Nibedita Pal and Nils Walter with the TERF microscope

They may work on motors but U-M chemist Nils G. Walter and physicist Nibedita Pal are more likely to be wearing lab coats than greasy overalls. You will not find them under the hood of a car but they may be at the hood in a chemistry lab. While car mechanics hook up an engine to a computer for diagnostics, Walter and Pal use a sophisticated microscope to watch what their tiny engines are doing.

The motors they build are nanoscale biological motors—the micro machines that living cells employ for cargo transport, cell division, energy generation and other tasks. “Many of the fundamental processes of life are driven by these tiny, sub-microscopic engines,” explains Walter, the Francis S. Collins Collegiate Professor of Chemistry, and Professor of Biophysics and Biological Chemistry.

Researchers envision a time when such biological motors may be components in nanoscale robots capable of performing ever more sophisticated tasks in medicine or other disciplines. For example, just like a real car can deliver cargo to your doorstep, the nanoengine could be used to “drive” drugs through a patient’s body for targeted delivery to cells.

Customized biological motors have been created using DNA nanostructures, biohybrid designs, or through synthetic organic synthesis. However, a major hurdle has been how to design a biological motor capable of moving in one direction by fueling it with chemical energy.

Most recently, Pal and Walter characterized a biohybrid rotor—the component of a bioengine akin to a flywheel--that could be made to move along predefined tracks. Pal is a research fellow in the Walter lab. For this work, they collaborated with Professor Michael Famulok of the University of Bonn and the Center of Advanced European Studies and Research (CAESAR) in Germany. The work is now found online in the journal Nature Nanotechnology.

“This mammoth nanoscale task required collaboration from two teams in powerhouses of the automobile industry--the US and Germany,” says Walter.

“We are using a motor protein (RNA polymerase) to drive a rotor and produce an exhaust (RNA) that drives the entire nanoengine forward on a ‘road’ built from DNA.

”The researchers designed their bioengine using a static building block of an engineered protein: T7 RNA polymerase fused to a DNA-binding motif. Their novel design harnesses the energy produced by hydrolysis of nucleoside triphosphates—RNA building blocks—for the continuous rotation of one DNA ring through another. The “exhaust” of RNA produced then drives unidirectional movement of the motor on a DNA “race track.” An important feature of the design is the ability to prescribe where the motor goes. Once the rotor moves along a DNA path that part of the path cannot be used again, which means the rotor must move ahead. Yet their design also has the potential to regenerate that path later by removing the RNA that was generated the first time through, say the researchers.

This nanoengine is just one of multiple components needed to build smart materials—materials that can automatically respond to changes in their environment, explains Walter. In an upcoming publication, the Walter Lab has also developed a fast random walker—a “cartwheeling” acrobat that rapidly moves around without being directed along a particular path. While evolution has had billions of years to employ biological molecular machines, chemists are just beginning to build machines for a toolbox that may ultimately prove useful in medicine, environmental monitoring, manufacturing or other disciplines.

Interested in the technical detail?

Dr. Pal explains:

“The nanoengine consists of a catalytic stator that unidirectionally rotates an interlocked rotor carrying an engineered T7 RNA polymerase (T7RNAP-ZIF) powered by NTP hydrolysis. The catenated stator and rotor are made of dsDNA wheels. Through NTP hydrolysis by T7RNAP-ZIF the wheel motor produces long RNA that remains attached to the engine and is further used to guide its movement along predefined ssDNA tracks arranged on a DNA nano-tube.

Single molecule experiments were carried out with a prism-type Total Internal Reflection Fluorescence Microscope (TIRFM) based on an inverted IX71, Olympus microscope to monitor the rotation of each nanoengine through single molecule Fluorescence Resonance Energy Transfer (smFRET). A 532 nm green laser (CrystaLaser, CL532-050-L) and a 638 nm red laser (Coherent, 1069415/AR) were used to excite fluorescent molecules (Cy3 and Cy5, respectively). The emitted fluorescence was collected through a 60X 1.2 NA water immersion objective (Olympus UplanApo). After passing through several optics (Thorlabs), the fluorescence was projected onto an intensified charged-coupled device (ICCD, I-Pentamax, Princeton Instruments). Later the data were analyzed using several analysis packages, e.g., Matlab, QuB, and IDL.”