Assessing Readability of an 8-Letter Expanded Deoxyribonucleic Acid Alphabet with Nanopores
Assessing Readability of an 8-Letter Expanded Deoxyribonucleic Acid Alphabet with Nanopores
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DOI：
10.1021/jacs.3c00829
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发表时间：
2023-04-10
影响因子：
15
通讯作者：
Laszlo，Andrew H.
中科院分区：
文献类型：
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作者：
Thomas，Christopher A.;Craig，Jonathan M.;Laszlo，Andrew H.
化学家们现在已经合成了新的DNA类型，在标准人类DNA中发现的四种标准核苷酸（鸟嘌呤，腺嘌呤，胞嘧啶和胸腺嘧啶）上添加核苷酸。这种“人工扩增的遗传信息系统”如今被用于分子诊断;支持定向进化以创造医学上有用的受体、配体和催化剂;以及探索与生命早期进化有关的问题。进一步的应用受到不能直接测序含有非标准核苷酸的DNA的限制。纳米孔测序非常适合此目的，因为它不需要酶促合成、扩增或核苷酸修饰。在这里，我们采取第一步，通过使用MspA（耻垢分枝杆菌A）纳米孔评估其纳米孔信号范围来实现8个字母的“hachimoji”扩展DNA字母表的纳米孔测序。我们发现thathachimojiDNA在纳米孔测序中表现出比单独的标准DNA更宽的信号范围，并且thathachimojiissingle-base取代具有高置信度。由于纳米孔测序依赖于分子马达来控制DNA的运动，因此我们随后通过跟踪单个Hel 308分子沿LonghachimojiDNA的易位、监测酶动力学和过早的酶从DNA解离来评估Hel 308马达酶与非标准核苷酸的相容性。我们发现，Hel 308是兼容的withhachimojiDNA，但更频繁地解离时，走过C-糖苷核苷，相比N-糖苷。通过Hel 308内的特定位点的C-杀糖核苷诱导更高的解离可能性。这突出了优化纳米孔测序马达以处理不同糖苷键的需要。它还可以为未来的替代DNA系统的设计提供信息，这些系统可以用现有的马达和孔进行测序。
Chemists have now synthesized new kinds of DNA that add nucleotides to the four standard nucleotides (guanine, adenine, cytosine, and thymine) found in standard Terran DNA. Such “artificially expanded genetic information systems” are today used in molecular diagnostics; to support directed evolution to create medically useful receptors, ligands, and catalysts; and to explore issues related to the early evolution of life. Further applications are limited by the inability to directly sequence DNA containing nonstandard nucleotides. Nanopore sequencing is well-suited for this purpose, as it does not require enzymatic synthesis, amplification, or nucleotide modification. Here, we take the first steps to realize nanopore sequencing of an 8-letter “hachimoji” expanded DNA alphabet by assessing its nanopore signal range using the MspA (Mycobacterium smegmatisporin A) nanopore. We find thathachimojiDNA exhibits a broader signal range in nanopore sequencing than standard DNA alone and thathachimojisingle-base substitutions are distinguishable with high confidence. Because nanopore sequencing relies on a molecular motor to control the motion of DNA, we then assessed the compatibility of the Hel308 motor enzyme with nonstandard nucleotides by tracking the translocation of single Hel308 molecules alonghachimojiDNA, monitoring the enzyme kinetics and premature enzyme dissociation from the DNA. We find that Hel308 is compatible withhachimojiDNA but dissociates more frequently when walking over C-glycoside nucleosides, compared to N-glycosides. C-glycocide nucleosides passing a particular site within Hel308 induce a higher likelihood of dissociation. This highlights the need to optimize nanopore sequencing motors to handle different glycosidic bonds. It may also inform designs of future alternative DNA systems that can be sequenced with existing motors and pores.