ALM

A pH Sensitive High-Throughput Assay for miRNA Binding of a Peptide-Aminoglycoside (PA) Library

Publication Date: December 11, 2015
Journal: PLOS ONE
Authors: Derrick Watkins, Liuwei Jiang, Smita Nahar, Souvik Maiti, et al
Volume: 10
Issue: 12
Pages: e0144251
DOI: https://dx.plos.org/10.1371/journal.pone.0144251
Publisher URL: http://journals.plos.org/plosone/article?id=10.1371%2Fjournal.pone.0144251
PubMed: http://www.ncbi.nlm.nih.gov/pubmed/26656788
PubMed Central: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4699463
Europe PMC: http://europepmc.org/abstract/MED/26656788
Web of Science: 000366903600032
Scopus: 84957108770
Mendeley: http://www.mendeley.com/research/ph-sensitive-highthroughput-assay-mirna-binding-peptideaminoglycoside-pa-library

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CiteULike11 May 22:26 UTC

CiteULike

Mendeley 728 Apr 16:05 UTC

Mendeley | Further Information

{"title"=>"A pH sensitive high-throughput assay for miRNA binding of a peptide-aminoglycoside (PA) library", "type"=>"journal", "authors"=>[{"first_name"=>"Derrick", "last_name"=>"Watkins", "scopus_author_id"=>"55430752900"}, {"first_name"=>"Liuwei", "last_name"=>"Jiang", "scopus_author_id"=>"56431703200"}, {"first_name"=>"Smita", "last_name"=>"Nahar", "scopus_author_id"=>"56118250800"}, {"first_name"=>"Souvik", "last_name"=>"Maiti", "scopus_author_id"=>"7202014988"}, {"first_name"=>"Dev P.", "last_name"=>"Arya", "scopus_author_id"=>"7004803271"}], "year"=>2015, "source"=>"PLoS ONE", "identifiers"=>{"doi"=>"10.1371/journal.pone.0144251", "issn"=>"19326203", "sgr"=>"84957108770", "scopus"=>"2-s2.0-84957108770", "pui"=>"607918471", "pmid"=>"26656788", "isbn"=>"1932-6203 (Electronic)\\r1932-6203 (Linking)"}, "id"=>"53a74dcb-105b-3b47-b9fa-dbd0ccc5a4f7", "abstract"=>"MicroRNAs (miRNA) are small RNAs that have a regulatory role in gene expression. Because of this regulatory role, miRNAs have become a new target for therapeutic compounds. Here, we outline an approach to target specific miRNAs using a high throughput capable assay and a 215 compound peptidic-aminosugar (PA) library. Aminosugars have been shown in a number of recent reports as important lead compounds that bind miRNA. In order to screen for compounds that bind miRNA, we have developed a high throughput displacement assay using a fluorescein-neomycin conjugated molecule (F-neo) as a probe for competitive miRNA binding compounds. We have applied the F-neo assay to four different miRNA constructs and the assay is applicable to most miRNAs, at various stages of processing. The results of the screen were validated by the determination of the IC50 for a select group of compounds from the library. For example, we identified eight compounds that bind to hsa-miR 504 with higher affinity than the parent neomycin. From the F-neo displacement assay we found that the number of binding sites differs for each miRNA, and the binding sites appear to differ both physically and chemically, with different affinity of the compounds resulting from the size of the molecule as well as the chemical structure. Additionally, the affinity of the compounds was dependent on the identity and position of the amino acid position of conjugation and the affinity of the compounds relative to other compounds in the library was miRNA dependent with the introduction of a second amino acid.", "link"=>"http://www.mendeley.com/research/ph-sensitive-highthroughput-assay-mirna-binding-peptideaminoglycoside-pa-library", "reader_count"=>7, "reader_count_by_academic_status"=>{"Unspecified"=>1, "Researcher"=>3, "Student > Ph. D. Student"=>1, "Student > Master"=>1, "Other"=>1}, "reader_count_by_user_role"=>{"Unspecified"=>1, "Researcher"=>3, "Student > Ph. D. Student"=>1, "Student > Master"=>1, "Other"=>1}, "reader_count_by_subject_area"=>{"Unspecified"=>2, "Biochemistry, Genetics and Molecular Biology"=>1, "Agricultural and Biological Sciences"=>3, "Pharmacology, Toxicology and Pharmaceutical Science"=>1}, "reader_count_by_subdiscipline"=>{"Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>3}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>1}, "Unspecified"=>{"Unspecified"=>2}, "Pharmacology, Toxicology and Pharmaceutical Science"=>{"Pharmacology, Toxicology and Pharmaceutical Science"=>1}}, "reader_count_by_country"=>{"Belgium"=>1}, "group_count"=>0}

ORCID09 May 08:14 UTC

Scopus | Further Information

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Web of Science®29 Mar 03:56 UTC

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Journal Comments13 Nov 23:18 UTC

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Wikipedia15 Mar 05:31 UTC

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Figshare 2004 May 07:11 UTC

Figshare

{"files"=>["https://ndownloader.figshare.com/files/2606938"], "description"=>"The preliminary miRNA (pre-miRNA) containing the mature 5’ (red) or 3’ (blue) strand is transported out of the nucleus by exportan5 in complex with RAN•GTP, and released into the cytoplasm. The pre-miRNA is cleaved by the endonuclease III, Dicer, to produce the mature duplex miRNA of ~22 bases per strand. The mature duplex miRNA is then loaded onto a argonaute (AGO) protein where it is unwound, the passenger strand is released, and the guide strand is arranged in the A-form conformation (modelled here as either or both the 5’ and 3’ strand is used as the guide strand). The target mRNA (lime green or magenta) is bound to the complex, resulting the suppression of its translation and protein synthesis. EXP5 (green) was modelled from PDB id:3A6P, Dicer (purple) was from PDB id:4NH5, and AGO (brown) was modelled from PDB id:4W5R.", "links"=>[], "tags"=>["binding sites", "pa", "miRNA binding compounds", "throughput displacement assay", "affinity", "ic", "bind miRNA"], "article_id"=>1623522, "categories"=>["Biological Sciences"], "users"=>["Derrick Watkins", "Liuwei Jiang", "Smita Nahar", "Souvik Maiti", "Dev P. Arya"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0144251.g001", "stats"=>{"downloads"=>1, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Schematic_of_miRNA_processing_/1623522", "title"=>"Schematic of miRNA processing.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-12-17 08:08:28"}
{"files"=>["https://ndownloader.figshare.com/files/2606939"], "description"=>"A) The chemical structures of neomycin, which was used as the positive control in the assay, B) the F-neo probe, C) the neomycin-amino acid conjugate, and D) the equilibrium between the monanion and dianion fluorescein and F-neo.", "links"=>[], "tags"=>["binding sites", "pa", "miRNA binding compounds", "throughput displacement assay", "affinity", "ic", "bind miRNA"], "article_id"=>1623523, "categories"=>["Biological Sciences"], "users"=>["Derrick Watkins", "Liuwei Jiang", "Smita Nahar", "Souvik Maiti", "Dev P. Arya"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0144251.g002", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_/1623523", "title"=>"A pH Sensitive High-Throughput Assay for miRNA Binding of a Peptide-Aminoglycoside (PA) Library", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-12-18 08:42:10"}
{"files"=>["https://ndownloader.figshare.com/files/2606940"], "description"=>"The miRNAs were screened for the change in fluorescence with the addition of equal molar concentrations of the probe F-neo. The 27 base model of the E. coli A-site is included as a positive control. The miRNA with the highest change in fluorescence, hsa-miR 504, the tumour suppressing miRNA with the lowest change in fluorescence, has-miR 142, and the oncogene with the lowest change in fluorescence, has-miR 335, were chosen for assay development and compound screening.", "links"=>[], "tags"=>["binding sites", "pa", "miRNA binding compounds", "throughput displacement assay", "affinity", "ic", "bind miRNA"], "article_id"=>1623524, "categories"=>["Biological Sciences"], "users"=>["Derrick Watkins", "Liuwei Jiang", "Smita Nahar", "Souvik Maiti", "Dev P. Arya"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0144251.g003", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_The_screening_of_the_20_miRNA_duplexes_containing_the_mature_miRNA_sequence_/1623524", "title"=>"The screening of the 20 miRNA duplexes containing the mature miRNA sequence.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-12-17 08:08:28"}
{"files"=>["https://ndownloader.figshare.com/files/2606941"], "description"=>"A) The absorbance spectra of 1 μM F-neo in 10 mM NaPO4, 25 mM KCl, and 0.05 mM EDTA pH 7.2, 6.4, and 5.0. B) The absorbance at 490 nm of F-neo as a function of pH in the absence (black) and presence (blue) of the mature duplex hsa-miR 504.", "links"=>[], "tags"=>["binding sites", "pa", "miRNA binding compounds", "throughput displacement assay", "affinity", "ic", "bind miRNA"], "article_id"=>1623525, "categories"=>["Biological Sciences"], "users"=>["Derrick Watkins", "Liuwei Jiang", "Smita Nahar", "Souvik Maiti", "Dev P. Arya"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0144251.g004", "stats"=>{"downloads"=>1, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_The_shift_in_the_pK_a_of_F_neo_in_the_presence_of_mature_duplex_miRNA_/1623525", "title"=>"The shift in the pKa of F-neo in the presence of mature duplex miRNA.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-12-17 08:08:28"}
{"files"=>["https://ndownloader.figshare.com/files/2606942"], "description"=>"Each miRNA was titrated into 100 nM F-neo. The titration was performed as the concentration of binding sites (Ψ). For (A) hsa-miR 142 and (B) hsa-miR 335, Ψ is equal to the number of moles of miRNA. (C) hsa-miR 504 has two binding sites on each molecule of miRNA, so Ψ is equal to two times the concentration of hsa-miR 504. (D) pre-hsa-miR 504 Ψ equals six times the concentration of miRNA.", "links"=>[], "tags"=>["binding sites", "pa", "miRNA binding compounds", "throughput displacement assay", "affinity", "ic", "bind miRNA"], "article_id"=>1623526, "categories"=>["Biological Sciences"], "users"=>["Derrick Watkins", "Liuwei Jiang", "Smita Nahar", "Souvik Maiti", "Dev P. Arya"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0144251.g005", "stats"=>{"downloads"=>0, "page_views"=>1, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_The_determination_of_the_apparent_dissociation_constant_of_F_neo_from_miRNA_binding_sites_/1623526", "title"=>"The determination of the apparent dissociation constant of F-neo from miRNA binding sites.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-12-17 08:08:31"}
{"files"=>["https://ndownloader.figshare.com/files/2606943"], "description"=>"The secondary structure predictions were taken miRbase [<a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144251#pone.0144251.ref056\" target=\"_blank\">56</a>–<a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144251#pone.0144251.ref060\" target=\"_blank\">60</a>]. Mature duplex miRNAs were constructed by the removal of bases from the pre-miRNA predictions not included in the mature sequence.", "links"=>[], "tags"=>["binding sites", "pa", "miRNA binding compounds", "throughput displacement assay", "affinity", "ic", "bind miRNA"], "article_id"=>1623527, "categories"=>["Biological Sciences"], "users"=>["Derrick Watkins", "Liuwei Jiang", "Smita Nahar", "Souvik Maiti", "Dev P. Arya"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0144251.g006", "stats"=>{"downloads"=>1, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_The_secondary_structure_of_mature_duplex_miRNAs_and_Pre_miRNA_screened_with_the_F_neo_displacement_assay_for_binding_with_PA_library_/1623527", "title"=>"The secondary structure of mature duplex miRNAs and Pre-miRNA screened with the F-neo displacement assay for binding with PA library.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-12-17 08:08:31"}
{"files"=>["https://ndownloader.figshare.com/files/2606944"], "description"=>"The fluorescence of F-neo (yellow, stick; fluorescence indicated when present) is quenched when bound in the groove of either the mature duplex or pre-miRNA. The addition of a miRNA binding compound, neomycin (cyan, stick), displaces the F-neo. The displacement of F-neo results in an increase in fluorescence. The mature miRNA is shown as a red (5’ strand) and blue (3’ strand) surface, and the addition of the loop region shown as a sky blue surface represents the pre-miRNA. The location of the binding site on the miRNA is highlighted with a black box was identified by docking experiments using Autodock Vina [<a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144251#pone.0144251.ref061\" target=\"_blank\">61</a>].", "links"=>[], "tags"=>["binding sites", "pa", "miRNA binding compounds", "throughput displacement assay", "affinity", "ic", "bind miRNA"], "article_id"=>1623528, "categories"=>["Biological Sciences"], "users"=>["Derrick Watkins", "Liuwei Jiang", "Smita Nahar", "Souvik Maiti", "Dev P. Arya"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0144251.g007", "stats"=>{"downloads"=>1, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Schematic_of_F_neo_displacement_assay_for_miRNA_binding_compounds_/1623528", "title"=>"Schematic of F-neo displacement assay for miRNA binding compounds.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-12-17 08:08:31"}
{"files"=>["https://ndownloader.figshare.com/files/2606945"], "description"=>"The IC50 for each compound was determined from a sigmoidal fit of a titration of the designated compound into 100 nM F-neo, 50 nM mature duplex hsa-miR 504 in 10 mM NaPO4, 25 mM KCl, 0.05 mM EDTA at pH 6.5. All measurements were taken in a 96-well black Greiner plate using a Tecan Infinite M-1000 plate reader with an excitation of 485 nm and an emission of 525 nm.", "links"=>[], "tags"=>["binding sites", "pa", "miRNA binding compounds", "throughput displacement assay", "affinity", "ic", "bind miRNA"], "article_id"=>1623529, "categories"=>["Biological Sciences"], "users"=>["Derrick Watkins", "Liuwei Jiang", "Smita Nahar", "Souvik Maiti", "Dev P. Arya"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0144251.g008", "stats"=>{"downloads"=>1, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_The_IC_50_determination_of_compounds_for_mature_duplex_hsa_miR_504_/1623529", "title"=>"The IC50 determination of compounds for mature duplex hsa-miR 504.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-12-17 08:08:30"}
{"files"=>["https://ndownloader.figshare.com/files/2606946"], "description"=>"The miRNA levels are normalized to U6 snRNA. Data are plotted as mean ± SEM *p < 0.05; **p < 0.01, unpaired two-tailed t test, n = 3.", "links"=>[], "tags"=>["binding sites", "pa", "miRNA binding compounds", "throughput displacement assay", "affinity", "ic", "bind miRNA"], "article_id"=>1623530, "categories"=>["Biological Sciences"], "users"=>["Derrick Watkins", "Liuwei Jiang", "Smita Nahar", "Souvik Maiti", "Dev P. Arya"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0144251.g009", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Relative_expression_of_miR_142_levels_after_treatment_of_20_uM_DPA_compounds_for_48_hrs_/1623530", "title"=>"Relative expression of miR-142 levels after treatment of 20 uM DPA compounds for 48 hrs.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-12-17 08:08:29"}
{"files"=>["https://ndownloader.figshare.com/files/2606947"], "description"=>"The miRNA levels are normalized to U6 snRNA. Data are plotted as mean ± SEM *p < 0.05; **p < 0.01, unpaired two-tailed t test, n = 3.", "links"=>[], "tags"=>["binding sites", "pa", "miRNA binding compounds", "throughput displacement assay", "affinity", "ic", "bind miRNA"], "article_id"=>1623531, "categories"=>["Biological Sciences"], "users"=>["Derrick Watkins", "Liuwei Jiang", "Smita Nahar", "Souvik Maiti", "Dev P. Arya"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0144251.g010", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Relative_expression_of_miR_335_levels_after_treatment_of_20_uM_DPA_compounds_for_48_hrs_/1623531", "title"=>"Relative expression of miR-335 levels after treatment of 20 uM DPA compounds for 48 hrs.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-12-17 08:08:41"}
{"files"=>["https://ndownloader.figshare.com/files/2606948"], "description"=>"The miRNA levels are normalized to U6 snRNA. Data are plotted as mean ± SEM *p < 0.05; **p < 0.01, unpaired two-tailed t test, n = 3.", "links"=>[], "tags"=>["binding sites", "pa", "miRNA binding compounds", "throughput displacement assay", "affinity", "ic", "bind miRNA"], "article_id"=>1623532, "categories"=>["Biological Sciences"], "users"=>["Derrick Watkins", "Liuwei Jiang", "Smita Nahar", "Souvik Maiti", "Dev P. Arya"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0144251.g011", "stats"=>{"downloads"=>2, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Relative_expression_of_miR_504_levels_after_treatment_of_20_uM_DPA_compounds_for_48_hrs_/1623532", "title"=>"Relative expression of miR-504 levels after treatment of 20 uM DPA compounds for 48 hrs.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2015-12-17 08:08:35"}
{"files"=>["https://ndownloader.figshare.com/files/2606949", "https://ndownloader.figshare.com/files/2606950", "https://ndownloader.figshare.com/files/2606951", "https://ndownloader.figshare.com/files/2606952", "https://ndownloader.figshare.com/files/2606953", "https://ndownloader.figshare.com/files/2606954", "https://ndownloader.figshare.com/files/2606955", "https://ndownloader.figshare.com/files/2606956", "https://ndownloader.figshare.com/files/2606957"], "description"=>"<div>MicroRNAs (miRNA) are small RNAs that have a regulatory role in gene expression. Because of this regulatory role, miRNAs have become a new target for therapeutic compounds. Here, we outline an approach to target specific miRNAs using a high throughput capable assay and a 215 compound peptidic-aminosugar (PA) library. Aminosugars have been shown in a number of recent reports as important lead compounds that bind miRNA. In order to screen for compounds that bind miRNA, we have developed a high throughput displacement assay using a fluorescein-neomycin conjugated molecule (F-neo) as a probe for competitive miRNA binding compounds. We have applied the F-neo assay to four different miRNA constructs and the assay is applicable to most miRNAs, at various stages of processing. The results of the screen were validated by the determination of the IC50 for a select group of compounds from the library. For example, we identified eight compounds that bind to hsa-miR 504 with higher affinity than the parent neomycin. From the F-neo displacement assay we found that the number of binding sites differs for each miRNA, and the binding sites appear to differ both physically and chemically, with different affinity of the compounds resulting from the size of the molecule as well as the chemical structure. Additionally, the affinity of the compounds was dependent on the identity and position of the amino acid position of conjugation and the affinity of the compounds relative to other compounds in the library was miRNA dependent with the introduction of a second amino acid.</div>", "links"=>[], "tags"=>["binding sites", "pa", "miRNA binding compounds", "throughput displacement assay", "affinity", "ic", "bind miRNA"], "article_id"=>1623533, "categories"=>["Biological Sciences"], "users"=>["Derrick Watkins", "Liuwei Jiang", "Smita Nahar", "Souvik Maiti", "Dev P. Arya"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0144251.s001", "https://dx.doi.org/10.1371/journal.pone.0144251.s002", "https://dx.doi.org/10.1371/journal.pone.0144251.s003", "https://dx.doi.org/10.1371/journal.pone.0144251.s004", "https://dx.doi.org/10.1371/journal.pone.0144251.s005", "https://dx.doi.org/10.1371/journal.pone.0144251.s006", "https://dx.doi.org/10.1371/journal.pone.0144251.s007", "https://dx.doi.org/10.1371/journal.pone.0144251.s008", "https://dx.doi.org/10.1371/journal.pone.0144251.s009"], "stats"=>{"downloads"=>12, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_A_pH_Sensitive_High_Throughput_Assay_for_miRNA_Binding_of_a_Peptide_Aminoglycoside_PA_Library_/1623533", "title"=>"A pH Sensitive High-Throughput Assay for miRNA Binding of a Peptide-Aminoglycoside (PA) Library", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2015-12-17 08:08:29"}

PMC Usage Stats 32917 Mar 21:27 UTC

PMC Usage Stats | Further Information

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Relative Metric10 May 18:29 UTC

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{"start_date"=>"2015-01-01T00:00:00Z", "end_date"=>"2015-12-31T00:00:00Z", "subject_areas"=>[]}

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