Department of Chemistry, Sarafan ChEM-H Institute, and Stanford Cancer Institute, Stanford University, Stanford, CA, 94305, USA.
Department of Chemistry, Le Moyne College, Syracuse, NY, 13214, USA.
Nat Commun. 2022 Aug 26;13(1):5043. doi: 10.1038/s41467-022-32494-8.
Fluorescence labeling of DNAs is broadly useful, but methods for labeling are expensive and labor-intensive. Here we describe a general method for fluorescence labeling of oligonucleotides readily and cost-efficiently via base excision trapping (BETr), employing deaminated DNA bases to mark label positions, which are excised by base excision repair enzymes generating AP sites. Specially designed aminooxy-substituted rotor dyes trap the AP sites, yielding high emission intensities. BETr is orthogonal to DNA synthesis by polymerases, enabling multi-uracil incorporation into an amplicon and in situ BETr labeling without washing. BETr also enables labeling of dsDNA such as genomic DNA at a high labeling density in a single tube by use of nick translation. Use of two different deaminated bases facilitates two-color site-specific labeling. Use of a multi-labeled DNA construct as a bright fluorescence tag is demonstrated through the conjugation to an antibody for imaging proteins. Finally, double-strand selectivity of a repair enzyme is harnessed in sensitive reporting on the presence of a target DNA or RNA in a mixture with isothermal turnover and single nucleotide specificity. Overall, the results document a convenient and versatile method for general fluorescence labeling of DNAs.
DNA 的荧光标记具有广泛的用途，但标记方法既昂贵又费时费力。在这里，我们描述了一种通过碱基切除捕获（BETr）简便且经济高效地对寡核苷酸进行荧光标记的通用方法，该方法利用脱氨 DNA 碱基标记标记位置，然后由碱基切除修复酶切除产生 AP 位点。专门设计的氨氧基取代转子染料捕获 AP 位点，产生高发射强度。BETr 与聚合酶的 DNA 合成正交，可在不进行洗涤的情况下将多尿嘧啶掺入扩增子中，并进行原位 BETr 标记。BETr 还可通过缺口平移在单个管中以高标记密度对 dsDNA（如基因组 DNA）进行标记。使用两种不同的脱氨碱基可促进双色定点标记。通过将多标记 DNA 构建体与抗体缀合来进行荧光标记，以用于蛋白质成像。最后，利用修复酶的双链体选择性，在等温循环和单核苷酸特异性下，对混合物中目标 DNA 或 RNA 的存在进行灵敏的报告。总体而言，这些结果证明了一种方便且通用的 DNA 通用荧光标记方法。
