Kazemier Hinke G, Paeschke Katrin, Lansdorp Peter M
European Research Institute for the Biology of Ageing, University of Groningen, University Medical Centre Groningen, A. Deusinglaan 1, NL-9713 AV Groningen, The Netherlands.
Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, BC V5Z 1L3, Canada.
Nucleic Acids Res. 2017 Jun 2;45(10):5913-5919. doi: 10.1093/nar/gkx245.
Previously we reported the production and characterization of monoclonal antibody 1H6 raised against (T4G4)2 intermolecular guanine quadruplex (G4) DNA structures (Henderson A. et al. (2014) Nucleic Acids Res., 42, 860-869; Hoffmann R.F. et al. (2016) Nucleic Acids Res., 44, 152-163). It was shown that 1H6 strongly stains nuclei and has exquisite specificity for heterochromatin by immuno-electron microscopy. Here we extend our studies of 1H6 reactivity using enzyme-linked immunosorbent assay (ELISA) and microscale thermophoresis (MST). As previously reported, 1H6 was found to strongly bind intermolecular G4 structures with a (T4G4)2 sequence motif. However, using both methods we did not detect significant binding to G4 structures without thymidines in their sequence motif or to G4 structures made with (T2G4)2 oligonucleotides. In addition, we observed strong, sequence-specific binding of 1H6 by ELISA to immobilized single stranded poly(T) DNA but not to immobilized poly(C) or poly(A) homo-polymers. Cross-reactivity of 1H6 to poly(T) was not measured in solution using MST. 1H6 was furthermore found to bind to selected areas on DNA fibers but only after DNA denaturation. Based on these observations we propose that 1H6 binds with high affinity to adjacent T's that are restricted in their movement in selected G4 structures and denatured DNA. Cross-reactivity of 1H6 to immobilized single stranded T-rich DNA next to its previously reported specificity for bona fide G4 structures needs to be taken into account in the interpretation of 1H6 binding to (sub-) cellular structures.
此前我们报道了针对（T4G4）2分子间鸟嘌呤四链体（G4）DNA结构产生的单克隆抗体1H6及其特性（亨德森A.等人（2014年），《核酸研究》，42卷，860 - 869页；霍夫曼R.F.等人（2016年），《核酸研究》，44卷，152 - 163页）。免疫电子显微镜显示1H6强烈染色细胞核，对异染色质具有极高的特异性。在此，我们使用酶联免疫吸附测定（ELISA）和微量热泳动（MST）扩展了对1H6反应性的研究。如先前报道，发现1H6能强烈结合具有（T4G4）2序列基序的分子间G4结构。然而，使用这两种方法，我们均未检测到其与序列基序中没有胸腺嘧啶的G4结构或由（T2G4）2寡核苷酸形成的G4结构有显著结合。此外，我们通过ELISA观察到1H6对固定化的单链聚（T）DNA有强烈的、序列特异性结合，但对固定化的聚（C）或聚（A）同聚物无结合。使用MST未在溶液中测定1H6与聚（T）的交叉反应性。此外，还发现1H6仅在DNA变性后才与DNA纤维上的选定区域结合。基于这些观察结果，我们提出1H6以高亲和力结合在选定的G4结构和变性DNA中移动受限的相邻T。在解释1H6与（亚）细胞结构的结合时，需要考虑1H6除了先前报道的对真正G4结构的特异性外，对固定化的富含单链T的DNA的交叉反应性。
