iBB

Description: Illustration of a DNA origami nanotetrahedron created with David Goodsell’s Illustrate software.

Context: BIOTecnico PhD student Ana Rita Santos, under the supervision of BERG-iBB researcher Miguel Prazeres is developing processes to produce and purify DNA scaffolds and DNA origami nano-objects.

DNA-origami biomanufacturing relies in many cases on the use of asymmetric PCR (aPCR) to generate 500-3500 base, single-stranded DNA (ssDNA) scaffolds that are then purified by agarose gel extraction, a technique that is laborious, not scalable and presents low recovery yields. In a paper just published in Separation and Purification Technology, Ana Rita Santos and Miguel Prazeres from BERG-iBB, in collaboration with Pedro Paulo from CQE, present alternative methods based on anion-exchange or multimodal chromatography that yield purified scaffolds from aPCR mixtures. The recovered scaffolds were further used to assemble DNA-origami nanostructures. The work indicates that chromatography could play an important role in scaling-up the purification of ssDNA scaffolds for the biomanufacturing DNA-origami nanostructures.

The ability to purify supercoiled minicircles (sc MC) from related miniplasmid (MP) and parental plasmid (PP) impurities is critical for a wider application of MC vectors in gene therapy research. BERG-iBB researchers developed a process that relies on multimodal chromatography with a 2-step NaCl elution to separate sc MC from open circular impurities. A detailed study of the DNA-ligand interactions underlying the separation shows that the order of elution reflects the increasing degree of base exposure of the species. Data further suggests that nucleic acid bases most likely engage in cation-π, π-π stacking and H-bonding with ligand. The work was published in Separation and Purification Technology.