Improving Aptamer Affinity and Determining Sequence-Activity Relationships via Motif-SELEX.

Area of Science:

• Biotechnology
• Molecular Biology
• Nucleic Acid Chemistry

Background:

• Systematic Evolution of Ligands by Exponential Enrichment (SELEX) is limited by incomplete screening of sequence space, often resulting in underperforming aptamers.
• Traditional post-SELEX optimization methods like doped SELEX and error-prone PCR introduce biases and limitations, hindering the discovery of superior aptamer sequences.

Purpose of the Study:

• To introduce Motif-SELEX, a novel method for optimizing nucleic acid aptamers by generating diverse sequence and length variations.
• To overcome the limitations of traditional methods in improving aptamer affinity and discovering novel binding sequences.

Main Methods:

• Developed Motif-SELEX to create libraries representing broad sequence and length variations of parent aptamers.
• Applied Motif-SELEX to DNA-based morphine and RNA-based apixaban aptamers for optimization.
• Analyzed Motif-SELEX pools to identify key sequence and structural elements for target binding.

Main Results:

• Discovered novel aptamer sequences with improved affinity, up to 10-fold higher than parent sequences.
• Identified sequences with optimized random domain lengths, inaccessible through traditional methods.
• Successfully predicted secondary and tertiary structures, including those with noncanonical nucleotide interactions.

Conclusions:

• Motif-SELEX is an effective and generalizable method for optimizing nucleic acid aptamer structure and binding affinity.
• The method enables the discovery of high-affinity aptamers and provides insights into structure-function relationships.
• Motif-SELEX offers a powerful tool for diverse applications requiring high-performance nucleic acid molecules.