Granted Patent B2

US 12,281,356 · App. 18/069,565 · Granted Apr 22, 2025

Inventors: Dwayne Dunaway (Seattle, WA); Elizabeth A. Manrao (Lake Forest Park, WA); Joseph M. Beechem (Piedmont, CA); Rustem Khafizov (Seattle, WA); Sanghamithra Korukonda (Seattle, WA); Yi Deng (Seattle, WA); Dae Kim (Bellevue, WA); Mark Gregory (Boise, ID); Margaret Hoang (Seattle, WA); Matthew Walsh (Seattle, WA); Gavin Meredith (Seattle, WA); Mark McElwain (Seattle, WA); Peter Skene (Issaquah, WA); Cassandra Burke (Seattle, WA)

Assignee: Bruker Spatial Biology, Inc.

C12Q1/6869C12Q1/6839C12Q1/6876

Abstract

Claims (49)

1. A probe comprising:

a target binding domain and a barcode domain;

wherein the target binding domain is at least 12 nucleotides in length;

wherein the barcode domain comprises a synthetic backbone, the barcode domain comprising at least two attachment positions, each attachment position comprising at least one attachment region comprising at least one nucleic acid sequence that hybridizes to a complementary nucleic acid molecule, and wherein the synthetic backbone comprises L-DNA,

wherein each of the at least two attachment positions has a different nucleic acid sequence,

wherein the at least two attachment positions correspond to the sequence of the target binding domain,

wherein said nucleic acid sequence of each position of the at least two attachment positions determines the identity of the target nucleic acid that is bound by said target binding domain, and

wherein each nucleotide of the at least one nucleic acid sequence of each attachment region is L-DNA.

2. The probe of claim 1 , wherein the synthetic backbone is single-stranded and is about 10 nucleotides to about 100 nucleotides in length.

3. The probe of claim 1 , wherein the barcode domain comprises:

a) at least three attachment positions; or

b) at least four attachment positions.

4. The probe of claim 1 , wherein each attachment position in the barcode domain comprises one attachment region, wherein the at least one nucleic acid sequence of each attachment position in the barcode domain is:

a) about 6 to about 20 nucleotides in length;

b) about 9 nucleotides in length;

c) about 12 nucleotides in length;

d) about 14 nucleotides in length; or

e) about 16 nucleotides in length.

5. The probe of claim 1 , wherein each nucleotide of the at least 12 nucleotides of the target binding domain is D-DNA.

6. A method for identifying the presence of a target nucleic acid in a sample comprising:

(1) hybridizing a target binding domain of the probe of claim 1 to the target nucleic acid;

(2) hybridizing a first complementary nucleic acid molecule comprising at least one first detectable label and at least one second detectable label to a first attachment position of the at least two attachment positions of the barcode domain;

(3) identifying the at least one first and the at least one second detectable label of the first complementary nucleic acid molecule hybridized to the first attachment position;

(4) removing the at least one first and the at least one second detectable label hybridized to the first attachment position;

(5) hybridizing a second complementary nucleic acid molecule comprising at least one third detectable label and at least one fourth detectable label to a second attachment position of the at least two attachment positions of the barcode domain;

(6) identifying the at least one third and the at least one fourth detectable label of the second complementary nucleic acid molecule hybridized to the second attachment position, and

(7) determining the presence of the target nucleic acid based on at least the identity of each of the identified detectable labels.

7. The method of claim 6 , wherein the barcode domain of the probe comprises at least three attachment positions or at least four attachment positions,

wherein the method further comprises, prior to step (7):

repeating steps (4) to (6) until each attachment position in the barcode domain has been bound by a complementary nucleic acid molecule comprising two detectable labels, and the two detectable labels of the bound complementary nucleic acid molecule have been detected.

8. The method of claim 6 , wherein the first and second detectable labels have the same emission spectrum or have different emission spectra.

9. The method of claim 6 , wherein the third and fourth detectable labels have the same emission spectrum or have different emission spectra.

10. The method of claim 6 , wherein at least the first complementary nucleic acid molecule comprises a cleavable linker.

11. The method of claim 10 , wherein the cleavable linker is a photocleavable linker.

12. The method of claim 10 , wherein each complementary nucleic acid molecule comprises a cleavable linker.

13. The method of claim 7 , wherein the first complementary nucleic acid molecule comprises a reporter probe comprising a primary nucleic acid,

wherein the primary nucleic acid molecule comprises at least two domains,

a first domain that hybridizes to the first attachment position of the barcode domain and

a second domain that is hybridized to six secondary nucleic acid molecules,

wherein each of the secondary nucleic acid molecules is hybridized to five tertiary nucleic molecules, wherein each of the tertiary nucleic acid molecules comprises a detectable label.

14. The method of claim 13 , wherein the primary nucleic acid molecule comprises a cleavable linker located between the first domain and the second domain.

15. The method of claim 14 , wherein the cleavable linker is a photocleavable linker.

16. The method of claim 13 , wherein each of the secondary nucleic acid molecules comprises at least two domains,

a first domain that hybridizes to the second domain of the primary nucleic acid molecule and

a second domain that hybridizes to the five tertiary nucleic acid molecules,

wherein each of the secondary nucleic acid molecules comprises a cleavable linker located between the first domain and the second domain.

17. The method of claim 16 , wherein the cleavable linker is a photocleavable linker.

18. The method of claim 13 , wherein removing the at least one first and the at least one second detectable label hybridized to the first attachment position comprises cleaving the cleavable linker between the first domain and the second domain of the primary nucleic acid molecule.

19. The method of claim 16 , wherein removing the at least one first and the at least one second detectable label hybridized to the first attachment position comprises cleaving the cleavable linkers between the first domains and the second domains of the secondary nucleic acid molecules.

Assignments (7)

Continuity (4)

References Cited (400)

Cited By (1)

US 12,630,869