Coded Molecules for Detecting Target Analytes
The present disclosure relates to methods of detecting target analytes based on single molecule detection of coded molecules.
1 . A method of detecting a target analyte, comprising:
(a) translocating a coded molecule through a nanopore, wherein the coded molecule comprises (i) an ordered plurality of code regions formed on a single-stranded nucleobase polymer scaffold, wherein one or more of the code regions is non-single-stranded and each code region has a detectable property such that detecting the ordered code regions generates a defined signal pattern, and (ii) a moiety capable of binding to a target analyte, and
wherein the nanopore is dimensioned for passage of the non-single-stranded region;
(b) sensing the detectable property of each code region as the coded molecule translocates through the nanopore to generate the defined signal pattern; and
(c) relating the signal pattern to presence of the moiety.
2 . The method of claim 1 , further comprising
(d) detecting the presence of a target analyte bound to the moiety.
3 . The method of claim 1 in which the coded molecule comprises a plurality of non-single-stranded code regions.
4 . The method of claim 3 in which at least two of the non-single-stranded code regions are adjacent.
5 . The method of claim 3 further comprising a single-stranded region separating at least two of the non-single-stranded code regions.
6 . The method of claim 5 in which the single-stranded region is a code region.
7 . The method of claim 3 in which the plurality of non-single-stranded code regions comprises at least a first and second non-single-stranded code regions, where the detectable property of the first and second code region is distinguishable.
8 . The method of claim 7 in which the first non-single-stranded code region has a nucleobase polymer sequence different from the second non-single-stranded code region.
9 . The method of claim 7 in which the first non-single-stranded code region comprises a nucleobase polymer different from the nucleobase polymer of the second non-single-stranded code region.
10 . The method of claim 1 in which the detectable property is a charge induced field effect.
11 . The method of claim 1 in which the detectable property is blockade of current through the nanopore.
12 . The method of claim 1 in which the detectable property is current across the coded molecule.
13 . The method of claim 1 in which the detectable property is current through the chain of the coded molecule.
14 . The method of claim 12 or 13 in which the current is electron tunneling current.
15 . The method of claim 1 in which the detectable property is the time of transit through the nanopore.
16 . The method of claim 1 in which the non-single-stranded code region is multistranded.
17 . The method of claim 16 in which at least one or more of the multistranded region is double-stranded.
18 . The method of claim 16 in which at least one or more of the multistranded region is triple-stranded.
19 . The method of claim 16 in which the coded molecule has a plurality of multistranded regions
20 . The method of claim 19 in which the plurality of multistranded regions comprises at least one double-stranded and at least one triple-stranded region.
21 . The method of claim 16 in which at least one strand of at least one of the multistranded region is a DNA.
22 . The method of claim 16 in which at least one strand of at least one of the multistranded region is an RNA.
23 . The method of claim 16 in which the at least one stand of at least one of the multistranded region is a polynucleotide analog or polynucleotide mimetic.
24 . The method of claim 23 in which the polynucleotide analog is PNA.
25 . The method of claim 16 in which at least one of the multistranded region comprises a detectable tag.
26 . The method of claim 25 in which the detectable tag is an electron transfer label.
27 . The method of claim 26 in which the electron transfer label is ferrocene.
28 . The method of claim 25 in which the detectable tag is a fluorophore.
29 . The method of claim 25 in which the detectable tag is a steric modifier.
30 . The method of claim 1 in which the binding moiety is attached to the coded molecule via a linker.
31 . The method of claim 1 in which the moiety comprises a nucleobase polymer probe capable of hybridizing to a target polynucleotide.
32 . The method of claim 1 in which the target polynucleotide is DNA
33 . The method of claim 1 in which the target polynucleotide is RNA.
34 . The method of claim 16 in which at least two strands of at least one of the multistranded region are crosslinked.
35 . The method of claim 34 in which all of the multistranded regions are crosslinked.
36 . A method of detecting a target analyte, comprising:
(a) translocating a coded molecule of a population of coded molecules through a nanopore, wherein the population of coded molecules comprises at least a first and second subpopulation, the coded molecule of the population comprising:
(i) an ordered plurality of code regions formed on a single-stranded nucleobase polymer scaffold, wherein one or more of the code regions is non-single-stranded and each code region has a detectable property such that detecting a combination of code regions generates a defined signal pattern distinguishable between the first and second subpopulations; and
(ii) a first moiety on the first subpopulation, wherein the first moiety is capable of binding to a first target analyte, and a second moiety on the second subpopulation, wherein the second moiety is capable of binding to a second target analyte, and
wherein the nanopore is of a sufficient dimension for transit of the non-single-stranded region;
(b) sensing the detectable property of each code region as the coded molecule translocates through the nanopore to generate the defined signal pattern; and
(c) relating the signal pattern to the presence of the moiety of the first or second subpopulation.
37 . The method of claim 36 , further comprising:
(d) detecting the presence of the target analyte bound to the first and second moiety.
38 . The method of claim 36 in which the first and second target analytes are different.
39 . The method of claim 36 further comprising:
(d) detecting the presence of the first and second target analytes.
40 . The method of claim 36 in which the coded molecule comprises a plurality of non-single-stranded code regions.
41 . The method of claim 40 in which the plurality of non-single-stranded code regions comprises at least a first and second non-single-stranded code region, wherein the detectable property of the first and second code region is different.
42 . The method of claim 41 in which the first non-single stranded code region has a nucleobase polymer sequence different from the second non-single stranded code region.
43 . The method of claim 41 in which the first non-single-stranded code region comprises a nucleobase polymer different from the nucleobase polymer of the second non-single-stranded code region.
44 . The method of claim 36 in which the coded molecule has adjacent non-single-stranded code regions.
45 . The method of claim 36 in which the coded molecule further comprises a single-stranded region separating at least two of the non-single-stranded code regions.
46 . The method of claim 45 in which the single-stranded region is a code region.
47 . The method of claim 36 in which the detectable property is a charge induced field effect.
48 . The method of claim 36 in which the detectable property is blockade of current through the nanopore.
49 . The method of claim 36 in which the detectable property is current across the coded molecule.
50 . The method of claim 36 in which the detectable property is current through the chain of the coded molecule.
51 . The method of claim 49 or 50 in which the current is electron tunneling current.
52 . The method of claim 36 in which the non-single-stranded region is multistranded.
53 . The method of claim 52 in which the multistranded region is double-stranded.
54 . The method of claim 52 in which the multistranded region is triple-stranded.
55 . The method of claim 52 in which at least one strand of at least one of the multistranded region is a DNA.
56 . The method of claim 52 in which at least one strand of at least one of the multistranded region is an RNA.
57 . The method of claim 52 in which the at least one stand of at least one of the multistranded region is a oligonucleotide analog or polynucleotide analog.
58 . The method of claim 57 in which the analog is PNA.
59 . The method of claim 36 in which at least one of the multistranded region comprises a detectable tag.
60 . The method of claim 59 in which the detectable tag is an electronic label.
61 . The method of claim 60 in which the electronic label is ferrocene.
62 . The method of claim 59 in which the detectable tag is a fluorescent label.
63 . The method of claim 59 in which the detectable tag is a bulky adduct.
64 . The method of claim 59 in which the detectable tag is on at least one strand of the multistranded region.
65 . The method of claim 36 in which the first moiety comprises a first nucleobase polymer probe capable of binding to the first target analyte, and the second moiety comprises a second nucleobase polymer probe capable of binding to a second target analyte, wherein in the first and second target analytes comprise different target nucleobase polymers.
66 . The method of claim 65 in which the sequences of the first and second target nucleobase polymers have a single nucleotide difference.
67 . The method of claim 66 in which the single nucleotide difference is a single nucleotide polymorphism.
68 . A method of detecting a target analyte, comprising:
(a) translocating a coded molecule of a population of coded molecules through a nanopore, wherein the population of coded molecules comprises a plurality of subpopulations, the coded molecule of each subpopulation comprising:
(i) an ordered plurality of code regions formed on a single-stranded nucleobase polymer scaffold, wherein one or more of the code regions is non-single-stranded and each code region comprises a detectable property such that the ordered code regions generates a defined signal pattern characteristic for each subpopulation; and
(ii) a moiety capable of binding to a target analyte, wherein the moiety of each subpopulation binds to a different target analyte, and
wherein the nanopore is dimensioned for transit of the non-single-stranded region;
(b) sensing the detectable property of each code region translocating through the nanopore to generate the signal pattern; and
(c) relating the generated signal pattern to the moiety of the subpopulation.
69 . The method of claim 68 , further comprising detecting the presence of the target analyte bound to the moiety.
70 . A method of forming a coded molecule, comprising:
(a) contacting a microcapsule with a first nucleobase oligomer, wherein the microcapsule comprises a single-stranded nucleobase scaffold; and
(b) hybridizing the first nucleobase oligomer to the scaffold, wherein the first nucleobase oligomer hybridizes to a first defined sequence on the scaffold to form a first non-single-stranded code region.
71 . The method of claim 70 , further comprising:
(c) contacting the microcapsule with an interstrand crosslinking agent to crosslink the hybridized first nucleobase oligomer to the scaffold.
72 . The method of claim 70 further comprising:
contacting the microcapsule with a second nucleobase oligomer, wherein the second nucleobase oligomer hybridizes to a second defined sequence on the scaffold to form a second non-single-stranded code region.
73 . The method of claim 72 , further comprising contacting the microcapsule with an interstrand crosslinking agent to crosslink the hybridized second nucleobase oligomer to the scaffold.
74 . The method of claim 72 in which the microcapsule is contacted with the second nucleobase oligomer subsequent to hybridization of the first nucleobase oligomer to the scaffold.
75 . The method of claim 70 in which the microcapsule comprises an inverse emulsion.
76 . The method of claim 72 in which the nucleobase oligomers are perfectly complementary to the defined sequences on the scaffold.
77 . A method of forming a coded molecule, comprising
(a) contacting a population of microcapsules with a first nucleobase oligomer, wherein the microcapsules comprise a single-stranded nucleobase scaffold and the first nucleobase oligomer hybridizes to a first defined sequence on the scaffold to form a first non-single-stranded code region;
(b) generating from the population of microcapsules at least a first and second subpopulation of microcapsules; and
(c) contacting the first subpopulation of microcapsules with a second nucleobase oligomer and the third subpopulation with a third nucleobase oligomer, wherein the second nucleobase oligomer hybridizes to a second defined sequence and the third nucleobase oligomer hybridizes to a third defined sequence on the scaffold to form a second non-single-stranded code region in each subpopulation.
78 . The method of claim 77 in which the second and third nucleobase oligomers are comprised of different nucleobase polymers.
79 . The method of claim 78 in which the second nucleobase oligomer comprises a PNA and the third nucleobase oligomer comprises a DNA.
80 . The method of claim 77 in which the second and third defined sequences are the same.
81 . The method of claim 77 in which each nucleobase oligomer is perfectly complementary to the defined sequence on the scaffold.
82 . The method of claim 77 in which the microcapsules with the hybridized first nucleobase oligomer are contacted with an interstrand crosslinking agent to crosslink the hybridized first nucleobase oligomer to the scaffold.
83 . The method of claim 77 in which the microcapsules with the hybridized second nucleobase oligomer and third nucleobase oligomers are contacted with an interstrand crosslinking agent to crosslink the hybridized second nucleobase oligomer to the scaffold.
84 . A method of forming a coded molecule, comprising
(a) contacting a population of microcapsules with a first nucleobase oligomer, wherein the microcapsules comprise a single-stranded nucleobase scaffold and the first nucleobase oligomer hybridizes to a first defined sequence on the scaffold to form a first non-single-stranded code region;
(b) generating from the population of microcapsules a plurality of microcapsule subpopulations;
(c) contacting each subpopulation with a second nucleobase oligomer that hybridizes to a second defined sequence on the scaffold to form a second non-single-stranded code region, wherein the second nucleobase oligomer of each subpopulation comprises a different nucleobase polymer.
85 . The method of claim 84 in which the microcapsules with the hybridized first nucleobase oligomer are contacted with an interstrand crosslinking agent to crosslink the hybridized first nucleobase oligomer to the scaffold.
86 . The method of claim 84 in which the microcapsules with the hybridized second nucleobase oligomer are contacted with an interstrand crosslinking agent to crosslink the hybridized second nucleobase oligomer to the scaffold.