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The N-(2-deoxy-d-erythro-pentofuranosyl)-urea DNA lesion forms following hydrolytic fragmentation of cis-5R,6S- and trans-5R,6R-dihydroxy-5,6-dihydrothymidine (thymine glycol, Tg) or from oxidation of 7,8-dihydro-8-oxo-deoxyguanosine (8-oxodG) and subsequent hydrolysis. It interconverts between α and β deoxyribose anomers. Synthetic oligodeoxynucleotides containing this adduct are efficiently incised by unedited (K242) and edited (R242) forms of the hNEIL1 glycosylase. The structure of a complex between the active site unedited mutant CΔ100 P2G hNEIL1 (K242) glycosylase and double-stranded (ds) DNA containing a urea lesion reveals a pre-cleavage intermediate, in which the Gly2 N-terminal amine forms a conjugate with the deoxyribose C1′ of the lesion, with the urea moiety remaining intact. This structure supports a proposed catalytic mechanism in which Glu3-mediated protonation of O4′ facilitates attack at deoxyribose C1′. The deoxyribose is in the ring-opened configuration with the O4′ oxygen protonated. The electron density of Lys242 suggests the ‘residue 242-in conformation’ associated with catalysis. This complex likely arises because the proton transfer steps involving Glu6 and Lys242 are hindered due to Glu6-mediated H-bonding with the Gly2 and the urea lesion. Consistent with crystallographic data, biochemical analyses show that the CΔ100 P2G hNEIL1 (K242) glycosylase exhibits a residual activity against urea-containing dsDNA.

In this report, we investigated the hexopyranose chemical modification Altriol Nucleic Acid (ANA) within small interfering RNA (siRNA) duplexes that were otherwise fully modified with the 2'-deoxy-2'-fluoro and 2'-O-methyl pentofuranose chemical modifications. The siRNAs were designed to silence the transthyretin (Ttr) gene and were conjugated to a trivalent N-acetylgalactosamine (GalNAc) ligand for targeted delivery to hepatocytes. Sense and antisense strands of the parent duplex were synthesized with single ANA residues at each position on the strand, and the resulting siRNAs were evaluated for their ability to inhibit Ttr mRNA expression in vitro. Although ANA residues were detrimental at the 5' end of the antisense strand, the siRNAs with ANA at position 6 or 7 in the seed region had activity comparable to the parent. The siRNA with ANA at position 7 in the seed region was active in a mouse model. An Oligonucleotide with ANA at the 5' end was more stable in the presence of 5'-exonuclease than an oligonucleotide of the same sequence and chemical composition without the ANA modification. Modeling studies provide insight into the origins of regiospecific changes in potency of siRNAs and the increased protection against 5'-exonuclease degradation afforded by the ANA modification. ispartof: NUCLEIC ACIDS RESEARCH vol:48 issue:8 pages:4028-4040 ispartof: location:England status: published

Chemical modification is a prerequisite of oligonucleotide therapeutics for improved metabolic stability, uptake and activity, irrespective of their mode of action, i.e. antisense, RNAi or aptamer. Phosphate moiety and ribose C2'/O2' atoms are the most common sites for modification. Compared to 2'-O-substituents, ribose 4'-C-substituents lie in proximity of both the 3'- and 5'-adjacent phosphates. To investigate potentially beneficial effects on nuclease resistance we combined 2'-F and 2'-OMe with 4'-Cα- and 4'-Cβ-OMe, and 2'-F with 4'-Cα-methyl modification. The α- and β-epimers of 4'-C-OMe-uridine and the α-epimer of 4'-C-Me-uridine monomers were synthesized and incorporated into siRNAs. The 4'α-epimers affect thermal stability only minimally and show increased nuclease stability irrespective of the 2'-substituent (H, F, OMe). The 4'β-epimers are strongly destabilizing, but afford complete resistance against an exonuclease with the phosphate or phosphorothioate backbones. Crystal structures of RNA octamers containing 2'-F,4'-Cα-OMe-U, 2'-F,4'-Cβ-OMe-U, 2'-OMe,4'-Cα-OMe-U, 2'-OMe,4'-Cβ-OMe-U or 2'-F,4'-Cα-Me-U help rationalize these observations and point to steric and electrostatic origins of the unprecedented nuclease resistance seen with the chain-inverted 4'β-U epimer. We used structural models of human Argonaute 2 in complex with guide siRNA featuring 2'-F,4'-Cα-OMe-U or 2'-F,4'-Cβ-OMe-U at various sites in the seed region to interpret in vitro activities of siRNAs with the corresponding 2'-/4'-C-modifications.

Euplotes eurystomus is a hypotrichous ciliate containing a transcriptionally active macronucleus (MAC) and a transcriptionally inactive micronucleus. Soluble MAC chromatin contains a normal complement of inner histones, an H1-like protein which is very sensitive to proteolysis, and a considerable proportion of non-histone proteins. A combination of N-Tosyl lysine chloromethyl ketone (TLCK) and PMSF was found to be most effective in preventing proteolysis. Microccocal nuclease digestion yielded an average nucleosome repeat length of 187 +/- 25 bp for soluble chromatin; and 190 +/- 9 bp for isolated macronuclei. Thermal denaturation profiles of MAC chromatin in 0.25 mM EDTA display two main transitions at about 76 and 83 degrees C, resembling the melting of soluble chicken erythrocyte chromatin. Circular dichroic spectra of MAC chromatin were compared to soluble chicken erythrocyte chromatin under the same ionic strength conditions and were found to be very similar. 2D chromatin/DNA agarose gel electrophoresis resulted in a diagonal line of DNA staining, which establishes a strict correlation between DNA and chromatin electrophoretic mobility.

Euplotes eurystomus is a hypotrichous ciliated protozoan possessing within one cytoplasm a transcriptionally-inactive micronucleus with chromosomal-size DNA and a transcriptionally active macronucleus with "gene-size" DNA fragments. The chromatin in the macronucleus can be isolated in a soluble form without prior treatment by nucleases. In this study, macronuclear, soluble chromatin was subfractionated using isokinetic sucrose density gradient ultracentrifugation in a buffer consisting of 50 mM NaCl, 1 mM Na2 EDTA, 1 mM TEA HCl, pH 7.0, 0.1 mM TLCK and 0.1 mM PMSF. Fractions were collected and analyzed by DNA and protein gel electrophoresis, dot blot hybridization with specific gene probes, and modified Miller chromatin spreads. Analysis of the chromatin spreads showed that the sizes of the chromatin fragments in the various fractions correlate with the DNA size of the fragments. When dot blots of the fractions were hybridized with 5S rRNA, tubulin and rRNA gene probes we obtained about a 6 to 14-fold enrichment of hybridizable sequences in individual fractions. There appear to be differences in the non-histones present on each fraction as well as some overall similarities in histone and non-histone proteins.