A high-affinity dopamine aptamer: implications of library diversity and negative selection

Area of Science:

• Biochemistry
• Molecular Biology
• Biotechnology

Background:

• A previous study isolated a DNA aptamer for dopamine in 2018 using extensive negative selection.
• A single nucleotide difference can significantly alter aptamer binding affinity and kinetics.

Purpose of the Study:

• To investigate the impact of negative selection stringency on aptamer discovery.
• To identify novel high-affinity dopamine aptamers by modifying selection strategies.

Main Methods:

• Performed a new aptamer selection experiment without negative selection steps.
• Characterized aptamer binding affinity using isothermal titration calorimetry (ITC).
• Assessed aptamer kinetics using fluorescence strand-displacement assays.

Main Results:

• A single family of binding sequences was identified with a K_d of 424 nM, showing 6-fold higher affinity than the previously reported aptamer.
• The newly selected aptamer exhibited a 4-fold faster release rate and a 15-fold lower apparent K_d in fluorescence strand-displacement assays.
• The improved properties suggest this aptamer was missed in prior work due to overly stringent negative selection.

Conclusions:

• Overly stringent negative selection can lead to the loss of high-affinity aptamers.
• Optimizing negative selection concentration and increasing library diversity are crucial for successful aptamer selection.
• This study identifies a high-affinity dopamine aptamer and provides insights into aptamer selection strategies.