# US 12,601,007 B2 · Multiplex labeling of molecules by sequential hybridization barcoding

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- Number: US 12,601,007 B2
- Document ID: US12601007B2
- Status: Granted
- Kind: B2
- Application number: 17/940,650
- Filed: 2022-09-08
- Published: 2023-09-21
- Granted: 2026-04-14
- Earliest claimed: 2013-04-30
- Assignees: California Institute of Technology
- Applicants: California Institute of Technology
- Inventors: Long Cai; Eric Lubeck; Timur Zhiyentayev; Ahmet Coskun; Ting-Fang He; Chang Ho Sohn; Sheel Shah
- Web page: https://patentgrep.com/patent/US12601007B2

## CPC

- C12Q 1/6841: In situ hybridisation
- C12N 15/1065: {Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags}
- G01N 21/6428: {Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" (in vivo A61B5/00; immunoassay G01N33/53)}
- G01N 21/6458: {Fluorescence microscopy (fluorescence microscopes per se G02B21/0076 and G02B21/16)}
- G02B 21/06: Means for illuminating specimens
- G02B 21/16: adapted for ultraviolet illumination {; Fluorescence microscopes (G02B21/0076 takes precedence)}
- G01N 2201/068: Optics, miscellaneous
- G01N 2201/10: Scanning
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- G02B: OPTICAL ELEMENTS, SYSTEMS OR APPARATUS

## Abstract

The present invention, among other things, provides technologies for detecting and/or quantifying nucleic acids in cells, tissues, organs or organisms. In some embodiments, through sequential barcoding, the present invention provides methods for high-throughput profiling of a large number of targets, such as transcripts and/or DNA loci.

## Claims

### Claim 1

A method, comprising steps of:
(a) performing a first contacting step that comprises contacting a cell comprising a plurality of target nucleic acids, the target nucleic acids comprising nucleic acids obtained by subjecting single stranded nucleic acids in the cell to amplification, with a first plurality of detectably labeled oligonucleotides, each of which targets a nucleic acid and is labeled with a detectable moiety, so that the first plurality comprises at least:
(i) a first oligonucleotide targeting a first nucleic acid in the cell, the first oligonucleotide being labeled with a first detectable moiety; and
(ii) a second oligonucleotide targeting a second nucleic acid in the cell, the second oligonucleotide being labeled with a second detectable moiety;
(b) imaging the cell, after the first contacting step so that interaction by hybridizations of oligonucleotides of the first plurality with their target nucleic acids are detected;
(c) performing a second contacting step that comprises contacting the cell, with a second plurality of detectably labeled oligonucleotides, which second plurality includes oligonucleotides targeting nucleic acids that are targeted by the first plurality so that the second plurality comprises at least:
(i) a third oligonucleotide, optionally identical in sequence to the first oligonucleotide, targeting the first nucleic acid; and
(ii) a fourth oligonucleotide, optionally identical in sequence to the second oligonucleotide, targeting the second nucleic acid, wherein the second plurality differs from the first plurality in that at least one of the oligonucleotides present in the second plurality is labeled with a different detectable moiety than the corresponding oligonucleotide targeting the same nucleic acid in the first plurality, so that, in the second plurality:
(iii) the third oligonucleotide is labeled with the first detectable moiety, the second detectable moiety or a third detectable moiety; and
(iv) the fourth oligonucleotide is labeled with the first detectable moiety, the second detectable moiety, the third detectable moiety, or a fourth detectable moiety, wherein either the third oligonucleotide is labeled with a different detectable moiety than was the first oligonucleotide, or the fourth oligonucleotide is labeled with a different detectable moiety than was the second oligonucleotide, or both;
(d) imaging the cell after the second contacting step so that interaction by hybridization of oligonucleotides of the second plurality with their target nucleic acids is detected; and
(e) optionally repeating the contacting and imaging steps, each time with a new plurality of detectably labeled oligonucleotides comprising oligonucleotides that target nucleic acids targeted by the first and second pluralities, wherein each utilized plurality differs from each other utilized plurality, due to at least one difference in detectable moiety labeling of oligonucleotides targeting the same nucleic acid,
so that the detected signals barcode the first target nucleic acid, and the second target nucleic acid in the cell, wherein the target nucleic acids can be differentiated by a difference in their barcodes.

### Claim 2 (depends on 1)

The method of claim 1, wherein the amplification is by a polymerase.

### Claim 3 (depends on 1)

The method of claim 1, wherein the amplification produces nucleic acids that are localized to the nucleic acids subjected to amplification.

### Claim 4 (depends on 1)

The method of claim 1, wherein the nucleic acids subjected to amplification are intermediate oligonucleotides.

### Claim 5 (depends on 1)

The method of claim 1, comprising N contacting steps, wherein N is at least two, and wherein each plurality of detectably labeled oligonucleotides comprises F detectable moieties, wherein F is at least two.

### Claim 6 (depends on 1)

The method of claim 1, further comprising a step of removing a plurality of detectably labeled oligonucleotides after each imaging step.

### Claim 7 (depends on 1)

The method of claim 1, wherein at least one detectably labeled oligonucleotide is labeled by hybridization chain reaction (HCR).

### Claim 8 (depends on 1)

The method of claim 1, wherein each plurality of detectably labeled oligonucleotides comprises two or more detectably labeled oligonucleotides targeting the same nucleic acid.

### Claim 9 (depends on 8)

The method of claim 8, wherein all detectably labeled oligonucleotides targeting the same target nucleic acids in the same contacting step are labeled with a fluorophore providing the same color or with the same fluorophore.

### Claim 10 (depends on 1)

The method of claim 1, wherein the third oligonucleotide is labeled with a different detectable moiety than was the first oligonucleotide, and the fourth oligonucleotide is labeled with the same detectable moiety as was the second oligonucleotide.

### Claim 11 (depends on 1)

The method of claim 1, wherein the third oligonucleotide is labeled with a different detectable moiety than was the first oligonucleotide, and the fourth oligonucleotide is labeled with a different detectable moiety than was the second oligonucleotide.

### Claim 12 (depends on 1)

The method of claim 1, wherein at least one barcode includes no signal for one or more of the contacting steps.

### Claim 13 (depends on 1)

The method of claim 1, wherein at least one contacting step differs from another contacting step in that at least one of the first and second target nucleic acids are contacted by an oligonucleotide having no signal for at least one of the first and second target nucleic acids.

### Claim 14 (depends on 1)

The method of claim 1, wherein at least one contacting step differs from another contacting step in that each of the first and second target nucleic acids are contacted by an oligonucleotide having a different detectable moiety.

### Claim 15 (depends on 1)

The method of claim 1, wherein the contacting and imaging steps are repeated a second and a third time.

### Claim 16 (depends on 1)

The method of claim 1, wherein at the least one contacting step differs from another contacting step in that the first and second target nucleic acids are contacted by oligonucleotides with different detectable moieties for the first target nucleic acid and for the second target nucleic acid.

### Claim 17 (depends on 1)

The method of claim 1, wherein the detectably labeled oligonucleotides comprise labels selected from three or four different labels.

### Claim 18

A method, comprising steps of:
(a) performing a first contacting step that comprises contacting a plurality of target nucleic acids, the target nucleic acids comprising transcripts or nucleic acids obtained by subjecting intermediate oligonucleotides to amplification, with a first plurality of detectably labeled oligonucleotides, each of which targets a nucleic acid and is labeled with a detectable moiety, so that the first plurality comprises at least:
(i) a first oligonucleotide targeting a first nucleic acid, the first oligonucleotide being labeled with a first detectable moiety; and
(ii) a second oligonucleotide targeting a second nucleic acid, the second oligonucleotide being labeled with a second detectable moiety;
(b) imaging the target nucleic acids, after the first contacting step so that interaction by hybridizations of oligonucleotides of the first plurality with their target nucleic acids are detected;
(c) performing a second contacting step that comprises contacting the target nucleic acids, with a second plurality of detectably labeled oligonucleotides, which second plurality includes oligonucleotides targeting nucleic acids that are targeted by the first plurality so that the second plurality comprises at least:
(i) a third oligonucleotide, optionally identical in sequence to the first oligonucleotide, targeting the first nucleic acid; and
(ii) a fourth oligonucleotide, optionally identical in sequence to the second oligonucleotide, targeting the second nucleic acid, wherein the second plurality differs from the first plurality in that at least one of the oligonucleotides present in the second plurality is labeled with a different detectable moiety than the corresponding oligonucleotide targeting the same nucleic acid in the first plurality, so that, in the second plurality:
(iii) the third oligonucleotide is labeled with the first detectable moiety, the second detectable moiety or a third detectable moiety; and
(iv) the fourth oligonucleotide is labeled with the first detectable moiety, the second detectable moiety, the third detectable moiety, or a fourth detectable moiety, wherein either the third oligonucleotide is labeled with a different detectable moiety than was the first oligonucleotide, or the fourth oligonucleotide is labeled with a different detectable moiety than was the second oligonucleotide, or both;
(d) imaging the nucleic acids after the second contacting step so that interaction by hybridization of oligonucleotides of the second plurality with their target nucleic acids is detected; and
(e) optionally repeating the contacting and imaging steps, each time with a new plurality of detectably labeled oligonucleotides comprising oligonucleotides that target nucleic acids targeted by the first and second pluralities, wherein each utilized plurality differs from each other utilized plurality, due to at least one difference in detectable moiety labeling of oligonucleotides targeting the same nucleic acid,
so that the detected signals barcode the first target nucleic acid, and the second target nucleic acid, wherein the target nucleic acids can be differentiated by a difference in their barcodes.

### Claim 19 (depends on 18)

The method of claim 18, wherein the amplification is by a polymerase.

### Claim 20 (depends on 18)

The method of claim 18, wherein the amplification produces nucleic acids that are localized to the intermediate oligonucleotides subjected to amplification.

### Claim 21 (depends on 20)

The method of claim 20, wherein the intermediate oligonucleotides are localized to the transcripts.

### Claim 22 (depends on 18)

The method of claim 18, comprising N contacting steps, wherein N is at least two, and wherein each plurality of detectably labeled oligonucleotides comprises F detectable moieties, wherein F is at least two.

### Claim 23 (depends on 18)

The method of claim 18, further comprising a step of removing a plurality of detectably labeled oligonucleotides after each imaging step.

### Claim 24 (depends on 18)

The method of claim 18, wherein each plurality of detectably labeled oligonucleotides comprises two or more detectably labeled oligonucleotides targeting the same nucleic acid.

### Claim 25 (depends on 18)

The method of claim 18, wherein all detectably labeled oligonucleotides targeting the same target nucleic acid in the same contacting step are labeled with a fluorophore providing the same color or with the same fluorophore.

### Claim 26 (depends on 18)

The method of claim 18, wherein the third oligonucleotide is labeled with a different detectable moiety than was the first oligonucleotide, and the fourth oligonucleotide is labeled with the same detectable moiety as was the second oligonucleotide.

### Claim 27 (depends on 18)

The method of claim 18, wherein the third oligonucleotide is labeled with a different detectable moiety than was the first oligonucleotide, and the fourth oligonucleotide is labeled with a different detectable moiety than was the second oligonucleotide.

### Claim 28 (depends on 18)

The method of claim 18, wherein at least one barcode includes no signal for one or more of the contacting steps.

### Claim 29 (depends on 18)

The method of claim 18, wherein at least one contacting step differs from another contacting step in that at least one of the first and second target nucleic acids are contacted by an oligonucleotide having no signal for at least one of the first and second target nucleic acids.

### Claim 30 (depends on 18)

The method of claim 18, wherein at least one contacting step differs from another contacting step in that each of the first and second target nucleic acids are contacted by an oligonucleotide having a different detectable moiety.

### Claim 31 (depends on 18)

The method of claim 18, wherein the contacting and imaging steps are repeated a second and a third time.

### Claim 32 (depends on 18)

The method of claim 18, wherein at the least one contacting step differs from another contacting step in that the first and second target nucleic acids are contacted by oligonucleotides with different detectable moieties for the first target nucleic acid and for the second target nucleic acid.

### Claim 33 (depends on 18)

The method of claim 18, wherein the intermediate oligonucleotides hybridized to a transcript.

### Claim 34 (depends on 18)

The method of claim 18, wherein the intermediate oligonucleotides comprise a sequence complementary to a transcript and an overhang sequence.

### Claim 35 (depends on 34)

The method of claim 34, wherein the intermediate oligonucleotides are preserved through multiple contacting and imaging steps.

### Claim 36 (depends on 18)

The method of claim 18, wherein the detectably labelled oligonucleotides interact with the transcripts through one or more of the intermediate oligonucleotides.

### Claim 37 (depends on 33)

The method of claim 33, wherein each of the one or more intermediate oligonucleotides remains hybridized to one of the transcripts through multiple contacting and imaging steps.

### Claim 38 (depends on 18)

The method of claim 18, wherein the detectably labeled oligonucleotides comprise labels selected from three or four different labels.

### Claim 39 (depends on 1)

The method of claim 1, wherein the nucleic acids in the cell subject to amplification are selected from DNA, transcripts, and intermediate oligonucleotides.

### Claim 40 (depends on 4)

The method of claim 4, wherein each of the intermediate oligonucleotides hybridized to a transcript.

### Claim 41 (depends on 4)

The method of claim 4, wherein each of the intermediate oligonucleotides comprises a sequence complementary or identical to a transcript.

### Claim 42 (depends on 4)

The method of claim 4, wherein the intermediate oligonucleotides are preserved through multiple contacting and imaging steps.

### Claim 43 (depends on 1)

The method of claim 1, wherein the detectably labelled oligonucleotides interact with the transcripts through one or more intermediate oligonucleotides.

### Claim 44 (depends on 4)

The method of claim 4, wherein the intermediate oligonucleotides remain hybridized to the transcript through multiple contacting and imaging steps.