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Aptabody-aptatope interactions in aptablotting assays.

Simranjeet Singh Sekhon1, Hyun-Ju Um, Woo-Ri Shin

  • 1School of Biological Sciences, Chungbuk National University, 1 Chungdae-Ro, Seowon-Gu, Cheongju 28644, South Korea. kyh@chungbuk.ac.kr jyahn@chungbuk.ac.kr.

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This study introduces aptabiotting, a novel assay using DNA aptamers as aptabodies for protein detection. Aptabodies effectively bind target proteins, offering a new tool for bioassays and potentially improving upon traditional immunoblot methods.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Biotechnology

Background:

  • Traditional immunoblot assays face limitations in specificity and accessibility.
  • Aptamers, short DNA or RNA molecules, offer unique binding capabilities.
  • Developing novel bioassay platforms is crucial for advancing molecular diagnostics.

Purpose of the Study:

  • To develop and validate a novel aptablotting assay method using DNA aptamers as aptabodies.
  • To characterize the binding interactions of aptabodies with target proteins.
  • To explore the potential of aptabodies in improving bioassay platforms.

Main Methods:

  • Utilized nanoscale single-stranded DNA aptamers as aptabodies against GST and DIG-tags.
  • Employed MOE-docking simulation to predict aptabody binding sites (aptatopes).
  • Assessed aptabody-aptatope interactions via in vitro enzyme inhibitory analysis, dot-blot, EMSA, and SDS-PAGE/electroblot.

Main Results:

  • Demonstrated direct and indirect aptabody recognition in the aptablotting assay.
  • Predicted aptatopes for GST-2 aptabody localized to the catalytically active region.
  • Confirmed aptabodies bind native and denatured forms of GST, and interact with membranes.
  • Showcased aptabodies' potential for overcoming immunoblot assay limitations and compatibility with ELISA.

Conclusions:

  • Aptablotting assays offer a versatile and effective method for protein detection using aptabodies.
  • Aptabodies demonstrate robust binding to target proteins in various forms.
  • The integration of aptabodies and antibodies (hybrid-aptablotting) holds promise for next-generation bioassay design.