Formation of G‑Wires: The Role of G:C-Base Pairing and G‑Quartet Stacking

G-wireslong, continuous G-quadruplexesare a promising element for use in nanotechnology, particularly in molecular electronics. The pairing of G-quadruplex structures through complementary GC-termini was tested. We designed four analogous G-quadruplex-forming oligonucleotides with the same core sequence d­(GG­T­G₄­T­GG) and with different number and position of GC-ends. We used circular dichroism to determine the topology and relative orientation of G-quadruplexes, ¹H NMR to identify Watson–Crick base pairing, and dynamic light scattering to examine the size of the G-quadruplex assemblies. The quadruplexes associated through GC-base pairs forming (G:C:G:C)-quartets and by stacking of unhindered terminal (G:G:G:G)-quartets. These two modes of association had different specificities: stacking occurred at both the 5′- and 3′- faces of the quadruplex, whereas GC-base pairing occurred only at the 5′-face of the quadruplex. These insights may be employed to design G-quadruplex-based nanowires or more complex architectures

Dot matrix of segmental duplications in the physical cluster 92.

<p>Physical cluster 92 is located on chromosome 18 and comprises twenty-two CYP82 sequences, one CYP74 sequence and one CYP704 sequence. The dots and the black lines represent the sequence similarities in cluster <b>92</b> compared to itself. The red rectangles on the sides of the graph represent cytochrome P450 sequences. Complete genes are labeled with their name and pseudogenes are labeled with “p” and the P450 family. A) The similarities for the whole cluster <b>92</b>. B) A zoom of the red squared region, which contains two 30kb blocks with very high similarity. Analysis of gene expression showed that CYP82D15 and CYP82D18 are co-expressed (expression cluster <b>A</b>, expression in ripe berries) as well as CYP82D17 and CYP82D20v2 (expression cluster <b>C</b>, expression in downy mildew infected leaves). The pseudogenes of the enlarged segment are not expressed.</p

Heatmap of the P450 sequences, clustered according to their expression profile.

<p>The expression levels were averaged over the experiments classified in one of the six experimental categories: leaves, downy mildew (<i>Plasmopara viticola</i>) infected leaves, powdery mildew (<i>Erysiphe necator</i>) infected leaves, flowers, young berries and ripe berries. This heatmap includes the 457 expressed cytochrome P450 sequences. The color scale for the expression level represents FPKM values normalized by row ((FPKM value − row minimum) / row maximum). The color bars on the left are showing the eight expression clusters, which are designated by the letters on the side.</p

Physical map of cytochrome P450 sequences on the 19 V. vinifera chromosomes.

<p>Yellow circles represent isolated annotations, light blue circles represent physical clusters composed of members of only one P450 family and the purple circles represent physical clusters composed of members of 2–3 P450 families. The circle size is proportional to the number of sequences in the cluster. The numbers 1–19 are chromosome numbers and “Un” is “Unknown chromosome” which contains sequences with unknown chromosome location.</p

Molecular phylogenetic analysis of grapevine cytochrome P450.

<p>The alignment of full-length cytochrome P450 protein sequences was used to generate a maximum likelihood tree. The dark blue clade is the clan 71, which often contains genes involved in specialized metabolism. The highlighted genes belong to the seven largest physical clusters.</p

Similarity of the P450 genes between and within clusters.

<p>For each circle, the grey bars correspond to the 19 grape chromosomes and the “Unknown chromosome”. The lines connect complete P450 genes according to their similarity. The lines outside the circles show the similarity between genes of the same cluster, whereas the lines in the circle connect similar genes of different clusters. Only P450 genes that form clusters composed of at least two complete genes are illustrated here. The seven largest clusters are labeled with numbers corresponding to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0199902#pone.0199902.t001" target="_blank">Table 1</a>. The lines are connecting the genes corresponding to the best BLAST hit (A), second best hit (B) or third best blast hit (C).</p

Description of the seven largest physical P450 clusters in the <i>V</i>. <i>vinifera</i> genome.

<p>Description of the seven largest physical P450 clusters in the <i>V</i>. <i>vinifera</i> genome.</p