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Matrix Imprinting and Clearing
| Content Provider | The Lens |
|---|---|
| Abstract | The present invention generally relates to systems and methods for imaging or determining nucleic acids or other desired targets, for instance, within cells or tissues. In one aspect, a sample is exposed to a plurality of nucleic acid probes that are determined within the sample. In some cases, however, background fluorescence or off-target binding may make it more difficult to determine properly bound nucleic acid probes. Accordingly, other components of the samples that may be contributing to the background, such as proteins, lipids, and/or other non-targets, may be “cleared” from the sample to improve determination. However, in certain embodiments, nucleic acids or other desired targets may be prevented from also being cleared, e.g., using polymers or gels within the sample. Other aspects are generally directed to compositions or kits involving such systems, methods of using such systems, or the like. |
| Related Links | https://www.lens.org/lens/patent/011-085-685-446-819/frontpage |
| Language | English |
| Publisher Date | 2019-08-29 |
| Access Restriction | Open |
| Content Type | Text |
| Resource Type | Patent |
| Jurisdiction | United States of America |
| Date Applied | 2017-11-08 |
| Applicant | Harvard College |
| Application No. | 201716347874 |
| Claim | A method, comprising: exposing a sample to a plurality of nucleic acid probes; polymerizing a gel within the sample; anchoring a target to the gel; clearing non-targets from the sample; and determining the targets within the gel by determining binding of the nucleic acid probes by imaging. The method of claim 1 , wherein the target is a nucleic acid. The method of any one of claim 1 or 2 , wherein the target comprises RNA. The method of any one of claim 1 or 2 , wherein the target comprises DNA. The method of any one of claims 1 - 4 , wherein anchoring the target to the gel comprises anchoring the target to a nucleic acid probe and covalently bonding the nucleic acid probe to the ge The method of any one of claims 1 - 5 , wherein anchoring the target to the gel comprises anchoring the target to a nucleic acid probe and noncovalently bonding the nucleic acid probe to the ge The method of any one of claims 1 - 6 , wherein anchoring the target to the gel comprises anchoring the target to the gel via hybridization to the nucleic acid probes. The method of any one of claims 1 - 7 , wherein anchoring the target to the gel comprises anchoring the target to the gel via covalently bonding the target to the nucleic acid probes. The method of any one of claims 1 - 8 , wherein anchoring the target to the gel comprises anchoring the target to the gel by physically entangling the target with the ge The method of any one of claims 1 - 9 , wherein anchoring the target to the gel comprises covalently binding the target directly to the ge The method of any one of claims 1 - 10 , wherein anchoring the target to the gel comprises noncovalently binding the target directly to the ge The method of any one of claims 1 - 11 , wherein anchoring the target to the gel occurs during polymerizing the gel within the sample. The method of claim 12 , wherein the target is anchored to a gel precursor prior to polymerizing the gel precursor to form the gel within the sample. The method of any one of claims 1 - 13 , wherein anchoring the target to the gel occurs after polymerizing the gel within the sample. The method of claim 14 , wherein after polymerizing the gel within the sample, the gel and/or the target is modified to anchor the target to the ge The method of any one of claims 1 - 15 , wherein clearing non-targets from the sample occurs after anchoring the target to the ge The method of any one of claims 1 - 16 , wherein exposing the sample to the plurality of nucleic acid probes occurs prior to clearing non-targets from the sample. The method of any one of claims 1 - 17 , wherein exposing the sample to the plurality of nucleic acid probes occurs after clearing non-targets from the sample. The method of any one of claims 1 - 18 , wherein the non-targets include proteins. The method of any one of claims 1 - 19 , wherein the non-targets include lipids. The method of any one of claims 1 - 20 , wherein the non-targets include nucleic acid The method of claim 21 , wherein the non-targets include DNA. The method of any one of claim 21 or 22 , wherein the non-targets include RNA. The method of any one of claims 1 - 23 , wherein the non-targets include a carbohydrate. The method of any one of claims 1 - 24 , wherein the non-targets include extracellular matrix. The method of any one of claims 1 - 25 , wherein during imaging, the gel has not expanded by more than 3×. The method of any one of claims 1 - 26 , wherein during imaging, the gel has not expanded by more than 1.5×. The method of any one of claims 1 - 27 , wherein the plurality of nucleic acid probes comprises smFISH probes. The method of any one of claims 1 - 28 , wherein the plurality of nucleic acid probes comprises MERFISH probes. The method of any one of claims 1 - 29 , wherein the plurality of nucleic acid probes comprises anchor probes able to polymerize with the ge The method of any one of claims 1 - 29 , wherein the plurality of nucleic acid probes comprises anchor probes able to associate with the target and polymerize into the ge The method of any one of claim 30 or 31 , wherein at least some of the anchor probes comprises a poly-dT portion. The method of any one of claims 30 - 32 , wherein at least some of the anchor probes comprises alternating dT and locked dT portions. The method of claim 33 , wherein at least some of the anchor probes comprises a 15-nt sequence of alternating dT and locked dT portions. The method of any one of claims 30 - 34 , wherein at least some of the anchor probes comprises an acrydite portion able to polymerize with the ge The method of claim 35 , wherein the acrydite portion is bound to the 5′ end. The method of claim 35 , wherein the acrydite portion is bound to the 3′ end. The method of claim 35 , wherein the acrydite portion is bound to an internal base. The method of any one of claims 1 - 38 , wherein the gel comprises polyacrylamide. The method of any one of claims 1 - 39 , wherein the gel comprises agarose. The method of any one of claims 1 - 40 , wherein clearing non-targets from the sample comprises exposing the gel to a proteinase. The method of claim 41 , wherein the proteinase comprises proteinase K. The method of any one of claims 1 - 42 , wherein clearing non-targets from the sample comprises exposing the gel to guanidine HC The method of any one of claims 1 - 43 , wherein clearing non-targets from the sample comprises exposing the gel to Triton X-100 (polyethylene glycol p-(1,1,3,3-tetramethylbutyl)-phenyl ether). The method of any one of claims 1 - 44 , wherein clearing non-targets from the sample comprises exposing the gel to sodium dodecyl sulfate. The method of any one of claims 1 - 45 , wherein clearing non-targets from the sample comprises exposing the gel to ethylenediaminetetraacetic acid. The method of any one of claims 1 - 46 , wherein clearing non-targets from the sample comprises removing proteins and/or lipids from the sample. The method of any one of claims 1 - 47 , wherein clearing non-targets from the sample comprises degrading proteins and/or lipids from the sample. The method of any one of claims 1 - 48 , wherein clearing non-targets from the sample comprises removing DNA from the sample. The method of claim 49 , wherein removing DNA from the sample comprises exposing the sample to a DNAse. The method of any one of claims 1 - 50 , wherein the nucleic acid probes comprise a first portion comprising a target sequence and a second portion comprising one or more read sequences. The method of claim 51 , further comprising determining read sequences based on determining binding of the read sequences bound to the ge The method of any one of claim 51 or 52 , comprising creating codewords or barcodes based on determination of the read sequences within the ge The method of any one of claims 51 - 53 , wherein the read sequences are taken from a set of orthogonal sequences, which have a homology of less than 15 basepairs with one another and with the nucleic acid species in a sample. The method of any one of claims 1 - 54 , wherein the sample comprises a cel The method of any one of claims 1 - 55 , wherein the sample comprises a tissue. The method of any one of claims 1 - 56 , comprising imaging using fluorescence microscopy. The method of any one of claims 1 - 57 , comprising imaging using epi-fluorescence microscopy, total-internal-reflectance microscopy, highly-inclined thin-illumination (HILO) microscopy, light-sheet microscopy, scanning confocal microscopy, scanning line confocal microscopy, or spinning disk confocal microscopy. The method of any one of claims 1 - 58 , comprising imaging using multiplexed fluorescence in situ hybridization. The method of any one of claims 1 - 59 , comprising imaging using multiplexed error robust fluorescence in situ hybridization (MERFISH). The method of any one of claims 1 - 60 , comprising imaging using multiple rounds of fluorescence in situ hybridization. The method of any one of claims 1 - 61 , comprising imaging using multiple rounds of fluorescence in situ hybridization wherein, in each round, one or more different nucleic acid probes, each conjugated to a spectrally distinct fluorescent dye are used to readout out multiple readout sequences simultaneously. The method of any one of claims 1 - 62 , comprising imaging at a resolution better than 500 nm. The method of any one of claims 1 - 63 , comprising imaging using a technique selected from the group consisting of STORM, PALM, FPALM, STED, SIM, RESOLFT, SOFI or SPDM. A method, comprising: exposing a sample to a plurality of nucleic acid probes; polymerizing a gel within the sample; anchoring a target to the gel; reducing background fluorescence within the sample; and imaging the nucleic acid probes. The method of claim 65 , wherein during imaging, the gel has not expanded by more than 3×. The method of any one of claim 65 or 66 , wherein during imaging, the gel has not expanded by more than 1.5×. The method of any one of claims 65 - 67 , wherein the plurality of nucleic acid probes comprises smFISH probes. The method of any one of claims 65 - 68 , wherein the plurality of nucleic acid probes comprises MERFISH probes. The method of any one of claims 65 - 69 , wherein the plurality of nucleic acid probes comprises anchor probes able to polymerize with the ge The method of claim 70 , wherein at least some of the anchor probes comprises a poly-dT portion. The method of claim 71 , wherein at least some of the anchor probes comprises alternating dT and locked dT portions. The method of claim 72 , wherein at least some of the anchor probes comprises a 15-nt sequence of alternating dT and locked dT portions. The method of any one of claims 70 - 73 , wherein at least some of the anchor probes comprises an acrydite portion able to polymerize with the ge The method of claim 74 , wherein the acrydite portion is bound to the 5′ end. The method of claim 74 , wherein the acrydite portion is bound to the 3′ end. The method of claim 74 , wherein the acrydite portion is bound to an internal base. The method of any one of claims 65 - 77 , wherein the gel comprises polyacrylamide. The method of any one of claims 65 - 78 , wherein the gel comprises agarose. The method of any one of claims 65 - 79 , wherein reducing background fluorescence comprises clearing cellular components. The method of any one of claims 65 - 80 , wherein reducing background fluorescence comprises clearing components that quench fluorescent molecules. The method of any one of claims 65 - 81 , wherein reducing background fluorescence comprises clearing autofluorescent components. The method of claim 82 , wherein clearing autofluorescent components comprises reacting the autofluorescent components. The method of any one of claim 82 or 83 , wherein reacting the autofluorescent components comprises exposing the gel to a proteinase. The method of claim 84 , wherein the proteinase comprises proteinase K. The method of any one of claims 82 - 85 , wherein reacting the autofluorescent components comprises exposing the gel to guanidine HC The method of any one of claims 82 - 86 , wherein reacting the autofluorescent components comprises exposing the gel to Triton X-100 (polyethylene glycol p-(1,1,3,3-tetramethylbutyl)-phenyl ether). The method of any one of claims 82 - 87 , wherein reacting the autofluorescent components comprises exposing the gel to sodium dodecyl sulfate. The method of any one of claims 82 - 88 , wherein reacting the autofluorescent components comprises exposing the gel to ethylenediaminetetraacetic acid. The method of any one of claims 82 - 89 , wherein reacting the autofluorescent components comprises removing proteins and/or lipids from the sample. The method of any one of claims 82 - 90 , wherein reacting the autofluorescent components comprises degrading proteins and/or lipids from the sample. The method of any one of claims 65 - 91 , wherein the nucleic acid probes comprise a first portion comprising a target sequence and a second portion comprising one or more read sequences. The method of claim 92 , further comprising determining read sequences based on determining binding of the read sequences bound to the ge The method of any one of claim 92 or 93 , comprising creating codewords based on determination of the read sequences within the ge The method of any one of claims 92 - 94 , wherein the read sequences are taken from a set of orthogonal sequences, which have a homology of less than 15 basepairs with one another and with the nucleic acid species in a sample. The method of any one of claims 65 - 95 , wherein the sample comprises a cel The method of any one of claims 65 - 96 , wherein the sample comprises a tissue. The method of any one of claims 65 - 97 , comprising imaging using fluorescence microscopy. The method of any one of claims 65 - 98 , comprising imaging using epi-fluorescence microscopy, total-internal-reflectance microscopy, highly-inclined thin-illumination (HILO) microscopy, light-sheet microscopy, scanning confocal microscopy, scanning line confocal microscopy, spinning disk confocal microscopy, or other comparable conventional microscopy techniques. The method of any one of claims 65 - 99 , comprising imaging using multiplexed fluorescence in situ hybridization. The method of any one of claims 65 - 100 , comprising imaging using multiplexed error robust fluorescence in situ hybridization (MERFISH). The method of any one of claims 65 - 101 , comprising imaging using multiple rounds of fluorescence in situ hybridization. The method of any one of claims 65 - 102 , comprising imaging at a resolution better than 500 nm. The method of any one of claims 65 - 103 , comprising imaging using a technique selected from the group consisting of STORM, PALM, FPALM, STED, SIM, RESOLFT, SOFI or SPDM. The method of any one of claims 65 - 104 , wherein anchoring the target to the gel comprises anchoring the target to a nucleic acid probe and covalently bonding the nucleic acid probe to the ge The method of any one of claims 65 - 105 , wherein anchoring the target to the gel comprises anchoring the target to a nucleic acid probe and noncovalently bonding the nucleic acid probe to the ge The method of any one of claims 65 - 106 , wherein anchoring the target to the gel comprises anchoring the target to the gel via hybridization to the nucleic acid probes. The method of any one of claims 65 - 107 , wherein anchoring the target to the gel comprises anchoring the target to the gel via covalently bonding the target to the nucleic acid probes. The method of any one of claims 65 - 108 , wherein anchoring the target to the gel comprises anchoring the target to the gel by physically entangling the target with the ge The method of any one of claims 65 - 109 , wherein anchoring the target to the gel comprises covalently binding the target directly to the ge The method of any one of claims 65 - 110 , wherein anchoring the target to the gel comprises noncovalently binding the target directly to the ge The method of any one of claims 65 - 111 , wherein anchoring the target to the gel occurs during polymerizing the gel within the sample. The method of claim 112 , wherein the target is anchored to a gel precursor prior to polymerizing the gel precursor to form the gel within the sample. The method of any one of claims 65 - 113 , wherein anchoring the target to the gel occurs after polymerizing the gel within the sample. The method of claim 114 , wherein after polymerizing the gel within the sample, the gel is modified to anchor the target to the ge The method of any one of claims 65 - 115 , wherein reducing background fluorescence occurs after anchoring the target to the ge The method of any one of claims 65 - 116 , wherein exposing the sample to the plurality of nucleic acid probes occurs prior to reducing background fluorescence. The method of any one of claims 65 - 117 , wherein exposing the sample to the plurality of nucleic acid probes occurs after reducing background fluorescence. A method, comprising: exposing a sample to a plurality of MERFISH nucleic acid probes; exposing a sample to a plurality of anchor nucleic acid probes; embedding at least a portion of the sample within a polyacrylamide gel; immobilizing at least some of the anchor nucleic acid probes to the polyacrylamide gel; clearing proteins and/or lipids and/or DNA and/or extracellular matrix and/or RNA molecules from the sample; and determining binding of the MERFISH nucleic acid probes by imaging the polyacrylamide ge The method of claim 119 , wherein the polyacrylamide gel comprises anchor probes incorporated within the polyacrylamide ge The method of any one of claim 119 or 120 , wherein clearing proteins and/or lipids from the sample comprises removing proteins and/or lipids from the sample. The method of any one of claims 119 - 121 , wherein clearing proteins and/or lipids from the sample comprises degrading proteins and/or lipids from the sample. The method of any one of claims 119 - 122 , wherein clearing removing DNA and/or RNA and/or extracellular matrix from the sample. The method of any one of claims 119 - 123 , wherein clearing comprises degrading DNA and/or RNA and/or extracellular matrix. The method of any one of claims 119 - 124 , wherein the nucleic acid probes comprise a first portion comprising a target sequence and a second portion comprising one or more read sequences. The method of claim 125 , further comprising determining read sequences based on determining binding of the read sequences bound to target RNAs. The method of any one of claim 125 or 126 , comprising creating codewords or barcodes based on determination of the read sequences within the ge The method of any one of claims 125 - 127 , wherein the read sequences are taken from a set of orthogonal sequences, which have a homology of less than 15 basepairs with one another and with the nucleic acid species in a sample. The method of any one of claims 119 - 128 , wherein at least some of the anchor probes comprises a poly-dT portion. The method of claim 129 , wherein at least some of the anchor probes comprises alternating dT and locked dT portions. The method of claim 130 , wherein at least some of the anchor probes comprises a 15-nt sequence of alternating dT and locked dT portions. The method of any one of claims 119 - 131 , wherein at least some of the anchor probes comprises an acrydite portion able to polymerize with the ge The method of claim 132 , wherein the acrydite portion is bound to the 5′ end. The method of claim 132 , wherein the acrydite portion is bound to the 3′ end. The method of claim 132 , wherein the acrydite portion is bound to an internal base. The method of any one of claims 119 - 135 , wherein clearing comprises exposing the gel to a proteinase. The method of claim 136 , wherein the proteinase comprises proteinase K. The method of any one of claims 119 - 137 , wherein clearing comprises exposing the gel to guanidine HC The method of any one of claims 119 - 138 , wherein clearing comprises exposing the gel to Triton X-100 (polyethylene glycol p-(1,1,3,3-tetramethylbutyl)-phenyl ether). The method of any one of claims 119 - 139 , wherein clearing comprises exposing the gel to sodium dodecyl sulfate. The method of any one of claims 119 - 140 , wherein clearing comprises exposing the gel to ethylenediaminetetraacetic acid. The method of any one of claims 119 - 141 , wherein clearing comprises removing proteins and/or lipids from the sample. The method of any one of claims 119 - 142 , wherein clearing comprises degrading proteins and/or lipids from the sample. The method of any one of claims 119 - 143 , wherein clearing non-targets from the sample comprises removing DNA from the sample. The method of claim 144 , wherein removing DNA from the sample comprises exposing the sample to a DNAse. The method of any one of claims 119 - 145 , wherein anchoring the target to the gel occurs during polymerizing the gel within the sample. The method of claim 146 , wherein the target is anchored to a gel precursor prior to polymerizing the gel precursor to form the gel within the sample. The method of any one of claims 119 - 147 , wherein anchoring the target to the gel occurs after polymerizing the gel within the sample. The method of any one of claims 119 - 148 , wherein the acts are performed in the order recited. The method of any one of claims 119 - 149 , wherein clearing occurs prior to exposing the sample to the plurality of anchor nucleic acid probes. A method, comprising: embedding at least a portion of a sample within a matrix; immobilizing targets to the matrix; clearing non-targets from the matrix; and imaging the targets within the matrix. The method of claim 151 , wherein the matrix comprises a polymer. The method of any one of claim 151 or 152 , wherein the matrix comprises a ge The method of any one of claims 151 - 153 , wherein the target comprises nucleic acids. The method of any one of claims 151 - 154 , wherein the target comprises proteins. The method of any one of claims 151 - 155 , wherein immobilizing targets to the matrix comprises incorporating an anchor probe to the matrix, wherein the anchor probe specifically binds the targets. The method of claim 156 , wherein the anchor probe comprises a nucleic acid able to specifically bind the targets. The method of any one of claim 156 or 157 , wherein the anchor probe comprises an antibody able to specifically bind the targets. The method of any one of claims 156 - 158 , wherein the anchor probe comprises a chemical crosslinker capable of covalently or non-covalently binding the specific targets and the matrix. The method of any one of claims 151 - 159 , wherein the target molecules are anchored to the matrix via physical entanglement within the matrix. The method of any one of claims 151 - 160 , wherein clearing non-targets comprises removing the non-targets from the matrix. The method of any one of claims 151 - 161 , wherein clearing non-targets comprises degrading the non-targets. The method of any one of claims 151 - 162 , wherein clearing non-targets comprises exposing the sample to an enzyme able to degrade a protein. The method of any one of claims 151 - 163 , wherein clearing non-targets comprises exposing the sample to a detergent. The method of any one of claims 151 - 164 , wherein clearing non-targets comprises exposing the sample to an enzyme able to degrade DNA. The method of any one of claims 151 - 165 , wherein clearing non-targets comprises exposing the sample to an enzyme able to degrade RNA. The method of any one of claims 151 - 166 , wherein clearing non-targets comprises exposing the sample to an enzyme able to degrade sugars or sugar-modified biomolecules. The method of any one of claims 151 - 167 , wherein imaging the targets comprises imaging using optical microscopy. The method of any one of claims 151 - 168 , wherein imaging the targets comprises imaging using fluorescence microscopy. |
| CPC Classification | MEASURING OR TESTING PROCESSES INVOLVING ENZYMES; NUCLEIC ACIDS OR MICROORGANISMS ;COMPOSITIONS OR TEST PAPERS THEREFOR;PROCESSES OF PREPARING SUCH COMPOSITIONS;CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES Bioinformatics; I.E. Information And Communication Technology [Ict] Specially Adapted For Genetic Or Protein-Related Data Processing In Computational Molecular Biology Investigating Or Analysing Materials By Determining Their Chemical Or Physical Properties |
| Extended Family | 009-756-799-518-684 063-745-539-110-767 190-852-819-772-237 049-113-402-135-610 191-923-904-399-037 040-933-650-438-270 052-916-163-879-248 129-932-435-285-593 127-434-143-802-793 050-802-467-179-533 054-663-538-801-296 155-678-401-703-98X 023-202-808-184-309 143-291-332-519-115 162-215-449-461-556 011-085-685-446-819 116-041-848-048-050 088-960-885-975-252 029-910-778-614-288 138-614-618-901-216 143-354-960-037-163 123-499-599-488-458 103-097-155-204-736 193-127-931-839-419 |
| Patent ID | 20190264270 |
| Inventor/Author | Zhuang Xiaowei Moffitt Jeffrey R Hao Junjie George Lu Tian |
| IPC | C12Q1/6841 G16B25/00 C12Q1/6806 C12Q1/6837 G01N21/64 |
| Status | Discontinued |
| Owner | Howard Hughes Medical Institute President and Fellows of Harvard College |
| Simple Family | 155-678-401-703-98X 162-215-449-461-556 049-113-402-135-610 011-085-685-446-819 138-614-618-901-216 052-916-163-879-248 143-354-960-037-163 129-932-435-285-593 127-434-143-802-793 193-127-931-839-419 |
| CPC (with Group) | C12Q1/6841 G16B25/00 C12Q1/6806 C12Q1/6837 C12Q2537/143 C12Q2543/10 C12Q2563/107 G01N21/6458 C12Q1/68 |
| Issuing Authority | United States Patent and Trademark Office (USPTO) |
| Kind | Patent Application Publication |
