Cyclic oligonucleotides

Other known oligonucleotide analogs include oligomers in which the heterocyclic bases have been modified [155] or replaced by other types of compound [156], Solid-phase syntheses of hybrids of DNA with peptides [157-163], with carbohydrates [164,165], and with PNA (peptide nucleic acids, see Section 16.4.1.2 [166-168]) have also been reported, and several strategies have been developed that enable the preparation of oligonucleotides with a modified 5 - or 3 -terminus [169-174] or of cyclic oligonucleotides [122,175], Various techniques for the parallel solid-phase synthesis of oligonucleotides have been developed for the preparation of compound libraries [176-181],... 

There have been two reports on branched or cyclic oligonucleotides. A series of V and Y shaped DNA and RNA oligonucleotides related to splicing intermediates of S. cerevisiae actin pre-mRNA has been used to study the effects... 

Applications of cyclised oligonucleotides are varied. They have been used to produce artificial human telomeres by rolling circle DNA synthesis/as inhibitors of viral replication in influenza virus and as structural motifs for quadruplex formation.A further form of cyclic oligonucleotide figures in a recently described method in which a self-complementary oligonucleotide, e.g., a hairpin structure, is denatured and allowed to re-anneal in the presence of circular DNA such as a plasmid (7). The effect is that the short oligonucleotide traps the plasmid in what has been termed a padlock. Such structures have been successfully used to inhibit transcription elongation reactions based on triple helix formation of the padlock structure. 

Current methods in synthesis of cyclic oligonucleotides and analogues 12COC1371. 

On use of N,N -dicyclohexylcarbodiimide instead of sulfonyl chlorides as condensation reagent in oligonucleotide synthesis, then the pyro-, tri- and tetraphosphate stages are again involved 124). The metaphosphate 183 a is found in small amounts by 3iP-NMR spectroscopy, but again no cyclic trimetaphosphate 184 can be detected, which would also be a possible phosphorylation reagent. 

Investigation of thiol- and disulfide-modified oligonucleotides with either 25 or 10 bases, or base pairs immobilized on polycrystaUine and Au(lll) electrodes has also been carried out [171]. In these studies, several techniques were employed, including X-ray photoelectron spectroscopy, cyclic and differential pulse voltammetry, interfacial capacitance data, and in situ STM. 

The PCR is a three-step cyclic process that repeatedly duplicates a specific DNA sequence, contained between two oligonucleotide sequences called primers (154,155). The two primers form the ends of the sequence of DNA to be amplified and are normally referred to as the forward and reverse primers. The forward primer is complementary to the sense strand of the DNA template and is extended 5 to 3 along the DNA by DNA polymerase enzyme (Fig. 27). The reverse primer is complementary to the antisense strand of the DNA template and is normally situated 200-500 base pairs downstream from the forward primer, although much longer sequences (up to 50 kbase) can now be amplified by PCR. The process employs a thermostable DNA polymerase enzyme (such as the Taq polymerase from Thermus aqualicus BM) extracted from bacteria found in hot water sources, such as thermal pools or deep-water vents. These enzymes are not destroyed by repeated incubation at 94 °C, the temperature at which all double stranded DNA denatures or melts to its two separate strands (155). 

Figure 10.14 (a) Schematic representation of hybridization between ferrocene-conjugated oligonucleotide immobilized on gold electrode and its complementary strand, (b) Cyclic voltammograms recorded at v — 2(X) V/s and dependence of the anodic peak current on scan rate, normalized versus v1/2. Reprinted with permission from Ref. 76. Copyright 2003 American Chemical Society. 

AH fungal RNases (T, T , Ni, Ui, and U2) treated in this section catalyze the reaction shown in Fig. 1. The first step (phosphate transfer) is the cleavage of the phosphodiester bond between the 3 and 5 positions of the ribose moities in the RNA chain with the formation of nucleoside 2, 3 -cyclic phosphates and oligonucleotides with 2, 3 -cyclic phosphate at 3 terminal. The nature of the phosphodiester bonds to be cleaved depends on the base specificity of the enzyme. This phosphoryl transfer step is reversible. In the second step (hydrolysis), these terminal cyclic phosphate groups are hydrolyzed with the formation of corresponding 3 -phosphates. Because the first-step is usually faster than the second step, more or less accumulation of the cyclic phosphate may be observed. 

Ribonuclease U2 is a novel enzyme found in the culture broth of Ustilago sphaerogena (7, 106). Ribonuclease U2 splits, practically specifically, the phosphodiester bonds of purine nucleotides in RNA with the intermediary formation of purine nucleoside 2, 3 -cyclic phosphates, indicating the specificity is complementary to that of pancreatic RNase A (106). Like RNase N, RNase U2 very slowly hydrolyzes the intermediate, nucleoside 2, 3 -cyclic phosphate, to 3 -nucleotides (80, 106). Thus, RNase U2 is a useful tool, not only for the analysis of nucleotide sequences of RNA (90, 92, 107, 108) but also for the synthesis of various oligonucleotides containing adenylyl or guanylyl residue (30) (T. Koike, T. Uchida, and F. Egami, unpublished). 

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