Construction of Foldback Intercoil DNA Nanostructures and Analysis of

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Construction of Foldback Intercoil DNA Nanostructures and Analysis of Their Vibrational Modes Junyoung Son, Soojin Jo, Yongwoo Song, Byung Ho Lee, Moon Ki Kim, Byung-Dong Kim, and Sung Ha Park J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/acs.jpcc.7b12169 • Publication Date (Web): 16 Jan 2018 Downloaded from http://pubs.acs.org on January 18, 2018

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The Journal of Physical Chemistry

Construction of Foldback Intercoil DNA Nanostructures and Analysis of their Vibrational Modes

Junyoung Son, #,† Soojin Jo,§,† Yongwoo Song,# Byung Ho Lee,§ Moon Ki Kim,*,§,% Byung-Dong Kim,*,⊥ Sung Ha Park*,#,%

#

§

Department of Physics, Sungkyunkwan University, Suwon 16419, Korea

School of Mechanical Engineering, Sungkyunkwan University, Suwon 16419, Korea %

Sungkyunkwan Advanced Institute of Nanotechnology (SAINT), Sungkyunkwan University, Suwon 16419, Korea

⊥

Department of Plant Science and Plant Genomics and Breeding Institute, Seoul National University, Seoul 08826, Korea

*E-mail: [email protected] (MKK) *E-mail: [email protected] (BDK) *E-mail: [email protected] (SHP), Phone: +82-31-299-4544

†

These authors contributed equally to this work.

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The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT: A foldback intercoil (FBI) DNA nanostructure has important biological functions that are closely related to specific life phenomena, and it has a geometrically unique four-stranded DNA configuration that consists of two folded-back anti-parallel DNA double helixes intertwining in the major groove by sharing the same helix axis. However, the geometrical complexity of its unusual FBI configuration has prohibited its in vitro formation from direct contact between B-form DNA duplexes. Although several efforts have been made to investigate its functionalities and configurations, the FBI structures have been rarely constructed (via structural DNA nanotechnology) and simulated (through computational biology) to determine their geometrical stability, experimental validity, and engineering feasibility. In this study, we designed an FBI configuration with a homologous DNA base sequence implemented on either a double-crossover DNA tile or a double-crossover DNA lattice which were observed using an atomic force microscope. In addition, we propose a 3dimensional FBI structural model and perform a normal mode analysis based on the massweighted chemical elastic network model. These results provide an implementation of a biological simulation in the design of unusual DNA nanostructures, a prediction of their corresponding biological functionality, and an assessment of the feasibility to construct naturally existing biological configurations through synthetic DNA molecules.


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INTRODUCTION DNA delivers the genetic information of life, and it is one of the most important biomolecules. Recently, DNA has been considered as a novel material for construction of various dimensional nanostructures through self-assembly and for fabrication of functionalized devices or sensors via the specific modification of DNA molecules.1–5 Structural DNA nanotechnology is a branch of DNA nanotechnology that focuses on the design of desired geometrical structures with nanoscale precision and the development of fabrication methodology to improve production yield. This field was first proposed by Seeman, and it has been developed and applied particularly toward nanotechnology and biotechnology.6 DNA nanotechnology has been recognized as one of the most promising technologies at present due to the significant features of DNA molecules, including the programmability of base sequences, high stability compared to the other biomolecules, and capability for a high information density. Foldback intercoil (FBI), guanine-quadruplex (G-Quad.), and paranemic helix (PX) DNA nanostructures have four-stranded DNA configurations that are geometrically distinct. Intercoil (intertwining DNA duplexes sharing the same helix axis, such as FBI) and supercoil (winding DNA duplexes without sharing their helix axes, such as G-Quad. and PX) structures are formed by a twist of the DNA double helix.7,8 Although the G-Quad. (supercoil) with its functions and the PX-DNA nanostructure (supercoil) have been extensively studied and have been synthetically constructed, researchers have not yet intensively studied intercoil DNA nanostructures and have rarely tried to construct them due to the difficulties in finding and purifying intercoiled DNA structures from biological species and in designing structures with two DNA duplexes sharing the same helical centers. However, FBI DNA nanostructures have unique geometrical features with important functionalities that are closely related to various in vivo phenomena, such as transposition, inversion, deletion, and insertion (Figure 1a). To be specific, this compact four-stranded DNA form has long been suggested to explain and 3 ACS Paragon Plus Environment


reconcile discrepancies in unusual biological phenomena (i.e., fast unwinding during replication, small-sized target site duplication (5-7 base pairs), and chromosomal pairing and synapsis without double-strand break enzymes) which are relatively hard to explain using canonical B-form duplexes.9–11 However, the geometrical complexity of this unusual FBI configuration has prohibited its formation from direct contact between B-form duplexes without any enzymatic assistance.12 Although efforts have been made in biology and biotechnology to explore the functionalities and geometrical configurations of these structures,13–16 there has rarely been an attempt to construct the physical FBI structure with the aid of DNA nanotechnology (to understand its geometrical stability, achieve in mass production, and improve the possibility of further study) and to simulate it through computational biology (to analyze its geometrical validity, predict biological phenomena, and verify engineering feasibility). Here, we have designed an FBI configuration with homologous (two duplexes in the stem region with repeat sequences) and palindromic (a forward 5' to 3' sequence on a strand complemented with a backward 5' to 3' sequence) DNA base sequences, implemented on double-crossover (DX) DNA tiles and lattices. We then verified their geometries via atomic force microscopy (AFM). The use of an individual DX DNA tile and periodic DX DNA lattices with the FBI configuration has two major advantages. First, the DX DNA tile and lattices can serve as appropriate templates to form the FBI configuration. Second, these DX DNA tile and lattices that are implemented with an FBI configuration can be used as an advanced testing platform to generate the FBI configuration in large quantity. In addition, we also propose a 3-dimensional FBI structure model and perform a mass-weighted chemical elastic network model (MWCENM)-based normal mode analysis (NMA) as the cornerstone to elucidate the folding mechanism of the FBI structure at an atomic level.


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EXPERIMENTAL METHOD Sequence-designed synthetic oligonucleotides purified via high performance liquid chromatography were purchased from Bioneer (Daejon, Korea) to fabricate the foldback intercoil/hairpin-embedded DX (FBI-DX/HP-DX) DNA motifs and lattices. For free-solution annealing, each strand with the same volume of equimolar concentration is put into a test tube containing a physiological buffer of 1× TAE/Mg2+ (40 mM tris, 20 mM acetic acid, 1 mM EDTA (pH 8.0), 12.5 mM magnesium acetate). The final DNA sample concentration and volume are 200 nM and 200 µL, respectively. The mixture is placed in a Styrofoam box with 2 L of boiling water and is then cooled down slowly from 95 to 25 °C during 24 hours for hybridization (see Figure 1c, Figure 2a, and Figure 3). The double-DX (DDX) DNA lattice is grown on a substrate by adding each strand with equimolar concentration into a test tube containing mica (size of 5 × 10 mm2) with 1× TAE/Mg2+. 50 nM of individual DNA strands in 200 µL are chosen to fully cover the given substrate. The mixture is placed in the Styrofoam box with 2 L of boiling water and is then cooled down slowly from 95 to 25 °C to hybridize the DNA motifs and provide for lattice growth on a given substrate (Figure 2e, Figure 2h, and Figure 2k). The stepwise assembly of FBI(HP)-DX motifs on a periodically holed DDX lattice is achieved by immersing mica covered with pre-formed DDX lattices in a test tube having FBI(HP)-DX motifs (200 µL with 200 nM), followed by slow cooling from 35 °C to room temperature for about 4 hours (Figure 2h and Figure 2k). A mica substrate with a DNA sample is glued onto a metal puck to conduct atomic force microscope (AFM) imaging. 30 µL of 1× TAE/Mg2+ is placed on the mica surface, and another 20 µL is wetted at the end of the non-conductive silicon nitride AFM tip (DNP-S10, Veeco Inc., California, USA). Liquid tapping mode images are then taken using a Digital Instruments Multimode Nanoscope III (Veeco Inc., California, USA) (Figure 2 and Figure 3). Next, an MWCENM-based NMA is performed to analyze the dynamic characteristics of the proposed model of the FBI configuration. MWCENM is an advanced molecular 5 ACS Paragon Plus Environment


modelling method in which a DNA molecule is represented by a mass-spring system, and the chemical bond information is reflected using various spring constants. For instance, the spring constants for the van der Waals interaction, hydrogen bond, and covalent bond are significantly different from one another by one or two orders of magnitude, such as 1:10:100.14,15 In addition, this method takes the inertia effect into account, so that one can predict both vibrational frequencies and the corresponding motions more precisely than the conventional elastic network model in which a uniform spring constant is applied and no inertia effect is considered.16 First, we extract representative atoms from the FBI configuration model. This coarse-graining is conducted by choosing six or seven atoms per nucleotide. That is, the sugar-phosphate backbone is commonly represented by three atoms (C1, C4, and P) and the pyrimidine (purine) base is coarse-grained by three (four) core atoms linking to the backbone or complementary base.17 Then, a lumped mass of the surrounding atoms is assigned to each representative atom. Next, a spring network is constructed among the representative atoms depending on the types of chemical bonds. Finally, vibrational features of the FBI configuration are revealed by solving a generalized eigenvalue problem for this MWCENM.

RESULTS AND DISCUSSION Figure 1 showed the schematics of the FBI configuration and FBI-implemented DX DNA tile that were designed. The four-stranded FBI DNA configuration could be analytically formed as follows: a small segment (loop and stem regions in Figure 1b) of the DNA duplex was unwound in the loop into the non-helical parallel track structure and then folded-back 180º, followed by intercoiling the repeat sequence in the stem duplexes. The known diameter of the FBI DNA configuration (~2.2 nm) was slightly larger than that of B-form DNA (known to be ~2.0 nm) due to the slight offset of the helix axis of stem duplexes along the dyad axis that was introduced during DNA intercoiling.9,10,18 6 ACS Paragon Plus Environment

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In order to construct the intercoiled configuration, we introduced a DX DNA motif that was constrained to grow two duplexes roughly sharing the same helical axis.1,19 The DX motif was composed of two 37 base-pair-long duplexes (a rectangular shape with 4.0 × 12.6 nm2), which were joined by two crossover junctions separated by 16 base-pair-long duplexes (Figure 1c).20,21 Intercoiled duplexes in the stem region were implemented onto one of the centers of the duplex in a DX through two pairs of duplex linkages. The blue arrows in Figure 1d indicate the linkages from 3' of a DX to 5' of an FBI, and the green ones indicate linkages from 5' of a DX to 3' of an FBI (with 2 extra Ts serving as spacers for stable formation without stress on an FBI configuration-embedded DX DNA motif). The separation of the linkage sites for blue and green along the duplex in a DX were 3 and 5 base-pairs long, respectively. The linkage sites of two pairs of duplexes (i.e., an FBI configuration) were close enough to share the axis of their helical centers (serving as a geometrical constraint) to form an intercoiled four-stranded DNA configuration. A double-DX (DDX) lattice was introduced in order to verify formation of the FBI configuration on a DX motif.22 The DDX lattice had periodic holes (length of 12.6 nm, the same as a DX duplex) and served as a template to attract FBI-DX DNA motifs via single stranded overhangs (complementary stick-ends to an FBI-DX motif) located at the terminus of each hole (a unit DDX building block and a DDX lattice were shown in Figures 2c and 2d). In addition, the HP-DX DNA motif (a duplex with a length of ~3.0 nm protruding up and downwards on the DX tile) was utilized to confirm the arrangement on the DDX lattice (Figures 2a and 2b). To provide sufficient space to accommodate the HP(FBI)-DX motifs (8 nm for HP and 6 nm for FBI were needed along the direction perpendicular to the DDX duplex), we introduced single duplexes of the same length and complementary sticky-ends as a DX duplex (Figures 2f and 2i). These single duplexes served as hole blockers and consequently provided room to align the HP(FBI)-DX motifs on a DDX lattice. After binding the HP(FBI)-DX motifs on a DDX lattice, the base distance between the closest crossover 7 ACS Paragon Plus Environment


junctions of the DDX and HP(FBI)-DX motifs was of 18 and 19 nucleotides for either of the sides (which made an angle, ~103° between a DDX lattice and an HP(FBI)-DX motif shown in Figures 2g and 2j). Figures 2e, 2h, and 2k showed the representative AFM images of a DDX lattice, HPembedded DX (HP-DX), and FBI-embedded DX (FBI-DX) DNA motifs on a DDX lattice, respectively. To construct the FBI(HP)-DX DNA motifs on a periodically holed DDX lattice, a substrate covered with pre-formed DDX lattices was added in a test tube containing FBI(HP)-DX motifs, followed by slow cooling from 35 °C to room temperature. After the formation of DDX lattices on a mica surface, the average characteristic length of periodic stripes between the holes was 26.5 nm, which is in good agreement with the length of 25.2 nm that was designed for the DDX scheme (Figures 2d and 2e). Dark trenches in the image indicated holes along the direction perpendicular to the duplex where FBI(HP)-DX DNA motifs were to be placed through sticky-end binding between a DDX lattice and DNA motifs. Line segments (produced by FBI(HP)-DX DNA motifs) were observed on the DDX lattices after implementation of the FBI(HP)-DX DNA motifs. The average distance between protruding neighbouring line segments along the duplex was of 25.5 ± 1.5 nm, which was expected and in agreement with the design scheme (Figure 2h and 2k). To verify the structural formation of the intercoiled configuration on a DX DNA tile, we designed and fabricated FBI configuration-embedded DX DNA lattices with normal (6 nucleotide-long, named as an FBI-DX DNA lattice) and doubled (12 nucleotide-long, a DFBI-DX) stem lengths and compared them with a dual HP embedded DX DNA lattice (8 nucleotide-long each, a DHP-DX). A connector DX DNA tile (37 nucleotide-long, same as an FBI-DX DNA motif) was introduced to construct a DX lattice (Figure 3a). Sticky end sets of a connector were carefully designed to form a lattice through complementary binding with the FBI(DFBI, DHP)-DX DNA motif. Cartoon representations of FBI-, DFBI-, and DHP-DX DNA motifs with specific base sequences and lattices were shown in Figures 3b ‒ 3d. 8 ACS Paragon Plus Environment

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With the FBI(DFBI)-DX DNA motifs and connectors, 2-dimensional FBI(DFBI)-DX DNA lattices with periodically aligned FBI(DFBI) configurations were observed with a distance of 25.7 nm (25.2), which was in good agreement with the expected distance (within 5%). There was a significant height suppression that was primarily due to the electrostatic interaction between the FBI(DFBI) configuration and a charged substrate since the designed heights of FBI and DFBI measured from the surface of the DX lattice were ~3 nm and ~5 nm respectively, but those that were experimentally measured for FBI and DFBI turned out to be ~0.6 nm and ~1.0 nm. However, the relative increment ratio of the heights (defined as the height of DFBI / height of FBI) was 0.6 for both the expected and measured heights (Figures 3e and 3f). The structural rigidity and geometrical stability of the FBI configuration could be indirectly confirmed by observing the DFBI-DX DNA lattices as well as the FBI-DX. Two hairpins with self-complementary sequences were implemented on the DX tile to compare the center-sharing characteristics of the intercoiled DNA duplexes (Figure 3d). As mentioned above, the major structural properties of the intercoiled configuration were a palindromic and homologous four-stranded structure with a shared helical axis of duplexes in the stem region and full complementarity of the plain duplex in the loop. Two hairpins (with no palindromic homologous sequence) sharing a helical axis with single four-T’s loops (without full complementarity of the plain duplex)-based DX DNA lattices were designed to compare the topological difference with the FBI(DFBI)-DX DNA lattices (Figures 3e and 3g). Although the diameter of the FBI(DFBI) configuration (~2.2 nm) was compatible enough with that of the single duplex (~2.0 nm) without a separation of the loop region, a clear separation was observed for the two hairpins (dual peaks observed through the section profile shown in inset, Figure 3g) in the DHP-DX DNA lattice. This meant that the major structural characteristics of an intercoiled configuration had to be met with the following conditions: a palindromic and homologous four-stranded structure with a common helical axis of duplexes in the stem region and full complementarity of the plain duplex in the loop. 9 ACS Paragon Plus Environment


To analyze the dynamic characteristics of the FBI configuration, a 3-dimensional computer model was constructed based on an inverted repeat sequence with 38 base pairs.9 The structure was composed of a loop, stem, and body (Figure 1b), and the diameter of the FBI model was of about 2.2 nm whereas that of the B-DNA was 2.0 nm. Both the FBI model and B-DNA model were compared by calculating distances between the adjacent phosphorous (P) atoms, hydrogen bond distance, and distances between neighbouring bases in order to confirm the validity of the proposed model, including the backbone construction, appropriate hydrogen bond, and structural deformation. The average distances of the adjacent P atoms were 0.347 nm in the loop and 0.668 nm in the stem. These values were almost identical to those of B-DNA, which were 0.340 nm and 0.667 nm. Also, the hydrogen bond distances of both FBI and B-DNA were the same at 0.288 nm. Finally, the unusual structural deformation and distortion were examined by evaluating the distances between consecutive bases, which were 0.385 nm of FBI and 0.376 nm of B-DNA. The difference in distance (of about 0.01 nm) was not significant due to the similarities in the helicity angles, ~34.3°. By using this model, we performed NMA based on MWCENM. Figure 4 illustrated the four major vibration modes of FBI. The first three lowest modes were the in-plane half sine bending, twisting, and out-of-plane half-sine bending modes, respectively. These motions were generally similar to the DNA rod motions that had already been reported in our previous study.13 However, the fourth lowest mode was quite different from that of the DNA rod structure. It was a mixture of both bending and twisting motions caused by the high rigidity of the stem region of the FBI configuration formed by four intercoiled DNA strands. Although the FBI configuration primarily followed the general vibration features of a DNA rod structure, such as bending and twisting, it could also generate more complicated mixed modes based on its unique stem structure.


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CONCLUSIONS We designed an FBI configuration with homologous and palindromic DNA base sequences implemented on double-crossover (DX) DNA tiles and lattices, and we then investigated their geometries via atomic force microscopy. By using FBI-DX DNA motifs, 2-dimensional FBIDX DNA lattices with periodically aligned FBI configurations were observed with distance of ~26.1 nm, which was in good agreement with the expected distance of 25.2 nm. In addition, we proposed a 3-dimensional FBI structure model and performed a mass-weighted chemical elastic network model based normal mode analysis to understand the dynamic characteristics of the FBI configuration. The three lowest modes consisted of in-plane half sine bending, twisting, and out-of-plane half-sine bending, respectively. In contrast, the fourth lowest mode was quite different, with a mixture of both bending and twisting motions caused by the rigidity of the stem region of the FBI configuration. The biological simulation predicts the biological functionality via geometrical shapes when designing DNA nanostructures, and it is also useful in assessing the feasibility to construct unusual biological configurations through synthetic DNA molecules.



Figure 1. Design of a Foldback Intercoil (FBI) configuration-implemented on a doublecrossover (DX) DNA tile. (a) Representative characteristics of an FBI configuration during the DNA transposition process (cut & paste and copy & paste).17 (b) The geometric formation of the FBI configuration (consisting of a loop and stems depicted in blue and red, respectively) can be described as follows: a double helix at the loop position untwists to the point of a parallel track and folds back 180°, and the two flanking stems intertwine in the major grooves to form an intercoiled structure. (c) Schematic of an FBI configuration ‒ stem length of 6 nucleotides ‒ implemented on a DX (FBI-DX) DNA motif with base sequences. Strands and sticky-ends are named as FBI-# and Sa# (red color), respectively. Sequences marked in blue (CAT-GTA) are complementary to each other. Green Ts served as spacers between the FBI configuration and a DX tile. (d) Cartoon representation for front and side views of an FBI-DX DNA motif. Blue and green arrows indicate 3' to 5' and 5' to 3' linkages, respectively, from a DX tile to an FBI configuration.


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Figure 2. Construction and verification of an FBI configuration-embedded DX (FBI-DX) DNA motif on a double-DX (DDX) lattice. (a) A schematic of a hairpin (HP) configuration (up and down)-embedded on a DX (HP-DX) DNA motif with base sequences. Strands and sticky-ends are named as DXhp-# and Sa# (in red), respectively. Single-stranded four (two) Ts are formed (served) as a loop (a spacer between an HP and a DX). (b) Cartoon representations for an HP-DX DNA motif displayed from top, front, and side views. (c) Schematic of a DDX DNA motif with base sequences. Strands and sticky-ends are named as T0-#, and Sa(A)# (in red), respectively. (d, e) 3D cartoon representation and representative atomic force microscope image of the DDX lattices. Periodic holes with single-strand 13 ACS Paragon Plus Environment


overhang sticky-ends are designed to bind with the complementary sticky-ends on either an HP-DX DNA motif or an FBI-DX. (f, g) 3D cartoons of individual HP-DX DNA motif, single duplex DNA blocker (provided binding space for HP-DXs), and an HP-DX DNA motif on a DDX lattice. (h) A representative atomic force microscope image of HP-DX DNA motifs on DDX lattices with the cartoon representation of the implemented HP-DX DNA motif. (i, j) Cartoons of individual FBI-DX DNA, single duplex DNA, and an FBI-DX DNA motif on a DDX lattice. (k) A representative atomic force microscope image of FBI-DX DNA motifs on DDX lattices with the cartoon of the implemented FBI-DX DNA. The scan sizes of all atomic force microscopic images are 500 × 500 nm2.


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Figure 3. Design, construction, and verification of an FBI configuration-embedded DX DNA lattice. (a) Schematic and a 3D cartoon of a connector DNA tile (named as DX0C) with base sequences. The strands and sticky-ends are named as DX0C-# and Sa#' (in red), respectively. (b) 3D cartoons of an FBI-DX DNA motif and an assembled lattice consisting of an FBI-DX DNA motif and a DX0C connector tile. (c) A cartoon of a long FBI configuration ‒ doubledstem length, 12 nucleotides ‒ embedded DX (DFBI-DX) DNA motif (to verify the FBI configuration stability with a different stem length) with the scheme of base sequences (consisting of a loop and a stem shaded in purple and red, respectively). Sequences in blue (CAT-GTA) are complementary to each other, and green Ts serve as spacers between the DX tile and an FBI DNA. (d) 3D cartoons of a dual HP embedded DX (DHP-DX) DNA motif (without full complementarity of the plain duplex in a loop region for geometrical comparison) and an assembled lattice consisting of a DHP-DX DNA motif and a DX0C connector tile. The scheme of the DHP base sequences is shown on bottom-right. Representative atomic force microscope images with section profiles of (e) FBI-DX DNA lattices, (f) DFBI-DX DNA, and (g) DHP-DX DNA lattices. The scan sizes of all atomic force microscopic images are 500 × 500 nm2.



Figure 4. Major vibrational modes of the proposed FBI configuration obtained through a normal mode analysis to study the vibrational characteristics. A coarse-grained FBI configuration is displayed from top (T), front (F), and side (S) views. The first and the second lowest modes are “in-plane” half-sine bending (1) and twisting (2), respectively, while the third lowest mode is “out-of-plane” half-sine bending (3). In addition, a mixed motion of bending and twisting appears at the fourth lowest mode (4). The computed natural frequencies of these four lowest modes are 8.45, 9.55, 9.69, and 12.12 cm-1, respectively.


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AUTHOR INFORMATION

Corresponding Authors *E-mail: [email protected] (MKK) *E-mail: [email protected] (BDK) *E-mail: [email protected] (SHP). Phone: 82-31-299-4544.

ORCID Sung Ha Park: 0000-0002-0256-3363

ACKNOWLEDGMENTS This research was supported by the National Research Foundation of Korea (NRF), funded by the Korean government, the Ministry of Science, ICT & Future Planning (MSIP) and the Ministry of Education (MOE) (2017R1D1A1B03035053 and 2015R1D1A1A01057280)



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(22) Son, J.; Lee, J.; Tandon, A.; Kim, B.; Yoo, S.; Lee, C.-W.; Park, S. H. Assembly of a Tile-Based Multilayered DNA Nanostructure. Nanoscale 2015, 7 (15), 6492–6497.

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Construction of Foldback Intercoil DNA Nanostructures and Analysis of

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