State Key Laboratory of Analytical Chemistry for Life Sciences, School of Chemistry and Chemical Engineering, Nanjing University, 210023 Nanjing, China.
Chemistry and Biomedicine Innovation Center (ChemBIC), Nanjing University, 210023 Nanjing, China.
ACS Sens. 2021 Aug 27;6(8):3082-3092. doi: 10.1021/acssensors.1c01212. Epub 2021 Jul 28.
Cisplatin, which selectively binds to N atoms of purines to inhibit normal replication and transcription, is a widely applied chemotherapeutic drug in the treatment of cancer. Though direct identification of cisplatin lesions on DNA is of great significance, existing sequencing methods have issues such as complications of preamplification or enrichment-induced false-positive reports. Direct identification of cisplatin lesions by nanopore sequencing (NPS) is in principle feasible. However, relevant investigations have never been reported. By constructing model sequences (83 nucleotides in length) containing a sole cisplatin lesion, identification of corresponding lesions by NPS is achieved with <10 ng of input sequencing library. Moreover, characteristic high-frequency noises caused by cisplatin lesions are consistently observed during NPS, clearly identifiable in corresponding high-pass filtered traces. This feature is, however, never observed in any other combinations of natural DNA bases and could be taken as a reference to identify cisplatin lesions on DNA. Further investigations demonstrate that cisplatin stalls the replication of phi29 DNA polymerase, which appears as a ∼5 pA level fluctuation in the single-molecule resolution. These results have confirmed the feasibility of NPS to identify cisplatin lesions at the genomic level and may provide new insights into understanding the molecular mechanism of platinum-based drugs.
顺铂选择性地与嘌呤的 N 原子结合，抑制正常的复制和转录，是一种广泛应用于癌症治疗的化疗药物。虽然直接鉴定 DNA 上的顺铂损伤具有重要意义，但现有的测序方法存在预扩增或富集诱导的假阳性报告等问题。纳米孔测序（NPS）直接鉴定顺铂损伤在原则上是可行的。然而，目前还没有相关的研究报道。通过构建包含单个顺铂损伤的模型序列（83 个核苷酸长），用<10ng 的输入测序文库即可实现 NPS 对相应损伤的鉴定。此外，在 NPS 过程中，顺铂损伤会产生特征性的高频噪声，在相应的高通滤波迹线中清晰可辨。然而，在任何其他天然 DNA 碱基的组合中都从未观察到这种特征，这可以作为识别 DNA 上顺铂损伤的参考。进一步的研究表明，顺铂会使 phi29 DNA 聚合酶的复制停滞，这在单分子分辨率下表现为约 5pA 水平的波动。这些结果证实了 NPS 在基因组水平上鉴定顺铂损伤的可行性，并可能为理解铂类药物的分子机制提供新的见解。
