Chemical compositions and methods of using same

Patent metadata

Field	Value
Publication number	US-11549139-B2
Application number	US-201916411394-A
Country	US
Kind code	B2
Filing date	May 14, 2019
Priority date	May 14, 2018
Publication date	Jan 10, 2023
Grant date	Jan 10, 2023

Abstract

The present disclosure relates to chemical compositions, kits, and apparatuses and methods for using these compositions, kits and apparatuses in various assays.

First claim

What is claimed is:

1. A probe comprising a target binding domain and a barcode domain;
wherein the target binding domain is at least 12 nucleotides in length and hybridizes to a target nucleic acid;
wherein the barcode domain comprises a synthetic backbone, the barcode domain comprising at least three 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 three attachment positions have a different nucleic acid sequence, and wherein each nucleotide of the at least one nucleic acid sequence of each attachment region is L-DNA,
wherein the at least one nucleic acid sequence of each attachment position comprises a 3′ terminal guanosine nucleotide.

2. The probe of claim 1 , wherein the probe comprises a single-stranded DNA synthetic backbone and a double-stranded DNA spacer between the target binding domain and the barcode domain.

3. The probe of claim 1 , wherein the synthetic backbone is a single-stranded DNA synthetic backbone that is about 10 nucleotides to about 100 nucleotides in length.

4. The probe of claim 1 , further comprising a first complementary primary nucleic acid molecule hybridized to a first attachment position of the at least three attachment positions,
wherein the first primary complementary nucleic acid molecule comprises at least two domains and a cleavable linker,
wherein the first domain is hybridized to the first attachment position of the barcode domain and the second domain is capable of hybridizing to at least one complementary secondary-nucleic acid molecule, and
wherein the cleavable linker is located between the first and second domains.

5. The probe of claim 1 , wherein the cleavable linker comprises:

6. The probe of claim 1 , wherein the number of nucleotides in the target binding domain is greater than the number of attachment positions in the barcode domain.

7. The probe of claim 1 , wherein the barcode domain comprises at least four attachment positions.

8. The probe of claim 1 , wherein each attachment position in the barcode domain comprises one attachment region.

9. The probe of claim 1 , wherein the at least one nucleic acid sequence of each attachment position in the barcode domain is about 9 nucleotides in length.

10. The probe of claim 1 , wherein the at least one nucleic acid sequence of each attachment position comprises at least one adenine nucleotide, at least one thymine nucleotide, at least one cytosine nucleotide or any combination thereof and a 3′ terminal guanosine nucleotide.

11. The probe of claim 1 , wherein each nucleotide of the at least one nucleic acid sequence of each attachment position is L-DNA.

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

13. The probe of claim 4 , wherein the first domain of the primary nucleic acid molecule comprises L-DNA.

14. The probe of claim 4 , wherein the second domain of the primary nucleic acid molecule comprises L-DNA.

15. The probe of claim 4 , wherein the first domain of the primary nucleic acid molecule comprises a 5′ terminal cytosine nucleotide.

16. The probe of claim 4 , wherein the first domain of the primary nucleic acid molecule comprises at least one adenine nucleotide, at least one thymine nucleotide, at least one guanine nucleotide or any combination thereof and a 5′ terminal cytosine nucleotide.

17. The probe of claim 4 , wherein the cleavable linker comprises at least one cleavable moiety.

18. The probe of claim 17 , wherein cleavable moiety is a photocleavable moiety.

19. The probe of claim 4 , wherein the primary nucleic molecule is hybridized to at least one secondary nucleic add molecule.

20. The probe of claim 4 , wherein the primary nucleic molecule is hybridized to at least four secondary-nucleic acid molecules.

21. The probe of claim 20 , wherein each of the secondary nucleic acid molecules comprise at least two domains,
a first domain capable of binding to a complementary sequence in at least one primary nucleic acid molecule; and
a second domain capable of binding to (a) a first detectable label and an at least second detectable label, (b) to at least one complementary tertiary nucleic acid molecule, or (c) a combination thereof.

22. The probe of claim 21 , wherein each of the secondary nucleic acid molecules comprise a cleavable linker.

23. The probe of claim 22 , wherein the cleavable linker is located between the first domain and the second domain.

24. The p e of claim 22 , wherein the linker is photo-cleavable.

25. The probe of claim 21 , wherein each of the secondary nucleic acid molecules are hybridized to at least one tertiary nucleic acid molecule.

26. The probe of claim 25 , wherein each of the secondary nucleic acid molecules are hybridized to (a) at least one tertiary nucleic acid molecule, and (b) a first detectable label and an at least second detectable label.

27. The probe of claim 26 , wherein each secondary nucleic acid molecule is hybridized to at least five tertiary nucleic acid molecules.

28. The probe of claim 26 , wherein the first and at least second detectable labels have the same emission spectrum or have different emission spectra.

29. The probe of claim 27 , wherein each of the tertiary nucleic acid molecules comprise at least two domains,
a first domain capable of binding to a complementary sequence in a secondary nucleic acid molecule; and
a second domain capable of binding to a first detectable label and an at least second detectable label.

30. The probe of claim 29 , wherein each of the tertiary nucleic acid molecules comprise a cleavable linker.

31. The probe of claim 30 , wherein the cleavable linker is located between the first domain and the second domain.

32. The probe of claim 30 , wherein the linker is photo-cleavable.

33. The probe of claim 27 , wherein each of the tertiary nucleic acid molecules comprise a detectable label.

34. The probe of claim 4 , wherein the primary nucleic acid molecule is hybridized to six secondary nucleic acid molecules,
wherein each of the six secondary nucleic acid molecules is hybridized to five tertiary nucleic acid molecules,
wherein each of the tertiary nucleic acid molecules comprise a detectable label.

35. A method for determining the presence of a target nucleic acid comprising:
(1) hybridizing the target binding domain of at least one first probe of claim 1 to a first region of a 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 three 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

Assignees

• Nanostring Technologies Inc

Inventors

• Dunaway Dwayne
• Manrao Elizabeth A
• Beechem Joseph M
• Khafizov Rustem
• Korukonda Sanghamithra
• Deng Yi
• Kim Dae
• Gregory Mark
• Hoang Margaret
• Walsh Matthew
• Meredith Gavin
• Mcelwain Mark
• Skene Peter
• Burke Cassandra

Classifications

• C12Q1/6839

  Triple helix formation or other higher order conformations in hybridisation assays · CPC title

• C12Q1/6869 Primary

  Methods for sequencing · CPC title

• C12Q1/6874 Primary

  involving nucleic acid arrays, e.g. sequencing by hybridisation · CPC title

• C12Q1/6876

  Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes · CPC title

• C12Q2563/107

  fluorescence · CPC title