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Title Mechanical Properties of DNA Origami Nanoassemblies Are Determined by Holliday Junction Mechanophores Author(S) Mechanical properties of DNA origami nanoassemblies are Title determined by Holliday junction mechanophores Shrestha, Prakash; Emura, Tomoko; Koirala, Deepak; Cui, Author(s) Yunxi; Hidaka, Kumi; Maximuck, William J; Endo, Masayuki; Sugiyama, Hiroshi; Mao, Hanbin Citation Nucleic Acids Research (2016), 44(14): 6574-6582 Issue Date 2016-08-19 URL http://hdl.handle.net/2433/216360 © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.; This is an Open Access article distributed under the terms of the Creative Commons Right Attribution License (http://creativecommons.org/licenses/by- nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. Type Journal Article Textversion publisher Kyoto University 6574–6582 Nucleic Acids Research, 2016, Vol. 44, No. 14 Published online 7 July 2016 doi: 10.1093/nar/gkw610 Mechanical properties of DNA origami nanoassemblies are determined by Holliday junction mechanophores Prakash Shrestha1, Tomoko Emura2, Deepak Koirala1, Yunxi Cui1, Kumi Hidaka2, William J Maximuck1, Masayuki Endo3,*, Hiroshi Sugiyama2,3,* and Hanbin Mao1,* 1Department of Chemistry and Biochemistry, Kent State University, Kent, OH 44242, USA, 2Department of Downloaded from Chemistry, Graduate School of Science, Kyoto University, Kitashirakawa-oiwakecho, Sakyo-ku, Kyoto 606-8502, Japan and 3Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Yoshida-ushinomiyacho, Sakyo-ku, Kyoto 606-8501, Japan Received November 29, 2015; Revised June 23, 2016; Accepted June 24, 2016 http://nar.oxfordjournals.org/ ABSTRACT process brings new strategies for the development of nano-sensors and actuators. DNA nanoassemblies have demonstrated wide ap- plications in various fields including nanomaterials, drug delivery and biosensing. In DNA origami, single- INTRODUCTION stranded DNA template is shaped into desired nanos- Because of the highly stable and specific recognition be- at Library of Research Reactor Institute, Kyoto University on August 22, 2016 tructure by DNA staples that form Holliday junctions tween two complementary DNA strands, DNA has been with the template. Limited by current methodolo- used as an attractive component in nanoassembly. In the gies, however, mechanical properties of DNA origami DNA origami nanoassembly, a long ssDNA serves as a tem- structures have not been adequately characterized, plate to fold into nanostructures through hundreds of Hol- which hinders further applications of these materi- liday junctions formed between short DNA staples comple- mentary to the template sequences at particular locations als. Using laser tweezers, here, we have described (1). The simple, robust and highly efficient synthesis strategy two mechanical properties of DNA nanoassemblies of DNA origami has established this structure as a highly represented by DNA nanotubes, DNA nanopyramids potent nanomaterial (2–10) yet to be fully characterized for and DNA nanotiles. First, mechanical stability of its properties. Among these unknown territories, mechan- DNA origami structures is determined by the effec- ical property is certainly a notable missing link with high tive density of Holliday junctions along a particular significance. Mechanical stability of the connecting regions stress direction. Second, mechanical isomerization in DNA nanoassemblies is essential to sustain robust in- observed between two conformations of DNA nan- teractions between biomolecules (11) and nanoassemblies. otubes at 10–35 pN has been ascribed to the collec- Likewise, mechanical rigidity of DNA nanocavities is crit- tive actions of individual Holliday junctions, which ical to define the morphology of inorganic nanoparticles are only possible in DNA origami with rotational sym- contained within (12,13). In the sensing applications using DNA nanoassemblies, (14–18), the mechanical rigidity and metric arrangements of Holliday junctions, such as stability of the DNA nanoassemblies can directly affect the those in DNA nanotubes. Our results indicate that accuracy in the signal output and the sensitivity in the ana- Holliday junctions control mechanical behaviors of lyte recognition (5). DNA nanoassemblies. Therefore, they can be con- So far, only a handful of investigations have been re- sidered as ‘mechanophores’ that sustain mechani- ported with a main focus on the mechanical functionalities cal properties of origami nanoassemblies. The me- of the DNA nanoassemblies (14,19–22). The insufficient in- chanical properties observed here provide insights formation on the mechanical rigidity and stability of DNA for designing better DNA nanostructures. In addi- nanoassemblies in general and DNA origami in particular tion, the unprecedented mechanical isomerization hinders rational design of DNA nanomaterials for applica- tions that exploit their mechanical properties. One reason for this lack of knowledge lies in the difficulty in the char- *To whom correspondence should be addressed. Tel: +1 330 672 9380; Fax: +1 330 672 3816; Email: [email protected] Correspondence may also be addressed to Masayuki Endo. Email: [email protected] Correspondence may also be addressed to Hiroshi Sugiyama. Tel: +81 075 753 4002; Email: [email protected] C The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected] Nucleic Acids Research, 2016, Vol. 44, No. 14 6575 acterization of individual nano-objects for their mechanical properties. Special tools with high resolution for mechani- cal force measurement, such as AFM and optical tweezers, must be employed to carry out the characterization (22,23) after immobilization of nanoparticles with different shapes, which is another challenging practice. We reasoned that due to the flexible nature in the design of a DNA origami with the single-nucleotide precision, it is possible to introduce two duplex DNA handles to tether a DNA origami. These handles are then linked to the two optically trapped polystyrene beads in laser tweezers, which allow the quantification of mechanical properties of DNA Downloaded from nanoassembly. Previously, with the aim to develop origami based nanomechanical devices, Sugiyama and coworkers have found that tubular designs of origami structures can have two stable conformations: a short and a long tubu- lar forms that may stem from different isomers of Holl- http://nar.oxfordjournals.org/ iday junctions contained inside the origami devices (10). Figure 1. Mechanoanalytical characterization of DNA origami nan- Given the potential applications of controlling conforma- otubes. (A) Schematic of the experimental set up for the mechanical iso- tions of DNA origami structures by mechanical factors as merization of DNA origami tubes. DNA origami tube is sandwiched discussed above, here we wish to understand whether it is between two long dsDNA handles and the whole construct is teth- a unique feature for the tubular-shaped origami to demon- ered between two optically trapped beads with affinity linkages. The mechanochemical property of DNA tubes is revealed by moving one of strate different structural isomers. To this purpose, we ap- the beads away from another at a loading rate of ∼5.5 pN/s. AFM image plied two duplex DNA handles to the tubular DNA origami of the eight-tube DNA for longitudinal (B) and horizontal stretching (C). structures for the comparison of their mechanical proper- Schematic pictures are shown to the right of corresponding AFM images. ties with those from other nanoassemblies, DNA nanopy- at Library of Research Reactor Institute, Kyoto University on August 22, 2016 ramids and DNA nanotiles. We found mechanical stabilities of DNA origami structures are correlated with the effective a home-built laser tweezers instrument (30,31) to character- density of Holliday junctions in a particular nanoassem- ize the mechanical properties of individual DNA origami bly. The mechanical stability is anisotropic in nature with nanoassemblies. To this end, we introduced two double- the short axis of a DNA nanotube resisting higher external stranded (ds) DNA handles to DNA origami structures (see stress than the long axis. Interestingly, mechanical isomer- Figure 1A, Supplementary Figure S1A and B and Mate- ization between two conformations of a DNA nanoassemby rials and Methods) (14). DNA handles were inserted into at 10–35 pN external force was observed only in DNA nan- the origami structure via overhang single-stranded staple otubes, which have unique symmetric arrangements of Hol- sequence, which contains 40 nucleotides complementary liday junctions. Similar to the mechanical stability, the me- to the single-stranded M13mp18 template (it has been re- chanical isomerization also showed anisotropic behavior. ported that the shearing force of 30 bp duplex DNA is >60 Given that individual Holliday junctions have isomeriza- pN) (32). The other end of the handle was labeled with a tion force in the sub-picoNewton range (24) and they are digoxigenin or biotin molecule. These DNA handles were anisotropically arranged in DNA nanotubes, we have at- mixed and incubated with rest of the DNA components
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