High-Affinity Six-Letter DNA XenoAptamer Generation by Genetic Alphabet Expansion

Area of Science:

• Biotechnology
• Molecular Biology
• Synthetic Biology

Background:

• Antibodies are widely used in diagnostics and therapeutics.
• Developing high-affinity aptamers is crucial for advancing molecular recognition technologies.
• Existing DNA aptamer generation methods have limitations in achieving high affinity.

Purpose of the Study:

• To develop a novel method for generating high-affinity DNA aptamers using an expanded genetic alphabet.
• To create DNA aptamers (XenoAptamers) with dissociation constants (Kd) in the picomolar range.
• To demonstrate the utility of these XenoAptamers in diagnostic and therapeutic applications.

Main Methods:

• Genetic alphabet expansion using a six-letter DNA incorporating two hydrophobic unnatural bases (Ds and Px/Pa').
• Optimization of the six-letter SELEX (Systematic Evolution of Ligands by Exponential Enrichment) process.
• Characterization of XenoAptamer binding affinity (Kd) and functional activity.

Main Results:

• Generated high-affinity XenoAptamers targeting interleukin-8 (IL8) with Kd = 61 pM and α-thrombin (thrombin) with Kd = 1.7 pM.
• Developed a sandwich-type ELISA using an anti-IL8 XenoAptamer-antibody combination with a lower limit of detection (LOD = 0.107 pg/mL) compared to antibody-antibody pairs (LOD = 1.227 pg/mL).
• Demonstrated that the antithrombin XenoAptamer inhibits thrombin-mediated fibrinogen cleavage to fibrin.

Conclusions:

• The six-letter ExSELEX method significantly enhances DNA structural and physicochemical diversity, enabling the generation of high-affinity aptamers.
• XenoAptamers show promise as effective tools for sensitive diagnostics and targeted therapeutics.
• This innovative aptamer generation strategy offers a viable alternative to antibody-based applications.