Increased demands for plasmid DNA (pDNA) for emerging nucleic acid-based vaccines and therapies exacerbate the need to remove bottlenecks from pDNA production. For industry, the problem has been that as pDNA is synthesized and purified, the plasmids sometimes come out of their supercoiled isoform. In therapeutics, the resulting heterogeneous sequence mixtures and uncontrolled transcripts may contribute to dangerous side effects.
Scientists led by Anubhav Tripathi, PhD, professor of biology and medical sciences, Brown University, developed a way to detect supercoiled, linear, and open circular pDNA isoforms using high-throughput microfluidic electrophoresis.
The method developed by Tripathi and colleagues detects isoforms as small as 0.1 ng/µL for supercoiled and linear isoforms, and 0.5 ng/µL for the circular isoform. With a sample volume of 10 µL and a turnaround time of one minute per sample, this method, they say, can help optimize processes and analyze batches.
As Tripathi tells GEN, “Where plasmids are part of the final product, it has been shown that supercoiled plasmids have a higher efficiency. Therefore, our method can be used to ensure no undesired single or double-stranded breaks in the plasmids have been induced.” That is true regardless of whether the plasmids are used as precursors, for transcription, or in the final product.
To achieve this, the scientists developed a microfluidic supercoiled pDNA assay, optimized the separation of supercoiled and linear isoforms, and validated separation efficacy.
Linearized pDNA is mobile
For the assay, they used a supercoiled pDNA ladder and optimized its electrokinetics, gel composition, and sample treatment parameters to maximize separation resolution and sensitivity.
The team chose a 0.10% gel concentration with a 2200 V separation for a pDNA script to ensure clear differentiation between the 8 and 10 kb peaks during multiple runs. This enabled optimal peak separation of 2.22 (± 0.61), and optimal peak height of 200 (± 107.07) relative fluorescent units (RFU). Ultimately, however, 0.07% gel concentration showed better resolution (and, thus, better separation between supercoiled and linear isoforms) until plasmids reached 7 kb.
This method, they report, generates consistent results and matches the length of supercoiled or linear plasmids that were determined by ladders.
“We were surprised to see that, beyond a specific size, linearized pDNA has a higher mobility than its supercoiled counterpart, since this provides insight into the length at which the supercoiled plasmids experience more drag than a linear conformation,” Tripathi says.
Currently, open circular isoform assessments are mainly qualitative, he adds. “The next step is to deepen our understanding of these isoforms to enable their quantitation…in terms of size and concentration.”
