Insertion-Site Proximity to AAV Inverted Terminal Repeats Increases Plasmid Recombination

Adeno-associated virus (AAV)-based massively parallel reporter assays (MPRA) have become an important platform for large-scale functional characterization of regulatory DNA elements. However, plasmids carrying AAV inverted terminal repeats (ITRs) are intrinsically unstable during propagation in Escherichia coli, potentially compromising library integrity before viral packaging. Although ITR-associated recombination has been recognized, the influence of cloning-site position relative to the ITR on plasmid stability has not been systematically investigated. Here, we examined the relationship between cloning-junction proximity to AAV2 ITRs and plasmid recombination using an AAV-MPRA reporter plasmid. We compared four restriction-ligation cloning strategies utilizing restriction sites at defined distances (4-543 bp) from the nearest ITR while preserving ITR integrity, and one strategy in which the ITR itself was disrupted. We observe that plasmid recombination exhibited a pronounced distance dependence. Constructs with ligation junctions located 4, 41, and 182 bp from an intact ITR showed recombination frequencies of 82.5%, 60%, and 20%, respectively, whereas a 0% recombination frequency was detected when the nearest ITR was positioned 543 bp from the cloning junction. In contrast, cleavage within ITR reduced recombination to 15%, demonstrating that preservation of the intact ITR secondary structure is required for efficient recombination. Whole-plasmid sequencing confirmed recurrent large-scale deletions in which the expression cassette and the downstream R-ITR were removed while the L-ITR and plasmid backbone were retained, consistent with preferential processing of the intact L-ITR region. These findings identify cloning-site proximity to an intact AAV ITR as a major determinant of plasmid stability during bacterial propagation and demonstrate that substantial loss of correctly assembled constructs can occur before AAV production. The results have direct implications for the design of AAV-based MPRA libraries and support positioning cloning sites as far as practical from the nearest ITR, together with routine validation of plasmid integrity prior to viral packaging.