Tag Archives: Jessica Luton

The secret lives of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas (CRISPR-associated) proteins

This research isn’t quite as exciting as the title promises but it is important as it attempts to answer some fundamental questions about Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-Associated (Cas).proteins. From a June 13, 2018 news item on phys.org,

Recently published research from the University of Georgia and UConn Health [University of Connecticu Health Center] provides new insight about the basic biological mechanisms of the RNA-based viral immune system known as CRISPR-Cas.

CRISPR-Cas, short for Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated, is a defense mechanism that has evolved in bacteria and archaea that these single celled organisms use to ward off attacks from viruses and other invaders. When a bacterium is attacked by a virus, it makes a record of the virus’s DNA by chopping it up into pieces and incorporating a small segment of the invader’s DNA into its own genome. It then uses this DNA to make RNAs that bind with a bacterial protein that then kills the viral DNA.

The system has been studied worldwide in hopes that it can be used to edit genes that predispose humans to countless diseases, such as diabetes and cancer. However, to reach this end goal, scientists must gain further understanding of the basic biological process that leads to successful immunity against the invading virus.

A June 12, 2018 University of Georgia news release by Jessica Luton and Jessica McBride, which originated the news item, provides more detail,

Distinguished Research Professor of Biochemistry and Molecular Biology in UGA’s Franklin College of Arts and Sciences and principal investigator for the project Michael Terns and UGA postdoctoral fellow Masami Shiimori collaborated with Brenton Graveley and Sandra Garrett at UConn Health to sequence millions of genomes to learn more about the process. Graveley is professor and chair of the Department of Genetics and Genome Sciences and associate director of the Institute for Systems Genomics at UConn Health, and Garrett is a postdoctoral fellow in his laboratory.

“This research is more fundamental and basic than studies that are trying to determine how to use CRISPR for therapeutic or biomedical application,” said Terns. “Our study is about the unique first step in the process, known as adaptation, where fragments of DNA are recognized and integrated into the host genome and provide immunity for future generations.”

Previously, researchers did not understand how the cell recognized the virus as an invader, nor which bacterial proteins were necessary for successful integration and immunity.

“In this project we were able to determine how the bacterial immune system creates a molecular memory to remove harmful viral DNA sequences and how this is passed down to the bacterial progeny,” said Graveley.

By looking at patterns in the data, the researchers discovered several new findings about how two previously poorly characterized Cas4 proteins function in tandem with Cas1 and Cas2 proteins found in all CRISPR-Cas systems.

In this initial adaptation phase, one of two different Cas4 proteins recognizes a signaling placeholder in the sequence that occurs adjacent to the snippet of DNA that is excised.

When the Cas1 and Cas2 proteins are present in the cell with either of two Cas4 protein nucleases, Cas4-1 and Cas4-2, they act like the generals of this army-based immune system, communicating uniform sized clipped DNA fragments, directions on where to go next and ultimately instructions that destroy the lethal DNA fragment.

In order for a cell to successfully recognize and excise strands of DNA, incorporate them into its own genome and achieve immunity, the Cas4 proteins must be present in conjunction with the Cas1 and Cas2 proteins.

“Cas4 is present in many CRISPR-Cas systems, but the roles of the proteins were mysterious,” said Terns. “In our system, there are two Cas4 proteins that are essential for governing this process so that functional RNAs are made and immunity is conferred”

To achieve these findings, the research team from the University of Georgia created strains of archaeal organisms with key genetic deletions.

Hundreds of millions of DNA fragments captured in the CRISPR loci were sent to the Graveley lab in Farmington, Connecticut, where they were sequenced with the Illumina MiSeq system. The researchers then used supercomputing for bioinformatics analysis and data interpretation.

While there is still much to learn about the biological mechanisms involved in CRISPR-Cas systems, this research tells scientists more about the way these proteins work together to save the cell and achieve immunity.

“The data are so clear. We sequenced millions and millions of DNA fragments captured in CRISPR loci in different genetic strains and found the same results consistently,” he said.

Here’s a link to and a citation for the paper,

Cas4 Nucleases Define the PAM, Length, and Orientation of DNA Fragments Integrated at CRISPR Loci by Masami Shiimori, Sandra C. Garrett, Brenton R. Graveley, Michael P. Terns.Molecular Cell Volume 70, Issue 5, p814–824.e6, 7 June 2018 DOI: https://doi.org/10.1016/j.molcel.2018.05.002

This paper is behind a paywall.

Making magnetic rust behave like gold and the nanoscale

Researchers at the University of Georgia (US) have found a way to combine gold nanoparticles with magnetic rust nanoparticles for a hybrid structure that behaves with the properties of both types of nanoparticles. From a Sept. 15, 2016 news item on ScienceDaily,

Researchers from the University of Georgia are giving new meaning to the phrase “turning rust into gold”—and making the use of gold in research settings and industrial applications far more affordable.

The research is akin to a type of modern-day alchemy, said Simona Hunyadi Murph, adjunct professor in the UGA Franklin College of Arts and Sciences department of physics and astronomy. Researchers combine small amounts of gold nanoparticles with magnetic rust nanoparticles to create a hybrid nanostructure that retains both the properties of gold and rust.

A Sept. 15, 2016 University of Georgia news release by Jessica Luton, which originated the news item, expands on the theme,

“Medieval alchemists tried to create gold from other metals,” she said. “That’s kind of what we did with our research. It’s not real alchemy, in the medieval sense, but it is a sort of 21st century version.”

Gold has long been a valuable resource for industry, medicine, dentistry, computers, electronics and aerospace, among others, due to unique physical and chemical properties that make it inert and resistant to oxidation. But because of its high cost and limited supply, large scale projects using gold can be prohibitive. At the nanoscale, however, using a very small amount of gold is far more affordable.

In the new study published this summer in the Journal of Physical Chemistry C, the researchers used solution chemistry to reduce gold ions into a metallic gold structure using sodium citrate. In this process, if other ingredients-rust in this case-are present in the reaction pot during the transformation process, the metallic gold structures nucleate and grow on these “ingredients,” otherwise known as supports.

“We are really excited to share our new discoveries. When researchers are looking at gold as a potential material for research, we talk about how expensive gold is. For the first time ever, we’ve been able to create a new class of cheaper, highly efficient, nontoxic, magnetically reusable hybrid nanomaterials that contain a far more abundant material-rust-than the typical noble metal gold,” said Murph, who is also a principal scientist in the National Security Directorate at the Savannah River National Laboratory in Aiken, South Carolina.

When materials are broken down in size to reach nanometer scale dimensions-1-100 nanometers, which is approximately 100,000 times smaller than the diameter of human hair-these substances can take on new properties. For example, bulk gold does not display catalytic properties; however, at the nanoscale, gold is an efficient catalyst, accelerating chemical change for many reactions including oxidation, hydrogen production or reduction of aromatic nitro compounds.

Gold nanoparticles of different sizes and shapes display different colors when impinged by light because they absorb and scatter light at specific wavelengths, known as plasmonic resonances. These plasmonic resonances are of particular interest for biological applications. If someone shines light on the gold nanoparticles, the absorbed light can be converted to heat in the surrounding media, and if bacteria or cancerous cells are in the vicinity of such gold nanoparticles, they can be destroyed by using light of appropriate wavelength. This phenomenon is known as photothermal therapy.

By replacing some of the nano-gold with magnetic nano-rust, researchers show that the hybrid gold and rust nanostructures are able to photothermally heat the surrounding media as efficiently as pure gold nanoparticles, even with a significantly smaller concentration of gold.

“In a way, we’ve done a little better than alchemy,” said George Larsen, co-investigator and postdoctoral researcher in the Group for Innovation and Advancements in Nano-Technology Sciences at the Savannah River National Laboratory, “because these new hybrid nanoparticles not only behave better than gold in some cases, but also have magnetic functionality.”

Murph and her team looked at three different shapes of hybrid nanoparticles in this research-spheres, rings and tubes.

“A differently shaped nanoparticle means that the atoms are arranged differently-into cubes, hexagons or triangles, for example,” she said. “A different atom arrangement means different packing densities, spacing between atoms, defects, surface area and surface energies. Different shapes lead to an increased atom area that is exposed to catalyze a chemical reaction. Scientifically speaking, different shape means different crystallographic facets and surface energy that could lead to higher catalytic activity and different catalytic products.

“The results of our research showed that the ring- and tube-shaped hybrid nanoparticles proved to be better catalytic materials than the sphere-shaped nanoparticles because of the way the atoms are arranged in the structure at this nanoscale. More importantly, the hybrid nanoparticles of gold and rust are better catalysts than gold nanoparticles alone, even with a significantly smaller amount of gold.

When these different shaped hybrid nanoparticles were exposed to light of specific wavelength, the spheres heated the solution up to slightly higher temperatures than the ring- or tube-shaped nanoparticles.

“This could have a variety of biological applications such as tracking, drug delivery or imaging inside the body,” Murph said. “If you feed these gold nanoparticles to bacteria and shine the light on them, you could destroy these by just using light.”

The hybrid structures could also be used for new application [sic], such as sensing, hyperthermia treatment, environmental cleaning and protection medical imaging applications including magnetic resonance imaging contrast agents, product detection and manipulation.

Here’s a link to and a citation for the paper/video,

Multifunctional Hybrid Fe2O3-Au Nanoparticles for Efficient Plasmonic Heating by Simona E. Hunyadi Murph, George K. Larsen, Robert J. Lascola. Journal of Visualized Experiments, 2016; (108) DOI: 10.3791/53598

This paper/video appears to be open access.