DNA Double Helix Does Double Duty in Assembling Arrays of Nanoparticles

A combination cryo-electron microscopy image of an octahedral frame with one gold nanoparticle bound to each of the six vertices, shown from three different angles

Synthetic pieces of biological molecule form framework and glue for making nanoparticle clusters and arrays

In a new twist on the use of DNA in nanoscale construction, scientists at the U.S. Department of Energy's (DOE) Brookhaven National Laboratory and collaborators put synthetic strands of the biological material to work in two ways: They used ropelike configurations of the DNA double helix to form a rigid geometrical framework, and added dangling pieces of single-stranded DNA to glue nanoparticles in place. 

The method, described in the journal Nature Nanotechnology, produced predictable clusters and arrays of nanoparticles—an important step toward the design of materials with tailored structures and functions for applications in energy, optics, and medicine.

"These arrays of nanoparticles with predictable geometric configurations are somewhat analogous to molecules made of atoms," said Brookhaven physicist Oleg Gang, who led the project at the Lab's Center for Functional Nanomaterials (CFN), a DOE Office of Science User Facility. "While atoms form molecules based on the nature of their chemical bonds, there has been no easy way to impose such a specific spatial binding scheme on nanoparticles. This is exactly the problem that our method addresses." 

Using the new method, the scientists say they can potentially orchestrate the arrangements of different types of nanoparticles to take advantage of collective or synergistic effects. Examples could include materials that regulate energy flow, rotate light, or deliver biomolecules. 

"We may be able to design materials that mimic nature's machinery to harvest solar energy, or manipulate light for telecommunications applications, or design novel catalysts for speeding up a variety of chemical reactions," Gang said.

Scientists built octahedrons using ropelike structures made of bundles 
of DNA double-helix molecules to form the frames (a). Single strands of 
DNA attached at the vertices (numbered in red) can be used to attach 
nanoparticles coated with complementary strands. This approach can 
yield a variety of structures, including ones with the same type of particle 
at each vertex (b), arrangements with particles placed only on certain 
vertices (c), and structures with different particles placed strategically 
on different vertices (d).

"We may be able to design materials that harvest solar energy, manipulate light, or speed up a variety of chemical reactions." — Brookhaven physicist Oleg Gang

The scientists demonstrated the technique to engineer nanoparticle architectures using an octahedral scaffold with particles positioned in precise locations on the scaffold according to the specificity of DNA coding. The designs included two different arrangements of the same set of particles, where each configuration had different optical characteristics. They also used the geometrical clusters as building blocks for larger arrays, including linear chains and two-dimensional planar sheets.

"Our work demonstrates the versatility of this approach and opens up numerous exciting opportunities for high-yield precision assembly of tailored 3D building blocks in which multiple nanoparticles of different structures and functions can be integrated," said CFN scientist Ye Tian, one of the lead authors on the paper.

Details of assembly

This nanoscale construction approach takes advantage of two key characteristics of the DNA molecule: the twisted-ladder double helix shape, and the natural tendency of strands with complementary bases (the A, T, G, and C letters of the genetic code) to pair up in a precise way. 

First, the scientists created bundles of six double-helix molecules, then put four of these bundles together to make a stable, somewhat rigid building material—similar to the way individual fibrous strands are woven together to make a very strong rope. The scientists then used these ropelike girders to form the frame of three-dimensional octahedrons, "stapling" the linear DNA chains together with hundreds of short complementary DNA strands.

"We refer to these as DNA origami octahedrons," Gang said.

To make it possible to "glue" nanoparticles to the 3D frames, the scientists engineered each of the original six-helix bundles to have one helix with an extra single-stranded piece of DNA sticking out from both ends. When assembled into the 3D octahedrons, each vertex of the frame had a few of these "sticky end" tethers available for binding with objects coated with complementary DNA strands.

"When nanoparticles coated with single strand tethers are mixed with the DNA origami octahedrons, the 'free' pieces of DNA find one another so the bases can pair up according to the rules of the DNA complementarity code. Thus the specifically DNA-encoded particles can find their correspondingly designed place on the octahedron vertices" Gang said.

The scientists can change what binds to each vertex by changing the DNA sequences encoded on the tethers. In one experiment, they encoded the same sequence on all the octahedron's tethers, and attached strands with a complementary sequence to gold nanoparticles. The result: One gold nanoparticle attached to each of octahedron's six vertices. 

In additional experiments the scientists changed the sequence of some vertices and used complementary strands on different kinds of particles, illustrating that they could direct the assembly and arrangement of the particles in a very precise way. In one case they made two different arrangements of the same three pairs of particles of different sizes, producing products with different optical properties. They were even able to use DNA tethers on selected vertices to link octahedrons end to end, forming chains, and in 2D arrays, forming sheets.

Visualization of arrays

By strategically placing tethers on particular vertices, the scientists
used the octahedrons to link nanoparticles into one-dimensional
chainlike arrays (left) and two-dimensional square sheets (right). 

Confirming the particle arrangements and structures was a major challenge because the nanoparticles and the DNA molecules making up the frames have very different densities. Certain microscopy techniques would reveal only the particles, while others would distort the 3D structures. 

To see both the particles and origami frames, the scientists used cryo-electron microscopy (cryo-EM), led by Brookhaven Lab and Stony Brook University biologist Huilin Li, an expert in this technique, and Tong Wang, the paper's other lead co-author, who works in Brookhaven's Biosciences department with Li. They had to subtract information from the images to "see" the different density components separately, then combine the information using single particle 3D reconstruction and tomography to produce the final images. 

"Cryo-EM preserves samples in their near-native states and provides close to nanometer resolution," Wang said. "We show that cryo-EM can be successfully applied to probe the 3D structure of DNA-nanoparticle clusters."

These images confirm that this approach to direct the placement of nanoparticles on DNA-encoded vertices of molecular frames could be a successful strategy for fabricating novel nanomaterials.

This research was supported by the DOE Office of Science.

Brookhaven National Laboratory is supported by the Office of Science of the U.S. Department of Energy.  The Office of Science is the single largest supporter of basic research in the physical sciences in the United States, and is working to address some of the most pressing challenges of our time.  For more information, please visit science.energy.gov.

Source link: http://www.bnl.gov/newsroom/news.php?a=11728

Repair protein’s DNA recognition motif

DNA replication – the process of copying the DNA each time a cell divides – must be completed accurately to avoid mutations that cause cancer and other diseases. The DNA damage response protein SMARCAL1 recognizes stalled replication “forks” and remodels the DNA to allow repair and restored replication. SMARCAL1 is essential to maintaining genome integrity during replication, but how it works is poorly understood.

Now, Brandt Eichman, Ph.D., and colleagues have determined the crystal structure of a region of SMARCAL1 (the HARP domain), which is fused to a motor domain. They used X-ray scattering to examine the conformation and assembly of the HARP domain in solution and found that the domain is conserved with DNA damage recognition domains from other DNA repair proteins. They showed that the HARP domain is a functional substitute for one of these regions and that mutations of predicted DNA-binding amino acids in the HARP domain reduced its ability to bind to replication forks and facilitate repair.

The studies, reported in the Proceedings of the National Academy of Sciences, uncovered a conserved recognition domain in DNA repair enzymes. This domain couples DNA recognition and remodeling and plays an important role in stabilizing replication forks and maintaining genome integrity.

The structure also illustrates the location of several SMARCAL1 mutations that cause Schimke immuno-osseous dysplasia (SIOD), a multi-system disorder characterized by growth defects, immune deficiencies and renal failure.

The findings are the latest in an ongoing collaboration between the teams of Eichman, associate professor of Biological Sciences and Biochemistry, David Cortez, Ph.D., professor of Biochemistry and Cancer Biology, and Walter Chazin, Ph.D., Chancellor’s Professor of Biochemistry and Chemistry. Together, the researchers aim to understand how DNA replication happens faithfully so that every cell ends up with exactly the same DNA – and without damaging mutations.

The research was supported by a pilot grant from the Vanderbilt Center in Molecular Toxicology and by National Institutes of Health grant CA136933.

http://news.vanderbilt.edu/2014/05/repair-proteins-dna-recognition-motif/

3D structure reveals protein’s Swiss-army knife strategy

The molecular machine that makes essential components of ribosomes – the cell’s protein factories – is like a Swiss-army knife, researchers at the European Molecular Biology Laboratory (EMBL) in Heidelberg, Germany, and the Centro de Investigaciones Biológicas in Madrid, Spain, have found. By determining the 3-dimensional structure of this machine, called RNA polymerase I, for the first time, the scientists found that it incorporates modules which prevent it from having to recruit outside help. The findings, published online today in Nature, can help explain why this protein works faster than its better-studied counterpart, RNA polymerase II. 

“Rather than recruiting certain components from outside, RNA polymerase I has them already built in, which explains why it is bigger, and less regulated, but at the same time more efficient,” says Christoph Müller from EMBL, who led the study. “Because everything is already assembled, there’s no time delay,” explains Maria Moreno-Morcillo, who carried out the work. 

There are three different RNA polymerases, each of which makes specific types of RNA molecule. For example, RNA polymerase II makes messenger RNA – the ‘middle-man’ that carries the information encoded in DNA to a ribosome where it can be used to make a protein. RNA polymerases I and III make parts of the machinery which reads that messenger RNA: I builds the RNA that will eventually form a ribosome, while III makes the transfer RNA that carries the protein building blocks to the ribosome for assembly. Scientists have known for over a decade what RNA polymerase II looks like and how it works, but obtaining detailed information on the structures of its counterparts has proven extremely difficult. Now that they have managed to do so for RNA polymerase I, Müller and colleagues have found explanations for some of the protein’s particularities.

Part of the difficulty in studying RNA polymerase I is that it is a larger molecule than RNA polymerase II. When they determined its 3-dimensional structure, the scientists found that some of the ‘extra’ modules in RNA polymerase I are remarkably similar to other, separate proteins that RNA polymerase II needs to do its job. It seems that RNA polymerase I has brought those helper modules permanently on board. In another part of the molecule, Müller and colleagues found that RNA polymerase I appears to have combined what in RNA polymerase II are two separate modules into a single, multi-tasking component. Together, these changes likely explain why RNA polymerase I can produce RNA molecules at a faster rate than RNA polymerase II.

The findings also imply that the cell has fewer ways of controlling RNA polymerase I’s activity, since it can’t influence it by changing the availability of helper proteins as it does in the case of RNA polymerase II. But here, too, RNA polymerase I’s Swiss-army knife strategy provides a solution. The structure showed that this molecular machine has a built-in regulatory mechanism: it can stop itself from attaching to DNA by bending a loop in its structure to block the space the DNA would usually dock onto.

The work was carried out in collaboration with Carlos Fernández-Tornero’s lab at the Centro de Investigaciones Biológicas in Madrid, Spain, as well as researchers at the University of Gӧttingen, Germany and the SOLEIL synchrotron in France, where some of the structural data was obtained. Structural data was also obtained at the Petra III ring at EMBL Hamburg, on the DESY campus in Germany.

Christoph Müller recently received an Advanced Grant from the European Research Council (ERC) to study RNA polymerase I and the proteins it interacts with.

Source Article

Fernández-Tornero, C., Moreno-Morcillo, M., Rashid, U.J., Taylor, N.M.I, Ruiz, F.M., Gruene, T., Legrand, P., Steuerwald, U. & Müller, C.W. Crystal structure of the 14-subunit RNA polymerase I. Published online in Nature on 23 October 2013. DOI: 10.1038/nature12636.

Article Abstract

Protein biosynthesis depends on the availability of ribosomes, which in turn relies on ribosomal RNA production. In eukaryotes, this process is carried out by RNA polymerase I (Pol I), a 14-subunit enzyme, whose activity is a major determinant of cell growth. Here, we present the crystal structure of Pol I fromSaccharomyces cerevisiae at 3.0 Å resolution. The Pol I structure shows a compact core with a wide DNA-binding cleft and a tightly anchored stalk. An extended loop mimics the DNA backbone in the cleft and may be involved in regulating Pol I transcription. Subunit A12.2 extends from the A190 jaw to the active site and inserts a TFIIS-like zinc ribbon into the nucleotide triphosphate entry pore, providing insight into the role of A12.2 in RNA cleavage and Pol I insensitivity to ␣-amanitin. The A49/A34.5 heterodimer embraces subunit A135 through extended arms thereby contacting and potentially regulating subunit A12.2.

Source: http://www.embl-hamburg.de/aboutus/communication_outreach/media_relations/2013/131023_Heidelberg/index.html