Customizing polyelectrolyte complex shapes through photolithographic directed assembly.

Polyelectrolyte complexes (PECs) form through the association of oppositely charged polymers and, due to their attractive properties, such as their mild/simple preparation and stimulus-sensitivity, attract widespread interest. The diverse applications of these materials often require control over PEC shapes. As a versatile approach to achieving such control, we report a new photolithographic directed assembly method for tailoring their structure. This method uses aqueous solutions of a polyelectrolyte, an oppositely charged monomer and a photoinitiator. Irradiation of these mixtures leads to site-specific polymerization of the ionic monomer into a polymer and, through this localized polyanion/polycation mixture formation, results in the assembly of PECs with 2-D and 3-D shapes that reflect the photoirradiation pattern. In addition to generating macroscopic PECs using photomasks, this photodirected PEC assembly method can be combined with multiphoton lithography, which enables the preparation of custom-shaped PECs with microscopic dimensions. Like other PECs, the custom-shaped structures formed through this photodirected assembly approach are stimulus-responsive, and can be made to switch shape or dissolve in response to changes in their external environments. This control over PEC shape and stimulus-sensitivity suggests the photopolymerization-based directed PEC assembly method as a potentially attractive route to stimulus-responsive soft device fabrication (e.g., preparation of intricately shaped, function-specific PECs through photolithographic 3-D printing).

Synthesis of damaged DNA containing the oxidative lesion 3'-oxothymidine.

Oxidative events that take place during regular oxygen metabolism can lead to the formation of organic or inorganic radicals. The interaction of these radicals with macromolecules in the organism and with DNA in particular is suspected to lead to apoptosis, DNA lesions and cell damage. Independent generation of DNA lesions resulting from oxidative damage is used to promote the study of their effects on biological systems. An efficient synthesis of oligodeoxyribonucleotides (ODNs) containing the oxidative damage lesion 3'-oxothymidine has been accomplished via incorporation of C3'-hydroxymethyl thymidine as its corresponding 5'-phosphoramidite. Through oxidative cleavage using sodium periodate in aqueous solution, the lesion of interest is easily generated. Due to its inherent instability it cannot be directly isolated, but must be generated in situ. 3'-Oxothymidine is a demonstrated damage product formed upon generation of the C3'-thymidinyl radical in ODN.

Photochemical Generation of a C5'-Uridinyl Radical.

It has been postulated that sugar radicals and related species are involved in oxidative events involving RNA. To determine the contribution, if any, of these species to the deleterious effects of the endogenous exposome, it is important to unambiguously identify their degradation products. C5'-Pivaloyl uridine was successfully synthesized and subsequently photolytically converted to a C5'-uridinyl radical. Generation of the radical under anaerobic conditions in the presence of glutathione led to the formation of the expected reduction product, uridine. However, regardless of the presence or absence of reductant, the base elimination product, uracil, was also observed. Mass balances and product distributions were dependent upon the pH of the photolysis mixture. At low pH, trapping with glutathione successfully competed with base loss. These results indicate that this precursor should function efficiently in an investigation of the fate of the C5'-uridinyl radical in RNA oligomers.

Thermodynamic and structural analysis of DNA damage architectures related to replication.

Damaged DNA, generated by the abstraction of one of five hydrogen atoms from the 2'-deoxyribose ring of the nucleic acid, can contain a variety of lesions, some of which compromise physiological processes. Recently, DNA damage, resulting from the formation of a C3'-thymidinyl radical in DNA oligomers, was found to be dependent on nucleic acid structure. Architectures relevant to DNA replication were observed to generate larger amounts of strand-break and 1-(2'-deoxy- ß -D-threo-pentofuranosyl)thymidine formation than that observed for duplex DNA. To understand how this damage can affect the integrity of DNA, the impact of C3'-thymidinyl radical derived lesions on DNA stability and structure was characterized using biophysical methods. DNA architectures evaluated include duplex DNA (dsDNA), single 3' or 5'-overhangs (OvHgs), and forks. Thermal melting analysis and differential scanning calorimetry measurements indicate that an individual 3'-OvHg is more destabilizing than a 5'-OvHg. The presence of a terminal 3' or 5' phosphate decreases the ΔG 25 to the same extent, while the effect of the phosphate at the ss-dsDNA junction of OvHgs is dependent on sequence. Additionally, the effect of 1-(2'-deoxy- ß -D-threo-pentofuranosyl)thymidine is found to depend on DNA architecture and proximity to the 3' end of the damaged strand.

The impact of structure on oxidatively generated DNA damage products resulting from the C3'-thymidinyl radical.

What's the damage? Trapping the C3'-thymidinyl radical in biologically significant architectures delivers both the repaired oligomer and 1-(2'-deoxy-ß-D-threo-pentofuranosyl)thymidine-containing substrates. The stereoselectivity of the reduction was found to be dependent upon the DNA structure.

Synthesis of C3' modified nucleosides for selective generation of the C3'-deoxy-3'-thymidinyl radical: a proposed intermediate in LEE induced DNA damage.

DNA damage pathways induced by low-energy electrons (LEEs) are believed to involve the formation of 2-deoxyribose radicals. These radicals, formed at the C3' and C5' positions of nucleotides, are the result of cleavage of the C-O phosphodiester bond through transfer of LEEs to the phosphate group of DNA oligomers from the nucleobases. A considerable amount of information has been obtained to illuminate the identity of the unmodified oligonucleotide products formed through this process. There exists, however, a paucity of information as to the nature of the modified lesions formed from degradation of these sugar radicals. To determine the identity of the damage products formed via the 2',3'-dideoxy-C3'-thymidinyl radical (C3'(dephos) sugar radical), phenyl selenide and acyl modified sugar and nucleoside derivatives have been synthesized, and their suitability as photochemical precursors of the radical of interest has been evaluated. Upon photochemical activation of C3'-derivatized nucleosides in the presence of the hydrogen atom donor tributyltin hydride, 2',3'-dideoxythymidine is formed indicating the selective generation of the C3'(dephos) sugar radical. These precursors will make the identification and quantification of products of DNA damage derived from radicals generated by LEEs possible.

The effect of reductant levels on the formation of damage lesions derived from a 2-deoxyribose radical in ssDNA.

Thiols play a major role in the outcome of oxidative damage to DNA when it is initiated through cellular exposure to ionizing radiation. DNA radicals formed under aerobic conditions are converted to peroxyl radicals through trapping by oxygen at a diffusion-controlled rate. As a primary source of cellular reductant, thiols are responsible for the conversion of these DNA-derived peroxyl radicals to their corresponding hydrogen peroxides and subsequent strand breaks. Through the use of modified nucleotides, which act as precursors to nucleic acid radicals, we have investigated the effect of varying amounts of the cellular thiol glutathione (GSH) on the distribution of damage products produced from a 2-deoxyribose radical in DNA: the C3'-thymidinyl radical. The C3'-thymidinyl radical results from the abstraction of a hydrogen atom from the C3'-position of DNA oligomers at a thymidine residue, and is known to deliver several DNA damage lesions including the 3'-phosphoglycolaldehyde, 3'-phosphoglycolate and a 5'-aldehyde. Here we show that the level of GSH present has an impact on the level of production of these C3'-thymidinyl radical derived damage products.

Aerobic fate of the C-3'-thymidinyl radical in single-stranded DNA.

Oxidative events that target the sugar-phosphate backbone of DNA can lead to reactive fragments that interfere with DNA repair, transcription and translation by the formation of cross-links and adducts of proteins and nucleobases. Here we report the formation of several such lesions through the aerobic degradation of an independently generated C-3'-thymidinyl radical in 2'-deoxyoligonucleotides. Individual fragments were identified by independent synthesis and comparison of retention times in high-performance liquid chromatography (HPLC) and/or matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-ToF MS) along with gel electrophoresis. The formation of this reactive intermediate in the presence of oxygen was found to produce 3'-phosphoglycolaldehyde (3'-PGA) as well as 3'-ketoenolether (3'-KEE), 3'-phosphoglycolate (3'-PG), and 5'-aldehyde terminated oligonucleotide fragments. Additionally, a significant outcome of C-3'-thymidinyl radical formation in DNA oligomers is a strand break resulting in one 3'- and two 5'-phosphate-terminated oligomers. These results suggest the involvement of several sugar derived reactive species upon C-3'-radical initiated scission of single-stranded DNA under aerobic conditions. The electrophilic nature of several of these products as well as their formation through a single oxidative event can make the presence of a C-3'-DNA radical more detrimental to the cell than products derived from more frequently occurring DNA sugar radicals.

Generation of a C-3'-thymidinyl radical in single-stranded oligonucleotides under anaerobic conditions.

[reaction: see text] A C-3'-thymidinyl radical has been photochemically generated site-specifically in DNA oligonucleotides. A nucleoside H-phosphonate bearing a C-3' acetyl group was incorporated into DNA oligomers using a hand-coupling technique. When nucleotides containing the modified monomer were photolyzed (> or =320 nm) in the presence of a hydrogen atom donor, reduction products were detected by RP-HPLC and MALDI-ToF MS analysis.