Shuo Huang's lab at State Key Laboratory of Analytical Chemistry for Life Sciences (http://hysz.nju.edu.cn/bionano/2.html) gained significant progress in detecting alkylated DNA damage using nanopore sequencing technology. The related paper entitled Nanopore sequencing accurately identifies the mutagenic DNA lesion O6-carboxymethyl guanine and reveals its behavior in replication was published on Angewandte Chemie International Edition on April 25, 2019 (DOI: 10.1002/anie.201902521, paper link: https://onlinelibrary.wiley.com/doi/abs/10.1002/anie.201902521).
Human genetic material DNA is always under the attack of endogenous stressors (active oxygen, nitroso compounds, etc.) and exogenous factors (UV, ionizing radiation, etc.). Those stressors may cause DNA damage, such as DNA double-strand breaks, abasic site, and various modified bases. If not properly repaired, DNA damage can negatively affect the accurate transmission of genetic material and even cause cell apoptosis. O6-carboxymethylguanine (O6-CMG) is one type of DNA damage. It can induce G-A base transversion during DNA replication, which may lead to permanent sequence changes and increase the probability of developing cancer. Thus, achieving precise positioning of O6-CMG at the gene level is important. It will provide valuable information for targeted repair and help establishing early tumor biomarkers.
Nanopore sequencing technology is an emerging single molecule sequencing method. In principle, this method can directly read the DNA sequence at the single molecule level without gene amplification. This unique advantage makes nanopore sequencing the most suitable candidates for detecting modified bases.
Shuo Huang's group and Dennis Gillingham's group (from the University of Basel) collaborated to use nanopore sequencing technology to identify O6-CMG at the single molecule level. The cooperative team designed a special DNA sequence and used chemical synthesis to introduce one or more O6-CMG at different positions. In the subsequent nanopore sequencing process, specific sequencing signals generated by O6-CMG were directly observed. The current signal of O6-CMG was 10 pA larger than that of canonic guanine. Based on this method, accuracy rate of O6-CMG recognition in single sequencing event can be as high as 95%. This method is the only way that can achieve accurate identification of O6-CMG while retains the original base modification and original whole gene information.
Figure 1. Molecular formula of O6-CMG and nanopore sequencing mechanism. Top left: chemical structure of O6-CMG. The carboxymethyl modification is marked in blue. Top right: nanopore sequencing mechanism. The narrowest part of the nanopore (blue) is the recognition site. When O6-CMG passes the recognition site, it reports the sequencing information. Bottom: Nanopore sequencing raw current data. The current signals in red are specific signals generated by O6-CMG. The subsequent signal reveals the process after the O6-CMG enters into the synthesis pocket of phi29 DNA polymerase.
During the nanopore sequencing of O6-CMG, the team also found that O6-CMG was a replication obstacle of phi29 DNA polymerase. The singular enzyme kinetic signal between modified bases and phi29 DNA polymerase may help to realize deep differentiation of a series of similarly modified bases.
This work was jointly completed by Professor Shuo Huang's lab at the School of Chemistry and Chemical Engineering of Nanjing University and Professor Dennis Gillingham's research group at School of Chemistry of University of Basel. Yu Wang, a graduate student from Shuo Huang's lab and Kiran M. Patil, a postdoc from Dennis Gillingham's research group, are co-first authors. Professor Shuo Huang and Professor Dennis Gillingham are co-corresponding authors of the thesis. Nanjing University is the first communication unit.
This work is supported by National Natural Science Foundation of China (Grant No.21327902, Grant No.21675083, Grant No.91753108), Fundamental Research Funds for the Central Universities (Grant No.020514380142, No. 020514380174), State Key Laboratory of Analytical Chemistry for Life Science (Grant No.5431ZZXM1804, No. 5431ZZXM1902), 1000 Plan Youth Talent Program of China, Program for high-step entrepreneurial and innovative talents introduction of Jiangsu Province, Technology innovation fund program of Nanjing University.