Systematic Investigation of Reaction Parameters and Purification Strategies for DNA-Polymer Conjugates

DNA-polymer conjugates are an emerging class of biohybrid materials with applications in therapeutics and biosensing, yet quantitative guidelines for optimizing conjugation conversion and purification remain unreported. Here, we systematically investigate the parameters governing the NHS ester-amine coupling of single-stranded DNA (ssDNA) to synthetic polymers and the subsequent purification of these DNA-polymer conjugates. Aqueous-phase size-exclusion chromatography was identified as the most reliable method to assess the conjugation conversion and purification efficacy. Using this approach, we found that increases in concentration, pH, and the ratio of polymer to ssDNA consistently improved conjugation conversion, while increasing the molar mass of either starting material decreased conversion. Subtle differences in the ssDNA sequence were also found to affect conjugation efficiency. In both low-throughput chromatographic and high-throughput spin-column purification settings, anion exchange achieved higher purity than all other techniques, although with reduced material recovery. Together, these findings provide a practical framework for reproducible coupling of chemically diverse macromolecules with broad implications for the development of oligonucleotide therapeutics and biosensors.