Granted Patent B2

US 12,391,988 · App. 18/958,854 · Granted Aug 19, 2025

Inventors: Andrew Pawlowski (San Diego, CA); Yeoan Youn (San Diego, CA); Michael Lawson (Pasadena, CA); Martin Maria Fabani (Encinitas, CA)

Assignee: Singular Genomics Systems, Inc.

C12Q1/6874C12Q1/25C12Q1/6841C12Q1/6862G01N2333/9015

Abstract

Claims (25)

1. A method of detecting a nucleic acid molecule in a cell, said method comprising amplifying a target sequence of the nucleic acid molecule, wherein amplifying comprises:

contacting a target polynucleotide complex in a cell with a probe oligonucleotide, thereby forming a probe polynucleotide complex;

wherein the target polynucleotide complex comprises a blocking oligonucleotide bound to a first sequence of a target polynucleotide, wherein the blocking oligonucleotide comprises a first target binding sequence, a first curl sequence, a second curl sequence, and a probe binding sequence, wherein said first curl sequence is complementary to said second curl sequence, wherein said probe polynucleotide complex comprises the probe oligonucleotide bound to a second sequence of the target polynucleotide and bound to the probe binding sequence, and wherein the target sequence is between said first and second sequence;

extending the probe oligonucleotide along the target sequence with a polymerase to generate an extended probe strand comprising a complement of the target sequence, and joining said extended probe strand to the probe oligonucleotide binding sequence, thereby generating a circular oligonucleotide;

extending an amplification primer bound to the circular oligonucleotide with a strand-displacing polymerase, thereby generating an amplification product comprising multiple copies of the target sequence; and

sequencing the target sequence in said cell and sequencing the second curl sequence, or a complement thereof, thereby detecting the nucleic acid molecule.

2. The method of claim 1 , wherein sequencing comprises sequencing by synthesis, sequencing by hybridization, sequencing by binding, or sequencing by ligation.

3. The method of claim 1 , wherein sequencing comprises hybridizing a sequencing primer to said amplification product, (a) extending the sequencing primer by incorporating a labeled nucleotide or labeled nucleotide analogue and (b) detecting the label for each incorporated nucleotide or nucleotide analogue.

4. The method of claim 1 , wherein the cell is permeabilized and immobilized to a solid support.

5. The method of claim 1 , wherein the first curl sequence is complementary to a sequence of the target polynucleotide.

6. The method of claim 1 , wherein the first curl sequence is about 4 to 15 nucleotides.

7. The method of claim 1 , wherein the blocking oligonucleotide comprises a modified nucleotide.

8. The method of claim 7 , wherein the modified nucleotide comprises a retarding moiety covalently bound to the modified nucleotide.

9. The method of claim 1 , wherein the first target binding sequence comprises locked nucleic acids (LNAs), Bis-locked nucleic acids (bisLNAs), twisted intercalating nucleic acids (TINAs), bridged nucleic acids (BNAs), 2′-O-methyl RNA:DNA chimeric nucleic acids, minor groove binder (MGB) nucleic acids, morpholino nucleic acids, C5-modified pyrimidine nucleic acids, peptide nucleic acids (PNAs), phosphorothioate nucleic acids, Zip nucleic acids (ZNAs), or combinations thereof.

10. The method of claim 1 , wherein the first target binding sequence comprises one or more locked nucleic acids (LNAs), Zip nucleic acids (ZNAs), 2-amino-deoxyadenosine (2-amino-dA), trimethoxystilbene-functionalized oligonucleotides (TFOs), Pyrene-functionalized oligonucleotides (PFOs), peptide nucleic acids (PNAs), or aminoethyl-phenoxazine-dC (AP-dC) nucleic acids.

11. The method of claim 2 , wherein the blocking oligonucleotide is the amplification primer, and amplifying comprises extending the probe binding sequence.

12. The method of claim 2 , wherein the target polynucleotide is the amplification primer.

13. The method of claim 1 , wherein the probe oligonucleotide further comprises a barcode sequence.

14. The method of claim 1 , wherein the target polynucleotide comprises RNA or cDNA.

15. The method of claim 1 , wherein the target polynucleotide comprises an IGH locus or a BCL-1, BCL-2, BCL-3, or BCL6 locus.

16. The method of claim 1 , wherein the target polynucleotide comprises a sequence encoding for a V region or a complement thereof and a J region or a complement thereof.

17. The method of claim 1 , wherein joining said extended probe strand to the probe oligonucleotide binding sequence comprises covalently binding adjacent sequences with a ligase.

18. The method of claim 17 , wherein the ligase is a pre-adenylated ligase.

19. The method of claim 17 , wherein the ligase is a PBCV-1 DNA Ligase.

20. The method of claim 17 , wherein the ligase is a TS2126 RNA ligase.

Assignments (2)

Continuity (3)

References Cited (284)