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. 2017 Jun 2;45(10):5913-5919.
doi: 10.1093/nar/gkx245.

Guanine quadruplex monoclonal antibody 1H6 cross-reacts with restrained thymidine-rich single stranded DNA

Hinke G Kazemier  1 , Katrin Paeschke  1 , Peter M Lansdorp  1   2   3
Affiliations

Guanine quadruplex monoclonal antibody 1H6 cross-reacts with restrained thymidine-rich single stranded DNA

Hinke G Kazemier et al. Nucleic Acids Res. .

Abstract

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.

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Figures

Figure 1.
Figure 1.
Denaturation of DNA fibers reveals novel 1H6 binding sites. Binding of 1H6 (light grey spots) to DNA fibers (dark grey lines) before (A) and after denaturation of DNA with 3 M HCl (B), heat and formamide (C) or 3 M NaOH (D).
Figure 2.
Figure 2.
1H6 cross reacts with single stranded poly-thymidine. (A) Binding of 1H6 to different oligonucleotides determined by ELISA. Results of three independent experiments performed in triplicate for each oligonucleotide or G4 structure. Error bars represent SEM. 1H6 binding to (T4G4)2 was set at 100% (A) Binding of 1H6 to (T4G4)2 G4 structures and poly(T) but not poly(A), poly(C) or random single stranded DNA. (B) Binding of 1H6 to poly(T) in ELISA requires more than eight thymidines. Statistical significance compared to T4G4 was determined by Student's t-test. All tested substrates showed a P ≤ 0.0001.
Figure 3.
Figure 3.
1H6 does not bind to G4 structures without thymidines, but binding of 1H6 to these G4 structures requires at least three thymidines in the oligonucleotides. (A–C) ELISA experiments of 1H6 binding to various G4 structures. All ELISA experiments were performed in triplicate and 1H6 binding to (T4G4)2 was set at 100%. Error bar represent SEM. (A) Comparison of 1H6 binding to (T4G4)2, versuss (A4G4)2 and (C4G4)2. (B) Comparison of 1H6 binding to (T4G4)2, versus (T3G4)2 (T2G4)2, (T4G3)2 (T4G2)2 by ELISA. (A and B) 1H6 does not bind to (A4G4)2, (C4G4)2, (T2G4)2, (T4G3)2 and (T4G2)2. Statistical significance compared to (T4G4)2 was determined by Student's t-test. **: P ≤ 0.0001, * P ≤ 0.001. (C) 1H6 binds to oligo's with 4 thymidines (poly(T4)) if these are bound to ELISA plate at both the 5΄ and the 3΄ end via biotin (bio-poly(T4)-bio) but not if the are bound only via the 5΄ end (bio-poly(T4)). Controls: (T4G4)2 G4 and T15 with just one biotin (bio-poly(T15)). (D) Cartoon showing how the presentation of short stretches of T's could determine whether 1H6 binds or not.
Figure 4.
Figure 4.
1H6 binds strongly to (T4G4)2 but not to poly(T) in solution. (A and B) MST measurements of 5΄Cy5-labeled oligonucleotides (A) (T4G4)2, (A4G4)2 and poly(T) and (B) (T4G3)2, (T3G4)2 (T3G3)2 and MYC (25 nM), binding with 1H6 at several concentrations (0.03 nM-1 μM). Error bars represent standard deviation with n = 3. (C) Table listing G4 motifs and their respective binding affinities. (D) Determination of the stoichiometry binding of 1H6, titrated in a narrow concentration range (50–330 nM) and 5΄Cy5-labeled (T4G4)2 (200 nM). Linear regression of the saturated and non-saturated data points reveal that 200 nM (T4G4)2 was bound by 215 nM 1H6, yielding a stoichiometry of 1:1. (n = 1).
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