Systems and methods for determining nucleic acids

Patent metadata

Field	Value
Publication number	US-11959075-B2
Application number	US-202318184967-A
Country	US
Kind code	B2
Filing date	Mar 16, 2023
Priority date	Jul 30, 2014
Publication date	Apr 16, 2024
Grant date	Apr 16, 2024

Abstract

The present invention generally relates to systems and methods for imaging or determining nucleic acids, for instance, within cells. In some embodiments, the transcriptome of a cell may be determined. Certain embodiments are directed to determining nucleic acids, such as mRNA, within cells at relatively high resolutions. In some embodiments, a plurality of nucleic acid probes may be applied to a sample, and their binding within the sample determined, e.g., using fluorescence, to determine locations of the nucleic acid probes within the sample. In some embodiments, codewords may be based on the binding of the plurality of nucleic acid probes, and in some cases, the codewords may define an error-correcting code to reduce or prevent misidentification of the nucleic acids. In certain cases, a relatively large number of different targets may be identified using a relatively small number of labels, e.g., by using various combinatorial approaches.

First claim

The invention claimed is:

1. A method for imaging RNA spatial organization in a sample comprising:
a. contacting the sample comprising a plurality of distinct RNA species in situ with a plurality of primary nucleic acid probe pools each of the nucleic acid probes comprising a target sequence and one or more read sequences, wherein each pool of nucleic acid probes hybridize to a distinct RNA species and each pool of probes encode a N-bit codeword with a Hamming weight of at least 2 that was assigned to each distinct RNA species, wherein each assigned N-bit codeword is a valid codeword with a Hamming distance equal to or greater than 2 between valid codewords and wherein each of the read sequences correspond to a bit value of 1 for the codewords assigned to the distinct RNA species;
b. contacting the bound primary nucleic acid probes with a plurality of readout probes comprising a fluorescent label, wherein the readout probes hybridize to the read sequences of the primary nucleic probes;
c. imaging the readout probes bound to the primary nucleic acid probes; and,
d. repeating steps b) and c) in one or more sequential hybridization and imaging rounds until all N positions in the N-bit codeword have been imaged providing an imaged codeword corresponding to each distinct RNA species in a spatial organization.

2. The method of claim 1 , wherein the assigned N-bit codewords have a Hamming distance equal to or greater than 4 between each of the valid codewords.

3. The method of claim 1 , wherein the N-bit codeword has a Hamming weight of 4.

4. The method of claim 1 , wherein the imaged codeword is matched to a valid codeword assigned to a distinct RNA species.

5. The method of claim 1 wherein the Hamming distance is 4 or greater and the imaged codewords are matched to valid codewords or discarded.

6. The method of claim 1 , wherein the N-bit codeword has a Hamming weight of 4 and the Hamming distance is 4 or greater and the imaged codewords are matched to valid codewords or discarded.

7. The method of claim 1 , wherein the RNA species is a RNA transcript.

8. The method of claim 1 , comprising determining the spatial organization of the transcriptome from a single cell.

9. The method of claim 1 , wherein each primary nucleic acid probe pool comprises at least 10 different primary nucleic acid probes.

10. The method of claim 1 , wherein each primary nucleic acid probe pool comprises at least four distinct read sequences.

11. The method of claim 1 , wherein each primary nucleic acid probe pool comprises four distinct read sequences.

12. The method of claim 1 , wherein each primary nucleic acid probe pool comprises at least eight distinct read sequences.

13. The method of claim 1 , wherein the primary nucleic acid probes comprise a target sequence, with an average length of between 10 and 200 nucleotides, that hybridize the distinct RNA species.

14. The method of claim 1 , wherein each primary nucleic acid probe pool comprises at least 10 different target sequences.

15. The method of claim 1 , wherein after each hybridization and imaging round the fluorescent read out probe is quenched to inactivate.

16. The method of claim 1 , wherein after each hybridization and imaging round the fluorescent read out probe is inactivated by chemically or enzymatically cleaving the fluorescent label from the read out probe.

17. The method of claim 1 , wherein the N-bit binary code comprises at least a 16-bit code.

18. The method of claim 1 , wherein the plurality of fluorescent readout probes comprise at least two distinct fluorescent labels.

19. The method of claim 1 , wherein the plurality of fluorescent readout probes comprise at least three distinct fluorescent labels.

20. The method of claim 1 , wherein the spatial organization of the distinct RNA species is imaged in 2 dimensions.

21. The method of claim 1 , wherein the spatial organization of the distinct RNA species is imaged in 3 dimensions.

22. The method of claim 1 , further comprising determining abundance for the distinct RNA species.

Assignees

• Harvard College

Inventors

• Zhuang Xiaowei
• Chen Kok-Hao
• Boettiger Alistair
• Moffitt Jeffrey R
• Wang Siyuan

Classifications

• C12N15/1065 Primary

  Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags · CPC title

• C07H21/02

  with ribosyl as saccharide radical · CPC title

• C07H21/04

  with deoxyribosyl as saccharide radical · CPC title

• C12N15/10

  Processes for the isolation, preparation or purification of DNA or RNA (chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34) · CPC title

• C12N15/1093

  General methods of preparing gene libraries, not provided for in other subgroups · CPC title