Aptamer Bioinformatics

Kinghorn, Andrew B.; Fraser, Lewis A.; Liang, Shuailong; Shiu, Simon Chi‐Chin; Tanner, Julian A.

doi:10.3390/ijms18122516

Cited by 130 publications

(76 citation statements)

References 118 publications

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“…The high repeat of a sequence in a cell-SELEX library does not guarantee specificity or affinity toward a receptor. For example, analysis of enriched aptamer libraries by HT sequencing, or Sanger sequencing, revealed that high-affinity sequences do not necessarily appear in high repeats, as determined in several studies 26, 27, 28. Outcompeted molecules in LIGS pools can be divided into three types of DNA ligands: those that specifically outcompeted DNA ligands resulting from specific mAb competition, those weak binding DNA ligands with high off-rates, and those nonspecifically eluted off-target sequences.…”

Section: Discussionmentioning

confidence: 99%

Integrating Ligand-Receptor Interactions and In Vitro Evolution for Streamlined Discovery of Artificial Nucleic Acid Ligands

Zumrut

Batool

Argyropoulos

et al. 2019

Molecular Therapy - Nucleic Acids

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To discover DNA ligands against a predetermined receptor protein complex, we introduce a comprehensive version of ligand-guided selection (LIGS). LIGS is, itself, a variant of systematic evolution of ligands by exponential enrichment (SELEX). Herein, we have optimized LIGS to identify higher affinity aptamers with high specificity. In addition, we demonstrate the expandability of LIGS by performing specific aptamer elution at 25°C, utilizing multiple monoclonal antibodies (mAbs) against cultured cells and primary cells obtained from human donors expressing the same receptor. Eluted LIGS libraries obtained through Illumina high-throughput (HT) DNA sequencing were analyzed by bioinformatics tools to discover five DNA aptamers with apparent affinities ranging from 3.06 ± 0.485 nM to 325 ± 62.7 nM against the target, T cell receptor-cluster of differentiation epsilon (TCR-CD3ε) expressed on human T cells. The specificity of the aptamers was validated utilizing multiple strategies, including competitive binding analysis and a double-knockout Jurkat cell line generated by CRISPR technology. The cross-competition experiments using labeled and unlabeled aptamers revealed that all five aptamers compete for the same binding site. Collectively, the data in this report introduce a modified LIGS strategy as a universal platform to identify highly specific multiple aptamers toward multi-component receptor proteins in their native state without changing the cell-surface landscape.

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Section: Discussionmentioning

confidence: 99%

Integrating Ligand-Receptor Interactions and In Vitro Evolution for Streamlined Discovery of Artificial Nucleic Acid Ligands

Zumrut

Batool

Argyropoulos

et al. 2019

Molecular Therapy - Nucleic Acids

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“…Therefore, most of the modifications aim at the improvement and acceleration of the aptamer selection process [ 12 , 13 ]. In this regard, NGS technologies have contributed substantially in the last years [ 15 , 18 , 19 , 20 , 21 ]. NGS has the potential to completely replace cloning and Sanger sequencing.…”

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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“…Although we started with a 60 nt library with 30 random sequences, the selected sequence was only a 41 nt. This sequence is generally thought to be a PCR error when amplified for the next round [21] and is excluded from the candidates. However, this sequence was confirmed extraordinarily, leading us to select it.…”

Section: Discussionmentioning

confidence: 99%

Detection of Nonylphenol with a Gold-Nanoparticle-Based Small-Molecule Sensing System Using an ssDNA Aptamer

Kim

et al. 2019

IJMS

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Endocrine-disrupting chemicals (EDCs) threaten many kinds of life throughout the world. These compounds function the same as sexual hormones, inducing precocious puberty, gynecomastia, etc., in the human body. To prevent excess exposure to nonylphenol (NP), a simple and rapid detection system is needed. In this study, we develop a nonylphenol-specific aptamer from a random single-stranded DNA library and test a rapid sensor system based on the aptamer and gold nanoparticles (AuNPs). The aptamer was screened by a methodology involving reduced graphene oxide (rGO). As a result of screening and sequencing, a DNA aptamer was developed that recognizes the target with high binding affinity (Kd = 194.2 ± 65.9 nM) and specificity. The sensor system developed using the aptamer and gold nanoparticles is sensitive (LOD = 2.239 nM). Circular dichroism (CD) spectrometry results show that the free aptamer binds to the target molecule. The aptamer was characterized using gold nanoparticles to measure UV absorbance. Our results suggest that the sensor system developed using this aptamer is useful for field diagnosis of small molecules.

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Aptamer Bioinformatics

Cited by 130 publications

References 118 publications

Integrating Ligand-Receptor Interactions and In Vitro Evolution for Streamlined Discovery of Artificial Nucleic Acid Ligands

Integrating Ligand-Receptor Interactions and In Vitro Evolution for Streamlined Discovery of Artificial Nucleic Acid Ligands

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

Detection of Nonylphenol with a Gold-Nanoparticle-Based Small-Molecule Sensing System Using an ssDNA Aptamer

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