Accepted Manuscript

Suspended DNA structural characterization by TEM diffraction

Monica Marini, Marco Allione, Sergei Lopatin, Manola Moretti,

Andrea Giugni, Bruno Torre, Enzo di Fabrizio

PII: S0167-9317(17)30396-9
DOI: doi:10.1016/j.mee.2017.11.020
Reference: MEE 10671
To appear in: Microelectronic Engineering
Received date: 12 October 2017
Revised date: 28 November 2017
Accepted date: 29 November 2017

Please cite this article as: Monica Marini, Marco Allione, Sergei Lopatin, Manola Moretti,
Andrea Giugni, Bruno Torre, Enzo di Fabrizio , Suspended DNA structural
characterization by TEM diffraction. The address for the corresponding author was
captured as affiliation for all authors. Please check if appropriate. Mee(2017), doi:10.1016/
j.mee.2017.11.020

This is a PDF file of an unedited manuscript that has been accepted for publication. As
a service to our customers we are providing this early version of the manuscript. The
manuscript will undergo copyediting, typesetting, and review of the resulting proof before
it is published in its final form. Please note that during the production process errors may
be discovered which could affect the content, and all legal disclaimers that apply to the
journal pertain.
 ACCEPTED MANUSCRIPT

Suspended DNA structural characterization by TEM diffraction.

Monica Marinia,*, Marco Allionea, Sergei Lopatinb, Manola Morettia, Andrea Giugnia, Bruno Torrea,
Enzo di Fabrizioa

a
SMILEs Lab, King Abdullah University of Science and Technology (KAUST), PSE divisions,
Thuwal 23955 - 6900, Kingdom of Saudi Arabia
b
Imaging and Characterization Core Lab, King Abdullah University of Science and Technology

PT
(KAUST), Thuwal 23955-6900, Kingdom of Saudi Arabia

RI
* Corresponding author: [email protected]

SC
Keywords: DNA, super-hydrophobic devices, diffraction, interbases distance.
NU
Abstract
In this work, micro-fabrication, super-hydrophobic properties and a physiologically compatible
MA

preparation step are combined and tailored to obtain background free biological samples to be
investigated by Transmission Electron Microscopy (TEM) diffraction technique. The validation was
performed evaluating a well-known parameter such as the DNA interbases value. The diffraction
D

spacing measured is in good agreement with those obtained by HRTEM direct metrology and by
E

traditional X-Ray diffraction. This approach addresses single molecule studies in a simplified and
PT

reproducible straightforward way with respect to more conventional and widely used techniques. In
addition, it overcomes the need of long and elaborated samples preparations: the sample is in its
CE

physiological environment and the HRTEM data acquisition occurs without any background
interference, coating, staining or additional manipulation. The congruence in the results reported in
AC

this paper makes the application of this approach extremely promising towards those molecules for
which crystallization remains a hurdle, such as cell membrane proteins and fibrillar proteins.

1. Introduction

The diffraction pattern taken by Raymond Gosling under Rosalind Franklin [1] supervision, shown
in the so-called “Photo 51”, has been a milestone for biology. The information contained in the
image was crucial for the correct determination of the helical structure of DNA by Watson and Crick
[2]. That moment changed the approach to biological sciences, highlighting the needed for new
techniques capable to investigate biological matter down to the single molecule scale and below.
 ACCEPTED MANUSCRIPT

X-Ray diffraction on crystallized biological systems is the state of the art, worldwide known
technique for structural investigation, and has been used to resolve the double helix main features
since the 50s, but the high-quality crystals needed still remains a major limitation. In addition, only a
fraction of the biomolecules can be reduced in fibers or crystallized and, even when possible, the
most flexible parts of the species cannot be resolved. A number of proteins cannot satisfy the X-Ray
diffraction requirements. For example, proteins embedded in the membrane phospholipid bilayers
have to be separated from their native environment, fibrillar proteins (e.g. amyloid proteins) cannot
be studied as single molecules [3,4] and not all the biologically interesting systems do crystallize.

PT
The above mentioned issues highlight the necessity to develop new methods to reveal
macromolecules structures and their relation to other compounds [5].

RI
In the last years, we reported a novel approach for structural characterization of biomolecules by

SC
HRTEM and spectroscopy, overcoming the limitations displayed by the conventional techniques [5–
10]. The first attempt to directly image DNA allowed researchers to distinguish the double helix
NU
period of 27 Å. The fine-tuning of the droplet dimension and deposition technique, micro-pillars
arrangement, buffer composition and samples preparation dramatically increased the resolution down
to the single base. Besides the nucleic acids helices, repair proteins and membrane proteins [7–9]
MA

direct imaging was performed by conventional Transmission Electron Microscopy (TEM) and High
Resolution TEM (HRTEM) to achieve a resolution to 3.3 and 1.5 Å, respectively.
D

Briefly, the method proposed by our group involves the use of biological molecules dissolved in
physiologically compatible buffers and super-hydrophobic devices. The super-hydrophobic surfaces
E

are composed by a regular pattern of cylindrical micro-pillars and holes between them. A few
PT

microliters of the solution containing the analyte of interest is deposited on the micro-structured
device under controlled temperature conditions, obtaining a droplet suspended on the micro-pillars
CE

with a quasi-spherical shape and a contact angle >150°. The evaporation of the buffer causes the
droplet volume reduction while maintaining its shape and shrinking towards the center. In this
AC

process, the DNA molecules contained in the droplet and attached to the top of a pillar are stretched
to the top of the consecutive one, accordingly to the dehydration movement. With this approach
double strand (ds) DNA, single strand (ss) DNA, neuronal cell membranes and related ion channels,
proteins such as Rad51 and lysozyme [8,11] have been suspended over the devices and
characterized, ensuring no background signal and no need of staining techniques prior the
visualization. The versatility of the device and the wide range of samples that can be tested on it,
make this system a suitable platform not only for HRTEM direct imaging and diffraction but also for
detection. Among the the recent literature available related to biosensing [12–14], the combination of
 ACCEPTED MANUSCRIPT

high resolution imaging, EM diffraction and Raman spectroscopy techniques applied to

biomolecules, highlights the strong potential of this approach in the biodetection field.
Herein we show the successful switch from the real space to the reciprocal space using HRTEM and
their application to biological materials suspended over dedicated super-hydrophobic devices. The
information obtained from the images acquired in the direct space strongly support the HRTEM
diffraction pattern of the DNA bundles studied, confirming the versatility and the wide applicability
of the newly developed approach. The combined method herein presented can be applied to a wide
range of biological molecules. This property gives to researchers the possibility to perform HRTEM

PT
diffraction and high resolution imaging also on the samples that cannot undergo to analysis with
conventional techniques, overcoming a significant hurdle of structural biology.

RI
SC
2. Materials
NU
2.1. Super-hydrophobic device realization

The devices used in this work were fabricated as previously reported [7,8]. Briefly, the super-
MA

hydrophobic samples were realized by using commercial Si <100> wafers, characterized by a

thickness of 50 μm. The device with holes is mostly defined with two steps of optical lithography
D

performed on the same side of the wafer. The first lithography step defines the pattern of pillars and
is followed by the deposition of thin metal layers: Titanium (10 nm), gold (50 nm) and Chromium
E

(50 nm) were evaporated in this order. The second lithography step is needed in order to obtain the
PT

holes through the device. Etching with Deep Reactive Ion Etching (DRIE) of silicon precedes the
oxygen plasma treatment done to remove the resists excesses. A second DRIE process is performed
CE

to create the pillars, using the previously deposited metal as a mask. The samples obtained are then
coated with a thin layer of Teflon-like fluorocarbon polymer (FDTS) of a final thickness of
AC

approximately 2 nm.

2.2. Sample preparation

The DNA of the lambda phage was used as a reference molecule for this work due to its commercial
availability (New England Biolabs, NEB UK) and due to its verified self-organization into
reproducible bundles of suitable length. An aliquot of the DNA sample was diluted in saline buffer
containing 6.5 mM NaCl and 10 mM Tris-HCl, pH 9.3, to reach the DNA concentration of 150
ng/l. A droplet of 5 l volume was pipetted on the super-hydrophobic device placed on a hot plate
 ACCEPTED MANUSCRIPT

at 25 ˚C and 50% of relative humidity until the complete evaporation of the buffer solution was
achieved.

2.2 Microscopy characterization of the DNA samples

After complete dehydration, samples were imaged by SEM (FEI, Quanta 200) working at an
acceleration voltage of 3 kV. No coating or additional treatment was performed, to avoid any
interference with the micrographs analysis. HRTEM imaging was performed working at 80 keV with

PT
a Titan Cs Image (FEI).

RI
3. Results and Discussion

SC
In this work, we applied TEM diffraction to the study of nucleic acids helix features in a single
NU
bundle of suspended dsDNA. The dsDNA form of the Lambda phage was chosen as a test system
due to its length, covering the pillar-pillar distance of 12 m. Lambda DNA was dissolved in a
physiological compatible saline buffer to reach a concentration of approximately 150 ng/l and a few
MA

microliters droplet of the freshly prepared solution was deposited on a super-hydrophobic device.
Recent literature shows several examples of devices able to repel water; the arrangement and the
D

shape of the micro-structures on the device are chosen on the basis of the sample and of the final
E

application, spanning from detection to visualization [15]. In our case, the device is designed to have
a circular pattern of cylindrical micro-pillar (diameter 6 m, distance 12 m, height 10 m). Among
PT

the purposes of the super-hydrophobic device herein proposed, the concentration of a diluted solution
CE

of macromolecules and their precise localization are two of the main objectives in this work. The
droplet starting dimensions of nearly 3 mm diameter decrease down to 400 m while the dehydration
occurs. Accordingly to the droplet shrinkage, three main concurrent effects take place: the non-
AC

suspended material (which includes nucleic acids and salts) become more concentrated; the buffering
salts excess is sieved from the biomolecules; the DNA helices self-arrange themselves in bundles
self-aligned between micro-pillars. The ordered network of DNA bundles obtained was imaged by
Scanning Electron Microscopy (SEM, Figure 1a-c). As reported in Figure 1a (top right) and in
Figure 1b (on the right) a saline residual corresponds to the final point of the evaporation where the
concentrated droplet collapses. Straight bundles of diameters ranging between 200 nm and 5 nm
were homogenously dispersed all around the salt residual. No crystals due to salts presence were
observed on the bundles, confirming the self-sieving effect and ensuring a DNA bundle free from
any additional contribution potentially interfering with the HRTEM electron beam.
 ACCEPTED MANUSCRIPT

Direct imaging and diffraction of the suspended DNA molecules by HRTEM is possible due to the
particular design of the silicon device. A regular pattern of holes is fabricated between the micro-
pillars allowing the electron beam to freely pass through the discontinuities of the device, resulting in
a background-free imaging.
Diffraction by HRTEM was performed working at an acceleration voltage of 80 keV, previously
reported as the best compromise between the radiation damage [16–18] occurring during suspended
DNA imaging and a good resolution and contrast of the bundle [7]. The background-free images
(Figure 2a, b) allowed directly analyzing sub-period details of DNA bundles without additional

PT
software processing. An interbase distance of ~2.7 Å was measured on the external edge of the
bundles as reported in the related plot in Figure 2c. The data obtained are in good agreement with

RI
the expected A-DNA features, which differentiates from the B-DNA due to the lower hydration of

SC
the helices. As reported by the milestone paper from Franklin and Gosling, in presence of a relative
humidity lower than 75% the DNA molecule assume an interbases distance of approximately 2.6 Å
NU
and a diameter of 23 Å [19]; in case of higher humidity rates (over the 75%), the rise per base pairs
increases to 3.4 Å while the diameter changes to 20 Å. [2].
Afterwards, TEM diffraction was performed on the bundles. The d-spacing obtained by the
MA

diffraction patterns resulted to be ranging between 2.8 and 3.2 Å, following bundle direction (Figure
3). These values are coherent with the previously reported data both for the A- (2.7 Å < h < 3.0 Å)
D

and the B-form (2.6 Å < h < 3.4 Å) of double strand DNA helices [20,21].
E

4. Conclusions
PT

In this paper, we showed the preliminary data obtained on the DNA diffraction performed by
CE

HRTEM. DNA in its double strand form is one of the most characterized biomolecules, and it was
chosen to perform this work as a test to evaluate the efficiency of the method. The data are extremely
AC

promising, however, a fine-tuning of the solution requirements, bundles dimension and HRTEM
measurements are still in progress.
We intend to develop this technique as a central part of a combined approach to effectively and
systematically address the study of the structural alterations to the pristine conformation of the
nucleic acids, revealing the presence of other compounds causing helix disruption such as heavy
metal ions or chemotherapeutic agents. In addition, the use of such a technique can be an innovative
tool for the investigation of the macromolecules (such as flexible proteins and cell membrane
proteins) that cannot be investigated with traditional methods.
 ACCEPTED MANUSCRIPT

In perspective of a systematic approach, a combination of this straightforward technique with more

refined ones like HRTEM direct imaging, capable of submolecular resolution and spectroscopic
studies to obtain valuable information on morphological and structural single point modifications and
interactions between biological molecules and the environment.

5. Acknowledgments

The authors acknowledge financial support from the KAUST start-up funding and from Italian

PT
Ministry of Health under the projects: Project no.: GR-2010-2320665 and Project no.: GR-2010-
2311677.

RI
SC
NU
MA
E D
PT
CE
AC
 ACCEPTED MANUSCRIPT

6. References

[1] R.E. Franklin, R.G. Gosling, Molecular Configuration in Sodium Thymonucleate, Nature. 171
(1953) 740–741. doi:10.1038/171740a0.
[2] J.D. Watson, F.H.C. Crick, Molecular structure of nucleic acids, Nature. 171 (1953) 737–738.
doi:10.1097/BLO.0b013e3181468780.
[3] M. Moretti, R. Proietti Zaccaria, E. Descrovi, G. Das, M. Leoncini, C. Liberale, F. de Angelis,
E. Di Fabrizio, Reflection-mode TERS on Insulin Amyloid Fibrils with Top-Visual AFM

PT
Probes, Plasmonics. 8 (2013) 25–33. doi:10.1007/s11468-012-9385-x.
[4] M. Moretti, C. Canale, C. Canale, M. Francardi, S. Dante, F. De Angelis, E. Di Fabrizio, AFM

RI
characterization of biomolecules in physiological environment by an advanced nanofabricated

SC
probe, Microsc. Res. Tech. 75 (2012) 1723–1731. doi:10.1002/jemt.22122.
[5] M. Marini, T. Limongi, M. Moretti, L. Tirinato, E. Di Fabrizio, The structure of DNA by
NU
direct imaging and related topics, La Riv. Del Nuovo Cim. 40 (2017) 241–277.
doi:10.1126/sciadv.1500734.
[6] M. Marini, M. Allione, B. Torre, M. Moretti, T. Limongi, L. Tirinato, A. Giugni, G. Das, E. di
MA

Fabrizio, Raman on suspended DNA: Novel super-hydrophobic approach for structural

studies, Microelectron. Eng. 175 (2017) 38–42. doi:10.1016/j.mee.2016.12.016.
D

[7] M. Marini, A. Falqui, M. Moretti, T. Limongi, M. Allione, A. Genovese, S. Lopatin, L.

Tirinato, G. Das, B. Torre, A. Giugni, F. Gentile, P. Candeloro, E. Di Fabrizio, The structure
E

of DNA by direct imaging, Sci. Adv. 1 (2015) e1500734–e1500734.

PT

doi:10.1126/sciadv.1500734.
[8] M. Marini, T. Limongi, A. Falqui, A. Genovese, M. Allione, M. Moretti, S. Lopatin, L.
CE

Tirinato, G. Das, B. Torre, A. Giugni, F. Cesca, F. Benfenati, E. Di Fabrizio, Imaging and

structural studies of DNA-protein complexes and membrane ion channels, Nanoscale. (2017).
AC

doi:10.1039/C6NR07958J.
[9] F. Gentile, M. Moretti, T. Limongi, A. Falqui, G. Bertoni, A. Scarpellini, S. Santoriello, L.
Maragliano, R. Proietti Zaccaria, E. Di Fabrizio, Direct imaging of DNA fibers: The visage of
double helix, Nano Lett. 12 (2012) 6453–6458. doi:10.1021/nl3039162.
[10] M. Marini, G. Das, R. La Rocca, F. Gentile, T. Limongi, S. Santoriello, A. Scarpellini, E. Di
Fabrizio, Raman spectroscopy for detection of stretched DNAs on superhydrophobic surfaces,
Microelectron. Eng. 119 (2014) 151–154. doi:10.1016/j.mee.2014.04.008.
[11] M. Moretti, M. Allione, M. Marini, B. Torre, A. Giugni, T. Limongi, G. Das, E. Di Fabrizio,
Raman study of lysozyme amyloid fibrils suspended on super-hydrophobic surfaces by shear
 ACCEPTED MANUSCRIPT

flow, Microelectron. Eng. 178 (2017) 194–198. doi:10.1016/j.mee.2017.05.045.

[12] L. Piantanida, D. Naumenko, E. Torelli, M. Marini, D.M. Bauer, L. Fruk, G. Firrao, M.
Lazzarino, Plasmon resonance tuning using DNA origami actuation, Chem. Commun. Chem.
Commun. 51 (2015) 4789–4792. doi:10.1039/c5cc00778j.
[13] V.K. Gupta, M.L. Yola, M.S. Qureshi, A.O. Solak, N. Atar, Z. Üstündaǧ, A novel
impedimetric biosensor based on graphene oxide/gold nanoplatform for detection of DNA
arrays, Sensors Actuators, B Chem. 188 (2013) 1201–1211. doi:10.1016/j.snb.2013.08.034.
[14] M.L. Coluccio, F. Gentile, G. Das, A. Nicastri, A.M. Perri, P. Candeloro, G. Perozziello, R.

PT
Proietti Zaccaria, J.S.T. Gongora, S. Alrasheed, A. Fratalocchi, T. Limongi, G. Cuda, E. Di
Fabrizio, Detection of single amino acid mutation in human breast cancer by disordered

RI
plasmonic self-similar chain., Sci. Adv. 1 (2015) e1500487. doi:10.1126/sciadv.1500487.

SC
[15] G. Ciasca, M. Papi, L. Businaro, G. Campi, M. Ortolani, V. Palmieri, A. Cedola, A. De Ninno,
A. Gerardino, G. Maulucci, M. De Spirito, Recent advances in superhydrophobic surfaces and
NU
their relevance to biology and medicine, Bioinspir. Biomim. 11 (2016) 11001.
doi:10.1088/1748-3190/11/1/011001.
[16] M. Mishyna, O. Volokh, Y. Danilova, N. Gerasimova, E. Pechnikova, O.S. Sokolova, Effects
MA

of radiation damage in studies of protein-DNA complexes by cryo-EM, Micron. 96 (2017)

57–64. doi:10.1016/j.micron.2017.02.004.
D

[17] R.F. Egerton, Mechanisms of radiation damage in beam-sensitive specimens, for TEM
accelerating voltages between 10 and 300 kV, Microsc. Res. Tech. 75 (2012) 1550–1556.
E

doi:10.1002/jemt.22099.
PT

[18] R.F. Egerton, P. Li, M. Malac, Radiation damage in the TEM and SEM, in: Micron, 2004: pp.
399–409. doi:10.1016/j.micron.2004.02.003.
CE

[19] R.E. Franklin, R.G. Gosling, The structure of sodium thymonucleate fibres. I. The influence of
water content, Acta Crystallogr. 6 (1953) 673–677. doi:10.1107/S0365110X53001939.
AC

[20] A.G.W. Leslie, S. Arnott, R. Chandrasekaran, R.L. Ratliff, Polymorphism of DNA double
helices, J. Mol. Biol. 143 (1980) 49–72. doi:http://dx.doi.org/10.1016/0022-2836(80)90124-2.
[21] S. Arnott, Polymorphism of nucleic acid duplexes as revealed by X-ray diffraction analysis of
oriented fibers, Biophys. J. 32 (1980) 249–250. doi:10.1016/S0006-3495(80)84951-4.
 ACCEPTED MANUSCRIPT

Figures

PT
RI
Fig. 1. Suspended dsDNA bundles: SEM imaging. a) As a result of the dehydration process, a salt
residual is segregated and confined in a limited area (located by design at the center of the circular

SC
structure, not shown). DNA bundles are homogeneously distributed in the region of interest around
the central residual area, in an oriented and regular manner. b) Higher magnification image showing
NU
a detail of the DNA bundle suspension. The DNA molecules are suspended over the holes of the
micro-patterned device, allowing the downstream TEM direct imaging. c) The pillars height of 12
m ensures the suspension of the macromolecules in a background-free environment. The sample in
MA

panels a and b was tilted of 25°.

E D
PT
CE
AC

Fig. 2. Suspended dsDNA bundles: HRTEM imaging. a,b) HRTEM micrographs of a DNA
bundle suspended over a super-hydrophobic device. Characteristics fringes related to sub-period
features are clearly visible in both images. The red rectangle highlights the area where the metrology
was performed for the plot in panel c). The distance between one fringe and the other is of 2.7 Å, in
good agreement with previous works on the direct metrology of the DNA helix [7].
 ACCEPTED MANUSCRIPT

PT
Fig. 3. Suspended dsDNA bundles: TEM diffraction. In the three panels the diffraction patterns

RI
obtained from three different suspended bundles of the same super-hydrophobic device are reported.
The d-spacing measured ranges between 2.8 Å and 3.2 Å and corresponds to the interbase distance of

SC
DNA.
NU
MA
E D
PT
CE
AC
 ACCEPTED MANUSCRIPT

PT
Graphical abstract

RI
SC
NU
MA
ED
PT
CE
AC
 ACCEPTED MANUSCRIPT

Highlights

Suspended DNA structural characterization by TEM diffraction.

Monica Marinia,*, Marco Allionea, Sergei Lopatinb, Manola Morettia, Andrea Giugnia, Bruno Torrea,
Enzo di Fabrizioa
a
SMILEs Lab, King Abdullah University of Science and Technology (KAUST), PSE divisions,
Thuwal 23955 - 6900, Kingdom of Saudi Arabia

PT
b
Imaging and Characterization Core Lab, King Abdullah University of Science and Technology
(KAUST), Thuwal 23955-6900, Kingdom of Saudi Arabia

RI
SC
* Corresponding author: [email protected]

1. Micro-fabrication, super-hydrophobic devices and physiologically compatible preparation used

NU
to obtain suspended and backgropund free DNA samples;

2. TEM diffraction technique applied to the suspended sample to obtain diffractograms of DNA
MA

fibers;

3. Super-hydrophobic devices with a circular pattern of micro-pillars to sieve and suspended nucleic
acids molecules;
D

4. TEM diffraction of suspended dsDNA show diffraction spacing in agreement with the previous
E

bibliography.
PT
CE
AC