High throughput screening of aptamer libraries for specific binding to proteins on viruses and other pathogens
First Claim
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1. A method for isolating pathogen-specific aptamers, comprising the steps of:
- contacting a pathogen sample comprising a target with an aptamer library; and
determining the sequence of the one or more aptamers that bind specifically to the target in the pathogen sample.
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Abstract
The present invention includes composition and methods for making and using a combinatorial library to identify thioaptamers that bind to targets on or about pathogens. Compositions, kits and methods are also provided for the identification of pathogens, e.g., viral, bacterial or other proteins related infectious disease, as well as, vaccines and vaccine adjuvants are provided that modify host immune responses.
85 Citations
179 Claims
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1. A method for isolating pathogen-specific aptamers, comprising the steps of:
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contacting a pathogen sample comprising a target with an aptamer library; and
determining the sequence of the one or more aptamers that bind specifically to the target in the pathogen sample. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45)
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- 46. A pathogen-specific aptamer comprising a partially thio-modified aptamer that specifically binds to a pathogen.
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57. A method for isolating a pathogen comprising the steps of:
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contacting a library of thioaptamer beads with a labeled pathogen sample; and
sorting the thioaptamer-beads that bind specifically to the labeled pathogen by flow cytometry. - View Dependent Claims (58, 59, 60, 61, 62)
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63. A method for identifying thioaptamers that bind to a target protein comprising the steps of:
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deproteinating with urea to remove bound protein from one or more thioaptamer beads that have bound specifically to the target protein; and
sequencing the aptamer to obtain the sequence of the aptamer on the one or more beads that bound the target protein.
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64. A method for identifying thioaptamers that bind to a target protein comprising the steps of:
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(a) deproteinating with urea to remove bound protein from one or more thioaptamer beads that have bound specifically to the target protein, (b) subjecting the deproteinated beads to PCR to produce a PCR product; and
(c) sequencing the PCR product to obtain the sequence of the aptamer on the one or more beads that bound the target protein. - View Dependent Claims (65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75)
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76. A method for determining which thioaptamers bound to a target protein comprising the steps of:
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(a) deproteinating with urea to remove bound protein one or more thio-aptamer beads that have bound specifically to the target protein, (b) subjecting the deproteinated beads to PCR to produce a PCR product;
(c) sequencing the PCR product to obtain the sequence of the aptamer on the one or more beads that bound the target protein; and
(d) contacting a one bead-one aptamer library comprising all the aptamers the thio-aptamer beads of step (a) to identify one or more thio-aptamers that bind specifically to the target protein. - View Dependent Claims (77)
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78. A method for isolating a pathogen comprising the steps of:
preparing a pathogen-specific aptamer array to screen one or more samples for the presence of a pathogen that binds specifically to the pathogen-specific aptamer array.
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79. A pathogen-specific aptamer array comprising:
one or more isolated nucleic acid aptamers that bind specifically to a pathogen on an array, wherein a pathogen that binds to the isolated nucleic acid aptamers on the array is detected to identify the presence of the pathogen in a sample.
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80. A pathogen-specific aptamer screening system comprising:
one or more beads comprising an immobilized pathogen-specific aptamer, wherein a pathogen that binds specifically to the immobilized pathogen-specific aptamer is detected upon binding to the immobilized pathogen-specific aptamer. - View Dependent Claims (81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92)
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93. A method for concentrating a pathogen out of a sample, comprising:
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immobilizing a pathogen-specific aptamer on a substrate; and
exposing a sample suspected of comprising the pathogen to the immobilized aptamers. - View Dependent Claims (94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108)
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110. A method for identifying a pathogen comprising the steps of:
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immobilizing a pathogen-specific aptamer on a substrate;
exposing a sample suspected of comprising at least a portion of the pathogen to the immobilized aptamers; and
identifying the aptamer that bound the portion of the pathogen, wherein the aptamer is specific to at least a portion of the pathogen.
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- 111. A filter for concentrating a pathogen, comprising a pathogen-specific aptamer immobilized on a substrate, wherein a sample suspected of comprising the pathogen is exposed to the immobilized pathogen-specific aptamers to concentrate the pathogen.
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118. A method of concentrating a pathogen comprising the step of:
immobilizing a pathogen-specific partially thio-modified aptamer on a substrate, wherein a sample suspected of comprising a pathogen is exposed to the immobilized pathogen-specific aptamers to concentrate the pathogen.
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119. A cancer-specific aptamer screening system comprising:
one or more beads comprising an immobilized cancer-specific aptamer, wherein at least a portion of a cancer cell that binds specifically to the immobilized cancer-specific aptamer is detected. - View Dependent Claims (120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131)
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132. A thioaptamer comprising:
a cancer-specific thioaptamer, wherein the aptamer binds to a specific cancer cell aptamer epitope. - View Dependent Claims (133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 148, 149)
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147. A pharmaceutical composition comprising a therapeutically effective amount of a thioaptamer specific for a virally-infected cell having SEQ ID NOS.:
- 30 to 107, or mixtures thereof.
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150. A method of treatment with a thioaptamer comprising:
identifying a patient in need of thioaptamer therapy and providing a therapeutically effective amount of the thioaptamer to the patient. - View Dependent Claims (151)
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152. A method of treating a target cell with a thioaptamer comprising:
contacting a target cell with a thioaptamer, wherein the thioaptamer binds specifically to a thioaptamer target within the target cell to modify the physiology of the target cell. - View Dependent Claims (153, 154, 155, 156, 157)
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158. A kit for detecting a pathogen comprising:
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a pathogen-specific aptamer; and
instructions for use of the pathogen-specific aptamer to detect a binding target. - View Dependent Claims (159, 160, 161, 162, 163, 164, 165, 166)
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167. A kit for concentrating a pathogen from a sample comprising:
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one or more immobilized pathogen-specific thioaptamers; and
instructions for use of the one or more immobilized pathogen-specific thioaptamers.
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168. A kit for concentrating a pathogen in a sample comprising:
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an immobilized pathogen-specific aptamer on a substrate; and
instructions for use of the immobilized pathogen-specific aptamer to concentrate the pathogen. - View Dependent Claims (169, 170, 171, 172, 173)
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174. A kit for identifying a target-specific aptamer comprising
a thioaptamer bead library; - and
instructions for isolating one or more of the thioaptamer beads from the thioaptamer bead library after contacting the thioaptamer beads to an thioaptamer binding target.
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175. A kit for treating a pathogenic infection comprising:
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a vial comprising one or more pathogen-specific thioaptamers in a pharmaceutically acceptable form; and
instructions for use of the pathogen-specific thioaptamers to treat infection with a pathogen.
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176. A kit for treating a cancer comprising:
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a vial comprising one or more cancer cell-specific thioaptamers in a pharmaceutically acceptable form; and
instructions for use of the cancer-specific thioaptamers to treat a cancer.
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177. A kit for cosmetic treatment comprising:
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a vial comprising one or more cell-specific thioaptamers attached to an agent that causes cell death in a pharmaceutically acceptable form; and
instructions for use of the cell-specific thioaptamers to kill an unwanted cell.
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178. A cosmetic composition comprising, in a cosmetically acceptable aqueous medium at least one target-cell specific thioaptamer, wherein the thioaptamer further comprises an agent that leads to cell death.
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179. A method for cosmetic treatment for eliminating unwanted cell comprising the administration of a composition comprising at least an effective amount of a thioaptamer conjugated to an agent that causes cell death, or a salt thereof.
Specification