Influence of thermal treatment on aptamer structure & Synthesis and purification of modified Acinetobacter baumanii-binding aptamers

Abstract

Aptamers are single-stranded nucleic acid sequences which bind to defined targets with high affinity and specificity, primarily through formation of unique secondary and tertiary structures. Aptamers are promising tools in biotechnology, with applications in diagnostics, biosensors, and therapeutics. However, their translation from research to application has been limited by recently reported issues with aptamer reproducibility – aptamers are often incompletely characterized in the literature. Since aptamers bind to their targets via unique secondary and tertiary structures, these binding structures need to be properly characterized. As nucleic acid structure may be highly flexible, it is important to outline the conditions under which the aptamer forms the correct ligand-binding structure. To this end, adenosine-, ampicillin-, and quinine-binding aptamers were subjected to different thermal pre-treatment conditions. Following thermal treatment, aptamers were analyzed using anion-exchange HPLC, circular dichroism and UV/vis spectroscopy, and thermal melt curves. Results clearly demonstrate the conformational flexibility of aptamer sequences depending on thermal pre-treatment, which may have implications for binding. These findings may help guide decisions for aptamer selection and optimization. Towards a different goal, solid-phase phosphoramidite chemistry was used for the synthesis of modified aptamers which are reported to bind Acinetobacter baumannii, and synthesis products were purified using anion-exchange HPLC. In one synthesis, the 80mer Aci55 was synthesized with a 5’-DBCO functionality for future coupling with azido-nanoparticles containing antibiotics as a therapeutic for the treatment of bacterial infections. A short-linker DBCO-Aci55 was synthesized with sufficient purity, but the long-linker DBCO-Aci55 synthesis conditions require further optimization. In a second synthesis, Aci55 and point mutation variants were synthesized with a 5’-FAM functionality to be analyzed for binding capabilities against different A. baumannii isolates. Nine FAM-modified Aci55 sequence variants were synthesized with sufficient purity as determined by MS and HPLC, with yields ranging from 6-9%. This work highlights the limitations of synthesis and purification of long oligonucleotides and proposes modifications to be made to the standard synthesis scheme to improve overall coupling efficiency.