University of Groningen Guanine quadruplex monoclonal antibody 1H6 cross-reacts with restrained thymidine-rich single stranded DNA Kazemier,

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. INTRODUCTION In vitro, single stranded guanine-rich RNA or DNA readily adopts higher order structures known as guanine quadruplex (G4) structures (1). Despite accumulating evidence supporting a role for G4 RNA and DNA in diverse biological processes (2,3) detection of G4 structures in situ has been problematic in part because suitable reagents to detect G4 structures have been lacking. We recently described 1H6, a mouse monoclonal antibody that strongly binds to synthetic interas well as intra-molecular G4 DNA structures (4,5). The specificity of 1H6 for various G4 DNA structures was validated in vitro and in vivo and epitopes recognized by 1H6 in cells were found to be sensitive to DNAse treatment but resistant to RNAse. Most nuclei of cultured cells were labeled by 1H6 and this staining intensity increased following incubation with G4 stabilizing ligands (4). Human and murine metaphase chromosomes were strongly labeled by 1H6 as were the nuclei of cells in most human tissues (4). In subsequent studies the binding to metaphase chromosomes was confirmed by immuno-electron microscopy and it was shown that 1H6 selectively binds to heterochromatic areas in the nucleus as well as heterochromatic bands in salivary gland polytene chromosomes from Drosophila (5). In this and other species nuclei of somatic cells stained much stronger with 1H6 than mitotic cells of the germline (5). In the current study, we extended our studies on the specificity of the 1H6 antibody following the observation that binding of 1H6 to DNA fibers is greatly increased upon DNA denaturation. MATERIALS AND METHODS Immunofluorescence on DNA fibers DNA fibers from mouse embryonic stem cells and HEK293 cells were obtained as described (6). Briefly, cells were harvested, 104–105 cells were spotted on a microscope slide and *To whom correspondence should be addressed. Tel: +31 50 3617300; Fax: +31 50 3617310; Email: k.paeschke@umcg.nl Correspondence may also be addressed to Peter M. Lansdorp. Tel: +31 50 3617300; Fax: +31 50 3617310; Email: p.m. lansdorp@umcg.nl C © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Downloaded from https://academic.oup.com/nar/article-abstract/45/10/5913/3752490/Guanine-quadruplex-monoclonal-antibody-1H6-cross by University of Groningen user on 15 September 2017 5914 Nucleic Acids Research, 2017, Vol. 45, No. 10 after 30–60 min cells were lysed (1% sodium dodecyl sulphate, 200 mM Tris and 50 mM ethylenediaminetetraacetic acid (EDTA), pH7.4). After 2 min the slide was tilted to a 15–30 degree angle to obtain DNA fibers. Slides with DNA fibers were typically air-dried and fixed with methanol and acetic acid (3:1). For experiments slides were rehydrated with phosphate buffered saline (PBS) and treated with RNAse A (Invitrogen, 0.4 mg/ml in PBS) for 1 h at 37◦C under a coverslip. DNA fibers were denatured by incubation for 15 min in 3 M HCl or 3 M NaOH or the slides were treated with 50% formamide in Na-citrate pH7.0 for 15 min at 75◦C on a slide heater. Slides were washed three times and blocked (5% bovine serum albumin (BSA) in PBS containing 300 mM glycine) for 30 min at room temperature followed by incubation with purified 1H6 or an isotype control antibody (mouse IgG2b, clone MOPC-141, Sigma) at 1 g/ml in blocking solution for 2 h at room temperature in a humidified slide incubator. Slides were incubated for 2–4 h with Alexa-Fluor-488 anti-mouse IgG at 1:2000 (Invitrogen) and DNA was counterstained using DAPI, NucRedTM Dead 647 or YOYO-1. Fluorescence microscopy was done using a Zeiss-LSM780 NLO confocal microscope. Oligonucleotides used All 5′-biotinylated, 5′-Cy5 labeled or unmodified oligonucleotides were from IDT (Leuven, Belgium). Only 5′-Cy5 labeled oligonucleotides were High-performance liquid chromatography (HPLC) purified, others were standard desalted. Lyophilized oligonucleotides were reconstituted to 100 M in water and were stored at −20◦C. See Supplementary Table S1 for sequences of oligonucleotides used in this study. In vitro formation of G4 structures G4 structures were folded as described previously (4). Briefly, oligonucleotides listed in Supplementary Table S1 were diluted to 10 M in TE buffer + 100 mM KCl (10 mM Tris–HCl pH = 7.5, 1 mM EDTA, 100 mM KCl). Following denaturation for 10 min at 95◦C, G4 structures could form overnight by slowly cooling to room temperature in the heating block. In Supplementary Table S1 oligonucleotides folded into G4 structures are marked in the seventh column.