Dataset Information

Controlling uncertainty in aptamer selection.

ABSTRACT: The search for high-affinity aptamers for targets such as proteins, small molecules, or cancer cells remains a formidable endeavor. Systematic Evolution of Ligands by EXponential Enrichment (SELEX) offers an iterative process to discover these aptamers through evolutionary selection of high-affinity candidates from a highly diverse random pool. This randomness dictates an unknown population distribution of fitness parameters, encoded by the binding affinities, toward SELEX targets. Adding to this uncertainty, repeating SELEX under identical conditions may lead to variable outcomes. These uncertainties pose a challenge when tuning selection pressures to isolate high-affinity ligands. Here, we present a stochastic hybrid model that describes the evolutionary selection of aptamers to explore the impact of these unknowns. To our surprise, we find that even single copies of high-affinity ligands in a pool of billions can strongly influence population dynamics, yet their survival is highly dependent on chance. We perform Monte Carlo simulations to explore the impact of environmental parameters, such as the target concentration, on selection efficiency in SELEX and identify strategies to control these uncertainties to ultimately improve the outcome and speed of this time- and resource-intensive process.

SUBMITTER: Spill F

PROVIDER: S-EPMC5087011 | biostudies-literature | 2016 Oct

REPOSITORIES: biostudies-literature

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Controlling uncertainty in aptamer selection.

Spill Fabian F Weinstein Zohar B ZB Irani Shemirani Atena A Ho Nga N Desai Darash D Zaman Muhammad H MH

Proceedings of the National Academy of Sciences of the United States of America 20161007 43

The search for high-affinity aptamers for targets such as proteins, small molecules, or cancer cells remains a formidable endeavor. Systematic Evolution of Ligands by EXponential Enrichment (SELEX) offers an iterative process to discover these aptamers through evolutionary selection of high-affinity candidates from a highly diverse random pool. This randomness dictates an unknown population distribution of fitness parameters, encoded by the binding affinities, toward SELEX targets. Adding to thi ...[more]

PMID: 27790993

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Project description:Brevetoxins (PbTxs) are very potent marine neurotoxins that can cause an illness clinically described as neurologic shellfish poisoning (NSP). These toxins are cyclic polyether in chemistry and have increased their geographical distribution in the past 2 decades. However, the ethical problems as well as technical difficulties associated with currently employed analysis methods for marine toxins have spurred the quest for suitable alternatives to be applied in a regulatory monitoring regime. In this work, we reported the first instance of concurrent aptamer selection of Brevetoxin-1 (PbTx-1) and Brevetoxin-2 (PbTx-2) and constructed a biolayer interferometry (BLI) biosensor utilizing PbTx-1 aptamer as a specific recognition element. Through an in vitro selection process, we have, for the first time, successfully selected DNA aptamers with high affinity and specificity to PbTx-1 and PbTx-2 from a vast pool of random sequences. Among the selected aptamers, aptamer A5 exhibited the strongest binding affinity to PbTx-1, with an equilibrium dissociation constant (KD) of 2.56 μM. Subsequently, we optimized aptamer A5 by truncation to obtain the core sequence (A5-S3). Further refinement was achieved through mutations based on the predictions of a QGRS mapper, resulting in aptamer A5-S3G, which showed a significant increase in the KD value by approximately 100-fold. Utilizing aptamer A5-S3G, we fabricated a label-free, real-time optical BLI aptasensor for the detection of PbTx-1. This aptasensor displayed a broad detection range from 100 nM to 4000 nM PbTx-1, with a linear range between 100 nM and 2000 nM, and a limit of detection (LOD) as low as 4.5 nM. Importantly, the aptasensor showed no cross-reactivity to PbTx-2 or other marine toxins, indicating a high level of specificity for PbTx-1. Moreover, the aptasensor exhibited excellent reproducibility and stability when applied for the detection of PbTx-1 in spiked shellfish samples. We strongly believe that this innovative aptasensor offers a promising alternative to traditional immunological methods for the specific and reliable detection of PbTx-1.

Dataset Information

Controlling uncertainty in aptamer selection.

Publications

Controlling uncertainty in aptamer selection.

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