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

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.