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Posts Tagged ‘Molecular machines and motors’


DNA-based nanomotor and chemomechanical crosstalk

Curators: Larry H. Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN

LPBI

 

Nano-walkers take speedy leap forward with first rolling DNA-based motor

“Ours is the first rolling DNA motor, making it far faster and more robust,” says Khalid Salaita, the Emory chemist who led the research. (Photos by Bryan Meltz, Emory Photo/Video.)  https://pharmaceuticalintelligence.files.wordpress.com/2016/05/c4943-khalid_salaita.jpg

hysical chemists have devised a rolling DNA-based motor that’s 1,000 times faster than any other synthetic DNA motor, giving it potential for real-world applications, such as disease diagnostics. Nature Nanotechnology is publishing the finding.

“Unlike other synthetic DNA-based motors, which use legs to ‘walk’ like tiny robots, ours is the first rolling DNA motor, making it far faster and more robust,” says Khalid Salaita, the Emory University chemist who led the research. “It’s like the biological equivalent of the invention of the wheel for the field of DNA machines.”

The speed of the new DNA-based motor, which is powered by ribonuclease H, means a simple smart phone microscope can capture its motion through video. The researchers have filed an invention disclosure patent for the concept of using the particle motion of their rolling molecular motor as a sensor for everything from a single DNA mutation in a biological sample to heavy metals in water.

“Our method offers a way of doing low-cost, low-tech diagnostics in settings with limited resources,” Salaita says.

The field of synthetic DNA-based motors, also known as nano-walkers, is about 15 years old. Researchers are striving to duplicate the action of nature’s nano-walkers. Myosin, for example, are tiny biological mechanisms that “walk” on filaments to carry nutrients throughout the human body.

“It’s the ultimate in science fiction,” Salaita says of the quest to create tiny robots, or nano-bots, that could be programmed to do your bidding. “People have dreamed of sending in nano-bots to deliver drugs or to repair problems in the human body.”

So far, however, mankind’s efforts have fallen far short of nature’s myosin, which speeds effortlessly about its biological errands. “The ability of myosin to convert chemical energy into mechanical energy is astounding,” Salaita says. “They are the most efficient motors we know of today.”

Some synthetic nano-walkers move on two legs. They are essentially enzymes made of DNA, powered by the catalyst RNA. These nano-walkers tend to be extremely unstable, due to the high levels of Brownian motion at the nano-scale. Other versions with four, and even six, legs have proved more stable, but much slower. In fact, their pace is glacial: A four-legged DNA-based motor would need about 20 years to move one centimeter.

Kevin Yehl, a post-doctoral fellow in the Salaita lab, had the idea of constructing a DNA-based motor using a micron-sized glass sphere. Hundreds of DNA strands, or “legs,” are allowed to bind to the sphere. These DNA legs are placed on a glass slide coated with the reactant: RNA.

The DNA legs are drawn to the RNA, but as soon as they set foot on it they destroy it through the activity of an enzyme called RNase H. As the legs bind and then release from the substrate, they guide the sphere along, allowing more of the DNA legs to keep binding and pulling.

“It’s called a burnt-bridge mechanism,” Salaita explains. “Wherever the DNA legs step, they trample and destroy the reactant. They have to keep moving and step where they haven’t stepped in order to find more reactant.”

The combination of the rolling motion, and the speed of the RNase H enzyme on a substrate, gives the new DNA motor its stability and speed.

“Our DNA-based motor can travel one centimeter in seven days, instead of 20 years, making it 1,000 times faster than the older versions,” Salaita says. “In fact, nature’s myosin motors are only 10 times faster than ours, and it took them billions of years to evolve.”

Emory post-doctoral fellow Kevin Yehl sets up a smart-phone microscope to get a readout for the particle motion of the rolling DNA-based motor.

The researchers demonstrated that their rolling motors can be used to detect a single DNA mutation by measuring particle displacement. They simply glued lenses from two inexpensive laser pointers to the camera of a smart phone to turn the phone into a microscope and capture videos of the particle motion.

“Using a smart phone, we can get a readout for anything that’s interfering with the enzyme-substrate reaction, because that will change the speed of the particle,” Salaita says. “For instance, we can detect a single mutation in a DNA strand.”

This simple, low-tech method could come in handy for doing diagnostic sensing of biological samples in the field, or anywhere with limited resources.

The proof that the motors roll came by accident, Salaita adds. During their experiments, two of the glass spheres occasionally became stuck together, or dimerized. Instead of making a wandering trail, they left a pair of straight, parallel tracks across the substrate, like a lawn mower cutting grass. “It’s the first example of a synthetic molecular motor that goes in a straight line without a track or a magnetic field to guide it,” Salaita says.

In addition to Salaita and Yehl, the co-authors on the Nature Nanotechnology paper include Emory researchers Skanda Vivek, Yang Liu, Yun Zhang, Megzhen Fan, Eric Weeks and Andrew Mugler (who is now at Purdue University).

Related:
Chemists reveal the force within you
Molecular beacons shine light on how cells ‘crawl’

 

High-speed DNA-based rolling motors powered by RNase H

Kevin YehlAndrew MuglerSkanda VivekYang LiuYun ZhangMengzhen FanEric R. Weeks & Khalid Salaita
Nature Nanotechnology   11;184–190 (30 Nov 2016)    doi:10.1038/nnano.2015.259

DNA-based machines that walk by converting chemical energy into controlled motion could be of use in applications such as next-generation sensors, drug-delivery platforms and biological computing. Despite their exquisite programmability, DNA-based walkers are challenging to work with because of their low fidelity and slow rates (∼1 nm min–1). Here we report DNA-based machines that roll rather than walk, and consequently have a maximum speed and processivity that is three orders of magnitude greater than the maximum for conventional DNA motors. The motors are made from DNA-coated spherical particles that hybridize to a surface modified with complementary RNA; the motion is achieved through the addition of RNase H, which selectively hydrolyses the hybridized RNA. The spherical motors can move in a self-avoiding manner, and anisotropic particles, such as dimerized or rod-shaped particles, can travel linearly without a track or external force. We also show that the motors can be used to detect single nucleotide polymorphism by measuring particle displacement using a smartphone camera.

 

 

 

 

http://www.nature.com/nnano/journal/v11/n2/full/nnano.2015.259.html

 

T cells use ‘handshakes’ to sort friends from foes

http://esciencecommons.blogspot.ca/2016/05/t-cells-use-handshakes-to-sort-friends.html

 

A 3-D rendering of a fluorescence image mapping the piconewton forces applied by T cells. The height and color indicates the magnitude of the applied force. (Microscopy image by Yang Liu.)

By Carol Clark

 

T cells, the security guards of the immune system, use a kind of mechanical “handshake” to test whether a cell they encounter is a friend or foe, a new study finds.

The Proceedings of the National Academy of Sciences (PNAS) published the study, led by Khalid Salaita, a physical chemist at Emory University who specializes in the mechanical forces of cellular processes.

“We’ve provided the first direct evidence that a T cell gives precise mechanical tugs to other cells,” Salaita says. “And we’ve shown that these tugs are central to a T cell’s process of deciding whether to mount an immune response. A tug that releases easily, similar to a casual handshake, signals a friend. A stronger grip indicates a foe.”

Salaita, from Emory’s Department of Chemistry, collaborated on the research with Brian Evavold in the Emory School of Medicine’s Department of Microbiology and Immunology.

T cells continuously patrol through the body in search of foreign invaders. They have molecules known as T-cell receptors (TCR) that can recognize specific antigenic peptides on the surface of a pathogenic or cancerous cell. When a T cell detects an antigen-presenting cell (APC), its TCR connects to a ligand, or binding molecule, of the APC. If the T cell determines the ligand is foreign, it becomes activated and starts pumping calcium. The calcium is part of a signaling chain that recruits other cells to come and help mount an immune response.

Scientists have known about this process for decades, but they have not fully understood how the T cell distinguishes small modifications to the antigenic ligand and how it decides to respond to it. “If you view this T cell response purely as a chemical process, it does not fully explain the remarkable specifity of the binding,” Salaita says. “When you take the two components – the TCR and the ligand on the surface of cells – and just let them chemically bind in a solution, for example, you can’t predict what will trigger a strong or a weak immune response.”

The researchers hypothesized that mechanical strain might also play a role in a T cell response, since the T cell continues to move even as it locks into a bind with an antigenic ligand.

To test this idea, the Salaita lab developed DNA-based gold nanoparticle tension sensors that light up, or fluoresce, in response to a miniscule mechanical force of a piconewton – about one million-millionth the weight of an apple.

The researchers designed experiments using T cells from a mouse and allowed them to test ligands containing eight amino acid peptides that had slight mutations.

“We swapped out the fourth amino acid position to create really subtle chemical changes in the ligand that would be very difficult to distinguish without a mechanical component,” Salaita says.

Some of the mutated ligands were given a firmer anchor to give them a tighter “grip” to the moving TCR.

Through the experiments, captured on microscopy video, the researchers were able to see, record and measure the responses of the T cells as they moved across the ligands.

“As a T cell moves across a cell’s surface and encounters a ligand, it pulls on it,” Salaita explains. “It doesn’t pull very hard, it’s a very precise and tiny tug that is not sustained. The T cell pulls and stops, pulls and stops, all across the surface. It’s like the T cell is doing a mechanical test of the ligand.”

During the experiments, the T cells did not activate fully when they encountered ligands with weak anchors. In contrast, when a T cell encountered a ligand with a firm anchor, the T cell became activated, showing that it experienced a piconewton level of resistance.

The amount of force that was applied by the T cell was mapped by using tension probes of different stiffness. Probes that responded to 19 piconewtons did not fluoresce, while softer, 12-piconewton probes produced high signal.

Following the fluorescence of the probe, the T cells switched on their calcium pumps and increased the calcium concentration within the cell, indicating that the T cell is mounting an immune response.

“We were able to map out the order of the cascade of chemical and mechanical reactions,” Salaita says. “First, the T cell uses a very specific and finely tuned mechanical tug to distinguish friend from foe. And when it senses a precise, piconewton level of force in response to that tug, the T cell realizes that it has encountered a foreign body and gives the signal for attack.”

The discovery could help in the search for treatments of auto-immune diseases and the development of immune therapies for cancer.

“Cancer cells have an extra molecule that can make T cell security guards ‘drunk’ or ‘sleepy’ so that they are not able to function properly,” Salaita says. “Learning more about the mechanical forces involved in an effective immune response may help us develop ways to evade this defense system of cancer cells.”

Co-authors on the study include Yang Liu, Victor Pui-Yan Ma, Kornelia Galior and Zheng Liu (from the Salaita lab); and Lori Blanchfield and Rakieb Andargachew (from the Evavold lab).

Related:
Chemists reveal the force within you
Molecular beacons shine light on how cells ‘crawl’
Nano-walkers take speedy leap forward with first rolling DNA-based motor

 

DNA-based nanoparticle tension sensors reveal that T-cell receptors transmit defined pN forces to their antigens for enhanced fidelity

Yang LiuaLori BlanchfieldbVictor Pui-Yan MaaRakieb AndargachewbKornelia GalioraZheng LiuaBrian Evavoldb, and Khalid Salaitaa,c,1
P
NAS May 2016; http://dx.doi.org:/10.1073/pnas.1600163113     

Significance

T cells protect the body against pathogens and cancer by recognizing specific foreign peptides on the cell surface. Because antigen recognition occurs at the junction between a migrating T cell and an antigen-presenting cell (APC), it is likely that cellular forces are generated and transmitted through T-cell receptor (TCR)-ligand bonds. Here we develop a DNA-based nanoparticle tension sensor producing the first molecular maps of TCR-ligand forces during T cell activation. We find that TCR forces are orchestrated in space and time, requiring the participation of CD8 coreceptor and adhesion molecules. Loss or damping of TCR forces results in weakened antigen discrimination, showing that T cells harness mechanics to optimize the specificity of response to ligand.

T cells are triggered when the T-cell receptor (TCR) encounters its antigenic ligand, the peptide-major histocompatibility complex (pMHC), on the surface of antigen presenting cells (APCs). Because T cells are highly migratory and antigen recognition occurs at an intermembrane junction where the T cell physically contacts the APC, there are long-standing questions of whether T cells transmit defined forces to their TCR complex and whether chemomechanical coupling influences immune function. Here we develop DNA-based gold nanoparticle tension sensors to provide, to our knowledge, the first pN tension maps of individual TCR-pMHC complexes during T-cell activation. We show that naïve T cells harness cytoskeletal coupling to transmit 12–19 pN of force to their TCRs within seconds of ligand binding and preceding initial calcium signaling. CD8 coreceptor binding and lymphocyte-specific kinase signaling are required for antigen-mediated cell spreading and force generation. Lymphocyte function-associated antigen 1 (LFA-1) mediated adhesion modulates TCR-pMHC tension by intensifying its magnitude to values >19 pN and spatially reorganizes the location of TCR forces to the kinapse, the zone located at the trailing edge of migrating T cells, thus demonstrating chemomechanical crosstalk between TCR and LFA-1 receptor signaling. Finally, T cells display a dampened and poorly specific response to antigen agonists when TCR forces are chemically abolished or physically “filtered” to a level below ∼12 pN using mechanically labile DNA tethers. Therefore, we conclude that T cells tune TCR mechanics with pN resolution to create a checkpoint of agonist quality necessary for specific immune response.

  1. Crystal structure of a complete ternary complex of T-cell receptor, peptide-MHC, and CD4.
    Yiyuan Yin et al., Proc Natl Acad Sci U S A, 2012
  2. T-cell antagonism by short half-life pMHC ligands can be mediated by an efficient trapping of T-cell polarization toward the APC.
    Leandro J Carreño et al., Proc Natl Acad Sci U S A, 2009
  3. T cell receptor binding kinetics required for T cell activation depend on the density of cognate ligand on the antigen-presenting cell.
    Pablo A González et al., Proc Natl Acad Sci U S A, 2005
  4. T-cell triggering thresholds are modulated by the number of antigen within individual T-cell receptor clusters.
    Boryana N Manz et al., Proc Natl Acad Sci U S A, 2011
  5. Quantum dot/peptide-MHC biosensors reveal strong CD8-dependent cooperation between self and viral antigens that augment the T cell response.
    Nadia Anikeeva et al., Proc Natl Acad Sci U S A, 2006
  6. Rachel A Gottschalk et al., Proc Natl Acad Sci U S A, 2012 
  7. Stage-dependent reactivity of thymocytes to self-peptide–MHC complexes.
    Qibin Leng et al., Proc Natl Acad Sci U S A, 2007  
  8. Maxim N Artyomov et al., Proc Natl Acad Sci U S A, 2010  
  9. Scott R Burrows et al., Proc Natl Acad Sci U S A, 2010  
  10. Andrej Kosmrlj et al., Proc Natl Acad Sci U S A, 2008
Larry H. Bernstein, MD, FCAP, Curator
LPBI
The two articles above are connected in an interesting way by the fact that cellular forces are generated and transmitted through T-cell receptor (TCR)-ligand bonds. The T-cell receptor (TCR) encounters its antigenic ligand, the peptide-major histocompatibility complex (pMHC), on the surface of antigen presenting cells (APCs).  The movement detected by the fluorescent sensor may be based on only a single amino acid at the cell surface ligand. The result is chemomechanical crosstalk between TCR and LFA-1 receptor signaling
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