INTELLECTUAL MERIT: The project will study the elasticity of single-stranded DNA (ssDNA) molecules of various sequences in a variety of solution conditions, and quantify the effects of multivalent counterions and crowding agents on dynamic aspects of biopolymer structure. The research program is designed to result in clear, quantitative physical principles governing ionic biopolymer behavior in the kind of crowded, salt-rich environments encountered in biological cells. The PI will directly quantify the physical parameters of ssDNA of various sequences, and attempt to resolve conflicting results on the sequence-dependence of ssDNA's local stiffness. He will investigate the ability of ssDNA base-stacking to create rod-like statistical monomers; rod-like polymers have been predicted (though not demonstrated) to have novel elasticity behaviors. Multivalent solution electrostatics represents a major unsolved theoretical problem, and this study of the effect of salts of various valencies will give clear data on complex issues of solution electrostatics. The approach will provide data that may validate or invalidate certain theories. Molecular crowding is a universal and non-specific interaction that promotes compact biomolecular conformations, thus affecting the stability of folded biopolymers and macromolecular binding equilibria. These are fundamental and ubiquitous biomolecular processes, and the proposed experimental approach to this problem will provide basic insight into this important problem. Specifically, the PI will measure the conformation of individual polymers in a crowded solution and will directly test recent theories that predict the magnitude of compaction at various crowded concentrations. All of the experiments are designed to help validate the link recently forged by the PI and coworkers between the scaling picture of polymers and single-molecule stretching experiments. The proposed experiments are based on the unique low-force stretching capabilities of the PI's lab. A polymer's low-force elasticity is directly sensitive to both the short- and long-range interactions between the monomers and thus can comprehensively probe the microscopic physics of the polymer. In this respect, the low-force technique is hypothesized to outperform traditional high-force methods, and may also have advantages over commonly-used scattering techniques.
BROADER IMPACTS: The mechanical properties of ssDNA underlie a variety of biological, biotechnological, and biomaterials problems. Any model including unstructured ssDNAs must accurately describe the sequence-dependent conformation of the unfolded state. Data from this project will provide basic input parameters for such models, and thus will impact our understanding of processes in molecular biology (e.g. RNA folding) and bubble formation and denaturation transitions of dsDNA. Further, the measurements of the mechanical response of ssDNAs could lead to their rational use within DNA-based nanostructures and better design of molecular beacons. Finally, parts of this work will be used as a summer project for undergraduate interns, who will be recruited from on-campus programs for students traditionally under-represented in the physical sciences. The interns will work directly alongside the graduate student working on this project. They will learn a wide variety of skills appropriate to this interdisciplinary effort, including biomolecular synthesis, coupling chemistries, and quantitative data acquisition and analysis. The interns will attend group meetings, give presentations, and write a final paper on their work. Thus, they will gain significant exposure and experience to a wide range of activities needed to carry out cutting-edge interdisciplinary research. Finally, the mentoring graduate student will receive invaluable experience as a teacher and director of a research project; this should directly prepare him for a future scientific career as a research group leader, as well as improve the quality of his present work through a better understanding of how to relate day-to-day research with big-picture goals.
Charged polymers are a class of molecules consisting of extremely long, string-like molecules carrying electrical charges along their length. This combination of length and charge makes charged polymers extremely sensitive to their environment: for example, the space occupied by a charged polymer in water can easily vary a thousand-fold when salt is added to the water. Such effects are exploited in a variety of technological materials/devices, and in biology. Technological examples include waste-water treatment, and diapers; both of these cases exploit the ability of charged polymers to swell, sponge-like, with water. Biology is full of charged polymers, inlcuding DNA, some proteins, and some carbohydrates. Here, charged polymer behavior underlies the molecular processes of life, including genetic processes that involve shape changes of DNA, and mechanical processes such as lubrication of the cartilage in joints. Despite their ubiquity and importance, aspects of the structure of charged polymers have been difficult to understand, essentially because commonly-used models and techniques developed to analyze uncharged polymers have difficulties handling the interactions among charged groups. The major goal of this project was to overcome these issues, and develop and apply a new experimental approach to charged polymer structure. Our approach involved high-precision experiments on the mechanics (elasticity) of single charged polymers, using mainly DNA as our test molecule. The major outcomes of this project involved both technical and scientific advances. Likely the most important advance was scientific: our measurements revealed key structural details of charged polymers. These measurements resolve long-standing disputes about the structure of these chains. The resulting improved understanding of charged polymer structure will enhance abilities to rationally engineer such systems for technological purposes. A second key advance is technical-- our work has established the elasticity approach as viable method to obtain molecular-scale information on these systems. Given this confidence in the method, we are now moving to broaden its application; for example, we have already started to apply the method to understanding the structure of polymers involved in joint lubrication, as well as to understanding the energetics of certain interactions of DNA molecules.
