Opportunities at Diamond

Learn more about career and student opportunities at Diamond:

Subscribe to our Corporate and Science Update mailing list

Keep up to date with the latest research and developments from Diamond. Sign up for news on our scientific output, facility updates and plans for the future.

Subscribe here

Building 3D nanomaterials with sticky DNA bricks

Several technological applications, spanning from energy storage to biomedicine, require the preparation of new materials with a finely organised 3D nanoscale structure. To achieve such delicate structures, they must be assembled from the bottom up, much like the iconic Lego bricks. However, unlike Lego, the building blocks in these scenarios are too small to be handled directly, so they must be encouraged to self-assemble.

Over recent years, some nanomaterials have been developed based on DNA, but they have been limited in size and shape. To overcome this barrier, a new type of DNA nanomaterial has been devised with the aid of the Small Angle X-ray Scattering (SAXS) beamline (I22).

The novel building material (termed C-Stars) was made from single-stranded DNA molecules that were adorned with cholesterol molecules to help them stick together. Through these sticky interactions, they self-assembled into crystals which were analysed at I22. The invaluable structural data showed that the C-Stars adopted a crystal lattice arrangement. This lattice geometry could be changed by altering the shape of the C-Stars, but more interestingly, the DNA network also could be engineered to change its structure in response to an external stimulus.

With such a flexible and robust building material, the resulting crystal arrangements are almost limitless. The 3D crystals are a completely new class of DNA nanostructures, which will have applications in advanced technologies such as biosensors, light harvesting and molecular sieving.

: Figure 1: Structure and self-assembly of C-Stars. (a) Each 4-arm C-Star assembles from 4 different ssDNA molecules forming the core of the junction (blue) and 4 identical cholesterol-functionalised strands (orange). (b) All single-stranded components are mixed in stoichiometric ratio and brought to high temperature, where cholesterol-DNA micelles coexist with free core strands. Upon slow cooling, the C-Star cores form and cross-link the micelles, leading to aggregates that grow over time into network. (c) Aggregation leads to the appearance of macroscopic single crystals, shown in bright-field light microscopy. Scale bar 50 μm.

Reliable self-assembly methods for the fabrication of nano-structured materials with prescribed properties would bring about a manufacturing revolution in many areas of advanced technology, including, but not limited to, (bio)sensing, photonics, light harvesting, energy storage, and molecular sieving.

Over the last two decades, the ever-expanding research field of DNA Nanotechnology has demonstrated how synthetic nucleic acid molecules can be used to create nanoscale motifs of near arbitrary shape1. DNA is indeed an ideal nanoscale construction material, mostly owing to the exquisite selectivity of the Watson-Crick base-pairing interactions, responsible for holding together double-helical DNA and enabling most of DNA’s biological functionalities. By carefully designing the base-sequence of synthetic DNA strands, it is possible to exploit base pairing to create complex shapes1. Furthermore, synthetic DNA can be commercially produced at relatively low cost, is robust against degradation, highly biocompatible, and can be connected to a plethora of other molecules that can impart advanced functionalities1.

A long-term goal of structural DNA Nanotechnology has been the creation of motifs that can support long-range order in three dimensions, forming macroscopic crystalline materials. Until recently, however, only very few examples of DNA nanostructures have proven capable of achieving this result2,3, substantially limiting our opportunity of tailoring the characteristics of the DNA crystals to specific technological needs.

Most of the available routes for creating ordered nanostructured materials rely on the self-assembly of amphiphilic molecules. These include lipids, soap molecules and specifically designed synthetic polymers, and feature, on each individual molecule, both hydrophobic and hydrophilic domains. When dispersed in water, the hydrophobic parts of amphiphilic molecules cluster with each other, leaving the hydrophilic segments in contact with the solvent. Depending on the geometry of the molecule, this frustrated phase separation, leads to a variety of different self-assembled structures, from small clusters, to filaments, to 2D membranes (e.g. lipid bilayers), to macroscopic 3D arrays4. Compared with the specific lock-and-key interactions between DNA Nanostructures, the self-assembly mechanism of amphiphiles is far more robust. However, the production of amphiphilic molecules often requires laborious chemical synthesis, and lacks all of the aforementioned practical advantages that make DNA-based materials so appealing.

$Figure 2: SAXS characterisation of C-Star crystals performed at Diamond I22 beamline. (a) SAXS powder diffraction pattern collected by illuminating a large number of individual crystallites. The black solid line shows the radial average of the 2D patterns shown in the inset. Red vertical lines mark the Bragg peaks. (b) Single crystal diffraction pattern collected using the microfocus end-station. Spots are highlighted by yellow circles. Both measurements are consistent with a BCC unit cell. (c) Plausible arrangement of the C-Stars in the BCC unit cell, with the micelle-like hydrophobic cores located at the lattice points, highlighted by red beads.$: Figure 2: SAXS characterisation of C-Star crystals performed at Diamond I22 beamline. (a) SAXS powder diffraction pattern collected by illuminating a large number of individual crystallites. The black solid line shows the radial average of the 2D patterns shown in the inset. Red vertical lines mark the Bragg peaks. (b) Single crystal diffraction pattern collected using the microfocus end-station. Spots are highlighted by yellow circles. Both measurements are consistent with a BCC unit cell. (c) Plausible arrangement of the C-Stars in the BCC unit cell, with the micelle-like hydrophobic cores located at the lattice points, highlighted by red beads.

In a recent contribution5, the team featuring Mr R. Brady, Prof P. Cicuta, Dr L. Di Michele (University of Cambridge), and Dr N. Brooks (Imperial College London), combined the best of both worlds, introducing a new type of amphiphilic DNA nanostructures, dubbed C-Stars5, capable of effectively forming macroscopic 3D crystalline aggregates. C-Stars are simple branched motifs self-assembled from a small number of synthetic single-stranded (ss)DNA molecules (Fig. 1a). Some of these strands are modified with a hydrophobic cholesterol molecule, ultimately positioned at the tip of each DNA arm. Since DNA itself is very hydrophilic, cholesterol modifications make C-Stars amphiphilic, and capable of self-assembling through the robust frustrated phase separation mechanism. At the same time, C-Stars retain all of the advantages of DNA-based materials, including the facile design and prototyping of different shapes, the easy functionalisation and the biocompatibility.

Figure 1 demonstrates the self-assembly principle of C-Stars. The ssDNA components are dispersed in buffered aqueous solution, and heated up to 90oC. At this high temperature, the DNA double helices (duplexes) cannot form, but the hydrophobic forces are still active, causing the cholesterol-bearing ssDNA strands to cluster into micelles (Fig. 1b). As the temperature is slowly decreased, the duplexes forming the C-Star junction start to self-assemble, linking together the micelles and forming a network whose structure is determined by the shape of the C-Stars, and thus encoded in the chosen sequence of DNA bases. The resulting aggregates grow over time and coalesce (Fig. 1b), eventually forming macroscopic networks exceeding 40 μm in size, equivalent to billions of individual C-Stars. When inspected with light microscopy, the aggregates display a polyhedral shape, strongly hinting at an underlying crystalline structure (Fig. 1c).

Small Angle X-ray Scattering (SAXS), performed at Diamond beamline I22, was used for the microscopic structural characterisationof aggregates self-assembled from 4-arm C-Stars. Powder diffraction experiments were performed at small-angle length scales by illuminating dense, millimeter-size, pellets prepared by concentrating a large number of DNA crystals. The generated diffraction patterns are the result of several crystals being exposed to the X-rays at once, and show well defined rings (Fig. 2b), which when averaged radially highlight clear Bragg peaks indicative of an ordered network.

Using the microfocus end-station available at I22, which can reduce the cross-section of the X-ray beam down to 10 μm, we could illuminate individual C-Star crystallites and obtain single crystal diffraction patterns (Fig. 2b).

$Figure 3: Self-assembly of C-Stars with 3 and 6 arms. Both 3-arm (a) and 6-arm (b) C-Stars form macroscopic single crystals, shown in bright field light microscopy. Scale bars 20 μm. SAXS powder diffraction patterns (insets) and radial intensity profiles (black solid lines) for 3-arm (c) and 6-arm (d) samples. The radial intensity profile for the 4-arm sample (dashed blue line), overlaid for easier visual comparison, highlights the difference in internal structure of the crystals formed by difference C-Star designs.$: Figure 3: Self-assembly of C-Stars with 3 and 6 arms. Both 3-arm (a) and 6-arm (b) C-Stars form macroscopic single crystals, shown in bright field light microscopy. Scale bars 20 μm. SAXS powder diffraction patterns (insets) and radial intensity profiles (black solid lines) for 3-arm (c) and 6-arm (d) samples. The radial intensity profile for the 4-arm sample (dashed blue line), overlaid for easier visual comparison, highlights the difference in internal structure of the crystals formed by difference C-Star designs.

The positions of the peaks in the powder diffraction and the spots of the single-crystal diffraction patterns are compatible with a body-centered cubic (BCC) lattice. Combining this information with the size of the individual C-Stars (imposed by design), and the coordination of cholesterol DNA micelles, we could identify the likely arrangement of the DNA building blocks in the BCC network (Fig. 2c). In our model, the unit cell, the elementary cube periodically tiling the 3D space, has a size (lattice parameter) of 23 Å, and contains 6 C-Stars. Interestingly, C-Star networks are extremely empty, with DNA and cholesterol occupying only about 15% of the volume of the crystal. In other words, C-Star aggregates have a very high porosity (~85%), which makes them ideal to encapsulate other molecules within the DNA.

Powder diffraction experiments were also performed on C-Stars featuring different numbers of arms. Remarkably, 3-arm and 6-arm building blocks are also capable of effectively supporting long-range 3D order, but form crystals with a different geometry compared to the 4-arm stars (Fig. 3).

That the large size of the unit cell, the intrinsic low contrast of DNA samples, and the very porous nature of C-Star crystals, make the use of synchrotron-based X-ray scattering strictly necessary. In view of this, the facilities of Diamond I22, optimized for the study of soft and biological matter, and the support of expert beamline scientists such as Dr. Andy Smith, played a crucial role in the study of this new class of nanomaterials.

Owing to their robust and flexible self-assembly mechanism, C-Stars will ultimately allow to significantly expand the range of shapes and sizes of DNA building blocks capable of sustaining long-range order in 3D. This design flexibility makes it possible to incorporate functional molecules within the DNA network, which can make the crystalline materials responsive to external stimuli. With the support of the I22 Diamond team, we are planning to demonstrate this possibility in an upcoming SAXS session.

References

Seeman, N. C. & Sleiman, H. F. DNA nanotechnology, Nature Reviews Materials 3, 17068, doi:10.1038/natrevmats.2017.68 (2017).
Zheng, J. et al. From molecular to macroscopic via the rational design of a self-assembled 3D DNA crystal, Nature 461, 74-77, doi:10.1038/nature08274 (2009).
Simmons, C. R. et al. Construction and Structure Determination of a Three-Dimensional DNA Crystal, J Am Chem Soc 138 (31), 10047-10054, doi:10.1021/jacs.6b06508 (2016).
Mai, Y. & Eisenberg, A. Self-assembly of block copolymers, Chem Soc Rev 41(18), 5969-85, doi:10.1039/C2CS35115C (2012).
Brady, R.A. et al. Crystallization of amphiphilic DNA nanostructures, Nano Letters 17 (5), 3276-3281, doi:10.1021/acs.nanolett.7b00980 (2017).

Funding acknowledgement:

The authors of the study (Ref. 5) acknowledge support from the EPSRC Programme Grant CAPITALS number EP/J017566/1, the Oppenheimer Fund, Emmanuel College Cambridge, the Leverhulme Trust, and the Isaac Newton Trust through an Early Career Fellowship (ECF-2015- 494) and the EPSRC CDT in Nanoscience and Nanotechnology (NanoDTC), grant number EP/L015978/1. The authors also acknowledge Diamond Light Source for provision of synchrotron beamtime (SM14217, SM15530, and SM15030), and thank Dr. Andy Smith for assistance in operating beamline I22.

Corresponding author: Dr Lorenzo Di Michele, Cavendish Laboratory, University of Cambridge, email: [email protected]

Related publication

Brady RA, Brooks NJ, Cicuta P, Di Michele L. Crystallization of amphiphilic DNA nanostructures, Nano Letters 17 (5), 3276-3281, doi:10.1021/acs.nanolett.7b00980 (2017).

Diamond Light Source is the UK's national synchrotron science facility, located at the Harwell Science and Innovation Campus in Oxfordshire.

Diamond Light Source Ltd
Diamond House
Harwell Science & Innovation Campus
Didcot
Oxfordshire
OX11 0DE

See on Google Maps

Diamond Light Source^® and the Diamond logo are registered trademarks of Diamond Light Source Ltd

Registered in England and Wales at Diamond House, Harwell Science and Innovation Campus, Didcot, Oxfordshire, OX11 0DE, United Kingdom. Company number: 4375679. VAT number: 287 461 957. Economic Operators Registration and Identification (EORI) number: GB287461957003.

feedback

On this website

About Diamond

For Users

Industry

Science

Instruments

Careers

Public

Procurement

Software & Tools

Sub-navigation

In This Section

Sub Navigation

Opportunities at Diamond

Subscribe to our Corporate and Science Update mailing list

Building 3D nanomaterials with sticky DNA bricks

References

Related publication