Kinetic Referencing Allows Identification of Epigenetic Cytosine Modifications by Single-Molecule Hybridization Kinetics and Superresolution DNA-PAINT Microscopy,ACS Nano

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Kinetic Referencing Allows Identification of Epigenetic Cytosine Modifications by Single-Molecule Hybridization Kinetics and Superresolution DNA-PAINT Microscopy
ACS Nano ( IF 15.8 ) Pub Date : 2023-12-29 , DOI: 10.1021/acsnano.3c08451
Julian Bauer ₁ , Andreas Reichl ₂ , Philip Tinnefeld ₁

Affiliation

We develop a DNA origami-based internal kinetic referencing system with a colocalized reference and target molecule to provide increased sensitivity and robustness for transient binding kinetics. To showcase this, we investigate the subtle changes in binding strength of DNA oligonucleotide hybrids induced by cytosine modifications. These cytosine modifications, especially 5-methylcytosine but also its oxidized derivatives, have been increasingly studied in the context of epigenetics. Recently revealed correlations of epigenetic modifications and disease also render them interesting biomarkers for early diagnosis. Internal kinetic referencing allows us to probe and compare the influence of the different epigenetic cytosine modifications on the strengths of 7-nucleotide long DNA hybrids with one or two modified nucleotides by single-molecule imaging of their transient binding, revealing subtle differences in binding times. Interestingly, the influence of epigenetic modifications depends on their position in the DNA strand, and in the case of two modifications, effects are additive. The sensitivity of the assay indicates its potential for the direct detection of epigenetic disease markers.

中文翻译：

动力学参考允许通过单分子杂交动力学和超分辨率 DNA-PAINT 显微镜鉴定表观遗传胞嘧啶修饰

我们开发了一种基于 DNA 折纸的内部动力学参考系统，具有共定位的参考分子和目标分子，以提高瞬态结合动力学的灵敏度和稳健性。为了展示这一点，我们研究了胞嘧啶修饰诱导的 DNA 寡核苷酸杂交体结合强度的微妙变化。这些胞嘧啶修饰，特别是 5-甲基胞嘧啶及其氧化衍生物，在表观遗传学背景下得到了越来越多的研究。最近揭示的表观遗传修饰与疾病的相关性也使它们成为早期诊断的有趣生物标志物。内部动力学参考使我们能够通过瞬时结合的单分子成像来探测和比较不同表观遗传胞嘧啶修饰对具有一或两个修饰核苷酸的 7 核苷酸长 DNA 杂交体强度的影响，揭示结合时间的细微差异。有趣的是，表观遗传修饰的影响取决于它们在 DNA 链中的位置，并且在两种修饰的情况下，效果是相加的。该测定的灵敏度表明其直接检测表观遗传疾病标志物的潜力。

更新日期：2023-12-29

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