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The reactions involving the 5-hexenyl or the 6-heptenyl radical cyclization of a distant double bond at C4' and the radical center at C2' of the ribofuranose ring of thymidine are used for synthesizing North-type conformationally constrained cis-fused bicyclic five-membered and six-membered carbocyclic analogues of LNA and ENA. Enhanced lifetimes have made the carbocyclic-LNA and carbocyclic-ENA modifications very good candidates as potential antisense therapeutic agents.

The molecular structures and dynamics of 1',2'-azetidine- and 1',2'-oxetane-fused thymidines, as well as their 2',4'-fused thymine (T) counterparts are determined to understand how the chemical nature of the conformational constraints of the sugar moiety in ON/RNA(DNA) dictates the duplex structure and reactivity. The results have shown that the nature of the sugar conformational constraints steer the physicochemical property of the constraint 1-thyminyl moiety, which plays a vital part in tuning the strength of hydrogen bonding in the basepairing.

The study describes the design, synthesis and phosphorylation pattern of the new bicyclo[3.1.0]hexane template that exhibits highly strong and increased chemical and enzymatic stability due to the absence of a true glycosyl bond. The template is found to be extremely rigid that makes it useful for sculpting other carbocyclic nucleosides, which also makes it easier to interact with the herpes simplex thymidine kinase.

Hybrid quantum mechanics/molecular mechanics methods are used to study the molecular mechanism of the thymidylate synthase-catalyzed reaction. It is revealed that there are several new features of substantial mechanistic interest in the complete multistep enzyme mechanism.

The synthesis of 2'-N,4'-C-ethylene-bridged thymidine (aza-ENA-T), physicochemical, nuclear magnetic resonance (NMR) and computational structural studies of aza-ENA-T and biochemical studies of aza-ENA-T containing oligonucleotides are reported. The aza-ENA-T modification in the antisense oligonucleotide (AON) has improved RNA target affinity, comparable RNase H cleavage rate and higher blood serum stability.

We examined the mechanism by which recombination between imperfectly matched sequences (homeologous recombination) is suppressed in mammalian chromosomes. DNA substrates were constructed, each containing a thymidine kinase (tk) gene disrupted by insertion of an XhoI linker and referred to as a 'recipient' gene. Each substrate also contained one of several 'donor' tk sequences that could potentially correct the recipient gene via recombination. Each donor sequence either was perfectly homologous to the recipient gene or contained homeologons sequence sharing only 80% identity with the recipient gene. Mouse L [tk.sup.-] fibroblasts were stably transfected with the various substrates and [tk.sup.+] segregants produced via intrachromosomal recombination were recovered. We observed exclusion of homeologous sequence from gene conversion tracts when homeologous sequence was positioned adjacent to homologous sequence in the donor but not when homeologous sequence was surrounded by homology in the donor. Our results support a model in which homeologous recombination in mammalian chromosomes is suppressed by a nondestructive dismantling of mismatched heteroduplex DNA (hDNA) intermediates. We suggest that mammalian cells do not dismantle mismatched hDNA by responding to mismatches in hDNA per se but rather rejection of mismatched hDNA appears to be driven by a requirement for localized homology for resolution of recombination.

A five-enzyme in vitro pathway is used for verifying that ORF36 has catalyzed oxidation of biogenic thymidine diphosphate (TDP)-L-epi-vancosamine and for determining whether ORF36 has displayed catalytic competence for any of its biosynthetic progenitors, which are candidate substrates for nitrososynthases in vivo. The studies have mapped substrate determinants and have described a minimal monooxygenase mechanism for amino sugar oxidation by ORF36.

A new class of conformationally constrained nucleosides, [alpha]-L-ribo-carbocyclic LNA thymidine ([alpha]-L-carba-LNA-T (LNA = locked nuclei acid) analogues and a novel ' double-locked' [alpha]-L-ribo-configured tetracyclic thymidine in which both the sugar puckering and glycosidic torsion are simultaneously constrained, are prepared by a key step involving 5-exo free-radical intramolecular cyclization. The improved nuclease resistances and efficient RNase H recruitment capabilities have elevated the [alpha]-L-carba-LNA-modified nucleotides into a new class of locked nucleic acids for RNA targeting therapeutics.

Two highly fluorescent, thermally stable diastereomeric photoadducts are formed when either 5-chloro-4-thiouridine (1), or 5-fluoro-4-thiouridine (2) are photoexcited with UV light in the presence of thymidine (T). The studies have shown that the geometry of the fluorescent photoadduct is consistent with formation of an interstrand cross-link in a DNA duplex if 1 or 2 is flanked by T in an opposite strand.

The photoelectron spectroscopy measurements in aqueous microjets were used to obtain vertical ionization energies of the nucleosides cytidine and deoxythymidine in water. Accurate aqueous-phase vertical ionization potentials provide a significant improvement to the corrected gas-phase values and could be applied to assess the threshold energies for photooxidation and oxidation free energies of solvent-exposed DNA components as well as delocalization and charge-hopping mechanisms in DNA ionized by radiation.

Nonpolar nucleoside analogues of systematically varied size and shape are used for probing the mechanisms by which the two human thymidine kinases (TK1 and TK2) recognize and phosphorylate their substrate, thymidine. The results have shown that nucleoside recognition mechanisms for the two classes of enzymes are very different and nonpolar nucleosides are likely to be active in the nucleotide salvage pathway in human cells.

The studies of comparative mutagenesis of [gamma]- or X-ray-induced tandem DNA lesions G[8,5-Me]T and T[5-Me,8]G intrastrand cross-links in simian (COS-7) and human embyronic (293T) kidney cells are presented. The results from in vitro bypass study of G[8,5-Me]T suggested that human polymerase [eta] (hpol [eta]) might be involved in the correct mutagenic bypass to incorporate dNMP opposite each cross-linked base.

A set of variably sized nonpolar thymidine and uracil are used as molecular rulers to probe the active site steric constraints of HIV-1 reverse transcriptase (RT) and the functional flexibility of these two activities are compared. The results have shown greater rigidity with a DNA template as compared with an RNA template, which has correlated directly with the higher fidelity of the DNA-templated synthesis.

The density functional theory treatment of a simple model for DNA, the nucleotide, 5'-thymidine monophosphate (5'-dTMPH) was employed to investigate the mechanisms of low-energy electron (LEE) attachment and subsequent single-strand break (SSB) formation. The results supports the hypothesis that transiently bound electrons (shape resonances) to the virtual molecular orbitals of the neutral molecule likely play a major role in the cleavage of the sugar-phosphate [C.sub.5']-[O.sub.5'] bond in DNA, resulting in the direct formation of single strand breaks without significant molecular relaxation.

Function and structure analysis investigates the Se derivatization on the nucleobases. The first synthesis of the 4-Se-thymidine phosphoramidite, its incorporation, and the thermostability and crystal structure studies of the DNAs containing 4-Se-thymidine changes, is described.

The photochemical behavior of the 3'-N-sulfamate analogue of thymidylyl(3'-5')thymidine (TnsoT) using a major C'3'-endo sugar pucker and an increased base stacking ability when compared to its natural counterpart thymidylyl(3'-5')thymidine (TpT) is investigated. The results have shown that the cyclobutane pyrimidine dimer (CPD) and the oligonucleotides containing (6-4) photoproduct surrogates arise from distinct minor stacked conformations.

A combination of optical and calorimetric techniques to investigate the incorporation of deoxythymidine ---> deoxyuridine (dT ---> dU) substitutions in the duplex and third strand of the parallel intramolecular triplex d(A(sub 7)C(sub 5)T(sub 7)C(sub 5)T(sub 7)) (ATT). The results suggest that the stabilizing effect of methyl groups might arise from the combination of both hydrophobic and electronic effect.

The first systematic test of steric effects in the active site of a Y-family DNA polymerase, Dpo4, is presented. The low steric selectivity of this enzyme is the result of a conformationally flexible or loose active site that adapts with small energetic costs to different base-pair sizes rather than a spatially large active site.