The biochemistry of dry state DNA is of interest to the fields of forensics, ancient DNA, and DNA storage. The exact chemical nature of the degradation of the DNA molecule in the dry state has not been studied prior. If determined what chemical changes the DNA molecule undergoes, to what degree and in what time frame then protocols can be implemented to bypass the impact of this damage or to repair it when necessary. It is suspected that similar reactions occur to the dry state DNA molecule as does to the hydrated molecule. It cannot be assumed, however that these types of chemical processes occur to the same extent and at the same rates. In general the generic process of hydrolysis encompasses two important reactions, that of deamination and of base loss from the 2’-deoxyribose backbone. Base loss is believed to ultimately lead to chain scission. It is also suspect that reactive oxygen species (ROS) have an important role in the chemistry associated with DNA. Species such as hydroxyl radicals (OH•) and singlet oxygen (1O2) can lead to strand scissions and chemically modified bases. Throughout this project various techniques were used to determine damage to DNA and its molecular constituents under conditions leading to hydrolytic and oxidative damage. Novel techniques used in this study include ionpairing chromatography and denaturing HPLC (DHPLC) to measure glycosidic bond cleavage and strand breaks. The extent to which the macromolecule haemoglobin (Hb) can lead to oxidative damage of DNA in dried blood stains by acting as a Fenton chemistry catalyst was evaluated. Additionally the enzymatic activity of the extracellular nuclease from Alteromonas espejiana, BAL 31 was studied as it pertains to the degradation of single-stranded short homopolymeric oligonucleotides. This study serves as the basis for future, more in depth experimentation into the more specific nature of dry state DNA biochemistry. It was found that to a large extent the same degradation reactions (base hydrolysis, base modifications, and strand breaks) do occur in the dry state as in the hydrated state when heat and UV radiation are used as energy sources. Reaction rates indicate that base hydrolysis and deamination occur much more slowly, yet have the same energies of activation in both states. Single strand breaks of dry state duplex DNA occur with a half life of 24 ± 2 days and appears to occur in a mechanistic manner which could be of interest when attempting to repair such damage. In addition, base loss alone does not correlate with the extent of single strand breaks detected. Thermodynamic data can lead to the conclusion that DNA degradation in both dry and hydrated states is not a spontaneous process. It is also concluded that though the Hb molecule undergoes oxidative changes over time, these changes do not impact its ability to become a more aggressive Fenton reagent. However, the presence of Hb in the vicinity of DNA does create the opportunity for OH•induced damage to the deoxyribose sugar, and most likely the DNA bases themselves. This study also reveals that the general purpose BAL 31 nuclease commonly used in molecular genetics exhibits a hithertofore non-characterized degree of substrate specificity with respect to single-stranded DNA oligomers. Specifically, BAL 31 nuclease activity was found to be affected by the presence of guanine in ssDNA oligomers.

<p>The first part of the work describes a procedure of oligonucleotide purification using a reversed-phase cartridge. The developed method employs a very efficient yet mild oligonucleotide detritylation on the cartridge support allowing fast purification of oligonucleotides regardless of their 5´-modification. Thiol- and amino-modified oligonuc-leotides were detritylated and purified with the same high efficiency as non-modified oligonucleotides. The method enables fast, parallel and automated purification of many oligonucleotide probes that was not possible before. In combination with the method of removal of tritylated failure fragments oligonucleotides were produced with purity superior to that of oligonucleotides purified using RP HPLC.</p><p>In the second part of the present study a method of solid-phase RNA synthesis using 2´-tert-butyldithiomethyl (2´-O-DTM) is discussed. The stability of the DTM group during oligonucleotide assembly and deprotection in ammonia, together with its ability for rapid deprotection under mild conditions, allowed the synthesis of RNA with the quality similar to that of synthetic DNA oligonucleotides. The advantage of the 2´-O-DTM group is that it is completely orthogonal to all protecting groups used for the traditional solid-phase DNA synthesis. Therefore, the synthesis can be performed using a standard DNA synthesis procedure – no changes are needed for the product assembly. RNA oligonucleotides synthesized with retained 5´-terminal trityl group can be subjected to a cartridge-based purification using the procedure described in the first part of the study. The phosphoramidite synthesis was optimized for a large scale preparation and gives versatility for introduction of other alkyldithiomethyl groups according to the preference to their certain properties.</p><p>The third part of the thesis describes the synthesis of a dithiomethyl linker and its utility for reversible conjugation of oligonucleotides. A dithiomethyl group, cleavable under mild conditions, was introduced onto 3´-OH of tritylated nucleosides via 3´-O-methylthiomethyl derivatives. The influence of different alkyl substituents on the disulfide bond stability was investigated, and stable analogues were employed in oligosyntheses. Two applications were developed using the present linker: 1) purification of oligonucleotides linked to the solid support; and 2) cartridge-based purification of tritylated oligonucleotides having an additional hydrophobic group on their 3´- terminus.</p>

The first part of the work describes a procedure of oligonucleotide purification using a reversed-phase cartridge. The developed method employs a very efficient yet mild oligonucleotide detritylation on the cartridge support allowing fast purification of oligonucleotides regardless of their 5´-modification. Thiol- and amino-modified oligonuc-leotides were detritylated and purified with the same high efficiency as non-modified oligonucleotides. The method enables fast, parallel and automated purification of many oligonucleotide probes that was not possible before. In combination with the method of removal of tritylated failure fragments oligonucleotides were produced with purity superior to that of oligonucleotides purified using RP HPLC. In the second part of the present study a method of solid-phase RNA synthesis using 2´-tert-butyldithiomethyl (2´-O-DTM) is discussed. The stability of the DTM group during oligonucleotide assembly and deprotection in ammonia, together with its ability for rapid deprotection under mild conditions, allowed the synthesis of RNA with the quality similar to that of synthetic DNA oligonucleotides. The advantage of the 2´-O-DTM group is that it is completely orthogonal to all protecting groups used for the traditional solid-phase DNA synthesis. Therefore, the synthesis can be performed using a standard DNA synthesis procedure – no changes are needed for the product assembly. RNA oligonucleotides synthesized with retained 5´-terminal trityl group can be subjected to a cartridge-based purification using the procedure described in the first part of the study. The phosphoramidite synthesis was optimized for a large scale preparation and gives versatility for introduction of other alkyldithiomethyl groups according to the preference to their certain properties. The third part of the thesis describes the synthesis of a dithiomethyl linker and its utility for reversible conjugation of oligonucleotides. A dithiomethyl group, cleavable under mild conditions, was introduced onto 3´-OH of tritylated nucleosides via 3´-O-methylthiomethyl derivatives. The influence of different alkyl substituents on the disulfide bond stability was investigated, and stable analogues were employed in oligosyntheses. Two applications were developed using the present linker: 1) purification of oligonucleotides linked to the solid support; and 2) cartridge-based purification of tritylated oligonucleotides having an additional hydrophobic group on their 3´- terminus.