This action funds an NSF Minority Postdoctoral Research Fellowship for FY 2008. The fellowship supports a research and training plan in a host laboratory for the Fellow who also presents a plan to broaden participation in biology. The title of the research and training plan for this fellowship to Elvin Aleman is "New Single-Molecule Fluorescence Assay to Study DNA Base Flipping." The host institution for this research is the Wayne State University and the sponsoring scientist is Dr. David Rueda.
DNA is a polymer with a backbone made of sugars and other chemical components. Attached to each sugar is one of four possible molecules called bases, whose sequences along the backbone encodes the genetic information for the synthesis of proteins in the cell in all living organisms. The chemical properties of the DNA bases can be changed when they are exposed to different chemical and non-chemical agents. Such damage can modify the genetic code and result in mutations that can lead to numerous diseases and cell death. Base-flipping is a phenomenon performed by some proteins to repair the mutated bases, but a detailed understanding of this process is lacking, impeding progress toward elucidating the prevention mechanisms that nature has developed to avoid the damage of bases. The project is developing a new single-molecule fluorescence assay to study the mechanisms of base-flipping and DNA protein interaction.
The Fellow is being trained in an area of biophysics and single-molecule fluorescence that allows him to develop new measurement techniques and permits him to introduce this expertise to underrepresented research communities in Puerto Rico, where expertise in such techniques is limited. The Fellow serves as a role model for underrepresented minority students interested in pursuing a career in science.
In nature, several enzymes participate in different repair processes by searching unusual aberrations in the structure of DNA. It has been suggested that some enzymes perform a base flipping process to recognize, gain access to, and repair damage sites in the DNA. The phenomenon of base flipping corresponds to the rotation of a DNA nucleotide out of the double helix, and its accommodation into a protein-binding pocket inside the enzyme. The fluorescent nucleotide analog 2-aminopurine (2AP) has been extensively used to study base flipping in ensemble average experiments. However, a detailed understanding of the base flipping phenomenon is lacking, impeding progress toward elucidating the mechanism that living organisms have developed to repair damage bases. Considering that base flipping plays a very important role in DNA repair, it is necessary to understand the biophysical basis of this phenomenon. The rationale of this project is developing a single-molecule fluorescence approach to understand and study local and global conformational changes after a protein-DNA interaction, simultaneously. The long-term goal is to understand at the molecular level how DNA-acting enzyme find their substrate sequence and induce base flipping as a recognition and catalytic mechanism over DNA molecules. The overall objective of this project is to elucidate the molecular mechanism of DNA base flipping. To study 2AP fluorescence at the single molecule level, we needed to immobilize the oligos to the surface of the probe volume. In this project, we found that the traditional streptavidin-biotin immobilization method produce a background signal upon excitation at 325 nm (wavelength used to excite the 2AP molecules). Initially, we attributed this background to the tryptophan amino acids in streptavidin. This background signal impeded the study of 2AP fluorescence at the single molecule level. The PI was trained in different molecular biology techniques to express the streptavidin protein and to substitute the tryptophans with a non-fluorescent analog 4-fluoro-tryptophan (4FW). Although the expression of the 4FW-streptavidin was not successful, after open discussions with experts in the field, the PI was able find evidence that explain the origin of the background signal from streptavidin. However, a new immobilization method was needed, and the PI developed new approaches based in click-chemistry and disulfide reactions. These new approaches required the synthesis of new DNA modification groups, and therefore the PI was also trained in different organic synthesis methods and different techniques for the purification and characterization of the synthesized molecules. The new immobilization methods have been presented in conferences and talks, and were published in the ChemBioChem Journal. Currently, the PI is advising other groups in the science community that are applying the click-chemistry approach in their single-molecule experiments. The click-chemistry immobilization method was used to study the fluorescence properties of 2AP at the single molecule level. The results from these experiments demonstrated that nucleotides have similar dynamic in single and double stranded DNAs. However, it was found that the number of nucleotides that are able to be dynamic in double stranded DNA is significantly reduced compared to the number of dynamic nucleotides in single-stranded DNA. Only through a single-molecule experiment these differences can be observed. A manuscript about this work is in preparation to be submitted in the Journal of the American Chemical Society. In order to study base flipping using the new 2AP single molecule approach, the PI used a protein (PspGI) that binds its canonical site and flips the central nucleotide. A DNA with 2AP incorporated into the canonical site and that substituted the nucleotide that flip out, were used in our single molecule experiment in presence and absence of PspGI. The results from these experiments demonstrate that in presence of PspGI, the nucleotide is most of the time in a flipped out state. To study local and global conformational changes in DNA, simultaneously, the PI designed and built a three color single molecule spectroscopy setup. However, the PI was not able to complete the dye labeling of the protein, which it would be used to study base flipping and DNA-protein binding events simultaneously. As part of the training process, the PI built different single molecule spectroscopy setups and studied other biomolecular and quantum dot complexes. The results obtained from these projects were presented in many conferences and talks in different universities. Soon after completion of the postdoctoral fellowship, the applicant began in a new position as an Assistant Professor in the Department of Chemistry at California State University Stanislaus, a Hispanic Serving Institution. The PI will have the opportunities to teach, train and perform research with students from underrepresented groups in the areas of biophysics and single molecule fluorescence. The knowledge gained during this fellowship will be used to continue this line of research and to develop new single molecule approaches to study base flipping in many DNA-protein complexes. The PI is very grateful to NSF for all the support.
