Microfluidic methods for aptamer selection and characterization

Dembowski, Sean K.; Bowser, Michael T.

doi:10.1039/c7an01046j

Cited by 49 publications

(35 citation statements)

References 110 publications

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“…Recent advances in aptamer technology have enabled increased selection throughput and high-resolution analysis of selection outcomes. 29 , 30 Microfluidic 31 and multiplexed selection platforms 32 , 33 facilitate efficient and simultaneous selection against numerous targets, and emulsion methods that encapsulate single genotypes into “monoclonal aptamer particles” can readily partition or sort according to phenotype. 34 , 35 Post-selection, HTS has empowered optimization of candidate aptamers on the basis of round-to-round enrichment of sequences and by providing large datasets that allow powerful bioinformatic analyses of sequence, structure, and function relationships.…”

Section: Discussionmentioning

confidence: 99%

Poly-Target Selection Identifies Broad-Spectrum RNA Aptamers

Alam

Chang

Lange

et al. 2018

Molecular Therapy - Nucleic Acids

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Aptamer selections often yield distinct subpopulations, each with unique phenotypes that can be leveraged for specialized applications. Although most selections aim to attain ever higher specificity, we sought to identify aptamers that recognize increasingly divergent primate lentiviral reverse transcriptases (RTs). We hypothesized that aptamer subpopulations in libraries pre-enriched against a single RT may exhibit broad-spectrum binding and inhibition, and we devised a multiplexed poly-target selection to elicit those phenotypes against a panel of primate lentiviral RTs. High-throughput sequencing and coenrichment/codepletion analysis of parallel and duplicate selection trajectories rapidly narrowed the list of candidate aptamers by orders of magnitude and identified dozens of priority candidates for further screening. Biochemical characterization validated a novel aptamer motif and several rare and unobserved variants of previously known motifs that inhibited recombinant RTs to varying degrees. These broad-spectrum aptamers also suppressed replication of viral constructs carrying phylogenetically diverse RTs. The poly-target selection and coenrichment/codepletion approach described herein is a generalizable strategy for identifying cross-reactivity among related targets from combinatorial libraries.

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Section: Discussionmentioning

confidence: 99%

Poly-Target Selection Identifies Broad-Spectrum RNA Aptamers

Alam

Chang

Lange

et al. 2018

Molecular Therapy - Nucleic Acids

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“…Significant progresses were achieved in optimizing specific technical protocols or introducing new methods and automatic processes. There are an ongoing number of comprehensive reviews addressing the current status of the SELEX technology and highlighting the recent progress but also giving main obstacles and limitations of the process [ 9 , 12 , 13 , 14 , 15 , 16 , 17 ].…”

Section: Introductionmentioning

confidence: 99%

Refining the Results of a Classical SELEX Experiment by Expanding the Sequence Data Set of an Aptamer Pool Selected for Protein A

Stoltenburg

Strehlitz

2018

IJMS

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New, as yet undiscovered aptamers for Protein A were identified by applying next generation sequencing (NGS) to a previously selected aptamer pool. This pool was obtained in a classical SELEX (Systematic Evolution of Ligands by EXponential enrichment) experiment using the FluMag-SELEX procedure followed by cloning and Sanger sequencing. PA#2/8 was identified as the only Protein A-binding aptamer from the Sanger sequence pool, and was shown to be able to bind intact cells of Staphylococcus aureus. In this study, we show the extension of the SELEX results by re-sequencing of the same aptamer pool using a medium throughput NGS approach and data analysis. Both data pools were compared. They confirm the selection of a highly complex and heterogeneous oligonucleotide pool and show consistently a high content of orphans as well as a similar relative frequency of certain sequence groups. But in contrast to the Sanger data pool, the NGS pool was clearly dominated by one sequence group containing the known Protein A-binding aptamer PA#2/8 as the most frequent sequence in this group. In addition, we found two new sequence groups in the NGS pool represented by PA-C10 and PA-C8, respectively, which also have high specificity for Protein A. Comparative affinity studies reveal differences between the aptamers and confirm that PA#2/8 remains the most potent sequence within the selected aptamer pool reaching affinities in the low nanomolar range of KD = 20 ± 1 nM.

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“…It offers a range of capabilities of high-resolution separation between oligonucleotide candidates using small quantities of reagents and samples. A single-round screening of aptamers was reported and this marked the innovation of a fully automated and integrated miniaturized SELEX process [ 10 ].…”

Section: The Selex Methods (In-vitro Selection)mentioning

confidence: 99%

Recent Microdevice-Based Aptamer Sensors

et al. 2018

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Since the systematic evolution of ligands by exponential enrichment (SELEX) method was developed, aptamers have made significant contributions as bio-recognition sensors. Microdevice systems allow for low reagent consumption, high-throughput of samples, and disposability. Due to these advantages, there has been an increasing demand to develop microfluidic-based aptasensors for analytical technique applications. This review introduces the principal concepts of aptasensors and then presents some advanced applications of microdevice-based aptasensors on several platforms. Highly sensitive detection techniques, such as electrochemical and optical detection, have been integrated into lab-on-a-chip devices and researchers have moved towards the goal of establishing point-of-care diagnoses for target analyses.

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Microfluidic methods for aptamer selection and characterization

Cited by 49 publications

References 110 publications

Poly-Target Selection Identifies Broad-Spectrum RNA Aptamers

Poly-Target Selection Identifies Broad-Spectrum RNA Aptamers

Refining the Results of a Classical SELEX Experiment by Expanding the Sequence Data Set of an Aptamer Pool Selected for Protein A

Recent Microdevice-Based Aptamer Sensors

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