DNA condensation is a fundamental process in all living organisms. The highly abundant nucleoid-associated proteins, HU and IHF, present in bacteria, have been shown to play an important role in shaping the nucleoid. However, the exact mechanism is not well understood. In this thesis, we have demonstrated that both HU and IHF guide DNA to condense into linear bundle-like structures in presence of cellular condensing components, but the proteins alone do not condense DNA into densely packed structures. Our results suggest a mechanism by which HU and IHF could act as architectural proteins during in vitro and in vivo DNA condensation. More recently, DNA condensation has attracted much attention for its relevance in optimizing artificial DNA delivery systems for gene therapy. The research presented in this dissertation provides in depth biophysical studies that demonstrate how local modulations in the nucleic acid structure can be used to control both the size and the morphology DNA condensates. We describe a novel strategy for improving the condensation of oligonucleotides that is based on the self-organization of half-sliding complementary oligonucleotides into long duplexes (ca. kb) with flexible sites at regular intervals along the duplex backbones, in the form of single-stranded nicks or single-stranded gaps. Our results also provide new insights into the role of DNA flexibility in condensate formation and suggest the potential for the use of this DNA structure in the design of vectors for oligonucleotide and gene delivery.

DNA bending plays a vital role in numerous cellular activities such as transcription, viral packaging, and nucleosome formation. Therefore, understanding the physics of DNA bending at the length scales relevant to these processes is one of the main keys to the quantitative description of life. However, previous studies provide a divided picture on how DNA should be modeled in strong bending condition relevant to biology. My thesis is devoted to answering how far an elastic rod model can be applied to DNA. We consider several subtle features that could potentially lead to the break-down of the worm-like chain model, such as local bendedness of the sequence and large bending angles. We used single-molecule Fluorescence Resonance Energy Transfer to track looping and unlooping of single DNA molecules in real time. We compared the measured looping and unlooping rates with theoretical predictions of the worm-like chain model. We found that the intrinsic curvature of the sequence affects the looping propensity of short DNA and an extended worm-like chain model including the helical parameters of individual base pairs could adequately explain our measurements. For DNA with random sequence and negligible curvature, we discovered that the worm-like chain model could explain the stability of small DNA loops only down to a critical loop size. Below the critical loop size, the bending stress stored in the DNA loop became less sensitive to loop size, indicative of softened dsDNA. The critical loop size is sensitive to salt condition, especially to magnesium at mM concentrations. This finding enabled us to explain several contrasting results in the past and shed new light on the energetics of DNA bending.

abstract: Conformational changes in biomolecules often take place on longer timescales than are easily accessible with unbiased molecular dynamics simulations, necessitating the use of enhanced sampling techniques, such as adaptive umbrella sampling. In this technique, the conformational free energy is calculated in terms of a designated set of reaction coordinates. At the same time, estimates of this free energy are subtracted from the potential energy in order to remove free energy barriers and cause conformational changes to take place more rapidly. This dissertation presents applications of adaptive umbrella sampling to a variety of biomolecular systems. The first study investigated the effects of glycosylation in GalNAc2-MM1, an analog of glycosylated macrophage activating factor. It was found that glycosylation destabilizes the protein by increasing the solvent exposure of hydrophobic residues. The second study examined the role of bound calcium ions in promoting the isomerization of a cis peptide bond in the collagen-binding domain of Clostridium histolyticum collagenase. This study determined that the bound calcium ions reduced the barrier to the isomerization of this peptide bond as well as stabilizing the cis conformation thermodynamically, and identified some of the reasons for this. The third study represents the application of GAMUS (Gaussian mixture adaptive umbrella sampling) to on the conformational dynamics of the fluorescent dye Cy3 attached to the 5' end of DNA, and made predictions concerning the affinity of Cy3 for different base pairs, which were subsequently verified experimentally. Finally, the adaptive umbrella sampling method is extended to make use of the roll angle between adjacent base pairs as a reaction coordinate in order to examine the bending both of free DNA and of DNA bound to the archaeal protein Sac7d. It is found that when DNA bends significantly, cations from the surrounding solution congregate on the concave side, which increases the flexibility of the DNA by screening the repulsion between phosphate backbones. The flexibility of DNA on short length scales is compared to the worm-like chain model, and the contribution of cooperativity in DNA bending to protein-DNA binding is assessed. / Dissertation/Thesis / Ph.D. Chemistry 2011

Nucleoside 5-triphosphates are the building blocks to synthesis of nucleic acids. Nucleic acids (RNA and DNA) participate in many important biological functions in living systems, including genetic information storage, gene expression, and catalysis. Nucleoside 5- triphosphates have many important therapeutic and diagnostic applications. To understand how these triphosphates are utilized in living systems, numerous synthetic mimics have been prepared and used as active metabolites of certain drugs and molecular probes. Over the years, nucleic acids have been modified at the nucleobase, sugar moiety and phosphate backbone with the aim of understanding their structures and functions. We have site-specifically replaced selected oxygen atoms of nucleosides and nucleotides with selenium atom in order to enzymatically synthesize selenium-derivatized DNAs for obtaining insights into the DNA flexibility, duplex recognition and stability. Although triphosphates have important biological and medicinal significance, they are however, very difficult to synthesize and isolate in high purity and yield. There are many approaches to the synthesis of nucleoside 5-triphosphates, but there is no general strategy that allows simple and direct synthesis of nucleoside triphosphates. To face the challenges, we have developed a new approach in the absence of protecting groups to quickly and efficiently synthesized native deoxynucleoside 5-triphosphates and deoxynucleoside 5-(α- P-seleno)- P-seleno)triphosphates. Syntheses of the triphosphates containing selenium-derivatized nucleobases were also successfully accomplished. After replacing the oxygen atoms at the 4-position of thymidine and uridine, and the 6-position of guanosine, we observed most strikingly, a large bathrochromic shift of over 100 nm, relative to their native counterparts of UV absorbance of 260 nm. Consequently, the synthesized selenium base modified triphosphates are yellow. We also synthesized 2-selenothymidine and 5-methylseleno thymidine 5-triphosphates. We conducted stability study on the colored 4-selenothymidine and used the 5- triphosphate analog (4-SeTTP) as substrate for polymerase recognition. The Klenow polymerase incorporated the 4-SeTTP with efficiency equal to that of the native counterpart. Finally, 4-SeTTP was used to demonstrate UVdamage resistance of selenium-derivatized DNAs and plasmid.