In Vitro Selection of RNA Aptamers to a Protein Target by Filter Immobilization

Hall, Bradley; Arshad, Seyed A.; Seo, Aaron; Bowman, Catherine M.; Corley, Meredith; Jhaveri, Sulay D.; Ellington, Andrew D.

doi:10.1002/0471142727.mb2403s88

Cited by 8 publications

(8 citation statements)

References 32 publications

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“…When the immobilisation of the target was performed at pH 7.4 but the pH was maintained at 4.5 during the SELEX process, no amplification was detected after a first round of selection. Another SELEX strategy without BACE1 biotinylation was then performed, with the complexes of bound ssXNA to the target protein being separated from unbound reagents by filtration . Unfortunately, no amplicon was obtained after the 11th round of selection despite several attempts.…”

Section: Resultsmentioning

confidence: 99%

Modulation of BACE1 Activity by Chemically Modified Aptamers

et al. 2018

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A modified DNA aptamer that binds BACE1, a therapeutic target involved in Alzheimer's disease has been developed. This ssXNA not only tightly binds to BACE1 but also inhibits its protease activity in vitro in the same range as a previously described unmodified aptamer. We report the in vitro selection of functional oligonucleotides incorporating two nucleobase modifications: 5-chlorouracil and 7-deazaadenine. The nucleoside analogue 5-chloro-2'-deoxyuridine has already been explored as a replacement for thymidine in a chemically modified genome of a bacterium. Thus, 5-chlorouracil modification is a good candidate to support genetic transfer in vivo as well as functional activity.

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Section: Resultsmentioning

confidence: 99%

Modulation of BACE1 Activity by Chemically Modified Aptamers

et al. 2018

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“…However, despite using the same N56 pool and other pools with random nucleotide lengths (N35 and N101) and the same selection conditions as used for Stx2, we could not identify aptamers that bound Stx1. Although reasons for not identifying Stx1-binding aptamers are unclear, it can be assumed that Stx1 may not be a “good” target as evidenced by other studies that not every target is suitable for aptamer selection [ 32 , 34 ].…”

Section: Discussionmentioning

confidence: 99%

Selective Evolution of Ligands by Exponential Enrichment to Identify RNA Aptamers against Shiga Toxins

Challa

Tzipori

Sheoran

2014

Journal of Nucleic Acids

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Infection with Shiga toxin- (Stx-) producing E. coli causes life threatening hemolytic uremic syndrome (HUS), a leading cause of acute renal failure in children. Of the two antigenically distinct toxins, Stx1 and Stx2, Stx2 is more firmly linked with the development of HUS. In the present study, selective evolution of ligands by exponential enrichment (SELEX) was used in an attempt to identify RNA aptamers against Stx1 and Stx2. After 5 rounds of selection, significant enrichment of aptamer pool was obtained against Stx2, but not against Stx1, using a RNA aptamer library containing 56 random nucleotides (N56). Characterization of individual aptamer sequences revealed that six unique RNA aptamers (mA/pC, mB/pA, mC, mD, pB, and pD) recognized Stx2 in a filter binding assay. None of these aptamers bound Stx1. Aptamers mA/pC, mB/pA, mC, and mD, but not pB and pD, partially blocked binding of Alexa 488-labeled Stx2 with HeLa cells in a flow cytometry assay. However, none of the aptamers neutralized Stx2-mediated cytotoxicity and death of HeLa cells.

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“…Subsequent CE-based partitioning reduces the number of rounds needed due to increase in separation efficiency and higher sensitivity of LIF detection and without the need for intermittent amplification. This is in comparison to other more traditional techniques such as using membrane filters on their own, magnetic beads or affinity columns that often require more than ten rounds of partitioning, amplification, and strand separation to enrich the library [16,17]. In addition, CE-based studies, can be carried out in free solution removing the need for target immobilization and because of the difference in migration times, negative selection is not required.…”

Section: Methodsmentioning

confidence: 99%

“…The original selection of aptamers by NC membrane filters involved immobilizing the protein onto the membrane filter and blocking the remaining sites with BSA to prevent nonspecific binding of DNA sequences to the filter [17]. In our procedure, we chose not to immobilize the protein onto the membrane and instead incubated the DNA library with the target protein in solution to form the complex before applying it directly onto the membrane.…”

Section: Selex Proceduresmentioning

confidence: 99%

Selection of cholesterol esterase aptamers using a dual‐partitioning approach

Ashley

2015

Electrophoresis

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Non-systematic evolution of ligands by exponential enrichment and other capillary-based methods have grown in popularity for selection of aptamers since they provide a fast and efficient partitioning method when compared to classical techniques. Despite promising developments in these techniques, a major obstacle needs to be overcome for capillary-based selections to be widely accepted. Due to the small injection volumes associated with CE, only a small proportion of the nucleic acid library can be partitioned at any one time. In this paper, we propose a new two-step method for the selection of aptamers which firstly incorporates a nitrocellulose membrane filter followed by CE. This technique allows for nonbinding sequences to be eliminated, reducing the library size before subsequent capillary-based partitioning, while still reducing the time taken for aptamers to be selected. We demonstrated this technique on the selection of aptamers for cholesterol esterase and the highest binding truncated aptamer CES 4T displayed a K(D) of 203 ± 14 nM. In addition, an increase in the number of sequences partitioned was estimated using spectrophotometry and capillary injection volumes. The results suggested that for successful selection a two-step approach is needed. This hybrid technique could be used to select aptamers that bind to targets both in solution and immobilized onto a stationary phase, allowing the aptamers to be used in different binding environments.

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In Vitro Selection of RNA Aptamers to a Protein Target by Filter Immobilization

Cited by 8 publications

References 32 publications

Modulation of BACE1 Activity by Chemically Modified Aptamers

Modulation of BACE1 Activity by Chemically Modified Aptamers

Selective Evolution of Ligands by Exponential Enrichment to Identify RNA Aptamers against Shiga Toxins

Selection of cholesterol esterase aptamers using a dual‐partitioning approach

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