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Regulation of Nucleic Acid Structure and Function with Peptoid, Small Molecules and Bpna(+)

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Regulation of nucleic acid structure and function with peptoid, small molecules and bPNA(+) DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Jie Mao Graduate Program in Chemistry The Ohio State University 2017 Dissertation Committee: Dr. Dennis Bong, Advisor Dr. Venkat Gopalan Dr. Psaras McGrier Copyrighted by Jie Mao 2017 Abstract Nucleic acids are not only central to the storage and expression of genomic information, but also key to many biological activities. Much effort has been dedicated to the research of molecular recognition of nucleic acids, to understand their structure and function, as well as possibly developing methods to regulate them. We have previously demonstrated that triaminotriazine (melamine, M*) moieties installed on peptide and polyacrylate backbones could trigger the formation of non-native secondary structures from unfolded T/U rich nucleic acids due to the Janus-Wedge type hydrogen bonding pattern between thymine/uracil and melamine. In this dissertation, we developed novel melamine displaying peptoids, small molecules and a new type of peptide called bPNA(+), all of which could trigger folding of T/U rich nucleic acids into defined triplex structures. In Chapter 2, we demonstrated the synthesis of melamine displaying peptoids, which are analogous to bPNAs. These peptoids could bind with oligo T DNAs, supported by CD spectra changes and corporative melting of the peptoid-DNA complexes. The backbone change from α- PNA (bPNA) to peptoid decreased the thermal stability of the complex, possibly due to the lack of backbone hydrogen bonding and the elimination of chiral centers. In Chapter 3, we reported that small molecule t3M, which was synthesized via one step arylation from tris(2-aminoethyl) amine (tren), could induce folding of oligo T DNAs. Further analysis ii showed that only two out the three melamines are needed to form hydrogen bonds with T rich DNA, thus creating the minimal binding unit t2M. It binds to a tetrathymidine bulge in DNA duplex with micromolar affinity. Functionalization of the tren backbone with t2M creates dendritic small molecule t4M, which exhibits much increased affinity towards oligo T buldges (50 nM) due to multivalency. These small molecules could act as allosteric switches for nucleic acid. Hammerhead ribozymes whose stem and/or loop regions are replaced with oligo U could refold in the presence of t4M, self-cleavage reactions are then initiated due to the restoration of both secondary and tertiary interactions. We also reported for the first time, the regulation of nucleic acid functions using small molecule as a molecular bridge, by mimicking crucial native tertiary interaction. This regulation pathway is similar with the chemically induced dimerization in protein regulation. The facile and general method describe here could be used for installation of t2M motif on any primary amine, thus stimulating the design of synthetic allosteric riboswitches and small molecule-nucleic acid complexes. In Chapter 4, we descried the selective fluorescence turn on of Spinach aptamer mutants and native human telomer G quadruplex with synthetic small molecules. Covalent linking of the triplex triggering small molecules melamine and fluorogenic moieties DFHBI yielded ligands that selectively turn on fluorescence of the U rich mutants of Spinach aptamer due to potential formation of U-M*-U triplex. Due to the structural similarities of Spinach and Mango (turns on thiazole orange fluorescence) aptamers, namely, both are composed of G quadruplexes in the fluorogenic dye binding interface, we created new aptamers for thiazole orange (TO) and its derivatives with melamine and t2M by rational redesign of Spinach aptamer, suggesting a new way of obtaining aptamers, through de novo designed instead of in vitro selection. t2M iii conjugates of TO was also found to selectively turn on human telomere core sequence (GGGTTA)3GGG, due to the possible triplex formation with the TTA loop region. Further optimizations and applications are currently underway. In Chapter 5, we synthesized a new class of peptide bPNA(+) by reductive alkylation on the amine side chains of native peptides, creating t2M motifs. The display of t2M motifs instead of single melamine in bPNA decreases the total chain length of the peptide, thus reducing the entropic cost when hybridizing with nucleic acids. We observed an increase affinity in binding with T rich DNAs and much higher thermal stability of the complex. Nucleic acid imaging and probing of structural change could be achieved thanks to this affinity enhancement. In summary, we demonstrated in this dissertation novel synthetic small molecules and oligomers that could trigger the folding of oligo T/U rich nucleic acids. The structural change induced by these melamine displaying compounds turns on nucleic acid functions such as hammerhead ribozyme catalysis and aptamer fluorescence turn on. Further applications such as nucleic acid packaging, delivery and labeling with the synthetic molecules discussed in this dissertation are currently underway. iv Acknowledgments I would like to express my sincere gratitude to my advisor, Dr. Dennis Bong, for his intelligence, motivation and patience as a mentor. His guidance over several exciting projects has helped me not only in learning new knowledge, but also in developing problem solving abilities. His thorough and careful attitude, together with his enthusiasm and persistence in research has inspired me and will continue beyond graduate school. I would also like to thank Drs. Venkat Gopalan and Psaras McGrier for serving on my committee, providing constructive and encouraging advice. Many thanks to the Bong group members, past and current, for creating a great environment to work in, for the helpful discussions and late night hangouts. Last by, I would like to thank my parents, father Huajun Mao and mother Shuqin Tao, for their endless support and unconditional love throughout the years that I was away from home. ii Vita 2004 to 2007 ..................................................Wuhu No.1 High School, Wuhu, China 2007 to 2011 ..................................................B.S. Chemistry, Tsinghua University, China 2011 to 2017 .................................................PhD. Chemistry, Department of Chemistry, The Ohio State University Publications Mao, J. and Bong, D*. (2015) “Synthesis of DNA-Binding Peptoids.” Synlett., 26, 1581- 1585. Piao, X.; Xia, X.; Mao, J. and Bong, D*. (2015) “Peptide ligation and RNA cleavage via an abiotic interface.” J. Am. Chem. Soc., 137, 3751-3754. Mao, J.; DeSantis, C. and Bong, D*. (2017) “Small molecule recognition triggers secondary and tertiary interactions in DNA folding and hammerhead ribozyme catalysis.” J. Am. Chem. Soc., DOI: 10.1021/jacs.7b05448. Fields of Study Major Field: Chemistry ii Table of Content Abstract ............................................................................................................................... ii Acknowledgments............................................................................................................... ii Vita ...................................................................................................................................... ii Table of Content ................................................................................................................ iii List of Tables ................................................................................................................... viii List of Figures .................................................................................................................... ix Chapter 1 : Structure and recognition of nucleic acids ...................................................... 1 1.1 Native nucleic acid structures .............................................................................. 2 1.1.1 DNA duplex .................................................................................................. 3 1.1.2 G quadruplex ................................................................................................. 3 1.1.3 RNA secondary structure .............................................................................. 4 1.2 Triple helical nucleic acids ................................................................................... 5 1.3 Peptide nucleic acid (PNA) for nucleic acid recognition ..................................... 6 1.4 Triplex forming artificial nucleobases ................................................................. 8 1.5 PNA with native α peptide backbone ................................................................. 10 iii 1.6 Melamine displaying bifacial peptide nucleic acid (bPNA) as allosteric switch 11 1.7 Small molecule nucleic acid binders .................................................................. 13 1.7.1 B-form DNA minor groove binders ............................................................ 13 1.7.2 G-quadruplex binders.................................................................................. 14 1.7.3 Mismatch sites targeting small molecules .................................................. 17 1.7.4 Small molecule RNA binders, aptamers, riboswitches ............................... 18 1.8 Small molecule induced dimerization ................................................................ 24 1.9 References. ........................................................................................................
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