Aptamer Selection by High-Throughput Sequencing and Informatic Analysis

Hoon, Shawn; Zhou, Bin; Janda, Kim D.; Brenner, Sydney; Scolnick, Jonathan

doi:10.2144/000113786

Cited by 81 publications

(64 citation statements)

References 13 publications

(13 reference statements)

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“…Aptamers are selected from libraries through a repetitive combinatorial process known as SELEX (Systematic Evolution of Ligands by Exponential Enrichment) (160,161). Recent improvements in SELEX technology have considerably accelerated the time in which aptamers can be selected, especially with the introduction of high throughput sequencing, which allows for the identification of high-affinity aptamers in early rounds of selection (162,163). This approach also reduces PCR biases that are introduced in each round of selection (164).…”

Section: Rna Aptamers As Novel Therapeutic For Costimulation Of 41bbmentioning

confidence: 99%

Agonists of Co-stimulation in Cancer Immunotherapy Directed Against CD137, OX40, GITR, CD27, CD28, and ICOS

Sanmamed

Pastor

Rodríguez

et al. 2015

Seminars in Oncology

176

141

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Section: Rna Aptamers As Novel Therapeutic For Costimulation Of 41bbmentioning

confidence: 99%

Agonists of Co-stimulation in Cancer Immunotherapy Directed Against CD137, OX40, GITR, CD27, CD28, and ICOS

Sanmamed

Pastor

Rodríguez

et al. 2015

Seminars in Oncology

176

141

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“…5) confirmed that the Sigma product contained HSA. This is an important observation because of the continuing widespread use of Sigma thrombin in aptamer research (for example, Lee et al, 2008;Yang et al, 2009;Miyachi et al, 2010;Diculescu et al, 2011;Hoon et al, 2011;Wang et al, 2010).…”

Section: Resultsmentioning

confidence: 99%

The Selection of DNA Aptamers for Two Different Epitopes of Thrombin Was Not Due to Different Partitioning Methods

Wilson

Cossins

Nicolau

et al. 2013

Nucleic Acid Therapeutics

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Nearly all aptamers identified so far for any given target molecule have been specific for the same binding site (epitope). The most notable exception to the 1 aptamer per target molecule rule is the pair of DNA aptamers that bind to different epitopes of thrombin. This communication refutes the suggestion that these aptamers exist because different partitioning methods were used when they were selected. The possibility that selection of these aptamers was biased by conflicting secondary structures was also investigated and found not to contribute. The preparation of protein-coated magnetic beads for systematic evolution of ligands by exponential enrichment (SELEX) and the different specificities of the thrombin aptamers for the a and b forms of thrombin are also reported.

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“…Many different approaches have been described in the literature to discriminate high-affinity aptamers from undesired background sequences arising from selection biases. These methods include enrichment fold (18), repeating motif (8), and copy number (19,20) analysis. We chose copy number analysis to test our capacity to directly distinguish high-affinity aptamers from background sequences via parallel binding measurements.…”

Section: Resultsmentioning

confidence: 99%

“…Traditional selection techniques depend on the convergence of the aptamer pool toward a relatively small number of sequences, such that consensus motifs can be obtained from a few hundred sequences. NGS can analyze millions of sequences, thus identifying enriched sequences much earlier in the selection process; this reduces the number of selection rounds required, and enables the identification and elimination of artifacts that typically arise over repeated rounds of PCR or during the cloning process (18)(19)(20). Despite these advances, the generation of high-quality aptamers remains a time-consuming and low-throughput process.…”

mentioning

confidence: 99%

Quantitative selection and parallel characterization of aptamers

Cho

Nie

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

115

114

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Aptamers are promising affinity reagents that are potentially well suited for high-throughput discovery, as they are chemically synthesized and discovered via completely in vitro selection processes. Recent advancements in selection, sequencing, and the use of modified bases have improved aptamer quality, but the overall process of aptamer generation remains laborious and low-throughput. This is because binding characterization remains a critical bottleneck, wherein the affinity and specificity of each candidate aptamer are measured individually in a serial manner. To accelerate aptamer discovery, we devised the Quantitative Parallel Aptamer Selection System (QPASS), which integrates microfluidic selection and nextgeneration sequencing with in situ-synthesized aptamer arrays, enabling simultaneous measurement of affinity and specificity for thousands of candidate aptamers in parallel. After using QPASS to select aptamers for the human cancer biomarker angiopoietin-2 (Ang2), we in situ synthesized arrays of the selected sequences and obtained equilibrium dissociation constants (K d ) for every aptamer in parallel. We thereby identified over a dozen high-affinity Ang2 aptamers, with K d as low as 20.5 ± 7.3 nM. The same arrays enabled us to quantify binding specificity for these aptamers in parallel by comparing relative binding of differentially labeled target and nontarget proteins, and by measuring their binding affinity directly in complex samples such as undiluted serum. Finally, we show that QPASS offers a compelling avenue for exploring structure−function relationships for large numbers of aptamers in parallel by coupling array-based affinity measurements with next-generation sequencing data to identify nucleotides and motifs within the aptamer that critically affect Ang2 binding.

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Aptamer Selection by High-Throughput Sequencing and Informatic Analysis

Cited by 81 publications

References 13 publications

Agonists of Co-stimulation in Cancer Immunotherapy Directed Against CD137, OX40, GITR, CD27, CD28, and ICOS

Agonists of Co-stimulation in Cancer Immunotherapy Directed Against CD137, OX40, GITR, CD27, CD28, and ICOS

The Selection of DNA Aptamers for Two Different Epitopes of Thrombin Was Not Due to Different Partitioning Methods

Quantitative selection and parallel characterization of aptamers

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