Direct Observation of the Formation and Dissociation of Double-Stranded DNA Containing G-Quadruplex/i-Motif Sequences in
We demonstrate the single-molecule operation and observation of the formation and resolution of double-stranded DNA (dsDNA) containing G-quadruplex (GQ)- and counterpart i-motif-forming sequences in the DNA nanostructure. Sequential manipulation of DNA st
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Introduction G-quadruplex (GQ) structures are of great interest because of their structural variations and biological functions. To understand the physical properties of individual molecules and their reactions, the formation of GQ structures and their variants should be directly observed. Atomic force microscopy (AFM) enables direct imaging of biomolecules in the physiological environment. This technique can be used to visualize molecules at nanometer-scale spatial resolution; however, an observation scaffold is required for the precise imaging of the formation of biomolecular structures. DNA origami technology allows precise placement of target molecules in the designed nanostructures and enables molecules to be detected at
Danzhou Yang and Clement Lin (eds.), G-Quadruplex Nucleic Acids: Methods and Protocols, Methods in Molecular Biology, vol. 2035, https://doi.org/10.1007/978-1-4939-9666-7_17, © Springer Science+Business Media, LLC, part of Springer Nature 2019
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Masayuki Endo et al.
the single-molecule level. Thus, DNA origami is applied for visualizing and analyzing the detailed movement of target molecules in a subsecond time resolution by using high-speed AFM (HS-AFM) [1]. Tandem G-rich repeat sequences, which form GQ structures, are often observed in promoter regions [2]. The GQ is thought to be closely involved in biological functions such as the regulation of gene expression and cell fate control [3]. Promoter sequences such as the c-myc promoter contain a G-rich sequence, which forms a GQ structure that is involved in the regulation of transcription [3]. In the dsDNA in this region, the complementary sequence contains a C-rich repeat sequence, which forms an i-motif structure [4]. A number of studies have described the formation of GQ and/or i-motif structures in the promoter regions containing GQ- and imotif-forming sequences [5–8]. The i-motif structure is physically induced under acidic conditions because its formation requires the formation of a hemiprotonated cytosine dimer [4]. However, i-motif formation at neutral pH has also been observed in dsDNA with negative superhelicity [6] and in a molecular crowding environment [9]. We demonstrate the single-molecule operation and describe the formation and resolution of DNA nanostructures composed of dsDNA containing GQ- and counterpart i-motif-forming sequences [10]. The formation and dissociation of the GQ/imotif complementary sequence in the insulin-linked polymorphic region (ILPR) core sequence were manipulated into the DNA frame structure (Fig. 1). The ILPR promoter region is thought to separate into GQ and i-motif structures in the regulation of transcription [8]. The GQ/i-motif sequence was introduced into the DNA frame for observation of GQ and i-motif formation under various conditions. In this system, topologically controlled dsDNA was prepared through sequential manipulation of the interaction of DNA strands in a series of programmed operations (Fig. 2). Using the strand displacement and the addition and removal of K+, topologic
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