GNG Motifs Can Replace a GGG Stretch during G-Quadruplex Formation in a Context Dependent Manner

<div><p>G-quadruplexes are one of the most commonly studied non-B DNA structures. Generally, these structures are formed using a minimum of 4, three guanine tracts, with connecting loops ranging from one to seven. Recent studies have reported deviation from this general convention. One such deviation is the involvement of bulges in the guanine tracts. In this study, guanines along with bulges, also referred to as GNG motifs have been extensively studied using recently reported <i>HOX11</i> breakpoint fragile region I as a model template. By strategic mutagenesis approach we show that the contribution from continuous G-tracts may be dispensible during G-quadruplex formation when such motifs are flanked by GNGs. Importantly, the positioning and number of GNG/GNGNG can also influence the formation of G-quadruplexes. Further, we assessed three genomic regions from <i>HIF1</i> alpha, <i>VEGF</i> and <i>SHOX</i> gene for G-quadruplex formation using GNG motifs. We show that <i>HIF1</i> alpha sequence harbouring GNG motifs can fold into intramolecular G-quadruplex. In contrast, GNG motifs in mutant <i>VEGF</i> sequence could not participate in structure formation, suggesting that the usage of GNG is context dependent. Importantly, we show that when two continuous stretches of guanines are flanked by two independent GNG motifs in a naturally occurring sequence (<i>SHOX</i>), it can fold into an intramolecular G-quadruplex. Finally, we show the specific binding of G-quadruplex binding protein, Nucleolin and G-quadruplex antibody, BG4 to <i>SHOX</i> G-quadruplex. Overall, our study provides novel insights into the role of GNG motifs in G-quadruplex structure formation which may have both physiological and pathological implications.</p></div

Summary of oligomeric DNA studied that support intramolecular G-quadruplex formation using GNG motifs and cartoon showing 2-D models of potential G-quadruplex structures.

<p>(A) Summary of oligomeric DNA of <i>HOX11</i> region I, and its mutants used in the study and their potential to fold into intramolecular G-quadruplex using GNG motifs. The red cross (from 2^nd to 7^th column) represents alterations in the DNA sequence and the black ‘tick mark’ represents no change in sequence. The red circle in the 8^th column indicates no formation of intramolecular G-qudraplex involving GNG motifs. The black circle in the 8^th column indicates formation of intramolecular G-quadruplex (Intra Gq) involving GNG motifs, under 100 mM KCl condition. The 9^th column provides an inference depending on the EMSA result, indicating whether GNG motifs are involved (black triangle) during intramolecular G-quadruplex structure formation and whether it is the 1^st GNG or the 2^nd GNG motif that is actually accommodated in the intramolecular structure. Black triangles for KD36, KD37, KD39, KD43 and KD44 suggest GNG involvement because their respective mutation abrogated intramolecular GNG motif involving G-quadruplex structure formation. The red triangle indicates no intramolecular G-quadruplex structure formation. (B) Left panel shows the mobility shift of MS113 intramolecular species in comparison to MN37 when resolved on a native PAGE containing KCl (100 mM). Unlabeled MS113 was loaded next to radioactive oligomer lane in the same gel and was electrophoresed. Based on the PI image, corresponding position of the two boxed bands (blue and red) in the unlabeled MS113 lane were excised out and intramolecular species were eluted subsequently. These two intramolecular species were then subjected to CD analysis (the spectrum and its colour correspond to the respective bands boxed in the gel). (C-E) 2-D model representation of G-quadruplexes involving GNG motifs based on the EMSA results. The pink circles indicate guanines involved in G-tetrad formation. 2-D model depicting intramolecular G-quadruplex formation on oligomers MS113 (C), KD42 (D) and MS115 (E) are shown.</p

Analysis of involvement of GNG motif on G-quadruplex structure formation, when second stretch of guanines was mutated at <i>HOX11</i> breakpoint region I.

<p>(A) The oligomeric DNA sequence spanning region I, a G-rich strand (MN38) and complementary C rich strand (MN37) were designed from the <i>HOX11</i> breakpoint region. Mutants of MN38 were also designed and named as MS104, MS115, MS116, MS117. The mutations were introduced such that the second G stretch is altered to thymine (indicated in red) and keeping that as backbone either the first GNG motif (blue) or the second GNG motif (blue) or both are mutated (indicated in red). (B) The radiolabeled G- and C-rich strands along with the mutants were incubated in the presence (+) or absence (-) of KCl (100 mM) and resolved in the absence (left panel) or presence (right panel) of KCl (100 mM), in the gel and running buffer. The substrate, intramolecular (Intra), and intermolecular (Inter) quadruplex structures are indicated. The red boxed ‘Intra’ indicate intramolecular G-quadruplex species involving GNG motifs.</p

Evaluation of role of individual guanines in a GNGNG motif during G-quadruplex formation.

<p>(A) The oligomeric DNA sequence with mutations were made using MS113 as template wherein the guanines in the first GNG motifs were altered sequentially to thymine (indicated in red) generating KD36 and KD37. (B) The G- and C-rich strands along with the mutants were incubated in the presence (+) or absence (-) of KCl (100 mM) and resolved in the absence (left panel) or presence (right panel) of KCl (100 mM), in the gel and running buffer. The substrate, intramolecular (Intra) and intermolecular (Inter) quadruplex structures are indicated. The red boxed “intra” indicate intramolecular G-quadruplex species involving GNG motifs.</p

Binding of Nucleolin to G-quartets with GNGs as a structural component.

<p>(A) SDS PAGE profile showing purified recombinant His-tagged Nucleolin (55 kDa) (upper panel) and corresponding western blot using anti-His (lower panel). (B) Binding of purified recombinant Nucleolin to oligomers KD52 (harbouring two G stretches and GNG motifs) and its reverse complement KD53 in the presence of KCl (100 mM). (C) Analysis of CD spectra of recombinant His-tagged Nucleolin (20 μg) and its ability to bind KD52.</p

Evaluation of effect of GNG motifs in G-quadruplex structure formation at HOX11 translocation breakpoint region.

<p>(A) The oligomeric DNA sequence spanning region I of chromosomal translocation breakpoint region from <i>HOX11</i> gene. A G-rich strand (MN38), its mutants (MS112, MS113, MS114, MS124) and complementary C rich strand (MN37) are designed based on sequence of <i>HOX11</i> breakpoint region and used for the study. The mutations were introduced such that the first G stretch is altered to thymines (indicated in red) and keeping that as the backbone, the first GNG motif (indicated in blue) or the second GNG motif (indicated in blue) or both are mutated (mutations shown in red). (B) Radiolabeled oligomeric DNA with G-rich, C-rich or mutant DNA sequence were incubated in the presence (+) or absence (-) of KCl (100 mM) and resolved in the absence (left panel) or presence (right panel) of KCl (100 mM) in the gel and running buffer. (C) Radiolabeled oligomeric DNA with G-rich, C-rich or mutant DNA sequence were heat denatured, gradually cooled and resolved in the presence of KCl in the gel and running buffer. For each oligonucleotide duplicate reactions are loaded in adjacent lanes. The substrate, intramolecular (Intra), and intermolecular (Inter) quadruplex structures are indicated. The red boxed intra indicate intramolecular G-quadruplex species involving GNG motifs. The blue coloured box indicate bimolecular G-quadruplex.</p

Evaluation of different genomic sequences harboring guanine stretches and GNG motifs for their ability to fold into G-quadruplex structures.

<p>(A) The oligomeric DNA spanning portion of <i>HIF1</i> alpha promoter and <i>VEGF</i> promoter. In both the cases, G-rich strand (RT17 and KD49 are from <i>HIF1</i> alpha and <i>VEGF</i>, respectively), its mutants (KD47 and KD48 are RT17 mutants; KD51 is the mutant of KD49) and complementary C-rich strand (MN89 and KD50 are reverse complement of RT17 and KD49, respectively) are depicted. In the case of <i>HIF1</i> alpha the mutations were made in the first and the fourth G stretch sequence, while it was the fourth G stretch in the case of <i>VEGF</i> sequence. (B) Native PAGE profile showing inter and intramolecular G-quadruplex formation at <i>HIF1</i> alpha promoter and <i>VEGF</i> promoter. The G- and C-rich strands along with the mutants were incubated in the presence (+) or absence (-) of KCl (100 mM) and resolved in the absence (left panel) or presence (right panel) of KCl (100 mM). For other details, refer <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158794#pone.0158794.g001" target="_blank">Fig 1</a> legend. The bar diagram at the lower portion of the right panel represents relative migration of the boxed bands for RT17, KD47 and KD48 with respect to MN89.</p

Impact of increase in concentration of oligomers on formation of inter and intramolecular G-quadruplex species.

<p>The wild type DNA sequence spanning region I of <i>HOX11</i> breakpoint region, MN38 (G-rich strand) or MS113 (mutant) was used for titration. MN37, the C-rich strand was used as a negative control. (A) EMSA gel shows formation of inter (boxed grey) and intramolecular G-quadruplexes. The intramolecular G-quadruplexes were further divided into species with (boxed blue) or without (boxed red) GNG motifs. (B) Bar diagram shows quantification of inter and intramolecular species over a range of oligonucleotide concentrations.</p

<i>SHOX</i> gene intramolecular G-quadruplex uses two GNG motifs for structure formation.

<p>(A) The oligomeric DNA sequence spanning portion of <i>SHOX</i> gene (KD52) and its complementary C rich strand (KD53). Native PAGE profile shows the formation of inter and intramolecular G-quadruplex in presence of KCl (100 mM). For other details, refer <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158794#pone.0158794.g001" target="_blank">Fig 1</a> legend. Quantification for inter and intra G-quadruplex species are also shown. (B) CD analysis of KD52 and KD53 in presence of KCl (100 mM). (C) DMS assay on KD52. Radiolabelled KD52 was treated with DMS in presence of either KCl (100 mM) or LiCl (100 mM). Chemically modified DNA was then cleaved using piperidine and the reaction products were resolved on 15% denaturing PAGE (left panel). Quantification for individual guanine bands (in LiCl and KCl cases) is depicted in the right panel. Individual bands corresponding to each guanines were quantified from the lanes 2 and 3 using Multi Gauge and relative band intensities are plotted pairwise. (D) 2-D model showing potential intramolecular G-quadruplex formation on KD52.</p

Evaluation of BG4 binding to G-quartets with GNGs as a structural component.

<p>(A) Binding of MN38 to increasing concentrations of BG4 (0.15, 0.3, 0.45, 0.6 and 0.9 μg; Lanes2-6) in presence of KCl (100 mM). (B) Binding of KD52 (G-quadruplex forming <i>SHOX</i> sequence) and a scrambled DNA control, BTM6 to BG4 (0.45 0078g) in presence of 100 mM KCl. (C) CD spectra of FLAG-tagged BG4 (5 μg) and its ability to bind KD52. (D) Immunoblot for BG4 (control and after IP) using anti-FLAG antibody. Quantification depicts relative reduction in BG4 level after IP. (E) Binding of BG4 to KD52. Upper panel depicts a representative gel image (6% native PAGE) for the relative binding of BG4 (between control and IP reaction) to KD52. Quantification based on multiple repeats is also shown.</p