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High-affinity one-step aptamer selection using a non-fouling porous hydrogel

Nature Biotechnology (2023)Cite this article

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Abstract

Aptamers, commonly referred to as chemical antibodies, are used in a wide range of applications including drug delivery and biosensing. However, the process of aptamer selection poses a substantial challenge, as it requires numerous cycles of enrichment and involves issues with nonspecific binding. We present a simple, fast instrument-free method for aptamer enrichment and selection based on a diffusion-binding process in a three-dimensional non-fouling porous hydrogel with immobilized target proteins. Low-affinity aptamer candidates can be rapidly released from the hydrogel, whereas high-affinity candidates are restricted due to their strong binding to the immobilized protein targets. Consequently, a one-step enriched aptamer pool can strongly bind the protein targets. This enrichment is consistent across five proteins with isoelectric points in varying ranges. With thrombin as a representative model, the anti-thrombin aptamer identified from an enriched aptamer pool has been found to have a binding affinity that is comparable to those identified over ten cycles of selection using traditional methods.

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Fig. 1: Hydrogel for aptamer selection (HAS).
Fig. 2: Hydrogel characterization and aptamer selection.
Fig. 3: Analysis of aptamer candidates acquired with one-step HAS.
Fig. 4: Selection and characterization of aptamers for GM-CSF, IL-12, IL-10 and BDNF.

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Data availability

Data supporting this new method are available within the article and its Supplementary Information. Parameters for simulation are provided in Supplementary Table 1. Sequences of ssDNA library, PCR primers and aptamer candidates are included in Supplementary Tables 2–5. All uncropped gel electrophoresis data are available in Source Data and Supplementary Information. Source data are provided with this paper.

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Acknowledgements

This work was partly supported by the National Institutes of Health (grants R01HL122311 and R01AR073364).

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Author notes

  1. Naveen K. Singh

    Present address: Centre for Biomedical Engineering, Indian Institute of Technology Delhi, Delhi, New Delhi, India

  2. These authors contributed equally: Naveen K. Singh, Yixun Wang.

Authors and Affiliations

  1. Department of Biomedical Engineering, The Penn State University, University Park, PA, USA

    Naveen K. Singh, Yixun Wang, Connie Wen, Brandon Davis, Xuelin Wang, Kyungsene Lee & Yong Wang

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Contributions

N.K.S. designed experiments, did experiments, analyzed data and wrote the manuscript. Yixun Wang studied the simulation of aptamer transport, did experiments, analyzed data and wrote the manuscript. C.W., B.D., X.W. and K.L. performed experiments and analyzed data. Yong Wang coined the concept, designed experiments, analyzed data and wrote the manuscript.

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Correspondence to Yong Wang.

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Competing interests

N.K.S. and Yong Wang filed a patent application (US patent 63/428,466) based on the method presented in this work.

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Nature Biotechnology thanks Honggang Cui, John Rossi and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–13 and Tables 1–6.

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Supplementary Video 1

Supporting video 1.

Source data

Source Data Fig. 2

Unprocessed gels.

Source Data Fig. 4

Unprocessed gels.

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Singh, N.K., Wang, Y., Wen, C. et al. High-affinity one-step aptamer selection using a non-fouling porous hydrogel. Nat Biotechnol (2023). https://doi.org/10.1038/s41587-023-01973-8

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