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This study introduces a novel, homogenous aptamer selection method for developing structure-switching biosensors. This breakthrough enables direct selection of aptamers for small molecule detection, exemplified by kanamycin A sensing.

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Area of Science:

  • Biotechnology
  • Molecular Biology
  • Biosensor Development

Background:

  • Aptamers are crucial recognition elements in small molecule biosensors, offering high affinity and selectivity.
  • Structure-switching aptamers are ideal for biosensing, as target binding induces conformational changes linked to functional output.
  • Existing selection methods for structure-switching aptamers require immobilization and do not directly report biosensor capacity.

Purpose of the Study:

  • To develop the first homogenous selection method for structure-switching aptamers that directly reports biosensor capacity.
  • To demonstrate a generalizable approach for selecting aptamers that undergo target-induced conformational changes for biosensing.

Main Methods:

  • A novel homogenous selection strategy was developed, exploiting restriction enzyme activity.
  • Aptamer candidates were isolated based on target-induced displacement of a complementary strand.
  • Selection was performed against kanamycin A, and candidate sequences were characterized as structure-switching biosensors.

Main Results:

  • Four enriched aptamer candidates were successfully identified and characterized as structure-switching biosensors for kanamycin A.
  • Optimized biosensor conditions enabled facile detection of kanamycin A within a concentration range of 90 μM to 10 mM.
  • The developed biosensors demonstrated high selectivity for kanamycin A over three other aminoglycosides.

Conclusions:

  • This research presents a generalizable and homogenous method for directly selecting structure-switching aptamers for biosensing.
  • The method overcomes limitations of traditional aptamer selection by eliminating the need for immobilization.
  • This approach is broadly applicable to the development of aptamer-based biosensors for various small molecule targets.