Quantitative selection of DNA aptamers through microfluidic selection and high-throughput sequencing

Cho, Minseon; Xiao, Yi; Nie, Jeff; Stewart, Ron; Csordas, Andrew T.; Oh, Seung Soo; Thomson, James A.; Soh, H. Tom

doi:10.1073/pnas.1009331107

Cited by 239 publications

(257 citation statements)

References 45 publications

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“…These higher sequence reads present a greater coverage of the total number of sequences in the pool, providing a more complete picture of the diversity of sequences. NGS also removes the cloning bias 32 , and allows for sequencing of multiple pools in a single batch with the addition of bar codes 24,33 . NGS was used to analyze the progression of enrichment from three separate pools in this sample selection (Figure 2).…”

Section: Representative Resultsmentioning

confidence: 99%

A Method for Selecting Structure-switching Aptamers Applied to a Colorimetric Gold Nanoparticle Assay

Martin

Smith

Warren

et al. 2015

JoVE

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Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health and performance. Nucleic acid aptamers have been increasingly applied as recognition elements on biosensor platforms, but selecting aptamers toward small molecule targets requires special design considerations. This work describes modification and critical steps of a method designed to select structure-switching aptamers to small molecule targets. Binding sequences from a DNA library hybridized to complementary DNA capture probes on magnetic beads are separated from nonbinders via a target-induced change in conformation. This method is advantageous because sequences binding the support matrix (beads) will not be further amplified, and it does not require immobilization of the target molecule. However, the melting temperature of the capture probe and library is kept at or slightly above RT, such that sequences that dehybridize based on thermodynamics will also be present in the supernatant solution. This effectively limits the partitioning efficiency (ability to separate target binding sequences from nonbinders), and therefore many selection rounds will be required to remove background sequences. The reported method differs from previous structure-switching aptamer selections due to implementation of negative selection steps, simplified enrichment monitoring, and extension of the length of the capture probe following selection enrichment to provide enhanced stringency. The selected structure-switching aptamers are advantageous in a gold nanoparticle assay platform that reports the presence of a target molecule by the conformational change of the aptamer. The gold nanoparticle assay was applied because it provides a simple, rapid colorimetric readout that is beneficial in a clinical or deployed environment. Design and optimization considerations are presented for the assay as proof-of-principle work in buffer to provide a foundation for further extension of the work toward small molecule biosensing in physiological fluids.

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Section: Representative Resultsmentioning

confidence: 99%

A Method for Selecting Structure-switching Aptamers Applied to a Colorimetric Gold Nanoparticle Assay

Martin

Smith

Warren

et al. 2015

JoVE

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“…We performed four rounds of M-SELEX using the micromagnetic separation (MMS) device previously developed by our group (16), which allowed us to uniformly control the selection and washing stringency (15)(16)(17)(18). Although the MMS device is capable of performing highly stringent washing, we used moderate selection conditions for this experiment.…”

Section: Resultsmentioning

confidence: 99%

“…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.…”

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confidence: 99%

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Quantitative selection and parallel characterization of aptamers

Cho

Nie

et al. 2013

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

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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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“…The basic approach named systematic evolution of ligands by exponential enrichment (SELEX) depicted in Fig. 1 can be considered an extremely powerful purification method in which very rare binding activities (with frequencies of 1 in 10 11 -1 in 10 13 ) are isolated by affinity purification (4,5). Aptamers isolated by this method can exhibit remarkable affinity and specificity for their targets comparable or exceeding those of antibodies.…”

Section: Oligonucleotide Aptamer Ligandsmentioning

confidence: 99%

Use of Oligonucleotide Aptamer Ligands to Modulate the Function of Immune Receptors

Gilboa

McNamara

Pastor

2013

Clinical Cancer Research

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The paucity of costimulation at the tumor site compromises the ability of tumor-specific T cells to eliminate the tumor. The recent U.S. Food and Drug Administration approval of ipilumimab, an antibody that blocks the inhibitory action of CTLA-4, and clinical trials targeting 4-1BB and PD-1 or PD-L1, have underscored the therapeutic potential of using immunomodulatory antibodies to stimulate protective immunity in human patients. Nonetheless, systemic administration of immunomodulatory antibodies has been associated with dose-limiting autoimmune pathologies, conceivably reflecting also the activation of resident autoreactive T cells. Arguably, targeting immunomodulatory ligands to the disseminated tumor lesions of the patient would reduce such drug-associated toxicities. We have recently developed a new class of inhibitory (CTLA-4) and agonistic (4-1BB and OX-40) ligands composed of short oligonucleotide (ODN) aptamers that exhibited bioactivities comparable or superior to that of antibodies. To reduce toxicity, the immunomodulatory aptamers were targeted to the tumor by conjugation to a second aptamer that bound to a product expressed on the surface of the tumor cell, the targeting aptamer, generating a bispecific aptamer conjugate analogous to bispecific antibodies. In a proof-of-concept study in mice, we have shown that an agonistic 4-1BB-binding aptamer conjugated to a prostate-specific membrane antigen (PSMA)-binding aptamer led to the inhibition of PSMA-expressing tumors, was more effective than, and synergized with, vaccination, and exhibited a superior therapeutic index compared with nontargeted costimulation with 4-1BB antibodies or 4-1BB aptamers. The cell-free chemically synthesized ODN aptamers offer significant advantages over antibodies in terms of synthesis, cost, as well as conjugation chemistry needed to generate bispecific ligand fusions.

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Quantitative selection of DNA aptamers through microfluidic selection and high-throughput sequencing

Cited by 239 publications

References 45 publications

A Method for Selecting Structure-switching Aptamers Applied to a Colorimetric Gold Nanoparticle Assay

A Method for Selecting Structure-switching Aptamers Applied to a Colorimetric Gold Nanoparticle Assay

Quantitative selection and parallel characterization of aptamers

Use of Oligonucleotide Aptamer Ligands to Modulate the Function of Immune Receptors

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