Antibodies, in charge of recognizing and homing in on molecular targets, are among the most useful tools in biology and medicine. Nanobodies – antibodies' tiny cousins – can do the same tasks, for example marking molecules for research or flagging diseased cells for destruction. But, thanks to their comparative simplicity nanobodies offer the tantalizing prospect of being much easier to produce.
Unfortunately, their promise hasn't been fully realized, because scientists have lacked an efficient way of identifying the nanobodies most closely tuned to their targets. However, a new system, developed by researchers at Rockefeller University and their collaborators and described today in Nature Methods, promises to make nanobodies dramatically more accessible for all kinds of research.
Antibodies are defensive proteins deployed by the immune system to identify and neutralize invaders. But their power can be harnessed in other ways as well, and they are used in biology and medicine for visualizing cellular processes, attacking diseased cells and delivering specific molecules to specific places. Like their larger cousins, nanobodies can also be used for these tasks but their small size makes nanobodies much easier to grow in bacterial factories. They can also access hard to reach places that may be off limits to larger molecules.
"Nanobodies have tremendous potential as versatile and accessible alternatives to conventional antibodies, but unfortunately current techniques present a bottleneck to meeting the demand for them," says study author Michael Rout, head of the Laboratory of Cellular and Structural Biology at Rockefeller. "We hope that our system will make high-affinity nanobodies more available, and open up many new possible uses for them."
In their first studies, the team generated high-affinity antibodies, those that are capable of most precisely binding to their targets, directed against two fluorescent proteins that biologists often use as markers to visualize activity within cells: GFP and mCherry. Their new system, like existing ones for generating antibodies, begins with an animal, in this case llamas housed in a facility in Connecticut.
Llamas were chosen because the antibody variants they produce are easily modified to generate nanobodies, which are only one-tenth the weight of a regular antibody. The llamas were immunized with GFP and mCherry, prompting their immune systems to generate antibodies against these foreign proteins.
"The key was to figure out a relatively fast way of determining the genetic sequences of the antibodies that bind to the targets with the greatest affinity. Up until now obtaining these high-affinity sequences has been something of a holy grail," says Brian Chait, Camille and Henry Dreyfus Professor and head of the Laboratory of Mass Spectrometry and Gaseous Ion Chemistry at Rockefeller. "Once those sequences are obtained, it's easy to engineer bacteria to mass produce the antibodies."
The researchers, led by graduate student Peter Fridy and postdoc Yinyin Li, started by making antibody sequence databases from RNA isolated out of antibody-producing cells in the llamas' bone marrow. Next, they picked out the tightest binding GFP and mCherry antibodies from blood samples from the same llamas, and chemically cut these into smaller pieces, keeping only the antigen-binding section to create nanobodies.
They then determined partial sequences of the amino acids that made up the protein of the nanobodies with a technique known as mass spectrometry. Using a computer algorithm called "llama magic," developed by David Fenyö and Sarah Keegan of New York University School of Medicine, they matched up the composition of the highest affinity nanobodies with the original RNA sequence. With this sequence, they could engineer bacteria to mass produce the nanobodies before putting them to use in experiments.
Antibodies are often used to isolate a particular structure within a cell so scientists can remove and examine it, and the team did just that with their new nanobodies. They purified various cellular structures tagged with GFP or mCherry, and also visualized these structures in situ.
All in all, their procedure generated 25 types of nanobodies capable of precisely targeting GFP and six for mCherry, a far more diverse set of high affinity nanobodies than is typically possible with conventional techniques.
This abundance opens up new options. Scientists can select only the best ones, eliminating nanobodies that by chance cross-react with other molecules, or string together two nanobodies that attach to different spots on the same target molecule to generate a super-high-affinity dimer, exactly as the researchers demonstrated for the GFP nanobodies. This super-high-affinity could be a powerful feature when delivering therapeutic or diagnostic molecules because it would lower the required dosage, and so reduce unwanted side effects.
"Given that we can now readily identify suites of high affinity nanobodies, the future for them as research tools, diagnostics and therapeutics looks bright," says Rout.
Zach Veilleux | EurekAlert!
Link Discovered between Immune System, Brain Structure and Memory
26.04.2017 | Universität Basel
Researchers develop eco-friendly, 4-in-1 catalyst
25.04.2017 | Brown University
More and more automobile companies are focusing on body parts made of carbon fiber reinforced plastics (CFRP). However, manufacturing and repair costs must be further reduced in order to make CFRP more economical in use. Together with the Volkswagen AG and five other partners in the project HolQueSt 3D, the Laser Zentrum Hannover e.V. (LZH) has developed laser processes for the automatic trimming, drilling and repair of three-dimensional components.
Automated manufacturing processes are the basis for ultimately establishing the series production of CFRP components. In the project HolQueSt 3D, the LZH has...
Reflecting the structure of composites found in nature and the ancient world, researchers at the University of Illinois at Urbana-Champaign have synthesized thin carbon nanotube (CNT) textiles that exhibit both high electrical conductivity and a level of toughness that is about fifty times higher than copper films, currently used in electronics.
"The structural robustness of thin metal films has significant importance for the reliable operation of smart skin and flexible electronics including...
The nearby, giant radio galaxy M87 hosts a supermassive black hole (BH) and is well-known for its bright jet dominating the spectrum over ten orders of magnitude in frequency. Due to its proximity, jet prominence, and the large black hole mass, M87 is the best laboratory for investigating the formation, acceleration, and collimation of relativistic jets. A research team led by Silke Britzen from the Max Planck Institute for Radio Astronomy in Bonn, Germany, has found strong indication for turbulent processes connecting the accretion disk and the jet of that galaxy providing insights into the longstanding problem of the origin of astrophysical jets.
Supermassive black holes form some of the most enigmatic phenomena in astrophysics. Their enormous energy output is supposed to be generated by the...
The probability to find a certain number of photons inside a laser pulse usually corresponds to a classical distribution of independent events, the so-called...
Microprocessors based on atomically thin materials hold the promise of the evolution of traditional processors as well as new applications in the field of flexible electronics. Now, a TU Wien research team led by Thomas Müller has made a breakthrough in this field as part of an ongoing research project.
Two-dimensional materials, or 2D materials for short, are extremely versatile, although – or often more precisely because – they are made up of just one or a...
20.04.2017 | Event News
18.04.2017 | Event News
03.04.2017 | Event News
26.04.2017 | Materials Sciences
26.04.2017 | Agricultural and Forestry Science
26.04.2017 | Physics and Astronomy