Nucleases modification

Figure 8.7 Modification of oligonucleotides to increase stability, (a) Oligonucleotides (here shown as DNA) with a phosphodiester backbone (X = 0) are rapidly degraded by nucleases. Modification to create phosphorothioate analogs (X = S ) greatly increases half-life, (b) Peptide nucleic acids represent another DNA analog that can be used to bind with complementary sequences of oligonucleotides. Dashed lines represent hydrogen bonding which follows Watson-Crick base pairs.

Staphylococcal nuclease Modification of the single Trp-residue results in 50% loss of activity various nitro-phenylsulfenyl chlorides are used (290)... 

CuATRECASAS, P., S. FucHS, and C. B. Anfinsen Tyrosyl Residues at the Active Site of Staphylococcal Nuclease. Modifications by Tetranitromethane. J. Biol. Chem. 243, 4787-4798 (1968). 

Modification of the Phosphodiester Backbone. Oligonucleotides having modified phosphate backbones have been extensively studied (46). Because altering the backbone makes derivatives generally more resistant to degradation by cellular nucleases, these materials have the potential to be more resilient antisense dmgs. 

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. 

Aptamers appear to display low immunogenicity but, when administered systemically, they are quickly excreted via size-mediated renal clearance. In order to prevent renal removal, such aptamers are usually conjugated to PEG. PEG may also help further protect the aptamers from degradation by serum nucleases native aptamers are prone to nuclease attack, but their half-lives can most effectively be extended via chemical modification, as discussed earlier in the context of antisense agents. 

Depletion of histone HI after covalent modification from chromatin is a key step in eukaryotic transcription (Lee et al, 1993 Juan et al, 1994 Rice and Allis, 2001). A comparison of the association of the antibiotic Mg + complexes with the normal and HI depleted chromatin suggests that smaller ligands, like anticancer drugs, have better accessibility for HI depleted chromatin compared to native chromatin. HI depleted chromatin is also more prone to aggregation upon association with the complex I of the antibiotic Mg + complexes. It is also less accessible to micrococcal nuclease. We propose that HI depleted chromatin is a better target of these antibiotics compared to native chromatin. This observation is particularly significant in case of neoplastic cells where most of the cell nuclei are transcriptionally active, and, therefore, contain HI depleted chromatin. 

The knowledge of the primary structure was the basis for the construction of models of the secondary structure of the RNA molecules. Different approaches have been used in several laboratories to get experimental support for developing secondary structure models for example, chemic modification of the RNA, treatment with single- or double-strand-specihc nucleases, intramolecular RNA cross-linking, isolation and sequence analysis of double-stranded RNA, and, last but not least, comparison of ribosomal RNA sequences from different organisms (reviewed by Brimacombe et al., 1983). 

To avoid the problem of chirality and to improve the potency and limit the non-specific actions of AS-ODN, new compounds are required. Synthesis of new AS-ODNs has further improved their nuclease stability, enhanced of cellular uptake and affinity through modification of the base, sugar and phosphate moieties of the oligonucleotides [105-108],... 

In spite of the improved stability to nucleases, achieved through chemical modification, AS-ODN degradation in plasma still occurs, predominantly from the 3 -terminus. In the liver and kidney, the major sites of metabolism, AS-ODNs are degraded from the 5 -terminus as well [127,128]. 

Before the hnRNA produced by RNA polymerase II (see p. 242) can leave the nucleus in order to serve as a template for protein synthesis in the cytoplasm, it has to undergo several modifications first. Even during transcription, the two ends of the transcript have additional nucleotides added (A). The sections that correspond to the intervening gene sequences in the DNA (introns) are then cut out (splicing see B). Other transcripts—e.g., the 45 S precursor of rRNA formed by polymerase I (see p. 242)—are broken down into smaller fragments by nucleases before export into the cytoplasm. 

Selected entries from Methods in Enzymology [vol, page(s)] Inhibitory properties, 68, 212 inactivator, of ribonucleases, 65, 681 lipase modification, 64, 390 nuclease inactivation, 79, 63 ribonuclease inactivation, 79, 52, 112-113, 267. 

A host of enzymes, which are described elsewhere in the book, act on DNA and RNA. They include hydrolytic nucleases, methyltransferases, polymerases, topoisomerases, and enzymes involved in repair of damaged DNA and in modifications of either DNA or RNA. While most of these enzymes are apparently proteins, a surprising number are ribozymes, which consist of RNA or are RNA-protein complexes in which the RNA has catalytic activity. 

In addition to the cutting and trimming of precursors by nucleases, extensive modification of purine and pyrimidine bases is required to generate mature tRNAs 235 Some of these modification reactions are... 

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