Oligonucleotides phosphorylation

For mutagenesis to introduce the stop codons, the protocols in Subheadings Oligonucleotide Phosphorylation with T4 Polynucleotide Kinase, Annealing of the Oligonucleotide to the Template, and step 1 of Enzymatic Synthesis of CCC-dsDNA can be scaled down by a factor of ten. The reaction product can be used directly to transform E. coli using any standard procedure. 

This amide, readily formed from an amine and the anhydride or enzymatically using penicillin amidase, is readily cleaved by penicillin acylase (pH 8.1, A -methylpyrrolidone, 65-95% yield). This deprotection procedure works on peptides, phosphorylated peptides, and oligonucleotides, as well as on nonpeptide substrates. The deprotection of racemic phenylacetamides with penicillin acylase can result in enantiomer enrichment of the cleaved amine and the remaining amide. An immobilized form of penicillin G acylase has been developed. ... 

As mentioned in last year s Report, aromatic phosphoramidates have been used to protect 5 -phosphoryl groups in the stepwise synthesis of oligodeoxyribonucleotides. The appropriate monomer units are coupled with DCC and the phosphoramidate protecting group is removed when required with isoamyl nitrite. A rapid and general preparative method for oligonucleotides has been developed based on phosphoramidates of the highly lipophilic 4-aminophenyltriphenylmethane (25). Purification of... 

The fact that the 3,P-NMR signal of 183a can only be observed in pyridine-containing solution provides food for thought124). Viewed in conjugation with the idea that alkyl metaphosphates could form adducts such as 173 and 174 U9,120) as discussed above, formulation as a zwitterionic pyridine/metaphosphate adduct (188) seems reasonable. Similar adducts have also been found in the reaction of TPS with dinucleotides and trinucleoside diphosphate 126). In any case, the reactions of 183 or 188 are in full accord with the expected properties of a monomeric metaphosphate and its reactivity towards alcohols is far greater than that of all other reactive phosphorylation intermediates which can arise on reaction of TPS with oligonucleotides 126). 

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. 

In an earlier study from Ken Beattie s group (then at the Houston Advanced Research Center), a similar conclusion was reached using a 9-mer oligonucleotide and triethylene glycol phosphoryl repeat unit as the spacer (1995). An optimal probe density leading to the greatest hybridization... 

The purchase of expensive phosphorylated oligonucleotides is unnecessary as the vector ends remain phosphorylated after the following ligation reaction. 

Another anti-herpes compound is the remarkably simple foscarnet (9.15, phospho-noformate). This is an inorganic pyrophosphate compound that inhibits viral DNA polymerase and RNA polymerase without requiring activation by phosphorylation. Fomivirsen is an oligonucleotide that inhibits human cytomegalovirus (CMV) via an... 

These considerations are consistent with the observation that 5 -phosphoryl oligo- and mononucleotides are inhibitory (3, 11), that the presence of a 3 -phosphate end group enhances cleavage of the adjacent bond of dinucleotides and oligonucleotides (11,22), and that endonucleolytic cleavage will readily occur in relatively random fashion in large oligonucleotides and polynucleotides. 

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. 

Spleen exonuclease is active on the 5 -OH oligonucleotides of both the ribo and the deoxyribo series. These are sequentially split from the 5 -OH end with formation of 3 -mononucleotides. It has been suggested that an enzyme-product intermediate may exist in the form of nucleoside-3 -phosphoryl-enzyme complex (3) since transfer of nucleoside-3 -phosphate to available 5 -hydroxyl functions (or other alcoholic functions) occurs at high substrate (or acceptor) concentrations 15, 18). 

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