Lučinskaitė Evelina, Bokhobza Alexandre F E, Stannard Andrew, Meletiou Anna, Estell Chris, West Steven, Michele Lorenzo Di, Soeller Christian, Clowsley Alexander H
Department of Physiology, University of Bern, Bern, 3012, Switzerland.
Department of Chemistry, Imperial College London, London, W12 OBZ, UK.
Small. 2024 Dec;20(51):e2405032. doi: 10.1002/smll.202405032. Epub 2024 Oct 18.
The DNA-based single molecule super-resolution imaging approach, DNA-PAINT, can achieve nanometer resolution of single targets. However, the approach can suffer from significant non-specific background signals originating from non-specifically bound DNA-conjugated DNA-PAINT secondary antibodies as shown here. Using dye-modified oligonucleotides the location of DNA-PAINT secondary antibody probes can easily be observed with widefield imaging prior to beginning a super-resolution measurement. This reveals that a substantial proportion of DNA probes can accumulate, non-specifically, within the nucleus, as well as across the cytoplasm, of cells. Here, Shielded DNA-PAINT labeling is introduced, a method using partially or fully double-stranded docking strand sequences, prior to labeling, in buffers with increased ionic strength to greatly reduce non-specific interactions in the nucleus as well as the cytoplasm. This new labeling approach is evaluated against various conditions and it is shown that applying Shielded DNA-PAINT can reduce non-specific events approximately five-fold within the nucleus. This marked reduction in non-specific binding of probes during the labeling procedure is comparable to results obtained with unnatural left-handed DNA albeit at a fraction of the cost. Shielded DNA-PAINT is a straightforward adaption of current DNA-PAINT protocols and enables nanometer precision imaging of nuclear targets with low non-specific backgrounds.
基于DNA的单分子超分辨率成像方法——DNA-PAINT，能够实现单个目标的纳米级分辨率。然而，如图所示，该方法可能会受到源自非特异性结合的DNA偶联DNA-PAINT二抗的显著非特异性背景信号的影响。在开始超分辨率测量之前，使用染料修饰的寡核苷酸，通过宽场成像可以轻松观察到DNA-PAINT二抗探针的位置。这表明，相当一部分DNA探针会非特异性地积聚在细胞的细胞核内以及整个细胞质中。在此，引入了屏蔽DNA-PAINT标记法，即在标记前，在离子强度增加的缓冲液中使用部分或完全双链的对接链序列，以大大减少细胞核和细胞质中的非特异性相互作用。针对各种条件对这种新的标记方法进行了评估，结果表明，应用屏蔽DNA-PAINT可以将细胞核内的非特异性事件减少约五倍。在标记过程中探针非特异性结合的显著减少与使用非天然左旋DNA获得的结果相当，尽管成本仅为其一小部分。屏蔽DNA-PAINT是对当前DNA-PAINT方案的直接改进，能够以低非特异性背景对核目标进行纳米级精确成像。
