Controlling the stoichiometry and strand polarity of a tetramolecular G-quadruplex structure by using a DNA origami frame

Abstract

Guanine-rich oligonucleotides often show a strong tendency to form supramolecular architecture, the so-called G-quadruplex structure. Because of the biological significance, it is now considered to be one of the most important conformations of DNA. Here, we describe the direct visualization and single-molecule analysis of the formation of a tetramolecular G-quadruplex in KCl solution. The conformational changes were carried out by incorporating two duplex DNAs, with G–G mismatch repeats in the middle, inside a DNA origami frame and monitoring the topology change of the strands. In the absence of KCl, incorporated duplexes had no interaction and laid parallel to each other. Addition of KCl induced the formation of a G-quadruplex structure by stably binding the duplexes to each other in the middle. Such a quadruplex formation allowed the DNA synapsis without disturbing the duplex regions of the participating sequences, and resulted in an X-shaped structure that was monitored by atomic force microscopy. Further, the G-quadruplex formation in KCl solution and its disruption in KCl-free buffer were analyzed in real-time. The orientation of the G-quadruplex is often difficult to control and investigate using traditional biochemical methods. However, our method using DNA origami could successfully control the strand orientations, topology and stoichiometry of the G-quadruplex.

Orienting Tetramolecular G‐Quadruplex Formation: The Quest for the Elusive RNA Antiparallel Quadruplex

“…The samples were prepared as described above and the concentration of each strand was 5 mm. Oligothymidylate markers, 9 to 100 nucleotide-long (dT 9 , dT 15 , dT 22 , dT 30 , dT 45 , dT 57 and dT 100 ) were also loaded on the gel. After electrophoresis, the gels were visualised by UV shadowing with a DNR bio-imaging, MF-ChemiBIS 3.2 system.…”

“…[14] However, the construction of nanostructures based on non-canonical pairing is gaining importance and nanodevices based on motifs such as quadruplexes or triplexes have been described. [3] Both motifs (duplexes and quadruplexes) may be combined and the synapsis between duplexes and quadruplex structures have been shown at the molecular level [15,16] and have already been applied as a tool for the construction of nanomaterials [17,18] . In order to expand the applications of this hybrid duplexquadruplex structure, a deep understanding of their properties and folding process is required.…”

HIV-1 Nucleocapsid Proteins as Molecular Chaperones for Tetramolecular Antiparallel G-Quadruplex Formation

“…The kinetics of NCp15-induced G-quadruplex formation is comparable with the salt-induced formation of (3+1)-type 40,41 and four-stranded G-quadruplex 36 structures as the X-shape was formed at 40 s in the former case and 85 s in the latter case, while faster than the G4-binding ligand (required 510 s in the presence of PDC–biotin). 39 …”

“…The minor but significant amount of X-shapes observed under this condition could possibly because of the Mg 2+ ions present in the buffer which can induce small amount of quadruplex structure. 36,37 The parallel-shape was also obtained for all other sequences with different number of G-repeats such as four and three guanines (representative zoom-in images in Figure 3a) and different length of the strands such as 67 and 77 bp.…”

“…We first characterized the topology of the long-strands with six G-repeats. 36 In the absence of the proteins and G4-promoting salts, the duplexes may not come into contact as the conditions are unfavorable for G-quadruplex formation. Therefore, the AFM topography of the incorporated duplexes within the origami frame structure was expected to be parallel.…”