Chemical compositions and methods of using same
The present disclosure relates to chemical compositions, kits, and apparatuses and methods for using these compositions, kits and apparatuses in various assays.
1. A complex comprising
a) a composition comprising a target binding domain and a barcode domain,
wherein the target binding domain comprises at least eight nucleotides and is capable of binding a target nucleic acid, wherein at least six nucleotides in the target binding domain are capable of identifying a corresponding nucleotide in the target nucleic acid molecule and wherein at least two nucleotides in the target binding domain do not identify a corresponding nucleotide in the target nucleic acid molecule;
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 capable of being bound by a complementary nucleic acid molecule, wherein the nucleic acid sequence of the at least three attachment positions determines the position and identity of the at least six nucleotides in the target nucleic acid that is bound by the target binding domain, and wherein each of the at least three attachment positions have a different nucleic acid sequence; and
a first complementary primary nucleic acid molecule hybridized to a first attachment position of the at least three attachment positions,
wherein the first complementary primary 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.
2. A complex comprising
a) a composition comprising a target binding domain and a barcode domain,
wherein the target binding domain comprises at least eight nucleotides and is capable of binding a target nucleic acid, wherein at least six nucleotides in the target binding domain are capable of identifying a corresponding nucleotide in the target nucleic acid molecule and wherein at least two nucleotides in the target binding domain do not identify a corresponding nucleotide in the target nucleic acid molecule;
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 capable of being bound by a complementary nucleic acid molecule, wherein the nucleic acid sequence of the at least three attachment positions determines the position and identity of the at least six nucleotides in the target nucleic acid that is bound by the target binding domain,
wherein each attachment position of the at least three attachment positions corresponds to two nucleotides of the at least six nucleotides in the target binding domain and each of the at least three attachment positions have a different nucleic acid sequence, and
wherein the nucleic acid sequence of each position of the at least three attachment positions determines the position and identity of the corresponding two nucleotides of the at least six nucleotides in the target nucleic acid that is bound by the target binding domain; and
a first complementary primary nucleic acid molecule hybridized to a first attachment position of the at least three attachment positions,
wherein the first complementary primary 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.
3. A probe comprising a target binding domain and a barcode domain;
wherein the target binding domain comprises at least eight nucleotides and is capable of binding a target nucleic acid, wherein at least six nucleotides in the target binding domain are capable of identifying a corresponding nucleotide in the target nucleic acid molecule and wherein at least two nucleotides in the target binding domain do not identify a corresponding nucleotide in the target nucleic acid molecule;
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 capable of being bound by a complementary nucleic acid molecule, wherein the nucleic acid sequence of the at least three attachment positions determines the position and identity of the at least six nucleotides in the target nucleic acid that is bound by the target binding domain, and wherein each of the at least three attachment positions have a different nucleic acid sequence.
4. A probe comprising a target binding domain and a barcode domain;
wherein the target binding domain comprises at least eight nucleotides and is capable of binding a target nucleic acid, wherein at least six nucleotides in the target binding domain are capable of identifying a corresponding nucleotide in the target nucleic acid molecule and wherein at least two nucleotides in the target binding domain do not identify a corresponding nucleotide in the target nucleic acid molecule;
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 capable of being bound by a complementary nucleic acid molecule,
wherein each attachment position of the at least three attachment positions corresponds to two nucleotides of the at least six nucleotides in the target binding domain and each of the at least three attachment positions have a different nucleic acid sequence, and
wherein the nucleic acid sequence of each position of the at least three attachment positions determines the position and identity of the corresponding two nucleotides of the at least six nucleotides in the target nucleic acid that is bound by the target binding domain.
5. The probe of claim 3 , wherein said synthetic backbone comprises a polysaccharide, a polynucleotide, a peptide, a peptide nucleic acid, or a polypeptide.
6. The probe of claim 3 , wherein said synthetic backbone comprises a single-stranded DNA.
7. The probe of claim 3 , wherein said probe comprises a single-stranded DNA synthetic backbone and a double-stranded DNA spacer between the target binding domain and the barcode domain.
8. The probe of claim 7 , wherein said double-stranded DNA spacer is from about 1 nucleotide to about 100 nucleotides in length.
9. The probe of claim 7 , wherein said double-stranded DNA spacer is about 25 nucleotides in length.
10. The probe of claim 3 , wherein the number of nucleotides in the target binding domain is greater than the number of attachment positions in the barcode domain.
11. The probe of claim 3 , wherein the number of nucleotides in the target binding domain is at least five more than the number of attachment positions in the barcode domain.
12. The probe of claim 3 , wherein the target binding domain comprises eight nucleotides and the barcode domain comprises three attachment positions.
13. The probe of claim 3 , wherein at least four nucleotides of the at least six nucleotides in the target binding domain are modified nucleotides or nucleotide analogues.
14. The probe of claim 3 , wherein at least six nucleotides of the at least six nucleotides in the target binding domain are modified nucleotides or nucleotide analogues.
15. The probe of claim 3 , wherein the modified nucleotide or nucleic acid analogue is a locked nucleic acid (LNA).
16. The probe of claim 3 , wherein at least one of the at least two nucleotides in the target binding domain that does not identify a corresponding nucleotide in the target nucleic acid molecule precedes the at least six nucleotides in the target binding domain and wherein at least one of the at least two nucleotides in the target binding domain that does not identify a corresponding nucleotide in the target nucleic acid molecule follows the at least six nucleotides in the target binding domain.
17. The probe of claim 3 , wherein each attachment position in the barcode domain comprises the same number of attachment regions.
18. The probe of claim 3 , wherein at least one of the at least three attachment positions in the barcode domain comprises a different number of attachment regions than the other two.
19. The probe of claim 3 , wherein each attachment position in the barcode domain comprises one attachment region.
20. The probe of claim 3 , wherein each attachment position in the barcode domain comprises more than one attachment region.
21. The probe of claim 3 , wherein when the attachment position in the barcode domain comprises more than one attachment region, the attachment regions are the same.
22. The probe of claim 3 , wherein when the attachment position in the barcode domain comprises more than one attachment region, the attachment regions are different.
23. The probe of claim 3 , wherein each nucleic acid sequence comprising each attachment region in the barcode domain is from about 8 nucleotides to about 20 nucleotides in length.
24. The probe of claim 3 , wherein each nucleic acid sequence comprising each attachment region in the barcode domain is about 12 nucleotides in length.
25. The probe of claim 3 , wherein each nucleic acid sequence comprising each attachment region in the barcode domain is about 14 nucleotides in length.
26. The probe of claim 3 , wherein at least one of the at least three attachment positions in the barcode domain is adjacent to at least one flanking single-stranded polynucleotide.
27. The probe of claim 3 , wherein each of the at least three attachment positions in the barcode domain is adjacent to at least one flanking single stranded polynucleotide.
28. The probe of claim 3 , wherein at least one attachment region in at least one attachment position is integral to the synthetic backbone.
29. The probe of claim 3 , wherein each attachment region in each of the at least three attachment positions is integral to the synthetic backbone.
30. The probe of claim 3 , wherein the complementary nucleic acid molecule is RNA, DNA or PNA.
31. The probe of claim 30 , wherein the complementary nucleic acid molecule is DNA.
32. The probe of claim 3 , wherein the complementary nucleic acid molecule is a primary nucleic acid molecule, wherein the primary nucleic acid molecule directly binds to at least one attachment region within at least one attachment position of the barcode domain.
33. The probe of claim 32 , wherein the primary nucleic acid molecule comprises at least two domains,
a first domain capable of binding to at least one attachment region within at least one attachment position of the barcode domain and
a second domain capable of binding to at least one complementary secondary nucleic acid molecule.
34. The probe of claim 33 , wherein the primary nucleic acid molecule comprises a cleavable linker.
35. The probe of claim 34 , wherein the cleavable linker is located between the first domain and the second domain.
36. The probe of claim 34 , wherein the linker is photo-cleavable.
37. The probe of claim 32 , wherein the primary nucleic acid molecule is hybridized to at least one attachment region within at least one attachment position of the barcode domain and comprises a first detectable label and an at least second detectable label.
38. The probe of claim 33 , wherein the primary nucleic acid molecule is hybridized to at least one attachment region within at least one attachment position of the barcode domain and is hybridized to at least one secondary nucleic acid molecule.
39. The probe of claim 38 , wherein the primary nucleic acid molecule is hybridized to at least one, two, three, four, five, or more secondary nucleic acid molecules.
40. The probe of claim 38 , wherein the primary nucleic acid molecule is hybridized to four secondary nucleic acid molecules.
41. The probe of claim 37 , wherein the first and at least second detectable labels have the same emission spectrum or have different emission spectra.
42. The probe of claim 33 , wherein the secondary nucleic acid molecule comprises 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.
43. The probe of claim 42 , wherein the secondary nucleic acid molecule comprises a cleavable linker.
44. The probe of claim 43 , wherein the cleavable linker is located between the first domain and the second domain.
45. The probe of claim 43 , wherein the linker is photo-cleavable.
46. The probe of claim 42 , wherein the secondary nucleic acid molecule is hybridized to at least one primary nucleic acid molecule and comprises a first detectable label and an at least second detectable label.
47. The probe of claim 42 , wherein the secondary nucleic acid molecule is hybridized to at least one primary nucleic acid molecule and is hybridized to at least one tertiary nucleic acid molecule.
48. The probe of claim 42 , wherein the secondary nucleic acid molecule is hybridized to (a) at least one primary nucleic acid molecule, (b) at least one tertiary nucleic acid molecule, and (c) a first detectable label and an at least second detectable label.
49. The probe of claim 47 , wherein the secondary nucleic acid molecule is hybridized to at least one, two, three, four, five, six, seven, or more tertiary nucleic acid molecules.
50. The probe of claim 47 , wherein the secondary nucleic acid molecule is hybridized to one tertiary nucleic acid molecule.
51. The probe of claim 46 , wherein the first and at least second detectable labels have the same emission spectrum or have different emission spectra.
52. The probe of claim 42 , wherein the tertiary nucleic acid molecule comprises at least two domains,
a first domain capable of binding to 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.
53. The probe of claim 52 , wherein the tertiary nucleic acid molecule comprises a cleavable linker.
54. The probe of claim 53 , wherein the cleavable linker is located between the first domain and the second domain.
55. The probe of claim 53 , wherein the linker is photo-cleavable.
56. The probe of claim 52 , wherein the tertiary nucleic acid molecule is hybridized to at least one secondary nucleic acid molecule and comprises a first detectable label and an at least second detectable label.
57. The probe of claim 52 , wherein the first and at least second detectable labels have the same emission spectrum or have different emission spectra.
58. The probe of claim 55 , wherein the at least first and second detectable labels located on the secondary nucleic acid molecule have the same emission spectra and the at least first and second detectable labels located on the tertiary nucleic acid molecule have the same emission spectra, and wherein the emission spectra of the detectable labels on the secondary nucleic acid molecule are different than the emission spectra of the detectable labels on the tertiary nucleic acid molecule.
59. A population of probes comprising a plurality of the probe of claim 3 .
60. A method for determining a nucleotide sequence of a nucleic acid comprising
(1) hybridizing a first probe of claim 3 to a first region of a target nucleic acid that is optionally immobilized to a substrate at one or more positions;
(2) hybridizing a first complementary nucleic acid molecule comprising a first detectable label and a second detectable label to a first attachment position of the at least three attachment positions of the barcode domain;
(3) identifying the first and the second detectable label of the first complementary nucleic acid molecule hybridized to the first attachment position;
(4) removing the first and the second detectable label hybridized to the first attachment position;
(5) hybridizing a second complementary nucleic acid molecule comprising a third detectable label and a fourth detectable label to a second attachment position of the at least three attachment positions of the barcode domain;
(6) identifying the third and the fourth detectable label of the second complementary nucleic acid molecule hybridized to the second attachment position;
(7) removing the third and fourth detectable label hybridized to the second attachment position;
(8) hybridizing a third complementary nucleic acid molecule comprising a fifth detectable label and a sixth detectable label to a third attachment position of the at least three attachment positions of the barcode domain;
(9) identifying the fifth and the sixth detectable label of the third complementary nucleic acid molecule hybridized to the third attachment position; and
(10) identifying the linear order of at least six nucleotides in the first region of the optionally immobilized target nucleic acid hybridized to the target binding domain of the first probe based on the identity of the first detectable label, second detectable label, third detectable label, fourth detectable label, fifth detectable label and sixth detectable label.
61. The method of claim 60 , wherein the target nucleic acid is obtained from a predetermined gene.
62. The method of claim 60 , wherein steps (4) and (5) occur sequentially or concurrently.
63. The method of claim 60 , wherein the first and second detectable labels have the same emission spectrum or have different emission spectra.
64. The method of claim 60 , wherein the third and fourth detectable labels have the same emission spectrum or have different emission spectra.
65. The method of claim 60 , wherein the fifth and sixth detectable labels have the same emission spectrum or have different emission spectra.
66. The method of claim 60 , wherein the first complementary nucleic acid molecule comprises a cleavable linker.
67. The method of claim 60 , wherein the second complementary nucleic acid molecule comprises a cleavable linker.
68. The method of claim 60 , wherein the third complementary nucleic acid molecule comprises a cleavable linker.
69. The method of claim 60 , wherein the first complementary nucleic acid molecule, the second complementary nucleic acid molecule and the third complementary nucleic acid molecule each comprise a cleavable linker.
70. The method of claim 66 , wherein the cleavable linker is photo-cleavable.
71. The method of claim 70 , wherein removing the first complementary nucleic acid molecule comprises contacting the first complementary nucleic acid molecule with light.
72. The method of claim 71 , wherein the light is provided by a light source selected from the group consisting of an arc-lamp, a laser, a focused UV light source, and light emitting diode.
73. The method of claim 60 , further comprising
(11) removing the first probe from the first region of the optionally immobilized target nucleic acid;
(12) hybridizing at least a second probe of claim 3 to a second region of the target nucleic acid that is optionally immobilized to a substrate at one or more positions, and wherein the target binding domain of the first probe and the at least second probe are different;
(13) hybridizing a first complementary nucleic acid molecule comprising a first detectable label and a second detectable label to a first attachment position of the at least three attachment positions of the barcode domain;
(14) detecting the first and the second detectable label of the first complementary nucleic acid molecule hybridized to the first attachment position;
(15) hybridizing a second complementary nucleic acid molecule comprising a third detectable label and a fourth detectable label to a second attachment position of the at least three attachment positions of the barcode domain;
(16) detecting the third and the fourth detectable label of the second complementary nucleic acid molecule hybridized to the second attachment position;
(17) removing the third and fourth detectable label hybridized to the second attachment position;
(18) hybridizing a third complementary nucleic acid molecule comprising a fifth detectable label and a sixth detectable label to a third attachment position of the at least three attachment positions of the barcode domain;
(19) identifying the fifth and the sixth detectable label of the third complementary nucleic acid molecule hybridized to the third attachment position; and
(20) identifying the linear order of at least six nucleotides in the second region of the optionally immobilized target nucleic acid hybridized to the target binding domain of the at least second probe based on the identity of the first detectable label, second detectable label, third detectable label, fourth detectable label, fifth detectable label and sixth detectable label.
74. The method of claim 73 , further comprising assembling each identified linear order of nucleotides in the at least first region and at least second region of the optionally immobilized target nucleic acid, thereby identifying a sequence for the optionally immobilized target nucleic acid.
75. An apparatus for performing the method of claim 60 .
76. A kit comprising a substrate, a population of probes of claim 59 , at least one capture probe, at least three complementary nucleic acid molecules comprising a first detectable label and at least two second detectable labels, and instructions for use.
77. The complex of claim 1 or claim 2 , wherein the cleavable linker is a photo-cleavable linker.
78. The complex of claim 1 or claim 2 , wherein the cleavable linker is