Title

Part I Nucleic Acid Site-Selective Binding Studies of Isomers of Dihydrodioxin-Masked Ortho-Quinones as Potential Antitumor Drugs Part II The Role of Non-Watson-Crick Base Pairs in Stabilizing Recurrent RNA Motif

Date of Award

2012

Document Type

Dissertation

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Photochemical Sciences

First Advisor

Neocles Leontis, Dr

Second Advisor

Robert Wilson, Dr (Committee Member)

Third Advisor

Zhaohui Xu, Dr (Committee Member)

Fourth Advisor

Tracy Huziak-Clark, Dr (Committee Member)

Abstract

This work comprises two parts: The first focuses on pyrene dihydrodioxins as potential antitumor drugs. The compound comprises effective DNA intercalation agents that are masked ortho - quinones and exists in two enantiomeric forms. Reactive quinone can be released by visible irradiation which is especially useful in photodynamic thera0py. The binding properties of PDHD enantiomers to herring sperm DNA as well as to short synthetic oligomers have been studied by spectroscopic methods and biochemical methods. Both PDHD enantiomers bind to double helical DNA with a high affinity. PDHD II has a slightly larger binding constant (Kb = 2.3 ± 0.8 x”105 M-1) than PDHD I (Kb = 1.6 ±0.15 x”105 M-1). Upon addition of DNA to the PDHD rac, CD spectra change dramatically: these results, together with UV titration experiments, reveal that DHDs intercalate to DNA double helix. Ability of DHDs to stabilize DNA was confirmed by UV-melting studies. DHD II enhances the melting temperature of 10 bps DNA by 15° C and, more surprisingly, it provides Δ”Tm of almost 29 °C for 12-mer DNA. This type of DNA binding is unique in that, it implements both the hydrophobic bonding characteristic of many aromatic hydrocarbons and ionic bonding. It reduces the ionic repulsion of the negatively charge phosphate backbones, inhibiting the separation of the two duplex chains. In addition, this type of duplex stabilizer is also photochemically active in the oxidation of ds-DNA, as it can be concluded from our φX 174 plasmid DNA photocleavage assay. Studies conducted to determine if there is any preference of PDHDs for base sequencing showed that PDHDs highly selective for guanine residues. iv The second project denotes to investigation of roles of non-Watson-Crick base pairs in stabilizing of RNA structural motif on the example of sarcin-ricin motif. Particularly, we address the question, is there a selection for particular base combinations in structurally conserved motifs, depending on the temperature regime to which an organism adapts. At present, the Protein Data Bank (www.pdb.org) contains atomic resolution of ribosomal RNA structures of three bacteria, one archaeon and two eukaryal organisms. To identify relevant sequence substitutions to study, we examined sequence variations of sarcin-ricin in 3D structures and in carefully selected aligned 16S rRNA sequences representing a range of phylogenetic and ecological groups (Sweeny B. data Ms. Thesis, BGSU 2011). It was found that only isosteric variations of base pairs are presented. Moreover, trans-Hoogsteen/sugar (tHS) AG is the most frequent base pair occurred in thermophilic and hyperthermophilic 16S sarcin-ricin (S/R) motif sequences. Thermodynamic analysis of SRM variations found in organisms occupying different temperature niches as well as those presented in 3D structures indicated that a single non-CW mutation considerably contributes to overall stability of the motif. In almost all studied S/R motif variations dependence of Mg ions was observed. Given the different stabilities of the motifs we have studied their solution structures by using single stranded specific nucleases T1, A, and the helix specific nuclease V1. All isosteric or neutral base pairs substitutions do not interrupt the overall conformation. We demonstrated that the compositions of non-canonical interactions in sarcin-ricin RNA motifs are very important. By changing nucleotides at certain non-canonical positions we can control the stability, and perhaps function of large RNAs.