Single Molecules Trapped for Study

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2 Single Molecules Trapped for Study Michelle D. Wang PHYSICS To help us understand some of life s mysteries, we have developed precision optical instruments and techniques to look at important molecules in biology particularly DNA molecules and their associated proteins one molecule at a time. A Focused Beam of Light When we highly focus a laser beam down to an extremely tight spot, it becomes very powerful. That tight spot becomes a trap for particles as small as a micron in diameter. If we move the trap, the particle follows. We can see the trapped particles, but at this point, we can t directly visualize molecules because they are around 10 nanometers. So we attach a molecule to a particle, trap the particle, and use the trapped particle as a handle to sense what s happening at the molecule and control it. More specifically, we have developed ways to exert controlled force and torque on the molecule and precisely measure its response. Our work opens up a new dimension for studying deoxyribonucleic acid (DNA), motor proteins that travel along DNA, and other motor proteins. Our particular ability to exert and measure torque has received tremendous attention, and many labs are duplicating it. Looking Inside a Cell Nucleus Take a look. If we were to line up end to end all the DNA molecules that are inside the nucleus of a single human cell, the lineup would be more than one meter long. But a cell nucleus is only a few microns in diameter. A micron is one millionth of a meter. All those DNA molecules are squeezed into this very tiny space, all tangled and twisted up. That s a lot of compaction. And a profusion of topological activity accompanies the compacting. What s more, biological molecules are all about motion. Some can translocate and rotate along DNA like tiny molecular motors. So we wanted to study the issues of topology, torque, and rotation, all at the same time. A DNA molecule is a right-handed double helix. Any motor proteins translocating along the DNA usually rotate around its backbone. As they move forward, we want to track their motion relative to the DNA. The ability to see all of this movement is immensely important to understanding what s going on inside cells.

3 Research in Progress T7 Helicase Few techniques exist to do this consequential work. Biochemical techniques are typically indirect and deal with very large numbers of molecules at the same time. These techniques therefore sometimes miss important behaviors of DNA-based molecules. location within one to two base pairs, or DNA units. Why Do We Care? The basic packing unit of DNA is a DNAprotein complex called a nucleosome. Wang Lab I have a large number of extremely talented students and postdocs working with me in my lab. They make a significant difference in my research. They make the research enjoyable. We observed the precise mechanics of how individual subunits of helicase enzymes that travel along one side of doublestranded DNA coordinate and physically cause the DNA unzipping mechanism. We were able to manipulate single DNA molecules to watch what happens when helicases encounter them and how different nucleotides that fuel the reactions affect the process. Fascinating! t We specialize in a technique called optical trapping, recording data from single molecules using a focused beam of light to trap microparticles attached to the molecules. This area of work is so big that we can work on many different problems. We can look at DNA compaction, transcription genetic information being copied from DNA into ribonucleic acid (RNA) and much more. Our work may enable solutions to problems in both biology and materials science. Conventional optical trapping is used to exert and measure a force on a biological molecule and monitor the molecule s displacement. But we also wanted to be able to see rotation and torque. No one had demonstrated that optical trapping could be used to see the rotation of biological molecules and measure the torque exerted on and by them. To solve all of these problems and tackle the work, we developed angular optical trapping. It s very direct and allows us to monitor rotation and torque. We literally watch how the molecules move and how they generate torque, and we can use torque to manipulate the molecule. Gene Information Storage and Retrieval In a very dense cell nucleus, tightly packed with a DNA-protein mixture, figuring out where a protein is bound on a DNA is essential to understanding how a gene is stored and how genetic information is retrieved. Our lab developed another technique, based on DNA unzipping, that allows us to see where a protein is bound on the DNA. We grab the two strands of the doublehelix DNA and pull them apart, like unzipping a zipper. We can unzip the DNA very easily until something gets caught in the zipper. We realize, Oh, it s likely a bound protein. We have to unzip harder to pop the protein off, which is a great way to verify that it s actually a bound protein and where it is. We can precisely determine its Generally, DNA that is packed in a nucleosome cannot be accessed by motor proteins that might need to, for example, read the genetic information stored in the DNA. The interactions of DNA molecules with many different types of protein molecules are exceedingly significant to defining where a nucleosome is on a DNA template, which in turn determines whether a motor protein can access that part of the DNA. If the motor proteins cannot access a certain region of DNA, then the corresponding gene may not be expressed. The fate of that cell may be different from the fate of a cell where that gene can be expressed. So even with the same genetic makeup, the cells may actually develop differently, based on whether the gene is expressed or not. The Basic Search and Where It Leads We investigate questions relating to gene expression and regulation: how DNA is packed and how that packing alters the ability of enzymes that need access to the DNA for information decoding. Our work is basic research. It s well understood in the scientific community that in order to find cures for a number of cancers, we must understand how cancers can get started. Understanding basic mechanisms lies at the heart of the development of a cure. How our research can be applied is unfathomable. Solutions to a multiplicity of problems may have a basis in this research. A variety of foundations have funded our work, including the Keck Foundation, the Beckman Foundation, 56

4 Wang Lab

5 Why this Research? I want to understand how nature works. Gene expression and regulation is very fundamental. I want to know how to understand it in a way that takes advantage of computing and precision instruments. We can exploit our. We can do modeling. We can create techniques and instrumentation to see what has never been seen before. And. 58

6 Research in Progress Simultaneously measuring force, extension, torque, and angular orientation Damon Runyon Cancer Foundation, the Sloan Foundation, the National Science Foundation, the Howard Hughes Medical Institute, and others. As a Howard Hughes Medical Investigator, I can explore whatever I want. I m looking at different ways to study similar phenomena and more. Now we do single-molecule studies and work on theoretical modeling. Next, I want to look at the cellular level and see what s going on in the complexity of cells. ` people.ccmr.cornell.edu/~mwang We have developed an angular optical trapping instrument that permits simultaneous and direct measurements of force and torque for concurrent observation of the tensile and torsional properties of biological molecules over broad ranges of forces and torques. In an angular optical trap, four signals are simultaneously and directly measured: torque, rotation, force, and position, all with high spatial and temporal resolution. This wide bandwidth is also well suited for detection of highly kinetic processes. To ensure controlled orientation of the trapping particle and its specific attachment to the molecule of interest, we nanofabricated quartz cylinders that proved to be ideal trapping particles. Using these cylinders has dramatically enhanced the precision of torque measurements, making the angular optical trap a powerful tool for biological torque measurements. This instrument is opening up new possibilities for experiments on biological molecules, many of which are known to generate rotational motions and work against topological obstacles (e.g., topoisomerases, RNA polymerases). 59