Generation of high-quality single-stranded DNA for full-length and truncated genome standards of recombinant adeno-associated viruses

The integrity of the single-stranded DNA (ssDNA) genome of recombinant adeno-associated viral vectors (rAAVs) for gene therapy applications is a critical quality attribute. To monitor this attribute, robust analytical methods, such as long-read sequencing or multiplex PCR, are needed. Due to the heterogeneity of the packaged rAAV payloads, the availability of appropriate ssDNA standards could clearly facilitate the development of these methods and may be used for system suitability testing. However, due to a genome length of up to approximately 4.7 kilobases and the secondary structure of the inverted terminal repeats, such standards are not trivial to produce. Here, we introduce a versatile method that starts with plasmid DNA containing the rAAV genome of interest. After its linearization, the sticky ends are filled in with biotinylated deoxynucleotide triphosphates, and the DNA is bound to streptavidin-coupled beads. With two further restriction enzymes, the plasmid backbone is removed, and finally, the ssDNA is obtained by alkaline strand separation. The quality and purity of the received ssDNA were verified using double-strand synthesis followed by size determination and long-read sequencing. With the presented method, it is possible to generate both full-length and truncated ssDNA standards within one day, contributing to improved quality control of rAAVs.