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PreprintCC BYChemRxiv

Looking Beyond KD for Aptamer Validation: Disentangling Specific Aptamer Binding from Electrostatic Association for Basic Protein Targets

  • Connor D. Flynn1,2,3
  • Shana O. Kelley1,2,3,4,5,6,7,8
  • Dingran Chang1,4,3,9,10,11
11 affiliations
  1. 1Chan Zuckerberg Biohub Chicago
  2. 2Department of Chemistry, Weinberg College of Arts & Sciences
  3. 3Northwestern University
  4. 4Department of Biomedical Engineering, McCormick School of Engineering
  5. 5Simpson Querrey Institute
  6. 6Department of Biochemistry, Feinberg School of Medicine
  7. 7International Institute for Nanotechnology
  8. 8Robert H. Lurie Comprehensive Cancer Center
  9. 9Ralph N. Adams Institute for Bioanalytical Chemistry
  10. 10University of Kansas
  11. 11Department of Chemistry
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Abstract

Nucleic acid aptamers provide a simple, cost-effective alternative to antibodies for targeting biomolecular analytes from ions to large protein complexes. However, the interactions of these nucleic-acid based structures in vivo remains largely uncharacterized. Here, we report the seemingly pervasive electrostatic association between nucleic acids and high isoelectric point proteins under biological conditions. Such non-specific binding runs the risk of convoluting aptamer selection and validation, while also potentially interfering with nucleic acid-based nanotechnologies in vivo. Through our explorations, we identify several methods to disentangle sequence-specific aptamer interactions from non-specific electrostatic association, allowing for discrimination between binding modes. We also identify dissociation rate (koff) and equilibrium binding response (Req) as potentially better discriminators than KD for isolating and validating true sequence-specific aptamers.

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Cite

BibTeXRIS
BibTeX
@misc{flynn2026looking,
  title = {{Looking Beyond KD for Aptamer Validation: Disentangling Specific Aptamer Binding from Electrostatic Association for Basic Protein Targets}},
  author = {Connor D. Flynn and Shana O. Kelley and Dingran Chang},
  howpublished = {ChemRxiv},
  year = {2026},
  month = oct,
  doi = {10.26434/chemrxiv.15010585/v1},
  url = {https://chemrxiv.org/doi/full/10.26434/chemrxiv.15010585/v1},
  note = {CC BY},
}
RIS
TY  - UNPB
TI  - Looking Beyond KD for Aptamer Validation: Disentangling Specific Aptamer Binding from Electrostatic Association for Basic Protein Targets
AU  - Connor D. Flynn
AU  - Shana O. Kelley
AU  - Dingran Chang
PY  - 2026
DA  - 2026/10/16
PB  - ChemRxiv
DO  - 10.26434/chemrxiv.15010585/v1
UR  - https://chemrxiv.org/doi/full/10.26434/chemrxiv.15010585/v1
AB  - Nucleic acid aptamers provide a simple, cost-effective alternative to antibodies for targeting biomolecular analytes from ions to large protein complexes. However, the interactions of these nucleic-acid based structures in vivo remains largely uncharacterized. Here, we report the seemingly pervasive electrostatic association between nucleic acids and high isoelectric point proteins under biological conditions. Such non-specific binding runs the risk of convoluting aptamer selection and validation, while also potentially interfering with nucleic acid-based nanotechnologies in vivo. Through our explorations, we identify several methods to disentangle sequence-specific aptamer interactions from non-specific electrostatic association, allowing for discrimination between binding modes. We also identify dissociation rate (koff) and equilibrium binding response (Req) as potentially better discriminators than KD for isolating and validating true sequence-specific aptamers.
ER  -