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

Rajendran A, Endo M, Hidaka K, Tran PL, Mergny JL, Sugiyama H - Nucleic Acids Res. (2013)

Bottom Line: 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.However, our method using DNA origami could successfully control the strand orientations, topology and stoichiometry of the G-quadruplex.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Graduate School of Science, Kyoto University, Kitashirakawa-oiwakecho, Sakyo-ku, Kyoto 606-8502, Japan, Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Yoshida-ushinomiyacho, Sakyo-ku, Kyoto 606-8501, Japan, CREST, Japan Science and Technology Corporation (JST), Sanbancho, Chiyoda-ku, Tokyo 102-0075, Japan and University of Bordeaux, INSERM, U869, ARNA Laboratory, 2 rue Robert Escarpit, Pessac, F-33607, France.

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.

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(a) The design of the DNA origami frame and the duplexes. (b) Graphical explanation of the DNA origami frame, insertion of the G–G mismatch repeats containing duplexes inside the frame and salt-mediated conformational changes of the incorporated strands. AFM image in each case is also given below the scheme. (c) Left: The length of the top duplex was 67-mer, while the bottom duplex used was either 67-mer (short duplex) or 77-mer (long duplex). Right: Schematic explanation of the DNA synapsis via the formation of G-quadruplex induced by KCl and the reverse conformation switching by the removal of the salt.
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gkt592-F1: (a) The design of the DNA origami frame and the duplexes. (b) Graphical explanation of the DNA origami frame, insertion of the G–G mismatch repeats containing duplexes inside the frame and salt-mediated conformational changes of the incorporated strands. AFM image in each case is also given below the scheme. (c) Left: The length of the top duplex was 67-mer, while the bottom duplex used was either 67-mer (short duplex) or 77-mer (long duplex). Right: Schematic explanation of the DNA synapsis via the formation of G-quadruplex induced by KCl and the reverse conformation switching by the removal of the salt.

Mentions: We have recently developed a defined DNA nanostructure, denoted as a ‘DNA origami frame’ (Figure 1a and b), to visualize the enzymatic reactions on double-stranded DNA (46,47). We used the same origami frame in the present study to observe the conformational switching and DNA synapsis with control over the stoichiometry and strand polarity. Briefly, this frame contains an inner vacant space of about 40 × 40 nm in which two sets of connection sites (A–B and C–D) were introduced to hybridize the duplex DNAs of interest. The length of each connection site is 32-mer (∼11 nm). The space between two connection sites (for example, A and B) is designed to be 64-mer double-stranded DNA, which corresponds to a length of ∼22 nm. To identify the orientation of the origami frame, a lacking corner at the right bottom of the frame was introduced (see Figure 1a and b).Figure 1.


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

Rajendran A, Endo M, Hidaka K, Tran PL, Mergny JL, Sugiyama H - Nucleic Acids Res. (2013)

(a) The design of the DNA origami frame and the duplexes. (b) Graphical explanation of the DNA origami frame, insertion of the G–G mismatch repeats containing duplexes inside the frame and salt-mediated conformational changes of the incorporated strands. AFM image in each case is also given below the scheme. (c) Left: The length of the top duplex was 67-mer, while the bottom duplex used was either 67-mer (short duplex) or 77-mer (long duplex). Right: Schematic explanation of the DNA synapsis via the formation of G-quadruplex induced by KCl and the reverse conformation switching by the removal of the salt.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3794576&req=5

gkt592-F1: (a) The design of the DNA origami frame and the duplexes. (b) Graphical explanation of the DNA origami frame, insertion of the G–G mismatch repeats containing duplexes inside the frame and salt-mediated conformational changes of the incorporated strands. AFM image in each case is also given below the scheme. (c) Left: The length of the top duplex was 67-mer, while the bottom duplex used was either 67-mer (short duplex) or 77-mer (long duplex). Right: Schematic explanation of the DNA synapsis via the formation of G-quadruplex induced by KCl and the reverse conformation switching by the removal of the salt.
Mentions: We have recently developed a defined DNA nanostructure, denoted as a ‘DNA origami frame’ (Figure 1a and b), to visualize the enzymatic reactions on double-stranded DNA (46,47). We used the same origami frame in the present study to observe the conformational switching and DNA synapsis with control over the stoichiometry and strand polarity. Briefly, this frame contains an inner vacant space of about 40 × 40 nm in which two sets of connection sites (A–B and C–D) were introduced to hybridize the duplex DNAs of interest. The length of each connection site is 32-mer (∼11 nm). The space between two connection sites (for example, A and B) is designed to be 64-mer double-stranded DNA, which corresponds to a length of ∼22 nm. To identify the orientation of the origami frame, a lacking corner at the right bottom of the frame was introduced (see Figure 1a and b).Figure 1.

Bottom Line: 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.However, our method using DNA origami could successfully control the strand orientations, topology and stoichiometry of the G-quadruplex.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Graduate School of Science, Kyoto University, Kitashirakawa-oiwakecho, Sakyo-ku, Kyoto 606-8502, Japan, Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Yoshida-ushinomiyacho, Sakyo-ku, Kyoto 606-8501, Japan, CREST, Japan Science and Technology Corporation (JST), Sanbancho, Chiyoda-ku, Tokyo 102-0075, Japan and University of Bordeaux, INSERM, U869, ARNA Laboratory, 2 rue Robert Escarpit, Pessac, F-33607, France.

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.

Show MeSH
Related in: MedlinePlus