Nanopore sequencing of DNA photoproducts

Cyclobutane pyrimidine dimers (CPDs) are the major photoproducts of DNA produced by direct absorption of UV light but can also be produced indirectly by photosensitization and chemiexcitation. Deamination of C-containing CPDs is responsible for the majority of C to T mutations caused by UV, which have been linked to skin cancer. Another frequent mutagenic photoproduct of DNA is the (6-4) photoproduct (64PP). Because of their roles in causing mutations, NextGen sequencing methods have been developed to determine the location and frequency of these photoproducts in chromosomal DNA. All these methods, however, rely on enzyme-coupled methods that can only detect one photoproduct at a time. There is evidence, however, that the 64PP and certain oxidized bases can photosensitize CPD formation to produce compound lesions. We propose that such rare but possibly important compound lesions can be detected by single-molecule sequencing using nanopores. Herein, we show that site-specific TT CPD and 64PP photoproducts cause a large current drop when sequenced by an Oxford Nanotechnologies R10-based sequencing device. Furthermore, we demonstrate that both single and multiple photoproducts can be detected in UVB-irradiated DNA substrates containing T₁₁- and (PuTT)₄Pu-tracts. We also provide a simple 9mer kmer model that can simulate the nanopore current data.