Machines that let you observe and manipulate a single biological molecule

Have you ever wondered how an individual molecule really behaves? Recent advances funded by the NIH and NSF have helped us reach the point where scientists can observe individual molecules act when they are isolated from other molecules. Let's take a look at a revolutionary and new way to perform scientific experiments.


How do single molecule experiments work?

There are several different types of single molecule experiments, but we'll focus on the most revolutionary, the optical tweezers experiment. This experimental method came to the forefront in the past decade and allows researchers to actually pull apart individual biological molecules.

Optical tweezers experiments hinge on the idea of a "radiation pressure trap" – the application of the fact that objects with a high refractive index (particles through which light travels significantly slower through than normal), can be held in place by a beam of light. Many plastics have this physical characteristic, and using light, tiny plastic beads can be trapped and manipulated. Once these beads are held in place, biological molecules like RNA and DNA can be attached between two beads. This results in one of the beads being held in place with a beam of light, while the other is held on a micro-pipette linked to a piezoelectric actuator.

The actuator allows for small, measurable movements to be made to the bead attached to the pipette, and in doing so, the molecule between the beads can be stretched and the applied force can be measured. A video of a typical optical tweezers experiment shows the moving of the pipette and bead (bottom) and the unfolding of a molecule (although the molecule, of course, is so small it cannot be seen). If you would like to follow along with the experiment going on in the video, a frame by frame breakdown can be read here.

What do single molecule experiments measure?

Biological molecules, particularly proteins, are very compact creatures, folded tightly into a three dimensional structure. Think of the bundle of iPhone and laptop chargers you have in your house – when tangled together, they are tightly wound and hard to distinguish, but when you begin to separate them, they are much longer. Because of increase in length of biological molecules as they are unfolded is substantial, this can be measured along with the amount of force applied.


Why would we want to look at the actions of a single molecule?

Optical tweezers experiments like the ones above are typically performed on pieces of RNA, a type of molecule that often codes for proteins. RNA is found in the cytoplasm of the cell and can take on a phenomenal number of shapes. RNA is folded as soon as it is created, but it is unfolded and folded many times throughout its existence. This leads to several opportunities for misfolding, thus creating a situation where the piece of RNA might fail to work properly. One of the most interesting discoveries in the course of single molecule studies is that molecules of the same type often take multiple paths to attain the same final structure.


Optical tweezers experiments can also look at larger biological molecules like proteins, with one pulling on one terminus of a protein and watching to see if it folds back to its normal three dimensional structure. The information for protein folding is built into the amino acid sequence that makes up the protein; however, we cannot successfully take an amino acid sequence and predict the final three dimensional structure, mathematically or computationally. Observing how a single molecule of a protein refolds after being pulled apart lends insight into this extremely important question, and predicting three dimensional structure from an amino acid sequence is one of the holy grails of structural biology. Other types of single molecule experiments that do not require the direct application of force are also being used to study to how a protein behaves in isolation.


Pioneers in Single Molecule Experiments

Born in Lima, Peru, Carlos Bustamante came to the United States in the late 1970s to pursue a Ph.D. in Biophysics at UC Berkley after finishing his undergraduate education. Bustamante returned to UC Berkley as a Professor of Molecular and Cell Biology, Chemistry, and Physics in 1998, and he has been member of a rather prestigious group as a Howard Hughes Medical Institute Investigator since 1994. He still works with closely with Tinoco Ignacio Jr., his doctoral thesis advisor. Bustamante has been the recipient (and still is) of a plethora of NIH and NSF grants. These grants have including ones vital to the field of single molecule instrument development which greatly benefited the field of biophysics and gave taxpayers an amazing bang for their buck.


A pioneer who became a Presidential Cabinet member

Carlos Bustamante's experiments build upon the work done by Dr. Steven Chu, a former professor at Stanford University and current Secretary of Energy in the Obama administration. Chu was an author on one of the most influential papers of the 20th Century, Observation of a single-beam gradient force optical trap for dielectric particles, which laid the foundation for Bustamante and others single molecule experiments. For his work in the field, Chu received a share of the 1997 Nobel Prize in Physics.


As Secretary of Energy, Dr. Chu has not given up on research, as he has recently co-authored two manuscripts published in the journal Nature, one expanding on Einstein's theory of relativity and the impact of gravity on slowing time and the other increasing the precision of earlier single molecule work to the sub-nanometer level. Chu worked on these scientific endeavors "during nights, weekends and on planes - after putting in 70-80 hours a week as energy secretary." Chu calls this work "my equivalent of ... vegging out in front of the TV."


Problems to overcome

Single molecule experiments are still in their infancy, with much more research (and money) needed to answer some of the questions of the field. One of the major issues preventing the proliferation of the optical tweezers experiments mentioned above is that these instruments have to be built individually from scratch, as there is no supplier that currently sells a "lab-ready" instrument. This should change in the coming years, allowing more to join in on this research, but at the moment, it is a significant hurdle to overcome. Even with this hurdle, the number of single molecule papers published has exploded from 100 at the beginning of the decade to over 1500 a year now. Observing larger individual molecules, like proteins, has its issues experimentally as well. Some proteins require multiple subunits or several of the same protein to be together in order to "assemble" into an active form. Other forms of single molecule experiments look at how a functional assembled protein in isolation from other common proteins or cellular components behaves.

The Future

Single molecule work lays the foundation for manipulating individual atoms, leading to the necessary contacts and finesse for the creation of the one of the hallmarks of science fiction lore, nano- assemblers. The finesse for moving individual pieces is certainly there, as shown in this video of a game of Tetris played with a set of plastic beads using optical tweezers, we just need to decrease the size a couple of million fold. A Nobel Prize might also be in Carlos Bustamante's future, especially with his research lab still going strong and looking at the application of a variety of single molecule techniques to biological molecules.


Top image from Shuttershock. Sources linked within article.

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