Nuclease resistance

The a-anomeric form of a 2 -deoxyribose, which has the base inverted with respect to the natural P-anomeric form, can be synthesized by using the phosphoramidite method sugar modification renders the derivatives nuclease-resistant. These analogues form parallel duplexes with complementary RNA... 

Another new modification is the 2 -deoxy-2 flouro-Darabinonucleic acid (2 F-ANA), which increases the strength of the oligonucleotide-mRNA hybrids, elicits efficient RNaseH-mediated degradation of the target, is more nuclease resistant and reaches high intracellular concentrations for prolonged time. Similar results could be obtained with oxetane modified ASONs. 

RNAi technology has obvious therapeutic potential as an antisense agent, and initial therapeutic targets of RNAi include viral infection, neurological diseases and cancer therapy. The synthesis of dsRNA displaying the desired nucleotide sequence is straightforward. However, as in the case of additional nucleic-acid-based therapeutic approaches, major technical hurdles remain to be overcome before RNAi becomes a therapeutic reality. Naked unmodified siRNAs for example display a serum half-life of less than 1 min, due to serum nuclease degradation. Approaches to improve the RNAi pharmacokinetic profile include chemical modification of the nucleotide backbone, to render it nuclease resistant, and the use of viral or non-viral vectors, to achieve safe product delivery to cells. As such, the jury remains out in terms of the development and approval of RNAi-based medicines, in the short to medium term at least. 

As to the stoichiometry of the H3-H4-DNA particle, two complexes were identified an H3-H4 tetramer and an H3-H4 octamer, each associated with about 140 base pairs of DNA. The complexing of 140 base pairs of DNA with H3 and H4 resulted in the formation of nucleosome-like particles, as observed by the EM, and reported to have an s20base pairs (Bina-Stein and Simpson, 1977 Bina-Stein, 1978). These results differ from those of Simon et al. (1978) who report that at least two complexes of H3 H4-DNA can be obtained upon reconstitution of H3, H4, and 150 bp DNA. In this experiment both an octamer and a tetramer of H3-H4 were found bound to 150 base pairs of DNA, having sM,w equal to 10.4 and 7.5 for the octamer and tetramer, respectively. The stoichiometry of the complexes obtained is dependent on the histone-to-DNA ratio. At low ratios of histone to DNA the predominant species contains an H3-H4 tetramer per 150 base pairs of DNA. At a histone-to-DNA ratio of 1 1 the octamer prevails. The nuclease and protease digestion experiments (Camerini-Otero et al., 1976 Sollner-Webb et al., 1976) were performed at a histone-to-DNA ratio of 0.5, conditions which for 140-base-pair DNA would lead primarily to a tetrameric complex. Therefore, it seems that a tetramer of H3 H4 is sufficient for the generation of nuclease-resistant fragments similar to those of complete nucleosomes. Upon addition of H2A and H2B to the tetrameric complex, nucleosomes are formed. Addition of H3-H4 to the tetrameric complex resulted in an octameric complex which is similar in compaction to nucleosomes. H3-H4 tetramers and octamers were similarly found complexed with about 140 base pairs of DNA upon reconstitution of H3-H4 with SV40 DNA. Both complexes were reported to be able to fold 140 base pairs of DNA (Thomas and Oudet, 1979). 

If smaller reaction products are present, purify the full-length RNA from the gel to avoid the selection and amplification of shorter sequences. Alternatively, for generation of nuclease-resistant transcripts substitute CTP and GTP by 2 -fiuoro- or 2 -amino-pyrimidines (TriLink BioTechnologies, San Diego, CA) at final concentrations of 1.2 mM. 

In the chromatin of eukaryotic cells DNA forms a coiled-coil structure with an approximately equal weight of a mixture of five basic proteins known as histones. Four of these histones in pairs form an octa-mer around which the DNA duplex occurs in a left-handed helix. The DNA octamer complex is called a nucleosome. Each nucleosome contains about 140 base pairs of DNA in a nuclease-resistant nucleosome core and approximately 60 base pairs of spacer between core particles. Histone HI binds to the chromatin independently of the octamer and is the first histone to dissociate from the chromatin when the ionic strength is raised. Beyond the nucleosome the higher order structure of the chromosome involves coiled-coil structures with varying degrees of regularity. 

The insufficient stability of RNA, mentioned above, limits the use of RNA aptamers. However, some established techniques yield nuclease-resistant aptamers. One alternative is called the Spiegelmer approach [29], another method involves incorporation of 2 -methoxy purine nucleotides [30]. 

Green, L.S., Jellinek, D., Bell, C., Beebe, L.A., Feistner, B.D., Gill, S.C., Jucker, F.M. and Janjic, N. (1995) Nuclease-resistant nucleic acid ligands to vascular permeability factor/vascular endothelial growth factor, Chem. Biol. 2, 683-695. 

Katayose, S. and K. Kataoka. 1998. Remarkable increase in nuclease resistance of plasmid DNA through supramolecular assembly with polyethylene glycol)-pD%(sine) block copolymerJ. Pharm. Sci. 

Matsufuji H, Shibamoto T (2004) The role of EDTA in malonaldehyde formation from DNA oxidized by Fenton reagent systems. J Agric Food Chem 52 3136-3140 McConlogue LC, Ward JF, Lewis HL, Norman A (1982) Radioimmune assay of induction and removal of UV lesions in total and staphylococcal nuclease-resistant DNA of mammalian chromatin. Radiat Res 89 381-395... 

Several other types of backbone modification have also been proposed, which produce nuclease-resistant oligos. Of these, a-oligos have been extensively studied. In a-oligos the base is transposed from the natural P-orientation to the unnatural a-orientation to form a parallel duplex with target sequence. This parallel duplex is nuclease-resistant, but does not elicit RNase H activity (Cazenave et al., 1989). These modifications have generated limited interest and application in antisense research. 

To enhance the cellular uptake and nuclease resistance of oligonucleotides, different terminal modifications at the 5 or 3 terminus of oligonucleotides have been attempted. Polylysine, avidin (such as acridine), and cholesterol have been used to improve cellular uptake and antisense effects of oligos (Nechers, 1989, 1993). However, the value of these approaches remains uncertain and needs to be further determined, especially in in vivo settings. 

Flory, C.M., Pavco, P.A., Jarvis, T.C., Lesch, M.E., Wincott, F.E., Beigelman, L. et al. (1996) Nuclease-resistant ribozymes decrease stromelysin mRNA levels in rabbit synovium following exogenous delivery to the knee joint. Proc. Natl. Acad. Sci. USA, 93, 754-758. Forster, A.C. and Altman, S. (1990) External guide sequences for an RNA enzyme. Science, 249, 783-786. 

Until recently, the phosphorothioate linkage had been the only modified linkage compatible with SELEX enzymes. But certain modifications at the 2 position, making RNA nuclease resistant, are accepted by polymerases. In particular, 2 -amino- and 2 -fluoro-ribopyrimidines (Figure 6.4) are substrates for the... 

In addition to increasing the nuclease resistance, the substitution at the 2 position of the deoxy-ribose has another interesting property it increases the molecular diversity of the pool by modifying the hydrogen-... 

Lee, S. and Sullenger, B. (1997) Isolation of a nuclease resistant decoy RNA that can protect human acetylcholine receptors from myasthenic antibodies. Nature, 15, 41 15. 

Harada, A., Togawa, H. and Kataoka, K. (2001) Physicochemical properties and nuclease resistance of antisense-oligodeoxynucleotides entrapped in the core of polyion complex micelles composed of poly(ethylene glycol)-poly (L-Lysine) block copolymers. Eur. J. Pharm. Sci., 13, 35—42. 

While isolation of a specific inhibitor will be necessary to assess the definitive role of the cytosolic nuclease in the low transfection efficiency in vivo, circumstantial evidence suggests that the metabolic instability of plasmid DNA represents one of the cellular barriers to gene transfer. Microinjection of DNA complexes with PEI has augmented the transfection efficiency (Pollard et al., 1998). Although the stability of the PEI-complexed DNA has not been determined in vivo, it has been demonstrated that the nuclease resistance of plasmid DNA is dramatically increased upon complex formation in vitro (Cappaccioli et al., 1993 Chiou et al., 1994 Thierry et al., 1997). Therefore, it is conceivable that faster diffusional mobility and decreased nuclease susceptibility jointly lead to the enhanced nuclear targeting efficiency of the PEI-condensed plasmid DNA. 

H. Sasmor, and H. Moser, E. 1996. Second generation of antisense oligonucleotides From nuclease resistance to biological efficacy in animals. Chimia 50 168-176. 

The cloned sequences often (but not always) have obvious secondary structures in common, leading to visual truncations by which the fixed sequences and other unnecessary sequences from the random regions are eliminated from the aptamer, often resulting in slightly increased affinity over the starting full-length aptamer. When visual truncation is not possible, experimental truncation is done instead. After truncation the aptamer may be further altered by substituting nuclease-resistant purines for the normal... 

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