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Introducing structure-switching functionality into small-molecule-binding aptamers via nuclease-directed truncation.

Zongwen Wang1,2, Haixiang Yu1, Juan Canoura1

  • 1Department of Chemistry and Biochemistry, Florida International University, 11200 SW 8th Street, Miami, FL 33199, USA.

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We developed a novel enzyme digestion method to create structure-switching aptamers for biosensors. This technique uses exonuclease III (Exo III) and target binding to generate functional aptamer sensors with high affinity.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Biosensor Technology

Background:

  • Aptamers are functional nucleic acid sequences that can bind to specific targets.
  • Developing structure-switching aptamers is crucial for creating sensitive biosensors.
  • Existing methods for generating structure-switching aptamers can be complex.

Purpose of the Study:

  • To develop a broadly applicable enzyme digestion strategy for creating structure-switching aptamers.
  • To demonstrate that target binding inhibits exonuclease III (Exo III) digestion of aptamers.
  • To generate and characterize structure-switching aptamers with retained target-binding affinity.

Main Methods:

  • Utilized exonuclease III (Exo III) digestion on pre-folded aptamers targeting cocaine and ATP.
  • Investigated the effect of target binding on Exo III digestion kinetics.
  • Confirmed structure-switching functionality using exonuclease I assays and sensor applications.

Main Results:

  • Exo III digestion of aptamers is significantly inhibited by target binding.
  • Digestion products exhibit target-induced structure-switching functionality.
  • Truncated aptamers retain high target-binding affinity and function in biosensor assays.

Conclusions:

  • The Exo III digestion strategy is a versatile method for generating structure-switching aptamers.
  • This approach simplifies the creation of functional sensor elements for aptasensors.
  • The method is applicable to various aptamers with specific secondary structures.