In the world of biomolecules, none is more iconic or more versatile than DNA. Nature uses the famous double helix to store the blueprints of all living forms, relying on a four-letter alphabet of nucleotides.
Researchers in the field of DNA nanotechnology have been inspired by the seemingly endless variety of living forms that nature has fashioned from this genetic raw material. The field seeks to emulate the creative enterprise of nature and even to extend the possibilities of DNA architecture beyond what nature has created.
In a new study, Hao Yan and his colleagues Nicholas Stephanopoulos and Petr Sulc explore a building block used in the manufacture of many DNA nanoforms. Known as the Holliday junction, this link of two segments of double-stranded DNA has been used to form elaborate, self-assembled crystal lattices at the nanometer scale (about 1:75,000e the width of a human hair).
The structures take their name from molecular biologist Robin Holliday, who first proposed their existence in 1964. Holliday junctions play a vital role in nature, where they are involved in a process known as homologous recombination, a driving force to generate new genetic variations in living organisms. things.
Since the beginning of DNA nanotechnology, the field has made remarkable progress, using DNA components to design tiny structures of intricate beauty as well as nanoscale devices with applications in fields as varied as photonics, computer storage, biosensing and tissue regeneration.
Yan has been at the forefront of rapid transformations in the field, designing a myriad of useful nanoarchitectural forms, cartwheel nanobots and DNA spiders at cancer research and destruction devices.
The new study uses crystallographic techniques to describe the characteristics of 36 base variants of the Holliday junction. The results show that the efficiency of a given Holliday junction for building crystalline nanoarchitectures substantially depends not only on the arrangement of the four nucleotide pairs forming the junction, but also on the sequences forming the four protruding arms of the junction. Certain DNA sequences act to enhance the crystallization process of these forms, while six of the 36 variants of the Holliday junction were found to be “fatal” due to their inability to form crystals.
Yan directs the Biodesign Center for Molecular Design and Biomimetics and holds the Milton D. Glick Distinguished Professorship at ASU’s School of Molecular Sciences. Stephanopoulos and Sulc are also faculty members at the center and the school.
The research results, which represent the first systematic study of Holliday junctions, were recently published in the journal Nature Communications.
DNA is proving to be an ideal material for designing and manufacturing nanoscale structures. The consistent and predictable nature of base pairing between the four nucleotides of DNA ensures that correctly designed shapes will reliably self-assemble into the desired structures. To this end, various elaborate nanoforms have been constructed using fundamental building blocks of DNA, one of the most popular and useful being the Holliday junction. DNA crystals composed of repeating structural units are key ingredients for nanotechnology applications, enabling versatile and scalable design features.
Vacation junctions are observed in nature as an intermediate step during the process of cell meiosis. The result of this transformation is an exchange of genes between the maternal and paternal chromosomes. This process, known as homologous recombination, takes place in four steps. (See illustration at right.)
First, a pair of double-stranded DNA helices come together. An enzyme called endonuclease then causes a single-strand break in each of the two double strands. The next stage, known as strand invasion, occurs when the free ends of each single-strand break come together, causing the originally separated double strands to intertwine.
This cross-shaped structure, which connects the two separate double strands of DNA, is the Holliday junction. In biological processes, the junction is then “resolved” when another enzyme cuts the Holliday junction in one of two ways, both resulting in two separate strands of DNA, which differ from the original strands because the Holliday junction introduced new DNA segments into both. Double strand of DNA.
This form of DNA recombination is a universal biological event of great importance. It simultaneously acts to preserve the integrity of the genome through DNA repair mechanisms while generating new variability, without which organisms would soon reach an evolutionary dead end. The key structure in the shuffling of the DNA package during cell division is the Holliday junction.
It was later noted that the Holliday junction motif could be used as a powerful building block for a multiplicity of artificial DNA structures. Although the Holliday junctions occurring during cell division can slip along the length of DNA, in a process known as branch migration the junctions used to build DNA nanostructures are immobilized because the flanking sequences are not complementary.
“The first stationary Holliday junction was described in 1982, and this sequence has since been used exclusively in self-assembling DNA crystals,” said Chad Simmons, the paper’s first author and lead scientist applying crystallography to crystals. X-rays for this study. “Our work sought to change this paradigm by probing the other 35 possible stationary junctions. As a result, we were able to identify several sequences that gave superior performance over their predecessors in terms of their ability to robustly crystallize and diffract at high resolution, and which allowed control over the symmetry of the grating arrangement. .
“This required an exhaustive effort that resulted in 134 new crystal structures, and we are very excited to share a comprehensive toolkit of sequence combinations to direct the design and construction of future self-directed DNA crystal systems. assembly.”
The new research demonstrates that most Holliday junction variants produce self-assembled crystals, although six fatal junction arrangements are incompatible with crystal formation. The common feature of these failed junctions was their lack of two critical ion binding sites, which are essential for crystal formation.
“This study was fascinating because it showed how subtle variations in Holliday junction geometries – which could be understood at the single nucleotide level – could have dramatic effects on crystal assembly and symmetry. This is truly ‘molecular science’, eventually allowing us to engineer molecular-level interactions that will give rise to exciting nanomaterials with unprecedented control,” Stephanopolous said.
“One of the challenges of this research was to determine why some Holliday junctions could produce crystals, but not others. Empirically, we could study the crystal structures of those junctions that crystallize, but to understand the behavior of arrangements of fatal junctions that don’t, computational chemistry was needed,” Sulc said.
“To this end, we teamed up with Dr. Miroslav Krepl and Prof. Jiri Sponer of the Czech Academy of Sciences, who simulated all Holliday junctions to atomistic resolution, and gained the critical insight that fatal junctions “were not able to bind to stabilizing ions. This effort provided an excellent example where computer modeling and experiments can jointly explain complex phenomena,” he said.
The new research provides valuable clues for the design and development of new forms to add to the ever-growing plethora of nanostructures and nanodevices serving a wide range of applications in electronics, imaging, computing and medicine.