i-Motifs (iMs) are non-canonical, four-stranded secondary structures formed by stacking of hemi-protonated CH+·C base pairs in cytosine-rich DNA sequences, predominantly at pH < 7. The presence of iM structures in cells was a matter of debate until the recent development of iM-specific antibody, iMab, which was instrumental for several studies that suggested the existence of iMs in live cells and their putative biological roles. We assessed the interaction of iMab with cytosine-rich oligonucleotides by biolayer interferometry (BLI), pull-down assay and bulk-FRET experiments. Our results suggest that binding of iMab to DNA oligonucleotides is governed by the presence of runs of at least two consecutive cytosines and is generally increased in acidic conditions, irrespectively of the capacity of the sequence to adopt, or not, an iM structure. Moreover, the results of the bulk-FRET assay indicate that interaction with iMab results in unfolding of iM structures even in acidic conditions, similarly to what has been observed with hnRNP K, well-studied single-stranded DNA binding protein. Taken together, our results strongly suggest that iMab actually binds to blocks of 2–3 cytosines in single-stranded DNA, and call for more careful interpretation of results obtained with this antibody.

中文翻译：

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iMab 抗体结合富含胞嘧啶的单链序列并展开 DNA i 基序


i-Motifs (iMs) 是非规范的四链二级结构，由富含胞嘧啶的 DNA 序列中半质子化的 CH+·C 碱基对堆叠而成，主要在 pH < 7 的情况下形成。细胞中 iM 结构的存在是直到最近 iM 特异性抗体 iMab 的开发，iMab 才成为争论的焦点，该抗体在多项研究中发挥了重要作用，这些研究表明活细胞中存在 iM 及其假定的生物学作用。我们通过生物层干涉测量 (BLI)、pull-down 测定和批量 FRET 实验评估了 iMab 与富含胞嘧啶的寡核苷酸的相互作用。我们的结果表明，iMab 与 DNA 寡核苷酸的结合受至少两个连续胞嘧啶运行的影响，并且通常在酸性条件下增加，无论序列是否采用 iM 结构的能力。此外，批量 FRET 测定的结果表明，即使在酸性条件下，与 iMab 的相互作用也会导致 iM 结构的展开，类似于在 hnRNP K（经过充分研究的单链 DNA 结合蛋白）中观察到的结果。总而言之，我们的结果强烈表明 iMab 实际上与单链 DNA 中的 2-3 个胞嘧啶块结合，并需要更仔细地解释使用该抗体获得的结果。