonuclease (e.g., endonuclease VIII).

[0125] In some embodiments, the kits are useful for profiling gene and protein expression in a cell. In some embodiments, the kits are useful for diagnosing a disease in a subject. In some embodiments, the kits are useful for screening for an agent capable of modulating RNA translation. In some embodiments, the kits are useful for diagnosing a disease or disorder in a subject. In some embodiments, the kits are useful for treating a disease or disorder in a subject. In certain embodiments, a kit described herein further includes instructions for using the kit.Systems

[0126] In one aspect, the present disclosure provides systems for profiling RNAs being translated in a cell. In some embodiments, such a system comprises: (a) a cell; (b) one or more sets of probes comprising a first probe, a second probe, a third probe, a fourth probe, a fifth probe, and a sixth probe, wherein (i) the first probe comprises a first oligonucleotide barcode sequence; an oligonucleotide portion that is complementary to a portion of the fifth probe; an oligonucleotide portion that is complementary to a portion of an RNA of interest; a second oligonucleotide barcode sequence; and an oligonucleotide portion that is complementary to a portion of the sixth probe; the second probe comprises a first oligonucleotide barcode sequence; an oligonucleotide portion that is complementary to a portion of the fifth probe; an oligonucleotide portion that is complementary to a portion of an RNA of interest; and a second oligonucleotide barcode sequence; (iii) the third probe comprises a portion that recognizes the 40S subunit of a ribosome and an oligonucleotide portion that is complementary to a portion of the sixth probe; (iv) the fourth probe comprises a portion that recognizes the 60S subunit of a ribosome and an oligonucleotide portion that is complementary to a portion of the sixth probe; (v) the fifth probe comprises an oligonucleotide portion that is complementary to the RNA of interest; an oligonucleotide portion that is complementary to a portion of the first probe; an oligonucleotide portion that is complementary to a portion of the second probe; and an oligonucleotide barcode sequence, wherein the oligonucleotide barcode sequence of the fifth probe is complementary to the first oligonucleotide barcode sequence of the first probe and the first oligonucleotide barcode sequence of the second probe; and (vi) the sixth probe comprises an oligonucleotide portion that is complementary to a portion of the first probe; an oligonucleotide portion that is584934-6856-6158.1Atty. Docket No. 114203-1551complementary to a portion of the third probe; and an oligonucleotide portion that is complementary to a portion of the fourth probe; (c) a microscope; and (d) a computer.

[0127] Any of the probes (i.e., the pairs of probes or sets of probes) described herein may be used in the systems contemplated by the present disclosure. In some embodiments, the microscope is a confocal microscope. In some embodiments, the system further comprises a CPU. In some embodiments, the system further comprises computer storage and / or memory, or a storage device. In some embodiments, the system further comprises a camera. In some embodiments, the system further comprises a CCD. In some embodiments, the system further comprises software for performing microscopy and / or for image analysis. In some embodiments, the system further comprises a ligase. In some embodiments, the system further comprises a polymerase. In some embodiments, the system further comprises amine-modified nucleotides. In some embodiments, the system further comprises reagents for making a polymeric matrix (e.g., a polyacrylamide matrix). The cell in the systems of the present disclosure may be of any of the cell types disclosed herein. In some embodiments, the system comprises multiple cells. In some embodiments, the cells are of different cell types. In certain embodiments, the cells are present in a tissue. In some embodiments, the tissue is a tissue sample provided by or from a subject. In certain embodiments, the subject is a human.Assembled ribosome complexes

[0128] In one aspect, the present disclosure provides assembled ribosome complexes. In some embodiments, such assembled ribosome complexes are not naturally-occurring. In some embodiments, an assembled ribosome complex provided herein comprises: (a) a first oligonucleotide probe, a second oligonucleotide probe, and a third oligonucleotide probe, wherein (i) the first probe comprises an oligonucleotide portion that recognizes the 40S subunit of a ribosome and an oligonucleotide portion that is complementary to a portion of the third probe; (ii) the second probe comprises an oligonucleotide portion that recognizes the 60S subunit of a ribosome and an oligonucleotide portion that is complementary to a portion of the third probe; and (iii) the third probe comprises an oligonucleotide portion that is complementary to a portion of the first probe and an oligonucleotide portion that is complementary to a portion of the second probe; (b) a 40S subunit of a ribosome; and (c) a 60S subunit of a ribosome.

[0129] In some embodiments, the assembled ribosome complex further comprises a target RNA of interest.594934-6856-6158.1Atty. Docket No. 114203-1551

[0130] In some embodiments, the assembled ribosome complex further comprises a fourth probe, wherein the fourth probe comprises a first oligonucleotide barcode sequence, an oligonucleotide portion that is complementary to a portion of an RNA of interest, a second oligonucleotide barcode sequence, and an oligonucleotide portion that is complementary to a portion of the third probe.

[0131] In some embodiments, the assembled ribosome complex further comprises a fifth probe, wherein the fifth probe comprises an oligonucleotide portion that is complementary to the RNA of interest, an oligonucleotide portion that is complementary to a portion of the fourth probe, and an oligonucleotide barcode sequence. In some embodiments, the portion of the first probe that recognizes the 40S subunit of a ribosome comprises an oligonucleotide that is complementary to a portion of rRNA within the 40S subunit of a ribosome. In some embodiments, the portion of the first probe that recognizes the 40S subunit of a ribosome comprises an oligonucleotide that is complementary to a portion of the 18s ribosomal RNA (rRNA). In some embodiments, the oligonucleotide that is complementary to a portion of the 18s rRNA is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more than 30 nucleotides in length. In some embodiments, the oligonucleotide that is complementary to a portion of the 18s rRNA is about 25 nucleotides in length. In some embodiments, the portion of the second probe that recognizes the 60S subunit of a ribosome comprises an oligonucleotide that is complementary to a portion of rRNA within the 60S subunit of a ribosome. In some embodiments, the portion of the second probe that recognizes the 60S subunit of a ribosome comprises an oligonucleotide that is complementary to a portion of the 28s ribosomal RNA (rRNA). In some embodiments, the oligonucleotide that is complementary to a portion of the 28s rRNA is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more than 30 nucleotides in length. In some embodiments, the oligonucleotide that is complementary to a portion of the 28s rRNA is about 25 nucleotides in length.

[0132] In some embodiments, the oligonucleotide barcode sequences are 3-13, 4-12, 5-11, 6-10, 7-9 nucleotides in length. In some embodiments, the oligonucleotide barcode sequences are 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 nucleotides in length. In some embodiments, the fourth and fifth probes are complementary to different portions of the RNA of interest. In some embodiments, the portion of the third probe that is complementary to a portion of the first probe is 9-15 nucleotides in length. In some embodiments, the portion of the third probe that is complementary to a portion of the second probe is 9-15 nucleotides in length. In some embodiments, the portion of the third probe that is complementary to a portion of the first 604934-6856-6158.1Atty. Docket No. 114203-1551probe is 9 nucleotides in length, and wherein the portion of the third probe that is complementary to a portion of the second probe is 11 nucleotides in length.

[0133] In some embodiments, the portion of the fourth probe that is complementary to a portion of the third probe is 4-20, 5-19, 6-18, 7-17, 8-16, 9-15, 10-14, or 11-13 nucleotides in length. In some embodiments, the portion of the fourth probe that is complementary to a portion of the third probe is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length. In some embodiments, the portion of the fourth probe that is complementary to a portion of the third probe is split between the 5' end and the 3' end of the fourth probe. In some embodiments, the portion of the fourth probe that is complementary to a portion of the fifth probe is split between the 5' end and the 3' end of the second probe. In some embodiments, the portion of the fourth probe that is complementary to a portion of the fifth probe is 5-15, 6-14, 7-13, 8-12, or 9-11 nucleotides in length. In some embodiments, the portion of the fourth probe that is complementary to a portion of the fifth probe is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides in length. In some embodiments, the portion of the fourth probe that is complementary to the RNA of interest is 10-30, 11-29, 12-28, 13-27, 14-26, 15-25, 16-24, 17-23, 18-22, or 19-21 nucleotides in length. In some embodiments, the portion of the fourth probe that is complementary to the RNA of interest is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

[0134] In some embodiments, the third probe further comprises a polymerization blocker. In some embodiments, the polymerization blocker is located at the 3' end of second probe. In some embodiments, the polymerization blocker comprises an inverted nucleic acid residue. In some embodiments, the inverted nucleic acid residue is an inverted thymine residue.

[0135] In some embodiments, the portion of the first probe that recognizes the 40S subunit of a ribosome and the portion of the first probe that is complementary to a portion of the third probe are joined by a poly-A nucleotide linker. In some embodiments, the poly- A nucleotide linker is 2-30, 3-25, 4-20, 5-15, or 8-12 nucleotides in length. In some embodiments, the poly-A nucleotide linker is about 10 nucleotides in length. In some embodiments, the portion of the second probe that recognizes the 60S subunit of a ribosome and the portion of the second probe that is complementary to a portion of the third probe are joined by a poly-A nucleotide linker. In some embodiments, the poly-A nucleotide linker is 2-30, 3-25, 4-20, 5-15, or 8-12 nucleotides in length. In some embodiments, the poly-A nucleotide linker is about 10 nucleotides in length.

[0136] In some embodiments, the portion of the fifth probe that is complementary to the RNA of interest is 10-30, 11-29, 12-28, 13-27, 14-26, 15-25, 16-24, 17-23, 18-22, or 19-21614934-6856-6158.1Atty. Docket No. 114203-1551nucleotides in length. In some embodiments, the portion of the fifth probe that is complementary to the RNA of interest is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some embodiments, the portion of the fifth probe that is complementary to a portion of the fourth probe is 5-15, 6-14, 7-13, 8-12, or 9-11 nucleotides in length. In some embodiments, the portion of the fifth probe that is complementary to a portion of the fourth probe is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides in length. In some embodiments, the portion of the fifth probe that is complementary to a portion of the fourth probe is 5-15, 6-14, 7-13, 8-12, or 9-11 nucleotides in length.

[0137] In some embodiments, the fourth probe comprises the structure:5 '-[portion complementary to third probe]-[portion complementary to portion of fifth probe]-[portion complementary to RNA of interest]-[second oligonucleotide barcode sequence]-[portion complementary to third probe]-3', wherein the first oligonucleotide barcode sequence is within portion complementary to portion of fifth probe.

[0138] In some embodiments, the fourth probe comprises the structure:5 '-[portion complementary to portion of fifth probe]-[portion complementary to RNA of interest] -[second oligonucleotide barcode sequence]-3', wherein the first oligonucleotide barcode sequence is within portion complementary to portion of fifth probe.

[0139] In some embodiments, the first probe comprises the structure: 5 '-[portion recognizing the 40S subunit of the ribosome]-[poly-A linker]-[portion complementary to the third probe]-3'.

[0140] In some embodiments, the second probe comprises the structure: 5 '-[portion complementary to the third probe]-[poly-A linker]-[portion recognizing the 60S subunit of the ribosome]-3'.

[0141] In some embodiments, the fifth probe comprises the structure: 5 '-[portion complementary to RNA of interest]-[portion complementary to fourth probe]-3', wherein the oligonucleotide barcode sequence is within the portion complementary to fourth probe.

[0142] In some embodiments, the third probe comprises the structure: 5'-[portion complementary to portion of second probe]-[portion complementary to portion of first probe]-3'.

[0143] In some embodiments, the third probe comprises the structure: 5'-[portion complementary to portion of second probe]-[portion complementary to portion of first probe]-[poly-A linker]-[portion complementary to portion of fourth probe]-3'.624934-6856-6158.1Atty. Docket No. 114203-1551

[0144] In some embodiments, the third probe comprises the structure: 5'-[portion complementary to portion of first probe]-[portion complementary to portion of second probe]-3'. In some embodiments, the third probe comprises the structure: 5'-[portion complementary to portion of first probe]-[portion complementary to portion of second probe]-[poly-A linker]-[portion complementary to portion of fourth probe]-3'.

[0145] In some embodiments, one or more of the first probe, the second probe, and the third probe comprises one or more deoxyuridine (dU) nucleotide. In some embodiments, one or more of the first probe, the second probe, and the third probe comprises dU nucleotides and does not comprise dT nucleotides. In some embodiments, the first probe and the second probe each comprise dU nucleotides in the oligonucleotide portion that is complementary to a portion of the third probe. In some embodiments, the third probe comprises dU nucleotides in the oligonucleotide portion that is complementary to a portion of the first probe and the oligonucleotide portion that is complementary to a portion of the second probe. In some embodiments, the first probe, the second probe, and the third probe each comprise dT nucleotides outside of the complementary portions. In some embodiments, the assembled ribosome complex further comprises an adapter probe comprising a portion that is capable of hybridizing to the fifth probe. In some embodiments, the fifth probe comprises an adapter sequence capable of binding to the adapter probe. In some embodiments, the adapter sequence is about 20 nucleotides in length. In some embodiments, the adapter sequence is separated from an RNA-complementary region of the fifth probe by an adenosine linker. In some embodiments, the adenosine linker comprises about 5 adenosine nucleotides. In some embodiments, the adapter probe comprises one or more acryl functional groups. In some embodiments, the third probe comprises one or more acryl functional groups. In some embodiments, the fifth probe comprises a photocrosslinking moiety. In some embodiments, the adapter probe comprises an acryl functional group on its 5’-end or 3’-end, wherein The assembled ribosome complex further comprises incubating the cell with methyl-acrylate NHS ester (MA-NHS) under conditions sufficient to permit the conversion of a remaining terminal amine group of the adapter probe to an acryl functional group, thereby generating an adapter probe comprising a 5’ acryl functional group and a 3’ acryl functional group.EXAMPLES

[0146] These examples are provided for illustrative purposes only and not to limit the scope of the claims provided herein.634934-6856-6158.1Atty. Docket No. 114203-1551Example 1: Simultaneous transcriptome and translatome profiling in single cells in situ

[0147] Translation regulation of mRNA is a critical step in cellular physiology. To respond to environmental stresses and maintain protein homeostasis, the fraction of translating mRNA relative to total mRNA is tightly controlled. Different cell types modulate translation efficiency (TE) differently, adding another layer of complexity to gene expression regulation (Schwanhausser et al. 2011; Hornstein et al. 2016; Gao, Gallardo-Dodd, and Kutter 2022). TE is commonly dysregulated in neurodegenerative diseases and aging conditions (Stein et al. 2022; Lehmkuhl and Zarnescu 2018). Therefore, profiling single-cell resolved translation efficiency is crucial to understanding cell-type or cell-state specific gene regulation and dysregulation. Previously, a series of ribosome profiling methods were developed to profile translation at bulk-cell and single-cell levels (Ingolia et al. 2009; Vanlnsberghe et al. 2021; Xiong et al. 2022; Ozadam et al. 2023). However, these methods require the dissociation of tissue in sample preparation, thus losing the information on the subcellular location of the mRNA as well as the location of the cells. Moreover, solid tissues such as the brain could not be dissociated into high-quality single cells. Recently, Applicant developed RIBOmap, an in situ technology that profiles single-cell and spatially resolved translatomics at subcellular level (Zeng et al. 2023). However, RIBOmap sometimes shows limited specificity, as it only relies on proximity ligation of padlock probes on mRNA near the 18s rRNA, which are not necessarily translating mRNA (hereinafter, RIBOmap using only the 18s rRNA splint probe is denoted as 40S RIBOmap, and RIBOmap using only the 28s rRNA splint probe is denoted as 60S RIBOmap). Moreover, because the translatome and transcriptome cannot be profiled in the same sample in RIBOmap, the single-cell information on TE is missing. Previous analysis on single-cell TE was based on the computational integration of two adjacent tissue slices, one of them profiled by RIBOmap, the other one profiled by STARmap (Tang et al.2024). However, computational integration can result in inaccurate quantification, especially on highly heterogeneous samples where alignment is suboptimal, such as brain slices, in vivo perturbed tissues ( in et al. 2020), and thick tissue blocks (Sui et al. 2024). Critical information about the interplay between translation and transcription cannot be accurately measured. Therefore, a new method that can experimentally co-profile the transcriptome, translatome, and other modalities on the same sample is needed (FIG. 1A).

[0148] Here, Applicant describes 80S qRIBOmap, which achieves highly specific and high-throughput co-mapping of gene expression level and RNA translation status in situ simultaneously at single-cell and subcellular resolution for thousands of genes. The methods address these two issues by integrating transcriptome and translatome profiling in one sample 644934-6856-6158.1Atty. Docket No. 114203-1551and utilizing an additional AND gate to ensure the detection of RNAs associated with intact fully assembled 80S ribosomes instead of individual ribosomal subunits (the 40S or 60S subunits) (FIG. 2). Moreover, the method easily enables the addition of other mapping modalities into the workflow as it relies on padlock probe competition from different modalities. Applicant envisions that the method can enable a wide variety of applications that elucidate RNA fate regulation at single-cell and subcellular level (FIG. IB).

[0149] In 80S qRIBOmap, each gene can be targeted by 3-6 pairs of primer and padlock probes (FIG. 2A). During hybridization, the RIBO padlock and STAR padlock can compete with each other and result in about half of the targeted locations occupied by RIBO probes and about half of the targeted locations occupied by STAR probes. To incorporate more modalities of RNA, padlock probes for profiling other modalities can also be added together to compete with RIBO and STAR padlock probes. 80S (fully-assembled) ribosomes can be simultaneously targeted by 18s arm probes and 28s arm probes, which hybridize to 18s rRNA in the 40S subunit and 28s rRNA hybridized to the 60S subunit, respectively. Fully assembled, actively translating 80S ribosomes will form a complete 20 nt bridge between the two arm probes and recruit the 80S splint probe. Dissociated, inactive ribosomes will only have either an 18s arm or a 28s arm, either of which by itself cannot stably recruit the 80S splint probe, as each side of the landing region of 80S probe is only 9-11 nucleotides long. This relatively short partial hybridization region for each individual subunit is not stable during subsequent washing steps, and any partial individual subunit probe binding will be removed. After hybridization and stringent washing, ligation can be performed by Salt-T4 DNA ligase in a high-salt condition to stabilize the ternary complex of 18s arm, 28s arm, and 80S splint probe. Notably, the 5’ phosphorylated 28s arm probe and 18s arm probe can be ligated, further increasing the stability of the 80S splint probe at the location. RIBO padlocks in proximity to fully assembled ribosomes can be ligated and circularized, mediated by the 80S splint probe. STAR padlocks will also be ligated and circularized if paired with the corresponding primers. Afterward, rolling circle amplification (RCA) can be performed to generate RIBO amplicons and STAR amplicons, and subsequent in situ sequencing steps can be performed, similarly to previous methods (Zeng et al. 2023). RIBO reads and STAR reads can be profiled in the same single cells. The ratio between RIBO reads and STAR reads reflects the TE of the RNA of interest.

[0150] To validate 80S qRIBOmap, Applicant visualized the active translation of ACTB, a housekeeping gene in HeLa cells. Applicant observed strong signals of ACTB in both RIBO and STAR channels (FIG. 3A). Under partial hybridization conditions (i.e. when only 18s 654934-6856-6158.1Atty. Docket No. 114203-1551arms or 28s arms are present), the decrease in RIBO reads was substantial and very few signals were left (FIG. 3B-C), confirming that 80S RIBOmap signals were generated only when both 40S and 60S subunits were associated with the RNA of interest. In addition, MALAT and vtRNAl-1, two different non-translating RNAs, generated very few RIBO reads in 80S qRIBOmap (FIG. 4), indicating that Applicant’s approach selectively detects translating RNAs but not non-coding RNAs.

[0151] The improved specificity on translating RNAs of 80S qRIBOmap, compared to 40S qRIBOmap and 60S qRIBOmap (co-profiling of STARmap and 40S or 60S RIBOmap in one sample), was further validated using translational drug inhibitors. Previous methods have shown that the combination of 4ElRCat, a eukaryotic initiation factor inhibitor, and puromycin inhibits translation (Cencic et al. 2011). Furthermore, harringtonine translation inhibitors are described in literature (Fresno, limenez, and Vazquez 1977). Both of these treatments were tested on NIH 3T3 cells. There was a significant decrease in the 80S RIBOmap signals ACTB mRNAs (FIG. 5A-B), suggesting that the signals of 80S qRIBOmap indeed come from translating mRNAs. In comparison, 40S and 60S qRIBOmap showed a slightly increased TE after translation inhibitor treatment (FIG. 5C-D), which might be due to increased 40S and 60S ribosomal association with mRNA after drug treatment that does not reflect active translation (Bohlen et al. 2020).

[0152] Moreover, Applicant’s method is applicable not only to cell cultures, but also to intact tissue slices. The feasibility of 80S qRIBOmap on tissues was validated through a 2-gene test targeting ACTB and MALAT 1 RNAs in a mouse brain slice at the midbrain region (FIG. 6).ACTB RIBO reads, ACTB STAR reads, and MALAT1 STAR reads indicated good sensitivity of 80S qRIBOmap in tissues, whereas few MALAT1 RIBO reads corroborated good specificity of 80S qRIBOmap in tissues.

[0153] The scalability of 80S qRIBOmap was demonstrated in Applicant’s large-scale data collection targeting 127 genes of interest in Fluorescent, Ubiquitination-based Cell Cycle Indicator (FUCCI) HeLa cells (Sakaue-Sawano et al. 2008). The data show that 80S qRIBOmap can robustly generate strong signals when scaled up (FIG. 7).

[0154] In addition to the aforementioned scheme, there are several exemplary alternative probe designs for 80S qRIBOmap (FIG. 8). Optimization of sensitivity and specificity was achieved by changing the length of the hybridization regions between 18s / 28s arms and the 80S splint probe. In the best-performing scheme, 9-nt complementary region with 18s arms / 11-nt complementary region with 28s arms yielded optimal results (FIG. 3A and FIG.8A).664934-6856-6158.1Atty. Docket No. 114203-1551

[0155] The 80S qRIBOmap technique is not limited to transcriptome and translatome profiling. Because the design is based on the competition binding of padlock probes, the method is generalizable to co-profiling of transcriptome and other modalities such as the nascent transcriptome and the RBP-bound transcriptome. For instance, co-profiling of the nascent and total transcriptome can be achieved by integrating TEMPOmap (Ren et al. 2023) and STARmap. The result can be useful for RNA velocity analyses (La Manno et al. 2018).

[0156] As Applicant moved to larger scale data collection of 80S qRIBOmap on thousands of genes, Applicant encountered issues in which a large number of amplicons could cause optical crowding and compromise reads assignment, leading to high drop-out rate and data sparsity. To address this issue, Applicant developed a new approach to compact the size of amplicons during the RCA step, in order to mitigate the optical crowding issue and enable the identification of more high quality amplicons. Briefly, a low concentration of azide-PEG4-dUTP and DBCO-PEG4-dUTP was spiked-in during RCA, together with dNTPs and aminoallyl-dUTP that were present in the traditional RCA reaction mixture. Due to the moderate kinetics (k ~ 10'1M^s'1) of the strain-promoted alkyne-azide cycloaddition (SPAAC) reaction between the azide and DBCO moiety(Ning et al. 2008), they will not react when freely diffusing in solution, but will react efficiently when incorporated into the amplicon due to much higher local concentration of the two click moieties in the amplicon. This will effectively crosslink the amplicon during amplification and limit its final size (FIG.9A). To validate this, Applicant performed DNA-PAINT (Jungmann et al. 2014) experiments on amplicons generated under different conditions by hybridizing the amplicons with a detection probe with a DNA-PAINT handle and showed that the SPAAC reaction resulted in smaller-sized amplicons with higher uniformity (FIG. 9B). In contrast, other conditions, including traditional RCA conditions, resulted in amplicons that are more elongated and irregularly shaped. Applicant further tested the performance of amplicon compaction in a 21-gene STARmap experiment on HeLa cells that included mostly abundant housekeeping genes to simulate a large-scale data collection experiment. As expected, the SPAAC reaction resulted in sharper amplicons and enabled us to use a higher detector gain setting without introducing too much optical crowding (FIG. 9C). At a similar image signal intensity, Applicant was able to identify about 1.6-fold more good quality reads in the SPAAC reaction condition than the traditional condition (FIG. 9D-E).Example 2: Optimization of splint probe hybridization in 80S qRIBOmap

[0157] Applicant optimized the lengths and configuration of complementary regions between the arm probes by performing 80S qRIBOmap on ACTB in HeLa cells while testing full 674934-6856-6158.1Atty. Docket No. 114203-1551hybridization (full arms) and partial hybridization (18s only and 28s only conditions). As shown in FIG. 10A, a splint probe can be designed to hybridize to both an 18s arm probe and a 28s arm probe. FIGs. 10B and IOC show partial hybridization schemes in which the 80S probe hybridizes to the 18s arm probe, or the 28s arm probe, respectively. Finally, FIG. 10D shows an alternative configuration in which the 80S splint probe hybridizes to both an 18s arm probe and a 28s arm probe.

[0158] FIG. 10E shows a quantification of RIBO reads normalized to STAR reads. x_y_splint indicates that there is an x nt complementary region between the 18s arm and 80S splint probe, and a y nt complementary region between the 28s arm and 80S splint probe. Probe configurations follow the configuration shown in FIG. 10A, except for the11 11 splint, which uses the configuration shown in FIG. 10D. The 9 11 splint and 10 11 splint achieve minimal partial hybridization while retaining high sensitivity of detecting translation. Other probe designs showed low sensitivity and / or high partial hybridization. Example 3: Optimization of sensitivity using probe digestion techniques

[0159] For thin-tissue samples and cell cultures, Applicant observed that sometimes the signals are sparse even though the cells have high translation activity (FIG. 11 and 12D, dT arms dT splint panel). Applicant hypothesized that the rolling circle amplification (RCA) step is the bottleneck. During RCA, the cytoplasmic space is occupied by highly abundant rRNA-targeting arm probes and 80S splint probes. Applicant hypothesized that the Phi29 DNA polymerase activity may be scavenged by these excessive oligonucleotides near the circularized padlock probes, especially in polysome-rich places, due to the intrinsic nonspecific DNA-binding activity of Phi29. The arm and splint probes are useless after the ligation step, and may form primer-template-like complexes that trap Phi29 enzymes in an unproductive state, decreasing the pool of active Phi29 molecules that can be used in cDNA amplicon production. To circumvent this issue, Applicant introduced a dU scheme, in which the deoxythymidines of arm and splint probes are fully or partially substituted by deoxyuridines (the dT-to-dU-replacement can be on the arm-splint complementary region, can be on the arm-rRNA hybridization region, and can be also on the splint-padlock complementary region). (Probe sequences are listed in Tables 1-5) These dU-containing probes will be digested by the USER II enzyme (a mixture of Antarctic uracil DNA glycosylase and Endonuclease III) after the ligation step to clear up the space for the next RCA step without inhibiting the Phi29 activity (FIG. 12A).

[0160] Applicant first validated the dU scheme in HeLa cells (FIG. 12B-D). In 80S qRIBOmap experiments in HeLa cells targeting ACTB, Applicant observed that the dU 684934-6856-6158.1Atty. Docket No. 114203-1551scheme where both the arms and splint are dU-substituted achieve the highest sensitivity based on the number of RIBO and STAR reads (amplicons) observed in each cell. The dU scheme exhibits ~6 fold signal increase in RIBO modality, as well as ~2-3 fold signal increase in STAR modality. Potential DNA fragments produced by USER II digestion do not serve as the primer for RCA (FIG. 12B and 12D). Interestingly, the specificity of 80S qRIBOmap is also improved by the dU scheme, as shown by the lower RIBO signal in 18S (and 28S) partial hybridization in the dU scheme compared to the dT scheme (FIG. 12C). This could be explained by the lack of 5’ methyl group in dU compared to dT, contributing to lower base stacking energy and thus lower melting temperature of dU-containing arm-splint annealing region. Noncoding RNA such as MAI AT! showed minimal RIBO signal compared to coding RNA ACTB (FIG. 12C).

[0161] The benefits of the dU scheme can be generalized to other cell culture or thin tissue (up to 30 pm) samples. In 20 pm mouse brain samples, Applicant observed that albeit 80S qRIBOmap with conventional dT probes have slightly lower sensitivity than the traditional 40S qRIBOmap and 60S qRIBOmap, 80S qRIBOmap with the dU scheme achieved much higher sensitivity than those conditions (FIG. 12E). Applicant observed that the USER digestion step is indispensable. Applicant showed that the USER II enzyme is slightly more efficient than the USER I enzyme (while having minimal impact on translation efficiency quantification) (FIG. 12E-12F), potentially due to their differences in processing deoxyuridines near the end of a DNA. As expected, non-80S-translated mitochondrial RNA Mt-Col showed minimal RIBO signal than the 80S-translated cytoplasmic RNA Actb (FIG.12F)Example 4: Deep-tissue qRIBOmap

[0162] Since the sensitivity of 80S qRIBOmap has been substantially improved (higher than traditional 40S and 60S-targeting) despite more AND gates, Applicant reasoned that thicktissue 80S Deep-qRIBOmap is feasible, enabling volumetric profiling of transcriptional and translational activity while visualizing cellular morphology and cell-cell interactions that are better represented in thick tissue up to 200 pm thickness. Applicant first validated SOS-targeting RIBOmap in thick tissue (FIG. 13A-13B). Applicant found that while the previous method (40S-targeting) Deep-RIBOmap (Sui et al, 2025) showed some non-specificity as it has high RIBO signal for mitochondrial RNA Mt-Col (potentially because of hydrogel deformation and minor drifting of 40S-targeting splint probes during tissue clearing), SOS-targeting Deep-RIBOmap was able to completely solve this issue, showing minimal Mt-Col RIBO reads while maintaining similar levels of Actb RIBO reads, indicating that 80S- 694934-6856-6158.1Atty. Docket No. 114203-1551targeting is key to specificity improvement in thick tissues. Applicant then applied the same probe competition mechanism to achieve 80S Deep-qRIBOmap, enabling simultaneous readout of translatome and total transcriptome signal in thick tissue blocks, showing high sensitivity and specificity towards translated RNAs over non-80S-associated RNAs (GIH.13C-13D)Example 5: Multimodal spatial and temporal profiling of the transcriptome and translatome

[0163] As the sensitivity of this spatial multi-omic platform has increased, Applicant reasoned that more modalities could be incorporated onto the same platform, including the temporal transcriptome (Ren et al, 2023) that contains RNAs synthesized at different time points (FIG. 14A), and RNAs bound by RNA-binding proteins or modified with epitranscriptomic modifications (FIG. 14B). These modalities could be simultaneously profiled on the same sample by designing splint probe and padlock probes that contain orthogonal ligation handle sequences. Different padlock probes for different modalities compete for the same RNA targeting region, enabling highly integrated spatial multi-omics (FIG. 14C)704934-6856-6158.1Atty. Docket No. 114203-1551EXEMPLARY SEQUENCESTable 1. dU-modified 18S arm, 28S arm, and 80S splint probes714934-6856-6158.1Atty. Docket No. 114203-1551724934-6856-6158.1Atty. Docket No. 114203-1551*dTs in 18S arm and 28S arm probes are replaced with dUs except when the dTs are the 5’ or 3’ terminal base. For 80S splint probe, the dTs in arm probe complementary region are fully replaced with dUs. The dTs in the padlock complementary ligation handle region are fully replaced with dUs (named as dU splint) or kept as dTs (named as dU dT hybrid). The dU dT hybrid splints usually result in a bit higher RIBO:STAR reads ratio than the dU splint.734934-6856-6158.1Atty. Docket No. 114203-1551Table 2. Primer sequences using mouse gene Actb and Mt-Col as examples (thin tissue)744934-6856-6158.1Atty. Docket No. 114203-1551Table 3. Adapter and primer sequences using mouse gene Actb and Mt-Col as examples (thick tissue)754934-6856-6158.1Atty. Docket No. 114203-155176 4934-6856-6158.1Atty. Docket No. 114203-1551Table 4. STAR padlock sequences using mouse gene Actb and Mt-Col as examples (for both thin and thick tissue)774934-6856-6158.1Atty. Docket No. 114203-1551784934-6856-6158.1Atty. Docket No. 114203-1551Table 5. RIBO padlock sequences using mouse gene Actb and Mt-Col as examples (for both thin and thick tissue)794934-6856-6158.1Atty. Docket No. 114203-1551804934-6856-6158.1Atty. Docket No. 114203-1551

[0168] The present application refers to various issued patent, published patent applications, scientific journal articles, and other publications, all of which are incorporated herein by reference. The details of one or more embodiments of the invention are set forth herein. Other features, objects, and advantages of the invention will be apparent from the Detailed Description, the Figures, the Examples, and the Claims.

[0169] Furthermore, the disclosure encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claims that is dependent on the same base claim. Where elements are presented as lists, e.g., in Markush group format, each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should it be understood that, in general, where the invention, or aspects of the invention, is / are referred to as comprising particular elements and / or features, certain embodiments of the disclosure or aspects of the disclosure consist, or consist essentially of, such elements and / or features. For purposes of simplicity, those embodiments have not been specifically set forth in haec verba herein. It is also noted that the terms “comprising” and “containing” are intended to be open and permits the inclusion of additional elements or steps. Where ranges are given, endpoints are included. Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0170] This application refers to various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. If there is a conflict between any of the incorporated references and the instant specification, the specification shall control. In addition, any particular embodiment of the present invention that falls within the prior art may be explicitly excluded from any one or more of the embodiments. Because such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the invention can be excluded from any embodiment, for any reason, whether or not related to the existence of prior art.814934-6856-6158.1Atty. Docket No. 114203-1551

[0171] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments described herein. The scope of the present embodiments described herein is not intended to be limited to the above Description, but rather is as set forth in the appended embodiments. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the present invention, as defined in the following claims.824934-6856-6158.1

Claims

1. Atty. Docket No. 114203-1551CLAIMS WHAT IS CLAIMED IS:

1. A method for simultaneous transcriptome and translatome profiling in a cell, the method comprising:(a) contacting the cell with one or more sets of probes, wherein each set of probes comprises a first probe, a second probe, a third probe, a fourth probe, a fifth probe, and a sixth probe, wherein(i) the first probe comprises(1) a first oligonucleotide barcode sequence;(2) an oligonucleotide portion that is complementary to a portion of the fifth probe;(3) an oligonucleotide portion that is complementary to a portion of an RNA of interest;(4) a second oligonucleotide barcode sequence; and(5) an oligonucleotide portion that is complementary to a portion of the sixth probe;(ii) the second probe comprises(1) a first oligonucleotide barcode sequence;(2) an oligonucleotide portion that is complementary to a portion of the fifth probe;(3) an oligonucleotide portion that is complementary to a portion of an RNA of interest; and(4) a second oligonucleotide barcode sequence;(iii) the third probe comprises an oligonucleotide portion that recognizes the 40S subunit of a ribosome and an oligonucleotide portion that is complementary to a portion of the sixth probe;(iv) the fourth probe comprises an oligonucleotide portion that recognizes the 60S subunit of a ribosome and an oligonucleotide portion that is complementary to a portion of the sixth probe;(v) the fifth probe comprises(1) an oligonucleotide portion that is complementary to the RNA of interest;834934-6856-6158.1Atty. Docket No. 114203-1551(2) an oligonucleotide portion that is complementary to a portion of the first probe;(3) an oligonucleotide portion that is complementary to a portion of the second probe; and(4) an oligonucleotide barcode sequence,wherein the oligonucleotide barcode sequence of the fifth probe is complementary to the first oligonucleotide barcode sequence of the first probe and the first oligonucleotide barcode sequence of the second probe; and (vi) the sixth probe comprises(1) an oligonucleotide portion that is complementary to a portion of the first probe;(2) an oligonucleotide portion that is complementary to a portion of the third probe; and(3) an oligonucleotide portion that is complementary to a portion of the fourth probe;(b) ligating the 5' end and the 3' end of the first probe together to produce a first circular oligonucleotide, and ligating the 5' end and the 3' end of the second probe together to produce a second circular oligonucleotide;(c) performing rolling circle amplification to amplify the first circular oligonucleotide and the second circular oligonucleotide, wherein the fifth probe is a primer for the amplification of each of the first circular oligonucleotide and the second circular oligonucleotide, thereby producing one or more concatenated amplicons from the first circular oligonucleotide and one or more concatenated amplicons from the second circular oligonucleotide;(d) embedding the one or more concatenated amplicons from the first circular oligonucleotide and one or more concatenated amplicons from the second circular oligonucleotide in a polymeric matrix; and(e) sequencing the concatenated amplicons embedded in the polymeric matrix to determine the identity, location, and translation status of each RNA of interest in the cell.

2. The method of claim 1, wherein the oligonucleotide portion that is complementary to a portion of an RNA of interest of the first probe and the oligonucleotide portion that is complementary to a portion of an RNA of interest of the second probe are identical.844934-6856-6158.1Atty. Docket No. 114203-15513. The method of claims 1 or 2, wherein the second oligonucleotide barcode sequence of the first probe and the second oligonucleotide barcode sequence of the second probe are not identical.

4. The method of any one of claims 1-3, wherein the portion of the third probe that recognizes the 40S subunit of a ribosome comprises an antibody, or an antibody variant or fragment.

5. The method of claim 4, wherein the antibody is a secondary antibody.

6. The method of claim 5, further comprising contacting the cell with a primary antibody that recognizes the 40S subunit of a ribosome and can be bound by the secondary antibody of the third probe.

7. The method of claim 6, wherein the primary antibody is an anti-40S ribosomal protein S3 (RPS3) antibody.

8. The method of any one of claims 1-3, wherein the portion of the third probe that recognizes the 40S subunit of a ribosome comprises an oligonucleotide that is complementary to a portion of rRNA within the 40S subunit of a ribosome.

9. The method of any one of claims 1-3, wherein the portion of the third probe that recognizes the 40S subunit of a ribosome comprises an oligonucleotide that is complementary to a portion of the 18s ribosomal RNA (rRNA).

10. The method of claim 8 or 9, wherein the oligonucleotide that is complementary to a portion of the 18s rRNA is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more than 30 nucleotides in length.

11. The method of any one of claims 8-10, wherein the oligonucleotide that is complementary to a portion of the 18s rRNA is about 25 nucleotides in length.

12. The method of any one of claims 1-11, wherein the portion of the fourth probe that recognizes the 60S subunit of a ribosome comprises an antibody, or an antibody variant or fragment.

13. The method of claim 12, wherein the antibody is a secondary antibody.854934-6856-6158.1Atty. Docket No. 114203-155114. The method of claim 13, further comprising contacting the cell with a primary antibody that recognizes the 60S subunit of a ribosome and can be bound by the secondary antibody of the fourth probe.

15. The method of claim 14, wherein the primary antibody is an anti-60S ribosomal protein L4 (RPL4) antibody.

16. The method of any one of claims 1-11, wherein the portion of the fourth probe that recognizes the 60S subunit of a ribosome comprises an oligonucleotide that is complementary to a portion of rRNA within the 60S subunit of a ribosome.

17. The method of any one of claims 1-11, wherein the portion of the fourth probe that recognizes the 60S subunit of a ribosome comprises an oligonucleotide that is complementary to a portion of the 28s ribosomal RNA (rRNA).

18. The method of claim 16 or 17, wherein the oligonucleotide that is complementary to a portion of the 28s rRNA is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more than 30 nucleotides in length.

19. The method of any one of claims 16-18, wherein the oligonucleotide that is complementary to a portion of the 28s rRNA is about 25 nucleotides in length.

20. The method of any one of claims 1-19, wherein the oligonucleotide barcode sequences are 3-13, 4-12, 5-11, 6-10, 7-9 nucleotides in length.

21. The method of any one of claims 1-20, wherein the oligonucleotide barcode sequences are 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13, nucleotides in length.

22. The method of any one of claims 1-21, wherein the first and fifth probes are complementary to different portions of the RNA of interest.

23. The method of any one of claims 1-22, wherein the second and fifth probes are complementary to different portions of the RNA of interest.

24. The method of any one of claims 1-23, wherein the portion of the sixth probe that is complementary to a portion of the third probe is 9-15 nucleotides in length.864934-6856-6158.1Atty. Docket No. 114203-155125. The method of any one of claims 1-24, wherein the portion of the sixth probe that is complementary to a portion of the fourth probe is 9-15 nucleotides in length.

26. The method of any one of claims 1-25, wherein the portion of the sixth probe that is complementary to a portion of the third probe is 9 nucleotides in length, and wherein the portion of the sixth probe that is complementary to a portion of the fourth probe is 11 nucleotides in length.

27. The method of any one of claims 1-26, wherein the portion of the first probe that is complementary to a portion of the sixth probe is 4-20, 5-19, 6-18, 7-17, 8-16, 9-15, 10-14, or 11-13 nucleotides in length.

28. The method of any one of claims 1-27, wherein the portion of the first probe that is complementary to a portion of the sixth probe is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length.

29. The method of any one of claims 1-28, wherein the portion of the first probe that is complementary to a portion of the sixth probe is split between the 5' end and the 3' end of the first probe.

30. The method of any one of claims 1-29, wherein the portion of the second probe that is complementary to a portion of the fifth probe is split between the 5' end and the 3' end of the second probe.

31. The method of any one of claims 1-30, wherein the portion of the first probe that is complementary to a portion of the fifth probe is 5-15, 6-14, 7-13, 8-12, or 9-11 nucleotides in length.

32. The method of any one of claims 1-31, wherein the portion of the first probe that is complementary to a portion of the fifth probe is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides in length.

33. The method of any one of claims 1-32, wherein the portion of the second probe that is complementary to a portion of the fifth probe is 5-15, 6-14, 7-13, 8-12, or 9-11 nucleotides in length.874934-6856-6158.1Atty. Docket No. 114203-155134. The method of any one of claims 1-33, wherein the portion of the second probe that is complementary to a portion of the fifth probe is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides in length.

35. The method of any one of claims 1-34, wherein the portion of the first probe that is complementary to the RNA of interest is 10-30, 11-29, 12-28, 13-27, 14-26, 15-25, 16-24, 17-23, 18-22, or 19-21 nucleotides in length.

36. The method any one of claims 1-35, wherein the portion of the first probe that is complementary to the RNA of interest is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

37. The method of any one of claims 1-36, wherein the portion of the second probe that is complementary to the RNA of interest is 10-30, 11-29, 12-28, 13-27, 14-26, 15-25, 16-24, 17-23, 18-22, or 19-21 nucleotides in length.

38. The method any one of claims 1-37, wherein the portion of the second probe that is complementary to the RNA of interest is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

39. The method of any one of claims 1-38, wherein the sixth probe further comprises a polymerization blocker.

40. The method of claim 39, wherein the polymerization blocker is located at the 3' end of second probe.

41. The method of claim 39 or 40, wherein the polymerization blocker comprises an inverted nucleic acid residue.

42. The method of claim 41, wherein the inverted nucleic acid residue is an inverted thymine residue.

43. The method of any one of claims 1-42, wherein the portion of the third probe that recognizes the 40S subunit of a ribosome and the portion of the third probe that is complementary to a portion of the sixth probe are joined by a poly-A nucleotide linker.884934-6856-6158.1Atty. Docket No. 114203-155144. The method of claim 43, wherein the poly-A nucleotide linker is 2-30, 3-25, 4-20, 5- 15, or 8-12 nucleotides in length.

45. The method of claim 43 or 44, wherein the poly-A nucleotide linker is about 10 nucleotides in length.

46. The method of any one of claims 1-45, wherein the portion of the fourth probe that recognizes the 60S subunit of a ribosome and the portion of the fourth probe that is complementary to a portion of the sixth probe are joined by a poly-A nucleotide linker.

47. The method of claim 46, wherein the poly-A nucleotide linker is 2-30, 3-25, 4-20, 5- 15, or 8-12 nucleotides in length.

48. The method of claim 46 or 47, wherein the poly-A nucleotide linker is about 10 nucleotides in length.

49. The method of any one of claims 1-48, wherein the portion of the fifth probe that is complementary to the RNA of interest is 10-30, 11-29, 12-28, 13-27, 14-26, 15-25, 16-24, 17-23, 18-22, or 19-21 nucleotides in length.

50. The method of any one of claims 1-49, wherein the portion of the fifth probe that is complementary to the RNA of interest is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

51. The method of any one of claims 1-50, wherein the portion of the fifth probe that is complementary to a portion of the first probe is 5-15, 6-14, 7-13, 8-12, or 9-11 nucleotides in length.

52. The method of any one of claims 1-51, wherein the portion of the fifth probe that is complementary to a portion of the first probe is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides in length.

53. The method of any one of claims 1-52, wherein the portion of the fifth probe that is complementary to a portion of the second probe is 5-15, 6-14, 7-13, 8-12, or 9-11 nucleotides in length.894934-6856-6158.1Atty. Docket No. 114203-155154. The method of any one of claims 1-53, wherein the portion of the fifth probe that is complementary to a portion of the second probe is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides in length.

55. The method of any one of claims 1-54, wherein the first probe comprises the structure:5 '-[portion complementary to sixth probe]-[portion complementary to portion of fifth probe]-[portion complementary to RNA of interest]-[second oligonucleotide barcode sequence]-[portion complementary to sixth probe]-3', wherein the first oligonucleotide barcode sequence is within the portion complementary to portion of fifth probe.

56. The method of any one of claims 1-55, wherein the second probe comprises the structure:5 '-[portion complementary to portion of fifth probe]-[portion complementary to RNA of interest] -[second oligonucleotide barcode sequence]-3', wherein the first oligonucleotide barcode sequence is within portion complementary to portion of fifth probe.

57. The method of any one of claims 1-56, wherein the third probe comprises the structure: 5'-[portion recognizing the 40S subunit of the ribosome]-[poly-A linker]- [portion complementary to the sixth probe]-3'.

58. The method of any one of claims 1-57, wherein the fourth probe comprises the structure: 5'-[portion complementary to the sixth probe]-[poly-A linker]-[portion recognizing the 60S subunit of the ribosome]-3'.

59. The method of any one of claims 1-58, wherein the fifth probe comprises the structure:5'-[portion complementary to RNA of interest]-[portion complementary to first probe or second probe]-3', wherein the oligonucleotide barcode sequence is within the portion complementary to first probe or second probe.

60. The method of any one of claims 1-59, wherein the sixth probe comprises the structure:5 '-[portion complementary to portion of fourth probe]-[portion complementary to904934-6856-6158.1Atty. Docket No. 114203-1551portion of third probe]-[poly-A linker]-[portion complementary to portion of first probe]-3'.

61. The method of any one of claims 1-59, wherein the sixth probe comprises the structure:5 '-[portion complementary to portion of third probe]-[portion complementary to portion of fourth probe]-[poly-A linker]-[portion complementary to portion of first probe]-3'.

62. The method of any one of claims 1-61, wherein RNAs are profiled in multiple cells simultaneously.

63. The method of claim 62, wherein RNAs are profiled in more than 100 cells, more than 200 cells, more than 300 cells, more than 400 cells, more than 500 cells, more than 1000 cells, more than 10,000 cells, more than 20,000 cells, more than 30,000 cells, more than 40,000 cells, or more than 50,000 cells simultaneously.

64. The method of claim 62 or 63, wherein the cells comprise a plurality of cell types.

65. The method of claim 64, wherein the cell types are selected from the group consisting of stem cells, progenitor cells, neuronal cells, astrocytes, dendritic cells, endothelial cells, microglia, oligodendrocytes, muscle cells, myocardial cells, mesenchymal cells, epithelial cells, immune cells, hepatic cells, smooth and skeletal muscle cells, hematopoietic cells, lymphocytes, monocytes, neutrophils, macrophages, natural killer cells, mast cells, adipocytes, and neurons.

66. The method of any one of claims 1-65, wherein the cell is present within an intact tissue.

67. The method of claim 66, wherein the intact tissue is a fixed tissue sample.

68. The method of claim 66 or 67, wherein the tissue is brain tissue.

69. The method of any one of claims 1-68, wherein translation of more than 1, more than 2, more than 3, more than 4, more than 5, more than 10, more than 20, more than 30, more than 40, more than 50, more than 100, more than 200, more than 500, more than914934-6856-6158.1Atty. Docket No. 114203-15511000, more than 2000, 3000 RNAs, more than 4000 RNAs, or more than 5000 RNAs are profiled simultaneously.

70. The method of any one of claims 1-69, wherein the first oligonucleotide barcode sequence of the first probe is a gene specific sequence used to identify an RNA of interest.

71. The method of any one of claims 1-70, wherein the first oligonucleotide barcode sequence of the second probe is a gene specific sequence used to identify an RNA of interest.

72. The method of any one of claims 1-71, wherein the second oligonucleotide barcode sequence of the first probe is a sequence used to identify the RNA of interest as a ribosome-associated actively translating RNA.

73. The method of any one of claims 1-72, wherein the second oligonucleotide barcode sequence of the second probe is a sequence used to identify the RNA of interest as likely to be a non-ribosome-associated RNA.

74. The method of any one of claims 1-73, wherein the step of sequencing comprises performing sequencing with error-reduction by dynamic annealing and ligation (SEDAL).

75. The method of claim 74, wherein SEDAL is performed, two, three, four, five, or more than five times.

76. The method of any one of claims 1-75, wherein the polymeric matrix is a hydrogel.

77. The method of claim 76, wherein the hydrogel is a polyvinyl alcohol hydrogel, a polyethylene glycol hydrogel, a sodium polyacrylate hydrogel, an acrylate hydrogel, or a polyacrylamide hydrogel.

78. The method of any one of claims 1-77, wherein the step of performing rolling circle amplification further comprises providing amine-modified nucleotides, wherein the amine-modified nucleotides are incorporated into the one or more concatenated amplicons.924934-6856-6158.1Atty. Docket No. 114203-155179. The method of claim 78, wherein the step of embedding the one or more concatenated amplicons in the polymeric matrix comprises reacting the amine-modified nucleotides of the one or more amplicons with acrylic acid N-hydroxysuccinimide ester and copolymerizing the one or more concatenated amplicons and the polymeric matrix.

80. The method of any one of claims 1-79, further comprising profiling additional molecules within the cell.

81. The method of claim 80, wherein the additional molecules are non-translating RNAs, RNAs in other subcellular locations, proteins, lipids, or small molecules.

82. The method of any one of claims 1-81, further comprising determining the cell type of the profiled cell, or the cell types of multiple profiled cells, by comparing the RNA translation profile of the cell or cells to reference data comprising RNA translation profiles of cells of various cell types.

83. The method of any one of claims 1-82, further comprising determining the cell state of the profiled cell, or the cell states of multiple profiled cells, by comparing the RNA translation profile of the cell or cells to reference data comprising RNA translation profiles of cells of various cell states.

84. The method of any one of claims 1-83, further comprising overexpressing or knocking out one or more genes in the cell to determine whether the one or more genes are involved in regulating the translation of the RNA of interest.

85. The method of any one of claims 1-84, wherein the method does not disrupt spatial information of subcellular structure, cell morphology, and / or tissue organization.

86. A method for diagnosing a disease or disorder in a subject, the method comprising:(a) contacting a cell obtained from the subject with one or more sets of probes, wherein each set of probes comprises a first probe, a second probe, a third probe, a fourth probe, a fifth probe, and a sixth probe, wherein(i) the first probe comprises(1) a first oligonucleotide barcode sequence;934934-6856-6158.1Atty. Docket No. 114203-1551(2) an oligonucleotide portion that is complementary to a portion of the fifth probe;(3) an oligonucleotide portion that is complementary to a portion of an RNA of interest;(4) a second oligonucleotide barcode sequence; and(5) an oligonucleotide portion that is complementary to a portion of the sixth probe;(ii) the second probe comprises(1) a first oligonucleotide barcode sequence;(2) an oligonucleotide portion that is complementary to a portion of the fifth probe;(3) an oligonucleotide portion that is complementary to a portion of an RNA of interest; and(4) a second oligonucleotide barcode sequence;(iii) the third probe comprises a portion that recognizes the 40S subunit of a ribosome and an oligonucleotide portion that is complementary to a portion of the sixth probe;(iv) the fourth probe comprises a portion that recognizes the 60S subunit of a ribosome and an oligonucleotide portion that is complementary to a portion of the sixth probe;(v) the fifth probe comprises(1) an oligonucleotide portion that is complementary to the RNA of interest;(2) an oligonucleotide portion that is complementary to a portion of the first probe;(3) an oligonucleotide portion that is complementary to a portion of the second probe; and(4) an oligonucleotide barcode sequence,wherein the oligonucleotide barcode sequence of the fifth probe is complementary to the first oligonucleotide barcode sequence of the first probe and the first oligonucleotide barcode sequence of the second probe; and (vi) the sixth probe comprises(1) an oligonucleotide portion that is complementary to a portion of the first probe;944934-6856-6158.1Atty. Docket No. 114203-1551(2) an oligonucleotide portion that is complementary to a portion of the third probe; and(3) an oligonucleotide portion that is complementary to a portion of the fourth probe;(b) ligating the 5' end and the 3' end of the first probe together to produce a first circular oligonucleotide, and ligating the 5' end and the 3' end of the second probe together to produce a second circular oligonucleotide;(c) performing rolling circle amplification to amplify the first circular oligonucleotide and the second circular oligonucleotide, wherein the fifth probe is a primer for the amplification of each of the first circular oligonucleotide and the second circular oligonucleotide, thereby producing one or more concatenated amplicons from the first circular oligonucleotide and one or more concatenated amplicons from the second circular oligonucleotide;(d) embedding the one or more concatenated amplicons from the first circular oligonucleotide and one or more concatenated amplicons from the second circular oligonucleotide in a polymeric matrix; and(e) sequencing the concatenated amplicons embedded in the polymeric matrix to determine the identity, location, and translation status of each RNA of interest in the cell,wherein a difference in the RNA translation profile of the cell relative to one or more non-diseased cells indicates that the subject has the disease or disorder.

87. The method of claim 86, wherein RNAs in one or more non-diseased cells are profiled as a control experiment alongside the cell obtained from the subject.

88. The method of claim 86, wherein the profile of RNAs being in one or more nondiseased cells comprises reference data.

89. The method of any one of claims 86-88, wherein the disease or disorder is a genetic disease, a proliferative disease, an inflammatory disease, an autoimmune disease, a liver disease, a spleen disease, a lung disease, a hematological disease, a neurological disease, a psychiatric disease, a gastrointestinal (GI) tract disease, a genitourinary disease, an infectious disease, a musculoskeletal disease, an endocrine disease, a metabolic disorder, an immune disorder, a central nervous system (CNS) disorder, or a cardiovascular disease.954934-6856-6158.1Atty. Docket No. 114203-155190. The method of any one of claims 86-89, wherein the cell is present in a tissue.

91. The method of claim 90, wherein the tissue is epithelial tissue, connective tissue, muscular tissue, or nervous tissue.

92. The method of claim 90 or 91, wherein the tissue is brain tissue.

93. The method of any one of claims 86-92, wherein the tissue is a tissue sample taken from a subject.

94. The method of claim 93, wherein the subject is a non-human experimental animal.

95. The method of claim 94, wherein the non-human experimental animal is a mouse, a rat, a dog, a pig, or a non-human primate.

96. The method of claim 93, wherein the subject is a human.

97. A method for screening for an agent capable of modulating translation of one or more RNAs, the method comprising:(a) contacting a cell that is being treated with or has been treated with a candidate agent with one or more sets of probes, wherein each set of probes comprises a first probe, a second probe, a third probe, a fourth probe, a fifth probe, and a sixth probe, wherein(i) the first probe comprises(1) a first oligonucleotide barcode sequence;(2) an oligonucleotide portion that is complementary to a portion of the fifth probe;(3) an oligonucleotide portion that is complementary to a portion of an RNA of interest;(4) a second oligonucleotide barcode sequence; and(5) an oligonucleotide portion that is complementary to a portion of the sixth probe;(ii) the second probe comprises(1) a first oligonucleotide barcode sequence;(2) an oligonucleotide portion that is complementary to a portion of the fifth probe;964934-6856-6158.1Atty. Docket No. 114203-1551(3) an oligonucleotide portion that is complementary to a portion of an RNA of interest; and(4) a second oligonucleotide barcode sequence;(iii) the third probe comprises a portion that recognizes the 40S subunit of a ribosome and an oligonucleotide portion that is complementary to a portion of the sixth probe;(iv) the fourth probe comprises a portion that recognizes the 60S subunit of a ribosome and an oligonucleotide portion that is complementary to a portion of the sixth probe;(v) the fifth probe comprises(1) an oligonucleotide portion that is complementary to the RNA of interest;(2) an oligonucleotide portion that is complementary to a portion of the first probe;(3) an oligonucleotide portion that is complementary to a portion of the second probe; and(4) an oligonucleotide barcode sequence,wherein the oligonucleotide barcode sequence of the fifth probe is complementary to the first oligonucleotide barcode sequence of the first probe and the first oligonucleotide barcode sequence of the second probe; and (vi) the sixth probe comprises(1) an oligonucleotide portion that is complementary to a portion of the first probe;(2) an oligonucleotide portion that is complementary to a portion of the third probe; and(3) an oligonucleotide portion that is complementary to a portion of the fourth probe;(b) ligating the 5' end and the 3' end of the first probe together to produce a first circular oligonucleotide, and ligating the 5' end and the 3' end of the second probe together to produce a second circular oligonucleotide;(c) performing rolling circle amplification to amplify the first circular oligonucleotide and the second circular oligonucleotide, wherein the fifth probe is a primer for the amplification of each of the first circular oligonucleotide and the second circular oligonucleotide, thereby producing one or more concatenated amplicons from the first 974934-6856-6158.1Atty. Docket No. 114203-1551circular oligonucleotide and one or more concatenated amplicons from the second circular oligonucleotide;(d) embedding the one or more concatenated amplicons from the first circular oligonucleotide and one or more concatenated amplicons from the second circular oligonucleotide in a polymeric matrix; and(e) sequencing the concatenated amplicons embedded in the polymeric matrix to determine the identity, location, and translation status of each RNA of interest in the cell, wherein a difference in the profile of RNAs being translated in the presence of the candidate agent relative to in the absence of the candidate agent indicates that the candidate agent modulates translation of one or more RNAs.

98. The method of claim 97, wherein the candidate agent is a small molecule, a protein, a peptide, a nucleic acid, a lipid, or a carbohydrate.

99. The method of any one of claims 97 or 98, where the candidate agent is a known drug or an FDA-approved drug.

100. The method of claim 98 or 99, wherein the protein is an antibody, or an antibody variant or fragment.

101. The method of claim 98 or 99, wherein the nucleic acid is an mRNA, an antisense RNA, a miRNA, an siRNA, an RNA aptamer, a double stranded RNA (dsRNA), a short hairpin RNA (shRNA), or an antisense oligonucleotide (ASO).

102. The method of any one of claims 97-101, wherein modulating RNA translation is associated with reducing, relieving, or eliminating the symptoms of a disease or disorder.

103. The method of claim 102, wherein the disease or disorder is a genetic disease, a proliferative disease, an inflammatory disease, an autoimmune disease, a liver disease, a spleen disease, a lung disease, a hematological disease, a neurological disease, a psychiatric disease, a gastrointestinal (GI) tract disease, a genitourinary disease, an infectious disease, a musculoskeletal disease, an endocrine disease, a metabolic disorder, an immune disorder, a central nervous system (CNS) disorder, or a cardiovascular disease.

104. A method for treating a disease or disorder in a subject, the method comprising:984934-6856-6158.1Atty. Docket No. 114203-1551(a) contacting a cell obtained from the subject with one or more sets of probes, wherein each set of probes comprises a first probe, a second probe, a third probe, a fourth probe, a fifth probe, and a sixth probe, wherein(i) the first probe comprises(1) a first oligonucleotide barcode sequence;(2) an oligonucleotide portion that is complementary to a portion of the fifth probe;(3) an oligonucleotide portion that is complementary to a portion of an RNA of interest;(4) a second oligonucleotide barcode sequence; and(5) an oligonucleotide portion that is complementary to a portion of the sixth probe;(ii) the second probe comprises(1) a first oligonucleotide barcode sequence;(2) an oligonucleotide portion that is complementary to a portion of the fifth probe;(3) an oligonucleotide portion that is complementary to a portion of an RNA of interest; and(4) a second oligonucleotide barcode sequence;(iii) the third probe comprises a portion that recognizes the 40S subunit of a ribosome and an oligonucleotide portion that is complementary to a portion of the sixth probe;(iv) the fourth probe comprises a portion that recognizes the 60S subunit of a ribosome and an oligonucleotide portion that is complementary to a portion of the sixth probe;(v) the fifth probe comprises(1) an oligonucleotide portion that is complementary to the RNA of interest;(2) an oligonucleotide portion that is complementary to a portion of the first probe;(3) an oligonucleotide portion that is complementary to a portion of the second probe; and(4) an oligonucleotide barcode sequence,994934-6856-6158.1Atty. Docket No. 114203-1551wherein the oligonucleotide barcode sequence of the fifth probe is complementary to the first oligonucleotide barcode sequence of the first probe and the first oligonucleotide barcode sequence of the second probe; and (vi) the sixth probe comprises(1) an oligonucleotide portion that is complementary to a portion of the first probe;(2) an oligonucleotide portion that is complementary to a portion of the third probe; and(3) an oligonucleotide portion that is complementary to a portion of the fourth probe;(b) ligating the 5' end and the 3' end of the first probe together to produce a first circular oligonucleotide, and ligating the 5' end and the 3' end of the second probe together to produce a second circular oligonucleotide;(c) performing rolling circle amplification to amplify the first circular oligonucleotide and the second circular oligonucleotide, wherein the fifth probe is a primer for the amplification of each of the first circular oligonucleotide and the second circular oligonucleotide, thereby producing one or more concatenated amplicons from the first circular oligonucleotide and one or more concatenated amplicons from the second circular oligonucleotide;(d) embedding the one or more concatenated amplicons from the first circular oligonucleotide and one or more concatenated amplicons from the second circular oligonucleotide in a polymeric matrix;(e) sequencing the concatenated amplicons embedded in the polymeric matrix to determine the identity, location, and translation status of each RNA of interest in the cell; and (f) administering a treatment for the disease or disorder to the subject if a difference in the profile of RNAs being translated in the cell relative to one or more non-diseased cells is observed.

105. The method of claim 104, wherein translation of one or more RNAs in one or more non-diseased cells is profiled simultaneously as a control experiment.

106. The method of claim 104 or 105, wherein the profile of translation of one or more RNAs in one or more non-diseased cells comprises reference data.1004934-6856-6158.1Atty. Docket No. 114203-1551107. The method of any one of claims 104-106, wherein the treatment comprises administering a therapeutic agent, surgery, or radiation therapy.

108. The method of any one of claims 104-107, wherein the therapeutic agent is a small molecule, a protein, a peptide, a nucleic acid, a lipid, or a carbohydrate.

109. The method of any one of claims 104-108, where the therapeutic agent is a known drug or an FDA-approved drug.

110. The method of claim 108 or 109, wherein the protein is an antibody, or an antibody variant or fragment.

111. The method of claim 108 or 109, wherein the nucleic acid is an mRNA, an antisense RNA, a miRNA, an siRNA, an RNA aptamer, a double stranded RNA (dsRNA), a short hairpin RNA (shRNA), or an antisense oligonucleotide (ASO).

112. The method of any one of claims 104-111, wherein the disease or disorder is a genetic disease, a proliferative disease, an inflammatory disease, an autoimmune disease, a liver disease, a spleen disease, a lung disease, a hematological disease, a neurological disease, a psychiatric disease, a gastrointestinal (GI) tract disease, a genitourinary disease, an infectious disease, a musculoskeletal disease, an endocrine disease, a metabolic disorder, an immune disorder, a central nervous system (CNS) disorder, or a cardiovascular disease.

113. The method of any one of claims 1-112, wherein one or more of the third probe, the fourth probe, and the sixth probe comprises one or more deoxyuridine (dU) nucleotide.

114. The method of claim 113, wherein one or more of the third probe, the fourth probe, and the sixth probe comprises dU nucleotides and does not comprise dT nucleotides.

115. The method of claim 113, wherein the third probe and the fourth probe each comprise dU nucleotides in the oligonucleotide portion that is complementary to a portion of the sixth probe.1014934-6856-6158.1Atty. Docket No. 114203-1551116. The method of claim 113 or 115, wherein the sixth probe comprises dU nucleotides in the oligonucleotide portion that is complementary to a portion of the third probe and the oligonucleotide portion that is complementary to a portion of the fourth probe.

117. The method of claim 115 or 116, wherein the third probe, the fourth probe, and the sixth probe each comprise dT nucleotides outside of the complementary portions.

118. The method of any one of claims 113-117, further comprising digesting one or more of the third probe, the fourth probe, and the sixth probe.

119. The method of claim 118, wherein digesting comprises contacting the one or more probes with an endonuclease enzyme.

120. The method of claim 118, wherein the endonuclease enzyme is a USER II enzyme.

121. The method of any one of claims 1-120, wherein the fifth probe comprises an adapter sequence capable of binding to an adapter probe.

122. The method of claim 121, wherein the adapter sequence is about 20 nucleotides in length.

123. The method of claim 121 or 122, wherein the adapter sequence is separated from a RNA-complementary region of the fifth probe by an adenosine linker.

124. The method of claim 123, wherein the adenosine linker comprises about 5 adenosine nucleotides.

125. The method of any one of claims 121-124, wherein the adapter probe comprises one or more acryl functional groups.

126. The method of any one of claims 121-125, wherein the sixth probe comprises one or more acryl functional groups.

127. The method of any one of claims 121-126, further comprising contacting the fifth probe with the adapter probe.

128. The method of claim 127, further comprising photocrosslinking the adapter probe to the fifth probe.1024934-6856-6158.1Atty. Docket No. 114203-1551129. The method of any one of claims 121-128, wherein the adapter probe comprises an acryl functional group on its 5 ’-end or 3 ’-end, wherein the method further comprises incubating the cell with methyl-acrylate NHS ester (MA-NHS) under conditions sufficient to permit the conversion of a remaining terminal amine group of the adapter probe to an acryl functional group, thereby generating an adapter probe comprising a 5’ acryl functional group and a 3’ acryl functional group.

130. The method of claim 129, further comprising embedding the cell in a hydrogel.

131. The method of claim 130, further comprising tissue clearing.

132. The method of claim 130 or 131, wherein ligation is performed after embedding and tissue clearing.

133. A set of probes comprising a first probe, a second probe, a third probe, a fourth probe, a fifth probe, and a sixth probe, wherein:(i) the first probe comprises(1) a first oligonucleotide barcode sequence;(2) an oligonucleotide portion that is complementary to a portion of the fifth probe;(3) an oligonucleotide portion that is complementary to a portion of an RNA of interest;(4) a second oligonucleotide barcode sequence; and(5) an oligonucleotide portion that is complementary to a portion of the sixth probe;(ii) the second probe comprises(1) a first oligonucleotide barcode sequence;(2) an oligonucleotide portion that is complementary to a portion of the fifth probe;(3) an oligonucleotide portion that is complementary to a portion of an RNA of interest; and(4) a second oligonucleotide barcode sequence;1034934-6856-6158.1Atty. Docket No. 114203-1551(iii) the third probe comprises a portion that recognizes the 40S subunit of a ribosome and an oligonucleotide portion that is complementary to a portion of the sixth probe;(iv) the fourth probe comprises a portion that recognizes the 60S subunit of a ribosome and an oligonucleotide portion that is complementary to a portion of the sixth probe;(v) the fifth probe comprises(1) an oligonucleotide portion that is complementary to the RNA of interest;(2) an oligonucleotide portion that is complementary to a portion of the first probe;(3) an oligonucleotide portion that is complementary to a portion of the second probe; and(4) an oligonucleotide barcode sequence,wherein the oligonucleotide barcode sequence of the fifth probe is complementary to the first oligonucleotide barcode sequence of the first probe and the first oligonucleotide barcode sequence of the second probe; and (vi) the sixth probe comprises(1) an oligonucleotide portion that is complementary to a portion of the first probe;(2) an oligonucleotide portion that is complementary to a portion of the third probe; and(3) an oligonucleotide portion that is complementary to a portion of the fourth probe.

134. The set of probes of claim 133, wherein the oligonucleotide portion that is complementary to a portion of an RNA of interest of the first probe and the oligonucleotide portion that is complementary to a portion of an RNA of interest of the second probe are identical.

135. The set of probes of claims 133 or 134, wherein the second oligonucleotide barcode sequence of the first probe and the second oligonucleotide barcode sequence of the second probe are not identical.1044934-6856-6158.1Atty. Docket No. 114203-1551136. The set of probes of any one of claims 133-135, wherein the portion of the third probe that recognizes the 40S subunit of a ribosome comprises an antibody, or an antibody variant or fragment.

137. The set of probes of claim 136, wherein the antibody is a secondary antibody.

138. The set of probes of any one of claims 133-136, wherein the portion of the third probe that recognizes the 40S subunit of a ribosome comprises an oligonucleotide that is complementary to a portion of rRNA within the 40S subunit of a ribosome.

139. The set of probes of any one of claims 133-136, wherein the portion of the third probe that recognizes the 40S subunit of a ribosome comprises an oligonucleotide that is complementary to a portion of the 18s ribosomal RNA (rRNA).

140. The set of probes of claim 138 or 139, wherein the oligonucleotide that is complementary to a portion of the 18s rRNA is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more than 30 nucleotides in length.

141. The set of probes of any one of claims 138-140, wherein the oligonucleotide that is complementary to a portion of the 18s rRNA is about 25 nucleotides in length.

142. The set of probes of any one of claims 133-141, wherein the portion of the fourth probe that recognizes the 60S subunit of a ribosome comprises an antibody, or an antibody variant or fragment.

143. The set of probes of claim 142, wherein the antibody is a secondary antibody.

144. The set of probes of any one of claims 133-143, wherein the portion of the fourth probe that recognizes the 60S subunit of a ribosome comprises an oligonucleotide that is complementary to a portion of rRNA within the 60S subunit of a ribosome.

145. The set of probes of any one of claims 133-143, wherein the portion of the fourth probe that recognizes the 60S subunit of a ribosome comprises an oligonucleotide that is complementary to a portion of the 28s ribosomal RNA (rRNA).

146. The set of probes of claim 144 or 145, wherein the oligonucleotide that is complementary to a portion of the 28s rRNA is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more than 30 nucleotides in length.1054934-6856-6158.1Atty. Docket No. 114203-1551147. The set of probes of any one of claims 144-146, wherein the oligonucleotide that is complementary to a portion of the 28s rRNA is about 25 nucleotides in length.

148. The set of probes of any one of claims 133-147, wherein the oligonucleotide barcode sequences are 3-13, 4-12, 5-11, 6-10, 7-9 nucleotides in length.

149. The set of probes of any one of claims 133-148, wherein the oligonucleotide barcode sequences are 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 nucleotides in length.

150. The set of probes of any one of claims 133-149, wherein the first and fifth probes are complementary to different portions of the RNA of interest.

151. The set of probes of any one of claims 133-150, wherein the second and fifth probes are complementary to different portions of the RNA of interest.

152. The set of probes of any one of claims 133-151, wherein the portion of the sixth probe that is complementary to a portion of the third probe is 9-15 nucleotides in length.

153. The set of probes of any one of claims 133-152, wherein the portion of the sixth probe that is complementary to a portion of the fourth probe is 9-15 nucleotides in length.

154. The set of probes of any one of claims 133-153, wherein the portion of the sixth probe that is complementary to a portion of the third probe is 9 nucleotides in length, and wherein the portion of the sixth probe that is complementary to a portion of the fourth probe is 11 nucleotides in length.

155. The set of probes of any one of claims 133-154, wherein the portion of the first probe that is complementary to a portion of the sixth probe is 4-20, 5-19, 6-18, 7-17, 8-16, 9-15, 10-14, or 11-13 nucleotides in length.

156. The set of probes of any one of claims 133-155, wherein the portion of the first probe that is complementary to a portion of the sixth probe is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length.

157. The set of probes of any one of claims 133-156, wherein the portion of the first probe that is complementary to a portion of the sixth probe is split between the 5' end and the 3' end of the first probe.1064934-6856-6158.1Atty. Docket No. 114203-1551158. The set of probes of any one of claims 133-157, wherein the portion of the second probe that is complementary to a portion of the fifth probe is split between the 5' end and the 3' end of the second probe.

159. The set of probes of any one of claims 133-158, wherein the portion of the first probe that is complementary to a portion of the fifth probe is 5-15, 6-14, 7-13, 8-12, or 9-11 nucleotides in length.

160. The set of probes of any one of claims 133-159, wherein the portion of the first probe that is complementary to a portion of the fifth probe is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides in length.

161. The set of probes of any one of claims 133-160, wherein the portion of the second probe that is complementary to a portion of the fifth probe is 5-15, 6-14, 7-13, 8-12, or 9-11 nucleotides in length.

162. The set of probes of any one of claims 133-161, wherein the portion of the second probe that is complementary to a portion of the fifth probe is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides in length.

163. The set of probes of any one of claims 133-162, wherein the portion of the first probe that is complementary to the RNA of interest is 10-30, 11-29, 12-28, 13-27, 14-26, 15-25, 16-24, 17-23, 18-22, or 19-21 nucleotides in length.

164. The set of probes any one of claims 133-163, wherein the portion of the first probe that is complementary to the RNA of interest is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

165. The set of probes of any one of claims 133-164, wherein the portion of the second probe that is complementary to the RNA of interest is 10-30, 11-29, 12-28, 13-27, 14- 26, 15-25, 16-24, 17-23, 18-22, or 19-21 nucleotides in length.

166. The set of probes any one of claims 133-165, wherein the portion of the second probe that is complementary to the RNA of interest is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.1074934-6856-6158.1Atty. Docket No. 114203-1551167. The set of probes of any one of claims 133-166, wherein the sixth probe further comprises a polymerization blocker.

168. The set of probes of claim 167, wherein the polymerization blocker is located at the 3' end of second probe.

169. The set of probes of claim 167 or 168, wherein the polymerization blocker comprises an inverted nucleic acid residue.

170. The set of probes of claim 169, wherein the inverted nucleic acid residue is an inverted thymine residue.

171. The set of probes of any one of claims 133-170, wherein the portion of the third probe that recognizes the 40S subunit of a ribosome and the portion of the third probe that is complementary to a portion of the sixth probe are joined by a poly-A nucleotide linker.

172. The set of probes of claim 171, wherein the poly-A nucleotide linker is 2-30, 3-25, 4- 20, 5-15, or 8-12 nucleotides in length.

173. The set of probes of claim 171 or 172, wherein the poly-A nucleotide linker is about 10 nucleotides in length.

174. The set of probes of any one of claims 133-173, wherein the portion of the fourth probe that recognizes the 60S subunit of a ribosome and the portion of the fourth probe that is complementary to a portion of the sixth probe are joined by a poly-A nucleotide linker.

175. The set of probes of claim 174, wherein the poly-A nucleotide linker is 2-30, 3-25, 4- 20, 5-15, or 8-12 nucleotides in length.

176. The set of probes of claim 174 or 175, wherein the poly-A nucleotide linker is about 10 nucleotides in length.

177. The set of probes of any one of claims 133-176, wherein the portion of the fifth probe that is complementary to the RNA of interest is 10-30, 11-29, 12-28, 13-27, 14-26, 15-25, 16-24, 17-23, 18-22, or 19-21 nucleotides in length.1084934-6856-6158.1Atty. Docket No. 114203-1551178. The set of probes of any one of claims 133-177, wherein the portion of the fifth probe that is complementary to the RNA of interest is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

179. The set of probes of any one of claims 133-178, wherein the portion of the fifth probe that is complementary to a portion of the first probe is 5-15, 6-14, 7-13, 8-12, or 9-11 nucleotides in length.

180. The set of probes of any one of claims 133-179, wherein the portion of the fifth probe that is complementary to a portion of the first probe is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides in length.

181. The set of probes of any one of claims 133-180, wherein the portion of the fifth probe that is complementary to a portion of the second probe is 5-15, 6-14, 7-13, 8-12, or 9- 11 nucleotides in length.

182. The set of probes of any one of claims 133-181, wherein the portion of the fifth probe that is complementary to a portion of the second probe is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides in length.

183. The set of probes of any one of claims 133-182, wherein the first probe comprises the structure:5 '-[portion complementary to sixth probe]-[portion complementary to portion of fifth probe]-[portion complementary to RNA of interest]-[second oligonucleotide barcode sequence]-[portion complementary to sixth probe]-3', wherein the first oligonucleotide barcode sequence is within the portion complementary to portion of fifth probe.

184. The set of probes of any one of claims 133-183, wherein the second probe comprises the structure:5 '-[portion complementary to portion of fifth probe]-[portion complementary to RNA of interest] -[second oligonucleotide barcode sequence]-3', wherein the first oligonucleotide barcode sequence is within portion complementary to portion of fifth probe.1094934-6856-6158.1Atty. Docket No. 114203-1551185. The set of probes of any one of claims 133-184, wherein the third probe comprises the structure: 5'-[portion recognizing the 40S subunit of the ribosome]-[poly-A linker]- [portion complementary to the sixth probe]-3'.

186. The set of probes of any one of claims 133-185, wherein the fourth probe comprises the structure: 5'-[portion complementary to the sixth probe]-[poly-A linker]-[portion recognizing the 60S subunit of the ribosome]-3'.

187. The set of probes of any one of claims 133-186, wherein the fifth probe comprises the structure:5'-[portion complementary to RNA of interest]-[portion complementary to first probe or second probe]-3', wherein the oligonucleotide barcode sequence is within the portion complementary to first probe or second probe.

188. The set of probes of any one of claims 133-187, wherein the sixth probe comprises the structure:5 '-[portion complementary to portion of fourth probe]-[portion complementary to portion of third probe]-[poly-A linker]- [portion complementary to portion of first probe]-3'.

189. The set of probes of any one of claims 133-187, wherein the sixth probe comprises the structure:5 '-[portion complementary to portion of third probe]-[portion complementary to portion of fourth probe]-[poly-A linker]-[portion complementary to portion of first probe]-3'.

190. The set of probes of any one of claims 133-189, wherein one or more of the third probe, the fourth probe, and the sixth probe comprises one or more deoxyuridine (dU) nucleotide.

191. The set of probes of claim 190, wherein one or more of the third probe, the fourth probe, and the sixth probe comprises dU nucleotides and does not comprise dT nucleotides.1104934-6856-6158.1Atty. Docket No. 114203-1551192. The set of probes of claim 190, wherein the third probe and the fourth probe each comprise dU nucleotides in the oligonucleotide portion that is complementary to a portion of the sixth probe.

193. The set of probes of claim 190 or 192, wherein the sixth probe comprises dU nucleotides in the oligonucleotide portion that is complementary to a portion of the third probe and the oligonucleotide portion that is complementary to a portion of the fourth probe.

194. The set of probes of claim 192 or 193, wherein the third probe, the fourth probe, and the sixth probe each comprise dT nucleotides outside of the complementary portions.

195. The set of probes of any one of claims 133-194, further comprising an adapter probe comprising a portion that is capable of hybridizing to the fifth probe.

196. The set of probes of any one of claims 195, wherein the fifth probe comprises an adapter sequence capable of binding to the adapter probe.

197. The set of probes of claim 195 or 196, wherein the adapter sequence is about 20 nucleotides in length.

198. The set of probes of any one of claims 195-197, wherein the adapter sequence is separated from a RNA-complementary region of the fifth probe by an adenosine linker.

199. The set of probes of claim 198, wherein the adenosine linker comprises about 5 adenosine nucleotides.

200. The set of probes of any one of claims 195-199, wherein the adapter probe comprises one or more acryl functional groups.

201. The set of probes of any one of claims 195-200, wherein the sixth probe comprises one or more acryl functional groups.

202. The set of probes of any one of claims 195-201, wherein the fifth probe comprises a photocrosslinking moiety.1114934-6856-6158.1Atty. Docket No. 114203-1551203. The set of probes of cany one of claims 195-202, wherein the adapter probe comprises an acryl functional group on its 5 ’-end or 3 ’-end, wherein The set of probes further comprises incubating the cell with methyl-acrylate NHS ester (MA-NHS) under conditions sufficient to permit the conversion of a remaining terminal amine group of the adapter probe to an acryl functional group, thereby generating an adapter probe comprising a 5’ acryl functional group and a 3’ acryl functional group.

204. A plurality of probes comprising multiple sets of probes of any one of claims 133- 203, and wherein each set of probes comprises oligonucleotide portions that are complementary to a different RNA of interest.

205. A kit comprising the set of probes of any one of claims 133-203.

206. A kit comprising multiple sets of probes of any one of claims 133-203, and wherein each set of probes comprises oligonucleotide portions that are complementary to a different RNA of interest.

207. The kit of claim 205 or 206, further comprising cells.

208. The kit of any one of claims 205-207, further comprising one or more enzymes.

209. The kit of claim 208, wherein the one or more enzymes comprises a ligase.

210. The kit of claim 208 or 209, wherein the one or more enzymes comprises a polymerase.

211. The kit of any one of claims 208-210, further comprising amine-modified nucleotides.

212. The kit of any one of claims 208-211, further comprising reagents and monomers for preparing a polymeric matrix.

213. The kit of any one of claims 205-212, further comprising an endonuclease enzyme.

214. The kit of claim 213, wherein the endonuclease enzyme is a USER II enzyme.

215. A system for profiling RNAs in a cell, comprising:(a) a cell;(b) one or more sets of probes comprising a first probe, a second probe, a third probe, a fourth probe, a fifth probe, and a sixth probe, wherein1124934-6856-6158.1Atty. Docket No. 114203-1551(i) the first probe comprises(1) a first oligonucleotide barcode sequence;(2) an oligonucleotide portion that is complementary to a portion of the fifth probe;(3) an oligonucleotide portion that is complementary to a portion of an RNA of interest;(4) a second oligonucleotide barcode sequence; and(5) an oligonucleotide portion that is complementary to a portion of the sixth probe;(ii) the second probe comprises(1) a first oligonucleotide barcode sequence;(2) an oligonucleotide portion that is complementary to a portion of the fifth probe;(3) an oligonucleotide portion that is complementary to a portion of an RNA of interest; and(4) a second oligonucleotide barcode sequence;(iii) the third probe comprises a portion that recognizes the 40S subunit of a ribosome and an oligonucleotide portion that is complementary to a portion of the sixth probe;(iv) the fourth probe comprises a portion that recognizes the 60S subunit of a ribosome and an oligonucleotide portion that is complementary to a portion of the sixth probe;(v) the fifth probe comprises(1) an oligonucleotide portion that is complementary to the RNA of interest;(2) an oligonucleotide portion that is complementary to a portion of the first probe;(3) an oligonucleotide portion that is complementary to a portion of the second probe; and(4) an oligonucleotide barcode sequence,wherein the oligonucleotide barcode sequence of the fifth probe is complementary to the first oligonucleotide barcode sequence of the first probe and the first oligonucleotide barcode sequence of the second probe; and (vi) the sixth probe comprises1134934-6856-6158.1Atty. Docket No. 114203-1551(1) an oligonucleotide portion that is complementary to a portion of the first probe;(2) an oligonucleotide portion that is complementary to a portion of the third probe; and(3) an oligonucleotide portion that is complementary to a portion of the fourth probe;(c) a microscope; and(d) a computer.

216. The system of claim 215, wherein the one or more sets of probes comprises the set of probes of any one of claims 133-1203.

217. The system of claim 215 or 216, wherein the one or more sets of probes comprises the plurality of probes of claim 204.

218. An assembled ribosome complex comprising:(a) a first oligonucleotide probe, a second oligonucleotide probe, and a third oligonucleotide probe, wherein(i) the first probe comprises an oligonucleotide portion that recognizes the 40S subunit of a ribosome and an oligonucleotide portion that is complementary to a portion of the third probe;(ii) the second probe comprises an oligonucleotide portion that recognizes the 60S subunit of a ribosome and an oligonucleotide portion that is complementary to a portion of the third probe; and (iii) the third probe comprises an oligonucleotide portion that is complementary to a portion of the first probe and an oligonucleotide portion that is complementary to a portion of the second probe;(b) a 40S subunit of a ribosome; and(c) a 60S subunit of a ribosome.

219. The assembled ribosome complex of claim 218, further comprising a target RNA of interest.

220. The assembled ribosome complex of claim 219, further comprising a fourth probe, wherein the fourth probe comprises a first oligonucleotide barcode sequence, an oligonucleotide portion that is complementary to a portion of an RNA of interest, a 1144934-6856-6158.1Atty. Docket No. 114203-1551second oligonucleotide barcode sequence, and an oligonucleotide portion that is complementary to a portion of the third probe.

221. The assembled ribosome complex of claim 220, further comprising a fifth probe, wherein the fifth probe comprises an oligonucleotide portion that is complementary to the RNA of interest, an oligonucleotide portion that is complementary to a portion of the fourth probe, and an oligonucleotide barcode sequence.

222. The assembled ribosome complex of any one of claims 218-221, wherein the portion of the first probe that recognizes the 40S subunit of a ribosome comprises an oligonucleotide that is complementary to a portion of rRNA within the 40S subunit of a ribosome.

223. The assembled ribosome complex of any one of claims 218-222, wherein the portion of the first probe that recognizes the 40S subunit of a ribosome comprises an oligonucleotide that is complementary to a portion of the 18s ribosomal RNA (rRNA).

224. The assembled ribosome complex of claim 222 or 223, wherein the oligonucleotide that is complementary to a portion of the 18s rRNA is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more than 30 nucleotides in length.

225. The assembled ribosome complex of any one of claims 222-224, wherein the oligonucleotide that is complementary to a portion of the 18s rRNA is about 25 nucleotides in length.

226. The assembled ribosome complex of any one of claims 222-225, wherein the portion of the second probe that recognizes the 60S subunit of a ribosome comprises an oligonucleotide that is complementary to a portion of rRNA within the 60S subunit of a ribosome.

227. The assembled ribosome complex of any one of claims 222-226, wherein the portion of the second probe that recognizes the 60S subunit of a ribosome comprises an oligonucleotide that is complementary to a portion of the 28s ribosomal RNA (rRNA).1154934-6856-6158.1Atty. Docket No. 114203-1551228. The assembled ribosome complex of claim 226 or 227, wherein the oligonucleotide that is complementary to a portion of the 28s rRNA is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more than 30 nucleotides in length.

229. The assembled ribosome complex of any one of claims 226-228, wherein the oligonucleotide that is complementary to a portion of the 28s rRNA is about 25 nucleotides in length.

230. The assembled ribosome complex of any one of claims 218-229, wherein the oligonucleotide barcode sequences are 3-13, 4-12, 5-11, 6-10, 7-9 nucleotides in length.

231. The assembled ribosome complex of any one of claims 218-230, wherein the oligonucleotide barcode sequences are 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 nucleotides in length.

232. The assembled ribosome complex of any one of claims 218-231, wherein the fourth and fifth probes are complementary to different portions of the RNA of interest.

233. The assembled ribosome complex of any one of claims 218-232, wherein the portion of the third probe that is complementary to a portion of the first probe is 9-15 nucleotides in length.

234. The assembled ribosome complex of any one of claims 218-233, wherein the portion of the third probe that is complementary to a portion of the second probe is 9-15 nucleotides in length.

235. The assembled ribosome complex of any one of claims 218-234, wherein the portion of the third probe that is complementary to a portion of the first probe is 9 nucleotides in length, and wherein the portion of the third probe that is complementary to a portion of the second probe is 11 nucleotides in length.

236. The assembled ribosome complex of any one of claims 218-235, wherein the portion of the fourth probe that is complementary to a portion of the third probe is 4-20, 5-19, 6-18, 7-17, 8-16, 9-15, 10-14, or 11-13 nucleotides in length.1164934-6856-6158.1Atty. Docket No. 114203-1551237. The assembled ribosome complex of any one of claims 218-236, wherein the portion of the fourth probe that is complementary to a portion of the third probe is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length.

238. The assembled ribosome complex of any one of claims 218-237, wherein the portion of the fourth probe that is complementary to a portion of the third probe is split between the 5' end and the 3' end of the fourth probe.

239. The assembled ribosome complex of any one of claims 218-238, wherein the portion of the fourth probe that is complementary to a portion of the fifth probe is split between the 5' end and the 3' end of the second probe.

240. The assembled ribosome complex of any one of claims 218-239, wherein the portion of the fourth probe that is complementary to a portion of the fifth probe is 5-15, 6-14, 7-13, 8-12, or 9-11 nucleotides in length.

241. The assembled ribosome complex of any one of claims 218-240, wherein the portion of the fourth probe that is complementary to a portion of the fifth probe is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides in length.

242. The assembled ribosome complex of any one of claims 218-241, wherein the portion of the fourth probe that is complementary to the RNA of interest is 10-30, 11-29, 12- 28, 13-27, 14-26, 15-25, 16-24, 17-23, 18-22, or 19-21 nucleotides in length.

243. The assembled ribosome complex any one of claims 218-242, wherein the portion of the fourth probe that is complementary to the RNA of interest is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

244. The assembled ribosome complex of any one of claims 218-243, wherein the third probe further comprises a polymerization blocker.

245. The assembled ribosome complex of claim 244, wherein the polymerization blocker is located at the 3' end of second probe.

246. The assembled ribosome complex of claim 244 or 245, wherein the polymerization blocker comprises an inverted nucleic acid residue.1174934-6856-6158.1Atty. Docket No. 114203-1551247. The assembled ribosome complex of claim 246, wherein the inverted nucleic acid residue is an inverted thymine residue.

248. The assembled ribosome complex of any one of claims 218-247, wherein the portion of the first probe that recognizes the 40S subunit of a ribosome and the portion of the first probe that is complementary to a portion of the third probe are joined by a poly-A nucleotide linker.

249. The assembled ribosome complex of claim 248, wherein the poly-A nucleotide linker is 2-30, 3-25, 4-20, 5-15, or 8-12 nucleotides in length.

250. The assembled ribosome complex of claim 248 or 249, wherein the poly-A nucleotide linker is about 10 nucleotides in length.

251. The assembled ribosome complex of any one of claims 218-250, wherein the portion of the second probe that recognizes the 60S subunit of a ribosome and the portion of the second probe that is complementary to a portion of the third probe are joined by a poly-A nucleotide linker.

252. The assembled ribosome complex of claim 251, wherein the poly-A nucleotide linker is 2-30, 3-25, 4-20, 5-15, or 8-12 nucleotides in length.

253. The assembled ribosome complex of claim 251 or 252, wherein the poly-A nucleotide linker is about 10 nucleotides in length.

254. The assembled ribosome complex of any one of claims 218-253, wherein the portion of the fifth probe that is complementary to the RNA of interest is 10-30, 11-29, 12-28, 13-27, 14-26, 15-25, 16-24, 17-23, 18-22, or 19-21 nucleotides in length.

255. The assembled ribosome complex of any one of claims 218-254, wherein the portion of the fifth probe that is complementary to the RNA of interest is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

256. The assembled ribosome complex of any one of claims 218-255, wherein the portion of the fifth probe that is complementary to a portion of the fourth probe is 5-15, 6-14, 7-13, 8-12, or 9-11 nucleotides in length.1184934-6856-6158.1Atty. Docket No. 114203-1551257. The assembled ribosome complex of any one of claims 218-256, wherein the portion of the fifth probe that is complementary to a portion of the fourth probe is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides in length.

258. The assembled ribosome complex of any one of claims 218-257, wherein the portion of the fifth probe that is complementary to a portion of the fourth probe is 5-15, 6-14, 7-13, 8-12, or 9-11 nucleotides in length.

259. The assembled ribosome complex of any one of claims 218-258, wherein the fourth probe comprises the structure:5 '-[portion complementary to third probe]-[portion complementary to portion of fifth probe]-[portion complementary to RNA of interest]-[second oligonucleotide barcode sequence]-[portion complementary to third probe]-3', wherein the first oligonucleotide barcode sequence is within portion complementary to portion of fifth probe.

260. The assembled ribosome complex of any one of claims 218-258, wherein the fourth probe comprises the structure:5 '-[portion complementary to portion of fifth probe]-[portion complementary to RNA of interest] -[second oligonucleotide barcode sequence]-3', wherein the first oligonucleotide barcode sequence is within portion complementary to portion of fifth probe.

261. The assembled ribosome complex of any one of claims 218-260, wherein the first probe comprises the structure: 5 '-[portion recognizing the 40S subunit of the ribosome]-[poly-A linker]-[portion complementary to the third probe]-3'.

262. The assembled ribosome complex of any one of claims 218-261, wherein the second probe comprises the structure: 5 '-[portion complementary to the third probe]-[poly-A linker]-[portion recognizing the 60S subunit of the ribosome]-3'.

263. The assembled ribosome complex of any one of claims 218-262, wherein the fifth probe comprises the structure:5 '-[portion complementary to RNA of interest]-[portion complementary to fourth1194934-6856-6158.1Atty. Docket No. 114203-1551probe]-3', wherein the oligonucleotide barcode sequence is within the portion complementary to fourth probe.

264. The assembled ribosome complex of any one of claims 218-263, wherein the third probe comprises the structure:5 '-[portion complementary to portion of second probe]-[portion complementary to portion of first probe]-3'.

265. The assembled ribosome complex of any one of claims 218-263, wherein the third probe comprises the structure:5 '-[portion complementary to portion of second probe]-[portion complementary to portion of first probe]-[poly-A linker]-[portion complementary to portion of fourth probe]-3'.

266. The assembled ribosome complex of any one of claims 218-265, wherein the third probe comprises the structure:5 '-[portion complementary to portion of first probe]-[portion complementary to portion of second probe]-3'.

267. The assembled ribosome complex of any one of claims 218-265, wherein the third probe comprises the structure:5 '-[portion complementary to portion of first probe]-[portion complementary to portion of second probe]-[poly-A linker]-[portion complementary to portion of fourth probe]-3'.

268. The assembled ribosome complex of any one of claims 218-267, wherein one or more of the first probe, the second probe, and the third probe comprises one or more deoxyuridine (dU) nucleotide.

269. The assembled ribosome complex of claim 268, wherein one or more of the first probe, the second probe, and the third probe comprises dU nucleotides and does not comprise dT nucleotides.1204934-6856-6158.1Atty. Docket No. 114203-1551270. The assembled ribosome complex of claim 268, wherein the first probe and the second probe each comprise dU nucleotides in the oligonucleotide portion that is complementary to a portion of the third probe.

271. The assembled ribosome complex of claim 268 or 270, wherein the third probe comprises dU nucleotides in the oligonucleotide portion that is complementary to a portion of the first probe and the oligonucleotide portion that is complementary to a portion of the second probe.

272. The assembled ribosome complex of claim 270 or 271, wherein the first probe, the second probe, and the third probe each comprise dT nucleotides outside of the complementary portions.

273. The assembled ribosome complex of any one of claims 221-272, further comprising an adapter probe comprising a portion that is capable of hybridizing to the fifth probe.

274. The assembled ribosome complex of any one of claims 273, wherein the fifth probe comprises an adapter sequence capable of binding to the adapter probe.

275. The assembled ribosome complex of claim 273 or 274, wherein the adapter sequence is about 20 nucleotides in length.

276. The assembled ribosome complex of any one of claims 273-275, wherein the adapter sequence is separated from a RNA-complementary region of the fifth probe by an adenosine linker.

277. The assembled ribosome complex of claim 276, wherein the adenosine linker comprises about 5 adenosine nucleotides.

278. The assembled ribosome complex of any one of claims 273-277, wherein the adapter probe comprises one or more acryl functional groups.

279. The assembled ribosome complex of any one of claims 273-278, wherein the third probe comprises one or more acryl functional groups.

280. The assembled ribosome complex of any one of claims 273-279, wherein the fifth probe comprises a photocrosslinking moiety.1214934-6856-6158.1Atty. Docket No. 114203-1551281. The assembled ribosome complex of cany one of claims 273-280, wherein the adapter probe comprises an acryl functional group on its 5 ’-end or 3 ’-end, wherein The assembled ribosome complex further comprises incubating the cell with methylacrylate NHS ester (MA-NHS) under conditions sufficient to permit the conversion of a remaining terminal amine group of the adapter probe to an acryl functional group, thereby generating an adapter probe comprising a 5’ acryl functional group and a 3’ acryl functional group.

282. A kit comprising a set of oligonucleotide probes, wherein the set of oligonucleotide probes comprises a first probe, a second probe, and a third probe, wherein(a) the first probe comprises an oligonucleotide portion that recognizes the 40S subunit of a ribosome and an oligonucleotide portion that is complementary to a portion of the third probe;(b) the second probe comprises an oligonucleotide portion that recognizes the 60S subunit of a ribosome and an oligonucleotide portion that is complementary to a portion of the third probe; and(c) the third probe comprises an oligonucleotide portion that is complementary to a portion of the first probe and an oligonucleotide portion that is complementary to a portion of the second probe.

283. A method for simultaneous transcriptome and translatome profiling in a cell, the method comprising:(a) contacting the cell with one or more sets of probes, wherein each set of probes comprises a first probe, a second probe, a third probe, a fourth probe, a fifth probe, and a sixth probe, wherein(i) the first probe comprises(1) a first oligonucleotide barcode sequence;(2) an oligonucleotide portion that is complementary to a portion of the fifth probe;(3) an oligonucleotide portion that is complementary to a portion of an RNA of interest;(4) a second oligonucleotide barcode sequence; and(5) an oligonucleotide portion that is complementary to a portion of the sixth probe;1224934-6856-6158.1Atty. Docket No. 114203-1551(ii) the second probe comprises(1) a first oligonucleotide barcode sequence;(2) an oligonucleotide portion that is complementary to a portion of the fifth probe;(3) an oligonucleotide portion that is complementary to a portion of an RNA of interest; and(4) a second oligonucleotide barcode sequence;(iii) the third probe comprises an oligonucleotide portion that recognizes a first subunit of a ribosome and an oligonucleotide portion that is complementary to a portion of the sixth probe;(iv) the fourth probe comprises an oligonucleotide portion that recognizes a second subunit of a ribosome and an oligonucleotide portion that is complementary to a portion of the sixth probe;(v) the fifth probe comprises(1) an oligonucleotide portion that is complementary to the RNA of interest;(2) an oligonucleotide portion that is complementary to a portion of the first probe;(3) an oligonucleotide portion that is complementary to a portion of the second probe; and(4) an oligonucleotide barcode sequence,wherein the oligonucleotide barcode sequence of the fifth probe is complementary to the first oligonucleotide barcode sequence of the first probe and the first oligonucleotide barcode sequence of the second probe; and (vi) the sixth probe comprises(1) an oligonucleotide portion that is complementary to a portion of the first probe;(2) an oligonucleotide portion that is complementary to a portion of the third probe; and(3) an oligonucleotide portion that is complementary to a portion of the fourth probe;(b) ligating the 5' end and the 3' end of the first probe together to produce a first circular oligonucleotide, and ligating the 5' end and the 3' end of the second probe together to produce a second circular oligonucleotide;1234934-6856-6158.1Atty. Docket No. 114203-1551(c) performing rolling circle amplification to amplify the first circular oligonucleotide and the second circular oligonucleotide, wherein the fifth probe is a primer for the amplification of each of the first circular oligonucleotide and the second circular oligonucleotide, thereby producing one or more concatenated amplicons from the first circular oligonucleotide and one or more concatenated amplicons from the second circular oligonucleotide;(d) embedding the one or more concatenated amplicons from the first circular oligonucleotide and one or more concatenated amplicons from the second circular oligonucleotide in a polymeric matrix; and(e) sequencing the concatenated amplicons embedded in the polymeric matrix to determine the identity, location, and translation status of each RNA of interest in the cell.

284. A method for diagnosing a disease or disorder in a subject, the method comprising:(a) contacting a cell obtained from the subject with one or more sets of probes, wherein each set of probes comprises a first probe, a second probe, a third probe, a fourth probe, a fifth probe, and a sixth probe, wherein(i) the first probe comprises(1) a first oligonucleotide barcode sequence;(2) an oligonucleotide portion that is complementary to a portion of the fifth probe;(3) an oligonucleotide portion that is complementary to a portion of an RNA of interest;(4) a second oligonucleotide barcode sequence; and(5) an oligonucleotide portion that is complementary to a portion of the sixth probe;(ii) the second probe comprises(1) a first oligonucleotide barcode sequence;(2) an oligonucleotide portion that is complementary to a portion of the fifth probe;(3) an oligonucleotide portion that is complementary to a portion of an RNA of interest; and(4) a second oligonucleotide barcode sequence;1244934-6856-6158.1Atty. Docket No. 114203-1551(iii) the third probe comprises a portion that recognizes a first subunit of a ribosome and an oligonucleotide portion that is complementary to a portion of the sixth probe;(iv) the fourth probe comprises a portion that recognizes a second subunit of a ribosome and an oligonucleotide portion that is complementary to a portion of the sixth probe;(v) the fifth probe comprises(1) an oligonucleotide portion that is complementary to the RNA of interest;(2) an oligonucleotide portion that is complementary to a portion of the first probe;(3) an oligonucleotide portion that is complementary to a portion of the second probe; and(4) an oligonucleotide barcode sequence,wherein the oligonucleotide barcode sequence of the fifth probe is complementary to the first oligonucleotide barcode sequence of the first probe and the first oligonucleotide barcode sequence of the second probe; and (vi) the sixth probe comprises(1) an oligonucleotide portion that is complementary to a portion of the first probe;(2) an oligonucleotide portion that is complementary to a portion of the third probe; and(3) an oligonucleotide portion that is complementary to a portion of the fourth probe;(b) ligating the 5' end and the 3' end of the first probe together to produce a first circular oligonucleotide, and ligating the 5' end and the 3' end of the second probe together to produce a second circular oligonucleotide;(c) performing rolling circle amplification to amplify the first circular oligonucleotide and the second circular oligonucleotide, wherein the fifth probe is a primer for the amplification of each of the first circular oligonucleotide and the second circular oligonucleotide, thereby producing one or more concatenated amplicons from the first circular oligonucleotide and one or more concatenated amplicons from the second circular oligonucleotide;1254934-6856-6158.1Atty. Docket No. 114203-1551(d) embedding the one or more concatenated amplicons from the first circular oligonucleotide and one or more concatenated amplicons from the second circular oligonucleotide in a polymeric matrix; and(e) sequencing the concatenated amplicons embedded in the polymeric matrix to determine the identity, location, and translation status of each RNA of interest in the cell,wherein a difference in the RNA translation profile of the cell relative to one or more non-diseased cells indicates that the subject has the disease or disorder.

285. A method for screening for an agent capable of modulating translation of one or more RNAs, the method comprising:(a) contacting a cell that is being treated with or has been treated with a candidate agent with one or more sets of probes, wherein each set of probes comprises a first probe, a second probe, a third probe, a fourth probe, a fifth probe, and a sixth probe, wherein(i) the first probe comprises(1) a first oligonucleotide barcode sequence;(2) an oligonucleotide portion that is complementary to a portion of the fifth probe;(3) an oligonucleotide portion that is complementary to a portion of an RNA of interest;(4) a second oligonucleotide barcode sequence; and(5) an oligonucleotide portion that is complementary to a portion of the sixth probe;(ii) the second probe comprises(1) a first oligonucleotide barcode sequence;(2) an oligonucleotide portion that is complementary to a portion of the fifth probe;(3) an oligonucleotide portion that is complementary to a portion of an RNA of interest; and(4) a second oligonucleotide barcode sequence;(iii) the third probe comprises a portion that recognizes a first subunit of a ribosome and an oligonucleotide portion that is complementary to a portion of the sixth probe;(iv) the fourth probe comprises a portion that recognizes a second subunit of a ribosome and an oligonucleotide portion that is complementary to a portion of the sixth probe;1264934-6856-6158.1Atty. Docket No. 114203-1551(v) the fifth probe comprises(1) an oligonucleotide portion that is complementary to the RNA of interest;(2) an oligonucleotide portion that is complementary to a portion of the first probe;(3) an oligonucleotide portion that is complementary to a portion of the second probe; and(4) an oligonucleotide barcode sequence,wherein the oligonucleotide barcode sequence of the fifth probe is complementary to the first oligonucleotide barcode sequence of the first probe and the first oligonucleotide barcode sequence of the second probe; and (vi) the sixth probe comprises(1) an oligonucleotide portion that is complementary to a portion of the first probe;(2) an oligonucleotide portion that is complementary to a portion of the third probe; and(3) an oligonucleotide portion that is complementary to a portion of the fourth probe;(b) ligating the 5' end and the 3' end of the first probe together to produce a first circular oligonucleotide, and ligating the 5' end and the 3' end of the second probe together to produce a second circular oligonucleotide;(c) performing rolling circle amplification to amplify the first circular oligonucleotide and the second circular oligonucleotide, wherein the fifth probe is a primer for the amplification of each of the first circular oligonucleotide and the second circular oligonucleotide, thereby producing one or more concatenated amplicons from the first circular oligonucleotide and one or more concatenated amplicons from the second circular oligonucleotide;(d) embedding the one or more concatenated amplicons from the first circular oligonucleotide and one or more concatenated amplicons from the second circular oligonucleotide in a polymeric matrix; and(e) sequencing the concatenated amplicons embedded in the polymeric matrix to determine the identity, location, and translation status of each RNA of interest in the cell,1274934-6856-6158.1Atty. Docket No. 114203-1551wherein a difference in the profile of RNAs being translated in the presence of the candidate agent relative to in the absence of the candidate agent indicates that the candidate agent modulates translation of one or more RNAs.

286. A method for treating a disease or disorder in a subject, the method comprising:(a) contacting a cell obtained from the subject with one or more sets of probes, wherein each set of probes comprises a first probe, a second probe, a third probe, a fourth probe, a fifth probe, and a sixth probe, wherein(i) the first probe comprises(1) a first oligonucleotide barcode sequence;(2) an oligonucleotide portion that is complementary to a portion of the fifth probe;(3) an oligonucleotide portion that is complementary to a portion of an RNA of interest;(4) a second oligonucleotide barcode sequence; and(5) an oligonucleotide portion that is complementary to a portion of the sixth probe;(ii) the second probe comprises(1) a first oligonucleotide barcode sequence;(2) an oligonucleotide portion that is complementary to a portion of the fifth probe;(3) an oligonucleotide portion that is complementary to a portion of an RNA of interest; and(4) a second oligonucleotide barcode sequence;(iii) the third probe comprises a portion that recognizes a first subunit of a ribosome and an oligonucleotide portion that is complementary to a portion of the sixth probe;(iv) the fourth probe comprises a portion that recognizes a second subunit of a ribosome and an oligonucleotide portion that is complementary to a portion of the sixth probe;(v) the fifth probe comprises(1) an oligonucleotide portion that is complementary to the RNA of interest;(2) an oligonucleotide portion that is complementary to a portion of the first probe;1284934-6856-6158.1Atty. Docket No. 114203-1551(3) an oligonucleotide portion that is complementary to a portion of the second probe; and(4) an oligonucleotide barcode sequence,wherein the oligonucleotide barcode sequence of the fifth probe is complementary to the first oligonucleotide barcode sequence of the first probe and the first oligonucleotide barcode sequence of the second probe; and (vi) the sixth probe comprises(1) an oligonucleotide portion that is complementary to a portion of the first probe;(2) an oligonucleotide portion that is complementary to a portion of the third probe; and(3) an oligonucleotide portion that is complementary to a portion of the fourth probe;(b) ligating the 5' end and the 3' end of the first probe together to produce a first circular oligonucleotide, and ligating the 5' end and the 3' end of the second probe together to produce a second circular oligonucleotide;(c) performing rolling circle amplification to amplify the first circular oligonucleotide and the second circular oligonucleotide, wherein the fifth probe is a primer for the amplification of each of the first circular oligonucleotide and the second circular oligonucleotide, thereby producing one or more concatenated amplicons from the first circular oligonucleotide and one or more concatenated amplicons from the second circular oligonucleotide;(d) embedding the one or more concatenated amplicons from the first circular oligonucleotide and one or more concatenated amplicons from the second circular oligonucleotide in a polymeric matrix;(e) sequencing the concatenated amplicons embedded in the polymeric matrix to determine the identity, location, and translation status of each RNA of interest in the cell; and (f) administering a treatment for the disease or disorder to the subject if a difference in the profile of RNAs being translated in the cell relative to one or more non-diseased cells is observed.1294934-6856-6158.1

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